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Background & objectives: Diverse mechanisms have been identified in enteric bacteria for their adaptation and survival against multiple classes of antimicrobial agents. Resistance of bacteria to the most effective fluoroquinolones have increasingly been reported in many countries. We have identified that most of the enterotoxigenic Escherichia coli (ETEC) were resistant to several antimicrobials in a diarrhoea outbreak at Ahmedabad during 2000. The present study was done to identify several genes responsible for antimicrobial resistance and mobile genetic elements in the ETEC strains. Methods: Seventeen ETEC strains isolated from diarrhoeal patients were included in this study. The antimicrobial resistance was confirmed by conventional disc diffusion method. PCR and DNA sequencing were performed for the identification of mutation in the quinolone resistance-determining regions (QRDRs). Efflux pump was tested by inhibiting the proton-motive force. DNA hybridization assay was made for the detection of integrase genes and the resistance gene cassettes were identified by direct sequencing of the PCR amplicons. Results: Majority of the ETEC had GyrA mutations at codons 83 and 87 and in ParC at codon 80. Six strains had an additional mutation in ParC at codon 108 and two had at position 84. Plasmid-borne qnr gene alleles that encode quinolone resistance were not detected but the newly described aac(6’)-Ib-cr gene encoding a fluoroquinolne-modifying enzyme was detected in 64.7 per cent of the ETEC. Class 1 (intI1) and class 2 (intI2) integrons were detected in six (35.3%) and three (17.6%) strains, respectively. Four strains (23.5%) had both the classes of integrons. Sequence analysis revealed presence of dfrA17, aadA1, aadA5 in class 1, and dfrA1, sat1, aadA1 in class 2 integrons. In addition, the other resistance genes such as tet gene alleles (94.1%), catAI (70.6%), strA (58.8%), blaTEM-1(35.2%), and aphA1-Ia (29.4%) were detected in most of the strains. Interpretation & conclusions: Innate gene mutations and acquisition of multidrug resistance genes through mobile genetic elements might have contributed to the emergence of multidrug resistance (MDR) in ETEC. This study reinforces the necessity of utilizing molecular techniques in the epidemiological studies to understand the nature of resistance responsible for antimicrobial resistance in different species of pathogenic bacteria.

Background & objectives: Bacillus cereus is one of the pathogens responsible for human diarrhoea, mainly due to consumption of contaminated food. The present study was undertaken to determine the occurrence of B. cereus among diarrhoeal patients and its phenotypic and genetic characteristics that determine the virulence and clonal features. Methods: Stool specimens were collected for two years from acute diarrhoeal patients attending the two referral hospitals in Kolkata. Presence of virulence genes in B. cereus was determined by PCR. Clonality was assessed by pulsed-field gel analysis (PFGE) by restriction digestion with SmaI and NotI enzymes. Enterotoxins were detected by haemolysin assay and using BCET-RPLA kit. Invasion assay was done on Hep-2 cell line. Antimicrobial susceptibility was tested by disc diffusion method. Results: B. cereus was identified in 54 (3.5%) of the 1536 diarrhoeal cases studied. Majority of the isolates were susceptible to many antibiotics but showed resistant to amoxyclav and cephalosporins. Six genes covering the two different enterotoxic complexes determining the pathogenicity of B. cereus have been characterized by PCR. The nhe genes were detected in a higher proportion than hbl. Except in two, clonal diversity was noticed among 21 B. cereus isolates. Haemolytic enterotoxin was detected in 76 per cent of the isolates. Majority of the isolates (67%) produced in vitro enterotoxin (BCET) confirming its involvement in the infection. Interpretation & conclusions: Though the presence of B. cereus was not high in patients with diarrhoea, several virulence factors confirm their association with diarrhoea. Distinct clonality was identified in majority of the isolates indicating their origin from different sources.

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.

An explosive epidemic of cholera in the district of Malda in the state of West Bengal, was induced by devastating floods resulting from overflowing of the two main rivers of the district, at the end of July 1998, affecting 15 blocks and 2 municipalities. Diarrhoeal outbreak occurred around the middle of August after receding of the flood waters. Within two weeks of its onset, the outbreak spread throughout the district. An investigation was conducted to understand the epidemiological characteristics, identify the etiological agent, rationalise clinical management and suggest control measures.The team visited the Block Primary Health Centres, surrounding the affected villages and also the district hospital. Morbidity and mortality data were collected and 88 patients were interviewed and examined clinically. Epidemiological and clinical observations were recorded. Rectal swabs were collected from both hospitalised and domiciliary cases.During the period between August and October 1998, 16,590 cases were reported with 276 deaths (case fatality rate of 1.7%). Twenty one of 29 (72%) rectal swabs were positive for Vibrio cholerae O1, biotype ElTor, serotype Ogawa. All the strains were sensitive to tetracycline, norfloxacilin, ciprofloxacilin, gentamycin, chloramphenicol but resistant to furazolidine, co-trimoxazole, nalidixic acid, streptomycin and ampicilin.Observations of the present study identified the epidemiological and clinical deficiencies in the management of the outbreak and recommendations were elaborated for its effective control.

Helicobacter pylori colonizes the gastric mucosa of more than half of all people worldwide and is the major cause of peptic ulcer disease and an early risk factor for gastric cancer, even though most infections are asymptomatic. Infection occurs preferentially in early childhood and once established tends to persist for years or decades. Much of the pathology H. pylori causes probably results from the host response to infection, which is affected by bacterial genotype, human host characteristics and environmental conditions. H. pylori is one of the most genetically diverse of bacterial species, with different genotypes predominating in different parts of the world. In particular, strains from India differ from those of Europe and East Asia in DNA sequence of several diagnostic gene segments. This outcome invites speculation about H. pylori origins and the possibility of Indian-specific genes that might be uncommon in Western strains. Much has been learned from H. pylori genome sequences, along with epidemiological, mutational, molecular and immunologic analyses. Candidate bacterial colonization and virulence genes and host responses are being identified, and the hypotheses being developed are amenable to tests in cell culture and animal models. These research efforts, many of which are collaborative and international, provide insights into mechanisms of establishment and persistence of H. pylori infection and virulence, and should lead to new, far more potent and cost effective anti-Helicobacter therapies or vaccines, and thereby major improvement in human health worldwide.

A highly sensitive bead enzyme-linked immunosorbent assay was applied for the quantitative determination of vacuolating cytotoxin (VacA) released in the culture supernatant of 40 well characterized Helicobacter pylori strains in order to clarify the significance of allelic combination of the vacA gene as the predictor of the level of toxin secretion and also to determine the most appropriate genotype of H. pylori associated with high VacA release. Attempts were also made for the detection of VacA in the gastric juice of patients for the rapid diagnosis of H. pylori infection.The genotypes of 40 H. pylori strains cultured from the gastric biopsy samples were determined by specific PCRs. The cell-free culture supernatant of the strains as well as the gastric juice of the patients were used for bead-ELISA and the purified VacA from the H. pylori strain ATCC49503 was used as positive control.Ninety per cent of the strains with vacAs1m1 allele combination secrete on an average 146.4 ng/ml of VacA while the corresponding value was 19.1 ng/ml for s1m2 strains. None of the s2m2 as well as the ice negative H. pylori strains produced detectable VacA in the medium while strains expressed the toxin irrespective of the presence or absence of cagA gene. Fifteen of 22 gastric juice samples yielded positive bead-ELISA results.vacAs1, vacAm1 and iceA1 could be considered as the determinants of high VacA secretion. Also, the detection of VacA by bead-ELISA in the gastric juice could be considered as an alternative approach in the diagnosis of H. pylori infection.

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.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.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.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.

Klebsiella pneumoniae strains occasionally cause diarrhoea in humans. This study was done to determine the involvement of calcium in the pathogenesis of aggregative K. pneumoniae strains.A total of nine strains of K. pneumoniae were tested for adherence assay in HeLa cell line. A representative strain CO-1215 was used for [Ca2+]i study using Fura-2 fluorescence.Infection of cultured HeLa cells with aggregative K. pneumoniae strain resulted in five-fold elevation of intracellular level of free calcium ([Ca2+]i) with maximum Ca2+ influx at 3 h after bacterial infection. Chelation of extracellular Ca2+ with [ethylenebis(oxyethylenenitrile)] tetraacetic acid and suspension of cells in Ca2+ free buffer suggested that the rise of Ca2+ in aggregative K. pneumoniae infected HeLa cells was due to influx of Ca2+ from extracellular medium.This study showed aggregative adherence in HeLa cells and this adherence leads to influx of extracellular Ca2+. The unrestricted passage of calcium ions across cell membranes could cause phosphorylation of proteins involved in ion transport across the membrane, which could result in secretory diarrhoea. Further work is in progress to study the enterotoxicity of these strains in an in vitro rabbit intestinal model.

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.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.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.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.

Detection of faecal leucocytes and RBCs in stool samples of cholera patients has been reported in a small number of studies. This study extends these observations by examining stool samples of cholera patients in Calcutta.Out of 1562 diarrhoeal stool samples, Vibrio cholerae was isolated in 266 cases. Stool samples obtained were examined microscopically within two hours of collection. RBCs and faecal leucocytes were examined by normal saline and methylene blue stain. Stool culture was performed using selective and differential media for isolation of V. cholerae.Among 266 cholera patients, RBCs was detected in 58 per cent and faecal leucocytes in 88 per cent respectively. The extent of the changes correlated with clinical severity.This study showed the presence of RBCs and faecal leucocytes in stools of patients of cholera caused by V. cholerae 01 and 0139 which indicates some inflammatory changes in the gut mucosa. Further study is required to elucidate the inflammatory mechanism involved in the underlying process(es).

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.

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.

Glucose-based or rice-based ORS is the standard treatment in acute dehydrating diarrhoea. However, glucose may not be easily available in remote villages and the rice needs to be cooked for rice-based ORS. We embarked on a study to examine whether uncooked rice powder could be used as an alternative to glucose or cooked rice powder in ORS. Initially, 50 adult male patients (aged 18 to 55 yr) were randomized to receive glucose-ORS or uncooked rice ORS, in two equal groups. Subsequently, 20 male children (aged 3 to 12 yr) were also enrolled in the study and received either WHO-ORS or study ORS. All the adult patients and the children could be successfully rehydrated with ORS containing uncooked rice powder. As compared to WHO-ORS, the study ORS significantly reduced stool output (6.60 +/- 1.24 vs. 5.88 +/- 1.34 l), ORS intake (9.17 +/- 1.54 vs 8.24 +/- 1.69 l) and duration of diarrhoea (45.68 +/- 6.91 vs 41.32 +/- 6.03 h). In children also similar results were obtained. No clinical complication (e.g., vomiting, abdominal pain etc.) or abnormality in serum electrolyte concentrations was encountered either in the adults or in the children. Uncooked rice powder containing ORS can be considered as an alternative to glucose-based ORS or rice-based ORS.

In the context of the reemergence of V. cholerae O1 in India and the recent evidence that O139 strains could have evolved from O1 E1 Tor strains, restriction fragment length polymorphism (RFLP) of the rRNA and the ctx genes and the antibiotic sensitivity profile of the two strains of V. cholerae, one an O1 and the other an O139, associated with mixed infection, were examined to determine their relatedness. Our results demonstrate that although the strains belonged to different clones of V. cholerae, they showed similar antibiotic sensitivity, profile indicating some exchange of genetic elements.

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.

In May 1996, a massive outbreak of cholera occurred in Alleppey district of Kerala which spread to Palghat district by July 1996. Of the 575 patients hospitalized at the Alleppey Medical College hospital between May 1 and August 2, 1996, 30 deaths occurred with a case fatality rate of 5.2 per cent while of the 638 diarrhoea patients admitted at Agali PHC of Attapadi area in Palghat district, 30 (4.7%) deaths were recorded. Clinically, the patients had profuse watery diarrhoea with vomiting. The epidemic of cholera in Alleppey and Palghat districts was caused by V. cholerae O1 of the EITor biotype, Ogawa serotype which possessed both the ctxA and tcpA genes when examined by multiplex PCR. Gross contamination of water sources was incriminated as the cause of the epidemic.

The past few years have witnessed a resurgence in the global incidence of cholera. The increasing application of procedures employing concepts and techniques assimilated from molecular biology has provided new means of discriminating V. cholerae. Such studies are providing a wealth of critical information that has assisted the epidemiologist in tracing the spread of epidemics and has provided new insights into the evolution and origin of newer variants of V. cholerae. In this article, an effort is made to collect all the recent information and present the impact this new information has made on our understanding of V. cholerae.

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.

We conducted studies to investigate the surface architecture of V. cholerae O139 using electron microscopy and compared it with O1 and other serogroups of V. cholerae. The bacterium is comma-shaped and has a single polar flagellum and morphologically resembles the classical and E1Tor biotypes of V. cholerae O1. High power electron microscopy showed a few pili, 5 to 7 nm in diameter, and 2 to 3 in number per bacterium. The presence of a capsule on electron microscopy of ultrathin sections of V. cholerae O139 treated with polycationic ferritin clearly distinguished the O139 serogroup from the O1 serogroup which are not encapsulated. Immunoelectron microscopy further revealed that an anti-O139 monoclonal antibody of the IgG2a isotype bound specifically only to an O139 strain but not to any other serogroup of V. cholerae indicating that O139 has unique epitopes not found in other serogroups of V. cholerae.

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.

The colonization ability of a representative epidemic strain of V. cholerae O139 Bengal was studied in the oral rabbit colonization model and the nature of colonization in the ileal and jejunal tissues was examined ultrastructurally. Results of the colonization study and ileal loop assay indicated that the strain proliferates and colonizes the small intestine of the rabbit mucosal surface. Further, the electronmicroscopic study revealed the disruptive effect of the strain on the apical membrane of the epithelial cells. The results of this study suggested that apart from colonization, invasion of the bacteria was important in the pathogenesis of V. cholerae O139 mediated infections.

Monoclonal antibodies to Ogawa-Inaba common antigenic determinant and Ogawa specific antigenic determinant of V. cholerae belonging to the serogroup O1 were generated from BALB/c mice immunized with V. cholerae O1 Eltor Ogawa strain. Reactivity and specificities of the monoclonal antibodies were examined by slide agglutination method. The monoclonal antibodies agglutinated all the V. cholerae O1 strains tested but did not agglutinate with any of the other currently recognized 140 serogroups of V. cholerae non-O1 as well as with a variety of other enteric pathogens. Diagnostic utility of the MAbs produced in this study as compared to polyclonal O1 and monospecific antisera showed that the MAbs were as good as the latter for serological diagnosis of the two serotypes of V. cholerae O1.

As one of large outbreaks of cholera-like illness in the Indian subcontinent, Calcutta and its neighbouring areas experienced an unprecedented epidemic due to a new strain of V. cholerae non-01, designated as V. cholerae 0139 Bengal, since January 1993. This epidemic predominantly affected the adult population of Calcutta as evidenced by the hospitalization of more adults at the Infectious Disease Hospital, Calcutta (IDH), which bore the main brunt of the epidemic in and around Calcutta. During the peak of the epidemic about 180 to 300 diarrhoea patients were admitted daily at the IDH. Of the 807 patients screened, 407 were positive for V. cholerae 0139 and majority (82.8%) of the cases were10 yr of age. Severe dehydration was recorded in 85.5 per cent of the cases.

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.

A simplified medium was developed for the detection of DNase produced by enteric campylobacters. Sensitivity and reproducibility of the test were similar to that of the improved toluidine blue DNA agar method. Logistically, the simplified DNA hydrolysis test was cheaper (5.5 times) than the earlier medium. Based on this study we recommend the routine use of the simplified medium to perform the DNase test for biotyping enteric campylobacters.

A recently developed charcoal-based, blood-free selective medium-charcoal cefoperazone deoxycholate agar (CCDA) was compared with the conventional Butzler's agar (BA) for primary isolation of enteric campylobacters from human stool specimens. CCDA yielded higher percentage (93.6%) of positive isolations as compared to BA (76.6%). The rate of isolation on CCDA was significantly higher (P less than 0.0001) than on BA. Cost-wise also, the CCDA medium was two times cheaper than BA. In addition to Campylobacter jejuni and C. coli, CCDA permitted the recovery of a recently described catalase-negative campylobacters which did not grow on BA. Based on these results the routine use of CCDA is recommended, particularly in developing countries, as blood is not required for this medium.

C. jejuni and C. coli isolates from diverse sources in Calcutta were serotyped and biotyped according to the Lior scheme. Of the 55 strains examined, 85.5 per cent reacted with one or another of the 73 antisera available. This included the formation of two new serogroups, LIO 67 and LIO 76. C. coli serogroup LIO 46 biotype II was the most frequently encountered strains (14.5%), followed by C. coli serogroup LIO 29, 55 biotype II (10.5%) and C. jejuni serogroup LIO 54, biotype I (5.5%). Serogroups recovered from animals and birds were also found to be prevalent in strains isolated from clinical sources, confirming the zoonotic implications of the disease.