Bacteriology and Micology [311704]

Bacteriology and Micology

Course book for Medical Students

Section I: BACTERIOLOGY

GENERAL BACTERIOLOGY

1.INTRODUCTION IN BACTERIOLOGY

2.BACTERIAL MORPHOLOGY. CLASSIFICATION. BACTERIA CELL STRUCTURE

3.BACTERIAL GENETICS

4.BACTERIAL GROWTH. CULTIVATION OF BACTERIA

5.PATHOGENESIS OF BACTERIAL INFECTION

6.NON-SPECIFIC HOST RESPONSE

7.SPECIFIC HOST RESPONSE: IMMUNITY

8.ACTION OF PHYSICAL FACTORS UPON BACTERIA

9.ANTIBACTERIAL THERAPY. RESISTANCE TO ANTIBACTERIAL DRUGS.

10.NORMAL FLORA OF THE HUMAN BODY

SECTION I

BACTERIOLOGY

CHAPTER 1

INTRODUCTION IN MEDICAL MICROBIOLOGY

Definition: Microbiology is the study of microorganisms: organisms too small to be seen with the naked eye.

Microbiology covers several disciplines:

bacteriology (study of bacteria)

parasitology (study of parasites)

mycology (study of fungi)

virology (study of viruses)

[anonimizat] –pathogenic (saprophytic), [anonimizat] .

The main methods of studying microorganisms are:

microscopy

cultures

immunologic methods

genetic studies (molecular biology)

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Table1.1 : Similarities and differences between eukaryotic cells and prokaryotic cells

CHAPTER 2

BACTERIAL MORPHOLOGY AND STRUCTURE

Bacterial morphology and structure are studied by optical and electronic microscopy.

[anonimizat] 900 – 1000 X, so structures of 0.1 – 0.2 micrometers may be distinguish. Photon microscope has a light source, a capacitor that adjusts the intensity of light, a [anonimizat].

[anonimizat], allows viewing transparent bacteria that are not stained by the usual methods (treponema, leptospira)

Phase contrast microscopy allows differentiation between living cells and the death ones.

[anonimizat], used the visible and near visible.

Electron microscopy allows visualization of 0.001 micrometers structures (nanometers). The electron microscope uses electronic lenses: magnetic, [anonimizat] a [anonimizat]. Such a microscope has a resolution power 100,000 times greater than the optical microscope.

Bacterial size: [anonimizat] 0.2 micrometers and 10 micrometers , usually around 1-2 micrometeres in diameter.

[anonimizat] (Coccus (singular), cocci (plural)

Cylindrical – Rod (Bacillus (singular), Bacilli (plural)

Curved coma shaped), Helical, Spiral .

[anonimizat], in chains or in clusters. [anonimizat]:

Single

Pairs: Diplo –eg. Streptococcus pneumoniae ( former Diplococcus pneumonia)

[anonimizat]. [anonimizat]. [anonimizat]. [anonimizat] a bacilli and coccus

Pleomorphic bacteria are those bacteria that cannot retain proper shape or proper stain. These could be very old or very young bacteria.

Figure 2.1. Bacterial morphology. Source:

Bacterial cell, a prokaryotic cell type consists of ( Figure 2.2. ) :

Internal ( mandatory ) structures:

Nucleus ( nucleoid )

Cytoplasm and ribosomes ,

Membrane

cell wall

The external ( optional ) structure are:

5. capsule

6. Flagella

7. Pili ( fimbriae)

8. spore

Internal structures

Nucleus (Nucleoid)

prockaryotes do not possess a distinct membrane enclosing the nucleus. The DNA is aggregated in one area known as a nucleoid .

Nucleotides comprises one single circular molecule of DNA, folded (aggregated), which in the unfolded state has a length of 1 m . This DNA molecule is considered to form one chromosome . Its role is to codify genetic information of the cell. The DNA can be extracted from the bacterial cell by gentle lysis followed by centrifugation.

Electron microscopy images show about the DNA is attached in one point of a cell membrane invagination called mesosome , with role during cell division .

Plasmids

Beside Chromosamal DNA, bacteria posess circular extrachromosomal DNA, smaller and separate from chromosome capable of self replicating – the plasmids. Plasmids carry supplemental genetic information (antibiotic resistance (R-plasmid),production of toxins, mating capabilities (F-plasmid -> F-pilus), tolerance to toxic metals .

Cytoplasm/cytosol

The cytoplasm contains water (70-80%) and a large number of organic and inorganic moecules . It has a granular appearance and is strongly basophilic due to the large number of ribosomes and RNA molecules. Other organelles are absent. Bacterial ribosomes are RNA/protein bodies, 70 S ribosomes made up of two subunits: 50S and and 30S. They are the site of protein synthesis .

Frequently, in the cytoplasm of bacteria there are storage granules – energy and nutrient stores (starch , glycogen , sulphur, hydroxybutyric acid ) and vacuoles ( liquid and gaseous ) .

Some bacteria shows specific inclusion bodies (eg. Babes – Ernst corpuscles or granular poly- metaphosphate, specific for Corynebacterium diphtheriae ).

3. Cell membrane – plasma membrane or Cytoplasmic membrane is a phospholipid bilayer 4-5nm thick, phospholipid 30-40%, and protein 60-70%, organised as “fluid mosaique “ model. Some proteic molecules are protruding at the outer surface and others at the internal surface, and others are tranpass the membrane . Functional proteins are in a permanent mobility. Membrane invaginasion form functional structures called mesosomes . They anchor DNA during cell division

Functions of cell membrane are:

semipermeable barrier and active transport , concentrate intracellulary the nutritive substances;

secretion of extracellular enzymes and toxins;

oxidative phosphorylation as bacteria do not have mitochondria;

biosynthesis and export cell wall components (phospholipid biosynthesis;

during cell division the membrane takes part to the division septum formation and anchors DNA (by the mesosomes);

chemotactic response – receptors located wthin the cytoplasmic membrane.

The Cell Wall surrounds all eubacteria is a specific resistance structure , located on the outside of the cell membrane . The cell wall composition is made of a specific bacterial polymer – peptidoglycan . Peptidoglycan (murein) is a complex polymer consisting of chains of N-acetyl muramic acid (NAM) and N – acetyl glucosamine (NAG), attached by tetrapeptide side chains joined by short peptide chains . In the tetrapeptide structure enters the diaminopimelic acid , lysine precursor seen only in prokaryotes .

Gram – pozotive bacteria cell wall consists of 40-50 peptidoglycan layers and teichoici acids .

Gram negative bacteria wall consists of 1-2 layers of peptidoglycan , but there is also an external membrane made of phospholipids and lipopolysaccharides ( endotoxin ) . The space between the peptidoglycan and outer membrane is called the periplasmic space .

Lipopolysaccharide LPS , structure is known as endotoxin – include

Lipid A anchors LPS to outer membrane (toxic)

Core polysaccharide portion external to lipid A

Terminal polysaccharide – repeating subunits known as O-antigen only in some bacteria

The functions of the cell wall are multiple:

– ensures cell shape and size , providing rigidity and strength in the conditions of a high osmotic pressure inside the bacterial cell (prevents osmotic lysis )

-it has a role in cell division , seving as primer for its own biosynthesis , and take part in the formation of the division septum .

-Is is the seat of surface antigens important in the induction of immune response.

-Is contains enzymes

– the lipopolysaccharide of Gram negative bacteria serves as endotoxin .

Protoplasts , spheroplasts and L forms

The bacteria lacking cell wall, either after hydrolysis by lysozyme , or after the action of some antibiotics blocking the synthesis of the cell wall, in conditions of favorable osmotic pressure can survive in the form of protoplasts ( Gram -positive ) or spheroplasts ( Gram -negative bacteria, which retain completely the outer membrane) .

Bacteria that keep debris of peptidoglycan, usually after being exposed to penicillin, can divide once reinnserted in a favorable environment without antibiotic, and return to their normal form, being called L forms. Lforms may produce chronic infections , or relapsing infection.

Figure 2.2. Bacterial structure

Figure 2.3. Gram positive bacilli ( left) and Gram negative bacilli ( right)

Source:

Gram’s staining

Invented in 1884 by Hans Christian Gram. This is the most important staining technique for identifying and classifying bacteria.. .
Steps:

Flood with Crystal violet (primary stain) 3-5 minutes

Flood with Gram's iodine (mordant) 1 minute

Drop wise add decoloriser (acetone and alcohol) 25 seconds

Immediately rinse with water

Flood with fuchsine (counter stain) 1 minute

Gram Positive Bacteria stain Purple

Gram Negative Bacteria stain Red

External structures ( Facultative structures)

Cilia or Flagella / Flagellum (singular form) are filamentous formations that extends outwards from within the cell; long and thin , they have proteic structure, composed of subunits of contractile proteins called flagellins . They are found in bacilli and spirilla .

After the number and arrangement of flagella, bacteria can be monotrichous (single polar flagellum);   amfitrichous ( two flagella); lophotrichous ( a cluster of flagella at one end); peritrichous ( several flagella all over the bacteria);

The role of cilia is to provide the mobility of bacteria. Even though they are not ot essential for bacterial survival , flagella have an essential role for colonization ,contributing to virulence

Having a protein structures, hey have also antigenic properties (H antigens).

Pili (fimbriae)    are short and very thin filamentous structures , made ​​up of proteins called pillines, and are common among Gram negative bacteria . There are two types of pili :

A. Common pili , numerous on bacterial cell , with a role in adherence to surfaces and tissues, being pathogenicity factors;

B. Sex pili , present less frequently ( eg . E. coli ) . They have a role in bacterial conjugation , forming connections between cells ; e.g. F pilus is a holoow tube that serves for attachment followed by unidirectional transfer of genetic material from one cell to another . Pili are plasmid encoded .

Figure 2.4. Flagella. Source: www.pubs.rsc.org

Figure 2.4. Pili ( fimbriae ) and flagella. Source: www.pubs.rsc.org

The capsule (slime layer)

The capsule is an external coating synthesized by some bacteria living in the natural environment ( in favorable conditions). Most often it has a polysaccharidic structure (pneumococci, Klebsiella), but it may be polypeptidic (Bacillus anthracis), or made of hyaluronic acid (Streptococcus pyogenes).

The ”true capsule” forms a dense, well-defined cell wall surrounding.

The glycocalix consists of a network of polysaccharidic fibers (Streptococcus mutans)

The slime is a thin layer, of discontinuous capsular material present on the surface of some bacteria.

The capsule has a major role in the invasiveness of pathogenic bacteria via protection against phagocytosis and against the action of antibiotics. Loss of capsule decreases virulence.

The capsular antigens have an important role in bacterial identification and classification of bacteria upon serotypes (pneumococcus)

Glicocalix has major role in adhesion ( Str. Mutans, which plays an important role in the development of dental plaque and tooth decay, adhere to the tooth enamel with the glicocalix ).

Figure 2.5. Capsulated bacteria

The spore

Bacterial spore is a form of resistance , and also is the only form of differentiation in bacteria. The spore is metabolically dormant body, without multiplication capacity.

In adverse environmental conditions some bacterial species sporulate: the nucleus and adjacent cytoplasm surround by a resistant thick cover- the spore cortex-, waterproof, consisting of several coatings and rich in calcium and dipicolinic acid (5 to 10% ). These are the endospores initially formed . Afterwards they are released from the rest of the cell, which lysis, becoming exospores.

The low water content and the presence of calcium dipicolinate, give to the bacterial spore an increased resistance to extreme temperatures, dryness and disinfectant substances.

In favorable conditions, the spore grows and germinate, transforming again in a vegetative cell.

Endospores location and size

The endospores position inside the cell can be : terminal , subterminal or central, and by its size it can be larger than the bacterial body, deforming it or smaller than the bacterial body. Eg. Bacillus anthracis, Clostridium spp.

References

Jawetz, Melnick, & Adelberg – Medical Microbiology, 25-th Edition, 2010, ISBN-978-0-07-162496-1;

Lennette, E.H., Ballows, A., Hausler, W.J.Jr., and Shadomy, H.J. Manual of ClinicalMicrobiology.Washington D.C.: American Society for Microbiology.

Murray P., Kobayashi G., Pfaller M., Rosenthal K. Medical Microbiology. Mosby Ed.

Mihaela Botnarciuc Curs de bacteriologie pentru colegii universitare si facultatea de moase

CHAPTER 3

BACTERIAL GENETICS

Genetics is the study of the science of heredity.

Bacteria have two types of DNA: chromosomal and extrachromosomal (plasmids).

Bacterial chromosome

Bacteria are prokatyotes which have a single circular chromosome and associated proteins, looped to create domains, supercoiled and attached at several points to the plasma membrane. The chromosome is located in the nucleoid region and attached to the cell membrane.

Chromosome is aDNA structure that carry hereditary information, codified in genes: sections or segments of DNA, composed of nucleotide sequences that code for functional products such as RNA, which in turn are used to make a polypeptide that could be an enzyme or a structural protein.

Figure 4.1. Bacteria genome . Source:www.biology.kenyon.edu

DNA structure consist of nucleotide macromolecule (N-base + 5C sugar + phosphate) on 2 stranded helix. Base pairing rules is purine to pyrimidine ( A- T and G-C). Bacterial DNA replication is semiconservative (new strand with old/parent strand). DNA is unwind via DNA helicases. Hydrogen bonds are broken. Replication can be bi-directional, creates a “Y” shaped replication fork. Leading strand synthesized continuously by DNA polymerase in 5’ to 3’ direction. After the fragments of DNA are synthesized they are then joined together by DNA ligase. Hydrogen bonds form between the "old" and "new" strands. After replication, each copy binds to plasma membrane at opposite poles.

Figure 4.2. Replication of bacterial DNA

Source: www.quia.com/jg/1275308list.html

Plasmids

Plasmids provide 2% of genetic information, within 5-200 genes. Plasmids consist of circular, double stranded, extrachromosomal DNA. They are not essential for normal bacterial growth and they multiply independently. They may contain information about selective advantages since encode for proteins not coded by the nucleoid.

R-plasmids contain genes that code resistance to antibiotics. F- factors (fertility factor) promote the conjugation pilus. Virulence factors are found in many species and produce extracellular toxins (exotoxins, endotoxins) or bacteriocins (toxins for other bacteria).

Multiple or single copies of the same plasmid may be present in each bacterial cell. Different plasmids co-exist in the same bacteria.

Figure 4.3 DNA of the bacteria http://bio1151.nicerweb.com

Bacterial RNA and protein synthesis

Protein synthesis represents the process in which the DNA is translated into proteins. In the process called transcription , RNA polymerase binds to site on the promoter region on DNA template strand, copies in 5’ to 3’ direction to create mRNA ( messenger RNA) , assembles free nucleotides matching N-Bases. The process stops when a terminator sequence on the DNA is reached, and mRNA is released.

Translation is the process in which mRNA codons are read by rRNA in a 5’->3’ direction. It begins at start codon and ends at stop (non-sense) codon. tRNA anticodon is matched to the mRNA codon at the P site. The next tRNA moves into the A site of the ribosome. Aminoacids brought by the tRNA are joined by peptide bonds via dehydration synthesis from ribozyme in the 50S subunit of the ribosome.

Figure 4.4. Transcription and translation in bacteria http://testrunofreality.tumblr.com/post/30858989632

Because mRNA is produced in the cytoplasm in bacteria, transcription and translation can occur simultaneously.

Mutations

Mutations are due to errors during DNA replication that result in the changed sequencing of DNA bases. They can occur in regulator genes, structural genes, RNA genes and in noncoding genes.

That explains why the mutations can be:

Silent (neutral): the change in DNA sequence cause no change in product activity;

Point mutation: base substitution in DNA sequence does cause a change in the activity of the end product;

Transition: allow for substituting a purine for a purine (A for T and vice versa)or a pyrimidine for pyrimidine (C for G and vice versa);

Transversion mutations: allow for substituting a purine for a pyrimidin (or vice versa);

Frameshift mutation: deletion or insertion of one or more nucleotides shifts the reading frame off from normal triplet base pairs that creates inactive protein due to change in amonoacids sequence.

Spontaneous mutations usually happens during replication and occur in absence of mutating causing agents. Spontaneous mutations can be: transition, transversion, or frameshift deletions, replication errors, and loss of the nitrogen in the nitrogen base.

Figure 4.5. Spontaneous mutations http://bsw3.naist.jp/eng/courses/courses301.html

Induced mutations appear when DNA is exposed to mutagens: (chemical or physical). Chemical mutagens are nitrous acid, toxins (smoke, soot, mold). Physical mutagens are X-rays, gamma rays (free radicals damage DNA base pairs and prevent repair or break sugar –phosphate backbone), UV light.

Conditional mutations are expressed under certain environmental conditions (auxotrophs: mutants that cannon grow with minimal requirements and prototrophs: mutants that can grow with minimal requirements)

Transposons are larger DNA fragments which carry genetic information. They are also called integrons that can be inserted and accumulate in the plasmid or chromosome.

Spontaneous mutation rate (probability that a gene will mutate when it divides) is very low rates: 10-9, allow for adaptation to environment. Mutagen increases rate of mutation by possibly doubling rate (10-3).

Normal genetic make up provides “wild type” of bacteria. Mutation from prevalent gene give a ”forward mutation”.

They are repair mechanisms for mutations:

DNA polymerase : reads the complimentary strands for control;

Nucleases: excise damaged DNA, allow for new DNA to form replacement as complementary strand;

DNA ligase: joins DNA fragments together;

DNA glycolases: removes damaged or unnatural DNA bases.

Genetic transfer and recombination

There are two types of gene transfer:

vertical gene transfer (genes passed from an organism to its offspring), and

horizontal gene transfer (transfer from one bacterium to another in the same generation).

Transfered DNA can be chromosomal or plasmid.

Genetic recombination refers the exchange of genes between two DNA molecules to form a new combination of genes on a chromosome.

Movement of DNA from a donor bacterium to a recipient takes place three ways:

Transformation,

Conjugation,

Transduction.

1.Transformation takes place in 1‰ of bacterial population.

Transformation is usually it is seen after lysis of some bacteria, resulting DNA fragments.

Any portion of the genome may be transferred to other bacteria in solution or environment.

Tranformed cells express the new genes. Transformation depends on certain conditions: stage of growth (exponential phase), ability to secrete a competence protein. It occurs naturally in a few genera: Bacillus, Haemophilus, Neisseria, Acinetobacter, some Streptococcus and Staphylococcus, Pseudomonas, Moraxella.

The result of the process is an increase of the organism’s pathogenicity.

Figure 4.6. Transformation in bacteria

http://www. bacterial-transformation-diagram /wp-content/uploads/2012/11/.png

2.Conjugation is transfer of DNA from a living donor bacterium to recipient one in close contact. Conjugation is the major way in wich bacteria aquire additional genes. Conjugation is mediated by a F (fertility) factor either as a plasmid [F+] or incorporated into the donor chromosomes [Hfr- high-frequency recombination] cell. For plasmid conjugation, a F+ transfers its plasmid to a F- to make it F+.

Gram-negative bacteria use sex pilus and Gram-positive bacteria use sticky surface. The sex pili form a channel or conjugation bridge between the adjacent cells.

If the exchanged plasmid has genes that code for antibiotic resistance, it is a resistance plasmid conjugation. After conjugation, both donor and recipient make the complementary copy of the R-plasmid.

Conjugation occurs between various genera: E.coli – Shigella or Salmonella; Serratia – Salmonella. The purpose of the conjugation is to increase the organism’s pathogenicity or virulence (Antibiotic resistance, adherence proteins, enterotoxin production), metabolic changes (production, enzyme degradation of substances, fixation of nitrogen), fertility factor (sex pili for future conjugation).

Figure 4.7. Conjugation in bacteria

http://www.scienceprofonline.com/microbiology/bacterial-genetics-plasmid-dna-conjugation-gene-transfer.html

Transduction is when bacterial DNA is transferred from a donor to recipient via a virus that infects the bacteria , viruses called bacteriophage or phage.

For this process, they are called transduction phages.

Phage can code for certain toxins produced by their bacterial hosts.

Replication cycle of the bacteriophage occurs one of two ways:

Lytic phase: virulent phages infect bacteria, replicate and lyse bacteria.

Lysogenic phase : temperate phages infect bacteria, replicates at later time. Cells appear normal even with viral replication. Incorporated viral genome is called a prophage.

.

Figure 4.8. Transduction in bacteria

http://www.quia.com/jg/1275308list.html

Phage attaches to bacterial cell, injects DNA into bacteria which acts as template for the synthesis of new phage proteins: capsid proteins and enzymes.

These enzymes can break bacterial DNA and some of it is incorporated into new phage protein capsids, so that phage DNA carries bacterial DNA instead of phage DNA.

Released phage can infect another bacteria and transfer bacterial genes.

Genetic engineering

Genetic engineering is the manipulation of the genetic material in the laboratory. The process consists of inserting genes of interest into bacterial DNA.

Figure 4.9. Production of insulin by genetic engineering

http://www.bbc.co.uk/bitesize/standard/biology/biotechnology/reprogramming_microbes/revision/2/

Enzymes used are:

Endonucleases: enzymes that cleave sugar-phosphate bonds;

Restriction enzymes: act at particular sequences of nucleotide bases usually 4, 6, or 8 base pairs long with staggered "sticky" ends;

DNA ligase: used to rejoin DNA pieces;

Reverse transcriptase: produce DNA copies from RNA genome.

Purpose of the genetic enineering is:

Production of vaccines,

Production of antibiotics,

Production of hormones – insulin, somatostatin, human growth hormone,

Production of interferon,

Production of vitamins,

Production of aminoacids.

CHAPTER 4

BACTERIAL PSYSIOLOGY. BACTERIAL GROWTH. CULTIVATION OF BACTERIA

Bacterial division

Prokaryotes use a relatively simple form of cell division – binary fission. Conceptually this is a simple process; a cell grows to twice its starting size and then split in two. But, to remain viable and competitive, a bacterium must divide at the right time, in the right place, and must provide each offspring with a complete copy of its essential genetic material. Before binary fission occurs, the cell must copy its genetic material (DNA) and segregate these copies to opposite ends of the cell. Then the many types of proteins that comprise the cell division machinery assemble at the future division site.[ micro.cornell.edu].

Figure 3.1. Binary fission.Source: https:// micro.cornell.edu/ research/ epulopiscium/binary-fission-and-other-forms-reproduction-bacteria

As division occurs, the cytoplasm is cleaved in two, and new cell wall is synthesized. The order and timing of these processes (DNA replication, DNA segregation, division site selection, invagination of the cell envelope and synthesis of new cell wall) are tightly controlled.

Figure 3.2. Bacterial division. Source: Murray: Medical Microbiology

The bacterial increase in vivo differs from that in vitro and it is influenced by factors such as:

– The patient's nutritional status;

– Factors humoral and cellular defense (immunoglobulines, complement);

– Host enzymes (protease, hyaluronidase);

– Environmental pH; temperature, atmosphere – O2 & CO2, H-ion concentration

– Availability of Nutrients & H2O

– Moisture or drying

– Osmotic pressure

– Radiation

– Mechanical & sonic stress.

Bacterial growth

Bacterial growth means increase in bacterial number.

It is an increase in all the cell components, which ends in multiplication of cell leading to an increase in population. It involves – an increase in the size of the cell and an increase in the number of individual cells.

The time required for a bacterial cell population to divide and double its number is called generation time or population doubling time

It can also be described as the time required for a bacterium to give rise to 2 daughter cells under optimum conditions.

Most bacteria, considered rapid growing bacteria, have a generation time of about 20 minutes. On solid media in vitro they grow producing visible colonies in 18-24 h. Slow growing bacteria have a generation time of about 20 hrs. On solid media in vitro they grow producing visible colonies in 2-8 weeks.

In liquid media, provided with all necessary nutriments, pH, temperature and oxygen or other factors, a typical bacterial growth curve may be measured.

When a bacterium is added to a suitable liquid medium and incubated, its growth follows a definite course. If bacterial counts are made at intervals after inoculation and plotted in relation to time, the result is the growth curve . Five phases are clearly seen:

Lag,

Log or Exponential,

Stationary

Decline and

Death

The LAG phase: bacteria do not divide immediately, but initially undergo a period of adaptation to the new environment, with active macromolecular synthesis. There may be an increase in the size of the cell, and the maximum cell size is reached towards the end of lag phase.

LOG /EXPONENTIONAL phase. Cell division is in constant rate, bacteria are metabolically very active, have typical characteristics and stain uniformly.

STATIONARY phase is reached when one or more essential nutrients become insufficient in the same time with toxic metabolic products accumulation. The number of dividing cells is equal to dead cells. Metabolism becomes slower. Irregular staining, sporulation and production of exotoxins and antibiotics are characteristic.

DECLINE. The number of deaths of deaths exceeds the number of new formed cells. Bacterial lysis and destruction cause accumulation of toxic products and autolytic enzymes.

DEATH phase. The majority of bacteria have died, but some survivors may persist

Figure 2.3 Growth curve of bacteria

Bacterial nutrition

Bacteria, like all cells, require nutrients for the maintenance of their metabolism and for cell division.

Essential nutrients (basic bioelements needed for bacterial cell growth):

H2O: universal solvent; hydrolyzing agent;

Carbon is an important food and energy source; in form of proteins, sugars, lipids;

Nitrogen is needed for protein synthesis andnucleic acid synthesis (purines and pyrimidines);

Sulfur (sulfate): aminoacids synthesis (cystine);

Phosphate: a key component of DNA, RNA, ATP, inner and outer membrane phospholipids;

Minerals are common component of enzymes.

Water constitutes 80% of the total weight of bacterial cells. Water is also the essential ingredient of bacterial protoplasm, hence drying is lethal to cells. Effect of drying varies : streptococci, pneumococci, Treponema. pallidum are highly sensitive, staphylococci are resistant and stand for months. The spores are resistant to desiccation and may survive for several decades. Dehydration by lyophylisation ( freeze-dry) is a method of preservation bacterial strains for long term

Proteins, polysaccharides, lipids, nucleic acids, mucopeptides and low molecular weight compounds make up the remaining 20% of the bacterial cell weight.

Some bacteria require certain organic compounds in minute quantities – Growth factors or bacterial vitamins. It can be :

Essential – when growth does not occur in their absence. The organism is unable to synthesize essential growth factors from available nutrients.

Accessory – when they enhance growth, without being absolutely necessary for it.

Essential factors may be:

purines and pyrimidines: required for synthesis of nucleic acids (DNA and RNA),

amino acids: required for the synthesis of proteins,

vitamins: needed as coenzymes and functional groups of certain enzymes: p-Aminobenzoic acid (PABA), biotin, nicotinic acid, pantothenic acid, riboflavin (B2), thiamine (B1), vitamin B12 (Cobalamine), vitamin K.

Bacteria require a source of energy.

Organisms that use radiant energy (light) are called phototrophs.

Organisms that use (oxidize) an organic form of carbon are called heterotrophs or (chemo)heterotrophs.

The carbon requirements of organisms must be met by organic carbon (a chemical compound with a carbon-hydrogen bond) or by CO2.

Organisms that use organic carbon are heterotrophs and organisms that use CO2 as a sole source of carbon for growth are called autotrophs.

Most human associated bacteria are chemoheterotrophs or heterotrophs. They use organic compounds both as energy source and as carbon source.

Primary gases = O2, N2, & CO2

O2 – greatest impact on microbial growth (even if the microorganism does not require it). Oxygen is readily converted into radicals (singlet oxygen, superoxide, hydrogen peroxide, hydroxyl radical). Most important detoxifying enzymes are superoxide dismutase and catalase.

Cells differ in their content of detoxifying enzymes and hence, ability to grow in the presence of oxygen. Classification of bacteria upon the respiration type (oxygen needs) is of a major importance :

Aerobic respiration – terminal electron acceptor is oxygen.

Anaerobic respiration – terminal electron acceptor is an inorganic molecule other than oxygen (e.g. nitrogen).

Strict (Obligate) Aerobic bacteria – grow only in the presence of O2. They require 20% O2 for survival and growth e.g. Pseudomonas aeruginosa.

Strict (Obligate) Anaerobic bacteria –grow in the absence of O2 and may even die on exposure to O2 e.g. Bacteroides fragilis

Aerobic Facultative Anaerobic bacteria – grow both in presence and in absence of O2; but grow best under aerobic conditions; e.g. Staphylococcus spp, Enterobacteriaceae.

Microaerophile – 4% O2: best growth with small amount O2

e.g. Campylobacter spp, Helicobacter spp. Aerotolerant anaerobe: anaerobes that “tolerate” or survive in O2, but do not utilize O2

Capnophilic organism – requires high CO2 levels eg Neisseria spps. Slow-growing or fastidious organisms may not generate enough carbon dioxide so that this must be supplied exogenously. Many pathogenic bacteria therefore require the addition of 5-10% carbon dioxide to the incubator atmosphere.

Figure 2.4. Clasiffication of bacteria acording on oxygen requirments

Temperature requirements of bacteria vary among species. Growth does not occur above the maximum or below the minimum, which is the temperature range. The optimum temperature favors the best growth and is around 37șC for most pathogenic bacteria. Classification of bacteria according to the temperature range:

Psychrophiles: -10 to 20C.

Mesophiles: 10 to 48C e.g. most bacterial pathogens.

Thermophiles: 40 to 72C.

Most bacteria require an isotonic environment or a hypotonic environment for optimum growth.

Osmotolerant – organisms that can grow at relatively high salt concentration (up tp 10%).

Halophiles – bacteria that require relatively high salt concentrations for growth, like some of the Archea that require sodium chloride concentrations of 20 % or higher.

Majority of bacteria are neutrophiles and grow best at neutral or slightly alkaline pH

pH 7.0 – 7.4 , like most normal body fluids.

Acidophiles are bacteria that grow best at low pH (acid: pH 0 – 1.0) M. tuberculosis. – pH 6.5-6.8.

Alkalophiles: grow best at high pH (alkaline: pH 10.0) V. cholerae – pH 8.4-9.2.

CULTIVATION OF BACTERIA

Bacteria can be isolated from clinical specimens by cultivation „in vitro”, in artificial culture media and incubated in specific conditions of temperature and atmposphere, a specific period of time.

Properties of Culture Media:

Must be nutritive , containing the required amount of nutrients proteins, carbohydrates, lipids, etc.

Must contain mineral salts – chlorides, sulphate, phosphates, carbonates of K, Ca, Mg

a proper moisture, water being indispensable for bacterial survival

a suitable pH – , generally of 7,2-7,4

accesory growth factors for some species ( eg. X & V factors for Haemophilus influenzae)

should be sterile (initially)

Classification of Culture Media

According to Consistency

Three physical forms are used:

liquid media- − water-based solutions that do not solidify at room temperature (eg.: Nutrient broth, Peptone solution);

semisolid media – used to determine the motility of bacteria and to localize a reaction at a specific site (ex.: Cystine trypticase agar medium);

solid media

– provide a firm surface

– usually they are used as: slants, stabs or poured in plates ( petri dishes. Eg: Nutrient agar, Blood agar

Both solid and semisolid media contain a solidifying agent (usually agar), whereas a liquid medium does not. Agar is an extract of seaweed that melts at 100ͦ C and solidifies at about 42ͦ C. Most pathogenic bacteria prefer to grow at 37ͦ C so agar allows for a solid medium at incubator temperatures.

According to complexity

Simple media – consists of only basic nutrients; they are used to culture non-fastidious bacteria (agar plate; nutrient broth; peptone water)

Complex media – for nutritionally more exigent bacteria, these media contain blood, serum, tissues extracts, etc.

According to application in microbiology

Usual media – for the general cultivation of bacteria (blood – agar plate, broth )

Transport media – media in which specimens are transported without changes in the microflora structure, eg. Carry Blair medium for the enteric flora, Amies medium, Stuart medium

Enrichment media – used to favourise a desired organism from a specimen with mixed flora , specially the growth of fastidious bacteria. Eg Alkaline Peptone Water , an enrichment media for Vibrio cholerae

Selective and differential media

Selective media –contain substances that inhibit the growth of unwanted organisms but permit the growth of the desired organism; uses certain dyes, high salt concentration, pH or antibiotics

Eg. Lowenstein-Jensen Medium, a Selective media for isolation of Mycobacterium tuberculosis

Differential media – supports the growth of several types of microorganisms, but it is designed to emphasize the differences among these microorganisms; contains a combination of nutrients, indicators and and pH indicators to visually differentiate bacteria

Examples of selective and differential media:

MacConkey agar (selects against gram-positives; for the selection of the Enterobacteriaceae and related gram-negative bacilli; highliht the lactose fermentation by changing the colour from light orange into red)

Bile Esculin Agar ( contains 40% bile, being a selective medium for the genus Enterococcus . Also tests the ability of organisms to hydrolyze esculin in the presence of bile by changing the coclou from brown to black.

Biochemical Test Media – used to isolate and identify bacteria from pure cultures; contain a sugar ( glucose, lactose, sucrose, etc.) or another biochemical substance such as: indole, methyl red, citrate , urea, and a colour indicator. Ex.: TSI (triple sugar iron), MIU (Motility Indole, Urea), Simmons citrate agar.

Culture media for anaerobic bacteria – are nutritional base media used for the cultivation of anaerobic bacteria (will grow only in the absence of O2) from clinical specimens They are liqiud media like broth containig Thioglycollate (0.1 %) or solid media – such as Schaedler blood agar

Media for antibiotic susceptibility testing – Mueller-Hinton agar

Storage media used for preseration of bacterial strains for a longer period of time.

Inoculation of culture media and bacterial growth

The artificial induction of microorganisms into a medium is called inoculation, aseptic technique for the transfer and maintenance of culture.

After innoculation, the cultures are incubated at a proper temperature, generally 37 O C, in aerobic , anaerobic or microaerophylic environment.

Inoculation of liquid media (broth) may be done with a sterile loop or with a pipette, if the specimen is also liquid. Growth of bacteria in liquid culture media is shown by increasing the turbidity of the medium.

Inoculation of solid media may be done by different techniques, most used being plateing by the streak plate technique or by the spread plate technique for culture media in Petri dishes. Inoculation is performed on the surface of the solid medium. Unlike cells in a liquid medium, cells on a solid medium are immobilized. Spreading the bacteria cells over the solid surface and then allow sufficient time so that each cell divides until it produces a colony of cells, a clone, that is visible to the naked eye .

For solid media in tubes, it is used to inoculate a deep , stabbing the loop in the middle of the deep and removing it through the same stab. Inoculating an agar slant means to spread the specimen with a loop using a zig-zag motion along the surface of the sterile agar slant from the bottom of the slant up to the top .

Isolation of anaerobic bacteria

On solid media

The anaerobic jar is the most common technique for maintaining an oxygen-free environment for the anaerobic culture. It is a glass or plastic jar with a tightly fitting lid.

Anaerobiosis is realised using a commercially available hydrogen and carbon dioxide generator envelope (GasPak) that is placed in the jar along with the culture plates. The generator is activated with water. Carbon dioxide, which is also generated, is required for growth by some anaerobes

An alternative method for achieving anaerobiosis in the jar consists of evacuation and replacement. Air is evacuated from the sealed jar containing the culture plates and is replaced with an oxygen-free mixture of 80 percent nitrogen, 10 percent hydrogen, and 10 percent carbon dioxide.

Bacteria  grow on solid media as colonies.  A colony is defined as a visible mass of microorganisms originating from a single mother cell, therefore a colony constitutes a clone of bacteria and are genetically alike.

Although bacterial colonies have many characteristics, there are a few basic elements that you can identify for all colonies:

The basic shape of the colony is circular, with regular or iregular edges.

The surface is an important character, and may be smooth (S), mucoid (M), rough (R), glistening (G).

Elevation – the cross sectional shape of the colony

Transparency – transparent (clear), opaque, translucent (like frosted glass), opaque,

Pigmentation – colourless or pigmented: white (Staphylococcus epidermidis), yellow ( Staphylococcus saprophyticus) etc.

Other characters: odor, consistency, emulsifiability

On blood agar medium, an important character is the presence or absence of haemolysis around the colonies

www. micro.cornell.edu/ research/ epulopiscium/ binary-fission-and-other-forms-reproduction-bacteria].

CHAPTER 5

BACTERIAL PATHOGENICITY

Bacteria-human host relationship

A pathogen is a microorganism that is able to cause disease in a host.

Infection means multiplication of a microorganism ( bacteria) in the organism of the host. It is followed or not by disease, depending on the outcome of dynamic relationship between:

A. Pathogenicity and virulence of the microorganism

B. Defence mechanisms of the host

A. Pathogenicity is the ability to produce a disease in the host organism which is in a conventional (normal) state of susceptibility or resistance . Pathogenicity is an atribute of a species.

Microorganisms express their pathogenicity by means of their virulence, a term which refers to the degree of pathogenicity of a specific strain. The determinants of virulence in a pathogen bacteria are any of its genetic, structural or biochemical features that enable it to produce disease in a host.

The determinants of virulence are:

1. Invasiveness is the ability to invade tissues. It encompasses

– mechanisms of colonization; adherence factors and initial multiplication capacity resulting microcolonies. Common pilli and bacterial capsule, hydrophobicity of bacterial surface and binding molecules corresponding to various tissue cell receptors play the most important role in adherence.

– production of extracellular substances which facilitate invasion (enzymes, invasins) : Hemolysins, leukocidins, Hyaluronidases, collagenase, coagulase, fibrinolysin, DNA-ase, which are tissue degrading enzymes . Beta – lactamases degrade beta-lactamic antibiotics. However, the mechanisms of bacterial invasiveness are not fully understood, and some bacteria can produce only or mainly lochalised infections while other bacteria are able to determine systemic or generalised infections.

– ability to bypass or overcome host defense mechanisms : antigenic heterogenicity and ability to make shift in the sutface antigens

2. Toxigenesis is the ability to produce toxins. Bacteria may produce two types of toxins : exotoxins and endotoxins.

Exotoxins are released from bacterial cells, enter general blood circulation and may act at specific tissue targets faraway from the site of bacterial growth. Usually, virulent strains of the bacterium produce the toxin (or toxins) while nonvirulent strains do not, such that the toxin is the major determinant of virulence. Both Gram-positive and Gram-negative bacteria produce soluble proteic toxins.

Endotoxins are cell-associated substances.  (lipopolysaccharide component of the outer membrane of Gram-negative bacteria).  They are released from cells that are lysed as a result of effective host defense (e.g. lysozyme) or the activities of antibiotics. Endotoxins may be transported by blood and lymph and cause general effects such as fever, leukopenia, disseminated intravascular coagulation , hipotension, shock.

Source www.slideshare.net

Bacterial exotoxins: are proteins, consist of two components:

subunit A is responsible for the activity of the toxin;

subunit B is concerned with binding to a specific receptor on the host cell membrane and transferring the enzyme across the membrane.

are denatured by heat, acid, proteolytic enzymes

have a high biological activity

The production of protein toxins is generally specific to a particular bacterial species (e.g. only Clostridium tetani produces tetanus toxin; only Corynebacterium diphtheriae produces the diphtheria toxin).

are highly specific in their target cells and in their mode of action. The denominations such as "enterotoxin", "neurotoxin", "leukocidin" or "hemolysin" are used to indicate the target site of some exotoxins. Certain exotoxins have very specific cytotoxic activity (for example, tetanus or botulinum toxins), or pore-forming hemolysins and leukocidins.

As non-self (foreign) substances to the host, most of the protein toxins are strongly antigenic. In vivo, specific antibody (antitoxin) neutralizes the toxicity of these bacterial proteins. Protein toxins are heat unstable: in time they lose their toxic properties but retain their antigenic ones. This was first discovered by Ehrlich who also introduced the term toxoid for this products. Toxoids are detoxified toxins which retain their antigenicity and their immunizing capacity, being used as vaccines.

Endotoxins are heat stable and resist to boiling for 30 minutes without any change in their structure. Some powerful oxidizing agents such as hydrogen peroxide, superoxide and hypochlorite may neutralize them. Although antigenic, endotoxins cannot be converted to toxoids.

The biological activity of endotoxins is due to the lipopolysaccharide. Toxicity is associated with the lipid component (Lipid A) and immunogenicity is associated with the polysaccharide. The somatic antigen (O antigen) of Gram-negative bacteria are components of the cell wall lipopolysaccharide. The lipopolysaccharide elicits an inflammatory response in the human host by the production of cytokines, including IL-1, IL-6, IL-8, tumor necrosis factor (TNF) and platelet-activating factor. It activates the complement by the alternative (properdin) pathway, so it plays an important role in the pathology of Gram-negative bacterial infections.

Figure Schematic of bacterial endotoxin (lipopolysaccharide)

Source www.sigmaaldrich.com/life-science/molecular-biology

Pathogenic bacteria fall into two groups with regard to their fate within phagocytes:

Extracellular bacteria are promptly killed after phagocytosis

Intracellular bacteria are resistant to intracellular killing unless macrophages are activated.

Antibacterial defence mechanisms of the host are also of two groups

a. Non-specific mechanisms

b. Specific mechanisms: immune mechanisms

CHAPTER 6

NON-SPECIFIC HOST RESPONSE

Nonspecific factors of antibacterial defence are innate (exist from birth). No contact with specific antigen is necessary for them to be expressed.

Non-specific defense mechanisms include:

-the skin forms a mechanical barrier, impervious to bacteria, both saprophytic and pathogenic ones. The fatty acids secreeted by sebaceous glands and the low pH ( between 3-5) have antibacterial activity.

– the mucous membranes of the respiratory tract, which mechanically remove dirt and bacteria by means of the ciliated epithelium and mucus secretion.

– the digestive tract secretions have antibacterial role: the hydrolytic enzymes in saliva, the gastric acid Ph and the various enzymes released in the small intestine.

– vaginal mucosa – has an acid pH, unfavourable to pathogenic microorganisms.

– the saprophytic flora opposes proliferation of pathogenic species through competition for cell receptors, competing for substrate nutrient, oxygen, and production of antibacterial substances (bacteriocins).

– Antimicrobial substances from blood and secretions:

Lysozyme is found in higher amount biological fluids tears, saliva, nasal secretions, mucus, milk. It is also present in cytoplasmic granules of the macrophages and the polymorphonuclear neutrophils (PMNs). Lysozyme has antibacterial role by hydrolysis of the bacterial wall.

Properdin is found in plasma and can activate the complement in the absence of immune complexes.

Beta-lysine is present in normal serum and platelets. It acts especially against Gram-positive bacteria.

Lactoferrin ( lactotransferrin ) and ferritin (apotransferrin) are iron-binding glycoproteins , that bind the seric and leukocytic iron ( Fe) and compete with bacteria for it. Lactoferrin has antimicrobial activity and is part of the innate defense, mainly in infants and at mucoses.

Fibronectin – a plasma glycoprotein that plays a role in opsonization, in the interaction between cells, and react with complement. Fibronectin is also found in human saliva, and prevent colonization  of the  oral and pharyngeal mucosa by potentially pathogenic bacteria

The complement family of proteins synthesized in the liver , spleen macrophages. They are part of the innate defence system, that enhances (complements) the capacity of phagocytic cells to clear pathogenic microorganisms  from the human organism. The complement proteins in blood, in general arenormally circulating as inactive precursors  and when are triggered ,activate each-other in cascade. There are three pathways that produce the complement activation; the classical pathway initiated by antibodies bound to the surface of foreign bodies and the alternative and lectin pathways that provide an antibody independent mechanism for complement activation, induced by the presence of bacteria and other micro-organisms.

The complement is a system of proteins that are proenzymes that are cleaved in cascades to be converted into active enzymes. Their role is to increase or "complement" the other defence mechanisms, mainly the immune response

The components of the complement are numbered from C1 to C9. Their activation may be done by three pathways: The classic pathway, the alternative pathway ant the lectin pathway ( Mannan-Binding lectin pathway)

The most important effects of complement activation are:

– lisys of bacteria and tumor cells

– opsonisation of microorganisms

– some complement subunits are mediators that participate in inflamation

– enhancement of antibofy-mediated immune response.

– anaphylatoxins

The classic pathway

The classic pathway convolve initiation of the complement activation by immune complexes. IgM and IgG can fix the complement after being bound by the antigen. At the complement binding site on the Fc region, Ciq is fixed ,then cleaved to C1, which cleaves C2 and C4 to C4b2b. This complex ( C4b2b) is an active enzyme: C3 convertase, which cleaves C3 into C3a and C3b. Further, C3b is bound to form C4b2b3b complex, enzyme that activate C5 , by cleaving it intoC5a and C5b. C5b links to C6, C7, C8 then to C9 and form the Membrane attack complex. This membrane attack complexes fix on the bacterial or other non-self cell membrane and produce pores or channels, allowing water and electrolytes passage, so the cell will be lysed.

The alternative pathway

Different substances produced in an injured tissue due to an infection or a wound can activate this pathway. They activate C3 and Properdin, factor B and factor D activate C3 into additional C3 convertase ( C3bBb), that generates more C3b. Extra C3b plus Bb Ceb results in C3bBbC3b complex, which is C5 convertase, generating C5b. C5b links to C6, C7, C8 then to C9 and form the membrane attack complex in the same way as in the classic pathway.

The Mannan-Binding lectin pathway

Mannan-binding lectin is a plasma protein that binds to polysaccharides residues on the surface of bacteria ( lipoplysaccharides) such as mannose, and can activate C4 to C4b and C2 to C2b. Then C4b2b complex acts in the same manner as in the classic pathway

The complement system activation is regulated by different proteins C1 inhibitor, factor H, factor I and fcator P ( properdin). Also there is a decay accelerating factor, bound on blood cells, that accelerate the dissociation of C3 convertase .

Figure …Complement activation

C-reactive protein (CRP ) is an acute phase protein, pentameric globulin with electrophoretic mobility near the gamma region. It is found in normal serum in quantities of less than 6 mg / l . PCR increases in infection or inflammation , being very useful in the diagnosis of occult infections . This protein binds to receptors on the surface of bacteria and promotes its destruction under the action of complement.

Interferons are produced by leukocytes, fibroblasts , NK cells , T-lymphocytes acts as immunomodulators ( IF- gamma ) , increasing activity of macrophages and NK cells IF alpha and beta antiviral activity in the infected cells by inducing the production of antiviral proteins .

SPECIFIC HOST RESPONSE: IMMUNITY

Specific host response, due to the immune system is only produced after the immune system had contact with the specific antigens of the microorganisms.

The immune system

Central (primary) lymphoid organs: the bone marrow and thymus. They appear in early embryogenesis and represent the headquarters of lymphopoiesis and maturation independent of antigen. Their removal is incompatible with human survival. Thymus lymphopoiesis lasts till the organ ‘s normal involution at the time of puberty.

Peripheral (secondary) lymphoid organs: lymph nodes, spleen, lymphoid structures associated respiratory and digestive tract: tonsils, Waldeyer ring, Peyer's patches, etc. They appear later in embryogenesis. The role of these organs is to ensure theat B and T lymphocytes become imunococmpetent: LB in lymphoid follicles, LT in paracortical areas of lymph and spleen periarteriolar sheaths. There is a permanent lymphocyte traffic between them. These organs are sites of the immune response, lymphocytes proliferation when activated by an antigen.

Immune cells. The main immune cells are granulocytes, monocytes, macrophages (phagocytic cells) and lymphocytes B and T.

B cell activation. The humoral (antibody) mediated immune response

After microorganisms being phagocyted, their antigens will undergo a partial degradation, followed by exposure of the epitopes coupled to class II MHC molecules on the surface of macrophages and B lymphocytes, expressing surface antigens ( sAG).

Surface Ag-II MHC complexes are recognized by T helper lymphocytes ( Th, CD4 +), releasing cytokines as interleukin IL-2 , which cause the activation of B lymphocytes. Activated B lymphocytes turn into plasma cells, which secrete antibodies with the same specificity as the presented Ag (primary immune response) .

Part of activated LB will be transformed into memory cells. At a subsequent penetration of the same antigen in the body, the immune response will be faster and will be more intense in terms of concentration of antibody – secondary or anamnestic immune response, which may persist for a relatively long period of time.

Source: courses.lumenlearning.net

The cell mediated immune response. T-cell activation.

The cell mediated immune response is realised by activated effector T lymphocytes. Activating T cytotoxic lymphocytes ( Tc, CD8+) requires two signals. The specific signal is given by the antigen ( Ag) coupled with the class I HLA molecule presented on a cell surface. The second signal is given by lymphocytes Th, by secretion of IL2. Lymphocytes Tc targets are represented by cells infected with viruses and foreign cells, which are destroyed by direct cytotoxic effect. Cytolysis produced by LTc is very specific.

Cell activation of Ts (suppressor) and lymphocytes B activation occur later, causing the inhibition of Tc and making an immunological control.

T-lymphocytes have also a subpopulation of memory. Memory lymphocytes T are activated faster than virgin cells in the presence of specific Ag. Their activation is followed quickly by transformation in effector cells.

Structure and functions of immunoglobulins

The immunoglobulins are a large family of glycoproteins with central role in the humoral immune response.

They are composed of polypeptides in about 85% and carbohydrates in about 15% of molecular weight and in plasma eletrophoresis , these glycoproteins migrate in the gamma band, being also called gamma globulins

Normal serum protein eletrophoresis source:htpp//synapse.koreamed.org

The immunoglobulin basic unit is made of four polypeptidic chains: a couple of heavy chains (H) and a couple of light chains ( L ), heach having a N-terminal and a COO- terminal end. The shape of the chains is held together by non-covalent forces and by covalent interchain disulfide bridges. All H chains and all L chains in an immunoglobulin are identical. Each H ans L chain contains folded globular regions of about 100 aminoacids bound by an intrachain disulfide bridge, called domains.

The structure is a bilaterally symmetrical molecule with „Y” shape .

Ig unit

The domains form the N-terminal end form the variable region (VL, VH), having a different aminoacid sequence, specific for each particular antigen. The variable regions within an immunoglobulin unit form the antigen-binding site (Fab). In this way, each immunoglobulin unit has two antigen binding sites.

The rest of the domains have a relatively constant structure, being called constant domains ( CL, CH1, CH2, CH3). The COO- end domains of the immunoglobuline unit form the cell binding site ( FC).

The hinge region is located between CH1 and CH2 and has a flexible structure, wich enable the immunoglobulin to change the position of the Fab regions. The hinge region is suceptible to enzymatic cleavage, by pepsin and papain.

The immunoglobulin classes and subclasses

There are 5 classes of immunoglobulines: IgM, IgG, IgA, IgE and IgD with 5 different isopypes of H chains (µ, γ, α, ε, respectively δ ). The light chains of any class may be one of the two types: kappa ( k) or lambda (λ). Immunoglobulins of all classes may be membrane-bound or secreted ( free).

As for their biological activities, immunoglobulines have two categories of functions:

1. specifically binding of antigens, and

2. other activities not related to the antigen-binding: opsonisation, complement activation.

Immunoglobulin M ( IgM) is large, with the structure made of 5 immunoglobulin units (is a penthamer) linked by a J-chain. The molecular weight is of about 900,000. It represents approximately 10% of total serum immunoglobulines.

Figure: IgM structure

The IgM is predominantely produced during the early primary immune response. That is why, the significance of detection of IgM antibodies against a specific pathogen in the serum of a patient generally is “recent infection”.

It is also the most efficient complement –fixing class of antibodies.

IgM and IgD are commonly expressed ( bound) on the surface of B lymphocytes.

Immunoglobulin G ( IgG)

Ig G is monomeric, but is the major immunoglobulin ( 75%) in the serum of adults. It is also the only class of antibody that can cross the placenta, ensuring the immune protection of the new born babies for the first 6 months of life.

IgG are produced later in the primary immune response and are predominantely produced during the secondary immune response . Their detection in a patient’s serum cannot distinguish a recent infection from a passed one (cannot differentiate acute from chronic infections), unless an important increase in titer of IgG is demonstrated.

Immunoglobulin A ( IgA)

IgA exist as a monomer but also as dimer, maid of two units joined by J chains a a secretory piece. IgA is abundantly present in secretions ( saliva, tears, bronchial secretion, intestinal mucus, prostatic secretion) , being secreted by B lymphocytes from tonsils, Peyer’s patches and other submucosal lymphatic structures.

In serum, it counts for 10-15 % of total immunoglobulines.

IgA is also present as monomer on the surface of B lymphocytes

Ig A dimer

Immunoglobulin E ( Ig E)

IgE immunoglobulins bind with their Fc regions on the surface of eosinophils, mast cells and basophils. When the specific antigens bind to the Fab of the IgE, the resulted immune complexes act as triggers for the degranulation of the mentioned cells. The result is an allergic reaction ( type I hypersensitivity). People with innate increased IgE concentrations are named atopic and are more exposed to allergic reactions to various antigens. Parasitic infections, specially due do helminthes have also increased serum IgE.

Immunoglobulin D ( Ig D)

IgD immunoglobulins are monomeric. They are present on the surface of B lymphocytes, as receptors. After antigen binding, IgD signals resulting in the activation of the lymphocytes. In serum they are only in very small amounts, less than 1%..

Human microbiome

8.ACTION OF PHYSICAL FACTORS UPON BACTERIA

Sterilisation, disinfection, antisepsis

Definitions of terms

Sterilisation refers to a physical or chemical process that completely destroys or removes all forms of viable microorganisms from an object, including resistance forms: bacterial spores or parasites’ cysts. An object that has been packaged so as to be contamination-proof, then subjected to a sterilization process, is considered sterile for a defined period of time. For medical equipment, it is compulsory to achieve a sterility assurance level.

Disinfection describes a process that eliminates many or all pathogenic microorganisms on inanimate objects (99%), with the exception of bacterial spores. The substances used to perform this process are named disinfectants. Whereas sterilisation is absolute, the disinfection is relative and may be realised in different levels: high, medium and low.

Antisepsis is the process of reducing the number of microorganisms on skin, mucous membranes or other body tissues. Antiseptic or antimicrobial agent (terms used interchangeably)-Chemicals that are applied to the skin or other living tissue to inhibit or kill microorganisms (both transient and resident) thereby reducing the total bacterial count.

Decontamination refers to destruction or removal of microorganisms to a lower level, such that there is no danger of infection to unprotected individuals.

Methods of Sterilisation

The   appropriate sterilization  method  is  determined  according  to the material of which the item is made, the sterilization methods available and how the item will be used.

Classification

There are two types of sterilisation: physical and chemical

Physical sterilisation methods include:

Heat sterilisation:

Dry heat sterilisation

Moist heat sterilisation

Filtration sterilisation

Radiation sterilisation

Chemical methods – ethylene oxide sterilisation

A.1.Dry heat sterilisation

A.1.1.Red heat flame

Red heat flame is done to loops and straight-wires in microbiology labs. Leaving the loop in the flame of a Bunsen burner or alcohol lamp until it glows red and another 5-10 sec. after that ensures that any infectious agent gets inactivated. This is commonly used for small metal or glass objects, but not for large objects. However, during the initial heating infectious material may be "sprayed" from the wire surface before it is killed, contaminating nearby surfaces and objects. To prevent this fenomen the loop will be put at the base of the flame.

A.1.2. Dry heat – Hot air ovens

In all the microbiological laboratories one of the common pieces of apparatus is hot air oven. Hot air oven is a sterilizer using dry heat.

Fig. 1.1. Hot oven

Their double walled insulation keeps the heat in and conserves energy, the inner layer being a poor conductor and outer layer being metallic. There is also an air filled space in between to aid insulation. An air circulating fan helps in uniform distribution of the heat. A thermostat regulates the temperature at the desired level and a thermometer is fitted for recording the temperature. The shelves within the hot air oven are perforated to allow proper air circulation.

Fig.1.2. Principle of sterilisation in hot oven

The dry heat oven is used for glassware, metal, and objects that won't melt. It cannot be used for culture media, liquids, textiles, rubber objects.

The standard setting for a hot air oven is one hour at 180 °C, for one hour effective time.

Before sterilisation all the galssware should be washed and wiped dry and then neatly wrapped in special paper. Only then they should be kept inside the chamber.

After the sterilisation , the door of oven must not be opened until the temperature has fallen under 100 ͦ C because the sudden rush of the air from outside into the oven may crack the glassware.

The objects will be extracted using sterile gloves and put on a sterile surface; they will be labeled. The time and the date should be noted.

A.2.1.Moist heat sterilisation – Autoclaves

The autoclave is a sterilizer using steam under pressure. It is the most dependable and economical method of sterilization. Autoclaving is the method of choice for metalware, glassware, most rubber  goods, media and equipment required for growing microorganisms, surgical steel instruments, textiles, fluids.

During autoclaving the contents (liquid or solid) become exposed to saturated steam at the required temperature for the appropriate length of time. Typical temperature/pressure/time sterilization parameters are 121 ͦC at 1 atm.of pressure for 30 minutes. Increasing the pressure at 1.5 atm. or 2 atm. will increase the temperature to 134 ͦC .

Fig. 3 Automatic and classical autoclaves

There are two types of autoclaves available:

Autoclaves with pre&post vacuum processes – used for textiles, rubber goods, glassware, surgical steel instruments, etc.

Autoclaves without postvacuum processes – used for media cultures and fluids.

After the sterilisation , the door of oven must not be opened until the temperature has fallen under 100 ͦ C .The objects will be extracted using sterile gloves and put on a sterile surface; they will be labeled. The time and the date should be noted.

To ensure the autoclaving process was able to cause sterilization, most autoclaves have meters and charts that record or display pertinent information such as temperature and pressure as a function of time. Indicator tape is often placed on packages of products prior to autoclaving. A chemical in the tape will change color when the appropriate conditions have been met. Some types of packaging have built-in indicators on them.

Biological indicators ("bioindicators") can also be used to independently confirm autoclave performance. Simple bioindicator devices are commercially available based on microbial spores. Most contain spores of the heat resistant microbe "Bacillus stearothermophilus", among the toughest organisms for an autoclave to destroy. Typically these devices have a self-contained liquid growth medium and a growth indicator. After autoclaving an internal glass ampule is shattered, releasing the spores into the growth medium. The vial is then incubated (typically at 56 °C (132 °F)) for 48 hours. If the autoclave destroyed the spores, the medium will remain its original color. If autoclaving was unsuccessful the "B. sterothermophilus" will metabolize during incubation, causing a color change during the incubation.

B.Filtration sterilisation

Liquids, such as solutions that would be damaged by heat, irradiation or chemical sterilization can be sterilized by mechanical filtration. A filter with pore size 0.2 µm will effectively remove bacteria. If viruses must also be removed, a much smaller pore size around 20 nm is needed.

Filters can be made of several different materials such as nitrocellulose or polyethersulfone (PES). The filtration equipment and the filters themselves may be purchased as pre-sterilized disposable units in sealed packaging, or must be sterilized by the user, generally by autoclaving at a temperature that does not damage the fragile filter membranes. For filtration of a liquid in which there is a microbial growth, the same filter should not be used for procedures lasting longer than one working day.

Fig. Filters

For reducing the danger of infection while working with pathogenic microbes is used a laminar air flow system. In this system , the air of a closed room is made to pass through high efficiency particulate air filters pack (HEPA).

The laminar air flow apparatus consist of a work table coverd from all sides , except the front portion. The top of the apparatus consist of HEPA filter through witch the air is blown. It has also a switch on/off UV light, tubelight and to turn on the air flow.

C.Radiation sterilisation

Nonionizing radiation. Ultra-violet (UV) rays (280–200 nm) are a type of nonionizing radiation that is rapidly absorbed by a variety of materials. UV germicidal lamps utilises short-wavelength ultraviolet radiation (UV-C) that is harmful to microorganismsdue to the destruction their  nucleic  acids. UV rays are therefore used only to reduce airborne pathogen counts (surgical theaters, filling equipment) and for disinfection of air and smooth surfaces. In laboratory, UV lamps are used for 10 minutes, for air and surfaces.

Ionizing radiation (α,β,γ,X). They have a good capacity for sterilisation , but are harmful to human organism . This is why they are used only in industrial activities.

II.Chemical sterilisation – Ethylene Oxide Sterilization

The most effective method of chemical sterilization presently available is used for devices that incorporate electronic components, plastic packaging or plastic containers is the use of ethylene oxide (ETO) gas. Materials such as polyethylene, polyamide (nylon), polytetrafluoroethylene (Teflon), silicone, aluminum, polypropylene, polystyrene, polyvinylchloride and compound products of various plastics may be sterilized without adverse effects if the necessary time for desorbtion is respected.

The gas can be used for sterilization at low temperatures (20–60 °C) and the sterilization periods range from 3 to 7 hours. The concentration of the gas and the temperature and humidity inside the sterilizer are vital factors that affect the gas sterilization process.

Finally, all items gas sterilized need to go through a degassing phase to remove any particle of EtO.

When using an ETO gas sterilizer, it is important to be extremely cautious because is inodorous, toxic and a strong mucosal irritant. This gas, mixed with air at a ratio of at least 3% EtO gas, forms an explosive mixture. Acute exposures to ETO gas may result in respiratory irritation and lung injury, central nervous system depression, headache, nausea, vomiting, diarrhea, shortness of breath, and cyanosis.

Disinfection

Chemical disinfection

There are a number of factors that may nullify or limit the efficacy of disinfection processes. These include:

prior cleaning of the object

the organic load present

the type and level of microbial contamination

the concentration of, and exposure time to the disinfectant

Microorganisms vary greatly in their resistance to chemical substances. There are three types of disinfectants ( Spaulding’s classification):

High-level disinfectants

Intermediate-level disinfectants

Low-level disinfectants

High-level disinfectants- are chemical substances, which when used for a shorter exposure period than would be required for sterilisation, kill all microorganisms with the exception of bacterial spores:

2% glutaraldehyde solution

8% formaldehyde solution

Sulfochromic mixture ( potasssium dichromate/sodium dichromate in concentrated sulfuric acid)

Peracetic acid or peroxyacetic

3-6% hydrogen peroxide

Sodium hypochlorite

The exposure time is at least 20 minutes.

These chemicals must be used in accordance with the relevant workplace health and safely legislation in each state.

Intermediate-level disinfectants- may kill mycobacteria, vegetative bacteria, most viruses, and most fungi but do not necessarily kill bacterial spores:

Ethyl or isopropyl alcohol (70-90%)

Sodium hypochlorite (5.25-6.15% household bleach diluted 1:500 provides >100 ppm available chlorine)

Phenolic germicidal detergent solution

Iodophor germicidal detergent solution

Surfactants. These substances (also known as surface-active agents, tensides, or detergents) include anionic, cationic, amphoteric, and nonionic detergent compounds, of which the cationic and amphoteric types are the most effective. Their efficacy is good against Gram-positive bacteria, but less so against Gram-negative rods. Their advantages include low toxicity levels, lack of odor, good skin tolerance, and a cleaning effect.

The exposure time is 10 minutes.

Low-level disinfectants may kill most vegetative bacteria, some fungi, and some viruses:

Quaternary ammonium germicidal detergent solution

Phenolic and Iodophor germicidal detergent solutions

Surfactants

The exposure time is 10 minutes.

Fig. Symbols for surface disinfectants and for instrumentary disinfectant

Antiseptics

An antiseptic is a chemical disinfectant that can be diluated sufficiently to be safe for application to living tissues ( intact skin, mucous membranes, wounds) while still retaining its antimicrobial property.

Fig. Symbol for an antiseptic

Antiseptic agents:

Alocohols: Ethyl or isopropyl alcohol ( 60-70% v/v)

Benzalkonium chloride

Hexachlorophene compounds

Iodophor compounds ( Betadine, Isodine)

Hydrogen peroxide 3%

1% silver nitrate

Potassium permanganate

Chlorine compounds: chlorhexidine (0,75-4%), hexachlorofene ( 1-3%)

Colored compounds: methylene blue, etc.

Physical disinfection

Disinfection by heat

Moist heat

Boiling water – produce a temperature of 100ͦ C at normal atmospheric pressure. It requires 10-20 min. exposure to this temperature, to kill many bacteria and some viruses ( including HIV and HBV).

Pasteurization – is the use of mild heat to reduce the number of microorganisms in a product or food. Milk is usually pasteurized by heating, typically at 63°C for 30 minutes (batch method) or at 71°C for 15 seconds (flash method), to kill bacteria and extend the milk's usable life. The process kills pathogens but leaves relatively benign microorganisms that can sour improperly stored milk.

During the process of ultrapasteurization, also known as ultra high-temperature (UHT) pasteurization, milk is heated to temperatures of 140 °C.

Dry heat – Flaming – a method used for microscope slides, glass tubes, etc.( flame de mouths of tubes for 2-5 sec. with the Bunsen burner)

Disinfection by radiation- using UV lamps for disinfection of surfaces and the rooms air as well as water.

Biological factors – bacteriocins

Bacteriocins are bactericidal substances. Mainly protein in nature which are produced by many bacteria. Have a killing action on strains of the same or closely related species. The narrow specificity of their action and their protein nature distinguish them from other(classical) antibiotics. The first discovery was reported in 1925 of a highly specific antibiotic produced by E. Coli – colicin – and active against other strains of the same species.

Bacteriocins are produced by several other bacteria also, and were given specific names based on the bacterial species of origin, for example Colicins – E. coli Pyocins – P.aeruginosa and Diphthericins – C.diphtheriae

Medical significance of Bacteriocins

– Bacteriocins produced by non-pathogenic bacteria kills other pathogenic bacteria. (Normal flora vs. Pathogens)

– Bacteriocins have also been suggested for certain cancer treatment.

– Bacteriocins have gained new attention particularly in the epidemiology of nosocomial infections by bacterial typing.

In humans ,probiotic therapy is a disease-precluding strategy used to ensure that 'good' bacteria (probiotics) complex in the gastrointestinal tract and prevent the accumulation of pathogenic bacteria. The probiotic strain's ability to produce antimicrobial compounds (such as bacteriocins, colicins, etc.) is the most important attribute that is extensively used. [16] Nonpathogenic colicin-producing strains that can kill, exclude or inhibit the sensitive target strain in its affinity have been added as probiotic,

Food-borne pathogens produce several toxic agents among which Shiga toxin producing E. coli O157: H7 is the most virulent one. Humans procure infection by eating raw meat, taking raw milk, person-to-person contact and sewage-contaminated water .

Several E. coli strains have been known to cause haemorrhagic colitis in humans; most of them have been reported in relation to O157:H7. [2] The patients have symptoms of gastroenteritis such as watery diarrhoea, vomiting and abdominal cramps. The later complications include bloody diarrhoea and kidney failure eventually leading to Human Uremic Syndrome (HUS), especially in children and aging people.

Administration of antibiotics (such as sulphamides) against this pathogen may increase the risk of uremic syndrome; especially in children and aged people.

In studies , alternate bio-therapeutics (probiotics) for the treatment of patients infected with E. coli O157:H7. were based on the identification of colicin-producing gram-negative bacteria (particularly enterobacteriaceae) which can competently exclude E. coli O157:H7 from the gut of the infected individual.

Detection of colicinogenic bacteria

E. coli strains and some closely related species including Citrobacter sp., Pantoea sp., Klyuvera sp and Shigella sonnei were tested for their ability to produce colicins

All colicins are organized into three domains, each corresponding to one step of colicin action:

The N-terminal domain is involved in translocation through the membrane.

The central domain is involved in binding to the receptor.

The C-terminal domain contains the active part.

Microcins are a peculiar class of gene-encoded low-molecular-mass antibacterial peptides secreted by enterobacteria. They contribute to the regulation of microbial competition within the intestinal microbiota. The genetic systems involved in microcin biosynthesis share a conserved organization. Similar to bacteriocins, microcins exert potent antibacterial activity directed against phylogenetically-related bacterial strains, with minimal inhibitory concentrations in the nanomolar range.

10.NORMAL FLORA OF THE HUMAN BODY

The human body is inhabited by thousands of different bacterial species (microbiota or normal flora) – some living transiently, others in a permanent parasitic relationship. Likewise, the environment that surrounds us, including air we breathe, water we drink, and food we eat, is populated with bacteria, many of which are relatively avirulent and some which are capable of producing life-threatening disease [1].

Pathogen or pathogenic-capable of producing disease.

Though only a minority of microorganisms are pathogenic, practical knowledge of microbes is necessary for their treatment so is highly relevant to medicine and related health sciences.

Normal flora [normal microbiota]- not typically-disease-causing

microorganisms normally found in and on healthy individuals.

on the skin,

in the eyes,

in the nose,

in the mouth,

in the upper throat,

in the lower urethra,

in the lower intestine.

Fig Normal flora of the human body

Fig Estimation of the organisms resident/cm2

Up until the time of birth, the human fetus lives in environment which is most of the part sterile. After the birth, the infant is exposed to bacteria, fungi and viruses from the mother, other close contacts, and the environment. The normal flora is not static; although a basic flora persist, it is subject to constant change [2].

With the exception of the alimentary tract, the internal organs of other systems are sterile in health, e.g. the bladder and kidneys, the bronchi and lungs, and the CNS.

Skin is constantly exposed to and is in contact with the environment, the skinis particularly apt to contain transient microorganisms. The predominantresident microorganisms of the skin are aerobic and anaerobic diphtheroid bacilli(eg, 61ropionibacteri, 61ropionibacterium); nonhemolytic aerobic and anaerobicstaphylococci (Staphylococcus epidermidis, occasionally S aureus, andpeptostreptococcus species); gram-positive, aerobic, spore-forming bacilli thatare ubiquitous in air, water, and soil; α-hemolytic streptococci (viridansstreptococci) and enterococci (enterococcus species); and gram-negative coliformbacilli and Acinetobacter. It is not evenly distributed.

Low pH, fatty acids in sebaceous secretions and presence of lysozymes areimportant factors for eliminating non-resident microorganisms from the skin.

Fig Culture on blood-agar of the oral flora

The mouth contains micrococci, gram positive aerobic spore bearing bacilli,coliforms, proteus and lactobacilli. The gums pockets between the teeth andcrypts of the tonsils have a wide spectrum of anaerobic flora like fusiform bacilli,treponemes, lactobacilli, etc. Candida is also found. Saliva contains about 109 bacteria/ml [1].

The oesophagus is colonized with viridans streptococci, commensal Neisseriae, Corynebacteria or Bacteroides. The empty stomach is sterile due the gastric acid.

Fig Electron microscopy of the intestinal flora

The normal flora of the large intestine consists of Bacteroides, bifidobacteria, anaerobic cocci, Escherichia coli, Streptococcus faecalis, clostridia, lactobacilli. Bacteria are mostly strict anaerobes, but some facultative anaerobes are also resident. The anaerobic environment of the colon is maintaining by aerobic bacteria utilizing free oxygen. Faeces contain enourmous numbers of bacteris which constitute up to one-third of the faecal weight.

Fig Flora of the digestive tract

The nose is cooler of the rest of respiratory system and has some unique microbiota. The nasopharynx is a naturalhabitat of the common pathogenic bacteria causing infection of the nose, throat,bronchi and lungs.The flora of nose harboursdiptheroids, Staphylococcus, Streptococcus, Haemophilus, andMoraxella lacunata. The alveoli of the lungs have no natural microbiota.

For anatomical reasons, the female genital tract is much more colonized than that of the male.main bacteria of the female vagina are: lactobacilli (Döderlein bacilli), Bacteroides, Streptococcus faecalis, corynebacteria, Gardnerella vaginalis, yeasts.Mycobacterium smegmatisa harmless commensal is found in the secretions(smegma) of both males and females genitalia. They may pose the confusionwith the tubercle bacilli.Strains of mycoplasma and ureaplasma are frequently present as part of normalflora.

Gardnerella vaginalis, bacteroidesand alpha streptococci have beenfound in penile urethra.Female urethra is either sterile or contains staphylococcus epidermidis. Thevagina of newly borne child is sterile and within 24 hours it colonizes withmicrococci, enterococci. In 2-3 days timeDöderlien’s bacillus appears. So theflora keeps on changing depending upon the pH of the vagina. Döderlien bacilliremain in the vagina till menopause. After menopause flora resembles thatbefore puberty.

Microorganisms that are constantly present on body surfaces are commensals.Their growth in a given area depends upon physiologic factors like temperature,moisture, and the presence of certain nutrients and inhibitory substances.Resident flora of certain areas plays a definite role in maintaining health andnormal function. Members of the resident flora in the intestinal tract synthesizevitamin K and and several B Groupvitamins, degrade mucins, epithelial cells and carbohydrate fibre. On mucous membranes andskin, the resident flora may prevent colonization by pathogens and possibledisease through “bacterial interference.” The mechanism of bacterial interferenceis not clear. It may involve competition for receptors or binding sites on hostcells, competition for nutrients, mutual inhibition by metabolic or toxicproducts, mutual inhibition by antibiotic materials or bacteriocins, or othermechanisms. The normal flora evokes the Antibodies production. These Antibodies crossreact with pathogens having related or shared antigens, thus raising theimmune status of the host against the invading pathogen.

Broad-spectrum drugs disrupt the composition of the normal flora by inhibiting sensitive organisms and allowing overgrowth of resistant bacteria.

Fig Infections produced by saprophytic flora

On the other hand, members of the normal flora may themselves produce diseaseunder certain circumstances and if removed from the restrictions of thatenvironment and introduced into the bloodstream or tissues, these organismsmay become pathogenic. For example, Escherichia coli resisdent in the colon entering and infecting the urinary tract. Small numbers of the viridans groupoccur transiently inthe bloodstream with minor trauma (eg, dental scaling or vigorous brushing) and they may settle on deformed or prosthetic heartvalves and produce infective endocarditis.

Bacteroides species are the commonest resident bacteria, if introduced into thefree peritoneal cavity or into pelvic tissues along with other bacteria as a resultof trauma, they cause suppuration and bacteremia.

There are many other examples, but the important point is that microbes of thenormal resident flora are harmless and may be beneficial in their normal locationin the host and in the absence of coincident abnormalities. They may producedisease if introduced into foreign locations in large numbers and if predisposingfactors are present.

Also, normal flora may cause confusion in diagnosis due to their ubiquitouspresence in the body and their resemblance to some of the pathogens.

Sleigh, J. Douglas; Timbury, Morag C. – Medical Bacteriology, 4th edition, Churchill Livingstone 1994;

Murray, Patrick R.; Rosenthal, Ken S.;Pfaller, Michael A.- Medical Microbiology, 8th edition, Elsevier 2016.

MECHANISMS OF ANTIBIOTIC ACTION AGAINST BACTERIAL CELLS:

The main mechanisms of activity of antibacterial drugs are:

Inhibition of Cell Wall Synthesis (most common mechanism)

Inhibition of Protein Synthesis (Translation) (second largest class)

Alteration of Cell Membranes

Inhibition of Nucleic Acid Synthesis

Antimetabolite Activity

Fig.1.1. Mechanisms of activity of antibacterial drugs. Source: Medical Laboratory Observer

Inhibition of Cell Wall Synthesis

Beta-Lactams –> Inhibition of peptidoglycan synthesis (bactericidal)

(1) fails to cross membrane (gram negatives)

(2) fails to bind to altered PBP’s

(3) hydrolysis by beta-lactamases

Vancomycin –> Disrupts peptidoglycan cross-linkage

Bacitracin –> Disrupts movement of peptidoglycan precursors (topical use)

Antimycobacterial agents –> Disrupt mycolic acid or arabinoglycan synthesis (bactericidal)

Inhibition of Protein Synthesis (Translation)

30S Ribosome site

Aminoglycosides –> Irreversibly bind 30S ribosomal proteins (bactericidal)

Resistance –>

(1) mutation of ribosomal binding site

(2) decreased uptake

(3) enzymatic modification of antib

Tetracyclines –> Block tRNA binding to 30S ribosome-mRNA complex (b-static)

Resistance –>

(1) decreased penetration

(2) active efflux of antibiotic out of cell

(3) protection of 30S ribosome

Tetracyclines –> Block tRNA binding to 30S ribosome-mRNA complex (b-static)

Resistance –>

(1) decreased penetration

(2) active efflux of antibiotic out of cell

(3) protection of 30S ribosome

50S Ribosome site

Chloramphenicol –> Binds peptidyl transferase component of 50S ribosome, blocking peptide elongation (bacteriostatic)

Resistance –>

(1) plasmid-encoded chloramphenicol transferase

(2) altered outer membrane (chromosomal mutations)

Macrolides –> Reversibly bind 50S ribosome, block peptide elongation (b-static)

Resistance –>

(1) methylation of 23S ribosomal RNA subunit

(2) enzymatic cleavage (erythromycin esterase)

(3) active efflux

Clindamycin –> Binds 50S ribosome, blocks peptide elongation; Inhibits peptidyl transferase by interfering with binding of amino acid-acyl-tRNA complex

Resistance –> methylation of 23S ribosomal RNA subunit

Alteration of Cell Membranes

Polymyxins (topical) –> Cationic detergent-like activity (topical use)

Resistance –> inability to penetrate outer membrane

Bacitracin (topical) –> Disrupt cytoplasmic membranes

Resistance –> inability to penetrate outer membrane

Inhibition of Nucleic Acid Synthesis

DNA Effects

Quinolones –> Inhibit DNA gyrases or topoisomerases required for supercoiling of DNA; bind to alpha subunit

Resistance –>

(1) alteration of alpha subunit of DNA gyrase (chromosomal)

(2) decreased uptake by alteration of porins (chromosomal)

Metronidazole –> Metabolic cytotoxic byproducts disrupt DNA

Resistance –>

(1) decreased uptake

(2) elimination of toxic compounds before they interact

RNA Effects (Transcription)

Rifampin –> Binds to DNA-dependent RNA polymerase inhibiting initiation & Rifabutin of RNA synthesis

Resistance –>

(1) altered of beta subunit of RNA polymerase (chromosomal)

(2) intrinsic resistance in gram negatives (decreased uptake)

Bacitracin (topical) –> Inhibits RNA transcription

Resistance –> inability to penetrate outer membrane

Antimetabolite Activity

Sulfonamides & Dapsone –> Compete with p-aminobenzoic acid (PABA) preventing synthesis of folic acid

Resistance –> permeability barriers (e.g., Pseudomonas)

Trimethoprim –> Inhibit dihydrofolate reductase preventing synthesis of folic acid

Resistance –>

(1) decreased affinity of dihydrofolate reductase

(2) intrinsic resistance if use exogenous thymidine

Trimethoprim-Sulfamethoxazole synergism

Macrolides

Erythromycin – obtained from Streptomyces erythreus

Clarythromycin, Azythromycin

Mechanism of cation: reversibly bind 50S subunit of the ribosome, and inhibit protein synthesis (block formation of initiation complexes and peptide elongation)

Bacteriostatic antibiotics. Their activity is enhanced at alkaline pH

They are active against Gram-positive cocci and bacilli and Gram negative cocci: staphylococci, streptococci and pneumococci, corynebacteria. Chlamydia, Legionella and Campylobacter are also sensitive.

Erythromycin and related drugs may be indicated as second choice, as substitute for Penicillin, for infections with the mentioned bacteria in patients allergic to beta-lactamines.

Clarythromycin has an enhanced activity against Helicobacter pylori, Chalmydia trachomatis, Moraxella catharalis and Borellia burgdorferi.

Azitromycin has a better activity against Neisseria Gonorrhoeae, Campylobacter jejuni, Haemophyllus influenzae and Mycoplasma pneumoniae

Administration is orally or intravenously

Side effects may be: mild gastrointestinal symptoms, fever, cholestatic hepatitis.

Resistance to macrolides results from alteration ( metilation) of ribosomal receptor, and is under controle of a transmissible plasmid .

Lincomycins: Lincomycin and Clindamycin

Lincomycin was obtained from Streptomyces lincolnensis. Clindamycin is semisynthetic derivative of lincomycin.

Both drugs inhibit bacterial protein synthesis by binding to the 50S subunit of the ribosomes

Antibacterial spectrum is similar to the erythromycin although they have different biochemical structures. Clindamycin is also very active against anaerobic bacteria, inclusive Bacteroides spp.

Lincomycins are very effective in bone infections , due to Staphylococcus aureus, MRSA included, and Clindamycin can be indicatd in skin infections with MRSA

Administration may be parenteral ( I.V.) or oral. Distribution in tissues is excellent, except CSF ( not indicated in menengitis)

Clindamycin in intravenous administration is indicated in severe infections with Bacteroides fragilis.

Side effects: lincomycins have been associated with antibiotic –associated collitis caused bt Clostridium difficile

Aminoglycosides

Aminoglycosides are a group of drugs structurally characterized by the presence of an aminocyclitol ring linked to amino sugars

Examples: Streptomycine, Kanamycine, Gentamycine, Tobramycine, Amikacine, , Netilmycine, Neomycine.

Streptomyicine (since 1944), was the first introduced in therapy. Newer preparations have a superior antibacterial activity, a better tolerance and reduced risks of adverse reactions.

Mechanism of activity: they concentrate in the bacterial cytoplasm and by binding to the 30 S subunit of ribosomes inhibit the protein synthesis.

Their activity is bactericidal, rapid and strong. Aminoglycosides act synergistic to antibiotics with activity on the bacterial cell wall (beta-lactamines and glycopeptides).

Spectrum : semisynthetic aminoglycosides (amkacine, netilmycine) have broad spectrum, being commonly used in the treatment of infections with gram-negative and gram-positive bacteria, specially Gram-negative bacilli.

The aminoglycosides have no activity against anaerobic bacteria, spirochetes, intracellular bacteria (chlamydia, rickettsia, legionella).

The gastro-intestinal absorbtion is low (administration is only parenteral).

Do not penetrate in CSF.

Toxic type side effects: ototoxic, nephrotoxic and neuro-muscular.

Fluoroquinolones

Fluoroquinolones are broad-spectrum antibiotics, active against many aerobic gram-positive and gram-negative bacteria. The mechanism of action is by inhibiting the activity of DNA gyrase and topoisomerase, essential enzymes for bacterial DNA replication.

Fluoroquinolones are bactericidal .

Oral and parenteral administration.

first generation fluoroquinolones: Nalidixic acid

second generation fluoroquinolones: Norfloxacin , Ciprofloxacin

third generation fluoroquinolones: Levofloxacin

fourth generation fluoroquinolones: Gatifloxacin , Moxifloxacin

They are indicated in bacterial infections of the respiratpry tract, sinuses, skin, and urinary tract, but also in sexually transmitted diseases due to susceptible strains (Chlamydia trachomatis and Ureaplasma urealyticum).

Fluoroquinolones have been contraindicated in children because they may cause cartilage lesions during growth. However, fluoroquinolones are indicated as a second-line antibiotic and restrictive use to a few specific situations, including P. aeruginosa infections in patients with cystic fibrosis, prophylaxis and treatment of bacterial infections in immunocompromised patients, life-threatening multiresistant bacterial infections in neonates and infants, and Salmonella or Shigella intestinal infections.

Fluoroquinolones are generally avoided in pregnancy as human and animal studies show some risks. Also, fluoroquinolones enter breast milk, so their use during breastfeeding must be avoided.

Adverse effects include gastro-intestinal and and central nervous system effects ( headache, dizziness) . Fluoroquinolone use has been strongly associated with Clostridium difficile–associated diarrhea (pseudomembranous colitis),

MECHANISMS OF ANTIMICROBIAL RESISTANCE

There is no doubt that antimicrobial agents have saved the human race from a lot of suffering due to infectious disease burden. Without antimi- crobial agents, millions of people would have succumbed to infectious diseases. Man has survived the accidental wrath of microorganisms using antimicrobial agents and other mechanisms that keep them at bay. Hardly years after the discovery and use of the first antibiotics was observation made of organisms that still survived the effects of the antimicrobial agents. That was the beginning of the suspicion that different microorganisms were getting a way around previously harmful agents that is known today as antimicrobial resistance.

Microbial resistance to antimicrobial agents was not a new phenomenon for it had been constantly used as competitive/survival mechanisms by microorgan- isms against others. These mechanisms have been well documented.

This chapter therefore gives a brief overview of the mechanisms of resistance by bacteria against antimicrobial agents, and the mechanisms, levels, and patterns of resistance to the different microorganisms in developing countries are dealt with in detail elsewhere in the book.

Understanding the mechanisms of resis- tance is important in order to define better ways to keep existing agents useful for a little longer but also to help in the design of better antimicrobial agents that are not affected by the currently known, predicted, or unknown mechanisms of resistance.

Microorganisms have existed on the earth for more than 3.8 billion years and exhibit the greatest genetic and metabolic diversity. They are an essential component of the biosphere and serve an important role in the maintenance and sustainability of ecosystems. It is believed that they compose about 50% ofthe living biomass. In order to survive, they have evolved mechanisms that enable them to respond to selective pressure exerted by various environments and competitive challenges.

The disease-causing microorganisms have particu- larly been vulnerable to man’s selfishness for survival who has sought to deprive them of their habitat using antimicrobial agents. These microorganisms have responded by developing resistance mechanisms to fight off this offensive. Currently antimicrobial resistance among bacteria, viruses, parasites, and other disease-causing organisms is a serious threat to infectious disease manage- ment globally.

Antibiotics were discovered in the middle of the nineteenth century and brought down the threat of infectious diseases which had devastated the human race. However, soon after the discovery of penicillin in 1940, a number of treatment failures and occurrence of some bacteria such as staphylococci which were no longer sensitive to penicillin started being noticed.

This marked the beginning of the error of antimicrobial resistance. Scientific antibiotic discovery started in the early 1900s by Alexander Fleming, who observed inhibition of growth on his agar plate on which he was growing Staphylococcus spp. It was later found that a microorganism that was later to be called

Penicillium notatum was the cause of the inhibition of the Staphylococcus around it as a result of excreting some chemical into the media. That marked the beginning of the discovery of penicillin which together with several other different antimicrobial agents was later to save millions of humans and animals from infectious disease-causing organisms. The detailed history and documen- tation of man’s search for agents to cure infectious disease has been described extensively elsewhere.

The observation of Staphylococci spp. that could still grow in the presence of penicillin was the beginning of the era of antimicrobial resistance and the realization that after all the drugs that were described as ‘‘magical bullets’’ were not to last for long due to the selective pressure that was being exerted by the use of these agents.

However, the complacency between the 1940s and the 1970s that infectious microorganisms had been dealt a blow was later proved to be a misplaced belief that available antibiotics would always effectively treat all infections. Nevertheless, antimicrobial agents have improved the management of infectious diseases up to date.

Increasing prevalence of resistance has been reported in many pathogens over the years in different regions of the world including developing countries (Byarugaba, 2005). This has been attributed to changing microbial character- istics, selective pressures of antimicrobial use, and societal and technological changes that enhance the development and transmission of drug-resistant organisms.

Although antimicrobial resistance is a natural biological phenom- enon, it often enhanced as a consequence of infectious agents’ adaptation to exposure to antimicrobials used in humans or agriculture and the widespread use of disinfectants at the farm and the household levels (Walsh, 2000). It is now accepted that antimicrobial use is the single most important factorresponsible for increased antimicrobial resistance (Aarestrup et al., 2001; Byarugaba, 2004).

In general, the reasons for increasing resistance levels include the following:

suboptimal use of antimicrobials for prophylaxis and treatment of infection,

noncompliance with infection-control practices,

prolonged hospitalization, increased number and duration of intensive- care-unit stays,

multiple comorbidities in hospitalized patients,

increased use of invasive devices and catheters,

ineffective infection-control practices, transfer of colonized patients from hospital to hospital,

grouping of colonized patients in long-term-care facilities,

antibiotic use in agriculture and household chores, and

increasing national and international travel.

The level of antibiotic resistance is dependent on the following:

the population of organisms that spontaneously acquire resistance mechanisms as a result of selective pressure either from antibiotic use or otherwise,

the rate of introduction from the community of those resistant organisms into health care settings, and

the proportion that is spread from person to person.

All of these factors must be addressed in order to control the spread of antimicrobial-resistant organisms within health care settings. Community- acquired antimicrobial resistance is increasing in large part because of the widespread suboptimal use of antibiotics in the outpatient settings and the use of antibiotics in animal husbandry and agriculture.

Mechanisms of Resistance to Antimicrobial Agents

Fig.2.1. Mechanisms of Resistance of Antimicrobial Agents. Source: Medical Laboratory Observer Source: Todar’s Online Textbook of Bacteriology

In order to appreciate the mechanisms of resistance, it is important to under- stand how antimicrobial agents act. Antimicrobial agents act selectively on vital microbial functions with minimal effects or without affecting host functions.

Different antimicrobial agents act in different ways. The understanding of these mechanisms as well as the chemical nature of the antimicrobial agents is crucial in the understanding of the ways how resistance against them develops. Broadly, antimicrobial agents may be described as either bacteriostatic or bactericidal.

Bacteriostatic antimicrobial agents only inhibit the growth or multiplication of the bacteria giving the immune system of the host time to clear them from the system. Complete elimination of the bacteria in this case therefore is dependent on the competence of the immune system. Bactericidal agents kill the bacteria and therefore with or without a competent immune system of the host, the bacteria will be dead.

However, the mechanism of actionof antimicrobial agents can be categorized further based on the structure of the bacteria or the function that is affected by the agents. These include generally the following:

Inhibition of the cell wall synthesis

Inhibition of ribosome function

Inhibition of nucleic acid synthesis

Inhibition of folate metabolism

Inhibition of cell membrane function

Mechanisms of Antimicrobial Resistance

Prior to the 1990s, the problem of antimicrobial resistance was never taken to be such a threat to the management of infectious diseases. But gradually treatment failures were increasingly being seen in health care settings against first-line drugs and second-line drugs or more. Microorganisms were increasingly becoming resistant to ensure their survival against the arsenal of antimicrobial agents to which they were being bombarded.

They achieved this through different means but primarily based on the chemical structure of the antimicro- bial agent and the mechanisms through which the agents acted. The resistance mechanisms therefore depend on which specific pathways are inhibited by the drugs and the alternative ways available for those pathways that the organisms can modify to get a way around in order to survive.

Resistance can be described in two ways:

intrinsic or natural whereby microorganisms naturally do not posses target sites for the drugs and therefore the drug does not affect them or they naturally have low permeability to those agents because of the differences in the chemical nature of the drug and the microbial membrane structures especially for those that require entry into the microbial cell in order to effect their action or

acquired resistance whereby a naturally susceptible microorganism acquires ways of not being affected by the drug.

Mechanisms for acquired resistance

the presence of an enzyme that inactivates the antimicrobial agent

the presence of an alternative enzyme for the enzyme that is inhibited by the antimicrobial agent

a mutation in the antimicrobial agent’s target, which reduces the binding of the antimicrobial agent

post-transcriptional or post-translational modification of the antimicro- bial agent’s target, which reduces binding of the antimicrobial agent

reduced uptake of the antimicrobial agent

active efflux of the antimicrobial agent

overproduction of the target of the antimicrobial agent

expression or suppression of a gene in vivo in contrast to the situation in vitro

previously unrecognized mechanisms

Resistance to b-Lactam Antibiotics

b-Lactam antibiotics are a group of antibiotics characterized by possession of a b-lactam ring and they include penicillins, cephalosporins, carbapenems, oxapenams, and cephamycins. The penicillins are one of the most commonly used antibiotics in developing countries because of their ready availability and relatively low cost.

The b-lactam ring is important for the activity of these antibiotics which results in the inactivation of a set of transpeptidases that catalyze the final cross-linking reactions of peptidoglycan synthesis in bacteria. The effectiveness of these antibiotics relies on their ability to reach the penicillin-binding protein (PBP) intact and their ability to bind to the PBPs.

Resistance to b-lactams in many bacteria is usually due to the hydrolysis of the antibiotic by a b-lactamase or the modification of PBPs or cellular perme- ability. b-Lactamases constitute a heterogenous group of enzymes which are classified according to different ways including their hydrolytic spectrum,susceptibility to inhibitors, genetic localization (plasmidic or chromosomal), and gene or amino acid protein sequence. The functional classification scheme of b-lactamases proposed by Bush, Jacoby and Medeiros (1995) defines four groups according to their substrate and inhibitor profiles:

Group 1 are cephalosporinases that are not well inhibited by clavulanic acid;

Group 2 are penicillinases, cephalosporinases, and broad-spectrum b-lacta- mases that are generally inhibited by active site-directed b-lactamase inhibitors;

Group 3 are metallo-b-lactamases that hydrolyze penicillins, cephalosporins, and carbapenems and that are poorly inhibited by almost all b-lactam-containing molecules;

Group 4 are penicillinases that are not well inhibited by clavulanic acid.

Tetracycline Resistance

Tetracyclines are another of the very commonly used antimicrobial agents in both human and veterinary medicine in developing countries because of their availability and low cost as well as low toxicity and broad spectrum of activity. The tetracyclines were discovered in the 1940s. They inhibit protein synthesis by preventing the attachment of aminoacyl-tRNA to the ribosomal acceptor (A) site.

They are broad-spectrum agents, exhibiting activity against a wide range of gram-positive and gram-negative bacteria, atypical organisms such aschlamydiae, mycoplasmas, and rickettsiae, and protozoan parasites. Examples of these include drugs such as tetracycline, doxycycline, minocycline, and oxtetracycline. Resistance to these agents occurs mainly through three mechan- isms (Roberts, 1996), namely

Efflux of the antibiotics,

Ribosome protection, and

Modification of the antibiotic.

These tetracycline resistance determinants are widespread in different micro- organisms (Levy, 1988).

Efflux of the drug occurs through an export protein from the major facil- itator superfamily (MFS). These export proteins are membrane-associated proteins which are coded for by tet efflux genes and export tetracycline from the cell. Export of tetracycline reduces the intracellular drug concentration and thus protects the ribosomes within the cell.

Tetracycline efflux proteins have amino acid and protein structure similarities with other efflux proteins involved in multiple-drug resistance, quaternary ammonium resistance, and chloramphenicol and quinolone resistance. The gram-negative efflux genes are widely distributed and normally associated with large plasmids, most of which are conjugative.

Ribosome protection occurs through ribosome protection proteins that protect the ribosomes from the action of tetracyclines (Taylor and Chau, 1996). Ribosome protection proteins are cytoplasmic proteins that bind to the ribosome and cause an alteration in ribosomal conformation which prevents tetracycline from binding to the ribosome, without altering or stopping protein synthesis. They confer resistance mainly to doxycycline and minocycline and confer a wider spectrum of resistance to tetracyclines than is seen with bacteria that carry tetracycline efflux proteins.

Modification of the antibiotic on the other hand occurs through enzy- matic alteration of the drugs. Some of these genes are coded for by tet(X) genes.

Chloramphenicol Resistance

Chloramphenicol binds to the 50S ribosomal subunit and inhibits the peptidyl transferase step in protein synthesis. Resistance to chloramphenicol is generally due to inactivation of the antibiotic by a chloramphenicol acetyltransferase (Traced et al., 1993). Various enzymes have been described and are coded for by the cat genes found in gram- negative and gram-positive bacteria and usually show little homology (Kehrenberg et al., 2001). Sometimes decreased outer membrane perme- ability or active efflux is responsible for the resistance in gram-negative bacteria (Butaye et al., 2003).

Aminoglycoside Resistance

Resistance to aminoglycosides such as gentamicin, tobramycin, amikacin, and streptomycin is widespread, with more than 50 aminoglycoside-modifying enzymes described (Schmitz and Fluit, 1999). Most of these genes are associated with gram-negative bacteria.

Depending on their type of modification, these enzymes are classified as aminoglycoside acetyltransferases (AAC), aminogly- coside adenyltransferases (also named aminoglycoside nucleotidyltransferases [ANT]), and aminoglycoside phosphotransferases (APH) (Shaw et al., 1993). Aminoglycosides modified at amino groups by AAC enzymes or at hydroxyl groups by ANT or APH enzymes lose their ribosome-binding ability and thus no longer inhibit protein synthesis. Besides aminoglycoside-modifying enzymes, efflux systems and rRNA mutations have been described (Quintiliani and Courvalin, 1995).

Quinolone Resistance

The first quinolone with antibacterial activity (nalidixic acid) was discovered in 1962 during the process of synthesis and purification of chloroquine (an anti- malarial agent). Since then several derivatives have been made available on the market, with the most important ones being fluoroquinolones which contain a substitution of a fluorine atom at position 6 of the quinolone molecule.

This greatly enhanced their activity against gram-positive and gram-negative bac- teria as well as anaerobes. These agents exert their antibacterial effects by inhibition of certain bacterial topoisomerase enzymes, namely DNA gyrase (bacterial topoisomerase II) and topoisomerase IV.

These essential bacterial enzymes alter the topology of double-stranded DNA (dsDNA) within the cell. DNA gyrase and topoisomerase IV are hetero- tetrameric proteins composed of two subunits, designated A and B.

Mechanisms of bacterial resistance to quinolones as described by Hooper (1999) fall into two principal categories:

alterations in drug target enzymes and

alterations that limit the permeability of the drug to the target. [1-4]

The target enzymes are most commonly altered in domains near the enzyme- active sites, and in some cases reduced drug-binding affinity. In gram-negative organisms, DNA gyrase seems to be the primary target for all quinolones.

In gram-positive organisms, topoisomerase IV or DNA gyrase is the primary target depending on the fluoroquinolones considered. In almost all instances, amino acid substitutions within the quinolone resistance-determining region (QRDR) involve the replacement of a hydroxyl group with a bulky hydropho- bic residue.

Mutations in gyrA induce changes in the binding-site conformation and/or charge that may be important for quinolone–DNA gyrase interaction (Everett and Piddock, 1998). Changes in the cell envelope of gram-negative bacteria, particularly in the outer membrane, have been associated with decreased uptake and increased resistance to fluoroquinolones, and this has not been demonstrated in gram-positive bacteria.

Macrolide, Lincosamide, and Streptogramin (MLS) Resistance

MLS antibiotics are chemically distinct inhibitors of bacterial protein synthesis. Intrinsic resistance to MLSB (including streptogramin B) antibiotics in gram- negative bacilli is due to low permeability of the outer membrane to these hydrophobic compounds. Three different mechanisms of acquired MLS resis- tance have been found in gram-positive bacteria (Johnston et al., 1998). These include the following:

Post-transcriptional modifications of the 23S rRNA by the adenine-N6- methyltransferase which alters a site in 23S rRNA common to the binding of MLSB antibiotics which also confers cross-resistance to MLSB antibiotics (MLSB-resistant phenotype) and remains the most frequent mechanism of resistance. In general, genes encoding these methylases have been designated erm (erythromycin ribosome methylation).

Efflux proteins, which pump these antibiotics out of the cell or the cellular membrane, keeping intracellular concentrations low and ribosomes free from antibiotic, and these have become more frequent in gram-positive populations and often coded for by mef, msr, and vga genes.

Hydrolytic enzymes which hydrolyze streptogramin B or modify the antibiotic by adding an acetyl group (acetyltransferases) to streptogramin A have also been described and these confer resistance to structurally related drugs.

Glycopeptide Resistance

Glycopeptides comprise peptide antibiotics of clinical interest such as vancomycin and teicoplanin. Their antimicrobial activity is due to binding to D-alanyl-D-alanine side chains of peptidoglycan or its precursors, therebypreventing cross-linking of the peptidoglycan chain and thus are largely effec- tive against gram-positive microorganisms which poses a bigger layer of the peptidoglycan although not all gram-positive organisms are susceptible to these agents.

High-level resistance to vancomycin is encoded by the vanA gene that results in the production of VanA, a novel D-Ala-D-Ala ligase resulting in the re- building of the peptidoglycan side chain to express D-alanyl-D-lactate type which has less affinity for glycopeptides (Leclerq and Courvalin, 1997).

There are also other proteins in this gene cluster that are necessary for resistance including VanH and VanX, as well as VanB which confers moderate levels of resistance to vancomycin and susceptibility to teicoplanin. Vancomycin gained clinical importance because it was traditionally reserved as a last resort treat- ment for resistant infections especially of methicillin-resistant Staphylococcus aureus (MRSA). The emergency of vancomycin-resistant organisms has deprived the usefulness of this drug.

Sulfonamides and Trimethoprim Resistance

Resistance in sulfonamides is commonly mediated by alternative, drug-resistant forms of dihydropteroate synthase (DHPS). Sulfonamide resistance in gram- negative bacilli generally arises from the acquisition of either of the two genes sul1 and sul2, encoding forms of dihydropteroate synthase that are not inhibited by the drug (Enne et al., 2001).

The sul1 gene is normally found linked to other resistance genes in class 1 integrons, while sul2 is usually located on small nonconjugative plasmids or large transmissible multi-resistance plasmids.

Trimethoprim is an analog of dihydrofolic acid, an essential component in the synthesis of amino acid and nucleotides that competitively inhibits the enzyme dihydrofolate reductase (DHFR). Trimethoprim resistance is caused by a number of mechanisms (Thomson, 1993) including

overproduction of the host DHFR,

mutations in the structural gene for DHFR, and

acquisition of a gene (dfr) encoding a resistant DHFR enzyme which is the most resistant mechanism in clinical isolates.

At least 15 DHFR enzyme types are known based on their properties and sequence homology (Schmitz and Fluit, 1999).

Multidrug Resistance

Multidrug resistance among many organisms has become a big challenge to infectious disease management. It is increasingly being reported in bacteria and is often mediated by genetic mobile elements such as plasmids, transposons, and integrons (Dessen et al., 2001). Integrons are mobile DNA elements with theability to capture genes, notably those encoding antibiotic resistance, by site- specific recombination, and they have an intergrase gene (int), a nearby recom- bination site (attI), and a promoter, Pant (Hall, 1997).

Integrons seem to have a major role in the spread of multidrug resistance in gram-negative bacteria but integrons in gram-positive bacteria have also been described (Dessen et al., 2001). Class 1 integrons are often associated with the sulfonamide resistance gene sulI and are the most common integrons. Class 2 integrons are associated with Tn7. The majority of genes encode antibiotic disinfectant resistance, including resistance to aminoglycosides, penicillins, cephalosporins, trimetho- prim, tetracycline, erythromycin, and chloramphenicol.

CHAPTER 17

GRAM-NEGATIVE BACILLI. ENTEROBACTERIACEAE

The Enterobacteriaceae are a large family of Gram-negative rods with natural habitat the intestinal tract of humans and animals. They have 10-16 m length and 0.3 m diameter, are aerobic facultative anaerobic, motile because of peritrichous flagella non-capsulated, but some being capsulated and non- motile. The most important biochemical characters are: fermentation of glucose, presence of catalase , absence of cytochrome oxidase (oxidase negative) .oxidase negative, and capacity to reduce nitrates in nitrites.

Other biochemical characters , which are used for biochemical identification of species are :

– Lactose fermentation , differentiating the lactose fermenting species (E. coli , Klebsiella ) and lactose non – fermenting ( Proteus speciesand the obligate pathogenic species )

– H2S production , the ability to use citrate as a sole carbon source , the production of indole, urea decomposition , lysine decarboxylase, etc.

In this family there are saprophytic species of the human digestive tract, conditioned pathogens and obligate human pathogenic species :

a. – saprophytic, conditioned pathogens

Genus escherichia, type specie E. Coli

Genus Klebsiella, type specie Kebsiella pneumoniae, other species

– K. ozenae

– K. Rhinoscleromatis

Genus proteus, species: Proteus vulgaris, Proteus mirabilis

Genera: Enterobacter, Citrobacter, Serratia, Providencia, Morganella, etc.

b. – obligate patogenic enterobacteria, belongigng to genera:

genus Salmonella, species

– S. Typhi

S. Paratyphi A, B, C

S. Enteritidis,

S. Cholerae suis

Genus Shigella, species

Shigella disenteriae

Sh. Flexneri

Sh. Boydii

Sh. Sonnei

Genus Yersinia, species

Y. Pestis

Y. Enerocolitica

Y. Pseudotuberculosis

Antigenic structure of Enterobacteriaceae is complex. The most important antigens are:

The somatic O antigens, lypopolisaccharidis, heat-stable, resistant to alcohol, and generally detectable by agglutination reactions. There are O antigens genus-specific, species specific and with other specificities. Cross reactivity is present among some species.

Capsular K antigens are present in some enterobacteriaceae (capsulated, uaually) and are exterior to O antigens, which they may cover and mask. K antigens are polysaccharides or proteins. They are type-specific and usually are associated with virulence, in Klebsiella , E.coli.

Flagellar H antigens have proteic structure and are heat-labile, alcohol sensitive, but formalin resistant. The H antigen agglutinate with specific type antibodies ( mainly Ig G) and may be present in two phases ( phase 1 and phase 2), that can change from one to other, function of the amino acid sequence in flagellines. Phase 1H antigens are designated by letters and phase 2 antigens are designated by numerals, eg. Salmonella typhi is O grup D, H type 9, 12, Vi, d-. E coli is O55; K5; H21.

Enterobacteria may produce bactericidal proteins, named bacteriocines, active agains other related species or other strains of the same species. E.coli produces colicines, Pseudomonas aeruginosa produces pyocine, all being codified by plasmid genes.

CHAPTER18. NON-FERMENTING GRAM-NEGATIVE BACILLI

Genus Pseudomonas

Pseudomonas genus ( the pseudomonads) comprises Gram -negative aerobic bacteria, measuring 0.5 to 0.8 µm by 1.5 to 3.0 µm, mobile due to a polar flagellum , glucose non -fermentative , oxidase positive , and producing water-soluble pigments . Pseudomonas can live without oxygen if they find NO3 as electron acceptor .

Pseudomonas are found in soil, water, plants, in the intestine and on the skin of humans and animals . They can survive in moist environments such as bathrooms , toilets , being present such places in in hospitals, even in the presence of weak disinfectants .

The species of the genus are generally saprophytic, conditioned pathogenic. Pseudomonas aeruginosa is the most important species and the major pathogen for humans.

Picture . Pseudomonas aeruginosa- E.M. Source : CDC

General characters

The main species of medical interest are the fluorochromes pigment producers Pseudomonas aeruginosa and Pseudomonas fluorescens and Pseudomonas putida . There alre also nonfluorescent species. Stenotrophomonas maltophilia is a species sometimes isolated in clinical specimens. The most important species with highest pathogen potential is P. aeruginosa.

Pseudomonads are saprophytic , conditioned pathogenic . In small number, they are found on the skin and among the intestinal flora of healthy subjects. In people with low immunity , with frequent and prolonged hospitalization, or following intravenous or urinary catheterizations, Pseudomonas produce infections . In the hospitals there are also selected particular antibiotic resistant strains, responsible for severe nosocomial (hospital aquired ) infections.

Culture characters

Pseudomonas species have minimal nutritional requirements, being a non-fastidous bacterium. They grow readily both in liquid and on solid culture media, commonly incubated at temperatures between 37 ° C – 42 ° C. They release in medium the water-soluble pigments and produce a specific odor. On blood-agar, some strains produce haemolysis.

Figure

Pseudomonas aeruginosa

Pseudomonas aeruginosa has the typical characters of the genus, Gram-negative rods, 0.6×2 µm, motile, occurring isolated, in short chains or in pairs, aerobic, oxydase-positive.

In liquid medium cultures, Ps.aeruginosa release the water-soluble pigments and produce a specific odor.

On solid culture media, the colonies may be type R , S or M , depending on the origin of the strain and age of the culture. Typical 24h or 48h colonies have a metallic sheen and release a distinct bitter odor , (acacia flower ) because the production of amino -acetophenone .

Ps. Aeruginosa can grow at 42O C, unlike other species from the fluorescent group ( differential character).

P.aeruginosa produces several pigments: a specific blue -green pigment, pyocyanine , which diffuses into the culture medium. Pyocyanin determines the "blue pus", which is a characteristic of suppurative infections caused by Pseudomonas aeruginosa. Other pigments produced by P. aeruginosa as well as by other species are pyoverdin – a yellow -greenish fluorescent pigment , and less frequent, a dark-red pigment pyorubrin and a black pigment pyomelanine (rarely).

Besides pigments, P. aeruginosa produces piocines, antibacterial substances that act against other species , especially against Gram -positive cocci .

Characters of pathogenicity

Pseudomonas aeruginosa is a conditioned pathogenic species, but having many virulence factors:

– Adhesion due to fimbriae ( common pilli), promoting attachment to the epithelial cells of the host;

– Invasiveness due extracellular proteolytic enzymes: elastase and alkaline protease that lyses the elastin, collagen, IgG, IgA and complement, but mainly the fibrin. It also produces two hemolysins: phospholipase and lecitinase.

– Mucopolysaccharides ( exopolysaccharides) forming a slime: encapsulated strains favor the formation of a biofilm that protects the bacteria from phagocytic activity and action of antibiotics. Mucoid strains of P. aeruginosa are frecquently isolated from patients with cystic fibrosis and of respiratory infections.

– the endotoxin ( lipopolysaccharide) responsible for typical endotoxic properties;

– Exotoxin production: exotoxin A and exoenzyme S. Exotoxin A, by means of blocking protein synthesis produces tissue necrosis at the site of colonization. There was demonstrated a systemic role of purified Exotoxin A , which is highly lethal for animals including primates. Exoenzyme S acts on phagocytic cells and impairs their functions.

– other enzymes are: two hemolysines, one heat- stable and one heat-labile ( phospholipase C). Also, there are produced and released proteases and elastases.

– Genetic factors: natural resistance to a number of antibiotics, presence of R factors and resistance plasmids, high variability mechanisms transduction and conjugation.

Pathogenesis

Most Pseudomonas infections are both invasive and toxinogenic. The Pseudomonas infection respects the three distinct stages: (1) bacterial attachment and colonization; (2) local invasion; (3) disseminated systemic disease.

Pseudomonas aeruginosa cause infections in patients with reduced defence : wound infections, skin infections in burns , urinary infections , respiratory infections , otitis media , post-operative conjunctivitis and post- traumatic meningitis, osteomyelitis . Bacteremia and septicemia may complicate the evolution of a localized infections in immunocompromised children and adults , particularly in patients with severe burns and in cancer and AIDS patients who are immunosuppressed. Pseudomonas aeruginosa infection is a serious problem in patients hospitalized with cancer, cystic fibrosis, and burns. The case fatality rate in these patients is near 50 percent.

Resistance to antibacterial drugs

Pseudomonas aeruginosa is wellknown for its resistance to antibiotics and is, therefore, a particularly dangerous pathogen. The bacterium is naturally resistant to many antibiotics Also, its capacity to colonize surfaces and form biofilms makes the cells impervious to therapeutic concentrations antibiotics. Since its natural habitat is the soil, living in association with the bacilli, actinomycetes and molds, it has developed resistance to a variety of their naturally-occuring antibiotics. Moreover, Pseudomonas maintains antibiotic resistance plasmids, both R-factors and RTFs, and it is able to transfer these genes mainly by transduction and conjugation. [K.Todar  Online Textbook of Bacteriology. "]
Only a few antibiotics are effective against Pseudomonas aeruginosa, The futility of treating Pseudomonas infections with antibiotics is most dramatically illustrated in cystic fibrosis patients, virtually all of whom eventually become infected with a resistant strain .

Diagnosis

Specimens are the most varied : secretions of the wound , pus , urine , ear secretion , conjunctival secretion , Cerebrospinal Fluid , blood for blood culture in systemic infections.

Culture is done on usual media : blood-agar and and differential media for Enterobacteriaceae. The culture characters are usually significant for diagnosis. Biochemical identification on classic or automatic devices finally establish the species diagnosis. (lack of fermenting glucose and lactose are the most characteristic ) .

Sensitivity tests to antibacterial drugs are mandatory due to variable sensitivity and high resistance to antibiotics. P. aeruginosa can be sensitive to certain beta – lactam antibiotics : carbenicillin , ticarcillin , azlocillin , mezlocillin , generation cephalosporins III and IV aminoglycosides , carbapenems ( imipenem ) and monobactams ( aztreonam ) , second and third generation fluoroquinolones: ciprofloxacin, levofloxacin .

19.VIBRIO, CAMPYLOBACTER, HELICOBACTER

In Campylobacter genus from the family of Campylobacteriaceae, there are gram-negative bacili, spiral shaped, 0.2 to 0.5 µm, very motile, micro-aerophilic, that grow only in the reduced oxygen level.

thefoodpoisoninglawyers.com

They can be easily eradicated by heat, drying, oxygen rich and acidic medium.

Campylobacter can be found in the intestine of human and most animals. The infection is more prevalent in the small intestine than large intestine. Most common for human pathology are C. Jejuni and C. fetus.

Campylobacter penetrate the mucus membrane due to vivid motility, like cork screw then it adhere to enterocytes producing diffuse, bloody, edematous, and exudative enteritis. There is an inflammatory infiltrate with neutrophils, mononuclear cells, and eosinophils. Some strains of C jejuni produce a heat-labile, choleralike enterotoxin, causing watery diarrhea. A cytotoxin production in some Campylobacter strains produce bloody diarrhea.

There are reported in a small number of cases of infection complications like hemolytic-uremic syndrome  and thrombotic thrombocytopenic purpura.

During infection, all the immunoglobulins rises but the most important is the IgA because it can cross the gut wall, this activates the complement that can give temporary immunity.Error: Reference source not found

The symptoms associated with Campylobacter food poisoning illness are diarrhea, which can be bloody, stomach cramps and pain, fever, nausea and vomiting.  Symptoms usually arise within two to five days after a person is exposed to the bacteria and persist for about one week, though some infected persons have no symptoms at all.

Illustration of H. Pylory also a gram-negative bacteria, penetrating the mucous membrane.

Photo credit. Error: Reference source not found All rights reserved to the owner.

The bacteria infect a person by:

C. Jejuni adheres to jejunum, ileum and colon epithelial and mucus using flagella.

The organism produces diffuse, bloody, edematous and exudative enteritis. Infiltration of lamina propia occurs with mononuclear cells and eosinophil.

It then proliferates and multiplies in the mesenteric lymph node and lamina propia. This result in extraintestional infections. C. Jejuni is also associated with Guillane-Barre syndrome.

C. Fetus is more likely to manifest as a systemic infection, bacteremia. S protein found in C. Fetus is the major virulence factor as it makes itself resistant to the bactericidal effect of the body.Error: Reference source not found

The human ingestion of as low as 500 – 10 000 organisms will give an infection.Error: Reference source not foundError: Reference source not found

TRANSMISSION

Campylobacter is widely spread in warm blooded animals. Animal act as a reservoir to infect human. Then it could also spread from person to person. It is usually more prevalent in food animals (cows, chicken, rabbit, ostrich, sheep etc.). It is believed to be spread from oral-fecal route as it is a food borne disease. This happen with undercooked meat and meat product. Washing the meat product could also spreads the disease easily by water splashing. It can also be easily spread from faeces during animal slaughtering. Drinking contaminated milk, raw milk, contaminated water and untreated water. Poulty meat can also spread the bacteria as it can be unproperly cured or treated.Error: Reference source not found

EPIDEMIOLOGY

Campylobacter infections are among the most common bacterial infections in humans. They produce both diarrheal and systemic illnesses. In industrialized regions, enteric Campylobacter infections produce an inflammatory, sometimes bloody, diarrhea or dysentery syndrome.

Campylobacter jejuni (see image below) is usually the most common cause of community-acquired inflammatory enteritis. In developing regions, the diarrhea may be watery.

Campylobacter is a leading cause of bacterial diarrheal disease worldwide; in the United States, it is estimated to cause 1.3 million human illnesses every year. Since 2008 to 2014 Campylobacter remained as the most reported pathogen of gastrointestinal disease in the European Union. Campylobacter is the most common laboratory-confirmed enteric pathogen reported in travelers returning to the United States from every region of the world. The risk of infection is highest in travelers to Africa and South America, especially in areas with poor restaurant hygiene and inadequate sanitation. The infectious dose is thought to be small as low as 500 organisms can cause disease.Error: Reference source not found

The known routes of Campylobacter transmission include fecal-oral, person-to-person sexual contact, unpasteurized raw milk and poultry ingestion, and waterborne (ie, through contaminated water supplies). Exposure to sick pets, especially puppies, has also been associated with Campylobacter outbreaks.

En the EU, incidence was highest among children under four years of age and, in particular, one-year-old boys were affected. This is in line with the epidemiology of other gastrointestinal infectious diseases showing a comparable demographic pattern, e.g., infections with Yersinia enterocolitica,Salmonella spp., and Shiga toxin-producing Escherichia coli (STEC) with peak incidence in young children [26,27, 28]. The immune response in infants has not fully established, which may explain their susceptibility to various infectious diseases. However, other age-specific risk factors, like e.g. insufficient hand hygiene or close contact to animals or to the environment, may also play a role.[ Anika Schielke]/

Transmission of Campylobacter organisms to humans usually occurs via infected animals and their food products. Most human infections result from the consumption of improperly cooked or contaminated foodstuffs. Chickens may account for 50-70% of human Campylobacter infections. Most colonized animals develop a lifelong carrier state.

The infectious dose is 1000-10,000 bacteria. Campylobacter infection has occurred after ingestion of 500 organisms by a volunteer; however, a dose of less than 10,000 organisms is not a common cause of illness. Campylobacter species are sensitive to hydrochloric acid in the stomach, and antacid treatment can reduce the amount of inoculum needed to cause disease.

Symptoms of Campylobacter infection begin after an incubation period of up to a week. The sites of tissue injury include the jejunum, the ileum, and can extend to involve the colon and rectum. C jejuni appears to invade and destroy epithelial cells. C jejuni are attracted to mucus and fucose in bile, and the flagella may be important in both chemotaxis and adherence to epithelial cells or mucus. Adherence may also involve lipopolysaccharides or other outer membrane components. Such adherence would promote gut colonization. PEB 1 is a superficial antigen that appears to be a major adhesin and is conserved among C jejuni strains.

Some strains of C jejuni produce a heat-labile, choleralike enterotoxin, which is important in the watery diarrhea observed in infections. Infection with the organism produces diffuse, bloody, edematous, and exudative enteritis. The inflammatory infiltrate consists of neutrophils, mononuclear cells, and eosinophils. Crypt abscesses develop in the epithelial glands, and ulceration of the mucosal epithelium occurs.

Cytotoxin production has been reported in Campylobacter strains from patients with bloody diarrhea. In a small number of cases, the infection is associated withhemolytic-uremic syndrome and thrombotic thrombocytopenic purpura through a poorly understood mechanism. Endothelial cell injury, mediated by endotoxins or immune complexes, is followed by intravascular coagulation and thrombotic microangiopathy in the glomerulus and the gastrointestinal mucosa.

Campylobacter species also produce the bacterial toxin cytolethal distending toxin (CDT), which produces a cell block at the G2 stage preceding mitosis. CDT inhibits cellular and humoral immunity via destruction of immune response cells and necrosis of epithelial-type cells and fibroblasts involved in the repair of lesions. This leads to slow healing and results in disease symptoms.[1]

In patients with HIV infection, Campylobacter infections may be more common, may cause prolonged or recurrent diarrhea, and may be more commonly associated with bacteremia and antibiotic resistance.

C fetus is covered with a surface S-layer protein that functions like a capsule and disrupts c3b binding to the organisms, resulting in both serum and phagocytosis resistance.

C jejuni infections also show recurrence in children and adults with immunoglobulin deficiencies. Acute C jejuni infection confers short-term immunity. Patients develop specific immunoglobulin G (IgG), immunoglobulin M (IgM), and immunoglobulin A (IgA) antibodies in serum; IgA antibodies also develop in intestinal secretions. The severity and persistence of C jejuni infections in individuals with AIDS andhypogammaglobulinemia indicates that both cell-mediated and humoral immunity are important in preventing and terminating infection.

The oral cavity contains numerous Campylobacter species, such as Campylobacter concisus, that have been associated with a subtype of inflammatory bowel disease.