Evaluation of the Colilert® Test for Detection of Verotoxin-Producing Escherichia coli in Water

ABSTRACT

The objective of this study was to estimate the sensitivity of the Colilert® test for detection of different isolates of Escherichia coli (E. coli) O157:H7 and other verotoxin-producing E. coli (VTEC) serotypes from water for potential on-farm food safety application. E. coli isolates evaluated were of human, bovine, porcine, or food origin. The sensitivity of the Colilert® test was 92% overall for the verotoxin-producing serotypes of E. coli. However, none of the E. coli O157:H7 isolates were identified as E. coli. No false-positive results for E. coli were detected. The Colilert® test could be used to screen water supplies for E. coli on the farm, however, an important pathogenic E. coli sero-type is not detected by the Colilert® test.     

Molecular and phylogenetic characterization of multidrug resistant extended spectrum beta-lactamase producing Escherichia coli isolated from poultry and cattle in Odisha,India

The present study was undertaken to determine the occurrence and characterization of extended spectrum beta-lactamase (ESBL) producing Escherichia coli isolated from cattle and poultry in Odisha, India. Of 316 E. coli isolated from 305 samples (170 fecal samples from poultry and 135 milk samples from cattle), a total of 18 E. coli isolates were confirmed as ESBL producers by combination disc method and ESBL E-test. The isolates were resistant to oxyimino cephalosporins and monobactam as revealed by disc diffusion assay and determination of minimum inhibitory concentration. Resistance against other antibiotics was frequently noted as well. Further, beta-lactamase genes viz., blaSHV, blaCTXM, blaTEM and blaampC were detected in 17, 13, 9 and 2 isolates, respectively in PCR. Of the 18 ESBL strains, 16 were positive for class I integron (int1), nine of them carried sulphonamide resistance gene (sul1) and one harbored quinolone resistance gene (qnrB). Virulence markers for extraintestinal pathogenic E. coli like astA, tsh and iucD were also present in 4, 3 and 3 isolates, respectively. All the PCR amplified products were cloned and subjected to sequencing for homology analysis and data were submitted to gene bank. Sequence analysis of the amplified variable regions of class 1 integron of four representative isolates revealed the presence of aadA2 and dfrA12 gene cassettes conferring resistance to aminoglycosides and trimethoprim, respectively. Most of the ESBL producing strains emerged as single lineage through phylogenetic analysis by RAPD and ERIC PCR. This is the first ever systemic study on multidrug resistant ESBL producing E. coli in food producing animals from India.     

Reduced faecal shedding of Escherichia coli O157:H7 in cattle following systemic vaccination with γ-intimin C₂₈₀ and EspB proteins

Enterohaemorrhagic Escherichia coli (EHEC) O157:H7 is the most prevalent EHEC serotype that has been recovered from patients with haemolytic uremic syndrome (HUS) worldwide. Vaccination of cattle, the main reservoir of EHEC O157:H7, could be a logical strategy to fight infection in humans. This study evaluated a vaccine based on the carboxyl-terminal fragment of 280 amino acids of γ-intimin (γ-intimin C₂₈₀) and EspB, two key colonization factors of E. coli O157:H7. Intramuscular immunization elicited significantly high levels of serum IgG antibodies against both proteins. Antigen-specific IgA and IgG were also induced in saliva, but only the IgA response was significant. Following experimental challenge with E. coli O157:H7, a significant reduction in bacterial shedding was observed in vaccinated calves, compared to control group. These promising results suggest that systemic immunization of cattle with intimin and EspB could be a feasible strategy to reduce EHEC O157:H7 faecal shedding in cattle.     

Veterinärmedizinische Aspekte der Infektionen durch enterohämorrhagische E.-coli-Stämme (EHEC)

Enterohaemorrhagic Escherichia coli (EHEC) strains can cause severe foodborne human diseases and even fatalities. They are a subgroup of verocytotoxin producing E. colis (VTEC) with EHEC expressing additional virulence factors. Most strains produce the adhesion factor “intimin”. The prototype of this pathogroup is the E. coli serovar O157:H7, which is predominant worldwide. Ruminants are considered as the primary reservoir. High prevalence rates are reported from nearly all EU countries. The primary circle of infection and contamination of cattle livestocks and the surroundings by living and non-living vectors can hardly be broken through. Pre-slaughter washing of cattle as well as decontamination procedures of carcasses were not able to prevent increase of O157 diseases in the USA. To reduce the EHEC hazard the trimming of carcasses can be recommended. Although raw food of animal as well as of plant origin may be an important source of EHEC infection, the direct contact with animals and transmissions from person to person have to be considered important infection routes.     

Similarity of Campylobacter coli from pigs,poultry and man

Campylobacteriosis is one of the most frequently occurring acute gastroenteritis in humans and 10% are caused by Campylobacter coli. A total of 136 isolates of C. coli from humans, poultry, and pigs were identified by multiplex polymerase chain reaction (PCR) and genetically characterized and compared by ribotyping. Automatic riboprints were performed with the PstI restriction enzyme and RiboPrinter®. All poultry, pig and human strains represented a heterogeneous spectre of ribotypes. Ten of 23 human strains (43%) could be given DUP-ID from the library represented by DUP-PSTI-1200 (n = 7), DUP-PST1-1201 (n = 2) and DUP-PSTI-1211 (n = 1). Eighteen of 28 (64%) poultry strains were given a DUP-ID. Three isolates were closely related to human strains DUP-PSTI-1201 (n = 2) and DUP-PSTI-1200 (n = 1) and may play an important role in the epidemiology of campylobacteriosis. Nineteen of 85 pig isolates (23%) could be given a DUP-ID, but none were common to human isolates. An overlap was found among poultry and pig isolates with DUP-PSTI-1182 and DUP-PSTI-1140.     

Sewage sludge and liquid pig manure as possible sources of antibiotic resistant bacteria

Within the last decades, the environmental spread of antibiotic resistant bacteria has become a topic of concern. In this study, liquid pig manure (n=305) and sewage sludge (n=111) - used as agricultural fertilizers between 2002 and 2005 - were investigated for the presence of Escherichia coli, Enterococcus faecalis and Enterococcus faecium. Bacteria were tested for their resistance against 40 chemotherapeutics including several “reserve drugs”. E. coli (n=613) from pig manure were at a significantly higher degree resistant to streptomycin, doxycycline, spectinomycin, cotrimoxazole, and chloramphenicol than E. coli (n=116) from sewage sludge. Enterococci (Ent. faecalis, n=387, and Ent. faecium, n=183) from pig manure were significantly more often resistant to high levels of doxycycline, rifampicin, erythromycin, and streptomycin than Ent. faecalis (n=44) and Ent. faecium (n=125) from sewage sludge. Significant differences in enterococcal resistance were also seen for tylosin, chloramphenicol, gentamicin high level, fosfomycin, clindamicin, enrofloxacin, moxifloxacin, nitrofurantoin, and quinupristin/dalfopristin. By contrast, aminopenicillins were more effective in enterococci from pig manure, and mean MIC-values of piperacillin+tazobactam and third generation cefalosporines were significantly lower in E. coli from pig manure than in E. coli from sewage sludge. 13.4% (E. coli) to 25.3% (Ent. faecium) of pig manure isolates were high-level multiresistant to substances from more than three different classes of antimicrobial agents. In sewage sludge, high-level-multiresistance reached from 0% (Ent. faecalis) to 16% (Ent. faecium). High rates of (multi-) resistant bacteria in pig manure emphasize the need for a prudent - cautious - use of antibiotics in farm animals.     

Bacterial quality of drinking water stored in containers by boat households in Hue City, Vietnam

Objectives  To examine the bacterial quality of drinking water stored in containers by boat households in the river basin of Hue City, and associated factors. Methods  Ready-to-drink water stored in containers on boats was collected from 766 households. Escherichia coli (E. coli), total coliforms, and Enterobacteriaceae in the water were examined by the rehydratable dry-film plating method. Socioeconomic characteristics, water source, handling practices, and proficiency of disease prevention of individual households were assessed. Results   E. coli, over ten counts of total coliforms, and over ten counts of Enterobacteriaceae were detected in 25.7, 44.5, and 51.5% of 1-ml samples of ready-to-drink water stored in containers on the boats. Bacterial contamination of the water stored in containers by boat households was significantly associated with use of river water as a source of drinking water, non-boiling before storing containers for drinking, and limited proficiency in disease prevention regardless of the influence of socioeconomic characteristics of the households (P < 0.01, P < 0.05, P < 0.01, respectively). Conclusions  Bacterial contamination of ready-to-drink water stored by boat households was indicated. The households’ proficiency in disease prevention buffered contamination. A comprehensive health promotion program with a wide range of contents is required for the communities of boat households.     

Comparison of Escherichia coli Isolates from humans, food, and farm and companion animals for presence of Shiga toxin-producing E. coli virulence markers

The objective of this study was to characterize Escherichia coli isolates from dairy cows/feedlots, calves, mastitis, pigs, dogs, parrot, iguana, human disease, and food products for prevalence of Shiga toxin-producing E. coli (STEC) virulence markers. The rationale of the study was that, isolates of the same serotypes that were obtained from different sources and possessed the same marker profiles, could be cross-species transmissible. Multiplex polymerase chain reaction (PCR) was used to detect presence of genes encoding Shiga toxin 1 and 2 (stx1 and stx2), H7 flagella (flicC), enterohemolysin (hly) and intimin (eaeA) in E. coli isolates (n = 400). Shiga toxin-producing isolates were tested for production of Shiga toxins (Stx1 and Stx2 and enterohemolysin. Of the E. coli O157:H7/H- strains, 150 of 164 (mostly human, cattle, and food) isolates were stx+. Sixty-five percent of O157 STEC produced both Stx1 and Stx2; 32% and 0.7% produced Stx2 or Stx1, respectively. Ninety-eight percent of O157 STEC had sequences for genes encoding intimin and enterohemolysin. Five of 20 E. coli O111, 4 of 14 O128 and 4 of 10 O26 were stx+ . Five of 6 stx+ O26 and O111 produced Stx1, however, stx+ O128 were Stx-negative. Acid resistance (93.3%) and tellurite resistance (87.3%) were common attributes of O157 STEC, whereas, non-O157 stx+ strains exhibited 38.5% and 30.8% of the respective resistances. stx-positive isolates were mostly associated with humans and cattle, whereas, all isolates from mastitis (n = 105), and pigs, dogs, parrot and iguanas (n = 48) were stx-negative. Multiplex PCR was an effective tool for characterizing STEC pathogenic profiles and distinguished STEC O157:H7 from other STEC. Isolates from cattle and human disease shared similar toxigenic profiles, whereas isolates from other disease sources had few characteristics in common with the former isolates. These data suggest interspecies transmissibility of certain serotypes, in particular, STEC O157:H7, between humans and cattle.     

Immunization of cattle with a combination of purified intimin-531, EspA and Tir significantly reduces shedding of Escherichia coli O157:H7 following oral challenge

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a human pathogen that can cause gastrointestinal disease with potentially fatal consequences as a result of systemic Shiga toxin activity. Cattle are the main reservoir host of EHEC O157 and interventions need to be developed that prevent cattle colonization or limit shedding of the organism from this host. EHEC O157 predominately colonizes the bovine terminal rectum and requires a type III secretion system (T3SS) for adherence and persistence at this site. A vaccine based on concentrated bacterial supernatant that contains T3S proteins has shown some efficacy. Here we have demonstrated that vaccination with a combination of antigens associated with T3S-mediated adherence; the translocon filament protein, EspA, the extracellular region of the outer membrane adhesin, intimin, and the translocated intimin receptor (Tir) significantly reduced shedding of EHEC O157 from experimentally infected animals. Furthermore, this protection may be augmented by addition of H7 flagellin to the vaccine preparation that has been previously demonstrated to be partially protective in cattle. Protection correlates with systemic and mucosal antibody responses to the defined antigens and validates the targeting of these colonization factors.     

Prevalence of CTX-M extended-spectrum beta-lactamases and sequence type 131 in Korean blood,urine, and rectal Escherichia coli isolates

A high proportion of extended-spectrum beta-lactamase (ESBL) producing Escherichia coli are of the ST131 lineage, but there are few estimates of ST131 prevalence among ESBL-negative E. coli. Without this information, it is difficult to evaluate the contribution of the ST131 lineage to the emergence and spread of ESBL E. coli. A total of 1658 E. coli isolates were collected at Gachon University Gil Medical Center in Korea from 2006 to 2008. The antibiotic resistance profile was determined for all isolates, and ESBL-positive isolates were screened for the presence of CTX-M-type ESBLs. All ESBL-positive (n = 84) and a representative sample of ESBL-negative (n = 100) isolates were screened for O25b-ST131 using a PCR-based assay. The isolates were further classified on the basis of fumC and fimH types, which allowed for a comparison of the two typing methods. 5.7% of isolates were ESBL-positive, 87% of which contained CTX-M-type ESBLs. There was no significant difference in the prevalence of ST131 between ESBL-positive and -negative groups; 14% of ESBL-positive isolates and 9% of tested ESBL-negative isolates were ST131 by CH-typing. ST131-positive isolates harbored CTX-M-1-group ESBLs (including CTX-M-15) more frequently than other CTX-M types, and exhibited greater levels of antibiotic resistance than non-ST131 isolates. Furthermore, a number of isolates identified as O25b-ST131 by PCR corresponded to non-ST131 sequence types by CH-typing, emphasizing the need to consider the testing method when comparing reported prevalences of ST131.     

Escherichia albertii in Wild and Domestic Birds

Escherichia albertii has been associated with diarrhea in humans but not with disease or infection in animals. However, in December 2004, E. albertii was found, by biochemical and genetic methods, to be the probable cause of death for redpoll finches (Carduelis flammea) in Alaska. Subsequent investigation found this organism in dead and subclinically infected birds of other species from North America and Australia. Isolates from dead finches in Scotland, previously identified as Escherichia coli O86:K61, also were shown to be E. albertii. Similar to the isolates from humans, E. albertii isolates from birds possessed intimin (eae) and cytolethal distending toxin (cdtB) genes but lacked Shiga toxin (stx) genes. Genetic analysis of eae and cdtB sequences, multilocus sequence typing, and pulsed-field gel electrophoresis patterns showed that the E. albertii strains from birds are heterogeneous but similar to isolates that cause disease in humans.     

Prevalence,characterisation and antimicrobial resistance of Campylobacter and Salmonella in raw poultrymeat in the UK, 2003–2005

This study was conducted to determine the prevalence and antimicrobial resistance of Campylobacter and Salmonella isolates from retail poultrymeat in the UK during 2003–2005. Poultrymeat (n = 2104) were more frequently contaminated with Campylobacter (57.3%) than with Salmonella (6.6%). Chicken exhibited the highest contamination from Campylobacter (60.9%), followed by duck (50.7%), turkey (33.7%) and other poultrymeat (34.2%). Duck had the highest contamination from Salmonella (29.9%), compared with chicken (5.6%), turkey (5.6%), and other poultrymeat (8.6%). C. jejuni predominated in raw chicken, whereas C. coli predominated in turkey and duck. C. coli isolates were more likely to exhibit antimicrobial drug resistance, including quinolones, than C. jejuni. Salmonella Enteritidis was the most frequent Salmonella serotype isolated. Salmonella isolates from turkey exhibited higher rates of multiple drug resistance (55.6%) than isolates from chicken (20.9%) and duck (13.6%). The findings reinforce the importance of thorough cooking of poultrymeat and good hygiene to avoid cross-contamination.     

Microbiological quality of water in a city with persistent and recurrent waterborne diseases under tropical sub-rural conditions: The case of Kikwit City,Democratic Republic of the Congo

The availability of safe drinking water in sub-Saharan countries remains a major challenge because poor sanitation has been the cause of various outbreaks of waterborne disease due to the poor microbiological quality of water used for domestic purposes. The faecal indicator bacteria (FIB) used in the present study included Escherichia coli (E. coli) and Enterococcus (ENT). FIB and aerobic mesophilic bacteria (AMB) were quantified during July 2015 (dry season) and November 2015 (rainy season) in order to assess the quality of drinking water from wells (n = 3; P1–P3), and two rivers, the River Lukemi (RLK, n = 3) and River Luini (RLN, n = 2) in the city of Kikwit, which is located in the province of Kwilu in the Democratic Republic of the Congo. Kikwit is well known for its outbreaks of persistent and recurrent waterborne diseases including Entamoeba, Shigella, typhoid fever, cholera, and Ebola Viral Hemorrhagic Fever. Consequently, E. coli, ENT, and AMB were quantified in water samples according to the standard international methods for water quality determination using the membrane filtration method. The FIB characterization was performed for human-specific Bacteroides by PCR using specific primers. The results obtained revealed high FIB concentrations in river samples collected during both seasons. For example, E. coli respectively reached 4.3 × 10⁴ and 9.2 × 10⁴ CFU 100 mL^?1 in the dry season and the wet season. ENT reached 5.3 × 10³ CFU 100 mL^?1 during the dry season and 9.8 × 10³ CFU 100 mL^?1 in the wet season. The pollution was significantly worse in the wet season compared to the dry season. Surprisingly, no faecal contamination was observed in well water samples collected in the dry season while E. coli and ENT were detected in all wells in the wet season with values of 6, 7, and 11 CFU mL^?1 for E. coli in wells P1–P3, respectively and 3, 5, 9 CFU mL^?1for ENT in the same wells. Interestingly, the PCR assays for human-specific Bacteroides HF183/HF134 indicated that 97–100% captured in all analyses of isolated FIB were of human origin. The results indicate that contamination of E. coli, ENT, and AMB in the studied water resources increases during the wet season. This study improves understanding of the microbiological pollution of rivers and wells under tropical conditions and will guide future municipal/local government decisions on improving water quality in this region which is characterised by persistent and recurrent waterborne diseases. Although the epidemiology can be geographically localised, the effects of cross border transmission can be global. Therefore, the research results presented in this article form recommendations to municipalities/local authorities and the approach and procedures can be carried out in a similar environment.     

Factors associated with post-treatment E. coli contamination in households practising water treatment: a study of rural Cambodia

The purpose of this study was to assess factors associated with Escherichia coli (E. coli) contamination in rural households in Cambodia that have adopted household water treatment. The following factors were significantly associated (α < 0.05) with apparent E. coli contamination: cleaning the drinking vessel with untreated water, not drying the cup (with a cloth), accessing treated water by the use of a scoop (ref: using a tap), having more than one untreated water storage container, having an untreated water storage container that appeared dirty on the outside, and cows living within 10 m of the household. This study provides further evidence confirming previous studies reporting an association between inadequate cleanliness of water storage containers and household drinking water contamination, and identifies practical recommendations statistically associated with reduced post-treatment E. coli contamination in the household setting in rural Cambodia.     

Molecular characterization of enterohemorrhagic Escherichia coli hemolysin gene (EHEC-hlyA)-harboring isolates from cattle reveals a diverse origin and hybrid diarrheagenic strains

In the present study we investigated the occurrence of Escherichia coli strains harboring the gene encoding enterohemorrhagic E. coli hemolysin (EHEC-HlyA) in cattle and the association of this gene with various diarrheagenic E. coli (DEC) pathotypes. First, the bovine E. coli isolates were screened for EHEC-hlyA gene by PCR, and then they were characterized for the phylogenetic groups and the presence of the major virulence genes of different DEC pathotypes. In total, 25 virulence gene profiles were observed in 54 EHEC-hlyA+ isolates that reflect a considerable heterogeneity. The EHEC-hlyA+ strains were mostly associated with EHEC (72%), while only 7.4% were enteropathogenic E. coli (EPEC). We also showed the presence of estA gene of enterotoxigenic E. coli (ETEC) in 6 isolates (11.1%). Interestingly, two of the estA+ strains showed hybrid pathotypes with one carrying eae/estA (EPEC/ETEC), and the other one stx2/astA/estA (EHEC/ETEC). None of the isolates were related to enteroaggregative E. coli (EAggEC), enteroinvasive E. coli (EIEC), and necrotoxigenic E. coli (NTEC). The EHEC-plasmid encoded genes occurred in seven different combinations with EHEC-hlyA/saa/subA/espP being the most prevalent (46.3%). All stx −/eae+ strains carried O island 57 (OI-57) molecular marker(s) that may indicate these to be the progenitors of EHEC or strains losing stx. The most prevalent phylogroup was B1 (61.1%), but the most heterogeneous strains including the hybrid strains belonged to A phylogroup. Overall, our results indicate that cattle EHEC-hlyA encoding E. coli isolates consist of diverse diarrheagenic strains with the possible existence of hybrid pathotypes. Future studies are required to clarify the evolutionary aspects and clinical significance of these strains in humans and domestic animals.     

Residue content of beef feedlot manure after feeding diethylstilbestrol,chlortetracycline and ronnel and the use of stirofos to reduce population of fly larvae in feedlot manure

Two beef cattle feedlot experiments were conducted to determine the amount of certain agricultural chemicals that are likely to be found in both fresh and stored feedlot manure and to investigate fly control in this manure. In experiment 1, diethylstilbestrol (DES), chlortetracycline (CTC), and ronnel were used as feed additives. Fresh manure, stored manure, runoff water, manure weathered on pasture, and soil from pasture fertilized with manure were analyzed for these additives. Stirofos was added to fresh manure as a larvicide for fly control. In experiment 2, the residue aspects of DES and CTC were repeated. In this experiment, stirofos instead of ronnel was fed with DES and CTC. Sixty-eight percent of the DES fed to cattle appeared in fresh manure and 52% in manure stored for 12 weeks. Comparable percentage values were 17 and 11% for CTC and 13 and 3% for ronnel; somewhat less DES and CTC were found when a concentrate diet was fed. Detectable amounts of DES, CTC, and ronnel were not found in runoff water, weathered manure, or soil. Adding an emulsifiable concentrate formulation of stirofos directly to manure at a rate of approximately 45 ppm of wet manure completely controlled the larvae of house fly (Musca domestica L.) whereas feeding stirofos at a rate of 1.5 mg per kg of body weight daily reduced larval counts 82% in manure from forage-fed heifers and 63% in manure from concentrate-fed heifers. Stirofos was not detected in runoff water, weathered waste, or soil     

Screening of tropical estuarine water in south-west coast of India reveals emergence of ARGs-harboring hypervirulent Escherichia coli of global significance

The goal of this study was to investigate the involvement of a tropical Indian estuary in the emergence of antibiotic resistance genes (ARGs)-harboring hypervirulent E. coli of global significance. A total of 300 E. coli isolates was tested for antibiotic susceptibility to β-lactams, aminoglycosides, chloramphenicol, quinolones, sulphonamides, tetracyclines, and trimethoprim. The E. coli isolates were screened for the presence of antibiotic resistance genes (bla_TEM, bla_CTX-M, tetA, tetB, sul1, sul2, strA, aphA2, catI, dhfr1, and dhfr7), integrase (int1, int2, and int3), Shiga toxin genes (stx1 and stx2) and extraintestinal virulence genes (papAH, papC, sfa/focDE, kpsMT II, and iutA). The highest prevalence of antibiotic resistance was observed for ampicillin, followed by tetracycline, and nalidixic acid. Among E. coli isolates, 64% were resistant to at least one of the 15 antibiotics tested, and approximately 40% were multiple antibiotic-resistant (MAR). More than 40% (n?=?122) of E. coli isolates had ARGs. Integrase 1 (int1) was found in 7.6% of E. coli isolates. Among E. coli isolates, 16.3% (n?=?49) were extraintestinal pathogenic E. coli (ExPEC), and approximately 34.6% (n?=?17) of ExPEC had ARGs. A hypervirulent ARGs-harboring STEC was isolated. The prevalence of Shiga toxin-producing E. coli (STEC) was low (n?=?1). The prevalence of ARGs-harboring pathogenic E. coli isolates was higher in stations close to the City (urban area), than that of other stations. ERIC-PCR (enterobacterial repetitive intergenic consensus sequence polymerase chain reaction) analysis revealed a high degree of genetic diversity among the ARGs-harboring E. coli isolates. The results demonstrate a high prevalence of ARGs-harboring E. coli in estuarine water and confirm the need for a better wastewater treatment facility and proper control measures to reduce the discharge of sewage and wastewater into the aquatic environments.     

Broiler Chickens as Source of Human Fluoroquinolone-Resistant Escherichia coli, Iceland

To investigate feed as a source for fluoroquinolone-resistant Escherichia coli in broiler chickens, we compared antimicrobial drug–resistant E. coli from broiler feed and broilers with ciprofloxacin-resistant human clinical isolates by using pulsed-field gel electrophoresis. Feed was implicated as a source for ciprofloxacin-resistant broiler-derived E. coli and broilers as a source for ciprofloxacin-resistant human-derived E. coli.     

Prevalence of protozoa, viruses, coliphages and indicator bacteria in groundwater and river water in the Kathmandu Valley, Nepal

Limited information is available on the prevalence of waterborne pathogens in aquatic environments in developing countries. In this study, water samples were collected from nine shallow wells and a river in the Kathmandu Valley, Nepal, during the rainy season in 2009 and were subjected to detection of waterborne protozoa, viruses and coliphages using a recently developed method for simultaneous concentration of protozoa and viruses in water. Escherichia coli and total coliforms were also tested as indicator bacteria. At least one type of the five pathogens tested (Cryptosporidium, Giardia, human adenoviruses, and noroviruses of genogroups I and II) was detected in five groundwater samples (56%) (1000 ml each) from shallow wells. Compared with groundwater samples, the pathogens were more abundant in the river water sample (100 ml); the concentrations of Cryptosporidium and Giardia were 140 oocysts/l and 8500 cysts/l, respectively, and the mean threshold cycle (Ct) values in real-time RT-PCR were 34.3, 36.8 and 34.0 for human adenoviruses and noroviruses of genogroups I and II, respectively. Genotyping of F-RNA coliphages by real-time RT-PCR was successfully used to differentiate human and animal faecal contamination in the samples. Moreover, for the groundwater samples, protozoa and viruses were detected only in E. coli-positive samples, suggesting that E. coli may be an appropriate indicator of pathogen contamination of valley groundwater.