Medicina (Kaunas) 201147(3)REVIEW [621990]

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Medicina (Kaunas) 2011;47(3)REVIEW
Medicina (Kaunas) 2011;47(3):137-46
Correspondence to A. Giedraitienė, Department of Microbiol-
ogy, Medical Academy, Lithuanian University of Health Sci-ences, Eivenių 4, 50161 Kaunas, LithuaniaE-mail: [anonimizat] susirašinėti: A. Giedraitienė, LSMU MA Mikrobiologi-
jos katedra, Eivenių 4, 50161 KaunasEl. paštas: [anonimizat] Resistance Mechanisms of Clinically
Important Bacteria
Agnė Giedraitien ė1, Astra Vitkauskien ė2, Rima Naginien ė3, Alvydas Pavilonis1
1Department of Microbiology, Medical Academy, Lithuanian Univers ity of Health Sciences,
2Department of Laboratory Medicine, Medical Academy, Lithuanian University of Health Sciences,
3Institute for Biomedical Research, Medical Academy, Lithuanian University of Health Sciences, Lithuania
Key words: bacteria; antibiotics; resistance mechanisms.
Summary. Bacterial resistance to antimicrobial drugs is an increasing he alth and economic
problem. Bacteria may be innate resistant or acquire resistance to one or few classes of antimicrobial
agents. Acquired resistance arises from: (i) mutations in cell genes (chromosomal mutation) leading
to cross-resistance, (ii) gene transfer from one microorganism to other by plasmids (conjugation or
transformation), transposons (conjugation), integrons and bacte riophages (transduction). After a
bacterium gains resistance genes to protect itself from various antimicrobial agents, bacteria can use
several biochemical types of resistance mechanisms: antibiotic inactivation (interference with cell
wall synthesis, e.g., β-lactams and glycopeptide), target modif ication (inhibition of protein synthe-
sis, e.g., macrolides and tetracyclines; interference with nucl eic acid synthesis, e.g., fluoroquinolones
and rifampin), altered permeability (changes in outer membrane, e.g., aminoglycosides; new mem-
brane transporters, e.g., chloramphenicol), and “bypass” metabo lic pathway (inhibition of metabolic
pathway, e.g., trimethoprim-sulfamethoxazole).
Introduction
Bacterial resistance is closely associated with
the use of antimicrobial agents in clinical practice. Prolonged therapy with antibiotics can lead to the development of resistance in a microorganism that initially is sensitive to antibiotics, but later it can adapt gradually and develop resistance to antibiotics. When an antibiotic attacks bacteria, bacterial cells susceptible to it will die, but those that have some insensitivity will survive. The emergence of a phe-notype resistant to antimicrobial agents depends on various factors of a host: degree of resistance expres-sion, capability of a microorganism to tolerate resist-ance mechanism, initial colonization site, and other factors. When resistance determinants are on plas-mids, they will spread quickly within the genus and even unrelated bacterial genera. When resistance is associated with genes on chromosomes, resistant mi-croorganisms will spread more slowly (1, 2).
An important cause of the spread of antimicro-
bial resistance is a failure to apply infection control measures in a hospital and outside it. It has been established that methicillin-resistant Staphylococcus
aureu s (S. aureus , MRSA) in a hospital and MRSA
in the community are often genetically related. Re-sistant bacteria are transmitted by aerosol transmis-sion, especially during periods of viral upper respir-atory infections, frequent hand-nose contacts, and
poor hand washing among health care workers (3).
Antibiotic use in nonhuman niches is another
important reason for the spread of resistant bacteria (4). It is known that the use of antimicrobial agents in animal food is related to bacterial resistance; for example, Salmonella and Campylobacter acquire re-
sistance to antibiotics and transfer genes of antibi-otic resistance to natural human fl ora, for example,
Enterococcus . High Escherichia coli (E. coli ) resist-
ance to ciprofl oxacin is associated with the use of
fl uoroquinolones in aviculture (1, 3).
Over the years, the continued use of various an-
tibacterial/antimicrobial agents has led microorgan-isms to develop resistance mechanisms, which are the cause of resistance to one or more drugs (mul-tidrug resistance, MDR) (5). Resistance mecha-nisms probably have evolved from genes present in organisms that produce antibiotics (6). Multidrug resistance has been demonstrated in Pseudomonas
aeruginosa (P . aeruginosa), Acinetobacter bauman-nii (A. baumannii ), E. coli, and Klebsiella pneumo-
niae (K. pneumoniae ), producing extended-spectrum
β-lactamases (ESBL), vancomycin-resistant ente-
rococci Enterococcus faecium (E. faecium ) (VRE),
MRSA, vancomycin-resistant S. aureus VRSA, ex-
tensively drug-resistant (XDR) Mycobacterium tu-

138
Medicina (Kaunas) 2011;47(3)berculosis (M. tuberculosis ) (5), Salmonella enterica
(S. enterica ) serovar Typhimurium , Shigella dysente-
riae (S. dysenteriae ), Haemophilus influenzae (H. in-
fluenzae ), Stenotrophomonas, and Burkholderia (1).
Antibiotic resistance can be acquired as a chromo-somal mutation, but usually resistance to antibiotics is associated with mobile extrachromosomal DNA elements – plasmids, transposons, and integrons – acquired from other bacteria. Effl ux pumps are rec-
ognized as the main multidrug resistance mecha-nism in bacteria (5).
Genetics of Antibiotic Resistance
Bacterial resistance to antibiotics can be intrinsic
or innate, which is characteristic of a particular bac-terium and depends on biology of a microorganism (E. coli has innate resistance to vancomycin), and
acquired resistance (2). Acquired resistance occurs from (i) acquisition of exogenous genes by plasmids (conjugation or transformation), transposons (con-jugation), integrons and bacteriophages (transduc-tion), (ii) mutation of cellular genes, and (iii) a com-bination of these mechanisms (3, 6–8).
Mutations. Spontaneous Mutations. Chromoso-
mal mutations are quite rare (one in a population of 10
6–108 microorganisms) and commonly determine
resistance to structurally related compounds (3). These mutations occur as errors of replication or an incorrect repair of damaged DNA. They are called spontaneous mutations or growth-dependent muta-tions. Resistance to quinolones in E. coli is caused
by changes in at least seven amino acids in the gyrA
gene or three amino acids in the parC gene (1, 6, 9),
whereas only a single point mutation in the rpoB gene
is associated with a complete resistance to rifampin (3). A chromosomal mutation in dihydropteroate synthetase results in a reduced affi nity for sulfona-
mides (7). Some biochemical resistance mechanisms are the result of mutations. Antibiotic uptake or ef-fl ux system can be modifi ed by mutations (10).
Hypermutators. According to the “hypermutable
state” model, a small bacterial population during a prolonged nonlethal selection of microorganisms may achieve a short-term state when the population mutates at a very high rate (hypermutable strains or mutators) (1). These cells can increase the rate of mutations from 10 to 50 up to 10 000 times (11). Most hypermutators are found in populations of E. coli, S. enterica , Neisseria meningitides (N. menin-
gitides ), H. influenzae, S. aureus, Helicobacter pylori
(H. pylori ), Streptococcus pneumoniae (S. pneumo-
niae), and P . aeruginosa (1).
Adaptive Mutagenesis. Most mutations occur in
dividing cells. However, they can also arise in non-dividing or slowly dividing cells. Mutations occur only during nonlethal selection of microorganisms and are called “adaptive mutations.” This adaptive process is the only and main source of the anti-
biotic-resistant mutants to originate under normal conditions. Streptomycin causes a hypermutable
phenotype in E. coli , and some antibiotics (quinolo-
nes) can induce the SOS mutagenic response and increase the rate of emergence of resistance to anti-
biotics (1, 12, 13).
Horizontal Gene Transfer. A transfer of resist-
ance genes from one bacterium to another is called a horizontal gene transfer (14). The main mecha-nisms of resistance gene transfer in a bacterium are plasmid transfer, transfer by viral delivery, and transfer of free DNA (Fig. 1). Genes can be trans-ferred by three main ways: transduction (via bacte-riophages and integrons), conjugation (via plasmids and conjugative transposons), and transformation (via incorporation of chromosomal DNA, plasmids into a chromosome) (mobile genetic elements are described in Table 1). Then genes are incorporated into the recipient chromosome by recombination or transposition and may have one or several changes in gene sequence (1, 5, 15).
Most plasmids are double-stranded circular DNA
whose size may vary from 2–3 kb to plasmids, which encode up to 10% of the host cell chromosome. The transfer of resistance genes is more effective than chromosomal mutation (5). Plasmids encode genes that confer resistance to main classes of antimicro-bial agents (cephalosporins, fl uoroquinolones, and
aminoglycosides) (14), toxic heavy metals (mercury, cadmium, silver), and virulence determinants that help a cell to survive in the environment of lethal antibiotic doses (15, 16).
MDR genes are located in a DNA sequence that
is transferred from one plasmid to another or to the genomes, which are called transposons or “jumping gene systems” (6). Transposons can be integrated into plasmids or the host’s chromosome, encompass small elements called insertion sequences (IS ele-ments), transposons, and transposing bacteriophag-es. They have terminal repeat sequences that play a role in recombination and recognize a protein (for example, transposase or recombinase) that is neces-sary to insert or remove a transposon from specifi c
genome regions (5, 14, 16). Transposons are trans-ferred by conjugation, transformation, or transduc-tion (e.g., mecA gene is found in MRSA) and spread
quicker than genes in chromosomes. Conjugative transposons have characteristic features of plasmids and can help to transfer endogenic plasmids from one microorganism to another (8, 15, 17).
Bacterial integrons are gene capture systems
(Fig. 2) that instead of transposition use a specifi c
recombination mechanism (14, 15). Integron en-codes three main components in the 5´ conserved segment: an enzyme integrase (gene int) that serves
as a specifi c recombination system to insert or to Agnė Giedraitienė, Astra Vitkauskienė, Rima Naginienė, Alvydas Pavilonis

139
Medicina (Kaunas) 2011;47(3)remove a new gene cassette, specifi c recombination
site ( attI site), and a promoter that starts gene tran-
scription. Most integrons of class I in the 3´ con-served segment have an additional gene suII respon-
sible for resistance to sulphonamides (10, 18, 19).
Biochemical Resistance Mechanisms
The main types of biochemical mechanisms that
bacteria use for defense are as follows: decreased up-take, enzymatic modifi cation and degradation, al-
tered penicillin-binding proteins (PBPs), effl ux, al-tered target, and its overproduction (Table 2) (3, 20,
21). Below we will describe main types of different biochemical mechanisms that are found in clinically
important bacteria.
Antibiotic Inactivation or Modification
There are three main enzymes that inactivate anti-
biotics: β-lactamases, aminoglycoside-modifying en-
zymes, and chloramphenicol acetyltransferases (7).
Antibiotic Modification by Hydrolysis. β -Lacta-
ma ses are broadly prevalent enzymes that are clas-
sifi ed using two main classifi cation systems: Ambler
and Bush-Jacoby-Medeiros (5). It is known about 300 different β-lactamases. The most clinically im-
portant are produced by gram-negative bacteria (22) and are coded on chromosomes and plasmids. Genes that encode β-lactamases are transferred by transpo-
sons but also they may be found in the composition of integrons (23). β-Lactamases hydrolyze nearly
all β-lactams that have ester and amide bond, e.g., Genetic Element General Characteristic Resistance Determinants Sp ecifi ed/Examples
Plasmid Variable size (1–>100 kb), conjugative, and mo-
bilizableR factor: multiple resistance
Insertion sequence Small (<2.5 kb), contains terminal inverted r e-
peats, and specifi es a transposaseIS1, IS3, IS4
Composite (compound)
transposonFlanked by insertion sequences and/or inverted
repeatsTn5: Kan, Bleo, Str
Complex
transposonLarge (>5 kb), fl anked by short terminal inverted
repeats, and specifi es a transposase and recom-
binaseTn1 and Tn3: β-lactamase
Tn7: Tmp, Str, SpcTn1546: glycopeptides
Conjugative transposon Promotes self-transfer Tn916: Tet and Mino
Tn1545: Tet, Mino, Ery, and Kan
Transposable bacteriophage A bacterial virus that can insert int o the chromo-
someMu
Other transposable
elementsOther than composite, complex, and conjugative
transposonsTn4: Amp, Str, Sul, and Hg
Tn1691: Gen, Str, Sul, Cm, and Hg
Integron Facilitates acquisition and dissemination of gene
cassettes; specifi es an integrase, attachment sites,
and transcriptional elements to drive expression of multiple resistance genesClass 1: multiple single determinants and MDR
effl ux pump (Qac)
Class 2: Tmp, Strp, Str, and Spc (Tn7)Class 3: carbapenemsClass 4: Vibrio spp. super-integronTable 1 . Mobile Genetic Elements (5)
Fig. 1 . Three main mechanisms of resistance gene transfer
in a bacterium (9)
a, plasmid transfer; b, transfer by viral delivery;
c, transfer of free DNA.
Fig. 2. Simplifi ed scheme of gene cassette capture
by a bacterial integron (14)
Integrase
Integron 1 Integron 2Gene
CassetteAntibiotic Resistance Mechanisms of Clinically Important Bacter ia

140
Medicina (Kaunas) 2011;47(3)Antibiotic
ClassResistance Type Resistance Mechanism Common Example(s)
Aminoglyco-
sidesDecreased
uptake
Enzymatic
modifi cation
(AMEs)Changes in outer membrane
permeabilityPhosphotransferaseAdenyltransferaseAcetyltransferaseBifunctional enzymeP . aeruginosa
Wide range of enteric negative bacteria
Wide range of enteric negative bacteriaWide range of enteric negative bacteriaS. aureus, E. faecium and E. faecalis aac(6´)-aph(2´´)
β-lactams Altered PBPs
Enzymatic deg-
radation(β-lactamases)PBP2a (additional PBP)
PBP2x, PBP2b, PBP1a
PBP5 (point mutation)Ambler class A
Ambler class B
Ambler class CAmbler class DmecA in S. aureus and coagulase-negative staphylococci
S. pneumoniaeE. faeciumTEM-1 in E. coli , H. influenzae, and
N. gonorrhoeae
SHV-1 in K. pneumoniae
K-1 (OXY -1) in K. oxytoca
Extended-spectrum β-lactamases (TEM – 3+, SHV – 2+,
and CTX-M types) K. pneumoniae and E. coli
BRO-1 in M. catarrhalis
PC1 in S. aureus
PSE-1 in P . aeruginosa
β-lactamases of C. koseri and P . vulgaris
L-1 in S. maltophilia
Ccr-A in B. fragilis
Amp C in E. cloacae , C. freundii
S. marcescens , M. morganii ,
P . stuartii and P . rettgeri
OXA-1 in E. coli
Chloram-
phenicolEnzymatic deg-
radationEffl uxCAT
New membrane transportersCAT in S. pneumonia
cmlA and flo-encoded effl ux in E. coli and Salmonella spp.
Glycopep-
tidesAltered target
Target overpro-
ductionAltered peptidoglycan cross-link
target (D-Ala-D-Ala to D-Ala-D-Lac or D-Ala-D-Ser) encoded by complex gene clusterExcess of peptidoglycanvanA and vanB gene clusters in E. faecium and E. faecalis
Glycopeptide “intermediate” strains of S. aureus
Fosfomycin Enzymatic deg-
radationThioltransferase fosA in negative bacteria and P . aeruginosa;
fosB in staphylococci and B.subtilis
Fusidic acid Altered target
Decreased per-
meabilityMutation leading to reduced
binding to active site(s)Chloramphenicol acetyltransfer-aseMutation in fusA in S. aureus
Mutation in fusB in S. aureus
Macrolides-
lincosamides- strepto-gramins BAltered target Methylation of ribosomal active
site with reduced bindingerm-encoded methylases in S. aureus , S. pneumoniae , and
S. pyogenes
Macrolides Effl ux Mef type pump mef-encoded effl ux in S. pneumoniae and S. pyogenes
Oxazolidi-
nonesAltered target Mutation leading to reduced
binding to active siteG2576U mutation in rRNR in E. faecium and S. aureus
Strepto-
graminsStrepto-gramin AEnzymatic deg-
radationAcetyltransferase vatA, vatB , and vatC in S. aureus
E. faecium vatD and vatE
Quinolones Altered target
Effl uxMutation leading to reduced
binding to active site(s)
New membrane transportersMutations in gyrA in enteric gram-negative bacteria and
S. aureus
Mutations in gyrA and parC in
S. pneumoniaeNorA in S. aureus
Rifampin Altered target Mutations leading to reduced
binding to RNA polymeraseMutations in rpoB in S. aureus and M. tuberculosis
Tetracyclines Effl ux
Altered targetNew membrane transporters
Production of proteins that bind
to the ribosome and alter the conformation of the active sitetet genes encoding effl ux proteins in gram-positive and
gram-negative bacteriatet(M) and tet(O) in diverse gram-positive and gram-nega-
tive bacteria speciesTable 2 . Biochemical Resistance Mechanisms (3, 20, 21) Agnė Giedraitienė, Astra Vitkauskienė, Rima Naginienė, Alvydas Pavilonis

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Medicina (Kaunas) 2011;47(3)penicillins, cephalosporins, monobactams, and car-
bapenems. Serine β-lactamases – cephalosporinases,
e.g. AmpC enzyme – are found in Enterobacter spp.
and P . aeruginosa and penicillases in S. aureus (5,
24–27). Metallo- β-lactamases (MBLs) found in P.
aeruginosa , K. pneumoniae , E. coli , Proteus mirabilis
(P . mirabilis ), Enterobacter spp. have the same role as
serine β-lactamases and are responsible for resist-
ance to imipenem, new-generation cephalosporins and penicillins. MBLs are resistant to inhibitors of β-lactamases but sensitive to aztreonam (24, 28).
Specifi c A. baumannii carbapenem-hydrolyzing
oxacillinase (OXA) enzymes that have low catalytic effi ciency together with porin deletion and other
antibiotic resistance mechanisms can cause high re-sistance to carbapenems (24). The resistance of K.
pneumoniae carbapenamases (KPC-1) to imipenem,
meropenem, amoxicillin/clavulanate, piperacillin/tazobactam, ceftazidime, aztreonam, and ceftriax-one is associated with the nonconjugative plasmid –
coded bla gene (29).
Extended-spectrum β-lactamases (ESBL) – TEM,
SHV, OXA, PER, VEB-1, BES-1, GES, IBC, SFO and CTX – mainly are encoded in large plasmids. They can be transferred in connection of two plas-mids or by transposon insertion. ESBL are resistant to penicillins (except temocillin), third-generation oxyimino-cephalosporins (e.g., ceftazidime, cefo-toxime, ceftriaxone), aztreonam, cefamandole, ce-foperazone, byt they are sensitive to methoxy-ceph-alosporins, e.g., cephamycins and carbapenems, and can be inhibited by inhibitors of β-lactamases,
e.g., clavulanic acid, sulbactam, or tazobactam (23, 30–34). Strains producing ESBL are commonly re-sistant to quinolones but their resistance depends not on multiple resistance plasmids but on muta-tions in gyrA and parC genes (35). Such strains are
found among E. coli , K. pneumoniae, and P . mirabilis
(1). The number of known ESBLs reaches 200 (32, 36).
Hydrolysis of antibiotics can be run by other
enzymes, e.g., esterases. E. coli gene ereB encodes
erythromycin esterase II that hydrolyzes a lactone ring of erythromycin A and oleandomycin. ereB
gene is prevalent in Enterobacteriaceae strain and is responsible for resistance to erythromycin A and
oleandomycin (37). Ring-opening epoxidases cause resistance of bacteria to fosfomycin (1).
Antibiotic Inactivation by Group Transfer. The
group of enzymes inactivating aminoglycosides, chloramphenicol, streptogramin, macrolides, or ri-fampicin is called transferases. Inactivation is made by binding adenylyl, phosphoryl, or acetyl groups to the periphery of the antibiotic molecule. These modifi cations are achieved in the process of trans-
port across the cytoplasmic membrane (co-substrate ATP, acetyl-CoA, NAD
+, UDP-glucose, or glu-
tathione) (1, 16). Aminoglycosides are neutralized by specifi c enzymes: phosphoryltransferases (APHs), nu-
cleotidyltransferases or adenylyltransferases (ANTs), and acetyltransferases (AACs). These aminogly-coside-modifying enzymes (AMEs) reduce affi n-
ity of a modifi ed molecule, impede binding to the
30S ribosomal subunit (38), and provide extended-spectrum resistance to aminoglycosides and fl uoro-
quinolones (39). AMEs are identifi ed in S. aureus ,
Enterococcus faecalis (E. faecalis ), and S. pneumoniae
strains. Presumably, they evolved from actinomy-cetes ( Streptomyces spp. and Micromonospora spp.)
that produce AMEs. Most AMEs are transferred by transposons (4).
Gram-positive and gram-negative bacteria and
some of H. influenzae strains are resistant to chloram-
phenicol and they have an enzyme chlorampheni-col transacetylase that acetylates hydroxyl groups of chloramphenicol. Modifi ed chloramphenicol is ena-
ble to bind to a ribosomal 50S subunit properly (17).
Antibiotic Inactivation by Redox Process. Oxida-
tion and reduction reactions are used by pathogenic bacteria as a resistance mechanism against antibi-otics. Streptomyces virginiae produces type A anti-
biotic virginiamycin M
1 and protects itself from its
own antibiotic by substituting a ketone group to an alcohol residue at position 16 (1, 6).
Target Modification
An interaction between an antibiotic and a target
molecule is very specifi c so even small changes in a
target molecule can infl uence antibiotic binding to a
target. Sometimes, in the presence of a modifi cation Antibiotic
ClassResistance Type Resistance Mechanism Common Example(s)
Sulfonamides Altered target Mutation or recombination of
genes encoding DHPSAcquisition of new low-affi nity
DHPS genesFound in a wide range of species : E. coli , S. aureus , S.
pneumoniae
sulI and sul II in enteric gram-negative bacteria
Trim-
ethoprimAltered target
Overproduction
of targetMutations in gene encoding
DHFRAcquisition of new low-affi nity
DHFR genesPromoter mutation leading to overproduction of DHFRS. aureus , S. pneumoniae , H. influenzae
dhfrI and dhfrII encoded, found in a wide range of species
E. coliTable 2 . Biochemical Resistance Mechanisms (3, 20, 21) (continuation)
Antibiotic Resistance Mechanisms of Clinically Important Bacter ia

142
Medicina (Kaunas) 2011;47(3)in a target, other changes in the cell are needed to
compensate an altered target (1, 40).
Peptidoglycan Structure Alteration. Inhibition of
cell wall synthesis is performed by β-lactams, e.g.,
penicillins, cephalosporins, carbapenems, mono-bactams, and glycopeptides, e.g., vancomycin and teicoplanin. The presence of mutation in PBPs leads to a reduced affi nity to β-lactam antibiotics. It re-
sults in resistance of E. faecium to ampicillin and
S. pneumoniae to penicillin. S. aureus resistance to
methicillin and oxacillin is associated with integra-tion of a mobile genetic element – “staphylococ-cal cassette chromosome mec” (SCC mec) – into the
chromosome of S. aureus that contains resistance
gene mecA. mecA gene encodes PBP2a protein, a
new penicillin-binding protein, that is required to change a native staphylococcal PBP (1, 5, 41). PB-P2a shows a high resistance to β-lactam antibiotics
(they do not bind to β-lactams) and ensures cell wall
synthesis at lethal β-lactam concentrations (6, 42).
S. aureus strains resistant to methicillin can be cross
resistant to all β-lactam antibiotics, streptomycin,
and tetracycline and in some cases to erythromy-cin (43). When lesions in membrane proteins are present, cross-resistance between β-lactam antibiot-
ics and fl uoroquinolones is possible (44). Cell wall
synthesis in gram-positive bacteria can be inhibited by glycopeptides, e.g., vancomycin or teicoplanin, by their binding to acyl-D-alanyl-D-alanine (acyl-D-Ala-D-Ala) residues of peptidoglycan precursors. Resistance to glycopeptides can be innate ( VanC –
type resistance) or acquired (1, 43). E. faecium and
E. faecalis strains have high resistance to vancomy-
cin and teicoplanin ( VanA -type resistance). VanA-
type resistance to glycopeptides is transferred from
E. faecalis to E. faecalis , S. pyogenes , S. sanguis ,
and Listeria monocytogenes (L. monocytogenes ) by
conjugation. E. faecium and E. faecalis strains that
have VanB -type resistance show resistance to van-
comycin, when its minimal inhibitory concentra-tion (MIC) varies from 4 to 1024 μg/mL, and are
sensitive to teicoplanin. Enterococcus gallinarum,
Enterococcus casseliflavus , and Enterococcus flaves-
cens have low innate resistance to vancomycin and
are sensitive to teicoplanin ( VanC -type resistance).
This type of resistance depends on a chromosomal gene (8, 17, 45). β
-Lactams (piperacillin, ceftazi-
dime, imipenem, meropenem, and aztreonam) in-hibit peptidoglycan-assembling transpeptidases that are located on the outer side of cytoplasmic mem-brane, whereas polymyxins (colomycin, colistin) bind to phospholipids (27).
Protein Synthesis Interference. Antibiotics (ami-
noglycosides, tetracyclines, macrolides, chloram-phenicol, fusidic acid, mupirocin, streptogramin, and oxazolidinones) can interfere with protein syn-thesis at its different stages; for example, during transcription via RNA polymerase, rifamycins mod-
ify a specifi c target (46). Aminoglycosides (gen-
tamicin, tobramycin, amikacin) bind to the 30S ri-bosomal subunit (27) while chloramphenicol binds to the 50S ribosomal subunit and suppresses protein synthesis (47).
Macrolides, lincosamides, and streptogramin B
(MLS antibiotics) block protein synthesis in gram-negative bacteria by binding to the 50S ribosomal subunit. Then the 50S subunit undergoes a post-transcriptional modifi cation (methylation). RNA
methyltransferase involves RNA that is close to or in the binding place of antibiotics. Mutations in 23S rRNA, the same as nonmethylated rRNA, are associated with resistance to MLS (1). Nonmethyl-ated 23S rRNA and 16S rRNA at U2584 position in Haloarcula marismortui cause resistance to kasuga-
mycin and sparsomycin. A nonreactive rluC gene is
responsible for resistance to clindamycin, linezolid, and tiamulin. Oxazolidinones interfere with pro-teins synthesis at several stages: (i) inhibit protein synthesis by binding to 23S rRNA of the 50S subu-nit and (ii) suppress 70S inhibition and interaction with peptidyl-tRNR (5, 7).
DNA Synthesis Interference. The mechanism of
resistance is a modifi cation of two enzymes: DNA
gyrase (also known as topoisomerase II) (genes gyrA
and gyrB) (37) and topoisomerase IV ( parC and
parE). Mutations in genes gyrA and parC are fol-
lowed by replication failure, and then quinolones/fl uoroquinolones cannot bind. The most common
mutation in E. coli gyrA causes a reduced drug affi n-
ity for modifi ed-DNA complex, and MIC is higher
(3, 5, 44, 48). Quinolones (ciprofl oxacin) bind to
DNA gyrase A subunit (26). Usually resistance to
quinolones is associated with mutations in chro-mosomes, but plasmid-mediated (49–51) and point mutation-related (in genes gyrA and parC) resist-
ance to quinolones (52) was reported as well.
Efflux Pumps and Outer Membrane
PermeabilityMembrane proteins that export antibiotics from
the cell and maintain their low intracellular concen-trations are called effl ux pumps (Fig. 3). Reduced
outer membrane (OM) permeability results in re-duced uptake of antibiotics (1).
Efflux Pumps. In analyzing resistance to antibi-
otics, identifi cation and characterization of effl ux
pumps is one of the most actual problems. Single-component effl ux systems transfer their substrates
across the cytoplasmic membrane. Multicomponent pumps found in gram-negative bacteria and to-gether with a periplasmic membrane synthesis pro-
tein (MFP) component and an OM protein (OMP) component transfer substrates across the cell enve-lope (1, 5, 6, 46). Antibiotics of all classes except Agnė Giedraitienė, Astra Vitkauskienė, Rima Naginienė, Alvydas Pavilonis

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Medicina (Kaunas) 2011;47(3)polymyxins are susceptible to the activation of ef-
fl ux systems (27). Effl ux pumps can be specifi c to
antibiotics. Most of them are multidrug transpor-ters (Table 3) that are capable to pump a wide range of unrelated antibiotics – macrolides, tetracyclines, fl uoroquinolones – and thus signifi cantly contribute
to MDR (1). Bacteria resistant to tetracyclines often produce increased amounts of membrane proteins that are used as export or effl ux pumps of antimi-
crobial drugs (53). To eliminate toxic compounds from the cytoplasm and periplasm, P . aeruginosa uses more than four powerful MDR effl ux pumps
(Mex) (38, 54, 55).
MexV-MexW-OprM MDR effl ux pumps are re-
sponsible for resistance to fl uoroquinolones, tetra-
cyclines, chloramphenicol, erythromycin, ethidium bromide, and acrifl avine (38). Increased expression
of MexAB-OprM effl ux pumps results in higher
inhibitory concentration against penicillins, broad-spectrum cephalosporins, chloramphenicol, fl uoro-
quinolones, macrolides, novobiocin, sulfonamides, tetracycline and trimethoprim, dyes and detergents Bacterium Effl ux System Representative Antibiotic Resistance
P . aeruginosa MexAB-OprM β-lactams, fl uoroquinolones
MexCD-OprJ fourth-generation cephalosporins
MexEF-OprN fl uoroquinolones, chloramphenicol, trimethoprim, triclosan
MexHI-OprD ethidium bromide
MexJK-OprM ciprofl oxacin, tetracycline, erythromycin, triclosan
MexVW-OprM fl uoroquinolones, chloramphenicol, tetracycline, erythromycin,
ethidium bromide, acrifl avine
MexXY -OprM aminoglycosides, tigecycline
A. baumannii AdeABC aminoglycosides, fl uoroquinolones, tetracycline, cefotaxime,
chloramphenicol, erythromycin, trimethoprim
S. maltophilia SmeABC aminoglycosides, β-lactams, fl uoroquinolones
SmeDEF macrolides, tetracycline, fl uoroquinolones, carbapenems,
chloramphenicol, erythromycin
B. cepacia CeoAB-OpcM chloramphenicol, ciprofl oxacin, trimethoprim
B. pseudomallei AmrAB-AprA macrolides, aminoglycosides
E. coli AcrB-Tolc fl uoroquinolones, β-lactams, tetracycline, chloramphenicol, acrifl avine, trimethoprim
K. pneumoniae AcrB-TolC fl uoroquinolones, β-lactams, tetracycline, chloramphenicol
S. aureus MepA tigecycline, minocycline, tetracycline, ciprofl oxacin, norfl oxacin ethidium bromide,
tetraphenylphosphonium bromide
E. faecalis EmeA norfl oxacin, ethidium bromide, clindamycin, erythromycin, novobioci n
Lsa clindamycin, quinupristin-dalfopristin
S. pneumoniae PmrA fl uoroquinolones, acrifl avine, ethidium bromideTable 3. Multidrug Resistance Effl ux System of Clinically Important Bacteria (5)Fig. 3 . Bacterial effl ux system
A, system for antibiotic pumping out of the cell; B, antibiotic interfering with ribosomes in protein biosynthesis (9).
ABAntibioticBacterial Outer
Membrane
Antibiotic-
Effl ux Pump
Nascent Bacterial
Protein
RibosomePump Blocker
Drug Blocks
Protein SynthesisAntibiotic Resistance Mechanisms of Clinically Important Bacter ia

144
Medicina (Kaunas) 2011;47(3)(24, 56). β-Lactam antibiotics in gram-negative
bacteria can penetrate through a membrane protein fi lled with water named porin. Absence of P . aerug-
inosa-specifi c OprD2 porin results in resistance to
imipenem, whereas resistance to meropenem occurs due to changes in MexAB-OprM effl ux system (1,
57). Overexpression of OprM, production of ac-quired β-lactamase, and overexpression of AmpC
cephalosporinase are attributed to P . aeruginosa re-
sistance to ticarcillin (58). MexZ, a transcriptional regulator of the
mexXY multidrug transporter op-
eron, confers resistance to aminoglycosides (59). Loss of 29 kDa OMP is responsible for A. baumannii
resistance to imipenem and meropenem. Loss of K.
pneumoniae OMP together with ampC β-lactamase
ad new generation carbapenemase A, KPC, re-sults in resistance to carbapenems (24), whereas overexpression of AdeABC effl ux pumps – resist-
ance to aminoglycosides and reduced sensitivity to fl uoroquinolones, tetracyclines, chloramphenicol,
erythromycin, trimethoprim, ethidium bromide, netilmicin, and meropenem. Chloramphenicol, li-pophilic β-lactams, fl uoroquinolones, tetracyclines,
rifampin, novobiocin, fusidic acid, nalidixic acid, ethidium bromide, acrifl avine, bile salts, short-chain
fatty acids, SDS, Triton X-100, and triclosan serve as substrates for E. coli AcrAB-TolC effl ux system.
The MtrCDE effl ux pump of penicillin-resistant
Neisseria gonorrhoeae (N. gonorrhoeae ) strains inter-
acts with porins ( penB) and low-affi nity PBPs, and
stimulates resistance to β-lactams. Homologues of
Mex and Acr effl ux systems are found in Entero-
coccus aerogenes , Klebsiella spp., P . mirabilis , Serra-
tia marcescens (S. marcescens ), Morganella morganii ,
H. influenzae , and H. pylori (60). The main elimi-
nation system for macrolides that is encoded by mef
gene is prevalent in gram-positive bacteria and can be used for the elimination of fl uoroquinolones and
aminoglycosides from the cell (61). An elimination system of tetracyclines and chloramphenicol that is
encoded by ramA gene is found in E. coli and K.
pneumoniae . This also might result in resistance to
norfl oxacin (43). Resistance to tetracyclines might
be encoded by tetK gene that is found in gram-
positive bacteria – Enterobacteriaceae , Haemophilus ,
Vibrio , Aeromonas , and Moraxella strains, whereas
tetL gene – in Streptococcus
spp. and Enterococcus
spp. Gram-positive cocci have both these genes: tetL
and tetK (61).
Changes in Outer Membrane Permeability. The
OM in gram-negative bacteria contains an inner layer that has phospholipids and an outer layer that has the lipid A. Such OM composition reduces drug uptake to a cell and transfer through the OM (through porin proteins, e.g., OmpF in E. coli and OprD in P . aeruginosa ). Drug molecules to a cell
can be transferred by the following mechanisms: (i) diffusion through porins, (ii) diffusion through the bilayer, and (iii) by self-promoted uptake. A type of entry depends on chemical composition of a drug molecule (1). Acquired resistance to all antibiotic classes in P . aeruginosa is due to low OM perme-
ability. Small hydrophilic molecules ( β-lactams and
quinolones) can cross the OM only through porins. Aminoglycosides and colistin cannot be transferred to the cell through porins; therefore, self-promot-ed uptake to the cell is initiated by binding to li-popolysaccharides of the outer side of the OM (27). Acquired resistance is characteristic of high resist-ance to almost all aminoglycosides (especially to to-bramycin, netilmicin, and gentamicin) (62).
Bypass of Antibiotic Inhibition
The fourth mechanism of bacterial resistance to
antibiotics is specifi c. Bacteria produce an alterna-
tive target (usually an enzyme) that is resistant to inhibition of antibiotic (for example, MRSA pro-duces an alternative PBP). At the same time, bac-teria produce a native target too, which is sensitive to antibiotics. An alternative target allows bacteria to survive by adopting the role of a native protein. Resistance to trimethoprim and sulphonamides is caused by reduced sensitivity and affi nity of altered
enzymes dihydropteroate synthetase (DHPS) and dihydropteroate reductase (DHFR) to trimethoprim and sulphonamides (16, 23).
Conclusions and recommendations
Massive usage of antibiotics in clinical practice
resulted in resistance of bacteria to antimicrobial agents. Bacteria use innate and acquired resistance mechanisms to protect themselves. Acquired resist-ance arises from mutations, gene transfer by con-jugation or transformation, transposons, integrons, and bacteriophages. The following biochemical types of resistance mechanisms are used by bacteria: antibiotic inactivation, target modifi cation, altered
permeability, and “bypass” metabolic pathway.
It is necessary to determine bacterial resistance
to antibiotics of all classes (phenotypes) and muta-tions that are responsible for bacterial resistance to antibiotics (genetic analysis). Better understanding of mechanisms of antibiotic resistance, location of genes in a chromosome and their expression would allow us to develop screening and control strategies that are needed to reduce the spread of resistant bacteria and their evolution.
Statement of Conflict of Interest
The authors state no confl ict of interest.Agnė Giedraitienė, Astra Vitkauskienė, Rima Naginienė, Alvydas Pavilonis

145
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Agnė Giedraitien ė1, Astra Vitkauskien ė2, Rima Naginien ė3, Alvydas Pavilonis1
¹Lietuvos sveikatos mokslų universiteto Medicinos akademijos Mikrobiologi jos katedra,
2Lietuvos sveikatos mokslų universiteto Medicinos akademijos Laboratorinė s medicinos klinika,
3Lietuvos sveikatos mokslų universiteto Medicinos akademijos Biomedicinin ių tyrimų institutas
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Received 11 May 2010, accepted 17 March 2011
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