Thandavarayan Ramamurthy

Genomic history of the seventh pandemic of cholera in Africa

Wave upon wave of disease The cholera pathogen, Vibrio cholerae, is considered to be ubiquitous in water systems, making the design of eradication measures apparently fruitless. Nevertheless, local and global Vibrio populations remain distinct. Now, Weill et al. and Domman et al. show that a surprising diversity between continents has been established. Latin America and Africa bear different variants of cholera toxin with different transmission dynamics and ecological niches. The data are not consistent with the establishment of long-term reservoirs of pandemic cholera or with a relationship to climate events. Science, this issue p. 785, p. 789 Multiple waves of local outbreaks and pandemic cholera indicate independence from climate change and marine reservoirs. The seventh cholera pandemic has heavily affected Africa, although the origin and continental spread of the disease remain undefined. We used genomic data from 1070 Vibrio cholerae O1 isolates, across 45 African countries and over a 49-year period, to show that past epidemics were attributable to a single expanded lineage. This lineage was introduced at least 11 times since 1970, into two main regions, West Africa and East/Southern Africa, causing epidemics that lasted up to 28 years. The last five introductions into Africa, all from Asia, involved multidrug-resistant sublineages that replaced antibiotic-susceptible sublineages after 2000. This phylogenetic framework describes the periodicity of lineage introduction and the stable routes of cholera spread, which should inform the rational design of control measures for cholera in Africa.

Attributes of carbapenemase encoding conjugative plasmid pNDM-SAL from an extensively drug-resistant Salmonella enterica Serovar Senftenberg.

A carbapenem resistant Salmonella enterica serovar Senftenberg isolate BCH 2406 was isolated from a diarrheal child attending an outpatient unit of B.C. Roy Hospital in Kolkata, India. This isolate was positive for the blaNDM-1 in the PCR assay, which was confirmed by amplicon sequencing. Except for tetracycline, this isolate was resistant to all the tested antimicrobials. The blaNDM-1 was found to be located on a 146.13-kb mega plasmid pNDM-SAL, which could be conjugally transferred into Escherichia coli and other enteric pathogens such as Vibrio cholerae O1 Ogawa and Shigella flexneri 2a. However, the expression of β-lactam resistance is not the same in different bacteria. The whole genome sequence of pNDM-SAL was determined and compared with other pNDM plasmids available in public domain. This plasmid is an IncA/C incompatibility type composed of 155 predicted coding sequences and shares homology with plasmids of E. coli pNDM-1_Dok01, Klebsiella pNDM-KN, and Citrobacter pNDM-CIT. In pNDM-SAL, gene cluster containing blaNDM-1 was located between IS26 and IS4321 elements. Between the IS26 element and the blaNDM-1, a truncated ISAba125 insertion sequence was identified. Downstream of the blaNDM-1, other genes, such as bleMBL, trpF, tat, and an ISCR1 element with class 1 integron containing aac(6′)-Ib were detected. Another β-lactacamase gene, blaCMY -4 was found to be inserted in IS1 element within the type IV conjugative transfer loci of the plasmid. This gene cluster had blc and sugE downstream of the blaCMY -4. From our findings, it appears that the strain S. Senftenberg could have acquired the NDM plasmid from the other members of Enterobacteriaceae. Transfer of NDM plasmids poses a danger in the management of infectious diseases.

Extraintestinal Infections Caused by Non-toxigenic Vibrio cholerae non-O1/non-O139

Vibrio cholerae is an aerobic, sucrose fermentative Gram-negative bacterium that generally prevails in the environment. Pathogenic V. cholerae is well-known as causative agent of acute diarrhea. Apart from enteric infections, V. cholerae may also cause other diseases. However, their role in causing extraintestinal infections is not fully known as it needs proper identification and evaluation. Four cases of extraintestinal infections due to V. cholerae non-O1/non-O139 have been investigated. The isolates were screened for phenotypic and genetic characteristics with reference to their major virulence genes. Serologically distinct isolates harbored rtx, msh, and hly but lacked enteric toxin encoding genes that are generally present in toxigenic V. cholerae. Timely detection of this organism can prevent fatalities in hospital settings. The underlying virulence potential of V. cholerae needs appropriate testing and intervention.

Molecular Insights into Antimicrobial Resistance Traits of Multidrug Resistant Enteric Pathogens isolated from India

Emergence of antimicrobial resistant Gram-negative bacteria has created a serious global health crisis and threatens the effectiveness of most, if not all, antibiotics commonly used to prevent and treat bacterial infections. There is a dearth of detailed studies on the prevalence of antimicrobial resistance (AMR) patterns in India. Here, we have isolated and examined AMR patterns of 654 enteric pathogens and investigated complete genome sequences of isolates from six representative genera, which in aggregate encode resistance against 22 antibiotics representing nine distinct drug classes. This study revealed that ~97% isolates are resistant against ≥2 antibiotics, ~24% isolates are resistant against ≥10 antibiotics and ~3% isolates are resistant against ≥15 antibiotics. Analyses of whole genome sequences of six extensive drug resistant enteric pathogens revealed presence of multiple mobile genetic elements, which are physically linked with resistance traits. These elements are therefore appearing to be responsible for disseminating drug resistance among bacteria through horizontal gene transfer. The present study provides insights into the linkages between the resistance patterns to certain antibiotics and their usage in India. The findings would be useful to understand the genetics of resistance traits and severity of and difficulty in tackling AMR enteric pathogens.

Rugose atypical Vibrio cholerae O1 El Tor responsible for 2009 cholera outbreak in India.

Vibrio cholerae causes cholera outbreaks in endemic regions where the water quality and sanitation facilities remain poor. Apart from biotype and serotype changes, V. cholerae undergoes phase variation, which results in the generation of two morphologically different variants termed smooth and rugose. In this study, 12 rugose (R-VC) and 6 smooth (S-VC) V. cholerae O1 Ogawa isolates were identified in a cholera outbreak that occurred in Hyderabad, India. Antimicrobial susceptibility results showed that all the isolates were resistant to ampicillin, furazolidone and nalidixic acid. In addition, R-VC isolates were resistant to ciprofloxacin (92u2009%), streptomycin (92u2009%), erythromycin (83u2009%), trimethoprim-sulfamethoxazole (75u2009%) and tetracycline (75u2009%). Based on the ctxB gene analysis, all the isolates were identified as El Tor variant with mutation in two positions of ctxB, similar to the classical biotype. The R-VC isolates specifically showed excessive biofilm formation and were comparatively less motile. In addition, the majority of these isolates (~83u2009%) displayed random mutations in the hapR gene, which encodes haemagglutinin protease regulatory protein. In the PFGE analysis, R-VC and S-VC were placed in distinct clusters but remained clonally related. In the ribotyping analysis, all the R-VC isolates exhibited R-III pattern, which is a prevailing type among the current El Tor isolates. A hapR deletion mutant generated using an S-VC isolate expressed rugose phenotype. To our knowledge, this is the first report on the association of rugose V. cholerae O1 in a large cholera outbreak with extended antimicrobial resistance and random mutations in the haemagglutinin protease regulatory protein encoding gene (hapR).

Comparative genome analysis of VSP-II and SNPs reveals heterogenic variation in contemporary strains of Vibrio cholerae O1 isolated from cholera patients in Kolkata, India

Cholera is an acute diarrheal disease and a major public health problem in many developing countries in Asia, Africa, and Latin America. Since the Bay of Bengal is considered the epicenter for the seventh cholera pandemic, it is important to understand the genetic dynamism of Vibrio cholerae from Kolkata, as a representative of the Bengal region. We analyzed whole genome sequence data of V. cholerae O1 isolated from cholera patients in Kolkata, India, from 2007 to 2014 and identified the heterogeneous genomic region in these strains. In addition, we carried out a phylogenetic analysis based on the whole genome single nucleotide polymorphisms to determine the genetic lineage of strains in Kolkata. This analysis revealed the heterogeneity of the Vibrio seventh pandemic island (VSP)-II in Kolkata strains. The ctxB genotype was also heterogeneous and was highly related to VSP-II types. In addition, phylogenetic analysis revealed the shifts in predominant strains in Kolkata. Two distinct lineages, 1 and 2, were found between 2007 and 2010. However, the proportion changed markedly in 2010 and lineage 2 strains were predominant thereafter. Lineage 2 can be divided into four sublineages, I, II, III and IV. The results of this study indicate that lineages 1 and 2-I were concurrently prevalent between 2007 and 2009, and lineage 2-III observed in 2010, followed by the predominance of lineage 2-IV in 2011 and continued until 2014. Our findings demonstrate that the epidemic of cholera in Kolkata was caused by several distinct strains that have been constantly changing within the genetic lineages of V. cholerae O1 in recent years.

Fostering research into antimicrobial resistance in India

Bhabatosh Das and colleagues discuss research and development of new antimicrobials and rapid diagnostics, which are crucial for tackling antimicrobial resistance in India

Molecular Insights into Antimicrobial Resistance Traits of Commensal Human Gut Microbiota

Antimicrobial resistance (AMR) among bacterial species that resides in complex ecosystems is a natural phenomenon. Indiscriminate use of antimicrobials in healthcare, livestock, and agriculture provides an evolutionary advantage to the resistant variants to dominate the ecosystem. Ascendency of resistant variants threatens the efficacy of most, if not all, of the antimicrobial drugs commonly used to prevent and/or cure microbial infections. Resistant phenotype is very common in enteric bacteria. The most common mechanisms of AMR are enzymatic modifications to the antimicrobials or their target molecules. In enteric bacteria, most of the resistance traits are acquired by horizontal gene transfer from closely or distantly related bacterial population. AMR traits are generally linked with mobile genetic elements (MGEs) and could rapidly disseminate to the bacterial species through horizontal gene transfer (HGT) from a pool of resistance genes. Although prevalence of AMR genes among pathogenic bacteria is widely studied in the interest of infectious disease management, the resistance profile and the genetic traits that encode resistance to the commensal microbiota residing in the gut of healthy humans are not well-studied. In the present study, we have characterized AMR phenotypes and genotypes of five dominant commensal enteric bacteria isolated from the gut of healthy Indians. Our study revealed that like pathogenic bacteria, enteric commensals are also multidrug-resistant. The genes encoding antibiotic resistance are physically linked with MGEs and could disseminate vertically to the progeny and laterally to the distantly related microbial species. Consequently, the AMR genes present in the chromosome of commensal gut bacteria could be a potential source of resistance functions for other enteric pathogens.

Involvement of topoisomerase mutations, qnr and aac(6′)Ib-cr genes in conferring quinolone resistance to the clinical isolates of Vibrio and Shigella spp. (1998 to 2009) from Kolkata, India

OBJECTIVESnQuinolone antibiotics have been widely used to treat diarrhoeal diseases caused by bacterial agents such as those belonging to the genera Vibrio and Shigella. As these pathogens are accumulating quinolone resistance, treating infections caused by them has become complicated.nnnMETHODSnIn this study, Vibrio and Shigella spp. isolates obtained from diarrhoeal patients from Kolkata, India, over a period of 12 years (1998-2009) were analysed for quinolone resistance. A total of 27 Vibrio spp. (9 Vibrio cholerae, 11 Vibrio fluvialis and 7 Vibrio parahaemolyticus) and 10 Shigella spp. isolates (7 Shigella flexneri, 2 Shigella dysenteriae and 1 Shigella sonnei) showing reduced susceptibility to quinolones were studied to unravel the genetic factors responsible for quinolone resistance.nnnRESULTSnAntimicrobial susceptibility testing showed a wide spectrum and varying degree of resistance to different generations of quinolones. Genotypic characterisation revealed the involvement of GyrA(S83I) and ParC(S85L) mutations in V. cholerae and V. fluvialis, whereas Shigella spp. isolates showed the mutations S83L and/or D87N/Y in GyrA and S80I or E84K in ParC. Analysis of plasmid-mediated quinolone resistance genes showed that qnrVC5 was detected in three V. fluvialis isolates, aac(6)-Ib-cr in one V. fluvialis isolate and qnrS1 in a S. flexneri isolate.nnnCONCLUSIONSnThese results emphasise that quinolone resistance is widespread and therefore quinolones should be used prudently. To the best of our knowledge, this is the first study where resistance to various generations of quinolones in Vibrio and Shigella spp. has been examined in terms of detailed genotype-phenotype correlation.