Under stress conditions, many species of bacteria enter into starvation mode of metabolism or a physiologically viable but non-culturable (VBNC) state. Several human pathogenic bacteria have been reported to enter into the VBNC state under these conditions. The pathogenic VBNC bacteria cannot be grown using conventional culture media, although they continue to retain their viability and express their virulence. Though there have been debates on the VBNC concept in the past, several molecular studies have shown that not only can the VBNC state be induced under in vitro conditions but also that resuscitation from this state is possible under appropriate conditions. The most notable advance in resuscitating VBNC bacteria is the discovery of resuscitation-promoting factor (Rpf), which is a bacterial cytokines found in both Gram-positive and Gram-negative organisms. VBNC state is a survival strategy adopted by the bacteria, which has important implication in several fields, including environmental monitoring, food technology, and infectious disease management; and hence it is important to investigate the association of bacterial pathogens under VBNC state and the water/foodborne outbreaks. In this review, we describe various aspects of VBNC bacteria, which include their proteomic and genetic profiles under the VBNC state, conditions of resuscitation, methods of detection, antibiotic resistance, and observations on Rpf.

A PCR-based assay was developed to discriminate the classical, El Tor, and Haitian types of ctxB alleles. Our retrospective study using this newly developed PCR showed that Haitian ctxB first appeared in Kolkata C holera still continues to be an important cause of human infection, especially in developing countries that lack access to safe drinking water and proper sanitation. The recent devastating cholera outbreak in Haiti (13), for the first time in almost a century, placed this ancient disease at the forefront of the global public health agenda. In May 2011, the World Health Assembly recognized the reemergence of cholera as a significant global public health problem and called for the implementation of an integrated and comprehensive global approach to cholera control (17). This dreadful diarrheal disease is caused by the Gram-negative toxigenic bacterium Vibrio cholerae (7). To date, more than 200 serogroups of V. cholerae are known, but only serogroups O1 and O139 cause epidemic and pandemic cholera (7, 16). To date, the world has experienced seven pandemics of cholera. Among these, the first six were caused by the classical biotype strains, whereas the ongoing seventh pandemic has been caused by the El Tor biotype (16). In recent years, the emergence and dissemination of novel pathogenic variants of V. cholerae O1 throughout many Asian and African countries (1,2,3,5,9,10,11,14,15) indicated a cryptic change in cholera epidemiology. Our recent study showed that the El Tor variant strains of V. cholerae O1 have replaced the prototype El Tor biotype strains in Kolkata, India, since 1995 (15). This report, together with the recent massive cholera outbreak in Haiti, caused by V. cholerae organisms with a mutation in the 58th nucleotide of ctxB (3), motivated us to investigate the emergence and dissemination of this new variant of V. cholerae O1 biotype El Tor strains, if any, in Kolkata.In this study, we have developed a double-mismatch-amplification mutation assay (DMAMA) to accurately discriminate the classical, El Tor, and Haitian type ctxB alleles through a rapid and simple PCR-based assay. A total of 142 V. cholerae O1 strains were included in this study. These strains were selected from the repository of the National Institute of Cholera and Enteric Diseases, Kolkata, India, covering different months of each year from 2004 to 2011. V. cholerae O1 strains O395 (serotype Ogawa), N16961 (serotype Inaba), and EL-1786 (Ogawa, El Tor) were used as standard strains for the classical, El Tor, and Haitian type, respectively.Development of the DMAMA-PCR. All 142 tested strains, along with the control strains, were grown in Luria-Bertani broth (Becton Dickinson, Sparks, MD) for 18 h and then streaked on Luria agar (LA) plates. In this study, we focused on ctxB in V. cholerae O1 strains to confirm the strains carrying Haitian, classical, and El Tor alleles in a simple PCR-based assay. Current methods for differentiating the biotype-specific cholera toxin B (CTB) subunit of V. cholerae O1 necessitate MAMA-PCR with ...

Together with plague, smallpox and typhus, epidemics of dysentery have been a major scourge of human populations for centuries(1). A previous genomic study concluded that Shigella dysenteriae type 1 (Sd1), the epidemic dysentery bacillus, emerged and spread worldwide after the First World War, with no clear pattern of transmission(2). This is not consistent with the massive cyclic dysentery epidemics reported in Europe during the eighteenth and nineteenth centuries(1,3,4) and the first isolation of Sd1 in Japan in 1897(5). Here, we report a whole-genome analysis of 331 Sd1 isolates from around the world, collected between 1915 and 2011, providing us with unprecedented insight into the historical spread of this pathogen. We show here that Sd1 has existed since at least the eighteenth century and that it swept the globe at the end of the nineteenth century, diversifying into distinct lineages associated with the First World War, Second World War and various conflicts or natural disasters across Africa, Asia and Central America. We also provide a unique historical perspective on the evolution of antibiotic resistance over a 100-year period, beginning decades before the antibiotic era, and identify a prevalent multiple antibiotic-resistant lineage in South Asia that was transmitted in several waves to Africa, where it caused severe outbreaks of disease.

Vibrio fluvialis is a pathogen commonly found in coastal environs. Considering recent increase in numbers of diarrheal outbreaks and sporadic extraintestinal cases, V. fluvialis has been considered as an emerging pathogen. Though this pathogen can be easily isolated by existing culture methods, its identification is still a challenging problem due to close phenotypic resemblance either with Vibrio cholerae or Aeromonas spp. However, using molecular tools, it is easy to identify V. fluvialis from clinical and different environmental samples. Many putative virulence factors have been reported, but its mechanisms of pathogenesis and survival fitness in the environment are yet to be explored. This chapter covers some of the major discoveries that have been made to understand the importance of V. fluvialis.

Shigella species represent one of the growing numbers of antimicrobial-resistant bacteria in developing countries. Fluoroquinolone-resistant strains of Shigella dysenteriae type1 and Shigella flexneri type 2a emerged in India during 2002 and 2003, respectively. Sixty strains of Shigella from different parts of India were analysed for antimicrobial susceptibility, the presence of the qnr plasmid, mutations in the quinolone resistance determining regions (QRDRs), fluoroquinolone accumulation, and the presence of other genes encoding resistance to various antimicrobials. Fluoroquinolone-resistant strains had mutations in gyrA and parC genes and had an active efflux system. They were also resistant to several other antimicrobials but were susceptible to azithromycin and ceftriaxone. The majority of the strains harboured genes encoding resistance to ampicillin (97 %), tetracycline (95 %), streptomycin (95 %) and chloramphenicol (94 %). PFGE analysis revealed clonality among strains of S. dysenteriae types 1 and 5, S. flexneri type 2a and Shigella boydii type 12. INTRODUCTIONShigellosis remains an important public health problem in developing countries with Shigella sonnei in Europe and the US and Shigella flexneri in Asian and African countries being of epidemiological importance. Antimicrobial therapy is advocated for shigellosis to shorten the duration of illness (Salam & Bennish, 1991). However, in Asia and Africa, antimicrobial resistance is an emerging problem among Shigella species (von Seidlein et al., 2006) and treatment options are becoming limited globally (Salam & Bennish, 1991;Kariuki & Hart, 2001). The World Health Organization has recommended that ciprofloxacin should be considered a first-line antibiotic for the treatment of shigellosis, and the use of nalidixic acid is not encouraged, even in areas where it is still effective against Shigella (WHO, 2004). Similar to the prevalence of different serotypes, antimicrobial-resistance patterns of strains also differ from country to country and even within the same country (Pazhani et al., 2005;von Seidlein et al., 2006), which may be due to the spread of resistant clones as found for multidrug-resistant strains of Shigella dysenteriae type 1 in eastern parts of India (Pazhani et al., 2004). In this study, we have investigated the mechanisms of antibiotic resistance and clonal relatedness of Shigella strains isolated from epidemic and endemic cases of shigellosis in different parts of India. METHODSBacterial strains. We examined 60 strains of Shigella species (20 S. dysenteriae, 16 S. flexneri, 7 Shigella boydii and 17 S. sonnei) isolated from dysentery outbreaks from different parts of India and sporadic hospitalized cases of shigellosis in Kolkata and Goa between 2001 and 2004. Strains were confirmed as Shigella spp. by standard biochemical tests (WHO, 1987) and serotyped using commercially available antisera (Denka Seiken).Antimicrobial susceptibility testing. Antimicrobial susceptibility tests were performed by a disc diffusion method in accordance with...

We identified 281 Vibrio cholerae non-O1, non-O139 strains from patients with diarrhea in Kolkata, India. Cholera-like diarrhea was the major symptom (66.0%); some patients (20.3%) had severe dehydration. These strains lacked the ctxA gene but many had hlyA, rtxA, and rtxC genes. Pulsed-field gel electrophoresis showed no genetic link among strains.

BackgroundTo analyse the trends in the prevalence of different pathogroups of diarrheagenic Escherichia coli (DEC) among hospitalized acute diarrheal patients.Methodology/Principal FindingsFrom the active surveillance of diarrheal disease at the Infectious Diseases Hospital, Kolkata, 3826 stool specimens collected during 2008–2011 were screened for DEC and other enteric pathogens. PCR was used in the detection of enterotoxigenic, enteropathogenic and enteroaggregative E. coli and 10 major colonization factor antigens (CFs) of enterotoxigenic E. coli. The relationship between DEC infected patient’s age group and clinical symptoms were also investigated. Multiplex PCR assay showed that the prevalence of EAEC was most common (5.7%) followed by ETEC (4.2%) and EPEC (1.8%). In diarrheal children >2 year of age, EAEC and EPEC were detected significantly (p = 0.000 and 0.007, respectively). In children >2 to 5 and >5 to 14 years, ETEC was significantly associated with diarrhea (p = 0.000 each). EAEC was significantly associated with diarrheal patients with age groups >14 to 30 and >30 to 50 years (p = 0.001, and p = 0.009, respectively). Clinical symptoms such as vomiting, abdominal pain, watery diarrhea, were recorded in patients infected with ETEC. Dehydration status was severe among patients infected by ST-ETEC (19%) and EPEC (15%). CS6 was frequently detected (37%) among ETEC.Conclusions/SignificanceHospital based surveillance reviled that specific pathogroups of DEC are important to certain age groups and among ETEC, CS6 was predominant.

A total of 178 strains of V. parahaemolyticus isolated from 13,607 acute diarrheal patients admitted in the Infectious Diseases Hospital, Kolkata has been examined for serovar prevalence, antimicrobial susceptibility and genetic traits with reference to virulence, and clonal lineages. Clinical symptoms and stool characteristics of V. parahaemolyticus infected patients were analyzed for their specific traits. The frequency of pandemic strains was 68%, as confirmed by group-specific PCR (GS-PCR). However, the prevalence of non-pandemic strains was comparatively low (32%). Serovars O3:K6 (19.7%), O1:K25 (18.5%), O1:KUT (11.2%) were more commonly found and other serovars such as O3:KUT (6.7%), O4:K8 (6.7%), and O2:K3 (4.5%) were newly detected in this region. The virulence gene tdh was most frequently detected in GS-PCR positive strains. There was no association between strain features and stool characteristics or clinical outcomes with reference to serovar, pandemic/non-pandemic or virulence profiles. Ampicillin and streptomycin resistance was constant throughout the study period and the MIC of ampicillin among selected strains ranged from 24 to >256 µg/ml. Susceptibility of these strains to ampicillin increased several fold in the presence of carbonyl cyanide-m-chlorophenyldrazone. The newly reported ESBL encoding gene from VPA0477 was found in all the strains, including the susceptible ones for ampicillin. However, none of the strains exhibited the β-lactamase as a phenotypic marker. In the analysis of pulsed-field gel electrophoresis (PFGE), the pandemic strains formed two different clades, with one containing the newly emerged pandemic strains in this region.