Genetic diversity of Nipah virus in Bangladesh.

BACKGROUND: Nipah virus (NiV) infection, often fatal in humans, is primarily transmitted in Bangladesh through the consumption of date palm sap contaminated by Pteropus bats. Person-to-person transmission is also common and increases the concern of large outbreaks. This study aimed to characterize the molecular epidemiology, phylogenetic relationship, and the evolution of the nucleocapsid gene (N gene) of NiV. METHODS: We conducted molecular detection, genetic characterization, and Bayesian time-scale evolution analyses of NiV using pooled Pteropid bat roost urine samples from an outbreak area in 2012 and archived RNA samples from NiV case patients identified during 2012-2018 in Bangladesh. RESULTS: NiV-RNA was detected in 19% (38/456) of bat roost urine samples and among them; nine N gene sequences were recovered. We also retrieved sequences from 53% (21 out of 39) of archived RNA samples from patients. Phylogenetic analysis revealed that all Bangladeshi strains belonged to NiV-BD genotype and had an evolutionary rate of 4.64 × 10-4 substitutions/site/year. The analyses suggested that the strains of NiV-BD genotype diverged during 1995 and formed two sublineages. CONCLUSION: This analysis provides further evidence that the NiV strains of the Malaysian and Bangladesh genotypes diverged recently and continue to evolve. More extensive surveillance of NiV in bats and human will be helpful to explore strain diversity and virulence potential to infect humans through direct or person-to-person virus transmission.

Clinical characteristics, virulence factors and molecular typing of methicillin-resistant Staphylococcus aureus infections in Shenzhen City, China.

Methicillin-resistant Staphylococcus aureus (MRSA) has emerged as a serious hospital and community-acquired infection and some strains are associated with greater severity. We investigated the clinical variability and molecular characteristics of MRSA infections in Shenzhen, China through a study at nine sentinel hospitals from January to December 2014. MRSA infections were classified as community-associated (CA-MRSA), healthcare-associated (HA-MRSA), and healthcare-associated community-onset (HACO-MRSA). In total, 812 MRSA isolates were collected and 183 of these were selected for further study. Patients with HA-MRSA infections were generally of greater age compared to other groups. Distinct body site and clinical presentations were evident in infected patients, e.g. CA-MRSA (skin and soft tissue, 53%), HA-MRSA (respiratory tract, 22%; surgical site, 20%; trauma wounds, 20%) and HACO-MRSA (mastitis, 47%). In contrast to HA-MRSA, other categories of strains were significantly more susceptible to gentamicin, sulfamethoxazole/trimethoprim, and tetracycline. No resistance to vancomycin or linezolid was recorded. The predominant clonal lineage within each strain category was CC59-t437-SCCmec IV/V-agr I (CA, 51·4%; HA, 28·9%; HACO, 52·9%) which exhibited characteristics of a traditional CA clone together with agr I which is more often associated with HA clones. In conclusion, for the three categories of MRSA infections, there were significant differences in clinical characteristics of patients, but the predominant clone in each category shared a similar genetic background which suggests that transmission of MRSA strains has occurred between the community and hospitals in Shenzhen.

Diarrhea burden due to natural infection with enterotoxigenic Escherichia coli in a birth cohort in a rural Egyptian community.

Enterotoxigenic Escherichia coli (ETEC) is commonly associated with diarrhea in Egyptian children. Children less than 3 years old in Abu Homos, Egypt, had approximately five diarrheal episodes per child every year, and at least one of these episodes was due to ETEC. The epidemiology of ETEC diarrhea among children living in a rural Egyptian community was further evaluated in this study. Between January 2004 and April 2007, 348 neonates were enrolled and followed for 2 years. Children were visited twice weekly, and a stool sample was obtained every 2 weeks regardless of symptomatology. A stool sample was obtained whenever a child had diarrhea. From the routine stool culture, five E. coli-like colonies were selected and screened for heat-labile and heat-stable toxins by GM1 enzyme-linked immunosorbent assay (ELISA) and further typed for colonization factor antigens by dot blot assay. Incidence of ETEC infection was estimated among children with diarrhea (symptomatic) and without diarrhea (asymptomatic). Incidence of diarrhea and ETEC-associated diarrhea was 7.8 and 1.48 per child-year, respectively. High risk of repeated ETEC diarrhea was associated with being over 6 months of age, warm season, male gender, and crowded sleeping conditions. Exclusive breast-feeding was protective for repeated ETEC infection. ETEC-associated diarrhea remains common among children living in the Nile Delta. The protective role of breast-feeding demonstrates the importance of promoting exclusive breast-feeding during, at least, the first 6 months of life.

Clinical characteristics of Clostridium difficile infection in hospitalized patients with antibiotic-associated diarrhea in a university hospital in China.

The purpose of this study was to identify clinical characteristics of Clostridium difficile infection (CDI) in patients with antibiotic-associated diarrhea (AAD). A prospective study was conducted among patients hospitalized in Fudan University Hospital Huashan from August 1, 2012 to July 31, 2013. Toxigenic C. difficile isolates were characterized by PCR ribotyping and multilocus sequence typing. AAD developed in 1.0 % (206/20437) of the antibiotic-treated hospitalized patients and toxigenic C. difficile was isolated from 30.6 % (63/206) of patients with AAD. The frequency of AAD was highest in the intensive care unit (10.7 %); however the proportion of CDI in AAD was highest in the Geriatric Unit (38 %). AAD ranged in severity from mild to moderate. One case with pseudomembranous colitis was identified. Use of carbapenems was found to significantly increase the risk of CDI (OR, 2.31; 95 % CI, 1.22-4.38; p = 0.011). Patient demographics, presumed risk factors, clinical manifestations and laboratory findings revealed no significant difference between patients with CDI and non-C. difficile AAD. Over 90 % of the patients with CDI or non-C. difficile AAD were cured. Two patients had CDI recurrence. Ribotype H was the dominant (18.8 %) genotype, followed by ribotype 012 and ribotype 017. C. difficile plays a significant role in AAD in our setting in China. Because the severity of diarrhea ranges from mild to moderate, it is difficult for Chinese clinicians to identify CDI from AAD patients, therefore CDI should be included in the routine differential diagnoses for hospitalized patients presenting with AAD.

Modifiable diarrhoea risk factors in Egyptian children aged <5 years.

By conducting a case-control study in two university hospitals, we explored the association between modifiable risk behaviours and diarrhoea. Children aged <5 years attending outpatient clinics for diarrhoea were matched by age and sex with controls. Data were collected on family demographics, socioeconomic indicators, and risk behaviour practices. Two rectal swabs and a stool specimen were collected from cases and controls. Samples were cultured for bacterial pathogens using standard techniques and tested by ELISA to detect rotavirus and Cryptosporidium spp. Four hundred cases and controls were enrolled between 2007 and 2009. The strongest independent risk factors for diarrhoea were: presence of another household member with diarrhoea [matched odds ratio (mOR) 4.9, 95% CI 2.8-8.4] in the week preceding the survey, introduction to a new kind of food (mOR 3, 95% CI 1.7-5.4), and the child being cared for outside home (mOR 2.6, 95% CI 1.3-5.2). While these risk factors are not identifiable, in some age groups more easily modifiable risk factors were identified including: having no soap for handwashing (mOR 6.3, 95% CI 1.2-33.9) for children aged 7-12 months, and pacifier use (mOR 1.9, 95% CI 1.0-3.5) in children aged 0-6 months. In total, the findings of this study suggest that community-based interventions to improve practices related to sanitation and hygiene, handwashing and food could be utilized to reduce the burden of diarrhoea in Egyptian children aged <5 years.

Comparative analysis of Shigella sonnei biotype g isolated from paediatric populations in Egypt, 1999-2005.

Strain characteristics of 51 Shigella sonnei isolates obtained from children seeking medical care (MC) and 48 isolates recovered during a prospective diarrhoea birth cohort (BC) study were compared. Biochemical characterization and antibiotic susceptibility testing determined that all S. sonnei isolates were biotype g and multidrug-resistant. Plasmid profiling identified 15 closely related patterns and XbaI pulsed-field gel electrophoresis confirmed the high degree of genetic similarity between isolates. All S. sonnei isolates harboured ipaH and class II integrase genes and 84∙3 and 80% of the MC and BC isolates, respectively carried the sen gene. Neither the class I integrase nor the set gene was detected. Our results indicate that S. sonnei isolates associated with severe diarrhoea were indistinguishable from those associated with mild diarrhoea. Additional genetic tests with greater discrimination might offer an opportunity to determine genetic differences within the globally disseminating biotype g clone.

Risk factors associated with tuberculosis infection among health care workers in Inner Mongolia, China.

SETTING: Health care workers (HCWs) are at increased risk for tuberculosis (TB) infection. In China, surveys examining TB infection among HCWs have not studied general health care facilities, compared tuberculin tests conducted using local protocols against an internationally accepted test or characterised risk factors. OBJECTIVE: To measure the prevalence of and risk factors for TB infection among HCWs in Inner Mongolia, China. DESIGN: Between April and August 2010, we administered QuantiFERON®-TB Gold In-Tube (QFT-GIT) tests, skin tests using Chinese tuberculin (TST) and surveys among HCWs at an infectious diseases hospital and a general medical hospital. We assessed whether demographic characteristics, personal exposure and work exposure were associated with QFT-GIT and TST positivity, and assessed agreement between test results. RESULTS: Of 999 HCWs, 683 (68%) were QFT-GIT-positive, which was associated with greater age, longer HCW career, TB disease in a co-worker and greater daily patient exposure using multivariable analysis. TST reactions ≥ 5 mm occurred in 69% of the HCWs; agreement between test results was low ( 0.22). CONCLUSIONS: The prevalence of TB infection among HCWs in Inner Mongolia is high; infection was associated with occupational exposure. Results from locally conducted TST are difficult to interpret. In China, TB infection control in health care facilities should be strengthened.

Epidemiological and molecular analysis of a waterborne outbreak of norovirus GII.4.

Contaminated water is one of the main sources of norovirus (NoV) gastroenteritis outbreaks globally. Waterborne NoV outbreaks are infrequently attributed to GII.4 NoV. In September 2009, a NoV outbreak affected a small school in Guangdong Province, China. Epidemiological investigations indicated that household use water, supplied by a well, was the probable source (relative risk 1·9). NoV nucleic acid material in concentrated well-water samples was detected using real-time RT-PCR. Nucleotide sequences of NoV extracted from diarrhoea and well-water specimens were identical and had the greatest sequence identity to corresponding sequences from the epidemic strain GII.4-2006b. Our report documents the first laboratory-confirmed waterborne outbreak caused by GII.4 NoV genotype in China. Our investigations indicate that well water, intended exclusively for household use but not for consumption, caused this outbreak. The results of this report serve as a reminder that private well water intended for household use should be tested for NoV.

Genetic diversity and antibiotic resistance in Shigella dysenteriae and Shigella boydii strains isolated from children aged <5 years in Egypt.

Diversity within Shigella dysenteriae (n=40) and Shigella boydii (n=30) isolates from children living in Egypt aged <5 years was investigated. Shigella-associated diarrhoea occurred mainly in summer months and in children aged <3 years, it commonly presented with vomiting and fever. Serotypes 7 (30%), 2 (28%), and 3 (23%) accounted for most of S. dysenteriae isolates; 50% of S. boydii isolates were serotype 2. S. dysenteriae and S. boydii isolates were often resistant to ampicillin, chloramphenicol and tetracycline (42%, 17%, respectively), although resistance varied among serotypes. Pulsed-field gel electrophoresis separated the isolates into distinct clusters correlating with species and serotype. Genetic differences in trimethoprim/sulfamethoxazole and ß-lactam-encoding resistance genes were also evident. S. dysenteriae and S. boydii are genetically diverse pathogens in Egypt; the high level of multidrug resistance associated with both pathogens and resistance to the most available inexpensive antibiotics underlines the importance of continuing surveillance.

Sentinel hospital-based surveillance of rotavirus diarrhea in iran.

BACKGROUND: Rotavirus is the most common causes of severe, acute diarrhea during childhood and is an important cause of morbidity and mortality in developing countries. We established active hospital-based surveillance of childhood diarrhea to assess the scope of severe rotavirus disease in Iran. METHODS: From May 2006 through April 2007, prospective surveillance of rotavirus diarrhea among children aged <5 years was conducted in 5 sentinel hospitals in Iran. Stool samples were tested for rotavirus using a commercially available enzyme immunoassay, and rotavirus-positive samples were genotyped using reverse-transcriptase polymerase chain reaction. RESULTS: Of 2198 children admitted to the hospital for acute gastroenteritis, 1298 (59.1%) had stool samples test positive for rotavirus by enzyme immunoassay. Of the rotavirus episodes, 85% occurred during the first 2 years of life, with the peak prevalence of severe rotavirus disease occurring from September through January. Among the 110 rotavirus-positive samples that were genotyped, G4P[8] was the most commonly detected rotavirus genotype (30.9% of strains). Other commonly detected genotypes included P[8] with G nontypeable (21.8%), G4 with P nontypeable (13.6%), G1[P8] (10.9%), and G2[P4] (5.5%). CONCLUSIONS: Rotavirus is the most common cause of severe diarrhea in Iran, which indicates that safe and effective rotavirus vaccination in Iran is a public health priority.

Phenotypic and genotypic analysis of enterotoxigenic Escherichia coli in samples obtained from Egyptian children presenting to referral hospitals.

Hospital surveillance was established in the Nile River Delta to increase the understanding of the epidemiology of diarrheal disease among Egyptian children. Between September 2000 and August 2003, samples obtained from children less than 5 years of age who had diarrhea and who were seeking hospital care were cultured for enteric bacteria. Colonies from each culture with a morphology typical of that of Escherichia coli were tested for the heat-labile (LT) and heat-stable (ST) toxins by a GM-1-specific enzyme-linked immunosorbent assay and colonization factor (CF) antigens by an immunodot blot assay. Enterotoxigenic E. coli (ETEC) isolates were recovered from 320/1,540 (20.7%) children, and ETEC isolates expressing a known CF were identified in 151/320 (47%) samples. ST CFA/I, ST CS6, ST CS14, and LT and ST CS5 plus CS6 represented 75% of the CFs expressed by ETEC isolates expressing a detectable CF. Year-to-year variability in the proportion of ETEC isolates that expressed a detectable CF was observed (e.g., the proportion that expressed CFA/I ranged from 10% in year 1 to 21% in year 3); however, the relative proportions of ETEC isolates expressing a CF were similar over the reporting period. The proportion of CF-positive ETEC isolates was higher among isolates that expressed ST. ETEC isolates expressing CS6 were isolated significantly less often (P < 0.001) than isolates expressing CFA/I in children less than 1 year of age. Macrorestriction profiling of CFA/I-expressing ETEC isolates by using the restriction enzyme XbaI and pulsed-field gel electrophoresis demonstrated a wide genetic diversity among the isolates that did not directly correlate with the virulence of the pathogen. The genome plasticity demonstrated in the ETEC isolates collected in this work suggests an additional challenge to the development of a globally effective vaccine for ETEC.

Surveillance and genotyping of Enterobacter sakazakii suggest its potential transmission from milk powder into imitation recombined soft cheese.

AIMS: To examine the presence of Enterobacter sakazakii in milk and milk-related products produced/distributed under Egyptian conditions and to probe possible transmission routes of the pathogen during the preparation of dairy products. METHODS AND RESULTS: One hundred and thirty-seven samples of milk and milk-related products were randomly collected from Egyptian markets and examined for the presence of Ent. sakazakii. The pathogen could be detected only in skimmed milk powder (SMP) and its related product, imitation recombined soft (IRS) cheese. Enterobacter sakazakii isolates recovered from these products were phenotypically similar and sensitive to all antibiotics examined in this study. They also showed indistinguishable banding patterns when subjected to macro-restriction profiling using pulsed-field gel electrophoresis (mrp-PFGE). One Ent. sakazakii isolate was inoculated into SMP that was used in the preparation of IRS cheese using two cheese making procedures. The pathogen could survive for up to 1 month in the IRS cheese prepared by either procedure. CONCLUSIONS: The simultaneous presence of Ent. sakazakii in SMP and IRS cheese samples collected within the same local market besides the phenotypic and genotypic similarities of isolates recovered from these samples suggested the possibility of Ent. sakazakii being transmitted from SMP into IRS cheese. This hypothesis was supported by the observation that the pathogen could survive in the IRS cheese prepared from contaminated SMP. SIGNIFICANCE AND IMPACT OF THE STUDY: The study highlights SMP and IRS cheese as potential transmission vehicles of Ent. sakazakii. It also raises concern on the microbiological safety of IRS cheese prepared from SMP.

Statistical comparison of Campylobacter jejuni subtypes from human cases and environmental sources.

AIM: To analyse Campylobacter jejuni typing data to define statistically which potential reservoirs and transmission sources contain isolates that are most similar to one another and to isolates from human infections. METHODS AND RESULTS: Serotyping and SmaI macrorestriction profiling data for C. jejuni isolates from human campylobacteriosis cases, chicken carcass rinses, duck, sheep, dairy and beef cattle faeces, river water, and sheep, beef and pork offal obtained from a defined rural area of New Zealand were compared using the Czekanowski proportional similarity index. Subtypes of isolates from ruminant animals, whether derived from their faeces or offals, were generally similar to one another. The spectrum of isolate subtypes from human cases was more similar to that from ruminant faeces than the other matrices considered. Isolate subtypes from chicken rinses, pork offal, water and duck faeces were not highly similar to those from other matrices. CONCLUSIONS: Results from a combination of phenotypic and genotypic approaches suggest that, for this rural population, exposures associated with a rural lifestyle may be significant sources of human campylobacteriosis. SIGNIFICANCE AND IMPACT OF THE STUDY: The Czekanowski index was applied to subtyping data and supported the greater importance of contact with livestock in campylobacteriosis cases associated with a rural setting, in comparison with urban studies that have identified poultry-related factors.

Epidemiology and genetic characterization of Shigella flexneri strains isolated from three paediatric populations in Egypt (2000-2004).

Ninety-seven isolates of Shigella flexneri from children seeking medical care from three sites in Egypt were characterized. Overall, 46.4% of children (median age 17 months) were febrile or reported blood in their stools, 25.8% were dehydrated and 16.5% were admitted to hospital. Serotypes 2a (37.1%), 1b (18.6%), 1c (17.5%), and 6 (15.5%) comprised over 88.7% of the total isolates. We observed marked resistance to ampicillin (87.6%), tetracycline (84.5%) and trimethoprim-sulfamethoxazole (63.9%). Pulsed-field electrophoresis grouped the majority of isolates within a serotype together, separately from isolates of an alternative serotype. The set gene was present in all serogroup 2a isolates, however, the sen gene was detected in every isolate. Our results show S. flexneri 1c has emerged as a dominant S. flexneri serotype in Egypt. Development and application of a Shigella vaccine should consider the diversity of Shigella serotypes within a geographical region prior to administration.

The occurrence of Campylobacter subtypes in environmental reservoirs and potential transmission routes.

AIM: To identify potential reservoirs and transmission routes of human pathogenic Campylobacter spp. METHODS AND RESULTS: An enrichment PCR method for the detection and identification of Campylobacter jejuni and/or Campylobacter coli in faecal, food and river water samples was applied to 1450 samples of 12 matrix types obtained from a defined geographical area. PCR-positive samples were cultured to yield isolates for typing, and the data for 616 C. jejuni isolates obtained. Serotyping and SmaI macrorestriction profiling using pulsed field gel electrophoresis revealed a high level of diversity within the isolates from each matrix. Campylobacter jejuni and C. coli subtypes indistinguishable from those obtained from human cases were detected in most of the matrices examined. No Campylobacter isolates were isolated from possum faeces. CONCLUSIONS: Ten of the 12 matrices examined may be involved in the transmission of human campylobacteriosis as they contained Campylobacter subtypes also isolated from clinical cases. SIGNIFICANCE AND IMPACT OF THE STUDY: Results indicate that, for this rural population, a range of potential transmission routes that could lead to campylobacteriosis exist. Their relative importance needs to be assessed from an exposure assessment standpoint.

Characterization of erythromycin resistance in Campylobacter coli and Campylobacter jejuni isolated from pig offal in New Zealand.

AIMS: To determine the level and mechanism(s) of antimicrobial resistance in Campylobacter isolates obtained from human and environmental sources from South Canterbury, New Zealand. METHODS AND RESULTS: A total of 251 Campylobacter isolates were tested for susceptibility to ciprofloxacin, erythromycin, nalidixic acid and tetracycline using disc diffusion assays. Five pig offal isolates were observed to be highly erythromycin resistant, with minimal inhibitory concentrations determined to be >/=256 microg ml(-1). Nucleotide sequencing of the 23S ribosomal DNA (rDNA) in these resistant isolates identified an A --> G change at Escherichia coli position 2059 that has been previously implicated in erythromycin resistance in Campylobacter coli. Macrorestriction profiling using pulsed-field gel electrophoresis showed these isolates were nonclonal. CONCLUSIONS: The majority of Campylobacter isolates from South Canterbury remain sensitive to the most clinically relevant antimicrobial agents. Our results support other reports showing that specific variations in the 23S rDNA contribute to erythromycin resistance. SIGNIFICANCE AND IMPACTS OF THE STUDY: This study defines the baseline frequency of antimicrobial resistance associated with Campylobacter isolates from South Canterbury, and discusses the likely molecular mechanisms conferring erythromycin resistance in this organism. Resistance to erythromycin in these isolates is not linked to a dominant Campylobacter clone and has likely arisen independently in different genetic lines exposed to selective antimicrobial pressure.

Genetic characterization of Pseudomonas 'NZI7'--a novel pathogen that results in a brown blotch disease of Agaricus bisporus.

AIMS: To characterize a novel pseudomonad isolate capable of causing brown blotch disease of Agaricus bisporus. METHODS AND RESULTS: Using the white-line-in-agar (WLA) assay, fluorescent pseudomonads isolated from a New Zealand mushroom farm were screened for the lipodepsipeptide tolaasin, a characteristic marker of Pseudomonas tolaasii. One isolate, NZI7, produced a positive WLA assay and caused brown lesions of A. bisporus comparable with those produced by Ps. tolaasii. However, genetic analysis suggested that Ps. tolaasii and NZI7 were genetically dissimilar, and that NZI7 is closely related to Pseudomonas syringae. Nucleotide sequence analyses of a gene involved in tolaasin production indicated that similar genes are present in both NZI7 and Ps. tolaasii. CONCLUSION: NZI7 represents a novel Pseudomonas species capable of causing brown blotch disease of A. bisporus. SIGNIFICANCE AND IMPACT OF THE STUDY: Phenotypic identification of Ps. tolaasii based on A. bisporus browning and positive WLA may have limited specificity.

Characterization by 16S rRNA sequence analysis of pseudomonads causing blotch disease of cultivated Agaricus bisporus.

Bacterial blotch of Agaricus bisporus has typically been identified as being caused by either Pseudomonas tolaasii (brown blotch) or Pseudomonas gingeri (ginger blotch). To address the relatedness of pseudomonads able to induce blotch, a pilot study was initiated in which pseudomonads were selectively isolated from mushroom farms throughout New Zealand. Thirty-three pseudomonad isolates were identified as being capable of causing different degrees of discoloration (separable into nine categories) of A. bisporus tissue in a bioassay. These isolates were also identified as unique using repetitive extragenic palindromic PCR and biochemical analysis. Relationships between these 33 blotch-causing organisms (BCO) and a further 22 selected pseudomonad species were inferred by phylogenetic analyses of near-full-length 16S rRNA gene nucleotide sequences. The 33 BCO isolates were observed to be distributed throughout the Pseudomonas fluorescens intrageneric cluster. These results show that in addition to known BCO (P. tolaasii, P. gingeri, and Pseudomonas reactans), a number of diverse pseudomonad species also have the ability to cause blotch diseases with various discolorations. Furthermore, observation of ginger blotch discoloration of A. bisporus being independently caused by many different pseudomonad species impacts on the homogeneity and classification of the previously described P. gingeri.

Enumeration of Campylobacter in New Zealand recreational and drinking waters.

AIMS: To use a published polymerase chain reaction (PCR) method for the detection and identification of thermotolerant Campylobacter species (Camp. jejuni, Camp. coli and Camp. lari) in tandem with a Most Probable Number (MPN) technique to enumerate these species in water samples. METHODS AND RESULTS: An initial study of 42 river water samples compared the use of conventional culture and PCR methods for the detection of Campylobacter in MPN enrichment tubes. It was found that all samples positive by culture were also positive by PCR. Thirty-seven percent more MPN tubes were positive by PCR compared with culture. The MPN/PCR technique was subsequently applied to 96 additional samples collected from rivers, drinking, roof and shallow ground water. Campylobacter was especially prevalent in river water (60% positive) and shallow ground water (75% positive) samples. Drinking water (29.2% positive) and roof water (37.5% positive) also contained Campylobacter, but the numbers detected were very low (maximum 0.3 and 0.56 MPN 100 ml-1, respectively). CONCLUSION: River waters contained Campylobacter at higher levels than any other water type and in a high percentage of the samples. Although Campylobacter was present in treated drinking water, the levels detected were low. SIGNIFICANCE AND IMPACT OF THE STUDY: These results suggest that water may act as a significant transmission route for human campylobacteriosis.

The rfaE gene from Escherichia coli encodes a bifunctional protein involved in biosynthesis of the lipopolysaccharide core precursor ADP-L-glycero-D-manno-heptose.

The intermediate steps in the biosynthesis of the ADP-L-glycero-D-manno-heptose precursor of inner core lipopolysaccharide (LPS) are not yet elucidated. We isolated a mini-Tn10 insertion that confers a heptoseless LPS phenotype in the chromosome of Escherichia coli K-12. The mutation was in a gene homologous to the previously reported rfaE gene from Haemophilus influenzae. The E. coli rfaE gene was cloned into an expression vector, and an in vitro transcription-translation experiment revealed a polypeptide of approximately 55 kDa in mass. Comparisons of the predicted amino acid sequence with other proteins in the database showed the presence of two clearly separate domains. Domain I (amino acids 1 to 318) shared structural features with members of the ribokinase family, while Domain II (amino acids 344 to 477) had conserved features of the cytidylyltransferase superfamily that includes the aut gene product of Ralstonia eutrophus. Each domain was expressed individually, demonstrating that only Domain I could complement the rfaE::Tn10 mutation in E. coli, as well as the rfaE543 mutation of Salmonella enterica SL1102. DNA sequencing of the rfaE543 gene revealed that Domain I had one amino acid substitution and a 12-bp in-frame deletion resulting in the loss of four amino acids, while Domain II remained intact. We also demonstrated that the aut::Tn5 mutation in R. eutrophus is associated with heptoseless LPS, and this phenotype was restored following the introduction of a plasmid expressing the E. coli Domain II. Thus, both domains of rfaE are functionally different and genetically separable confirming that the encoded protein is bifunctional. We propose that Domain I is involved in the synthesis of D-glycero-D-manno-heptose 1-phosphate, whereas Domain II catalyzes the ADP transfer to form ADP-D-glycero-D-manno-heptose.