Antimicrobials & cholera: are we stranded?

(i) Resistance for quinolone and fluoroquinolone

In India and other countries, emergence of fluoroquinolone resistance has been reported from early 2000s as this drug was in extensive use for the treatment of different infectious diseases, including cholera. In many bacteria, resistance to the quinolones is generally associated with amino acid substitutions in portions of GyrA and ParC proteins called quinolone resistance-determining regions (QRDRs). Quinolone resistance in vibrios is mainly due to the occurrence of mutation in gyrA, which encodes a subunit of DNA gyrase, followed by mutations in parC, which encodes a subunit of DNA topoisomerase IV. In addition to the mutations in gyrA and parC, proton motive force-dependent efflux is also involved in quinolone resistance in clinical isolates of V. cholera4. Extensive use of ciprofloxacin has compromised its effectiveness in many countries due to the decreasing susceptibility of V. cholerae.

(ii) Integrons

Integrons are naturally occurring gene acquisition systems which “help” bacteria capture exogeneous genes and incorporate them into their genomes56. Integrons consist of a gene intI encoding a site-specific recombinase belonging to tyrosine-recombinase family called “integrase”, a recombination site att1 into which the exogenous gene cassettes, harbouring the recombination site attC, are inserted through site-specific recombination and a promoter Pc, located within intI that drives transcription of the captured gene. Integrons play a prominent role in the dissemination of drug resistance because these frequently carry drug resistance genes and are often associated with mobile genetic elements. Based on the sequence of intI, integrons have been divided into five classes. Out of these, class 1 integrons are found widely among the clinical isolates of pathogenic bacteria. IntI integrase of class 1 integrons possesses all the features needed for performing recombination between att1 and attC, however, the rate of cassette recombination is controlled by the transcriptional repressor LexA7. Till date more than 100 different types of integron-borne gene cassettes, most of which code for antibiotic resistance, have been discovered. Out of these, only a limited few have been found in V. cholerae even though integrons have been detected in a large number of V. cholerae strains isolated all over the world8. Besides the five classes of integrons alluded to above, there exists another class called “Superintegrons”. These are chromosomally located and are sedentary in the sense that these do not move. A superintegron was first discovered in V. cholerae but are now known to be present in many g-protobacteria5. Superintegrons harbour hundreds of genes but the functions of most of which are unknown. In V. cholerae O1, a few display significant homology to a number of drug resistance genes, suggesting that under appropriate conditions these could “become” drug resistance genes and confer upon its host the ability to express resistance phenotype. It may be mentioned here that in two strains of V. cholerae O1, one isolated in Brazil and the other in Vietnam, an integron borne qnr gene responsible for resistance to ciprofloxacin, have been detected910.

(iii) Integrative and conjugative element (ICE)

The newly discovered integrating conjugative elements are movable linear DNA elements, which can integrate into bacterial genome and “move” through conjugation. These have the capacity to incorporate genes encoding many functions from drug-resistance to DNA repair pathways11. SXT in V. cholerae is an ICE, which carries resistance genes for sulphamethoxazole-trimethoprim, streptomycin and chloramphenicol11. SXT exconjugants may contain tandem SXT arrays and promote the formation of novel ICEs12. The array formation appears to depend on conjugative transfer and is recA-independent. SXT excises from the chromosome to form a circular but non-replicative extrachromosomal molecule that is required for its transfer. The IncJ elements such as R391 are now found to be closely related to SXT11. Till date, more than 25 members of the SXT/R391 family of ICEs have been identified in environmental and clinical isolates of diverse species of gamma-proteobacteria worldwide.

The 100-kb ICE was first identified in a V. cholerae O139 clinical strain isolated in 1992 in India. Prior to the emergence of V. cholerae O139, SXT was rarely detected in O1 serogroup13. After the emergence of the serogroup O139, however, ICEs began to be detected frequently in Asian V. cholerae strains1113. Even among O139 strains, the SXT elements are not stably maintained and this results in periodic change in the strains in their resistance to sulphamethoxazole-trimethoprim, chloramphenicol, and streptomycin. SXT or closely related ICEs are now being reported in most clinical and some environmental strains of V. cholerae isolated in Asia and Africa. An SXT-related ICE, ICEVchMex1 identified in a Mexican environmental V. cholerae isolate provided the first description of an SXT-related ICE in the Western Hemisphere14. The significant difference between the SXT and ICEVchMex1 suggests that these ICEs evolved independently.

It was discovered by Beaber et al15 that ‘SOS response’ can enhance the conjugative transfer of SXT by promoting the expression of genes necessary for the transfer of SXT through the inactivation of SetR, an SXT encoded repressor that keeps the transfer genes repressed. Ciprofloxacin which can induce SOS response was found to be able to promote the transfer of SXT and thus facilitate its dissemination. In other words, it was found that therapeutic agents too can aid in the spread of antibiotic resistance. Kim et al10 found that ciprofloxacin activity is further compromised in strains habouring qnrVC3, which encodes a pentapeptide repeat protein of the Qnr subfamily as this protein protects topoisomerases from quinolone action. The gene qnrVC3 is present within a member of the SXT integrating conjugative element family found commonly on the chromosomes of multidrug-resistant strains of V. cholerae.

(iv) Extended-spectrum beta-lactamases

Beta-lactamase production could be demonstrated in 92.8 per cent of the ampicillin resistant V. cholerae strains isolated in south India16. The CTX-M-ases, which hydrolyze cefotaxime efficiently, are mostly encoded by transferable plasmids, and the enzymes have been found predominantly in many members of Enterobacteriaceae and V. cholera17. The CTX-M-ases belong to the molecular class-A beta-lactamases, and the enzymes are functionally characterized as extended-spectrum beta-lactamases which confer resistance to penicillin, extended-spectrum cephalosporins, and monobactams. Class I integron-associated orf513 also seems to be involved in the mobilization of bla^CTX-M genes. CTX-M-type, PER-2-type and TEM-1-like enzymes were identified in V. cholerae strains isolated from cholera cases in Argentina18. Plasmid profile analysis and Southern blotting revealed the presence of plasmids of about 150 kb with genes encoding CTX-M-type or PER-2-type ESBLs.

(v) Plasmids

One of the common modes of the dissemination of drug resistance is through the plasmids. V. cholerae O1 strains isolated before 1970s were susceptible to tetracycline. Subsequently, due to extensive use of this drug, resistance to tetracycline was reported in many African countries. Strains isolated in Angola co-transferred tetracycline and chloramphenicol phenotype to E. coli. Plasmids belonging to incompatibility group (Inc) C and J were detected in V. cholera1920. Some of the V. cholerae O139 from India showed multidrug resistance. These strains harboured a 200 kb self-transmissible plasmid that mediated resistance to tetracycline, ampicillin, chloramphenicol, kanamycin, gentamicin, sulphamethoxazole and trimethoprim21. V. cholerae O1 El Tor isolated from patients in Uganda possessed a 130-MDa plasmid of incompatibility group 6-C that conferred resistance to trimethoprim (mediated by a dfrI gene), sulphonamides (suII), tetracycline (tetC), chloramphenicol (catI), ampicillin (a beta-lactamase gene different frombla^TEMor bla^SHV), and streptomycin22. This plasmid was transferred from V. cholerae to many other enteric bacteria indicating its potential to spread.

The emergence of pMRV150-like or pIP1202-like plasmids in many bacterial pathogens and non-pathogens in many geographical areas pose an increasing health risk, as these mediate antimicrobial resistance to at least six antibiotics, ampicillin, streptomycin, gentamicin, tetracycline, chloramphenicol, and trimethoprim-sulphamethoxazole23. The plasmid pMRV150 was being found with increasing frequency in V. cholerae O139 from Hangzhou, eastern China from 1994 onwards and was found to be similar to the plasmid pIP1202, an IncA/C plasmid detected in an MDR Yersinia pestis isolated from a bubonic plague patient in Madagascar23.

(vi) Efflux systems

Bacteria may acquire efflux pumps that export antibacterial agents before it can reach its target site and exert its effect. In V. cholerae, efflux pumps responsible for resistance to many antimicrobials have been demonstrated. In many Gram-negative bacteria, resistance to antimicrobial agents is mediated by resistance-nodulation-division (RND) family efflux systems. Deletion of six genes encoding RND efflux pumps from the genome of the V. cholerae O1 El Tor strain N16961 made this strain sensitive to multiple antimicrobial compounds, including bile acids, antimicrobial peptides and antibiotics24. Colmer et al25 identified two open reading frames (ORFs) in V. cholerae with high degree of similarity with the conserved regions of the E. coli efflux pump proteins, EmrA and EmrB. NorM, a putative efflux pump of V. cholerae, is a member of the multidrug and toxic compound extrusion family of transporters. Singh et al26 demonstrated that NorM confers resistance to norfloxacin, ciprofloxacin, and ethidium bromide. When the “selected” amino acids in the periplasmic and cytoplasmic loops of NorM was mutated, V. cholerae became hypersensitive towards norfloxacin, there- by showing the importance of NorM in norfloxacin resistance.

V. cholerae chromosome contains many putative genes of the multidrug and toxic compound extrusion (MATE) family. These include vcrM vcmB, vcmD, vcmH and vcmN27. Elevated MICs of multiple antimicrobial agents, such as fluoroquinolones, aminoglycosides, ethidium, etc. were observed in a drug hypersusceptible strain of E. coli when these genes were introduced into it through transformation. It was further shown that efflux activities of VcmB, VcmD and VcmH were Na+-dependent. Besides these, an operon VccCAB consisting of three genes involved in multiple-drug resistance (MDR) through efflux has been indentified in V. cholerae. This operon is negatively regulated by a transcriptional autoregulatory protein belonging to the TetR family of transcriptional regulators28. As mentioned earlier, there are six operons of putative RND-type efflux transporters present in the chromosome of V. cholerae O1. Out of these, vexAB, vexCD or vexEF together with tolC(Vc) of V. cholerae NCTC4716 elevated MICs of various antimicrobial agents when introduced into a susceptible E. coli strain29. Though all these efflux pumps have been studied well, epidemiological significance of these in the dissemination of drug resistance remains unexplored.