evolution of mdrs

Evolution of MDRs

Ashima Kushwaha Bhardwaj and Kittappa Vinothkumar

Introduction

With the evolution of multidrug-resistantbacteria at threatening rates, the mankind haswitnessed the glorious rise and fall of antibiotics.Antibiotics, the miracle drugs and the magicbullets that appeared to have marked the endof the infectious diseases, are fast losing theircharm and effectiveness in human medicine.The swift and untimely demise of these wondermolecules has been attributed chiefly to theresistance mounted by bacteria against them.The phenomenon of antibiotic resistance isinevitable and was something that was cautionedin the Noble Prize lecture by Sir AlexanderFleming in 1945. Dr. Joshua Lederberg veryaccurately fathomed the seriousness of theseresistant bacteria whom he considered muchmore dangerous a threat as compared to Ebolaand West Nile virus. Resistance to any moleculeor drug intended to kill a target organism isa very natural phenomenon for the survival ofthat organism; a cancer cell being subjectedto chemotherapeutic treatment, a fungal cellsubjected to anti-fungals and, similarly, anti-parasitic and antibacterial compounds are alllikely to face resistance from their target

A.K. Bhardwaj (�) • K. VinothkumarDepartment of Human Health and Disease, School ofBiological Sciences and Biotechnology, Indian Instituteof Advanced Research, Koba Institutional Area,Gandhinagar 382007, Gujarat, Indiae-mail: [email protected]

cells. Thus, all the popular drugs includingantimalarials, anti-tuberculosis, anti-parasitic,antivirals, anti-fungals and antibacterial drugsare facing the risk of becoming obsolete.Consequently, the human race faces the risk ofan apocalypse in the hands of these invinciblebugs that no drug is able to kill. This chapterdescribes various genetic and some of the non-genetic factors such as environmental, social andpolitical factors that have led to the evolutionof a phenomenon called multidrug resistance(MDR). The threat of antibiotic resistance nowspans a wide range of infectious agents includingGram-positive and Gram-negative bacteria, allthe infectious diseases and all the geographicallocations on this planet.

There has been an evolution of a myriad ofresistant bacteria such as methicillin-resistantStaphylococcus aureus (MRSA), vancomycin-resistant Enterococci (VRE), vancomycin-resistant Staphylococcus aureus (VRSA),extremely drug-resistant tuberculosis (XDR),totally drug-resistant tuberculosis (TDR), NewDelhi metallo-“-lactamases (NDM)-carryingsuperbugs, extended spectrum “-lactamases(ESBLs)-carrying bugs and carbapenem-resistantKlebsiella pneumoniae (CRKP) to name a few.Having thrived in hospital settings at operationtheatres and intensive care units or in communitysettings, these superbugs have wreaked havocand led to the number of deaths spirallinghigh. This exhaustive list also deserves themention of major threats posed by Pseudomonasaeruginosa and Acinetobacter baumannii in

V.C. Kalia (ed.), Quorum Sensing vs Quorum Quenching: A Battle with No End in Sight,DOI 10.1007/978-81-322-1982-8__2, © Springer India 2014

9

mailto:[email protected]

10 A.K. Bhardwaj and K. Vinothkumar

nosocomial infections. In addition to this,Vibrio cholerae actually stands at the top ofthe superbug list (Davies and Davies 2010). Theworld has witnessed seven pandemics due to thispathogen which has shown continuous evolutionin terms of virulence factors and antibioticresistance traits.

Causes of MDR

MDR is a complex phenomenon that arises dueto interplay of large number of factors that workin synergy to manifest this problem. Some of thecauses of MDR are described below.

Genetic Factors

There is a plethora of genes responsible for theevolution and dissemination of MDR. Thesegenes could be chromosome borne or carriedby molecular vehicles such as plasmids, viruses,integrons and transposons, some of which willbe described in sections “General mechanismsinvolved in the evolution of antibiotic resistance”and “Role of MGEs in evolution of MDR”.

Social and Political Factors

The underuse, overuse as well as misuse ofantibiotics can lead to serious consequences intreatment of infectious diseases. Inappropriateprescriptions due to callousness of the medicaldoctors like prescribing drugs with improperdosage or prescribing wrong drugs could beone of the reasons. Using antibiotics to treatviral infections could lead to the developmentof resistance in bacteria residing in the humanbody. Poor compliance to the drug courses,sub-inhibitory concentrations or prematureabrogation of antibiotic usage by humansserve as crucial factors for the developmentof resistant bugs. Profligate use of antibioticsin human medicine, aquaculture, agricultureand poultry adds to the reservoir of resistance-conferring genes from drug-resistant bacteria inenvironment, also termed as resistome. Through

the extensive and persistent use of antibiotics,the selective pressures continue to be exerted onthe bacterial communities. In this scenario, theprobability of infection of any individual witha drug-resistant bacterium is much higher ascompared to infection with a drug-susceptiblebacterium. Therefore, the problem that startedwith a single patient or a small group of peopleassumes the proportion of a public healthproblem. Lack of government policies for properdisease surveillance, antibiotic usage as well ascontainment of the infectious diseases often leadsto the spread of MDR. Pharmaceutical industriesare losing interest in the antibiotic developmentdue to the lack of government policies thatcould give incentives to the pharmaceuticalsector for the research and development of newantibiotics. Inability to detect the newer andmore subtle antibiotic resistance phenotypes withthe available laboratory diagnostic techniquesmay lead to longer survival and circulation ofMDR pathogens in human populations oftenhindering successful treatment regimens. Forexample, pneumococcal resistance to “-lactamsand staphylococcal resistance to vancomycin arethe difficult phenotypes to detect.

Environmental Factors

Natural disasters or calamities like earthquakes,floods, tsunamis and famines and politicalsituations like civil wars or unrest where themedical facilities are heavily impaired can alsolead to the high case fatality rates due to thethriving MDR bacteria. Many drugs such asantibiotics, antidepressants, chemotherapeuticsand their residues often escape purification bywater treatment plants and, therefore, contam-inate drinking water supplies. Considerableamounts of these antibiotics are released intothe biosphere by hospitals, research laboratories,pharmaceutical industries and domestic use. Itis not surprising that the microbial world insoil, water and food has to resort to myriadresistance determinants to avert the catastrophedue to these contaminants. The EnvironmentalProtection Agency (EPA) and Food and Drug

Evolution of MDRs 11

Administration (FDA) have not yet formulatedany rules and regulations on this aspect of drugcontamination in drinking water. This has seriousconsequences not only for humans but also forthe aquatic ecosystems. At many places suchas the United States and India, drugs have beendumped by pharmaceutical companies in therivers (http://www.purewaterfreedom.com/osc/pharma_water_contamination.php). In additionto the spurt in the appearance and disseminationof drug resistance genes, their toxicity to all theorganisms in water or land or air is probablyunfathomable.

Consequences of MDR

The dissemination of MDR traits in bacteria hasdifferent consequences for mankind as well as thepathogens.

For the human hosts that fall prey to the superbugs, it leads to:• Treatment failure• Prolonged stays in the hospitals escalating the

health-care budgets• Reduction in manpower that has both social as

well as economical consequencesFor the bacterial populations, this MDR trans-

lates into:• Increased virulence of the bacterium. For ex-

ample, the studies on A. baumannii have re-vealed that genomic islands in this organismalso harbour virulence determinants in addi-tion to the antibiotic resistance determinants(Barbe et al. 2004). Similarly, the community-acquired MRSA has equipped itself with awide range of genes that endow the bacteriawith pathogenicity genes as well as antibioticresistance genes (DeLeo and Chambers 2009).

• More efficient transmission of bacterium.• Dissemination of the resistance genes to all

other pathogens in their vicinity leading toamplification of the resistance genes in thenature.

• Transfer of resistance genes to the commensalorganisms residing in the host affecting the mi-croflora often leading to some other outcomesin the host health.

General Mechanisms Involved in theEvolution of Antibiotic Resistance

The evolutionary history of resistant bacteria pre-dates the introduction of the antibiotic era. It isunderstandable that the antibiotic producers wereactually the reservoirs of drug resistance genes.These antibiotic resistance genes were part ofthe paraphernalia involved in the production ofantibiotics by the bacteria where they providedprotection to the producers.

As antibiotics target the vital processes of abacterial cell, they create a do-or-die situationfor a bacterium. Hence, it is indispensable forthe bugs to resist the action of antibiotics at anycost by devising various tactics. The molecularmechanisms of resistance exerted by bacteria toovercome drugs have been well studied, and theyemploy any one or a combination of the followingstrategies (Alekshun and Levy 2007).

Chromosomal Mutationsat the Target Sites of Antibiotics

Mutations at the antibiotic target sites arethe main mode of resistance to most of theantibiotics. Mutations occurring as a resultof replication errors reduce the affinity ofthe antibiotics to their targets resulting in theresistant phenotype. For example, quinolone andfluoroquinolone resistance occurs through themutations at the DNA gyrase and topoisomeraseIV genes. Similarly, mutations in the geneencoding dihydropteroate synthase decreasethe enzyme affinity to the sulphonamides. InMycobacterium tuberculosis, resistance to thecommon drugs such as rifampin, streptomycin,ethambutol and fluoroquinolones used to treatthe pathogen arises due to mutations in thegenes that are involved in metabolic pathwaysor in housekeeping. Additional mutations inthe already mutated genes result in increasingthe minimum inhibitory concentration (MIC) ofthe antibiotic for the pathogen or extending thespectrum of resistance such as the developmentof extended spectrum “-lactamases (ESBLs) inthe pneumococcus (Medeiros 1997).

http://www.purewaterfreedom.com/osc/pharma_water_contamination.php

http://www.purewaterfreedom.com/osc/pharma_water_contamination.php


Increased Efflux and Reduced Influxof Antibiotics in the Bacterial Cell

Efflux pumps play a major role in conferringresistance to antibiotics by efficiently recognisingand throwing them out of the cells. Efflux pumpsin bacteria can be classified into five differentfamilies, namely, the resistance nodulation celldivision (RND), major facilitator super family(MFS), small multidrug resistance (SMR), ATP-binding cassette (ABC) and multidrug and toxiccompound extrusion (MATE) families (Bhardwajand Mohanty 2012). Among these five pumps,ABC pumps utilise ATP as their energy source,whereas others are driven by the proton-motiveforce (PMF). Generally efflux pumps are knownto extrude out a wide range of substancesincluding antibiotics, and therefore, this is a non-specific mechanism of resistance. However, fewpumps are shown to have high specificity towardsparticular drugs. For example, TetA and NorMare found to be more specific towards tetracyclineand norfloxacin, respectively, whereas AcrB,VcmA and MdfA have multiple substratespecificities. Efflux pumps confer only lowlevel resistance to the bacteria towards drugs buttheir over-expression or cooperativity with othermechanisms could result in moderate to high-level resistance (Bhardwaj and Mohanty 2012).

Porins present in the cell membrane of bac-teria are the passages which facilitate the entryand exit of antibiotics and other small organicmolecules. Decrease in the expression of porinsresults in reduced uptake of antibiotics. For ex-ample, mutations that caused reduced expressionof OprD porins contributed to imipenem resis-tance (Alekshun and Levy 2007).

Enzymatic Drug Modificationor Degradation

This mechanism of resistance involves enzymesthat either degrade or chemically modify theantibiotics so that they cannot exert their action.“-lactamases are the well-known examples forthe enzymes that degrade “-lactam antibiotics.

Few of them behave as extended spectrum“-lactamases (ESBLs) and as carbapenemasesand show wider spectrum of resistance tonewer generation “-lactam antibiotics (Alekshunand Levy 2007). There are a large numberof aminoglycoside-modifying enzymes whichchemically modify (acetylate or adenylate orphosphorylate) the aminoglycosides. Similarly,chloramphenicol is inhibited by chloramphenicolacetyltransferases and tetracycline by a flavin-dependent monooxygenase TetX (Alekshun andLevy 2007).

Protection and Alteration of DrugTarget

Resistance to fluoroquinolones is mediated by alarge number of pentapeptide repeat proteins,quinolone resistance (Qnr) proteins, whichprotect target DNA gyrase and topoisomeraseIV from the antibiotic action. As these proteinsmimic the structure of DNA, they occupy theDNA-binding portion of the topoisomerasesand prevent the antibiotics from exertingtheir effect on these protected topoisomerasetargets. The altered penicillin-binding protein(PBP) of methicillin-resistant S. aureus, PBP2a,confers resistance to most of the “-lactams bycontributing the transpeptidase activity whenexposed to methicillin (Fig. 1a).

Other Mechanisms

Sometimes resistance to different antibiotics canbe conferred by a single determinant. For ex-ample, aminoglycoside acetyl transferase (aac(60)-Ib) generally acetylates aminoglycosides likeamikacin, kanamycin and tobramycin. But itsmutant form aac (60)-Ib-cr is known to acety-late quinolones like ciprofloxacin also (Robicseket al. 2006). Therefore, a single protein ren-ders resistance to aminoglycosides as well asquinolone class of antibiotics (Fig. 1b).

The tandem duplication of the resistance-conferring gene results in overexpression which


Staphylococcuschromosomalcassette(scc)

a b

Production of PBP2a toactas transpepetidasethat is not inhibited bymethicillin

X

X

MRSA

Methicillin-sensitivestaphylococcus aureus(MSSA)

Aminoglycoside 6’-N-acetyltransferase(Aac (6’) Ib)

Ciprofloxacin(active) Aac (6’) Ib-cr

CH3

O N

N N

F

O O

OH

OH

HN

N

F

O O

N

N-acetyl Ciprofloxacin(inactive)

Mutations and Asp179→Tyr

Trp102→Arg

SCCmecA

S. Sciuri

mecA

SCCmecA

Fig. 1 Evolution of methicillin-resistant Staphylococcusaureus (MRSA) and Aac (60) Ib-cr, an enzyme showingpromiscuous drug resistance. (a). The evolution of MRSAby the successful acquisition and expression of mecA fromStaphylococcus sciuri. The evolved S. aureus expresses

mecA-derived PBP2a that acts as an alternate transpepti-dase that is not inhibited by methicillin; (b). By acquiringmutations at the active sites, the modifying enzyme Aac(60)-Ib evolves as Aac (60)-Ib-cr with the additional abilityto modify ciprofloxacin

eventually helps the bacteria to exhibit a high-level resistance to the antibiotics. In one instance,the overexpression of tandem duplicated genesof AcrAB drug efflux pumps in E. coli led to anMDR phenotype (Alekshun and Levy 2007).

Processes That Drive Evolutionof MDR

There are chiefly two processes through whichthe mechanisms of resistance described in section“General mechanisms involved in the evolutionof antibiotic resistance” lead to the evolution andpersistence of MDR. These processes describedbelow are either the horizontal gene transfers orthe pressures due to environment. SOS responsesmounted in a bacterium due to antibiotic expo-sure or HGT are also related to these processesand therefore deserve a special mention in thissection (Fig. 2).

Horizontal Gene Transfer (HGT)

The process of HGT enables bacteria to exchangegenetic material within themselves without

the requirement of cell division. Differentkinds of mobile genetic elements (MGEs)are transferred between bacteria through thisprocess leading to the adaptation and evolutionof bacteria/bacterial communities in tune withthe changing environments. HGT is mediated bythe processes of transformation, transduction orconjugation, and different types of MGEs couldmove through these processes of HGT (Fig. 2).These MGEs as agents of evolution will bedescribed in section “Role of MGEs in evolutionof MDR”.

Selective Pressure dueto Environment

Environment plays a vital role in the selectionand spread of antibiotic resistance among bac-terial communities that would be discussed atmany places in this chapter with examples. Theseselective pressures lead to induction of mutationsin the drug target genes conferring the mutantbacteria, a resistant phenotype (Fig. 2). The trans-mission dynamics of MDR is hugely responsiveto the environmental factors at the hospitals or thecommunities.


Horizontal gene transfer

Antibiotic exposureSelection pressure

Bacteriophage

Conjugation Transduction Transformation

Horizontal gene transfer

Surviving cells

Survivingmutant cells

Dead non-mutant cells

Mutation induction

Transformed plasmid

Transformed plasmid

TransducedDNAConjugated

plasmid

Susceptible bacterium

Non-mutantcells

Mutant cells

plasmids

Mutations

SOS response

Fig. 2 The process of evolution of MDR bugs. The sus-ceptible bacteria attain some mutations at their antibiotictarget sites prior to the exposure of antibiotics. Thesemutants are selected under antibiotic pressure when theyemerge, persist and disseminate as resistant bugs. Theacquisition of resistance genes by the bacteria through

horizontal gene transfer (HGT) also helps bacteria to resistantibiotics under selection pressure. Antibiotic exposureelicits SOS responses which facilitate both mutationsand HGT processes. Therefore, evolution of MDR is aninterplay of three processes: HGT, selection pressure andSOS responses

SOS Responses in Bacteriumon HGT/Antibiotic Exposure

Any type of HGT through conjugation, transfor-mation and transduction or any type of antibioticchallenge induces SOS response (Fig. 2) throughevents mediated by single-stranded DNA, RecAprotein and LexA repressor (Baharoglu et al.2013). On antibiotic exposure/HGT, RecA getsactivated which leads to autoproteolysis of LexArepressor that keeps the SOS regulon in therepressed state under normal conditions. LexAinactivation thus leads to the expression of adiverse array of genes that were repressed by it.

Integrases associated with integrons and integrat-ing conjugative elements (ICEs) are examplesof the genes that are induced during SOS dueto LexA inactivation (Baharoglu et al. 2013).This leads to the escape of integrons and ICEsfrom the bacterial cell under crisis. Similarly, theregulation of expression of qnrB2 (a quinoloneresistance determinant) through SOS responseis induced by ciprofloxacin in LexA-/RecA-dependent manner. Even sub-inhibitory concen-tration of ciprofloxacin was found to cleave LexArepressor so that it was prevented from bindingon the LexA binding site present in the promoterregion of qnrB2 gene (Da Re et al. 2009).


Therefore, under ciprofloxacin pressure, thebacterial cell expressed resistance gene for thisantibiotic through SOS-mediated pathway.

Role of MGEs in Evolution of MDR

MDR evolves through a large spectrum of ge-netic elements that could either reside on thechromosomes of a bacterium or reside on thepieces of DNA that are mobile. The latter typesare called mobile genetic elements (MGEs) andinclude a diverse class of genetic elements suchas integrons, bacteriophages, integrating conjuga-tive elements (ICE) and conjugative plasmids(Fig. 3). These MGEs play an important role inreshaping and in the evolution of the bacterialgenomes enabling bacteria to thrive in a varietyof ecological niches. In the subsequent sections,the MGEs that have resulted in fast acquisi-tion and dissemination of MDR genes have beendescribed.

Integrons

These MGEs are capable of capturing the genecassettes by site-specific recombination, integrat-ing them and expressing them using a commonpromoter (Stokes and Hall 1989; Recchia andHall 1995). Integrons therefore convert the ac-quired open reading frames (ORFs) into theirfunctional form. Integrons consist of an integrasegene (intI), a recombination site (attI) and apromoter Pc. There are numerous classes of in-tegrons known that are classified based on thesequences of their integrase genes. Class 1 in-tegrons have been studied most extensively, andthese integrons have been characterised vis-à-vistheir role in dispersal of the MDR genes in clin-ical isolates of Gram-negative bacteria. Integronshave been an important part of bacterial evolu-tion, are widespread among all the bacteria andhave a wider role to play in bacterial physiologyand adaptation than simply antibiotic resistance(Rapa and Labbate 2013). The structure of a class

5’CS

a

b c

Donor chromosome transposon

Integron

R-determinants

Transfer factorsdeterminants

Recipient chromosome

ICE(circular intermediate)

prfC

attL

attBprfC

attP

attRICE

Yr

Zr

Xr

Ori

Intl 1 Gene X Gene Y Gene Z sul1qacEΔ1

attl 1 attC attC attC

Variable region 3’CS

Fig. 3 MGEs that facilitate evolution of MDR. (a). Struc-ture of a class 1 integron having conserved segmentsat its 50 and 30 ends (50CS and 30CS, respectively) andvariable region consisting of extraneous gene cassettesthat encode various functions including antibiotic resis-tance. intI 1 encodes an integrase and attI 1, and attCsites are formed with the insertion of the extraneous gene

cassettes. (b). Insertion and excision of integrating con-jugative elements (ICEs) in the prfC region of a bacterialgenome. The recombination process is mediated by acircular intermediate. (c). A conjugative R plasmid thatmay carry resistance genes, integrons and transposonsalong with transfer factor determinants


1 integron consists of the conserved segments atthe 50 and 30 ends of the integron (50 CS and30CS). These conserved segments encompass avariable region which varies with the numberand nature of the gene cassettes captured by theintegrons (Fig. 3a). The 50 CS consists of intIgene, the attI site and the promoter, whereas the30CS consists of two genes that encode resistancefor ethidium bromide and sulphonamides. Theextraneous gene cassettes captured by integronsare usually promoterless, and they recombinewith the attI site through a recombination sitecalled attC or 59-base element (Fig. 3a). Eachgene cassette captured in an integron is thusbound by the attI site on its 50 end and an attCsite on its 30 end. The attC sites share a com-mon set of characteristics that enable them to beidentified despite the diversity of their sequencesand sizes (Hall et al. 1991). They are charac-terised by a palindrome of variable length andsequence between the RYYYAAC (R D Purines;Y D Pyrimidine) inverse core site and the GTTR-RRY core site. The size of these recombinationsites vary in length from 57 to 141 bp.

Integrons have been reported from a wide vari-ety of bacteria such as V. cholerae, V. fluvialis, V.parahaemolyticus, P. aeruginosa and K. pneumo-niae. They have been shown to harbour a diversearray of genes including antibiotic resistancegenes. Resistance genes for chloramphenicol(catB, cmlA), trimethoprim (dfrA, dfrB), “-lactamantibiotics (bla, oxa), aminoglycosides (aac andaad) and many ORFs of unknown functions havebeen observed in integrons. Two types of inte-grons are known to exist: chromosomal integrons(CIs) or superintegrons (SIs) that are sedentaryin nature and the mobile integrons (MIs) that areassociated with mobile DNA elements and in-volved in the spread of antibiotic resistance genes(Rowe-Magnus et al. 2002). CIs are located onthe chromosomes of a large number of bacteria.MIs usually contain less than 20, while SIs/CIscontain more than twenty-gene cassettes. Thenature of gene cassettes harboured by MIs and SIsalso varies. While MIs usually contain antibioticresistance gene cassettes, majority of cassettesassociated with SIs are of unknown functions.

Integrating ConjugativeElements (ICEs)

ICEs are a type of conjugative transposons thatintegrate and replicate with the chromosomalDNA of the host bacterium (Burrus and Waldor2004). ICEs are not capable of autonomous repli-cation, and therefore, they have to depend on thehost cell machinery for its survival. They excisethemselves from the host chromosome, form acircular intermediate and then get transferred tothe recipient cell during conjugation (Fig. 3b).OriT, a cis-acting site, is required on ICEsfor their translocation to the recipient throughthe mating bridge formed during conjugation.ICE known as SXT element was first reportedfrom Madras, India, in 1992, in V. choleraeO139 strains where they imparted resistanceto drugs like trimethoprim, sulphamethoxazole,streptomycin and chloramphenicol (Waldoret al. 1996). Since then, these elements havebeen reported from a large number of bacteriasuch as V. cholerae, Providencia alcalifaciensand P. rettgeri at many places as importantvehicles for spreading of antibiotic resistance.The integration in the host genome is mediatedby an integrase, and ICEs also encode otherfunctions required for their maintenance. Thesefunctions include conjugative transfer of theseelements, their excision and integration andregulation of the events related to ICE transferand maintenance. ICEs harbour a wide array ofgenes for diverse functions such as antibioticresistance, heavy metal resistance and complexdegradation pathways for toxic compounds.Two different ICE elements can also recombineto produce a tandem array of ICE elementscalled hybrid ICEs. One such hybrid is anSXT/R391 family of ICEs which is the largestfamily of ICEs detected in clinical as well asenvironmental strains of many bacteria. Throughthe process of recombination mediated mainlyby RecA protein, these hybrids are known topromote their own diversity resulting in theformation of novel mosaics with new combi-nations of antibiotic resistance genes (Garrisset al. 2009).


Plasmids

Plasmids are autonomously replicating extrachro-mosomal DNA molecules that are transferredfrom donor to the recipient bacterium throughconjugation. Resistance plasmids also knownas R plasmids, harbouring genes conferringantibiotic resistance, have been well known fortheir role in the transfer of resistance traits froma drug-resistant bacterium to a drug-sensitivebacterium (Fig. 3c). Plasmids appear to have amajor contribution in the spread of drug resis-tance, and several pathogens have been reportedthat harbour plasmids with multiple resistancetraits. Bacteria carry either conjugative plasmidsthat are large in size or non-conjugative small-sized plasmids. Non-conjugative plasmids can bemobilised with the help of other conjugative plas-mids present in the same cell or by the processof transformation. In some cases, these plasmidsmay carry integrons or transposons on them facil-itating the dissemination of antibiotic resistancegene cassettes in different species of bacteria(Fig. 3c). In enteric pathogens V. cholerae andShigella dysenteriae, the multidrug resistanceplasmids have been responsible for MDR thuscomplicating the treatment of diarrhoeal diseases(Ries et al. 1994; Sack et al. 2001). In anotherpathogen V. fluvialis, plasmids have been shownto confer resistance to a large number of drugs(Rajpara et al. 2009; Singh et al. 2012).

Role of Environmental Factorsin Evolution of MDR

The presence of an antibiotic in the environmentaccentuates the appearance of bacteria resistantto this antibiotic. Often, there is a direct correla-tion between the antibiotic consumption and theappearance of strains resistant to that antibiotic.Antibiotics promote evolution of MDR by therandom genetic drift or by induction of largemutational events selecting for the survival ofresistant bacteria (Baquero et al. 1998). Ran-dom genetic drift occurs during the crisis sit-uations where the random variations acquiredby the bacteria may improve the chances of

bacterial survival. Apart from antibiotic usage,other environmental factors such as epidemiolog-ical features, other drugs being used at the timeof study, host immunity and pollutants presentin an environment also induce selective pres-sures for the development of MDR (Baqueroet al. 1998). The resistant strain may have higherpossibility of surviving in an immunocompro-mised host as compared to an immunocompe-tent host. Similarly, the presence of some othernon-antibiotic drugs could alter the expressionof porins or efflux pumps of bacteria eventuallyaffecting the antibiotic concentrations inside thebacterial cell. This will lead to a change/evolutionin the resistance phenotype of this bacterium.For example, drugs such as salicylate lead tothe increase in efflux pump expression. Cumu-lative effect of all these environmental factorsallows the survival of bacteria which have ac-quired the mutations to face the antibiotic pres-sure. These factors also promote the prolifera-tion and dissemination of such novel bacterialmutants. Profligate use of antibiotics in all thespheres of life including human health, veterinarymedicine, food industry and aquaculture actuallyseems to have provided selective pressures forthe evolution of MDR in frightening proportionsas we witness it today. Each new generation ofantibiotics has spawned new generations of bac-terial proteins/mechanism to thwart the effect ofantibiotics. As described in Sect. “Extended spec-trum “-lactamase (ESBL)-producing bacteria”,when new “-lactams such as cefotaxime wereproduced to face the challenges imposed by early“-lactam-resistant bacteria, the “-lactamases ac-quired some additional mutations to inactivatethese newer drugs. Higher mutated variants likeTEM-10 of the “-lactamase TEM were evolved toprovide higher resistance. Especially interestingis the scenario in intensive care units where multi-ple antibiotics are used at varying concentrationsfor different patients and different pathogens.This leads to selection of a large number ofMDR bacteria due to the vast availability ofresistome (the population of resistance genes innature) with the potential to get incorporated intothe genome of any bacterial cell and to expressthe trait.


Case Studies on the Evolutionof MDR Bugs

As described in the earlier sections, the evolutionof MDR bugs is mediated by the processes suchas HGT, selective pressure and SOS response,and these processes are induced by several ge-netic and environmental factors. The antibioticera has witnessed the evolution of several resis-tant bugs, and the following examples can explainthe evolution of MDR bugs as a result of interplayof the above-mentioned factors.

Methicillin-Resistant Staphylococcusaureus (MRSA)

The rise of MRSA could explain the extraordi-nary ability of bacteria to evolve as a menacefor public health. S. aureus is an omnipresentbacterium mostly found in the human nostrilsand skin. They often cause respiratory diseases(e.g. nosocomial pneumonia) and skin diseases(e.g. impetigo) in humans. During early 1940s,the infections caused by this bacterium weretreated using penicillin as they were extremelysensitive to these wonder drugs at that time.But soon the upsurge of penicillinase-mediatedpenicillin-resistant strains of S. aureus led tothe arrival of an alternative drug, methicillin,a semisynthetic penicillin. Methicillin with apower to resist the penicillinase action came totherapeutic use in 1959. But within a short timespan, the first case of MRSA was reported. Thespectacular mechanism of resistance exhibited byMRSA to fight methicillin was a new penicillin-binding protein, PBP2a. PBPs, the targets ofpenicillin, methicillin and other “-lactams, aretranspeptidases which are responsible for thecross-linking of the cell wall of bacteria. Butthe new variant of PBP, PBP2a, has low affinityfor methicillin and other “-lactams and couldsubstitute the role of native PBPs for cell wallformation (Fig. 1a). PBP2a was encoded bymecA gene which is a distinctive feature ofMRSA and hence the methicillin resistance. ThemecA gene was found in the chromosome of

MRSA but associated with a large mobile geneticelement called staphylococcal chromosomecassette [SCC] (Pantosti and Venditti 2009).The mecA gene seemed to have originated fromStaphylococcus sciuri and then got incorporatedinto SCC to become SCCmec (Fig. 1a). Thesuccessful acquisition and expression of SCCmecin S. aureus gave rise to the strain of MRSA (deLencastre et al. 2007). The first MRSA cloneappeared in the 1960s, spread widely in thehospitals and clinical settings for about 17 yearsand new clonal types with different SCCmecelements were reported subsequently. Theepidemic hospital-acquired MRSA (HA-MRSA)clones reported so far mainly fall into threetypes (type I, II and III) based on the multilocussequence typing (MLTS) method. Subsequentto the acquisition of SCCmec, MRSA furtherevolved to resist other classes of antibiotics suchas aminoglycosides, tetracycline, sulphonamidesand quinolones as a result of selective pressure onexposure to antibiotics and acquisition of variousresistance genes through HGT. In the 1990s theenigmatic emergence of community-acquiredMRSA (CA-MRSA) has been reported withdifferent epidemiological and molecular profilethan that of HA-MRSA. Initially CA-MRSAclones carried the single trait of mecA mainly intwo SCCmec element types (type IV and V) andwere susceptible to non-“-lactam antibiotics. Butfew typical CA-MRSA have been reported nowto evolve as multidrug-resistant strains (e.g. USA300 and ST80 clone) (Pantosti and Venditti 2009;de Lencastre et al. 2007).

Vancomycin-ResistantStaphylococcus aureus (VRSA)and Vancomycin-ResistantEnterococci (VRE)

Vancomycin served as a possible alternate ther-apy for the infections caused by the MRSA.Vancomycin inhibits cell wall synthesis by block-ing the transglycosylation and transpeptidationreactions as it binds to the C-terminal peptide ofD-Ala-D-Ala of pentapeptide precursor for theformation of bacterial peptidoglycan. The van


gene mediating vancomycin resistance was firstobserved in enterococci only. These genes areof seven types (vanA, vanB, vanC, vanD, vanE,vanG, vanL) which are known to synthesise anew target (peptidoglycan precursor) which re-places the normal D-Ala-D-Ala precursor, andhence, the antibiotic cannot find its target. ThevanA-, vanB- and vanD-type genes produce theD-Ala-D-Lac target, whereas vanC, vanE, vanGand vanL gene types synthesise the D-Ala-D-Ser target. The acquisition of plasmid-borne vanAgene through conjugation from Enterococcus toS. aureus resulted in the development of VRSA.The evolution of S. aureus which were alreadyresistant to multiple drugs into VRSA furthercomplicated the treatment of infections caused bysuch bacteria (Perichon and Courvalin 2009).

Extended Spectrum “-Lactamase(ESBL)-Producing Bacteria

The emergence of “-lactamases served as a com-mon mechanism of resistance for “-lactam an-tibiotics in Gram-negative bacteria. In the 1970sto 1980s, “-lactamases such as TEM-1, TEM-2 and SHV-1 that hydrolysed penicillin, ampi-cillin and early generation cephalosporins weredetected. TEM-1 and TEM-2 were predominantin E. coli and SHV-1 was prevalent in K. pneumo-niae (Chong et al. 2011). During the early 1980s,the emergence of modified “-lactamases carryingamino acid mutations in TEM-1, TEM-2 andSHV-1 enzymes was detected. As they were ableto hydrolyse the third-generation cephalosporinssuch as cefotaxime, ceftriaxone, ceftazidime, ce-furoxime and cefepime, apart from penicillin andampicillin, they were termed as ESBLs. TheTEM and SHV ESBLs were genetically evolvedby amino acid substitutions from their non-ESBLprogenitors TEM-1, TEM-2 and SHV-1, whereasanother ESBL called CTX-M evolved indepen-dently of this lineage. Some other ESBLs dif-ferent from TEM, SHV and CTX-M are OXA,BEL-1, BES-1, GES/IBC, SFO-1, TLA-1, TLA-2, PER and VEB enzyme families. ESBLs soonbecame pervasive and were reported all acrossthe globe within two decades. So far more than

300 ESBLs have been described (Lynch et al.2013). The increased incidents of disseminationof ESBL genes among bacteria through vari-ous MGEs which carry other antibiotic resis-tance genes have reduced the therapeutic op-tions and caused an emerging threat to publichealth.

Quinolone-Resistant Bacteria

The increased drug resistance among bacteriatowards various natural and semisynthetic antibi-otics led to the introduction of synthetic drugslike quinolones and fluoroquinolones due to theirbroad spectrum of activity and possibilities of theabsence of resistance mechanisms in bacteria tothese synthetic drugs. Quinolones inhibit nucleicacid synthesis in bacteria by targeting DNAgyrase and topoisomerase IV enzymes which areinvolved in the essential activities of bacterial cellsuch as replication, transcription, recombinationand repair. The mutations at the quinoloneresistance determining regions (QRDRs) ofsubunits of DNA gyrase (GyrA, GyrB) and topoi-somerase IV (ParC, ParE) enzymes cause theirreduced affinity towards quinolones which lead toquinolone resistance phenotypes. Mutations thatoccur at the target sites of the antibiotics priorto the exposure of antibiotics help in selectionand subsequent evolution of resistant bacteria.The accumulation of multiple mutations in thedrug target facilitates the development of high-level resistance to quinolones in bacteria. Thequinolone resistance can also be mediated byefflux pumps encoded by chromosome-bornegenes such as norM, norA, vcmA and bmrA(Bhardwaj and Mohanty 2012). When quinoloneswere introduced, it was imprudently predictedthat there would not be any quinolone resistancegenes as these antibiotics were not naturallyproduced by any bacteria (Hernandez et al.2011). Hence, the dissemination of resistanceamong bacterial communities through HGT wasnot expected. But the emergence of factors liketarget-protecting quinolone resistance proteins(Qnr), drug-modifying enzyme and plasmid-borne efflux pump genes falsified the latter belief.


Qnr proteins are pentapeptide repeat proteinswhich are believed to be evolved from theproteins like McbG that protect topoisomerasesfrom the naturally occurring toxins like microcinB17, a topoisomerase poison. These pentapeptiderepeat proteins occupy the DNA-binding regionof the enzyme and protect them from thedrug action. Though the Qnr proteins areof chromosomal origin, they are more oftenfound in plasmids through which they tend todisseminate among different bacterial species.Similarly, as shown in Fig. 1b, a variantaminoglycoside acetyl transferase (AAC(60)-Ib-cr) borne on plasmid was found to inactivate(by acetylation) ciprofloxacin and norfloxacinapart from aminoglycosides due to two aminoacid changes (Trp102Arg and Asp179Tyr) in theactive site of the enzyme (Robicsek et al. 2006).Two plasmid-mediated quinolone transporters(OqxAB and QepA) have been described toeffectively efflux out quinolone antibiotics. Allthe above-mentioned resistance mechanisms maywork alone or in synergy to combat the quinolonedrugs. Apart from the mutation in the target sites,other genetic factors of quinolone resistance suchas qnr genes, aac(60)Ib-cr gene and oqxAB andqepA genes are often harboured by plasmids andcause plasmid-mediated quinolone resistance(PMQR). Though higher-level resistance throughPMQR has not been reported, they could help theisolates to attain clinical breakpoint of resistancein combination with other mechanisms. Hence,the great plasticity of the bacterial systems allowsthem to educe their armaments to battle againstthese drugs.

Conclusions

Evolution of MDR is a very natural process,and from the discussions above, it can beconcluded that the resistance determinants forantibiotics were always present in nature evenbefore the miracle drugs were introduced forhuman use. These determinants just made theirpublic appearances with the escalating useof antibiotics in many applications. With theselective pressures rising due to antibiotic use,the resistance genes kept appearing in their

new avatars. Evolution of antibiotic resistancemechanisms paralleled evolution of antibiotics.Therefore, new strategies need to be used forkeeping one-step ahead of the MDR pathogens.The advent of technologies based on microbialgenomics, proteomics, combinatorial chemistryand high-throughput screening could lead tosuccess stories in the development of new anti-infectives inspite of the large funds required forthem. There are many innovative strategies beingused to curb the problem of MDR (Breithaupt1999; Tegos and Hamblin 2013). In pathogensNeisseria gonorrhoeae and N. meningitides,lipooligosaccharides on the bacterial surfacehave been shown to be crucial for their virulence-associated functions such as colonisation andimmune evasion. The glycosyltransferases andhydrolases involved in the synthesis of theselipooligosaccharides have been used as targetsfor synthesis of small inhibitors as antibiotics.Companies such as TerraGen Diversity Inc.(Canada), ChromaXome Corp.(California) andGLYCODesign (Toronto) have been activelyinvolved in the innovations pertaining toantibiotic research and development. Strategiesof quorum-sensing inhibition and efflux pumpinhibition also provide attractive alternatives tosolve the problems of MDR (Bhardwaj et al.2013; Kalia 2013; Bhardwaj and Mohanty 2012)though the possibility of evolution of resistanceto these new molecules cannot be ruled out(Bhardwaj et al. 2013; Kalia et al. 2014). Thegovernments should realise the seriousness ofimpending disaster due to MDR bacteria andurgency of the situation (Finch and Hunter 2006).Accordingly, new policies should be made todeal with this problem. In India, a national policyhas been made for containment of antimicrobialresistance by the Directorate General ofHealth Services (2011), and a task force wasconstituted to work on various aspects related toantimicrobial resistance and its monitoring.

Opinion

From the concepts and facts presented in thischapter and reiterated throughout this book,it would be amply clear that the problem of


multidrug resistance is real and threatening andin all possibilities here to stay. Prudent choicesneed to be made to keep this problem to its lowestor least dangerous level where solutions for thisproblem are easier to make. As Dr. Stuart Levypointed out in one of his writings, the mankindshould understand how this equation of drugresistance should be balanced. Improving labora-tory techniques for diagnosis of drug resistanceprofiles of prevailing pathogens or environmentalorganisms, proper surveillance and molecularepidemiology studies, striking a balance betweenthe antibiotic use and abuse and utilisation ofnovel anti-virulent and anti-infective strategiesare some of the approaches that should be utilisedin synergy to keep the magic of antibioticsalive. Obviously, this herculean task can onlybe realised with the concerted efforts by peoplefrom different spheres of life including clinicians,policymakers, academicians, researchers, clinicalmicrobiologists, citizens and pharmaceuticalcompanies.

Acknowledgements The laboratory is supported by thegrants from the Department of Biotechnology (DBT),Ministry of Science and Technology, Government ofIndia (No. BT/PR/11634/INF/22/104/2008), GujaratState Biotechnology Mission (GSBTM), Departmentof Science and Technology, Government of Gujarat(No. GSBTM/MD/PROJECTS/SSA/1535/2013–14) andIndian Council of Medical Research, New Delhi, India(No. AMR/49/11-ECDI). K. Vinothkumar is a JRF in theabove-mentioned GSBTM grant. The authors are gratefulto Dr. Amit Ghosh, Dr. T. Ramamurthy and Dr. S. K.Niyogi, National Institute of Cholera and Enteric Diseases(NICED), Kolkata, India, for their support and advice. Theauthors thankfully acknowledge the Puri Foundation forEducation in India for providing infrastructure facilitiesand Ms. Neha Rajpara, Mr. Priyabrata Mohanty, Mr. BrajM. R. N. S. Kutar and Ms. Aneri Shah for their invaluablehelp, advice and support.

References

Alekshun MN, Levy SB (2007) Molecular mechanisms ofantibacterial multidrug resistance. Cell 128:1037–1050

Baharoglu Z, Garriss G, Mazel D (2013) Multiple path-ways of genome plasticity leading to development ofantibiotic resistance. Antibiotics 2:288–315

Baquero F, Negri MC, Morosini MI, Blazquez J (1998)Antibiotic-selective environments. Clin Infect Dis27(Suppl 1):S5–S11

Barbe V, Vallenet D, Fonknechten N, Kreimeyer A,Oztas S, Labarre L et al (2004) Unique featuresrevealed by the genome sequence of Acinetobac-ter sp. ADP1, a versatile and naturally transforma-tion competent bacterium. Nucleic Acids Res 32:5766–5779

Bhardwaj AK, Mohanty P (2012) Bacterial efflux pumpsinvolved in multidrug resistance and their inhibitors:rejuvinating the antimicrobial chemotherapy. RecentPat Antiinfect Drug Discov 7:73–89

Bhardwaj AK, Vinothkumar K, Rajpara N (2013) Bacte-rial quorum sensing inhibitors: attractive alternativesfor control of infectious pathogens showing multipledrug resistance. Recent Pat Antiinfect Drug Discov8:68–83

Breithaupt H (1999) The new antibiotics. Nat Biotechnol17:1165–1169

Burrus V, Waldor MK (2004) Shaping bacterial genomeswith integrative and conjugative elements. Res Micro-biol 155:376–386

Chong Y, Ito Y, Kamimura T (2011) Genetic evolution andclinical impact in extended-spectrum beta-lactamase-producing Escherichia coli and Klebsiella pneumoniae.Infect Genet Evol 11:1499–1504

Da Re S, Garnier F, Guerin E, Campoy S, Denis F,Ploy MC (2009) The SOS response promotes qnrBquinolone-resistance determinant expression. EMBORep 10:929–933

Davies J, Davies D (2010) Origins and evolution ofantibiotic resistance. Microbiol Mol Biol Rev 74:417–433

de Lencastre H, Oliveira D, Tomasz A (2007) Antibioticresistant Staphylococcus aureus: a paradigm of adap-tive power. Curr Opin Microbiol 10:428–435

DeLeo FR, Chambers HF (2009) Reemergence ofantibiotic-resistant Staphylococcus aureus in the ge-nomics era. J Clin Invest 119:2464–2474

Directorate General of Health Services (2011) Nationalpolicy for containment of antimicrobial resistance,India. http://nicd.nic.in/ab_policy.pdf

Finch R, Hunter PA (2006) Antibiotic resistance-action topromote new technologies: report of an EU Intergov-ernmental Conference held in Birmingham, UK, 12–13 December 2005. J Antimicrob Chemother 58(Suppl1):i3–i22

Garriss G, Waldor MK, Burrus V (2009) Mobile antibioticresistance encoding elements promote their own diver-sity. PLoS Genet 5:e1000775

Hall RM, Brookes DE, Stokes HW (1991) Site-specificinsertion of genes into integrons: role of the 59-baseelement and determination of the recombination cross-over point. Mol Microbiol 5:1941–1959

Hernandez A, Sanchez MB, Martinez JL (2011)Quinolone resistance: much more than predicted. FrontMicrobiol 2:22

Kalia VC (2013) Quorum sensing inhibitors: an overview.Biotechnol Adv 31:224–245

Kalia VC, Wood TK, Kumar P (2014) Evolution ofresistance to quorum sensing inhibitors. Microb Ecol68(1):13–23

http://nicd.nic.in/ab_policy.pdf


Lynch JP 3rd, Clark NM, Zhanel GG (2013) Evolu-tion of antimicrobial resistance among Enterobacte-riaceae (focus on extended spectrum beta-lactamasesand carbapenemases). Expert Opin Pharmacother 14:199–210

Medeiros AA (1997) Evolution and dissemination ofbeta-lactamases accelerated by generations of beta-lactam antibiotics. Clin Infect Dis 24(Suppl 1):S19–S45

Pantosti A, Venditti M (2009) What is MRSA? Eur RespirJ 34:1190–1196

Perichon B, Courvalin P (2009) VanA-type vancomycin-resistant Staphylococcus aureus. Antimicrob AgentsChemother 53:4580–4587

Rajpara N, Patel A, Tiwari N, Bahuguna J, Antony A,Choudhury I et al (2009) Mechanism of drug resistancein a clinical isolate of Vibrio fluvialis: involvementof multiple plasmids and integrons. Int J AntimicrobAgents 34:220–225

Rapa RA, Labbate M (2013) The function of integron-associated gene cassettes in species: the tip of theiceberg. Front Microbiol 4:385

Recchia GD, Hall RM (1995) Gene cassettes: a newclass of mobile element. Microbiology 141(Pt 12):3015–3027

Ries AA, Wells JG, Olivola D, Ntakibirora M, Nyandwi S,Ntibakivayo M et al (1994) Epidemic Shigella dysente-riae type 1 in Burundi: panresistance and implicationsfor prevention. J Infect Dis 169:1035–1041

Robicsek A, Strahilevitz J, Jacoby GA, Macielag M,Abbanat D, Park CH et al (2006) Fluoroquinolone-modifying enzyme: a new adaptation of a commonaminoglycoside acetyltransferase. Nat Med 12:83–88

Rowe-Magnus AD, Davies J, Mazel D (2002) Impactof integrons and transposons on the evolution of re-sistance and virulence. Curr Top Microbiol Immunol264:167–188

Sack DA, Lyke C, Mc Laughlin C, Suwanvanichkij V(2001) Antimicrobial resistance in shigellosis, choleraand campylobacteriosis. World Health Organization,Geneva, Switzerland, pp 1–20

Singh R, Rajpara N, Tak J, Patel A, Mohanty P, Vinothku-mar K et al (2012) Clinical isolates of Vibrio fluvialisfrom Kolkata, India, obtained during 2006: plasmids,the qnr gene and a mutation in gyrase A as mecha-nisms of multidrug resistance. J Med Microbiol 61:369–374

Stokes HW, Hall RM (1989) A novel family of po-tentially mobile DNA elements encoding site-specificgene-integration functions: integrons. Mol Microbiol3:1669–1683

Tegos GP, Hamblin MR (2013) Disruptive innovations:new anti-infectives in the age of resistance. Curr OpinPharmacol 13:673–677

Waldor MK, Tschape H, Mekalanos JJ (1996) A newtype of conjugative transposon encodes resistance tosulfamethoxazole, trimethoprim, and streptomycin inVibrio cholerae O139. J Bacteriol 178:4157–4165

http://www.springer.com/978-81-322-1981-1

evolution of mdrs

Documents