Microbiological, biochemical and antibiotic resistance characterization of some Gram negative bacilli isolated from different patients [306388]
[anonimizat]. Mariana Carmen Chifiriuc
Scientific advisor
Dr. Irina Gheorghe
STUDENT: [anonimizat]
2017
Contents
Chapter I
1.1. Introduction………………………………………………………………
1.2. Escherichia coli …………………………………………………………………………..
1.2.1. Urinary tract infections (UTI)…………………………………………………………
1.2.2. Neonatal meningitis……………………………………………………………………..
1.2.3. Intestinal diseases……………………………………………………………………….
1.3 Klebsiella pneumoniae…………………………………………………………………….
1.4. Pseudomonas aeruginosa…………………………………………………………………
1.4.1. P. aeruginosa the opportunistic pathogen……………………………………………..
1.5. Acinetobacter baumannii…………………………………………………………………
1.5.1 Clinical Manifestations of Acinetobacter baumannii Infections………………………
1.5.1.1 Hospital-Acquired Pneumonia…………………………………………………..……
1.5.1.2 Community-Acquired Pneumonia……………………………………………………
1.5.1.3 Urinary tract infection ………………………………………………………………..
1.5.1.4 Meningitis………………………………………………………………………………
1.5.1.5 Bloodstream infections…………………………………………………………………
1.5.1.6 Burn and soft tissue infections………………………………………………………..
Chapter II
2.1. The history of antibiotic……………………………………………………………..……
2.2. Beta-lactam antibiotics …………………………………………………………………..
2.3. Mechanisms of antimicrobial resistance in Gram‑negative bacilli…………………….
2.3.1. Antibiotic resistance mechanisms in Enterobacteriaceae…………………………….
2.3.1.1. [anonimizat]………………………………………………
2.3.1.2. [anonimizat]……………………………………………………………
2.3.1.3. [anonimizat] (ESBLs) ……………………………………….
2.3.1.4. Carbapenemases………………………………………….…………………………….
2.3.2. Resistance to aminoglycosides……………………………………………………………
2.3.3. Resistance to polymyxins…………………………………………………………………
2.3.2. Antimicrobial resistance in non fermenting GNB………………………………………
2.3.2.1. Pseudomonas aeruginosa……………………………………………………………….
2.3.2.2. Acinetobacter baumannii………………………………………………………………
2.4. Genetic support of Carbapenemase Genes in Gram negative bacteria…………………
2.4.1. Classification of Carbapenemases……………………………………………………….
Class A carbapenemases………………………………………………………………………….
Class B carbapenemases………………………………………………………………………..
Class D carbapenemases………………………………………………………………………..
2.4.2. Genetic Platforms of Carbapenemase Genes in Enterobacteriaceae and in P. aeruginosa, A. baumanni, Species………………………………………………………………
2.5. P. aeruginosa virulence factors: Secreted proteins………………………………………..
2.5.1. Inner membrane translocation……………………………………………………………
2.5.1.1. The sec Translocon………………………………………………………………………
2.5.1.2. The Tat Translocon………………………………………………………………………
2.5.2. The Type I Secretion System (TISS)………………………………………………………
2.5.3. The Type II Secretion System (T2SS)…………………………………………………….
2.5.4. The Type III Secretion System (T3SS)…………………………………………………….
Chapter III
Aim………………………………………………………………………………………………….
Objectives………………………………………………………………………………………….
Materials and Methods…………………………………………………………………………..
Results and Discussions …………………………………………………………………………
Conclusion………………………………………………………………………………………..
References………………………………………………………………………………………..
Chapter I
[anonimizat]. [anonimizat] on the rise. For example, between 1986 and 2003, the ratio of Enterobacteriaceae sp resistant to third-generation cephalosporins has raised by more than tenfold; resistant Klebsiella pneumoniae and Escherichia coli have increased by two fold (1).The rate of antibiotic resistance has increased from 4% in 1986 to 7% in 2003 for Pseudomonas aeruginosa (2) and Acinetobacter species (1). Multidrug resistant Gram-negative bacteria (MDR-GNB) are responsible for a broad diversity of infections encountered in the intensive care unit (ICU) such as ventilator-associated pneumonia, vascular catheter-related infection and urinary tract infection.
Infections gained from clinically relevant Gram-negative bacilli involve a broad diversity of species. Urinary tract infections (Proteus species, E.scherichia coli, Serratia marcescens, Enterobacter species), respiratory infections (Pseudomonas aeruginosa, Haemophilus species, Legionella pneumophila, Klebsiella species), and gastrointestinal infections (Salmonella enteritidis ,Salmonella typhi , Helicobacter pylori, Shigella species) are the primary Gram negative infections in most hospital settings (3).In recent times, a non fermentative Gram negative organism, Acinetobacter baumannii has been associated with hospital-acquired infections. Similarly, other less encountered Gram-negative bacteria such as Stenotrophomonas maltophilia and Aeromonas species have been increasingly reported in abundant episodes of bacteraemia, meningitis, surgical site or wound problems in hospital intensive-care settings, pneumonia (4). Most of these Gram-negative bacteria are resistant to multiple drug agents and are increasingly becoming resistant to almost all available antimicrobial drugs worldwide (5).
1.2. Escherichia coli
E. coli was first described in 1885 by Theodor Escherich as Bacterium commune, which he isolated from the feces of newborns. It was renamed E. coli and considered to be a commensal of the large intestine. Escherichia species are Gram-negative bacilli that exist singly or in pairs and are commonly present in the intestines of humans and animals. E. coli are facultative anaerobes and have the ability undergo both respiratory and fermentative metabolisms. They are motile by peritrichous flagella.
E. coli is nonspore-forming and beta hemolytic. On MacConkey Agar, it generally ferments lactose or produces pink colonies with surrounding areas of precipitated bile salts. It also displays with a green sheen on eosin methylene blue agar. E. coli strain will production indole from tryptophan; it does not produce hydrogen sulfide, Urease, and do not has the ability to use citrate as sole carbon source (6). Pathogenic strains of E. coli are responsible for three types of infections in humans; urinary tracts infections, neonatal meningitis, and intestinal diseases (7).
1.2.1. Urinary tract infections (UTI)
E. coli strains that cause UTIs are termed uropathogenic E. coli (UPEC) which causes 90 % of urinary tract infections in anatomically normal and unobstructed urinary tracts. The (UPEC) strains have an adherence factor called P fimbriae, or pili, which have the ability binds to the P blood group antigen and mediates the attachment of E. coli to uroepithelial cells. Thus, patients with intestinal carriage of this strain are at higher risk of developing UTI than the general population. Complicated UTI and pyelonephritis caused by E. coli are observed in old patients with structural abnormalities or obstruction such as neurogenic bladders, prostatic hypertrophy or in patients with urinary catheters. The UTI is the most common site of E. coli infection and the uropathogenic strain is usually implicated in most (90 %) of all uncomplicated UTIs including uncomplicated urethritis/cystitis, symptomatic cystitis, pyelonephritis, acute prostatitis, prostatic abscess, and urosepsis (8).
1.2.2. Neonatal meningitis
Neonatal meningitis is a life-threatening disease which affects infants. The disease is transmitted from mothers who are colonized with the K1 strain of E. coli during pregnancy to their infants (8).
1.2.3. Intestinal diseases
As a cause of enteric infections, six various mechanisms of 6 different varieties of E coli have been reported. Enterotoxigenic E. coli (ETEC) causes traveller’s diarrhea. Enteropathogenic E. coli (EPEC) is responsible for childhood diarrhea. Enter invasive E coli (EIEC) causes a Shigella -like dysentery. Enterohemorrhagic E. coli (EHEC) causes hemorrhagic colitis or hemolytic-uremic syndrome (HUS). Enter aggregative E. coli (EAggEC) is firstly related to persistent diarrhea in children in developing states, and enter adherent E. coli (EAEC) is the cause of childhood diarrhea and traveller’s diarrhea in Mexico and North Africa. All the diverse varieties colonize the small bowel, except EIEC and EHEC which preferentially colonize the large bowel prior to causing diarrhea (9).
1.3 Klebsiella pneumoniae
Klebsiella pneumoniae is an encapsulated Gram-negative bacterium that is frequently found in the flora of the mouth, skin and intestines as well as in natural environments (10). It is the most important clinical member of the Klebsiella genus of Enterobacteriaceae, sp and is the third most commonly isolated microorganism in blood cultures from sepsis patients that can cause serious epidemic and endemic nosocomial infections (10, 11). In humans, immunocompromised or immunodeficient individuals have a significantly increased probability of catching Klebsiella pneumoniae in the lung, urinary tract, blood (sepsis), liver and other organs (12). Globally, K. pneumoniae is always encountered hospital acquired opportunistic pathogen which typically infects patients with indwelling medical devices. In this environment, it is often severe, persistent, and hard to eradicate. This bacterium is rapidly developing resistance to multiple antibiotics, including broad-spectrum cephalosporins and β-lactams (13).Consequently, Klebsiella infections are usually associated with high mortality rates, particularly in infected immunodeficient individuals (14).Among the Enterobacteriaceae ,sp K. pneumoniae is the leading cause of nosocomially acquired pneumonias (15) and recent epidemiological evidence suggests that the occurrence rates of Klebsiella infections are significantly higher than in reported data (16) Due to their clinical significance, the underlying virulence factors have been actively investigated, and a variety of attributes have been characterized and implicated in the pathogenicity of K. pneumoniae (17). The selective infection in immunodeficient populations indicates that the host environment and immunity competency play an important role in the outcomes of the infection progression. Two surface structural composites of K.pneumoniae, capsular polysaccharides (CPS) and lipopolysaccharide (LPS) are important virulence factors. These two antigens are also used for serotyping of pathogenesis potency, K antigens for CPS structure, and O antigens for LPS structure (18).
Non fermentative Gram negative bacilli
1.4. Pseudomonas aeruginosa
P. aeruginosa is a Gram negative, rod-shaped bacterium, with a cell diameter of 0.5-1.0 μm and length of 1.5-3.0 μm. It has an optimum growth temperature of 37 °C, but it has the ability to grow between 4 °C and 42 °C. P. aeruginosa has comparatively simple nutritional requirements, often reported living in distilled water. P. aeruginosa is bacterium of the Pseudomonadaceae family (a member of the Gammaproteobacteria) (19). P. aeruginosa contains 12 other members of its family. Similar to other members of the genus, P. aeruginosa is commonly found in water and soil and also in humans and plants. Importantly, P. aeruginosa has become an emerging opportunistic pathogen in the clinics. Lately, epidemiological studies show its nosocomial pathogen status, particularly those strains with increased antibiotic resistance.
P. aeruginosa exploits weaknesses in host defense to mount an infection. Indeed, P. aeruginosa is the epitome of an opportunistic pathogen of humans. P. aeruginosa hardly infects uncompromised tissues, but has the ability to invade any tissue beleaguered by immunodeficiency. Many infections caused by P. aeruginosa infection the respiratory system, urinary tract, dermis, soft tissue, bacteraemia, joint and bone, gastrointestinal and blood, particularly in patients with severe burns, tuberculosis, cancer and AIDS. Importantly, P. aeruginosa cause a significant trouble in patients hospitalized with cancer, cystic fibrosis, and burns, with 50% fatality (20) According to the Centers for Disease Control and Prevention, the total incidence of P. aeruginosa infections in united states hospitals medium about 0.4% (4 per 1000 discharges), and the bacterium is the fourth most prevalent isolated nosocomial pathogen, accounting for 10% of all hospital-acquired infections..The bacterial strains are motile with a single polar flagellum. The metabolism is oxygen-based respiratory, but this ubiquitous bacterium will grow in the absence of O2 but in the presence of NO3 (21).
P. aeruginosa isolates demonstrate three types of colonies. Natural isolates from water or soil typically are a small, rough colony, while clinical isolates are probably to smooth colony types, sometimes with a fried-egg appearance that is large, smooth, with flat edges and an elevated appearance. Respiratory and urinary tract secretions may display a mucoid-type (alginate slime). P. aeruginosa contains two different soluble pigments, the blue pyocyanin. Pyocyanin refers to blue pus and fluorescent pigment pyoverdin, a typical feature of suppurative infections caused by P. aeruginosa. Pyochelin (a derivative of pyocyanin) is a siderophore and can get iron from the host or in low-iron environments to maintain the pathogen growth. Pyocyanin can cause a problem for the normal function of human nasal cilia and the respiratory epithelium, thereby igniting pro-inflammatory responses (22).
1.4.1. P. aeruginosa the opportunistic pathogen
In addition to being an environmental organism, P. aeruginosa is a common opportunistic pathogen of humans, and infections can be acquired in the community or in the hospital. Community-acquired infections include outer ear, skin and soft tissue infections in those with diabetes mellitus, ulcerative keratitis in contact lens users, and soft tissue infections associated with burn wounds (23).
Hospital-acquired P. aeruginosa infections can be life threatening with high levels of morbidity and mortality. P. aeruginosa infects a range of sites, including the lungs, surgical wounds, urinary tract, and can cause septicaemia (23). P. aeruginosa is responsible for 11-13.8% of nosocomial infections where a microbiological cause is identified in three studies from Korea, Italy and Switzerland (24, 25, 26), and is the second most common cause of healthcare-associated pneumonias in the United States (27) P. aeruginosa is therefore clearly responsible for a large proportion of the infectious disease burden in hospitals.
Infections caused by P. aeruginosa are a particular problem for those with immune deficiencies including cancer, HIV, and recipients of organ transplantation. P. aeruginosa is responsible for 14-21% of bacteraemia in patients with acute leukemia, a higher rate compared to non-cancerous patients (28). In individuals infected with HIV, the incidence of P. aeruginosa bacteraemia is ten times higher compared to those not infected by the virus (29).
1.5. Acinetobacter baumannii
Acinetobacter baumannii is a Gram-negative, aerobic, non-motile, nonfastidious, and nonfermenting coccobacilli (30). Acinetobacter spp. normally form smooth, sometimes mucoid, pale yellow to grayish-white colonies on solid media, although some environmental strains that produce a diffusible brown pigment (31).The colonies are comparable in size to those of enterobacteria. Acinetobacters are strictly aerobic, non fastidious organisms that can grow on common laboratory media. Most of the Acinetobacters grow between 20Co and 41Co with most strains having an optimum at 37 °C.Growth at 44 °C which is a characteristic feature of A. baumannii. The pH range for growth of Acinetobacters spp is 5.5to6.0 (32). On nutrient agar media Acinetobacter sp grows to form smooth, mucoid, pale yellow to greyish white colonies about 1-2mm in diameter (33).
This opportunistic pathogen has emerged as one of the most problematic microorganisms in causing nosocomial infections globally (30). Most of skin and blood infections, as well as pneumonia and meningitis, are caused by A. baumannii (30). Patients in military hospitals and intensive-care units are commonly vulnerable to infections caused by A. baumannii and these infections often have a very high mortality rate (30, 34). This bacterium is especially troublesome because it has the ability to survive for prolonged periods of time on dry surfaces and is hard to remove with disinfectants, which contributes to its spread (35). In addition, A. baumannii has the remarkable ability to up-regulate its antibiotic resistance systems and get resistance determinants from other species, making it difficult to treat with antibiotics (30).
1.5.1 Clinical Manifestations of Acinetobacter baumannii Infections
1.5.1.1 Hospital-Acquired Pneumonia
In most institutions, the majority of A. baumannii isolates are from the respiratory tracts of hospitalized patients. In many circumstances, it is so hard to differentiate upper airway colonization from true pneumonia. There is no doubt, however, that true ventilator-associated pneumonia (VAP) due to A. baumannii happens. In large surveillance studies from the United States, between 5 and 10% of cases of ICU-acquired pneumonia were due to A. baumannii (35). It is highly likely that in certain institutions, the ratio of intensive care units (ICU)-acquired pneumonia due to A. baumannii is much higher. Typically, patients with A. baumannii infections have had prolonged ICU stays (36).
1.5.1.2 Community-Acquired Pneumonia
Community-acquired pneumonia due to A. baumannii described for tropical regions of Australia and Asia (37, 38). The illness most typically happens during the rainy season among people with a history of alcohol abuse also sometimes require admission to an ICU. It is characterized by a fulminant clinical course, secondary bloodstream infection, and mortality rate of 40 to 60% (325).The source of infection may be throat carriage, which occurs in up to 10% of community residents with excessive alcohol consumption (37).
1.5.1.3 Urinary tract infection
A. baumannii is an occasional cause of urinary tract infection (UTI), being responsible for just 1.6% of ICU-acquired UTIs in one study (35). Typically, the organism is associated with catheter-associated infection or colonization. It is not usual for this organism to cause uncomplicated UTI in healthy outpatients.
1.5.1.4 Meningitis
Nosocomial, post neurosurgical A. baumannii meningitis is an increasingly important structural. The microbial epidemiology of nosocomial meningitis is developing to include further Gram negative pathogens (39).
1.5.1.5 Bloodstream infections
A. baumannii is associated with bloodstream infections in the intensive care units. The common sources of bloodstream infections are lower respiratory tract infections and intravascular devices, although other primary infections, such as wound infections and urinary tract infections have also been associated with BSI (40). The crude mortality associated to bloodstream infections ranges between 28% and 43% (41)
1.5.1.6 Burn and soft tissue infections
A. baumannii is also an important cause of soft tissue and burns infections, which are complex to treat because the strains causing these infections are usually MDR and some antibiotics have a poor penetration to the infected sites (40). Interestingly, A. baumannii has been associated with wound infections in military personnel. Specifically, in a study carried out at the US Army Institute of Surgical Research Burn Center, A. baumannii represented 22% of the microorganisms recovered from burn injuries, where 56% of the isolates were MDR (42)
However, these infections are uncommon outside the military environment (43).These infections are hard to treat, as they could produce necrosis and cellulitis, requiring surgical removal of the infected tissue and require antibiotic therapy (44).
Chapter II
2.1. The history of antibiotic
Alexander Fleming and his group in 1928 did one of the most important contributions in the history of modern medicine. He and his colleagues studied the antibiotic properties of Penicillium notated, a mold able to produce a molecule which inhibits the bacterial growth. This compound had a stronger activity than sulfonamides and influenced a wide spectrum of microorganism. (45). It was not until 1940 that a chemistry group led by Ernest Chain and Howard Florey purified the antibiotic compound, called penicillin and by 1945 they were able to produce large quantities of this new drug (46).
The first use of penicillin in England was in 1941. A patient with acute infection caused by Staphylococcus aureus, previously treated with sulfonamides, was then treated with a limited amount of penicillin, and the infection was controlled (47). After this success, penicillin usage increased broadly and the improvement of the chemical operations to find and synthesize antibiotics enabled the discovery of a number of new agents. The 1945-1960 times is known as the “golden era” of antibiotics, where most of the classes of antibiotics, currently in clinical utilize, were at first characterized (48).
The following decade, between 1970-1980, was a period where a considerable number of new antibiotics were produced using the natural scaffold of the previously describe antibiotics, in order to overcome bacterial resistance and increased their effectiveness. The manufacture of these semi-synthetic antibiotics led to this period being known as the “golden age of antibiotic medicinal chemistry” (48).Nonetheless, after this period, the number of new antibiotics approved for clinical use decreased alarmingly, mainly due to the rapid appearance of resistant bacteria. Only a few new antibiotics were approved in the late 1990’s and early 2000’s, such as linezolid, tigecycline, and daptomycin, resulting in a reduction of therapeutic options to treat microbial infections. In this sense, Sir Alexander Fleming, during his Nobel lecture in 1945, expressed a major concern about the misuse of antibiotics, saying that “there may be a danger in under dosage. It is not hard to make microbes resistant to penicillin in the laboratory by exposing them to concentrations not sufficient to kill them, and the same thing has occasionally occurred in the body” .This message was the first warning of a phenomenon that is affecting us worldwide today.
2.2. Beta-lactam antibiotics
Beta-lactams are a group of antibiotics (Table 1) that share a common structural feature, a highly reactive four-member, or beta-lactam, ring (49). Their antibacterial effect is achieved by interfering with the final stage of bacterial cell wall synthesis. Peptidoglycan is the main component of bacterial cell walls and is made up of alternating units of N-acetylmuramic acid (NAM) and N-acetylglucosamine (NAG); adjacent strands are cross-linked to give the peptidoglycan a flexible, ‘net-like’ structure. The final cross-linking step is catalyzed by transpeptidases (also known as penicillin-binding proteins or PBPs). Beta-lactam antibiotics are competitive inhibitors of the bacterial transpeptidase because of their constructional similarity to the terminal D-Ala-D-Ala motif of NAM. When a transpeptidase uses a β-lactam as a substrate it becomes acylated and so is unavailable to perform its usual biological function and cell wall synthesis is hindered. And a result of this, the cell wall becomes compromised allowing a flow of water into the cell then leads to cell lysis and cell death (50, 51).
Table 1 Beta-lactam antibiotics
Natural penicillins 1st generation (narrow spectrum) meropenem Aztreonam Cefmetazole
Benzylpenicillin Cephalothin Ertopenem Cefoxitin
(Penicillin G) cephalexin Imipenem
Phenoxymethylpenicillin
(Penicillin V)
Extended spectrum penicillins 2nd generation
Aminopenicillins (extended spectrum)
(Ampicillin, Amoxicillin) Cefuroxime
Carboxypenicillins Cefamandole
(Carbenicillin, Ticarcillin) Cefaclor
Anti-staphylococcal 3rd generation (broad spectrum)
(To target staphylococcal Cefotaxime
Penicillinase producers) Ceftriaxone
Cloxacillin
Meticillin 4th generation (broad spectrum)
Oxacillin Cefepime
Cefpirome
5th generation (anti-MRSA
Ceftobiprole
2.3. Mechanisms of antimicrobial resistance in Gram‑negative bacilli
2.3.1. Antibiotic resistance mechanisms in Enterobacteriaceae
2.3.1.1. Resistance mechanisms to β-lactams
Resistance to β-lactams in Enterobacteriaceae is mainly conferred by β-lactamases. These enzymes inactivate beta-lactam antibiotics by hydrolysis. 2 classifications of beta-lactamases are known, namely the Bush-Jacoby-Medeiros and the Ambler (Table 2). The Ambler classes are based on the amino acid homology, where they are classified in 4 molecular classes namely, A, B, C, and D. Molecular classes A, C, and D include the β -lactamases with serine at their active site, whereas molecular class B stands for metallo-beta-lactamases (MBLs), enzymes with zinc molecule in the active site. The Bush-Jacoby-Medeiros classification grouped the β -lactamases in 3 major groups and sixteen subgroups. The classification is based on the inhibitors and substrates of the enzymes (52, 53).
Table 2 β-lactamases occurring in Enterobacteriaceae.
2.3.1.2.. AmpC type beta-lactamase
AmpC beta-lactamases are mainly chromosomally encoded in Enterobacteriaceae and they confer resistance to cephalothin, cefazoline, cefoxitin, most penicillin and to β -lactamase inhibitor (clavulanic acid). Chromosomal AmpC enzymes are inducible and can be expressed at high levels by a mutation in ampD leading to AmpC hyper inducibility or constitutive hyperproduction (54). Overexpression confers resistance to extended-spectrum cephalosporins including cefotaxime, ceftazidime, and ceftriaxone. AmpC enzymes existing on transmissible plasmids are commonly constitutively expressed and show in bacteria that do not have or weakly expressing a chromosomal AmpC gene, such as E. coli, K. pneumoniae, and P. mirabilis. AmpC enzymes encoded by both chromosomal and plasmid genes are capable of hydrolyzing broad-spectrum cephalosporins more efficiently (55).
2.3.1.3. Extended spectrum beta-lactamases (ESBLs)
ESBLs are β -lactamases have the ability to confer bacterial resistance to the penicillins, early and extended-spectrum cephalosporins, and aztreonam (but not to cephamycins or carbapenems) by hydrolysis of these antibiotics, and are inhibited by β -lactamase inhibitors like sulbactam, clavulanic acid, and tazobactam (52). The most known ESBLs are SHV-, TEM-, and CTX-M. Each of these enzymes derives from its own progenitor. Interestingly, SHVs are more diffuse in Europe, TEMs are dominantly existing in the USA while the CTX-Ms are being increasingly detected worldwide (56).The origine of SHV-1 (sulphydryl variable) is the chromosome of Klebsiella spp. and has only a narrow β-lactam hydrolyzing activity conferring resistance to penicillin and ampicillin (52). The amino acid sequence of SHV-1 has two hot spots where a single amino acid replacement extends its hydrolysis spectrum to early and extended spectrum cephalosporins and aztreonam. These hot spots are detached in position 179 and glycin in position 238 (57). Altogether 171 SHV-type β-lactamases are known most of them are ESBLs (58). They differ from an SHV-1 maximum in five amino acid positions. Interestingly, SHV-38 is a chromosomal SHV enzyme with ESBL and carbapenemase efficiency as it gives resistance to extended-spectrum cefalosporins and to imipenem. Although it has a decreased activity against amoxicillin and cefalothin (59).
TEM-1 and TEM-2 (patient’s name: Temoneira) are usually found in E. coli and both have hydrolytic activity mainly to ampicillin. TEM-3 has the activity of ESBL, and it different from TEM-2 by two amino acid substitutions (60). The two hot spots in the amino acid sequence determining ESBL activity are the arginine in position 164 and glycin in position 238. Both amino acids change to serine extends the hydrolytic activity to ESBL. Over 200 type TEM β-lactamases are known and the majority of them are ESBLs (58). However, they also vary maximum in five positions from the progenitor TEM-1 or TEM-2. The TEM-type ESBLs are derivatives of TEM-1 while the TEM-2 analogous has only a broad-spectrum beta-lactamase effect. In TEM-1 the amino acid changes in positions of 39, 69, 165, 182, 244, 261, 275, and 276 determines the IRT (inhibitor resistant TEM) type β-lactamases. These TEMs hydrolytic activity immediately do not change but are resistant to beta-lactamase inhibitors (61).
CTX-M beta-lactamases (cefotaximase-Munich) are derived from Kluyvera sp. where it is chromosomally coded. In Enterobacteriaceae sp usually E. coli and Klebsiella sp. carry the gene of this β-lactamase on plasmids. These enzymes were named after the hydrolytic activity of cefotaxime although their spectrum involves aztreonam and extended spectrum cephalosporins. Altogether 140 CTX-M enzymes were identified and all of them are ESBLs (56, 58).These enzymes are comprised in five sub-groups as CTX-M-1, -2, -8, -9 and -25 whereas the most dominant is CTX-M- 15 which belongs to CTX-M-1 sub-group (62).
OXA β-lactamases (Bush-Jacoby-Medeiros group 2d and Ambler class D) were named after their oxacillin hydrolyzing abilities; actually, they inactivate benzylpenicillin, cloxacillin, and oxacillin (52). They predominantly happen in P. aeruginosa (63) but have been discover in many other Gram negative bacteria especially in Enterobacteriaceae sp (64). OXA-1 and OXA-10 β-lactamases have only a tight hydrolytic spectrum, however; another OXA beta-lactamases are ESBLs including OXA-11, -14, -15,-16, -28, -31, -35 and -45 as they confer resistance toncefotaxime, ceftazidime, and aztreonam. Altogether 311 OXA-type β-lactamses were discovered including narrow spectrum and extended-spectrum-β-lactmases (58, 65).
PER (Pseudomonas extended resistance) β-lactamase hydrolyzes penicillins and cephalosporins and inhibited by clavulanic acid. PER-1 was first detected in Pseudomonas aeruginosa (66) and later in Salmonella sp, and in Acinetobacter isolates as well (67, 68, 69, and 70). VEB (Vietnam extended-spectrum β-lactamase) has 38% homology with PER. It confers resistance to cefotaxime, ceftazidime, and aztreonam while inhibited by clavulanic acid. The gene encoding VEB-1 was found to be plasmid mediated and such plasmids frequently carry non-β-lactam resistance determinants (71). The minor ESBLs involve GES, BES, TLA, SFO and BEL as they are a few identified and geographically localized (72).
2.3.1.4. Carbapenemases
Carbapenemases are β-lactamases with a broad hydrolytic spectrum. Almost all hydrolyzable β-lactams including the carbapenems inactivate by the action of these enzymes (73). Carbapenemases are in β-lactamases from Ambler classes A, B and D (53).
In class A, the prevalent carbapenemase is Klebsiella pneumoniae carbapenemase which was mainly detected on plasmids of K. pneumoniae (74, 75). In previous years, sporadic cases of Klebsiella pneumoniae carbapenemase producing E. coli, Enterobacter cloacae, Serratia marcescens, and Citrobacter freundii were detected (76). Till today, Klebsiella pneumoniae carbapenemase enzymes have fifteen different amino-acid variants (58) and possess hydrolytic activity on the extended-spectrum cephalosporins, carbapenems and aztreonam (52). The NMC (non-metallo carbapenemase), IMI (imipenem hydrolyzing β-lactamase) and SME (Serratia marcescens enzyme) carbapenemases part also of Ambler class A and 2f in Bush-Jacoby-Medeiros classification. These enzymes are chromosomally situated in Enterobacter sp and in S. marcescens while they are closely related to each other as imipenem hydrolyzing β-lactamase and non-metallo carbapenemase have 97% amino acid similarity and they are homolog 70% to Serratia marcescens enzyme (77, 78). All the 3 enzymes have a wide hydrolysis spectrum that includes the penicillins, early cephalosporins, carbapenems, and aztreonam (73, 79).
Ambler class B and Bush-Jacoby-Medeiros group 3a include the metallo-β -lactamases (MBLs) all capable of hydrolyze not only the carbapenems but all the hydrolysable β-lactams (52). The technique of hydrolysis is depending on the interaction of zinc ions in the enzyme’s active site and this is the side inhibited by EDTA (73). Non-fermentative bacteria as A. baumannii and P. aeruginosa inherit (metallo-beta-lactamase) generally chromosomally, where their genes are incorporated in integrons as gene cassettes. Conservated sequences in the integrons enable for recombination crossover. In Enterobacteriaceae sp, MBL genes are located on transferable plasmids thus disseminate by conjugation. The first transferable MBL was IMP metallo-beta-lactamase (“active on imipenem”) detected in P. aeruginosa, later on it was detected in Enterobacteriaceae sp (80,81). VIM (Verona integron-encoded metallo-beta-lactamase) was also first detected in P. aeruginosa, although it is widely disseminated in Klebsiella spp, and E. coli. in the chromosome of A. baumannii founded, The currently emerging MBL is the NDM (New Delhi metallo-beta-lactamase) but detected in Enterobacteriaceae mainly in Klebsiella sp. and E. coli (82, 83, 84, and 85). OXA-48 is a class D carbapenemase that belongs to OXA-type β-lactamases with a hydrolyzing spectrum including penicillins and carbapenems, but excluding extended-spectrum cephalosporins (ceftazidime, ceftriaxone) and aztreonam (86). OXA-48 has only been detected in Enterobacteriaceae sp isolates and mainly in K. pneumoniae and E. coli although OXA-type β-lactamases are dominant in Acinetobacter species (87).
2.3.2. Resistance to aminoglycosides
Resistance to aminoglycosides is mainly explained by the antibiotic modification. Aminoglycoside- modifying enzymes stimulating the modification at −OH or −NH2 groups of the 2-deoxystreptamine nucleus or the sugar moieties and can be aminoglycoside acetyltransferases (AACs), aminoglycoside O-nucleotidyltransferases (ANTs), or aminoglycoside Ophosphotransferases (APHs). aminoglycoside acetyltransferases catalyze the acetylation of –NH2 groups in the aminoglycoside molecule using acetyl coenzyme A as donor substrate. aminoglycoside O-nucleotidyltransferases mediate inactivation of aminoglycosides by stimulating the transport of an AMP group from the donor substrate ATP to a hydroxyl group in the aminoglycoside molecule (88). Other well described mechanisms are a modification of target molecule due to the methylation of 16S rRNA by Arm and Rmt methyltransferases (89). Efflux pump AcrD in E. coli can extrude amikacin, gentamicin, neomycin, kanamycin, and tobramycin from the bacterial cell (90). The genes of these aac, arm and rmt are usually a member of integrons where they are associated with β-lactam and quinolone resistance determinants (91).
2.3.3. Resistance to polymyxins
Enterobacteriaceae strains have the ability to develop resistance to polymyxins because of the alteration of lipopolysaccharide molecule. The addition of 4-amino-4-deoxy-l-arabinose (LAra4N) to a phosphate group in lipid A lead to modification of the lipopolysaccharide. This addition causes an absolute increase in lipid A charge, making the positively charged polymyxins inactive (92). The biosynthesis of 4-amino-4-deoxy-l-arabinose is mediated by PmrA/PmrB and PhoP/PhoQ 2 component regulatory system. PmrD has a protective role by inhibiting PmrA dephosphorylation. While PmrA exerts negative feedback by repressing pmrD transcription (93, 94). These mechanisms are common in gram-negative bacteria and well described in Salmonella enterica serotype thyphimurium, but in Yersinia pestis PmrD is not present, in E. coli PhoP/PhoQ exists but does not activate PmrA/PmrB. Moreover, modifications of the bacterial outer membrane include the increased production of capsule polysaccharide in K. pneumoniae. CPS limits the interaction of polymyxins with their target sites. Thus, up regulation of CPS production leads to increased polymyxin resistance (95). Interestingly, Proteus spp, Providentia spp, and Serratia spp. are intrinsically resistant to polymyxins, wherease P. mirabilis owns 4-amino-4-deoxy-L-arabinopyranose on LPS phosphate thus conferring resistance (96).
2.3.2. Antimicrobial resistance in non fermenting GNB
2.3.2.1. Pseudomonas aeruginosa
AmpC-type cephalosporinase which is P. aeruginosa harbors an inducible that can be derepressed following mutations in the regulation system (97). Wild-type strains of P. aeruginosa are resistant to amoxicillin (with or without clavulanate), first-generation cephalosporins, second-generation cephalosporins, cefotaxime, ceftriaxone and ertapenem, while they remain susceptible to ticarcillin, piperacillin, ceftazidime, cefepime, imipenem, meropenem and doripenem. Aztreonam activity is variable. Unlike tazobactam, clavulanate is a strong inducer of AmpC in P. aeruginosa, and experimental data suggest a risk of clinical failure with the ticarcillin–clavulanate association (98). AmpC hyperproducing strains remain susceptible to carbapenems only.
P. aeruginosa has several 3-component efflux systems, some of which give resistance to β-lactams when highly expressed after mutations in their promoter regions (99). The most generally involved system is MexAB-OprM, whose over-expression give resistance to ticarcillin, aztreonam, cefepime and meropenem the major determinants of the multidrug resistance phenotypes are Efflux pumps that are increasingly observed in P. aeruginosa. The diverse antimicrobial classes may be substrates of a single pump: exposure to a given class like beta-lactams may thereby select mutants with resistance to other classes like aminoglycosides or betalactams and fluoroquinolones (100).
Imipenem resistance in otherwise β-lactam-susceptible strains of P. aeruginosa indicates the functional loss of OprD, a porin which manages the passage of imipenem through the outer membrane (100,101). The development of imipenem resistance under therapy results from the selection of OprD mutants, maybe from previously imipenem-susceptible inoculums or, more after cross-transmission of another clone (102). The risk appears notably high in clinical practice. Indeed, in 4 randomized controlled experiments also patients with hospital-acquired P. aeruginosa pneumonia, the average rate of resistance emergence under therapy was 30% (range, 6–53%) for imipenem while only 15% (range, 6–36%) for other β-lactams (103). P. aeruginosa can develop resistance to all β-lactams as the one result of chromosomal mutations. Nonetheless, the species can gain Mobile genetic elements (MGE) encoded β-lactamases, including Extended-Spectrum β-Lactamase and carbapenemases (101).Main hospital outbreaks have been seen with VIM or IMP carbapenemases- producing clones (104, 105).
Resistance to tobramycin as result of aminoglycosides modifying enzymes in P. aeruginosa whiles the over-expression of efflux pumps mostly lead to resistance to amikacin (106). Mobile genetic elements -borne 16S rRNA methylases such as ArmA, RmtA and RmtD are also reported as an emerging mechanism of aminoglycoside resistant in P. aeruginosa (107). The Fluoroquinolone resistance occurs as result of Mutations in the topoisomerase-encoding genes or the hyper-expression of efflux syste (100). Lastly, and as for Enterobacteriaceae, colistin-resistant mutants of P. aeruginosa could arise in settings with a high frequency of colistin use (108).
2.3.2.2. Acinetobacter baumannii
A non inducible AmpC-type cephalosporinase (ACE-1 or ACE-2) and an OXA-51-like oxacillinase that produce naturally in Acinetobacter baumannii which confer, at basal levels of expression, intrinsic resistance to aminopenicillins, first-generation cephalosporins, second-generation cephalosporins and aztreonam (109). Ertapenem naturally lacks activity against A. baumannii. Together with the expression of multiple efflux systems and a marked impermeability, the plasticity of its genome enables the types to gather many resistance mechanisms, leading easily to multidrug resistance. Most of the time, gain resistance to carboxypenicillins, ureidopenicillins and third-generation cephalosporins rests on the overproduction of the AmpC-type cephalosporinase. However, in addition to plasmidic narrow-spectrum β-lactamases, A. baumannii: PER and VEB which acquired several ESBLs are the most frequently encountered types, particularly within pandemic clones (110). In both cases, imipenem and meropenem remain the drugs of choice. Although carbapenem resistance can result from the over-expression of the chromosomal OXA-51-like enzyme (111), this phenotype is mostly due to the acquisition of plasmid-borne OXA-23-like, IMP, VIM, SIM or, more recently, NDM-type carbapenemases (107). Of note, the spread of such carbapenemases producing strains rise steadily from Northern to Southern European countries (110). Acquired resistances to fluoroquinolones (mutations in gyrA and/or parC) and aminoglycosides (plasmid-borne Aminoglycosides-modifying enzymes AMEs—particularly aminoglycoside acetyltransferase AAC (3), AAC (6′), aminoglycoside phosphotransferase, APH (3′)—and 16S rRNA methylases) are commonly observed in ESBL- as well as Carbapenemase-producing A. baumannii strains.
Colistin stands as the main therapeutic option for ICU-acquired infections due to extensively drug-resistant A. baumannii, and should be considered as part of the empirical antibiotic regimen in settings with high densities of carbapenem-resistant strains (112). Nevertheless, colistin-resistant isolates are now increasingly reported worldwide, especially in patients previously exposed to this drug (113). This phenotype mainly depends on the loss of lipopolysaccharide (LPS) production secondary to the insertion of the ISAba11 sequence in genes encoding the lipid A biosynthesis (114). Increased expression of the PmrAB two-component regulatory system is another mechanism of LPS alteration resulting in colistin resistance (115). Interestingly, the reduction of the negative charge of the lipid A, which lowers the affinity for colistin (positively charged), may also induce cross-resistance to host cationic antimicrobials such as lysozyme (116). Furthermore, colistin exposure may select for a resistant fraction among an otherwise colistin-susceptible A. baumannii population (117, 118). The prevalence of this mechanism of resistance—referred as hetero resistance— is poorly documented due to missed detection by conventional microbiological methods but could have significant clinical consequences (119). For infection due to colistin-susceptible A. baumanni strains, the benefit of combination with rifampin has not been confirmed by a recent RCT (120). Sulbactam, a BLI with intrinsic activity against A. baumannii, may be useful alone or in combination (109), although clinical data are still scarce. Clinical experience is also limited for minocycline, despite a high in vitro activity against multidrug-resistant isolates (121). The use of tigecycline may be discussed in the absence of other option (i.e., colistin resistance or toxicity) (110). Double-dose regimens appear well tolerated and could be more active than standard dosing owing to pharmacokinetic considerations, notably in patients with VAP (122).
2.4. Genetic support of Carbapenemase Genes in Gram negative bacteria.
The emergence of resistance to carbapenems in Gram-negative bacteria, including Enterobacteriaceae, Pseudomonas and Acinetobacter (EPA) species, over the last decade has become a major public health crisis worldwide, because of their rapid spread and the lack of development of new antimicrobial drugs. Since the description of a metallo-β-lactamase, IMP-1, in P. aeruginosa (123). A serine carbapenemase, OXA-23, in A. baumannii sp (124) and a serine carbapenemase, KPC-1, in K. pneumonia sp (125). Carbapenemase-encoding genes have spread worldwide, and are now distributed through the main Gram-negative multidrug-resistant bacteria, which are responsible for a large number of hospital-acquired and nosocomial infections. Carbapenemases are enzymes that inhibit almost all β-lactam antibiotics, including carbapenems, and have now been reported mainly in Enterobacteriaceae, A. baumannii, and P. aeruginosa sp (126). Although carbapenemases were reported to be species-specific in the 1990s, the identification and spread of plasmid-encoded carbapenemases has recently changed our view of the magnitude of the problem in Gram-negative bacteria, especially the rapid and extensive worldwide dissemination of carbapenem-resistant bacteria carrying the KPC-type, VIM-type, NDM-type and OXA-type carbapenemases (127,128).
2.4.1. Classification of Carbapenemases
Classification of carbapenemases can be achieved both functionally and genetically, and is summarized in (Fig. 1) Carbapenemases are divided into two groups according to their active sites: (i) serine carbapenemases belonging to the class A penicillinases and class D oxacillinases, which contain a serine in the active site and can be inactivated by β-lactamase inhibitors, including clavulanic acid and tazobactam; and (ii) metallo-b-lactamases belonging to the class B carbapenemases, which contain one or more zinc atoms at the active site, allowing them to hydrolyze the bicyclic β-lactam ring. These enzymes are inhibited by EDTA (127,129,130) (Fig. 1).
Class A carbapenemases
Class A carbapenemases include the IMI/NMC, SME, KPC and GES enzymes that confer resistance to carbapenems at various levels, from reduced susceptibility to full resistance (127). SME, NMC and IMI enzymes are usually chromosomally encoded, whereas KPC and GES enzymes are plasmid-encoded (127) (Fig. 1). SME enzymes are usually restricted to Serratia marcescens, whereas IMI and NMC enzymes are sporadically detected in Enterobacter cloacae (127). The fact that these latter three types of enzyme are chromosomally encoded probably explains why they are rarely reported worldwide. Conversely, the genes for KPC enzymes are found on transferable plasmids, and are highly prevalent, mainly in K. pneumoniae, but have also been detected in other Enterobacteriaceae, P. aeruginosa, and A. baumannii (126). The genes for GES enzymes are found in integrons on transferable plasmids in P. aeruginosa and K. pneumoniae (127).
Class B carbapenemases
Class B metallo-β-lactamases include the IMP, VIM, GIM, SIM and NDM enzymes, whose genes are mainly found in transferable plasmids in Enterobacteriaceae sp, but are also found in P. aeruginosa and A. baumanni sp. The IMP-type enzymes, initially reported in 1991 in an S. marcescens clinical isolate from Japan (131).Have now been reported all over the world in Enterobacteriaceae, P. aeruginosa, and A. baumanni sp (128). Among the VIM-type enzymes, which now comprise >30 variants, blaVIM-1 was reported for the first time in Verona, Italy, in a P. aeruginosa clinical isolate recovered in 1997, and was integrated as a gene cassette located in a class I integron along with an aacA4 gene responsible for aminoglycoside resistance (132). This gene was also reported in an Escherichia coli isolate from Greece in November 2001 that carried blaVIM-1 along with aacA7, dhfrI and aadA located in a class I integron (133). Since then, both E. coli and K.pneumoniae with blaVIM-1 have spread in Greece (134,135), and they have now been reported all over the world (128). The VIM-2 variant was first reported in a P. aeruginosa
Clinical isolate from France, the gene also being integrated as a gene cassette in a class I integron (136), and is now endemic in many countries worldwide (128). NDM enzymes have been reported recently (137), and the first large series of clinical isolates from Asia and UK were reported in August 2010 (138). Since this report, NDM-1 has spread worldwide (138), and it is now one of the most common carbapenemases in all Enterobacteriaceae and in A. baumanni sp (126,128, 139).
Figure 1. Phylogenetic tree of the metallo-carbapenemase and serine carbapenemase genes with their mean percentage GC content, according to groups and phenotypic properties. The tree was constructed from the amino acid sequences aligned with the free ClustalX software version 2.0 and MEGA software version 6.06, by use of the neighbour-joining method with the amino acid Poisson correction model with 1000 bootstrap replicates. Bootstrap values are expressed as percentage of the 1000 replicates, and only those up to 50% are kept and shown at branch points. aOXA genes described overall in Gram-negative bacteria. bOXA genes described only in Acinetobacter species. The percentage GC values correspond to the averages of each subgroup. ATM, aztreonam; CLA, clavulanic acid; R, resistant; S, susceptible (https://www.ncbi.nlm.nih.gov/pubmed/24766097).
Class D carbapenemases
Class D metallo-β-lactamases are also known as OXA β-lactamases, for ‘oxacillin-hydrolysing’, and their genes are found both on plasmids and in the chromosome. Basically, the OXA-23, OXA-24 and OXA-58 groups are mainly found in A. baumannii, whereas OXA-48 is increasingly being reported in K. pneumoniae (127).
2.4.2. Genetic Platforms of Carbapenemase Genes in Enterobacteriaceae and in P. aeruginosa, A. baumannii, Species
The EPA species are today well recognized to constitute the most common source of both community-acquired and hospital-acquired infections. These bacteria can spread easily and rapidly between humans, via the hands, materials used (in hospital settings), contaminated foods, or water, and can therefore exchange genetic material by lateral gene transfer, mainly mediated by plasmids and transposons (141). However, these carbapenemases can be divided into those that are usually chromosomally encoded and those that are encoded by mobile genetic elements, including transposons, integrons, and plasmids (127). Indeed, the serine carbapenemase enzymes, including SME, NMC-A, and IMI, which have been less well described than the other carbapenemases in EPA species, are chromosomally encoded in some species, including S. marcescens and Enterobacter cloacae. These enzymes are extremely rarely associated with mobile genetic elements, and perhaps this has contributed to their rarity in EPA species (127).despite the report of IMI-2 being encoded by a plasmid in Enterobacter cloacae (142). In contrast to the enzymes cited above, the KPC and GES enzymes are well described as being mostly encoded by transferable plasmids (127). Indeed, as reported, the first KPC gene was discovered in a large plasmid that encodes the KPC-1 carbapenemase enzyme. Shortly after this, a variant of KPC-1, i.e. KPC-2, which differs by a single amino acid was reported, and now represents the most common KPC enzyme worldwide in Gram-negative bacteria (143). Interestingly, a single K. pneumoniae clone, ST-258, harbouring KPC-2 has been extensively reported worldwide, suggesting wide spread of blaKPC-2 (143). However, it has recently been reported that KPC-2 is mainly associated with a single mobile genetic element, transposon Tn4401 that is capable of a high frequency of transposition (Fig. 2a) (144). In addition, the transposon Tn4401, which is identical to those identified in K. pneumoniae, has been recently described in a P. aeruginosa plasmid, pCOL-1 (accession number: KC609323), suggesting the possibility of this transposon being transferred among EPA species. In contrast to the KPC genes, the OXA Carbapenemase genes have been reported to be associated with both bacterial chromosomes and plasmids (145). As shown in (Fig. 2b), the most well described OXA gene in A. baumannii, blaOXA-23, is generally located on transposon Tn2006, which is almost always bracketed on both sides by the insertion sequence ISAba1 (146). The transposon Tn2006 reported in transferable plasmids is also often identified in the bacterial chromosome of A. baumannii, inserted in the hot spot of the integration comm. gene (147). As reported in multidrug-resistant A. baumannii, this comM gene is a hot spot for the integration of genomic islands of various sizes carrying antimicrobial resistant determinants, including those responsible for antibiotic, antiseptic and heavy metal resistance (147). Like blaOXA-23, which is associated with ISAba1 (Fig. 2b), blaOXA-58 is often bracketed by two copies of ISAba3, forming a composite transposon, whereas blaOXA-48, discovered in a K.pneumoniae plasmid, is associated with IS1999, located upstream of the transposon Tn1999 (145). Concerning the metallo-carbapenemase genes, as described for the first acquired metallo-β-lactamase identified in Pseudomonas species, IMP-1 (131), IPM and VIM genes are mainly found in a class I integron, characterized by an integrase intlI gene associated with a transposase tnpA located upstream of the integron (Fig. 2c). As shown in Fig. 3c, these metallo-carbapenemase genes are often associated on this class I integron with genes that encode aminoglycoside resistance (aacA4, aadA1, and/or aadB), class D β-lactamases (OXA genes), antiseptic resistance (qacΔG), or chloramphenicol resistance (catB) (148). The class I integron has been reported to belong to the mobile integron elements, which are divided into five groups and are always plasmid-mediated; however, some large integrons, classified as superintegrons, have been detected in the chromosome (149). In addition to the important role of class I integrons in the dissemination of IMP and VIM genes, these mobile genetic elements are also widely associated with the wide spread of antibiotic resistance genes in Enterobacteriaceae sp (149). The blaNDM-1 gene, the latest metallo-carbapenemase gene to be found, has now disseminated intercontinentally (138).In the study describing the discovery of blaNDM-1 (136), it was found in a plasmid on a 4.3-kb region linked to the 4.8-kb complex class I integron. Interestingly, it has been reported that blaNDM-1 can be associated with different types of insertion sequence, and this characteristic of blaNDM-1 and the other metallo-β-lactamase genes partly accounts for their different propensities to disseminate (128). Indeed, as shown in (Fig. 3d), in A. baumannii, blaNDM-1 is often bracketed by two copies of the insertion sequence ISAba125, located on the transposon Tn125 (150). This transposon Tn125 carrying blaNDM-1 has also been described in the A. baumannii chromosome, supporting the ability of this gene to move between both bacterial DNA molecules (151) In Enterobacteriaceae, blaNDM-1 is also associated with complete or truncated ISAba125, but is mainly located upstream (150). Interestingly, bleMBL, a bleomycin resistance gene, is always coupled with blaNDM-1, which regulates the RecA-dependent mutation rate, and therefore plays a role in the stabilization of blaNDM-1-positive isolates (150). The same genetic structure of blaNDM-1 as characterized in Enterobacteriaceae has been in identified in P. aeruginosa, where it is located in the variable region of a new complex class 1 integron bearing the insertion sequence ISCR1 and localized in the bacterial chromosome (152).
Fig .2. Genetic platforms of the most reported carbapenemases in Enterobacteriaceae, Pseudomonas and Acinetobacter species. (a) Class I integron: mobile genetic element harbouring the metallo-β-lactamase-encoding gene blaVIM-2 from P. aeruginosa; ISPa, transposase; intL1, integrase; blaOXA-2, oxacillinase; aacA4, aminoglycoside-60-N-acetyltransferase; aadB, aminoglycoside-modifying enzyme; qaΔG, quaternary ammonium resistance protein; ISPa (tniC), invertase. (b) Transposon Tn4401a: mobile genetic element from Klebsiella pneumoniae harbouring the class A carbapenemase-encoding gene blaKPC-2; IRL, inverted repeat left; tnpR, resolvase; tnpA, transposase; ISKpn7, insertion sequence; istA, transposase A; istB, transposase B; ISKpn6, insertion sequence; IRR, inverted repeat right. (c) Transposon Tn2006: mobile genetic element from A. baumannii harbouring the class D serine carbapenemase-encoding gene blaKPC-23; ISba1, insertion sequence; tnpB, transposase subunit; tnpA, transposase 1; orfX, hypothetical protein; atpase, AAA ATPase superfamily; tnpA, transposase 1; tnpB, transposase subunit. (d) Transposon Tn125 from A.baumannii harbouring the metallo-carbapenemase-encoding gene blaNDM-1; ISAba125, transposase; bleMBL, bleomycine resistance protein; iso, similar to delta-phosphoribosylanthranilate isomerase; tat, twin-arginine translocation pathway signal sequence; dvt, divalent cation tolerance protein; groES, chaperonin protein GroES; groEL, chaperonin protein GroEL; ISCR21, transposase; pac, similar to delta-PAC; truncated phospholipidacetyltransferase. Conserved genes in these mobile elements are boxed and highlighted (https://www.ncbi.nlm.nih.gov/pubmed/24766097).
2.5. P. aeruginosa virulence factors: Secreted proteins
A large number of secreted proteins contribute to the pathogenicity of P. aeruginosa, and are delivered from the bacterial cytoplasm by dedicated protein secretion systems. Protein secretion systems in Gram negative bacteria have been classified, and there are currently six types. These are named Type I Secretion System (T1SS) through to the Type VI Secretion System (T6SS). Secretion systems export proteins in one- or two-steps, as shown in (Fig. 3) One-step mechanisms involve secretion of proteins across both bacterial membranes in a single step by dedicated machinery, and periplasmic intermediates do not accumulate in a secretion mutant. The T1SS, T3SS, T4SS and T6SS work in this way. Two-step mechanisms involve initial secretion across the membrane using universal transporters, and separate dedicated machinery is used for transport across the outer membrane. The T2SS and T5SS secrete proteins in two steps.
Fig.3. One and Two-step protein secretion mechanisms
Gram negative protein secretion systems are either a one- or two-step process. One-step secretion (left) involves transport across the bacterial inner membrane (IM) and outer membrane (OM) in a single step, while two-step secretion (right) involves initial transport into the periplasm via Sec or Tat, and dedicated machinery for outer membrane transport (http://orca.cf.ac.uk/89225/).
2.5.1. Inner Membrane Translocation
Protein export by two-step mechanisms (T2SS and T5SS) first requires translocation across the bacterial inner membrane. The Sec (secretion) and Tat (twin arginine-translocation) systems provide a route across the inner membrane for substrates of both the T2SS and T5SS, as well as for periplasmic-localised or outer membrane proteins. Once in the periplasmic, T2SS and T5SS substrates are available for secretion across the outer membrane by dedicated machinery. Despite their common function, the mechanisms used by the Sec and Tat systems to transport proteins are different. The Sec system transports unfolded single linear polypeptides and is essential for viability, while the substrates of the Tat system are folded in the cytoplasm prior to transport including folded proteins carrying co-factors (153) and multi-protein complexes (154) . Proteins are targeted to the Sec or Tat systems by a cleavable N terminal signal sequence. The signal sequences for targeting to the Sec and Tat systems are structurally similar, but not identical, as illustrated by (Fig .4) Targeting to the Sec translocon is mediated by a 20 amino acid N terminal sequence, made up of three parts. The amino terminal n-region of the signal sequence is positively charged, and followed by a hydrophobic core h-region, terminating with a polar carboxyl-terminal c-region (155). The c-region is required for recognition by signal peptidase which cleaves the signal peptide to yield the mature protein, typically at an ‘A-x-A’ motif (156). Proteins targeted to the Tat translocon possess an N terminal signal sequence which also possesses a positively charged n-region which is much longer compared to the sec sequence. Tat substrates also have a hydrophobic h-region and a short polar c-region for recognition by the signal peptidase. Proteins targeted to the Tat machinery have two conserved arginine residues within their signal 39 sequence, located between the n and h regions, and important for substrate recognition (157).
Fig.4. Sec and Tat signal sequences.
Amino terminal n-regions, hydrophobic h-region and carboxy terminal c-regions shown for Sec and Tat signal sequences. Black arrow indicates the site of cleavage by signal peptidase, white arrow indicates the location of the Tat specific twin arginine sequences as shown (http://orca.cf.ac.uk/89225/).
2.5.1.1. The Sec Translocon
Proteins can be targeted to the Sec translocon co-translationally or post-translationally, as indicated by (Fig. 5) the co-translational route involves a protein known as signal recognition particle, SRP, which binds to the signal sequence of a Sec targeted protein as it emerges from the ribosome. The entire Ribosome-SRP-nascent polypeptide complex is targeted to the Sec machinery (158). A signal recognition particle receptor transfers the nascent polypeptide to the Sec machinery. Proteins transported in this way generally end up inserted into the inner membrane (158). The post-translational route is used by the majority of Sec-targeted secreted, periplasmic and outer membrane proteins, and involves recognition of substrates by the secretion specific chaperone SecB. SecB binds to proteins post-translationally, following release from the ribosome complex, and maintains them in a secretion competent unfolded state as well as targeting them to the Sec machinery at the inner membrane (159).
Fig. 5. Targeting of proteins to the Sec translocon
Proteins can be targeted to the Sec machinery co-translationally (left) or post-translationally (right). IM denotes the inner membrane, where the SecYEG channel is located (green ring). The details of each mechanism are included in the text. Red oval: signal recognition particle (SRP), green oval: SecB. Blue rectangle represents SRP receptor, and the yellow pentagon represents SecA. The white stacked ovals represent the ribosome, with an emerging Sec targeted polypeptide (http://orca.cf.ac.uk/89225/).
The Sec translocon consists of three integral membrane proteins, SecY, SecE and SecG, and a cytoplasmic ATPase, SecA (160). SecA associates with SecYEG and receives the Sec-targeted polypeptide from the ribosome or the SecB chaperone. SecA threads the unfolded protein through the SecYEG membrane channel, using energy from ATP hydrolysis and the proton motive force (161). The SecYEG channel is only opened once a Sec-targeted polypeptide is bound, and unwanted leakage through the pore is avoided by providing a tight seal around the translocating protein (162). Sec targeted polypeptides are exported in a ‘step-wise’ manner into the periplasm, with multiple cycles of SecA binding and ATP hydrolysis contributing to translocation (163).
2.5.1.2. The Tat Translocon
Since the Sec translocon is limited to unfolded protein transport, the Tat system provides a vital route across the periplasm for folded proteins. These include proteins containing co-factors or multi-protein complexes which are incompatible with the Sec translocon (164,153). During the study of periplasmic hydrogenases from Desulfovibrio vulgaris it became clear that a Sec-independent system was present, since only one subunit of this hydrogenase contained a signal sequence, but both subunits were required for their translocation. This indicated that these proteins were secreted as a complex in a folded state (165). The mechanism of translocation by the Tat system is summarised in (Fig. 6).The minimal Tat translocon is composed of two subunits, TatA and TatC, while a third component, TatB, is also found in the more complex Proteobacteria (166,167). TatB is very similar to TatA, and may be specialised for more stable TatC interactions (168). TatB is not essential, since TatA can complement for the lack of TatB (169). TatC is an integral membrane protein, with six transmembrane domains, able to bind twin arginine signal sequence domains (170,171). Following substrate binding a conformational change occurs in TatC that pulls the substrate through the membrane, facilitated by weakening of the membrane by TatA and the proton motive force. TatA proteins, when purified following overexpression, form ring-like structures with a variable diameter, which may allow transient weakening and disruption of the membrane for substrate transport (172,173). Transport via the Tat translocons is proposed to be in a single step, after which the membrane quickly re-seals, consistent with a large proton flux accompanying Tat transport (174).
Fig. 6.Targeting of proteins to the tat translocon
Tat targeted proteins, containing a twin-arginine motif (RR) are released from the ribosome (white ovals) and are folded in the cytoplasm. The signal sequence is then recognised by TatBC (blue) and exported across the inner membrane (IM) in a Tat A dependent manner (green). Once in the periplasmic space, the signal sequence is cleaved by signal peptidase (red) to release the mature protein (http://orca.cf.ac.uk/89225/).
2.5.2 .The Type I Secretion System (T1SS)
T1SS is among the simplest protein secretion systems, delivering unfolded proteins through the bacterial envelope in a single step, thus independent of the Sec and Tat translocons. The Type I machinery is a three component translocator, illustrated by (Fig. 7) The T1SS spans the bacterial inner and outer membranes, allowing transport of proteins from the cytoplasm directly into the extracellular medium. Two components, located at the inner membrane, are the ABC (ATP binding cassette) protein and an adaptor protein which extends into the periplasm. The third component, located at the outer membrane, forms a beta barrel pore with α-helical projections into the periplasm (175). The periplasmic sections of the outer membrane pore and the inner membrane adaptor protein interact and form a continuous channel across the cell envelope (176) Proteins targeted to the T1SS contain a non-cleavable C terminal secretion signal, although the amino acid sequence of this signal is not highly conserved (177). The signal is believed to partly rely on the helical secondary structure of the C terminal 60 residues of the protein (178).This C terminal signal sequence interacts with the ABC protein and the adapter protein (179). which is thought to induce a conformational change in the ABC transporter, promoting ATP binding and hydrolysis, and providing the energy required for the translocation of the substrate through the pore(1). P. aeruginosa encodes two T1SSs. The first is encoded by the apr genes, responsible for the secretion of an alkaline protease AprA, and a protein of unknown function AprX, (181,182). AprA alkaline protease is responsible for the degradation of host tissues, allowing bacterial invasion and subsequent systemic infection (183,184). The apr T1SS is composed of the AprD ABC transporter, the AprE adapter protein and the AprF outer membrane component.
Fig. 7. Models of the T1SS
The three components of the P. aeruginosa Apr T1SS. The AprD ATPase (green) is located in the inner membrane (IM), while AprF (blue) is located in the outer membrane (OM) and forms a β-barrel with alpha helical projections. ArpF and ArpE are linked by the AprE adaptor protein (red) (http://orca.cf.ac.uk/89225/).
The second characterised P. aeruginosa T1SS is the HasAp system, which is involved in the secretion of a haemophore (180). HasAp is important for the scavenging of iron from haemoglobin, allowing iron acquisition by P. aeruginosa in the iron limited environment of host tissues (180).In this system the ABC transporter is HasD, the adaptor is HasE, and the outer membrane protein is HasF. A fourth protein, HasR, is an outer membrane receptor which allows for the internalisation of the HasAp-heam complex (180).
2.5.3. The Type II Secretion System (T2SS)
A second T2SS is encoded on the P. aeruginosa genome, known as the Hxc T2SS (Homologous to Xcp). The Hxc system has been shown to be active under phosphate limiting conditions, and is responsible for the secretion of low molecular weight alkaline phosphates (185). exoproteins secreted by the Xcp T2SS have a broad spectrum of activity including, but not limited to, proteolytic enzymes and lipid metabolism. The secreted substrates are important in the infection process causing tissue damage, and allowing nutrient acquisition (186). These 49 include LasA and elastase (LasB), which are able to degrade elastin, a major component of lung connective tissue (187). Elastase (LasB) is also able to cleave surfactant protein D, disrupting several immune functions and aiding in P. aeruginosa survival during infections (188). LasA has additionally been shown to have specific activity against staphylococci (189). The P. aeruginosa Xcp T2SS also secretes a range of lipases and phospholipases which are targeted to the eukaryotic membrane. The secreted phospholipases (Plc) are able to cause tissue destruction by hydrolysing phospholipids in the host epithelium or erythrocyte membranes (190). Phospholipases also play a role in disease through over-stimulating the immune response and promoting inflammation (191). The secreted alkaline phosphatase PhoA is capable of liberating free phosphate for utilisation by P. aeruginosa (192). A secreted chitin binding protein (CbpD) is degraded by a second T2SS secreted effector, elastase. When bound to chitin, CbpD is protected from proteolysis. Since CbpD is not able to degrade chitin, its role in pathogenicity is proposed to be as a chitin binging adhesin and not a chitinase, although the precise role of this effector is yet to be shown (193). A well characterised Xcp T2SS substrate from P. aeruginosa is the AB toxin exotoxin A (ToxA), which is a highly toxic virulence factor. The catalytic activity of ToxA is responsible for ADP ribosylation of elongation factor 2, which disrupts the protein synthesis of host cells, ultimately leading to cell death (194). Although the protein is secreted into the extracellular milieu, it is capable of self-targeting to eukaryotic cell membranes. The linear protein sequence can be divided into several domains, including N terminal cell recognition and translocation domains (154) and a C terminal catalytic domain, responsible for the observed toxicity (195).
2.5.4. The Type III Secretion System (T3SS)
The T3SS further extends the secretion capability of P. aeruginosa, allowing injection of proteins from the bacterial cytoplasm directly into host cells. The T3SS translocates proteins across the bacterial inner and outer membranes, and the host cell plasma membrane, in a single step. These proteins, called effectors, interfere with host cell signalling. The T3SS apparatus is known as the ‘injectisome’, and is evolutionarily and structurally related to the flagellum basal body apparatus (196). The observation of a needle like injection apparatus from the T3SS of Salmonella spp. The T3SS was first characterized in P. aeruginosa by Yahr and colleagues (197). A single copy of the T3SS is encoded within the P. aeruginosa genome, and the structural components of the system are contained within 5 operons containing 36 genes, clustered within one genetic locus. These structural genes can be categorised into structural components of the needle complex, 51 proteins involved in the translocation of effectors, regulatory elements and chaperones. T3SS effector genes are scattered throughout the P. aeruginosa chromosome.
There are four identified T3SS effectors in P. aeruginosa strains, known as exoenzymes; ExoS, ExoT, ExoU and ExoY. These effectors are targeted in an unfolded state to the cytoplasm of eukaryotic cells, and play an important role in P. aeruginosa pathogenicity. The nature of the T3SS signal is controversial, and proposed to be a non-cleavable N-terminal signal sequence. However, the signal may be encoded within the corresponding mRNA molecule (198). The trigger for T3SS effector delivery is contact with eukaryotic cells. T3SS can be induced in-vivo under conditions of low Ca2+ availability, which results in the secretion of T3SS effectors into the supernatant (199). P. aeruginosa strains typically carry only three T3SS effectors, normally ExoT and ExoY, and either ExoS or ExoU (200). The subset of T3SS effectors encoded by a P. aeruginosa strain determines its infectious potential. Strains carrying exoU, encoded within a pathogenicity island, are cytotoxic and cause rapid host cell lysis (201). Strains carrying exoS, cause delayed cell death (202).
ExoS and ExoT are 48 kDa and 49 kDa respectively, and share 76% amino acid identity and common domain architecture. The extreme amino termini of these two effectors encode amino acids required for targeting to the T3SS apparatus (197) and a membrane localisation domain for eukaryotic plasma membranes follows the signalling domain (203). Both ExoS and ExoT are bifunctional enzymes, and have GTPase activating (GAP) domains and ADP ribosyl transferase activity (ADPRT). The ExoS and ExoT GTPase activating proteins (GAP) domains target Rho, Rac and CDC42 host cell proteins, which are small GTPases (204). These proteins normally switch between a GTP-bound active state and a GDP-bound inactive state. ExoS and ExoT render these proteins inactive in the GDP-bound state (205). As GTPases normally regulate intracellular actin dynamics, the activity of ExoS and ExoT on GAPs disrupts the actin cytoskeleton, leading to cell rounding and decreased internalisation into epithelial cells and macrophages (206). The ADPRT domain activity is located towards the C termini of ExoS and ExoT. This domain requires the binding of a eukaryotic co-factor, thus self-toxicity in the bacterial cytoplasm is prevented. The ADPRT domains are able to catalyse the transfer of ADP-ribose to specific target host proteins, interfering with signalling pathways involved in actin processes (207,208). The end results include cell rounding, inhibition of vesicular trafficking and inhibition of endocytosis (209). Both ExoS and ExoT result in cell death, however, ExoS is a more potent toxin, mediating cell death in 2-5h compared to 10h for ExoT (210). ExoY is a 42 kDa secreted protein with adenylyl cyclase activity (211). Injection of ExoY into host cell leads to an accumulation of cAMP, a global regulator of gene expression. This cAMP accumulation leads to the differential expression of multiple genes, resulting in disruption of the actin cytoskeleton, inhibition of bacterial uptake, and increased membrane permeability (212,213). Expression of ExoY in PAO1 promotes the formation and trafficking of P. aeruginosa to respiratory epithelial membrane blebs, dependent on the adenlylate cyalase activity of this protein. ExoY adnelyate cyclase activity also promotes P. aeruginosa survival in mice cornea (214). The largest P. aeruginosa T3SS effector is ExoU, which is a 74 kDa protein and a potent phospholipase (215).ExoU is able to cause rapid death of eukaryotic cells within 1-2h due to the rapid loss of plasma membrane integrity (216). Comparison to the other Exo proteins suggests it also has a secretion signal at its extreme N terminus. The catalytic activity of this toxin is mediated by a patatin like phospholipase A2 domain, capable of the hydrolysis of a wide range of substrates (217). This domain is followed by a C terminal domain which has no homology to known motifs, but is essential for the cytotoxicity of the protein (218). Three classes of chaperones are associated with the T3SS. Some of the P. aeruginosa T3SS toxins interact with chaperones prior to secretion, which facilitate storage of effectors in the bacterial cytoplasm and correct delivery to the secretion apparatus, known as Class I chaperones (219). ExoS and ExoT share the SpcS chaperone, which is required for their maximal secretions (220). While SpcU is the cognate chaperone of ExoU (221) and both interact with the effectors via interaction with the chaperone binding domain within the first 100 amino acids of the effector. No chaperone has yet been identified for ExoY. Class II chaperones neutralise the potentially damaging effects of the hydrophobic translocators, such as PcrH binding of PopB and PopD (222). Class III chaperones bind to structural subunits and prevent their polymerisation by masking polymerisation domains, known as PscE and PscG in P. aeruginosa, which bind to the PscF subunit of the T3SS needle in a 1:1:1 ratio (223).
Chapter III
EXPERIMENTAL SECTION
HERE I WANT A SHORT INTRODUCTION (MAX 1 PAGE)
Aim
Our purpose was to investigate at phenotypical and genotypic level the antibiotic resistance mechanisms and virulence factors identified in nosocomial and community acquired infections during 2015-2016 in two big hospitals from Bucharest, Romania.
Objectives
Biochemical characterization of the identified strains.
Phenotypic study of soluble virulence factors for Gram negative bacilli isolated from different patients hospitalized in one big hospital from Bucharest and from community acquired infections;
Investigate the antibiotic resistance and virulence markers in Gram negative bacilli isolated during 2015 and 2016 from patients hospitalized in the National Institute for Cardiovascular Diseases Prof. C.C. Iliescu and from Synevo Central Reference Laboratory from Bucharest.
Materials and methods
Materials:
Bacterial strains – this study was conducted on a total of 46 strains isolated during 2015 and 2016 from patients hospitalized in the National Institute for Cardiovascular Diseases Prof. C.C. Iliescu and Synevo Central Reference Laboratory from Bucharest: P. aeruginosa (n=15), A. baumannii (n=20), K. pneumoniae (n=10) and E.coli (n=1). The selected strains were isolated from different clinical sources most of them being from urine, tracheal secretion, catheter liquid, secretion, wound, umbilicus, feaces, blood cultures.
Methods:
3.1. Identification of the strains
The strains identification and the antibiotic susceptibility testing of the respective strains were performed in the Microbiology Laboratory of the above mentioned hospital with the automated VITEK 2 system. Some supplementary tests were also realized in Microbiology Laboratory of Faculty of Biology. The phenotypic identification of 46 strains is based on their characteristics on the culture media and biochemical features.
3.1.1. Culture-based methods
In order to identify pigment production by P. aeruginosa, the strains were refreshed on a pure agar media and incubated 18-24h at 370C. With a sterile loop few colonies were pick from pure culture and were streaked onto Pseudomonas isolation agar F (King B medium) to detect fluorescing production, onto Pseudomonas isolation agar P (King A medium) to detect pyocyanin production and also onto Cetrimide agar plate, a medium used for the selective isolation and identification of Pseudomonas aeruginosa. Any colours produced by the isolates were recorded.
Also, all the strains were cultured on MacConkey Agar media (for Gram-negative rods), for detection of lactose fermentative species and incubated for 24 hours at 37˚C in aerobic condition. Lactose fermenting strains grow as red or pink and may be surrounded by a zone of acid precipitated bile. The red colour is due to production of acid from lactose, absorption of neutral red and a subsequent colour change of the dye when the pH of medium falls below 6.8. Lactose non-fermenting strains, such are colourless and transparent and typically do not alter appearance of the medium.
3.1.2. Biochemical tests
Triple Sugar Iron test (TSI) – A wire loop of Klebsiella pneumoniae culture was inoculated by stabbing method into the Triple sugar iron gel and streaking over the surface of a slope of the agar and incubated at 37˚C overnight to examine.
If lactose (or sucrose) is fermented, a large amount of acid is produced, which turns the phenol red indicator yellow both in butt and in the slant. Gases may be percent. If lactose is not fermented but the small amount of glucose is, the oxygen deficient butt will be yellow, but on the slant the acid will be oxidized to carbondioxide and water by the organism and the slant will be red. If neither lactose/sucrose nor glucose is fermented, both the butt and the slant will be red. If H2S is produced, the black color of ferrous sulfide is seen.
Simmon’s (Citrate Agar) test – A wire loop of culture was inoculated by streaked over the surface of a slope of the agar and was incubated overnight to examine the colour changes. Blue colour indicates the citrate was used (positive reaction) and original green colour indicates the citrate was not used (negative reaction).
Lysine iron agar (LIA) slant- A wire loop of culture was inoculated by stabbing method into LIA medium and streaked over the surface of a slope of the agar and was incubated for 24 h at 37˚C to examine lysine decarboxylation, lysine deamination and hydrogen sulphide based on the colour changes:
Lysine decarboxylation Positive (+): purple slant / purple butt with or without H2S blackening and Negative (-): purple slant / yellow butt.
Lysine deamination Positive (+): red slant / yellow butt and Negative (-): no red slant. Hydrogen sulfide H2S if produce show by blackening at the bottom area of lysine slant.
MIU medium is recommended for detection of motility, urease and indole production. The culture was inoculated by stabbing method and was incubated for 24 h at 37˚C in order to read the result. Motility and urease reactions are read before testing indole production. If the organism growth away from the stab line is a positive test for motility. Growth only along the stab line is a negative test for motility. Organisms that utilize urea produce ammonia which makes the medium alkaline, showing pink-red colour by change in the phenol red indicator. For the indole reaction 2-3 drops of Kovac's reagent were added, and if a red layer on a surface of the medium appears the reaction is positive.
API 20NE – The API 20 NE system was chosen as it provides identification of non-enteric, non-fastidious Gram-negative rods including Pseudomonas sp., Acinetobacter sp. The system uses a strip consisting of 20 microtubules each containing dehydrated substrates. With a pipette 1-4 colonies from agar plate were pick up and added to API NaCl 0.85 % Medium (2 ml) and made the bacterial suspension. The suspension turbidity was equal to a 0.5 McFarland standard. With this suspension tube section of the NO3 until PNPG were inoculated. Then 200 uL of the bacterial suspension was added to ampule of AUX Medium and loaded the tubes of tests GLU to PAC; after that mineral oil was added to the 3 underlined tests (GLU, ADH, and URE). About 5 ml of distilled water was added to an incubation box and the strip then incubated for 24 hours. After 24 hours of incubation performed the Nitrate test and the TRP test. One drop of Nitrate 1 and one drop of Nitrate 2 reagents were added to NO3. After 5 minutes a red color appears if it a positive reaction. A negative reaction may be due to the production of nitrogen. Zinc dust was added to the NO3. After 5 minutes if the test is colorless positive reaction occurs. A pink-red color means negative reaction. For TRP test one drop of JAMES Reagent was added. The reaction takes place immediately; pink color appears if the reaction is positive. Positive results were recorded on the result sheet. API 20NE analytical profile index had been used to identify the isolate.
Rapid API 20E- Rapid 20 E is a system which enables the identification of Enterobacteriaceae in only 4 hours after the microorganism has been inoculated on this gallery.. With a pipette 1-4 colonies from agar plate were picked up and added to API NaCl 0.85 % Medium (2 ml) and made the bacterial suspension. The suspension turbidity was equal to a 0.5 McFarland standard. With this suspension were loaded the tests tube according the manufacturer’s protocol. After that mineral oil was added to the 3 underlined tests LDC, ODC and URE. About 5 ml of distilled water added to an incubation box, and incubated the strip for 4 hours. After 4 hours of incubation the test was read. For VP test one drop of each potassium hydroxide was added and after 5-10 minutes if the red color occurs the result is positive reaction. For IND test one drop of Kovac’s reagent was added then after 3 minutes if the red color occurs the result is positive reaction. Positive results were recorded on the result sheet. API 20NE analytical profile index had been used to identify the isolate.
The detection of virulence factors expression
Each agar plate with different substrate (described below) was divided into six parts. A wire loop was used to pick a few colonies from each strain (P. aeruginosa, A, baumannii, K.pneumoniae and E.coli) and inoculated by the stabbing method on the different plates agar. The plates were then incubated for 24 -48 h at 37˚C.
Amylase agar plate (starch hydrolysis):
A starch-nutrient agar plate was stabbed once with the bacteria. After incubation for up to 48 h at 37˚C, plates were flooded with 5-10 ml of iodine solution. If any clear area around the growth the culture indicated the breakdown of starch by the bacteria due to its production of amylase. Unhydrolyzed starch formed a blue color with the iodine (negative reaction).
Lipase agar (lipid hydrolysis):
Fats are broken down into fatty acids and glycerol by an enzyme called lipase. If the bacteria express this enzyme, a clear zone around the growth area on the agar appears. This is indicated as a positive result.
Casein agar (casein hydrolysis):
Agar which use to determine if the bacteria can produce the exoenzyme caseinase. This test is conducted on milk agar. A positive result determines a clearing zone around the growth area, meaning the presence of the enzyme caseinase.
Lecithinase activity
The nutrient agar plate was supplemented with egg yolk emulsion. After incubation for up to 48 h at 37 ˚C, if organisms produce lecithinase precipitation around the growth area occurs, and the result considered positive.
DNA-se agar plate
After incubation for up to 48 h at 37 ˚C, the hydrolysis of DNA in the agar by bacterial DNA-se activity reduces the agar pH. positive result if appear a clear zone around growth area. If there is no colour change, the result should be considered negative.
Gelatinase agar plate (gelatine liquefaction):
The Gelatinase agar was used to determine the ability of an organism to produce proteolytic enzymes like (gelatinases). After incubation for up to 48 h at 37 ˚C, the plates were placed in a refrigerator for about (1) hour; the positive reaction is considered if an opaque clearing zone around the stabbing area occurs.
Esculin agar plate ((Esculin hydrolysis):
The esculin agar plate was used to detect the bacterial capacity to hydrolyse esculin. After incubation for up to 48 h at 37 ˚C. If organisms hydrolyse esculin, a black pigment around the colony appears.
Blood agar plate (Hemolytic reaction):
The blood agar was divided into four parts. The plates were inoculated by picking with a sterile loop few colonies from culture and streaked onto blood agar then incubated for (1-2) days at 37˚C. The results were interpreted as follows: Alpha (α) hemolysis if the formation of a green or brown zone around the colonies. Beta (β) hemolysis- complete lysis of cells and a clear zone appears around isolated colonies. Gamma (γ) hemolysis no hemolytic reaction.
Molecular detection of ARGs (antibiotic resistance genes) and virulence factors
The genetic support of the ARGs and virulence factors was investigated by simplex and multiplex PCR, using a reaction mix of 20 or 25 µl (PCR Master Mix 2x, Thermo Scientific) containing 1 µl of bacterial DNA extracted by the alkaline extraction method (table 3).
Table 3. The composition of the reaction mix.
3.3.1. DNA extraction
In this purpose, 1-5 colonies of bacterial cultures were suspended in 1.5 ml tubes containing 20 µl solution of NaOH (sodium hydroxide) and SDS (sodium dodecyl sulphate) and heated on a thermo block at 95°C for 15 min. for the permeabilization of bacterial wall. The following step was the addition of 180 µl of TE buffer (TRIS+EDTA) 1X and centrifugation at 13000 rpm for 3 minutes then take the supernatant and transfer in a new tube freezes at -4o-20 °C .
3.3.2. PCR assay
All PCR reactions were performed using the Thermal Cycler machine Corbet. Genomic DNA was used as a template for the PCR screening of (5) virulence genes encoding for protease IV, tow exoenzymes – exoT, exoU, two phospholipases – plcH (haemolytic phospholipase C) and plcN (non-haemolytic phospholipase C) and (9) resistance carbapenemases genes and (ESBL) which encoded by blaIMP, blaVIM, blaTEM, blaCTX-M, blaOXA-48, blaNDM , blaOXA-48, blaOXA-23 and blaOXA-24.for. The parameters for the amplified cycles used in each experiment are presented in (table. 4) The amplification products were visualized by electrophoresis on a 1% agarose gel, stained with the specific weight marker (1kb, Ladder Thermo Scientific). Primers sequences used in simplex and multiplex PCR assays for carbapenemases and (ESBL) in K. pneumoniae, A. baumannii and P. aeruginosa and virulence genes in P. aeruginosa strains are presented in (table. 5).
Table.4. The amplification program.
Table 5. The nucleotide sequence of the primers.
Chapter IV
Results and discussions
4.1. Identification of the strains
This study was conducted on a total of 46 clinical strains isolated during 2015 and 2016 from patients hospitalized in the National Institute for Cardiovascular Diseases Prof. C.C. Iliescu and Synevo Central Reference Laboratory from Bucharest. The selected strains were isolated from different clinical sources as urine, tracheal secretion, catheter, liquid, secretion, wound, umbilicus, feces or blood cultures (table.6).
The strains identification and the antibiotic susceptibility testing of the respective strains were performed in the Microbiology Laboratory of the above mentioned hospital with the automated VITEK 2 system. Some supplementary tests were also realized in Microbiology Laboratory of Faculty of Biology. The phenotypic identification of 46 strains is based on their characteristics on the culture media and biochemical features.
In Microbiology Laboratory of Faculty of Biology for the confirmation of bacterial strains different culture media and biochemical test were used (King A, King B, Cetrimide Agar for pigment production by P. aeruginosa, MacConkey Agar for differentiate lactose fermentative species by lactose non-fermentative ones, and mini galleries ( API 20NE and API RapiD20E) (fig. 8 A-F and 9 A-D).
Fig.8. Pigment production by Pseudomonas aeruginosa grown on selective agars: A, B and C- negative control, D positive Agar P (king A) the blue/green pigment, E positive Agar F (king B) the fluorescent yellow/green pigment and D positive Cetrimide agar the blue/green pigment.
Fig.9. MacConkey Agar media (For gram-negative rods), For detection of lactose fermentation. Figure (A) negative control, (B) lactose fermentation – Klebsiella pneumoniae, (C) non -lactose fermentation – Pseudomonas aeruginosa and (D) non lactose fermentation- A, baumannii.
Fig.10.Triple Sugar Iron test for three Klebsiella pneumoniae strains: B = strain 40, C = strain 41 and D = strain 42. (A = negative control). Glucose, lactose and sucrose fermented. Gas (+). H2s (-).
Fig.11. Simmon’s (Citrate Agar) test for three Klebsiella pneumoniae strains: B = strain 40, C = strain 41 and D = strain 42. (A = negative control). Citrate –negative
Fig.12. Lysine Iron Agar test for three Klebsiella pneumoniae strains: B = strain 40, C = strain 41 and D = strain 42. (A = negative control). Lysine decarboxylation positive and Hydrogen sulfide -negative.
Fig.13. MIU medium test for three Klebsiella pneumoniae strains:A = strain 40, B =strain 41 and C= strain 42.
(A) Urease activity (++), Motility (negative), Indole reaction (positive)
(B) Urease activity (++), Motility (negative), Indole reaction (positive)
(C) Urease activity (-), Motility (negative), Indole reaction (positive).
Fig.14. API tests: A- P. aeruginosa strain 29, B- A, baumannii strain 14 identified by API 20NE and C- Klebsiella pneumoniae strain 40 identified by Rapid API 20E.
The majority of investigated isolates of A, baumannii were MDR showing, colistin resistance also four isolates and only one strain carbapenemase producer. 33% of Pseudomonas aeruginosa were carbapenemases producers; regarding Klebsiella pneumoniae strains 3 isolates revealed exhibited Extended spectrum β-lactamases (ESBLs).
Table 6. The bacterial strains and their isolation source.
The most frequent isolated strain was A. baumannii (43 %) followed by P. aeruginosa (33%), Klebsiella pneumoniae (22%) and E.coli (2%) (Fig .15).
Fig.15. The frequencies of the identified bacterial species.
Also, our results revealed that the most frequent strains were isolated from tracheal secretion (28%), followed by urine (20%), other secretions (20%), catheter (11%), wound (7%), blood cultures (4%), feces (4%), liquid (2%) and umbilicus (2%)(Fig.16).
Fig.16.The isolation sources of bacterial strains.
4.2. The virulence factors expression
All the strains presented some virulence factors, their presence depending on the strains and the isolation source. P. aeruginosa strains proved to be most virulent strains followed by K. pneumoniae. Although A. baumannii were the most frequent isolated strains, their virulence was lower expressed comparing with other mentioned species. Although A. baumannii were the most frequent isolated strains, their virulence was lower expressed comparing with other mentioned species.
Caseinase, a protease that contribute to tissue degradation was predominant (60% of the strains) followed by esculin hydrolysis (52% of the isolates forming a black-brown pigment around colonies), and lecithinase (50% of the isolates), an enzyme involved in dissemination of the infections. 28% of the isolates showed hydrolyzed gelatin, another protease as an opaque clearing zone around the stabbing area, 15% of the strains were DNA-se positive, and 13% showed lipase production by formed an opaque clearing zone around the stabbing zone. Regarding hemolytic activity, 63.04% of the isolates showed α haemolysis and 13.04% of the strains were β haemolytic. None of the strains expressed amylase activity (table.7).
Table 7. The identified virulence factors expressed by isolated strains
(-) negative result, (+) positive result, (++) average result, (+++) strong result, (α) alfa haemolytic, (β) beta haemolytic, (γ) gamma haemolytic
Fig.17. some aspects of media for detection of enzymatic virulence factors: A-Casein agar, B -Esculin agar, C- DNA-se agar, D- Blood agar
4.3. Molecular assays in investigated strains
The results of PCR assays revealed different distribution of carbapenemases genes (blaOXA 23, blaOXA24) in A. baumannii (Fig.18, 19). In P. aeruginosa we observed different distribution of ARGs (blaTEM, blaCTX-M, blaIMP, blaVIM, blaOXA-48 and blaNDM); (Fig 20,22,26) and virulence genes in P.aeruginosa (PlcH , PlcN, ExoU , ExoT and protease IV (TCF/TCR); (Fig. 23,24,25,26). In K.pneumoniae were demonstrated different distribution of carbapenemases genes and (ESBLs); (Fig. 21,22,27).
Fig.18. Electrophoresis gel for carbapenemases (blaOXA-23 and OXA-24) : well no1 – strain no1; 2-2; 3-3; 4-4; 5-5; 6-6; 7-7; 9-9; 10-10; 12-12; 13-13; 14-14; 15-15; L- Marker (Thermo Scientific) – 1kb ; 16-16; 17-17; 18-18; 19-19; 20-20; 21-21; 22-22; – negative control. Positives isolates for OXA-23 gene: no. 1, 2, 3, 7, 9, 10, 16, 21, 22 and OXA-24gene no. 4, 5, 12; 14, 15, 17, 18, 19.
Fig.19. Distribution of carbapenemases in A,baumannii were (53%) OXA-23 gene and (47%) OXA-24 gene.
Fig.20. Electrophoresis gel for carbapenemases (IMP and VIM) : well no 11- strain no11;25-25;26-26;27-27;28-28;29-29;30-30;31-31;32-32;33-33;34-34; L- Marker (Thermo Scientific) – 1kb ;35-35;36-36;37-37;38-38;39-39;40-40;41-41;42-42;43-43;44-44;45-45;46-46 ; – negative control .positive isolates for VIM gene no .32,38.IMP gene negative for all.
Fig.21. Electrophoresis gel carbapenemases (OXA48 and NDM) : will no 8 – strain no 8;11-11;23-23;24-24;25-25;26-26;27-27;28-28;29-29;30-30;31-31;32-32;33-33; L- Marker (Thermo Scientific) – 1kb ;34-34;35-35;36-36;37-37;38-38;39-39;40-40;41-41;42-42;43-43;44-44;46-46; – negative control. Positives isolates for OXA-48 gene: no 39, 40, 41, 42,43,44,46. For NDM gene negative for all.
Fig.22. Electrophoresis gel for (ESBLs) (CTX-M and TIM ) : will no 8 – strain no 8; 11-11;23-23;24-24;25-25;26-26;27-27;28-28;29-29;30-30;31-31;32-32;33-33;35-35;34-34; L- Marker (Thermo Scientific) – 1kb ;36-36;37-37;38-38;39-39;40-40;41-41;42-42;43-43;64-64; – negative control .positives isolates for CTX-M gene ; no 11,23,24, 25,26,27,28,29,30,31, 33,35,34, 36, 38,39,40,41,42,43,44 and TEM gene ; 8,26,33,35, 36.
Fig.23. Electrophoresis gel for phospholiphases (PlcH and PlcN) : will no 11-strain no 11;25-25;26-26;27-27;28-28;29-29;30-30;31-31;32-32;33-33;34-34; ; L- Marker (Thermo Scientific) – 1kb ;35-35;36-36;37-37;38-38;- negative control. Positives isolates for PlcH gene .no 11,25,26,27,28,29,30,31 ,33,34,35,36,37,38 and for PlcN gene no 11,25,26,27,28,29,30,31 ,33,34,35,36,37,38 .
Fig.24. Electrophoresis gel for exoenzymes (ExoU and ExoT) : will no 11-strain no 11;25-25;26-26;27-27;28-28;29-29;30-30;31-31; L- Marker (Thermo Scientific) – 1kb ;32-32;33-33;34-34;35-35;36-36;37-37;38-38;- negative control. Positives isolate for ExoU gene no .28, 30, 31, 35; and ExoT – negative for all
Fig.25. Electrophoresis gel for protease IV (TCF/TCR) : will no 11-strain no 11;25-25;26-26;27-27;28-28;29-29;30-30;31-31;32-32;33-33;34-34; L- Marker (Thermo Scientific) – 1kb ;35-35;36-36;37-37;38-38;- negative control. Positives isolates for TC gene no 11, 25,26,27,28,29,30,31,33,34,35,36,37,38.
Detection of blaCTX-M gene responsible for 3rd generation cephalosprine resistance in our study revealed the presence of 8.66% of blaCTX-M gene in P. aeruginosa, compared with Al-Grawi result from Baghdad wich demonstrated that 80% of P. aeruginosa isolates experessed blaCTX-M (230). 33.33% of P.aeruginosa showed the presence blaTEM and this disagrees with the result of Al-Marjani in Baghdad Who didn't found any blaTEM gene in P.aeruginosa isolates (231). Previous studies showed that 17.3% of P.aeruginosa isolates from northwest of Iran and 19.51% from Southwest of Iran were VIM type positive (234) and it closer to our result blaVIM gene in 13.33% of P.aeruginosa.
No imipenem resistance genes were found in the investigated strains and this similar with studies from Ahwaz and Tehran Provinces, the scientists could not find any IMP-type MBL producing P.aeruginosa strains (232, 233).
Concerning the virulence profiles the molecular analysis through PCR arrays showed that 93.33% of P.aeruginosa revealed the plcH gene, plcN gene, and protease IV gene, and only 26.66% of P. aeruginosa revealed ExoU gene. No ExoT virulence genes were found in the investigated of P.aeruginosa strains (Fig. 26). Porumbel et al. In 2015 revealed closely percentages of virulence genes in P. aeruginosa from ,,C.C. Iliescu” showing the orizontal transfer of the ARGs.
Fig.26.Distribution of antibiotic resistance genes and virulence genes in P.aeruginosa
.
The results of PCR analysis for ESBLs and carbapenemases revealed the presence of blaCTX-M gene in 80% of K. pneumoniae isolates. High prevalence of blaCTX-M were also reported from other parts of the world; 71.4% in Sudan, 83.3% in Spain, 41.3% in Mongolia, 59.0% in Bangladesh, 48.5% in India, 50.0% in Taiwan, 84.0% in Thailand and 35.9% in Russia [235, 236,237). Co-producing NDM-1 and OXA-48 carbapenemases (one K. pneumoniae strain) in Turkey was reported (238). In this study blaOXA-48 gene detected in 70% 0f K. pneumoniae but no NDM resistance genes were found in the investigated of Klebsiella pneumoniae strains. blaTEM gene in 10% of K. pneumoniae (Fig. 27).
Fig. 27.Distributions of carbapenemases and (ESBL) in Klebsiella pneumoniae
CONCLUSIONS
Our results showed that all the strains presented at last 3-4 virulence factors, their presence depending on the strains and the isolation source. P. aeruginosa strains proved to be most virulent strains followed by K. pneumoniae. Although A. baumannii were the most frequent isolated strains, their virulence was lower expressed comparing with other mentioned species. Although A. baumannii were the most frequent isolated strains, their virulence was lower expressed comparing with other mentioned species.
The molecular screening of carbapenem resistance genes revealed the presence of OXA-23 gene (53%) and OXA-24 gene(47%) in A. baumannii isolates.
The most prevalent ESBLs and carbapenemases encoding genes among P.aeruginosa were blaCTX-M followed by blaTEM gene and blaVIM gene.
Concerning the virulence profiles showed that 93.33% of P.aeruginosa revealed the plcH gene, plcN gene, and protease IV gene, and only 26.66% of P. aeruginosa revealed ExoU gene. The diversity of virulence factors expression determines a large of clinical manifestations of infection with Pseudomonas.
The results of PCR analysis for ESBLs and carbapenemase revealed the presence of blaCTX-M gene in 80% of K. pneumoniae isolates and blaOXA-48 gene in 70% of K. pneumoniae and blaTEM gene in 10% of K. pneumoniae, an aspect which demonstrates the involvement of these strains in nosocomial infections.
In conclusion, we report here the characterization of phenotypic and genotypic β-lactam-resistance in NFGNR and Enterobacteriaceae strains isolated in 2015-2016 from ICU and ambulatory in Bucharest, Romania.
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