Hybrid & El Tor variant biotypes of Vibrio cholerae O1 in Thailand.

Background & objectives: El Tor Vibrio cholerae O1 carrying ctxBC trait, so-called El Tor variant that causes more severe symptoms than the prototype El Tor strain, first detected in Bangladesh was later shown to have emerged in India in 1992. Subsequently, similar V. cholerae strains were isolated in other countries in Asia and Africa. Thus, it was of interest to investigate the characteristics of V. cholerae O1 strains isolated chronologically (from 1986 to 2009) in Thailand. Methods: A total of 330 V. cholerae O1 Thailand strains from hospitalized patients with cholera isolated during 1986 to 2009 were subjected to conventional biotyping i.e., susceptibility to polymyxin B, chicken erythrocyte agglutination (CCA) and Voges-Proskauer (VP) test. The presence of ctxA, ctxB, zot, ace, toxR, tcpAC, tcpAE, hlyAC and hlyAE were examined by PCR. Mismatch amplification mutation assay (MAMA) - and conventional- PCRs were used for differentiating ctxB and rstR alleles. Results: All 330 strains carried the El Tor virulence gene signature. Among these, 266 strains were typical El Tor (resistant to 50 units of polymyxin B and positive for CCA and VP test) while 64 had mixed classical and El Tor phenotypes (hybrid biotype). Combined MAMA-PCR and the conventional biotyping methods revealed that 36 strains of 1986-1992 were either typical El Tor, hybrid, El Tor variant or unclassified biotype. The hybrid strains were present during 1986-2004. El Tor variant strains were found in 1992, the same year when the typical El Tor strains disappeared. All 294 strains of 1993-2009 carried ctxBC ; 237 were El Tor variant and 57 were hybrid. Interpretation & conclusions: In Thailand, hybrid V. cholerae O1 (mixed biotypes), was found since 1986. Circulating strains, however, are predominantly El Tor variant (El Tor biotype with ctxBC).

Shiga toxin producing Escherichia coli infection: current progress & future challenges.

Shiga toxin producing Escherichia coli (STEC) is a newly emerged pathogen that has been the focus of immense international research effort driven by its recognition as a major cause of large scale epidemics and thousands of sporadic cases of gastrointestinal illness. It produces a severe bloody diarrhoea that is clinically distinct from other types of diarrhoeal diseases caused by other enteric pathogens. One of the most important areas of current exploration concerns how STEC enters our food chain, an investigational avenue that begins with the ecology of STEC in animals and in the environment. A variety of foods have been identified as vehicles of STEC-associated illness and this makes the organism one of the most serious threats to the food industry in recent years. The pathogenesis of STEC is multifactorial and involves several levels of interaction between the bacterium and the host. STEC strains carry a set of virulence genes that encode the factors for attachment to host cells, elaboration of effective molecules and production of two different types of Shiga toxins. These genes are found in the locus of enterocyte effacement (LEE), lamboid phages, and a large virulence associated plasmid. The publication of the complete genome sequence of Esch. coli O157:H7 chromosome offers a unique resource that will help to identify additional virulence genes, to develop better methods of strain detection and in the understanding of the evolution of Esch. coli through comparison with the genome of the non-pathogenic laboratory strain Esch. coli K-12. These research efforts in turn, should lead to development of new potent and cost effective anti-Stx therapies or vaccines and thereby major improvement in human health world-wide.

Isolation and identification of aeromonas from patients with acute diarrhoea in Kolkata, India.

Isolation of diarrhoea causing Aeromonas was carried out in the division of Active Surveillance, National Institute of Cholera and Enteric Diseases (NICED), Kolkata for a period of 12 months from January 1999 to December 1999. Out of 602 stool samples collected from patients with acute diarrhoea admitted in Infectious Diseases (ID) Hospital, Kolkata, 64 (10.6%) samples were identified positive for Aeromonas as the pathogen. The different isolated and identified species from patients with acute diarrhoea were A. hydrophila (60%), A. caviae (20%), A. veronii (10%), A. schubertii (4%), A. jandaei (3%), and A. trota (3%). Most of the isolated Aeromonas strains showed resistance to commonly employed antibiotics. All the clinical isolates of Aeromonas possessed virulence genes encoding for aerolysin and cytotonic enterotoxin genes. Except A. schubertii and A. jandaei, all the other species possessed the gene for haemolysis.

Molecular comparison of toxigenic clinical & non-toxigenic environmental strains of Vibrio cholerae O1 Ogawa isolated during an outbreak of cholera in south India.

BACKGROUND & OBJECTIVES: While investigating a cholera outbreak in south India, toxigenic and nontoxigenic strains of Vibrio cholerae O1 were isolated from patients and from the environment, respectively. This study was performed to compare the genetic relatedness of the patient and environmental strains to determine clonal relationships among these strains and thereby determine the source of the cholera outbreak. METHODS: The 16 strains of V. cholerae isolated from hospitalized patients and 8 environmental V. cholerae strains isolated from the environment were phenotypically and genotypically characterized using a variety of standard techniques. RESULTS: Sixteen toxigenic clinical strains and 2 nontoxigenic environmental strains belonged to O1 serogroup, Ogawa serotype and El Tor biotype. The remaining 6 nontoxigenic environmental strains were classified as non-O1, non-O139 V. cholerae. The drug resistance pattern of the clinical and environmental strains of V. cholerae showed marked differences with the patient strains being resistant to more number of drugs as compared to the environmental strains. DNA fingerprinting of the strains showed considerable diversity between toxigenic clinical and nontoxigenic environmental O1 Ogawa isolates and between the O1 and non-O1, non-O139 isolates. INTERPRETATION & CONCLUSION: In this outbreak of cholera, the O1 strains of V. cholerae from clinical and environmental sources belonged to two different clones and the environmental strains could perhaps be the future cholera outbreak causing clones.

Modification of the multiplex PCR for unambiguous differentiation of the El Tor & classical biotypes of Vibrio cholerae O1.

BACKGROUND & OBJECTIVES: Biotyping of Vibrio cholerae O1 using multiplex PCR (ctxA-tcpA) exploits the nucleotide sequence differences of the major subunit protein of the toxin co-regulated pilus (TCP) gene (tcpA) to differentiate between the classical and El Tor biotypes. However, the presence of classical biotype specific tcpA amplicon with the El Tor strains often complicates the interpretation. The effect of PCR variables on the amplification of biotype specific tcpA in the multiplex PCR has been investigated. METHODS: Reference strains of toxigenic V. cholerae O1 belonging to classical and El Tor biotypes were selected to optimize the PCR variables for the unambiguous biotype determination by multiplex PCR. RESULTS: In the multiplex PCR assay, a reduction in the reaction volume from 100 microliters to 25 microliters and the annealing temperature of 64 degrees C, the El Tor strain produced ctxA amplicon (302 bp) along with tcpA amplicons of 618 bp and 472 bp which are specific for classical and El Tor tcpA respectively. The simplex PCR with biotype specific tcpA primer pairs showed the amplification of either 472 bp or 618 bp tcpA amplicon with El Tor template. With the classical biotype strain, the specific primer pair yielded tcpA amplicon of the expected size. Lowering of PCR annealing temperature from 64 to 60 degrees C resulted in the elimination of the amplification of the nonspecific tcpA amplicon with El Tor strain. INTERPRETATION & CONCLUSION: A comparison of the theoretical melting temperature (Tm) values of the reacting primers, and their alignment to the biotype specific tcpA revealed the basis of unambiguous biotyping of V. cholerae O1 at a PCR annealing temperature of 60 degrees C.

Cluster-analysis & patterns of dissemination of multidrug resistance among clinical strains of Vibrio cholerae in Calcutta, India.

BACKGROUND & OBJECTIVES: Antimicrobial resistance among Vibrio cholerae has been monitored for several years in Calcutta. To investigate the changing trends in multidrug resistance (MDR) among different serogroups of V. cholerae and to perform software assisted cluster analysis the current study was undertaken. METHODS: Strains isolated from patients with cholera and "cholera-like" diarrhoea admitted in the Infectious Diseases Hospital, Calcutta were analysed. Eight hundred and forty V. cholerae strains isolated from 1992 through 1997 were tested for susceptibility to 11 antibiotics. Cluster analysis was done using SPSS software. RESULTS: Most of the strains exhibited MDR with fluctuating trends as the resistance profile diverged each year. A total of 119 different resistance profiles exhibited by V. cholerae O1, O139 and non-O1, non-O139 serogroups were analysed by cluster combination method. During 1993 and 1994, 53 per cent of V. cholerae O139 and 82 per cent of V. cholerae O1 serogroups, respectively, exhibited maximal number of new resistance patterns. The frequency of new resistance patterns among V. cholerae non-O1, non-O139 was constantly high (33-47%) during 1995 to 1997. INTERPRETATION & CONCLUSIONS: With a few exceptions, preponderance of the resistance profiles was generally not confined to any serogroup. The cluster analysis depicted dissemination of some of the resistance patterns commonly found among V. cholerae non-O1, non-O139 belonging to different serogroups to the O139 serogroup in the succeeding years. In this study we have shown that the V. cholerae strains are resistant to several antibiotics with constant change in the MDR profiles. It is imperative to define the susceptibility pattern of the strains to determine the effective drug of choice for the treatment of cholera.

Shiga-toxin producing Escherichia coli from healthy cattle in a semi-urban community in Calcutta, India.

From October to December 1998, single faecal samples from 67 healthy cattle in a semi-urban community near Calcutta were examined for Shiga toxin producing Esch. coli (STEC) using a multiplex PCR primary screen followed by plating on sorbitol MacConkey agar. STEC was isolated from the faeces of 7 (10.5%) animals. The eight strains isolated belonged to eight serotypes viz, O146:H1, O149:HNT, ONT:H34, ONT:H19, O88:HN, ONT:H2, O82:H8 and O28ac:H21. Bead enzyme linked immunosorbent assay showed that three strains produced Shiga toxin 1, one produced Shiga toxin 2 and four produced both.

Clonal analysis of non-toxigenic Vibrio cholerae O1 associated with an outbreak of cholera.

We examined the clonal relationships among eight clinical isolates of non-toxigenic (NT) V. cholerae O1 associated with a cluster of cases of cholera in Warangal, Andhra Pradesh in south India and compared their relatedness to toxigenic O1 strains of classical and E1Tor biotypes and with O139 Bengal strains of V. cholerae by pulsed-field gel electrophoresis (PFGE). Phylogentic analysis of the NotI restriction fragment length polymorphism showed that all the NT. V. cholerae O1 strains formed a tight cluster with more than 80 per cent similarity. Interestingly, the NT V. cholerae O1 cluster was more closely related to V. cholerae O139 than to classical and E1Tor biotypes of V. cholerae O1 indicating closer genetic relationships between NT V. cholerae 01 and O139 Bengal strains that were isolated during the same time-frame.

Comparison between the multiplex PCR, sensitivity to biotype specific phages & polymyxin B for biotyping of Vibrio cholerae O1.

A total of 196 Vibrio cholerae O1 strains isolated between 1970 and 1996 were biotyped by multiplex PCR, susceptibility to polymyxin B and sensitivity to biotype specific phages. We modified the multiplex PCR by increasing the primer concentration of tcpA to improve the results. Comparison of the results of modified multiplex PCR and sensitivity to biotype specific phages and to polymyxin B showed that multiplex PCR was as efficient as phage typing for biotyping of V. cholerae O1. All the strains of V. cholerae O1 could be accurately distinguished based on polymyxin B sensitivity. Thus our results show that susceptibility of strains of V. cholerae O1 to polymyxin B is the easiest method to biotype V. cholerae O1 and is feasible in most laboratories when compared with multiplex PCR and sensitivity to biotype specific phages.

Comparison of the sensitivity & specificity of a polyclonal versus monoclonal capture antibody based Bead ELISA for direct detection of cholera toxin from stool specimens.

This study was conducted to examine whether substitution of polyclonal anti-CT IgG (PAb) coated beads with monoclonal anti-CT IgG, (MAb) coated beads would enhance the sensitivity and specificity of the Bead ELISA for direct detection of CT in stool samples of cholera patients. Sensitivity of MAb Bead ELISA was found to be more efficient (92.7%) than the PAb Bead ELISA (88.2%) in comparison to the 'gold standard' method of conventional culture method (CM). However, the MAb based Bead ELISA had lower specificity (45%) than that of PAb Bead ELISA (51.8%). Further, the positive predictive value was also lower in MAb Bead ELISA (69.9%) as compared to PAb Bead ELISA (82.1%). Generally, all the statistical evaluation demonstrated better agreement between PAb (77.9%) and MAb (72.6%) Bead ELISAs in direct detection of free CT and culture method in confirming the causative organism i.e., Vibrio cholerae in stool specimens. When the two assay systems, viz., PAb and MAb Bead ELISAs were compared, the superiority of the PAb Bead ELISA over MAb Bead ELISA was clearly evident as it had 82.4 per cent of agreement value.

Comparative analysis of factors promoting optimal production of cholera toxin by Vibrio cholerae O1 (classical & E1Tor biotypes) & O139.

Various culture media [AKI, Brain heart infusion broth (BHI), Casamino acid-yeast extract broth (CAYE), Casamino acid-yeast extract broth supplemented with 90 micrograms/ml of lincomycin (CAYE-L), Tryptic soy broth (TSB) and Yeast extract peptone (YEP)], cultural conditions (stationary and shaking) and incubation temperatures (30 degrees C and 37 degrees C) were evaluated to determine optimal conditions for production of cholera toxin (CT) by different biotypes (classical and E1Tor) and serogroups (O1 and O139) of V. cholerae. It was found that V. cholerae O1 E1Tor grown in CAYE-L and incubated at 30 degrees C with constant shaking was optimal for production of CT, while for the classical biotype and for the O139 serogroup, CT was maximally produced when grown in YEP and incubated at 30 degrees C in a shaker. Temperature appeared to be a prominent factor affecting the production of CT by the O1 E1Tor biotype when the media used were AKI, CAYE-L and YEP and also for the classical biotype when the media used were the AKI, BHI, CAYE and YEP. In the case of the O1 E1Tor biotype, CAYE-L was the best medium for CT production whereas for the classical biotype, CAYE-L was a poor medium as far as CT production was concerned. Irrespective of the media used, 30 degrees C shake culture condition seemed to be more favourable for supporting CT production except in CAYE medium for the O1 E1Tor biotype where incubation at 37 degrees C in a shaker was as good as incubation at 30 degrees C.

Serovar, biotype, phage type, toxigenicity & antibiotic susceptibility patterns of Vibrio cholerae isolated during two consecutive cholera seasons (1989-90) in Calcutta.

Characteristics of V. cholerae isolated from patients of acute secretory diarrhoea admitted to the Infectious Diseases Hospital, Calcutta during two consecutive cholera seasons (1989 and 1990), with special emphasis on biotyping and toxigenicity, were investigated. The isolation rates of V. cholerae during 1989 and 1990 were 78 and 85.1 per cent respectively, with Inaba serotype dominating in 1989 and Ogawa in 1990. All the V. cholerae 01 strains isolated in this study belonged to biotype Eltor with phage type 4 dominating (48.8%). Most of the strains of V. cholerae were resistant to 10 and 150 micrograms/ml of 0/129 vibriostatic agent. Similarly, majority of the V. cholerae strains were resistant to furazolidone (95.7%), cotrimoxazole (83%) and tetracycline (63.1%) and several resistance patterns were encountered. All the V. cholerae 01 strains examined produced cholera toxin (CT) in amounts ranging between greater than 70 pg/ml and greater than 2.5 ng/ml. In contrast, all but one of the non-01 strains isolated in this study did not produce CT. Further studies are required to elucidate the mechanism involved in the pathogenesis of non-01 V. cholerae mediated diarrhoea.

Detection of pathogenic of bacteria in children with diarrhoea

86 children ranging from one to three years old with diarrhoea from Yangon Children's Hospital and respective controls were included in this study. Isolation rates of major pathogens responsible for acute diarrhoea in children were Enterotoxigenic Escherichia coli (EPEC) 8.1 per cent , Salmonella 1.2 per cent, Shigella sonnei 1.2 per cent, Plesiomonas 1.2 per cent and vibrios 1.2 per cent. In the control group the prevalence was found to be ETEC 9.3 per cent, EPEC 7.0 per cent, Salmonella 12.0 per cent , S. sonnei 1.2 per cent, Plesiomonas 7.0 per cent and vibrios 1.2 per cent. The total pathogen isolated from diarrhoea and control cases were 46.5 per cent and 38.4 per cent respectively. It was also noted that Salmonella and Plesiomonas isolation rates were higher in the control group.

Detection of Eschericia Coli enterotoxin from stool sample directly by Bead ELISA method

Bacterial protein toxins as Escherichia coli enterotoxigenic heat labile toxin (LT), E. coli shigella like toxin I (VT1), E. coli shegella like toxin 2 (VT2) and cholera toxin where detected directly from stool using Bead ELISA method. A total of 390 stools from adults with diarrhoea and dysentery from 2 Military Hospital, Workers Hospital, Yangon General Hospital and Infectious Diseases Hospital were included in this study. It was observed that 9, 5 and 10 samples of stools showed to possess LT, VT1, VT2 toxin respectively. Some of the samples showed to possess either 2 or 3 toxins in combination. Of these, 2 samples showed to possess LT and VT1 toxins; 3 samples showed to possess LT and VT2 toxin; 11 samples showed to possess VT1 and VT2 toxin and 14 samples showed to possess LT, VT1 and VT2 toxins. Thus a total of 54 samples (13.8 per cent) showed to possess bacterial toxins. It was also observed that 28 samples out of 199 cases (14.1 per cent); 20 out of 109 cases (18.3 per cent) and 6 out of 82 cases (7.3 per cent) showed to possess bacterial toxin in January, June and November 1988 respectively. Moreover, cholera toxin was detected from 5 out of 25 cases tested.

Microbiologia da diarréia aguda endêmica em crianças do Recife / Microbiology of endemic acute diarrhoea in children of Recife

Entre fevereiro e junho de 1989, investigaram-se 126 crianças diarréicas e 126 controles, pareados pela idade, para a detecçäo de bactérias, vírus e parasitos enteropatogênicos. Identificaram-se um ou mais patógenos em 81,8% dos pacientes e em 61,9% dos controles. Escherichia coli enteropatogênica(EPEC) foi o patógeno mais frequentemente identificado. EPEC mostrando aderência localizada, E.coli enteroaderente agregativa, Shigella, Rotavírus e Cryptosporidium foram associados com os casos significativamente. Por outra parte, EPEC näo aderente, E.Coli exibindo aderência difusa, E.coli enterotoxigênica (ETEC) produtora de LT, Salmonella, Campylobacter, Adenovírus, Giardia lamblia e Endamoeba histolytica näo mostraram diferenças significativas entre casos e controles. ETEC produtoras de ST,E.coli enteroinvasiva e enterohemorrágica, Aeromonas, Pleisaomonas e Vibrio foram isolados em taxas muito baixas para permitir avaliar seus papéis na doenças diarréica, no Recife