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  ISSN 1330-9862  review (FTB-1931) Antibiotic Resistance Mechanisms in Bacteria:Biochemical and Genetic Aspects Senka D`idi} 1 , Jagoda [u{kovi} 2*  and Bla`enka Kos 2 1 Ru|er Bo{kovi} Institute, Department of Molecular Genetics, POB 180, HR-10 002 Zagreb, Croatia 2 Laboratory for Antibiotic, Enzyme, Probiotic and Starter Culture Technology, Faculty of FoodTechnology and Biotechnology, University of Zagreb, POB 625, HR-10 001 Zagreb, CroatiaReceived: June 5, 2007Accepted: November 19, 2007 Summary Since the discovery and subsequent widespread use of antibiotics, a variety of bacte-rial species of human and animal srcin have developed numerous mechanisms that ren-der bacteria resistant to some, and in certain cases to nearly all antibiotics. There are manyimportant pathogens that are resistant to multiple antibiotic classes, and infections caused by multidrug resistant (MDR) organisms are limiting treatment options and compromisingeffective therapy. So the emergence of antibiotic-resistant pathogens in bacterial popula-tions is a relevant field of study in molecular and evolutionary biology, and in medicalpractice. There are two main aspects to the biology of antimicrobial resistance. One is con-cerned with the development, acquisition and spread of the resistance gene itself. The ot-her is the specific biochemical mechanism conveyed by this resistance gene. In this reviewwe present some recent data on molecular mechanisms of antibiotic resistance. Key words:  antibiotic resistance, multidrug resistance, antibiotic inactivation, target modifi-cation, drug efflux, membrane permeability changes, hypermutators, horizontal gene transfer Introduction Infections have been the major cause of diseasethroughout the history of human population. With theintroduction of antibiotics, it was thought that this prob-lem should disappear. However, bacteria have been ableto evolve to become resistant to antibiotics ( 1–3 ). The in-crease in antibiotic resistance has been attributed to acombination of microbial characteristics, the selectivepressure of antibiotic use and social and technical chan-ges that enhance the transmission of resistant organisms.The growing threat from resistant organisms calls forconcerted action to prevent the emergence of new resis-tant strains and the spread of existing ones ( 4 ).Recent extensive reviews on the application of anti- biotics in human and veterinary medicine ( 5–7 ), agricul-ture ( 8 ) and aquaculture ( 9 ) have documented the en-richment of antibiotic-resistant bacteria. Many procedures,use and misuse of antibiotics in man have resulted inantibiotic-resistant bacteria. The nutritive and therapeu-tic antibiotic treatment of farm animals amounts to a half of the world’s antibiotic output and has also resulted inantibiotic-resistant bacteria. Evidence is accumulating tosupport the hypothesis that antibiotic-resistant bacteriafrom poultry, pigs and cattle enter the food supply, can be found in human food ( 10–13 ), colonize human diges-tive tract and transfer resistance genes to human com-mensals.There have been very few systematic studies to in-vestigate the acquired antibiotic resistance in lactic acid bacteria (LAB) of food srcin. However, they are latelyexpanding due to increased interest in probiotic lacticacid bacteria and genetic modification of LAB. WhenLAB live in a biotope regularly challenged by antibiotics(human or animal intestine, bovine udder), the acquired 11 S. D@IDI]  et al. : Antibiotic Resistance in Bacteria,  Food Technol. Biotechnol. 46  (1) 11–21 (2008)*Corresponding author; Phone: ++385 1 4605 291; Fax: ++385 1 4836 424; E-mail: jsusko @ pbf.hr  antibiotic resistance is found in  Enterococcus, Lactococcus and  Lactobacillus  species ( 14–16 ). The resistant bacteria mayinteract with the resident human microflora and possi- bly transfer or acquire antibiotic resistance determinants by horizontal gene transfer. Large numbers of probiotic bacteria are consumed to maintain and restore the mi-crobial balance in the intestines. It must be kept in mindthat they have a potential to transfer antibiotic resistan-ces to pathogenic bacteria. For these and other applica-tions the safety aspects of these bacteria are of concern,including the presence of potentially transferable antibi-otic resistances ( 14–17 ) . Bacteria that normally reside in the human coloncan transfer resistance genes among themselves ( 18–21 ).This type of transfer becomes a huge problem whenthese harmless commensal bacteria transform into patho-gens ( 22 ). The environment is replete with drug resis-tance genes, among both pathogen and commensal bac-teria. Once acquired, resistance genes are not easily lost.Instead, they become a relatively stable part of a ge-nome. Additional resistance determinants may join thosealready prevailing, thus broadening the multidrug resis-tance phenotype and further diminishing treatment op-tions ( 23–25 ). An increasing number of bacterial isolatesis resistant to practically all available therapeutic agents.Multidrug resistance has been demonstrated in  Escheri-chia coli ,  Salmonella enterica  serovar Typhimurium,  Shigel-la dysenteriae, Enterococcus faecium ,  Staphylococcus aureus ,  Mycobacterium tuberculosis ,  Haemophilus influenzae, Pseu-domonas aeruginosa ,  Klebsiella pneumoniae ,  Acinetobacterbaumannii, Stenotrophomonas maltophilia, Xanthomonas  and Burkholderia  ( 26 ).Thus, the emergence of antibiotic resistance in bac-terial populations is a relevant field of study in molecu-lar and evolutionary biology as well as in medical prac-tice. Here we present recent data on bacterial resistanceto antibiotics. We will focus on the molecular mecha-nisms of antibiotic resistance and genetic parameters in-volved in development, acquisition and spread of resis-tance genes. Modes of Antibiotic Action Three conditions must be met for an antibiotic to beeffective against bacteria:  i)  a susceptible antibiotic tar-get must exist in the cell,  ii)  the antibiotic must reach thetarget in sufficient quantity, and  iii)  the antibiotic mustnot be inactivated or modified ( 27,28 ).Understanding antibiotic resistance mechanisms re-quires an understanding of where antibiotics exert theireffect. There are five major modes of antibiotic mecha-nisms of activity and here are some examples. Interference with cell wall synthesis b -lactam antibiotics such as penicillins and cephalo-sporins interfere with enzymes required for the synthe-sis of the peptidoglycan layer. Glycopeptides (vancomy-cin, teicoplanin, oritavancin) target the bacterial cell wall by binding to the  D -alanyl- D -alanine termini of the pep-tidoglycan chain, thereby preventing the cross-linkingsteps. Telavancin, a novel rapidly bactericidal lipoglyco-peptide, inhibits peptidoglycan biosynthesis throughpreferential targeting of transglycosylation ( 29,30 ). Inhibition of protein synthesis Macrolides bind to the 50S ribosomal subunit andinterfere with the elongation of nascent polypeptidechains. Aminoglycosides inhibit initiation of protein syn-thesis and bind to the 30S ribosomal subunit. Chlor-amphenicol binds to the 50S ribosomal subunit blockingpeptidyltransferase reaction. Tetracyclines inhibit pro-tein synthesis by binding to 30S subunit of ribosome,thereby weakening the ribosome-tRNA interaction. Thesemisynthetic tetracycline derivatives, colloquially termedthe glycylglycines, act at the bacterial ribosome to arresttranslation. The glycylglycines bind the ribosome moretightly than previous tetracyclines, so that the TetM re-sistance factor is unable to displace them from this site,hence TetM is unable to protect the ribosomes from theaction of these new drugs. The TetA-mediated effluxsystem is ineffective against the glycylglycines, as theyare not substrates for the transporter. The oxazolidino-nes, one of the newest classes of antibiotics, interact withthe A site of the bacterial ribosome where they shouldinterfere with the placement of the aminoacyl-tRNA ( 29,31 ). Interference with nucleic acid synthesis Rifampicin interferes with a DNA-directed RNA po-lymerase. Quinolones disrupt DNA synthesis by inter-ference with type II topoisomerases DNA gyrase and to-poisomerase IV during replication and by causing doublestrand breaks ( 29 ). Inhibition of a metabolic pathway The sulfonamides ( e.g.  sulfamethoxazole) and trime-thoprim each block the key steps in folate synthesis,which is a cofactor in the biosynthesis of nucleotides, the building blocks of DNA and RNA ( 29 ). Disorganizing of the cell membrane The primary site of action is the cytoplasmic mem- brane of Gram-positive bacteria, or the inner membraneof Gram-negative bacteria. It is postulated that polymy-xins exert their inhibitory effects by increasing bacterialmembrane permeability, causing leakage of bacterial con-tent. The cyclic lipopeptide daptomycin displays rapid bactericidal activity by binding to the cytoplasmic mem- brane in a calcium-dependent manner and oligomeri-zing in the membrane, leading to an efflux of potassiumfrom the bacterial cell and cell death ( 32,33 ). Biochemistry of Antibiotic Resistance Understanding the mechanisms of resistance has be-come a significant biochemical issue over the past sev-eral years and nowadays there is a large pool of infor-mation about how bacteria can develop drug resistance( 34–36 ). Biochemical and genetic aspects of antibiotic re-sistance mechanisms in bacteria are shown in Fig. 1.Although the manner of acquisition of resistancemay vary among bacterial species, resistance is created by only a few mechanisms: ( i ) Antibiotic inactivation – 12  S. D@IDI]  et al. : Antibiotic Resistance in Bacteria,  Food Technol. Biotechnol. 46  (1) 11–21 (2008)  direct inactivation of the active antibiotic molecule ( 36 );( ii ) Target modification – alteration of the sensitivity tothe antibiotic by modification of the target ( 37 ); ( iii )Efflux pumps and outer membrane (OM) permeabilitychanges – reduction of the concentration of drug with-out modification of the compound itself ( 38 ); or ( iv ) Tar-get bypass – some bacteria become refractory to specificantibiotics by bypassing the inactivation of a given en-zyme. This mode of resistance is observed in many trime-thoprim- and sulfonamide-resistant bacteria. The exam-ple is in bypassing inhibition of dihydrofolate reductase(DHFR) and dihydropteroate synthase (DHPS) enzymes(involved in tetrahydrofolate biosynthesis). They are in-hibited by trimethoprim and sulfonamides, respectively.In several trimethoprim- and sulfonamide-resistant strains,a second enzyme that has low affinity for the inhibitorsis produced ( 34 , 39 ).There is an amazing diversity of antibiotic resistancemechanisms within each of these four categories and asingle bacterial strain may possess several types of resis-tance mechanisms. Which of these mechanisms prevailsdepends on the nature of the antibiotic, its target site,the bacterial species and whether it is mediated by a re-sistance plasmid or by a chromosomal mutation.  Antibiotic inactivation The defence mechanisms within the category of an-tibiotic inactivation include the production of enzymesthat degrade or modify the drug itself. Biochemical stra-tegies are hydrolysis, group transfer, and redox mecha-nisms.Antibiotic inactivation by hydrolysisMany antibiotics have hydrolytically susceptible che-mical bonds ( e.g.  esters and amides). Several enzymesare known to destroy antibiotic activity by targeting andcleaving these bonds. These enzymes can often be ex-creted by the bacteria, inactivating antibiotics beforethey reach their target within the bacteria. The classicalhydrolytic amidases are the  b -lactamases that cleave the b -lactam ring of the penicillin and cephalosporin antibi-otics. Many Gram-negative and Gram-positive bacteriaproduce such enzymes, and more than 200 different b -lactamases have been identified.  b -Lactamases areclassified into four groups on the basis of functionalcharacteristics, including preferred antibiotic substrate.Clinical isolates often produce  b -lactamases belonging todifferent functional groups. They can be both chromo-somal and plasmid-encoded  b -lactamases transferredfrom different bacteria ( 40–43 ).Extended-spectrum  b -lactamases (ESBLs) mediate re-sistance to all penicillins, third generation cephalospo-rins ( e.g.  ceftazidime, cefotaxime, ceftriaxone) and az-treonam, but not cephamycins (cefoxitin and cefotetan)and carbapenems. ESBLs are very diverse: more than180 different ESBLs have been identified. They are mostcommonly detected in  Escherichia coli ,  Klebsiella pneumo-niae  and  Proteus mirabilis , but have also been found inother  Enterobacteriaceae  ( 44,45 ) .  The website  http://www.lahey.org/Studies/   was established to standardize the no-menclature for the growing number of   b -lactamases andprovide references to sources for nucleotide and aminoacid sequence information ( 46 ).Other hydrolytic enzyme examples include esterasesthat have been linked to macrolide antibiotic resistanceand ring-opening epoxidases causing resistance to fos-fomycin ( 47–49 ).Antibiotic inactivation by group transferThe most diverse family of resistant enzymes is thegroup of transferases. These enzymes inactivate antibiot-ics (aminoglycosides, chloramphenicol, streptogramin,macrolides or rifampicin) by chemical substitution (ade-nylyl, phosphoryl or acetyl groups are added to the pe-riphery of the antibiotic molecule). The modified antibi-otics are affected in their binding to a target. Chemicalstrategies include  O -acetylation and  N  -acetylation ( 50– 52 ),  O -phosphorylation ( 53–55 ),  O -nucleotidylation ( 56,57 ), O -ribosylation ( 58 ),  O -glycosylation, and thiol transfer.These covalent modification strategies all require a co--substrate for their activity (ATP, acetyl-CoA, NAD + ,UDP-glucose, or glutathione) and consequently theseprocesses are restricted to the cytoplasm.Antibiotic inactivation by redox processThe oxidation or reduction of antibiotics has beeninfrequently exploited by pathogenic bacteria. However,there are a few of examples of this strategy ( 59–61 ). Oneis the oxidation of tetracycline antibiotics by the TetXenzyme.  Streptomyces virginiae , producer of the type Astreptogramin antibiotic virginiamycin M 1 , protects itself from its own antibiotic by reducing a critical ketonegroup to an alcohol at position 16. Target modification The second major resistance mechanism is the mod-ification of the antibiotic target site so that the antibioticis unable to bind properly. Because of the vital cellularfunctions of the target sites, organisms cannot evadeantimicrobial action by dispensing with them entirely. 13 S. D@IDI]  et al. : Antibiotic Resistance in Bacteria,  Food Technol. Biotechnol. 46  (1) 11–21 (2008) Biology of antibiotic resistance  Antibiotic inactivation - hydrolysis- group transfer - redox process Genetic aspectsBiochemical aspectsTarget modification - peptidoglycan structure alteration- protein structure interference- DNA synthesis interference Efflux pumps and OMpermeability changesTarget bypassMutations - spontaneous mutations- hypermutators- adaptive mutagenesis Horizontal genetransfer  - plasmids- (conjugative) transposons- integrons Fig. 1.  Biochemical and genetic aspects of antibiotic resistancemechanisms in bacteria  However, it is possible for mutational changes to occurin the target that reduce susceptibility to inhibitionwhilst retaining cellular function ( 62 ).In some cases, the modification in target structureneeded to produce resistance requires other changes inthe cell to compensate for the altered characteristics of the target. This is the case in the acquisition of the peni-cillin-binding protein 2a (PBP2a) transpeptidase in  Sta- phylococcus aureus  that results in resistance to methicillin(methicillin-resistant  S. aureus , MRSA) and to most other b -lactam antibiotics. To save the efficiency of peptido-glycan biosynthesis, PBP2a needs alterations in the com-position and structure of peptidoglycan, which involvesfunctioning of a number of additional genes ( 39,63,64 ).Peptidoglycan structure alterationThe peptidoglycan component of the bacterial cellwall provides an excellent selective target for the antibi-otics. It is essential for the growth and survival of most bacteria. Consequently, enzymes involved in synthesis andassembly of the peptidoglycan component of the bacte-rial cell wall provide excellent targets for selective inhi- bition. The presence of mutations in the penicillin-bind-ing domain of penicillin-binding proteins (PBPs) resultsin decreased affinity to  b -lactam antibiotics. Alterationsamong PBPs result in ampicillin resistance among  En-terococcus faecium , and penicillin resistance among  Strep-tococcus pneumoniae  ( 65–67 ). Resistance to methicillin andoxacillin in  S. aureus  is associated with acquisition of amobile genetic element called SCC mec , which containsthe  mecA  resistance gene. The  mecA  determinant encodesPBP2a, a new penicillin-binding protein distinct from thePBPs normally found in  S. aureus.  PBP2a is highly resis-tant to inhibition by all clinically used  b -lactams and re-mains active to maintain cell wall synthesis at normallylethal  b -lactam concentrations ( 32 ).Glycopeptides such as vancomycin inhibit cell wallsynthesis of Gram-positive bacteria by binding C-termi-nal acyl- D -alanyl- D -alanine (acyl- D -Ala- D -Ala)-containingresidues in peptidoglycan precursors. Resistance isachieved by altering the target site by changing the  D --Ala- D -Ala to  D -alanyl- D -lactate ( D -Ala- D -Lac) or  D -alanyl-- D -serine ( D -Ala- D -Ser) at the C-terminus, which inhibitsthe binding of vancomycin ( 68-70 ). As a consequence,the affinity of vancomycin for the new terminus is 1000times lower than for the native peptidoglycan precursorin the case of   D -Ala- D -Lac. Dissemination of glycopep-tide resistance in Gram-positive cocci can occur at thelevel of the bacteria (clonal spread), replicons (plasmidepidemics) or of the genes (transposons). Glycopeptide(vancomycin) resistance can be intrinsic (VanC-type re-sistance) or acquired, present only in certain isolates be-longing to the same species (VanA, B, D, C, E and Gtypes of vancomycin resistance) ( 71 ).Protein synthesis interferenceA wide range of antibiotics interfere with proteinsynthesis on different levels of protein metabolism. Theresistance to antibiotics that interfere with protein syn-thesis (aminoglycosides, tetracyclines, macrolides, chlo-ramphenicol, fusidic acid, mupirocin, streptogramins,oxazolidinones) or transcription  via  RNA polymerase(the rifamycins) is achieved by modification of the spe-cific target ( 39 ).The macrolide, lincosamide and streptogramin Bgroup of antibiotics block protein synthesis in bacteria by binding to the 50S ribosomal subunit ( 72–74 ). Resis-tance to these antibiotics is referred to as MLS(B) typeresistance and occurs in a wide range of Gram-positive bacteria. It results from a post-transcriptional modifica-tion of the 23S rRNA component of the 50S ribosomalsubunit ( 75 ). Mutations in 23S rRNA close to the sites of methylation have also been associated with resistance tothe macrolide group of antibiotics in a range of organ-isms. In addition to multiple mutations in the 23S rRNA,alterations in the L4 and L22 proteins of the 50S subunithave been reported in macrolide-resistant  S. pneumoniae ( 76 ). The mechanism of action of oxazolidinones (for ex-ample, linezolid) involves multiple stages in the proteinsynthesis ( 77 ). Although they bind to the 50S subunit,the effects include inhibition of formation of the initia-tion complex and interference with translocation of pep-tidyl-tRNA from the A site to the P site. Resistance has been reported in a number of organisms including en-terococci and is linked to mutations in the 23S rRNA re-sulting in decreased affinity for binding ( 78 ).Mutations in the 16S rRNA gene confer resistance tothe aminoglycosides ( 79 ). Chromosomally acquired strep-tomycin resistance in  M. tuberculosis  is frequently due tomutations in the  rpsL  gene encoding the ribosomal pro-tein S12. Microorganisms that produce aminoglycosideshave developed mechanism of high level antibiotic resis-tance by posttranscriptional methylation of 16S rRNA inthe aminoglycoside binding site. This mechanism of re-sistance has recently been reported in human pathogensfrom nosocomial infections and animal isolates ( 80 ).DNA synthesis interferenceFluoroquinolones interact with the DNA gyrase andtopoisomerase IV enzymes and prevent DNA replicationand transcription. Resistance is conferred by mutationsin specific regions of the structural genes that sufficient-ly alter these enzymes preventing the binding of antibi-otics ( 81,82 ). The most common mutations in this regioncause resistance through decreased drug affinity for thealtered gyrase–DNA complex ( 83–85 ). Efflux pumps and outer membrane (OM) permeability The efflux pumps are the membrane proteins thatexport the antibiotics out of the cell and keep its intra-cellular concentrations at low levels. Reduced outer mem- brane (OM) permeability results in reduced antibiotic up-take. The reduced uptake and active efflux induce lowlevel resistance in many clinically important bacteria ( 86 ).Efflux pumpsEfflux pumps affect all classes of antibiotics, espe-cially the macrolides, tetracyclines, and fluoroquino-lones because these antibiotics inhibit different aspectsof protein and DNA biosynthesis and therefore must beintracellular to exert their effect. Efflux pumps vary in both their specificity and mechanism ( 87,88 ). Althoughsome are drug-specific, many efflux systems are multi-drug transporters that are capable of expelling a widespectrum of structurally unrelated drugs, thus contribut- 14  S. D@IDI]  et al. : Antibiotic Resistance in Bacteria,  Food Technol. Biotechnol. 46  (1) 11–21 (2008)  ing significantly to bacterial multidrug resistance (MDR)( 89 ). Inducible multidrug efflux pumps are responsiblefor the intrinsic antibiotic resistance of many organisms,and mutation of the regulatory elements that control theproduction of efflux pumps can lead to an increase inantibiotic resistance. For example, the MexAB-OprM eff-lux pump in  Pseudomonas aeruginosa  is normally posi-tively regulated by the presence of drugs, but mutationsin its regulator ( mexR ) lead to the overexpression of MexAB-OprM, which confers increased resistance to an-tibiotics such as  b -lactams ( 90–92 ). Both Gram-positiveand Gram-negative bacteria can possess single-drugand/or multiple drug efflux pumps ( 93,94 ).Bacterial drug efflux transporters are currently clas-sified into five families ( 95,96 ). The major facilitator su-perfamily (MFS) and the adenosine triphosphate (ATP)--binding cassette (ABC) superfamily are very large andthe other three are smaller families: the small multidrugresistance (SMR) family, the resistance-nodulation-cell di-vision (RND) superfamily and the multidrug and toxiccompound extrusion (MATE) family. Efflux transporterscan be further classified into single or multicomponentpumps ( 97–99 ). Single component pumps transport theirsubstrates across the cytoplasmic membrane. Multicom-ponent pumps, found in Gram-negative organisms, func-tion in association with a periplasmic membrane fusionprotein (MFP) component and an outer membrane pro-tein (OMP) component, and efflux substrates across theentire cell envelope.Furthermore, the regulators of efflux systems may be attractive drug targets themselves. The regulators in-volved in efflux gene expression are either local or glo- bal regulators. Many pump component-encoding ope-rons contain a physically linked regulatory gene. Someefflux pumps are known to be regulated by two-compo-nent systems. These systems mediate the adaptive respon-ses of bacterial cells to their environment. Expression of various efflux pumps is also controlled by different glo- bal regulators. So far, several global transcriptional acti-vators, including MarA, SoxS and Rob, have been shownto be involved in the regulation of expression of this sys-tem ( 97–99 ).Outer membrane (OM) permeability changesGram-negative bacteria possess an outer membraneconsisting of an inner layer containing phospholipids andan outer layer containing the lipid A moiety of lipopoly-saccharides (LPS). This composition of the outer mem- brane (OM) slows down drug penetration, and transportacross the OM is achieved by porin proteins that formwater-filled channels. Drug molecules can penetrate theOM employing one of the following modes: by diffusionthrough porins, by diffusion through the bilayer or byself-promoted uptake. The mode of entry employed by adrug molecule largely depends on its chemical composi-tion. For example, hydrophilic compounds either enterthe periplasm through porins ( e.g.  b -lactams) or self-pro-moted uptake (aminoglycosides). Antibiotics such as  b --lactams, chloramphenicol and fluoroquinolones enter theGram-negative outer membrane  via  porins. As such,changes in porin copy number, size or selectivity will al-ter the rate of diffusion of these antibiotics ( 100–104 ).The role of LPS as a barrier to antibiotics is welldocumented. Mutations in LPS that result in antibiotichypersusceptibility have been reported. Strains of   E. coli and  S. enterica  serovar Typhimurium defective in LPShave been found to be at least 4-fold more susceptible toerythromycin, roxithromycin, clarithromycin and azithro-mycin than the wild-type strains ( 105,106 ). Genetics of Antibiotic Resistance Studies of a wide variety of bacterial pathogens haveidentified numerous genetic loci associated with antibi-otic resistance. For some types of resistance there is alarge diversity of responsible genetic determinants.Resistance can be an intrinsic property of the bacte-ria themselves or it can be acquired. Acquired bacterialantibiotic resistance can result from a mutation of cellu-lar genes, the acquisition of foreign resistance genes or acombination of these two mechanisms. Thus, there aretwo main ways of acquiring antibiotic resistance:  i) through mutation in different chromosomal loci and  ii) through horizontal gene transfer ( i.e.  acquisition of resis-tance genes from other microorganisms). This raises sev-eral questions about the evolution and ecology of antibi-otic resistance genes. Phylogenetic insights into the evo-lution and diversity of several antibiotic resistance genessuggest that at least some of these genes have a longevolutionary history of diversification that began well before the  ð antibiotic era’ ( 107 ).  Mutations Spontaneous mutationsExploring the srcins of resistant mutants beganwith the antibiotic era in 1940s, when researchers per-formed classical experiments proving that mutationsconferring resistance to certain antibiotics arise prior toor in the absence of any selective pressure. These muta-tion events occur randomly as replication errors or anincorrect repair of a damaged DNA in actively dividingcells. They are called growth dependent mutations (spon-taneous mutations) and present an important mode of generating antibiotic resistance ( 108 ).Antibiotic resistance occurs by nucleotide point mu-tations which are at the same time growth permissiveand are able to produce a resistance phenotype ( 109 ).For instance, quinolone resistance phenotype in  Esche-richia coli  is a result of changes in at least seven posi-tions in the  gyrA  gene, but in only three positions in the  parC  gene ( 110,111 ). A variety of genes can be involvedin antibiotic resistance either because there are severaldifferent targets, access, or protection pathways for theantibiotic in the bacterial cell or because each pathwayrequires the expression of several genes.There is a substantial numberof biochemical mecha-nisms of antibiotic resistance that are based on mutatio-nal events, like the mutations of the sequences of genesencoding the target ofcertain antibiotics (forinstance, re-sistance to rifamicins and fluoroquinolones are caused by mutations in the genes encoding the targets of thesetwo molecules, RpoB and DNA-topoisomerases, respec-tively) ( 112,113 ). The variation in the expression of anti- biotic uptake or of the efflux systems may also be modi- 15 S. D@IDI]  et al. : Antibiotic Resistance in Bacteria,  Food Technol. Biotechnol. 46  (1) 11–21 (2008)
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