Flooding adds pathogenic Escherichia coli strains to the water sources in southern Khyber Pakhtunkhwa,Pakistan

Purpose: Seasonal rains in Pakistan result in heavy floods across the country, whereby faecal contaminants will be added to the water bodies and cause numerous food-borne outbreaks. The present study was aimed to determine the prevalence of diarrheagenic Escherichia coli (DEC) strains in the water sources. Materials and Methods: Two hundred water samples collected during (2011–2012) were processed for the isolation of E. coli (EC) strains. EC strains were further analysed for antibiotic susceptibility patterns, and pathogroups-specific virulence factors stx1, stx2, stx2c, eae, tir, hlyA, bfpA, estA and eltA were detected using multiplex polymerase chain reaction. Results: Thirty-three percent of the water samples were contaminated with EC pathotypes. Fifty percent (33/66) of the DEC pathotypes were identified as enterotoxigenic EC (ETEC). Seventy-two percent (13/18) of the enteropathogenic EC (EPEC) strains were identified as typical EPEC and 28% (5/18) as atypical EPEC. Eleven percent (7/66) of the Shiga toxin EC (STEC) isolates carried a combination of stx1 and stx2 genes. Summer was found as a peak season with 47% (31/66) for EC pathogroups’ activities. Eighty-nine percent of the strains showed resistance against tetracycline. Conclusion: ETEC and EPEC are the primary causes of water contamination in southern regions of Khyber Pakhtunkhwa province, Pakistan. Firm adherence to the prescribed drugs can decrease trends in antibiotic resistance.     

Molecular detection and antibacterial susceptibility of enteropathogenic Escherichia coli (EPEC) and shigatoxigenic Escherichia coli (STEC) strains isolated from healthy and diarrhoeic dogs

Animal contacts have been regarded as an emerging rout of Shiga toxin-producing Escherichia coli (STEC) infection in humans. Diarrhoeic and asymptomatic dogs have been recognised as a reservoir of atypical enteropathogenic Escherichia coli (EPEC), and STEC in some investigations. In this study E. coli isolates from 100 faecal samples of healthy (n = 50) and diarrhoeic (n = 50) dogs were screened by polymerase chain reaction (PCR) for the presence of determining virulence genes of STEC and EPEC pathotypes including stx and eaeA. The confirmed virulence-positive strains were subjected to antimicrobial susceptibility testing against 12 antibacterial using disc diffusion method. Resistance profiles were also determined for the STEC and EPEC strains. Ten isolates from 10 dogs (10%) were shown to possess at least one of the tested virulence genes. Six of these isolates (6%) harboured only the eaeA gene and were considered as EPEC. Four isolates (4%) were stx+ and regarded as STEC, of which two were stx+/eae+. The resistance was specially observed against penicillin, ampicillin, sulfomethoxazole, streptomycin and oxytetracyclin. Altogether, nine resistance profiles were observed among 10 isolates. In conclusion, dogs can act as a reservoir for EPEC and STEC strains, and close contacts of children with companion animals can be a potential risk factor in development of diarrhoea and haemolytic uremic syndrome. In rural areas shepherd dogs can also be a transient carrier of STEC strains that they may acquire from ruminants. To our knowledge this is the first study which reports the faecal shedding of STEC and EPEC from dogs in Iran.     

Diarrhoeagenic Escherichia coli isolated from children with acute diarrhoea at Rakai hospital,Southern Uganda

BackgroundDiarrhoeagenic Escherichia coli (DEC) is a leading cause of childhood diarrhoea. This study estimated the prevalence of DEC and DEC pathotypes among children with acute diarrhoea in Southern Uganda.MethodsA cross-sectional study was conducted on 267 children less than 5 years with acute diarrhoea, admitted to Rakai General Hospital in Southern Uganda. Faecal samples were collected from the children and processed for isolation of E. coli. The presence of DEC and the distribution of DEC pathotypes were determined by polymerase chain reaction.ResultsA total of 102 (38.2%, 102/267) children had DEC of various pathotypes – enteroaggregative E. coli (EAEC) (14.2%); enteropathogenic E. coli (EPEC) (6.7%); enterotoxigenic E. coli (ETEC) (6%); enteroinvasive E. coli (EIEC) (7.5%); enterohemorrhagic E. coli (EHEC) (3%); and cell-detaching E. coli (CDEC) (0.75%). The difference in the overall prevalence of DEC was not significant regarding HIV but individually, EAEC and CDEC were associated with HIV-positive status while ETEC was associated with HIV-negative status.ConclusionsDEC is prevalent in children with acute diarrhoea in Southern Uganda and its identification in children should be considered among strategies for combatting childhood diarrhoea in Africa.     

Detection of Escherichia coli O157:H7 virulence genes in isolates from beef, pork, water, human and animal species in the northwest province, South Africa: public health implications

The aim of the present study was to isolate and identify Escherichia coli O157:H7 from pigs, cattle, humans, beef, pork and water samples and to determine their putative virulence genes by PCR analysis. A total of 220 samples were analysed; 5600 presumptive E. coli O157:H7 were screened for the presence of rfb_O157 and fliC_H7 gene fragments by PCR and 130 isolates were confirmed. The prevalence of E. coli O157:H7 was higher in pigs and pork 88(67.7%) than in cattle and beef 36(27.7%), water 3(2.3%) or humans 1 (0.77%). Moreover, the pathogen was more frequently isolated from faecal (16.9%-43.1%) than from meat samples (10.8%-24.6%). A large proportion—73 (56.2%)—of the isolates possessed the hlyA gene, while 48 (36.9%) harboured the eaeA gene. Although there were no major differences in the number of isolates harbouring the stx₁ and stx₂ genes, respectively, only a small proportion 13(10%) harboured both shiga toxin genes. Despite this, the proportion of isolates that possessed the stx₁ 29(22.3%) was higher than those possessing the stx₂ gene. None of the E. coli O157:H7 isolates harboured all four shiga-toxin producing E. coli (STEC) virulence genes investigated. When comparing the proportion of isolates obtained from the different sample sources and/or stations, significant positive correlations were observed between isolates from Mafikeng and Lichtenburg (r = 0.981, p < 0.05) and those from Mafikeng and Rustenburg (r = 0.991, p < 0.05). These results therefore indicate that meat and faeces samples obtained from major cities in the northwest province were contaminated with E. coli O157:H7. We suggest that there is a need for improving the sanitary conditions of farms, abattoirs and butcher shops. This could reduce transmission of E. coli O157:H7 to humans.     

Real-Time Multiplex Polymerase Chain Reaction with High-Resolution Melting-Curve Analysis for the Diagnosis of Enteric Infections Associated with Diarrheagenic Escherichia coli

Introduction: Although diarrheagenic Escherichia coli (DEC) strains are important bacterial causative agents of diarrhoea, they are not routinely sought as stool pathogens in clinical laboratories as conventional microbiological testing are unable to distinguish between normal flora and pathogenic strains of E. coli. This study was undertaken to determine the prevalence of DEC pathotypes amongst children with and without diarrhoea and to detect specific virulent genes present in different DEC pathotypes, using real-time multiplex polymerase chain reaction (PCR) with high-resolution melting (HRM) technology. Materials and Methods: Stool samples were obtained from cases and controls. Using a set of conventional biochemical tests, E. coli strains were identified. Further, these isolates were subjected to multiplex PCR system for the detection of virulence genes of different pathotypes of DEC. Real-time multiplex PCR was performed for the detection of specific virulent genes of DEC pathotypes, using Rotor-Gene Q instrument (Qiagen) having High-resolution Melt analyser using Type-it HRM PCR kit (Qiagen) containing EvaGreen fluorescent intercalating dye. Results: In this study, we had successfully standardised two multiplex PCR assays which were found to be effective for direct detection of enteropathogenic E. coli (EPEC), enteroaggregative E. coli (EAEC), enterotoxigenic E. coli (ETEC) and enteroinvasive E. coli (EIEC). A total of 42 DEC strains were detected at an overall rate of 19.3% (n = 42), from the total 217 E. coli isolates recovered from the cases (n = 39, 17.9%) and control (n = 3, 3.8%) groups. Amongst the 42 DEC pathotypes (39 from cases and 3 from controls), EPEC (10%), EAEC (8.82%), ETEC (2.94%) and EIEC (1.18%) were found in children with diarrhoea (cases) and in children without diarrhoea (control) only EAEC (2.13%) and EPEC (4.26%) were detected. Age distribution, gender variation, seasonal variation and clinical features were also analysed Conclusion: This study helped evaluate the prevalence of DEC amongst children (<18 years of age) with and without diarrhoea using multiplex real-time PCR with HRM analysis.     

Clonal Diversity of Chilean Isolates of Enterohemorrhagic Escherichia coli from Patients with Hemolytic-Uremic Syndrome, Asymptomatic Subjects, Animal Reservoirs, and Food Products

To determine clonal relationship among Chilean enterohemorrhagic Escherichia coli (EHEC) strains from different sources (clinical infections, animal reservoirs, and food), 54 EHEC isolates (44 of E. coli O157, 5 of E. coli O111, and 5 of E. coli O26) were characterized for virulence genes by colony blot hybridization and by pulsed-field gel electrophoresis (PFGE). By colony blotting, 12 different genotypes were identified among the 44 E. coli O157 isolates analyzed, of which the genetic profile stx₁⁺ stx₂⁺ hly⁺ eae⁺ was the most prevalent. All human O157 strains that were associated with sporadic cases of hemolytic-uremic syndrome (HUS) carried both the stx₁ and stx₂ toxin-encoding genes and were eaeA positive. Only 9 of 13 isolates from human controls were stx₁⁺ stx₂⁺, and 8 carried the eaeA gene. Comparison of profiles obtained by PFGE of XbaI-digested genomic DNA showed a great diversity among the E. coli O157 isolates, with 37 different profiles among 39 isolates analyzed. Cluster analysis of PFGE profiles showed a wide distribution of clinical isolates obtained from HUS cases and asymptomatic individuals and a clonal relationship among O157 isolates obtained from HUS cases and pigs. Analysis of virulence genes showed that a correlation exists among strains with the genotype stx₁⁺ stx₂⁺ eae⁺ and pathogenic potential. A larger difference in the PFGE restriction patterns was observed among the EHEC strains of serogroups O26 and O111. These results indicate that several different EHEC clones circulate in Chile and suggest that pigs are an important animal reservoir for human infections by EHEC. Guidelines have been proposed for better practices in the slaughter of animals in Chile.     

Virulence versus fitness determinants in Escherichia coli isolated from asymptomatic bacteriuria in healthy nonpregnant women

Purpose: Escherichia coli isolated from asymptomatic bacteriuria (ABU) correlated genotypically and phenotypically with cystitis isolates may help in distinguishing urovirulence determinants from ‘fitness factors’, latter necessary only for survival of E. coli in urinary tract; for gaining insight into the pathogenesis of urinary tract infection. Materials and Methods: In this cross-sectional study, we compared genotypic (phylogroups and 15 putative virulence genes), and phenotypic profiles of ABU E. coli strains with our previously genotyped collection of cystitis isolates. Virulence score was calculated for each isolate as a number of virulence genes detected. Results: Significant differences were observed in the proportion of four phylogenetic groups (P = 0.009) amongst cystitis and ABU isolates. Average virulence score was higher for ABU isolates (6.6) than cystitis strains (4.2); and hlyA (P = 0.001), cytotoxic necrotising factor 1 (P = 0.00), fyuA (P = 0.00), ibeA (P = 0.00), kpsMII (P = 0.01), and malX/pathogenicity-associated island (P = 0.01) were more frequently present in ABU strains. Conclusions: The expression of adhesins, haemolysin, aerobactin, and capsule synthesis gene were similar in two groups suggesting their role as fitness factors. ABU isolates were better biofilm producers, reflecting its importance in silent persistence. Serum resistance gene which was more expressed in cystitis isolates may represent virulence determinant. Genetic makeup of E. coli does not change much rather genes helping in survival and colonisation are expressed equally in ABU and cystitis isolates as opposed to phenotypic attenuation of those that helps in invasion or inflammation in ABU isolates.     

Detection and Characterization of Shiga Toxigenic Escherichia coli by Using Multiplex PCR Assays for stx1, stx2, eaeA, Enterohemorrhagic E. coli hlyA, rfbO111, and rfbO157

Shiga toxigenic Escherichia coli (STEC) comprises a diverse group of organisms capable of causing severe gastrointestinal disease in humans. Within the STEC family, certain strains appear to be of greater virulence for humans, for example, those belonging to serogroups O111 and O157 and those with particular combinations of other putative virulence traits. We have developed two multiplex PCR assays for the detection and genetic characterization of STEC in cultures of feces or foodstuffs. Assay 1 utilizes four PCR primer pairs and detects the presence of stx₁, stx₂ (including variants of stx₂), eaeA, and enterohemorrhagic E. coli hlyA, generating amplification products of 180, 255, 384, and 534 bp, respectively. Assay 2 uses two primer pairs specific for portions of the rfb (O-antigen-encoding) regions of E. coli serotypes O157 and O111, generating PCR products of 259 and 406 bp, respectively. The two assays were validated by testing 52 previously characterized STEC strains and observing 100% agreement with previous results. Moreover, assay 2 did not give a false-positive O157 reaction with enteropathogenic E. coli strains belonging to clonally related serogroup O55. Assays 1 and 2 detected STEC of the appropriate genotype in primary fecal cultures from five patients with hemolytic-uremic syndrome and three with bloody diarrhea. Thirty-one other primary fecal cultures from patients without evidence of STEC infection were negative.     

Incidence of blaNDM-1 gene in Escherichia coli isolates at a tertiary care referral hospital in Northeast India

Purpose: Increasing reports on New Delhi metallo-β-lactamase-1 (NDM-1) producing Escherichia coli constitute a serious threat to global health since it is found to be highly resistant to most of the currently available antibiotics including carbapenems. This study has been performed to find out the incidence bla_NDM-1 in E. coli isolates recovered from the various clinical samples at a tertiary care referral hospital in Northeast India. Materials and Methods: A total of 270 non-duplicated E. coli isolates were recovered from the various clinical samples at a tertiary care referral hospital in Northeast India. All isolates with reduced susceptibility to meropenem or ertapenem (diameter of zones of inhibition, ≤21 mm) were further phenotypically confirmed for carbapenemase production by modified Hodge test. All screened isolates were also subjected to the polymerase chain reaction detection of bla_NDM-1 gene and additional bla genes coding for transmission electron microscopy, SHV, CTX-M, and AmpC. Results: Out of 270 E. coli isolates, 14 were screened for carbapenemase production on the basis of their reduced susceptibility to meropenem or ertapenem. All screened isolates were found to be positive for bla_NDM-1. Each of the bla_NDM-1 possessing isolate was also positive for two or more additional bla genes, such as bla_TEM, bla_CTX-M and bla_AmpC. Phylogenetic analysis showed very less variation in bla_NDM-1 gene with respect to bla_NDM-1 possessing E. coli isolates from other parts of India and abroad. Conclusions: Our findings highlight the incidence of bla_NDM-1 in E. coli isolates with a reduced susceptibility to meropenem or ertapenem.     

Diarrheagenic Escherichia coli pathotypes investigation revealed atypical enteropathogenic E. coli as putative emerging diarrheal agents in children living in Botucatu,São Paulo State,Brazil

The aim of the present study was to investigate the prevalence of Diarrheagenic Escherichia coli (DEC) pathotypes, a leading cause of diarrhea worldwide, among diarrheal and healthy children, up to 5 years of age, living in the city of Botucatu, São Paulo, Brazil. DEC, investigated by PCR detection of virulence factor‐encoding genes associated with the distinct pathotypes, was isolated from 18.0% of the patients, and 19.0% of the controls, with enteroaggregative E. coli (EAEC), the most frequent pathotype, being detected in equal proportion between patients and controls (10.0%). Among the enteropathogenic E. coli (EPEC) isolates, only one isolate was able to produce the localized adherence pattern to HeLa cells, being thus the only typical EPEC identified. All the remaining EPEC were classified as atypical (aEPEC), and detected in 8.0% and 8.5% of the patients and controls, respectively. Regarding the serotypes, 26.5% of the analyzed EPEC isolates belonged to classical EPEC‐serogroups, and the only two STEC found were serotyped as O26:H11 (patient) and O119:H7 (control). Antimicrobial susceptibility tests revealed that 43.6%, 29.5% and 2.6% of the DEC isolates were resistant to ampicillin, cotrimoxazole and gentamicin, respectively. Our data indicate that EAEC remains prevalent among children living in Botucatu, and revealed atypical EPEC as emerging putative diarrheal agents in this geographical region.     

Molecular Characterization of Human Atypical Sorbitol-Fermenting Enteropathogenic Escherichia coli O157 Reveals High Diversity

Alongside the well-characterized enterohemorrhagic Escherichia coli (EHEC) O157:H7, serogroup O157 comprises sorbitol-fermenting typical and atypical enteropathogenic E. coli (EPEC/aEPEC) strains that carry the intimin-encoding gene eae but not Shiga toxin-encoding genes (stx). Since little is known about these pathogens, we characterized 30 clinical isolates from patients with hemolytic uremic syndrome (HUS) or uncomplicated diarrhea with respect to their flagellin gene (fliC) type and multilocus sequence type (MLST). Moreover, we applied whole-genome sequencing (WGS) to determine the phylogenetic relationship with other eae-positive EHEC serotypes and the composition of the rfbO157 region. fliC typing resulted in five fliC types (H7, H16, H34, H39, and H45). Isolates of each fliC type shared a unique ST. In comparison to the 42 HUS-associated E. coli (HUSEC) strains, only the stx-negative isolates with fliCH7 shared their ST with EHEC O157:H7/H⁻ strains. With the exception of one O157:H⁻_fliCH16 isolate, HUS was exclusively associated with fliCH7. WGS corroborated the separation of the fliCH7 isolates, which were closely related to the EHEC O157:H7/H⁻ isolates, and the diverse group of isolates exhibiting different fliC types, indicating independent evolution of the different serotypes. This was also supported by the heterogeneity within the rfbO157 region that exhibited extensive recombinations. The genotypic subtypes and distribution of clinical symptoms suggested that the stx-negative O157 strains with fliCH7 were originally EHEC strains that lost stx. The remaining isolates form a distinct and diverse group of atypical EPEC isolates that do not possess the full spectrum of virulence genes, underlining the importance of identifying the H antigen for clinical risk assessment.     

Class 1 integrons contributes to antibiotic resistance among clinical isolates of Escherichia coli producing extended-spectrum beta-lactamases

Objectives: The objective of this study is to determine the prevalence of antibiotic resistance factors, including the production of extended-spectrum beta-lactamases (ESBLs) and the presence of class 1 integrons among Escherichia coli isolated from clinical specimens. Materials and Methods: Bacterial species identification was performed using a VITEK-2 system (VITEK2 GN-card; bioMérieux, France). Antimicrobial susceptibility testing was determined using the disk diffusion method according to the 2010 Clinical and Laboratory Standards Institute guidelines. Polymerase chain reaction (PCR) was used to detect integrons and amplify variable regions of the bla_TEM, bla_SHV and blaCTX-M genes. Gene cassettes were detected by deoxyribonucleic acid sequencing. Results: In this study, 58% (100/172) of clinical E. coli isolates were identified as ESBL producers. We found that 90% of the ESBL-producing E. coli isolates harbored the bla_CTX-M gene, whereas only 59% and 32% possessed the bla_TEM and bla_SHV genes respectively. The presence of class 1 integrons was based on the detection of the integrase gene by PCR. A total of 69% of the ESBL-producing isolates were integron-positive. Resistance to 10 antibiotics, including quinolones, sulfonamides and β-lactam/enzyme inhibitors, was significantly higher in the class 1 integron-positive isolates (P < 0.05). The occurrence of class 1 integrons in bla_TEM, bla_SHV and bla_CTX-M gene carriers was 72.9%, 84.4% and 68.9%, respectively. Class 1 integrons were detected in 61.5% of the isolates with only one ESBL genotype, but in 69.0% and 92.3% of the isolates with two or three different ESBL genotypes, respectively. Conclusions: Our findings indicate that clinical strains of bacteria with multiple ESBL genotypes may have greater opportunities to carry class 1 integrons.     

Prolonged and mixed non-O157 Escherichia coli infection in an Australian household

An Australian family was identified through a Public Health follow up on a Shiga-toxigenic Escherichia coli (STEC) positive bloody diarrhoea case, with three of the four family members experiencing either symptomatic or asymptomatic STEC shedding. Bacterial isolates were submitted to stx sequence sub-typing, multi-locus variable number tandem repeat analysis (MLVA), multi-locus sequence typing (MLST) and binary typing. The analysis revealed that there were multiple strains of STEC being shed by the family members, with similar virulence gene profiles and the same serogroup but differing in their MLVA and MLST profiles. This study illustrates the potentially complicated nature of non-O157 STEC infections and the importance of molecular epidemiology in understanding disease clusters.     

Diarrhoeagenic microbes by real-time PCR in Rwandan children under 5 years of age with acute gastroenteritis

Acute gastroenteritis is a main cause of disease and death among children in low-income countries. The causality rates and pathogenic characteristics of putative aetiological agents remain insufficiently known. We used real-time PCR targeting 16 diarrhoeagenic agents to analyse stool samples from children ≤5.0 years old with acute diarrhoea in Rwanda. Among the 880 children (median age 14.2 months; 41% female) at least one pathogen was detected in 92% and two or more agents in 63% of cases. Rotavirus was detected in 36.9%, adenovirus in 39.7%, enterotoxigenic Escherichia coli (ETEC) with genes for labile (eltB) or stable (estA) toxin in 31.3% and 19.0%, E. coli with eae or bfpA genes in 25.2% and 14.2%, Shigella in 17.5% and Cryptosporidium in 7.8%. Rotavirus and ETEC-estA were associated with more severe dehydration than diarrhoea due to other causes. Shigella was associated with bloody stools and higher CRP. Microbial loads (C_t values) of rotavirus, ETEC-estA and Shigella were associated with severity of symptoms. Rotavirus, ETEC-estA and E. coli with bfpA were associated with younger age, Shigella with older age. Antibiotic treatment was given to 42% and was associated with dehydration, fever and CRP, but not with pathogen. We conclude that rotavirus and ETEC-estA were the most important causes of diarrhoea with dehydration, that Shigella caused bloody diarrhoea but less severe dehydration, that microbial loads of rotavirus, ETEC-estA and Shigella were associated with severity of symptoms, and that antibiotic use was frequent and in poor agreement with microbiological findings.     

Towards a Molecular Definition of Enterohemorrhagic Escherichia coli (EHEC): Detection of Genes Located on O Island 57 as Markers To Distinguish EHEC from Closely Related Enteropathogenic E. coli Strains

Among strains of Shiga-toxin (Stx) producing Escherichia coli (STEC), seven serogroups (O26, O45, O103, O111, O121, O145, and O157) are associated with severe clinical illness in humans. These strains are also called enterohemorrhagic E. coli (EHEC), and the development of methods for their reliable detection from food has been challenging thus far. PCR detection of major EHEC virulence genes stx₁, stx₂, eae, and O-serogroup-specific genes is useful but does not identify EHEC strains specifically. Searching for the presence of additional genes issued from E. coli O157:H7 genomic islands OI-122 and OI-71 increases the specificity but does not clearly discriminate EHEC from enteropathogenic E. coli (EPEC) strains. Here, we identified two putative genes, called Z2098 and Z2099, from the genomic island OI-57 that were closely associated with EHEC and their stx-negative derivative strains (87% for Z2098 and 91% for Z2099). Z2098 and Z2099 were rarely found in EPEC (10% for Z2098 and 12% for Z2099), STEC (2 and 15%), and apathogenic E. coli (1% each) strains. Our findings indicate that Z2098 and Z2099 are useful genetic markers for a more targeted diagnosis of typical EHEC and new emerging EHEC strains.     

Molecular characterisation of enteroinvasive Escherichia coli O136:K78 isolates from patients of a diarrhoea outbreak in China

Purpose: A diarrhoea outbreak occurred in a kindergarten, which caused 21 relevant infected cases. Our object was to confirm the pathogens and their molecular characterisation. Materials and Methods: Faecal samples from 21 patients were collected on the 3^rd day after their symptom onset, and a regular epidemiological investigation was conducted. Bacterial isolation was performed in accordance with standard laboratory protocol, serological and molecular characterisations were determined by serum agglutination test and real-time polymerase chain reaction (PCR) method, respectively. The pulsed field gel electrophoresis (PFGE) and 16S rRNAs were conducted to determine the homology. Results: Eleven enteroinvasive Escherichia coli (EIEC) O136:K78 strains were isolated. The serum agglutination test showed that all strains’ serotypes were E. coli (EIEC) O136:K78. Real-time PCR showed that 10 (91%) strains carried the invasion plasmid antigen H gene (ipaH), carried by all four Shigella species and EIEC. The strain that didn’t carry the ipaH gene had different biochemical reactions of L-lizyna and L-rhamnose with the other strains. The complete 16S rRNA sequences showed 98.4% identity between ipaH-negative isolate and the others, and the PFGE indicated that the ipaH-negative isolate was not homological with other isolates in this diarrhoea outbreak. Conclusions: The diarrhoea outbreak was caused by E. coli (EIEC) O136:K78.     

A cross-sectional study on aetiology of diarrhoeal disease,India

Background: Global, regional and national estimates clearly place diarrhoeal diseases as a major, albeit to an extant neglected public health problem. Deaths of children aged <5 years owing to diarrhoea was estimated to be 1.87 million at the global level (uncertainty range from 1.56 to 2.19 million), which is approximately 19% of total child deaths. Objectives: The present report is a cross-sectional study undertaken to estimate the role of various aetiological agents causing diarrhoea in North Karnataka and adjoining areas of Maharashtra and Goa. Methods: Three hundred stool samples were collected from patients seeking health care at KLES Dr. Prabhakar Kore Hospital and Medical Research Centre, Belgaum; and processed for detection of various bacterial, viral and parasitic agents. Results: Bacterial pathogens attributed to 65.7% of diarrhoea cases, followed by viral infection (22%), parasitic infection (16.3%) and infection by Candida spp. (5.6%). The study identified Escherichia coli in general and Enteropathogenic E. coli in particular, and Group A Rotavirus to be the most frequently isolated pathogens among diarrhoea patients. Conclusion: The data generated from the current study will help the health officials for better interventional and treatment strategies for diarrhoeal diseases.     

Characterization of Enterohemorrhagic Escherichia coli O111 and O157 Strains Isolated from Outbreak Patients in Japan

In April and May 2011, there was a serious food-poisoning outbreak in Japan caused by enterohemorrhagic Escherichia coli (EHEC) strains O111:H8 and O157:H7 from raw beef dishes at branches of a barbecue restaurant. This outbreak involved 181 infected patients, including 34 hemolytic-uremic syndrome (HUS) cases (19%). Among the 34 HUS patients, 21 developed acute encephalopathy (AE) and 5 died. Patient stool specimens yielded E. coli O111 and O157 strains. We also detected both EHEC O111 stx₂ and stx-negative E. coli O111 strains in a stock of meat block from the restaurant. Pulsed-field gel electrophoresis (PFGE) and multilocus variable-number tandem-repeat analysis (MLVA) showed that the stx-negative E. coli O111 isolates were closely related to EHEC O111 stx₂ isolates. Although the EHEC O157 strains had diverse stx gene profiles (stx₁, stx₂, and stx₁ stx₂), the PFGE and MLVA analyses indicated that these isolates originated from a single clone. Deletion of the Stx2-converting prophage from the EHEC O111 stx₂ isolates was frequently observed during in vitro growth, suggesting that strain conversion from an EHEC O111 stx₂ to an stx-negative strain may have occurred during infection.     

Real-Time Multiplex PCR Assay and Melting Curve Analysis for Identifying Diarrheagenic Escherichia coli

A real-time multiplex PCR assay was designed to amplify the virulence genes eae, pEAF, aatA, daaC, elt, est, ipaH, stx₁, and stx₂ for the detection of all diarrheagenic Escherichia coli pathotypes. This assay proved to be more sensitive and rapid than a conventional multiplex PCR for diarrheagenic E. coli isolates from children with diarrhea.