Survival of Indicator and Pathogenic Bacteria in Bovine Feces on Pasture

The survival of enteric bacteria was measured in bovine feces on pasture. In each season, 11 cow pats were prepared from a mixture of fresh dairy cattle feces and sampled for up to 150 days. Four pats were analyzed for Escherichia coli, fecal streptococci, and enterococci, and four inoculated pats were analyzed for Campylobacter jejuni and Salmonella enterica. Two pats were placed on drainage collectors, and another pat was fitted with a temperature probe. In the first 1 to 3 weeks, there were increases (up to 1.5 orders of magnitude) in the counts of enterococci (in four seasons), E. coli (three seasons), fecal streptococci (three seasons), and S. enterica (two seasons), but there was no increase in the counts of C. jejuni. Thereafter, the counts decreased, giving an average ranking of the times necessary for 90% inactivation of C. jejuni (6.2 days from deposition) < fecal streptococci (35 days) < S. enterica (38 days) < E. coli (48 days) < enterococci (56 days). The pat temperature probably influenced bacterial growth, but the pattern of increases and decreases was primarily determined by desiccation; growth occurred when the water content was greater than 80%, but at a water content of 70 to 75% counts decreased. E. coli and enterococcus regrowth appeared to result from pat rehydration. Of 20 monthly leaching losses of E. coli, 16 were <10% of the total counts in the pat, and 12 were <1%. Drainage losses of C. jejuni (generally <1%) were detected for only 1 to 2 months. Although enterococci exhibited the best survival rate, higher final counts suggested that E. coli is the more practical indicator of bovine fecal pollution.     

Prevalence of Campylobacter spp. in Cattle in Finland and Antimicrobial Susceptibilities of Bovine Campylobacter jejuni Strains

The study investigated the prevalence of Campylobacter spp. in Finnish cattle at slaughter and carcass contamination after slaughter. During the period January to December 2003, bovine rectal fecal samples (n = 952) and carcass surface samples (n = 948) from 12 out of 15 Finnish slaughterhouses were examined. In total, campylobacters were detected in 31.1% of fecal samples and in 3.5% of carcass surface samples. Campylobacter jejuni was isolated from 19.5%, Campylobacter coli from 2.2%, and presumptive Campylobacter hyointestinalis from 10.8% of fecal samples. Campylobacters were detected in 4.4% and 37.4% of the fecal samples examined both by direct culture and by enrichment (n = 730), respectively, suggesting a low level of campylobacters in the intestinal content. A slightly increasing trend was observed in the overall prevalence of campylobacters towards the end of summer and autumn. Seventeen different serotypes were detected among the fecal C. jejuni isolates using a set of 25 commercial antisera for serotyping heat-stable antigens (Penner) of C. jejuni by passive hemagglutination. The predominant serotypes, Pen2 and Pen4-complex, were isolated from 52% of the fecal samples. Subtyping by pulsed-field gel electrophoresis (SmaI) yielded 56 and 20 subtypes out of 330 fecal and 70 carcass C. jejuni isolates, respectively. MICs of ampicillin, enrofloxacin, erythromycin, gentamicin, nalidixic acid, and oxytetracycline for 187 C. jejuni isolates were determined using a commercial broth microdilution method. Sixteen (9%) of the isolates were resistant to at least one of the antimicrobials tested. Resistance to nalidixic acid was most commonly detected (6%). No multiresistance was observed.     

Health Risk from the Use of Roof-Harvested Rainwater in Southeast Queensland,Australia, as Potable or Nonpotable Water,Determined Using Quantitative Microbial Risk Assessment

A total of 214 rainwater samples from 82 tanks were collected in urban Southeast Queensland (SEQ) in Australia and analyzed for the presence and numbers of zoonotic bacterial and protozoal pathogens using binary PCR and quantitative PCR (qPCR). Quantitative microbial risk assessment (QMRA) analysis was used to quantify the risk of infection associated with the exposure to potential pathogens from roof-harvested rainwater used as potable or nonpotable water. Of the 214 samples tested, 10.7%, 9.8%, 5.6%, and 0.4% were positive for the Salmonella invA, Giardia lamblia β-giardin, Legionella pneumophila mip, and Campylobacter jejuni mapA genes, respectively. Cryptosporidium parvum oocyst wall protein (COWP) could not be detected. The estimated numbers of Salmonella, G. lamblia, and L. pneumophila organisms ranged from 6.5 × 10¹ to 3.8 × 10² cells, 0.6 × 10° to 3.6 × 10° cysts, and 6.0 × 10¹ to 1.7 × 10² cells per 1,000 ml of water, respectively. Six risk scenarios were considered for exposure to Salmonella spp., G. lamblia, and L. pneumophila. For Salmonella spp. and G. lamblia, these scenarios were (i) liquid ingestion due to drinking of rainwater on a daily basis, (ii) accidental liquid ingestion due to hosing twice a week, (iii) aerosol ingestion due to showering on a daily basis, and (iv) aerosol ingestion due to hosing twice a week. For L. pneumophila, these scenarios were (i) aerosol inhalation due to showering on a daily basis and (ii) aerosol inhalation due to hosing twice a week. The risk of infection from Salmonella spp., G. lamblia, and L. pneumophila associated with the use of rainwater for showering and garden hosing was calculated to be well below the threshold value of one extra infection per 10,000 persons per year in urban SEQ. However, the risk of infection from ingesting Salmonella spp. and G. lamblia via drinking exceeded this threshold value and indicated that if undisinfected rainwater is ingested by drinking, then the incidences of the gastrointestinal diseases salmonellosis and giardiasis are expected to range from 9.8 × 10° to 5.4 × 10¹ (with a mean of 1.2 × 10¹ from Monte Carlo analysis) and from 1.0 × 10¹ to 6.5 × 10¹ cases (with a mean of 1.6 × 10¹ from Monte Carlo analysis) per 10,000 persons per year, respectively, in urban SEQ. Since this health risk seems higher than that expected from the reported incidences of gastroenteritis, the assumptions used to estimate these infection risks are critically examined. Nonetheless, it would seem prudent to disinfect rainwater for use as potable water.Roof-harvested rainwater has received significant attention as a potential alternative source of potable and nonpotable water in regions where water is scarce (37). To encourage the use of roof-harvested rainwater, governmental bodies of many countries, such as Australia, Denmark, Germany, India, and New Zealand, are providing subsidies to residents to encourage the use of rainwater for domestic purposes. The use of rainwater is quite common in Australia, particularly in rural and remote areas, where reticulated mains or town water is not available. Recent water scarcity in several capital cities prompted the use of rainwater as an alternative source. For instance, the Queensland State Government initiated the “Home Water Wise Rebate Scheme,” which provides subsidies to Southeast Queensland (SEQ) residents who use rainwater as nonpotable water for domestic purposes (49). Over 260,000 householders were granted subsidies up to December 2008, when the scheme was concluded.There is a general community feeling that roof-harvested rainwater is safe to drink, and this is partially supported by limited epidemiological evidence (26). Some studies have reported that roof-harvested rainwater quality is generally acceptable for use as potable water (13, 29). In contrast, the presence of potential pathogens, such as Aeromonas spp. Campylobacter spp., Campylobacter jejuni, Salmonella spp., Legionella pneumophila, Giardia spp., Giardia lamblia, and Cryptosporidium spp., in roof-harvested rainwater samples has been reported (2, 9, 34, 45, 47, 48). Such pathogens can cause gastrointestinal illness in humans, with nausea, vomiting, and/or diarrhea occurring within 12 to 72 h (Salmonella enterica serovar Typhimurium) to 9 to 15 days (Giardia lamblia) after ingestion of contaminated water. L. pneumophila can cause the respiratory infection pneumonia, and the fatality rate can be 50% in immunocompromised patients (57).Direct routine monitoring of the microbiological quality of source water for all possible pathogens is not economically, technologically, or practically feasible. Consequently, traditional fecal indicators, such as fecal coliforms, Escherichia coli, and enterococci, have long been used to determine the presence of pathogens. Most studies assess the quality of roof-harvested rainwater based on the numbers of these fecal indicators (13, 30). However, the major limitation in using fecal bacteria as indicators is their poor correlation with the presence of pathogenic microorganisms in water (2, 30). An alternative is the measurement of pathogens using traditional culture-based methods. However, there are several limitations of such methods, including the underestimation of the bacterial number due to the presence of injured or stressed cells (10) and the fact that certain microorganisms in environmental waters can be viable but not culturable (39). Culture-based methods are also generally laborious and costly. Recent advances in molecular techniques such as PCR technology enable rapid, specific, and sensitive detection of many pathogens. Advances in PCR methodology also enable the quantification of potential pathogens in source water that are otherwise difficult and/or laborious to culture using traditional microbiological methods. In view of this, we used binary PCR (presence/absence)- and quantitative PCR (qPCR)-based assays to first detect and then quantify zoonotic pathogens in samples from roof-harvested rainwater in SEQ residential houses.The aims of the research study were 2-fold: (i) to quantify the number and frequency of occurrence of Salmonella, G. lamblia, and L. pneumophila organisms in a range of domestic water tanks in SEQ by using qPCR-based methods and (ii) to apply quantitative microbial risk assessment (QMRA) analysis in order to estimate the risk of infection from exposure to these pathogens found in roof-harvested rainwater. The uniqueness of this study stems from the fact that instead of measuring fecal indicators, the pathogens that are capable of causing illness were quantified and this information was combined with QMRA to assess the human health risk of using roof-harvested rainwater as potable or nonpotable water.     

Influx of Enterococci and Associated Antibiotic Resistance and Virulence Genes from Ready-To-Eat Food to the Human Digestive Tract

The influx of enterococcal antibiotic resistance (AR) and virulence genes from ready-to-eat food (RTEF) to the human digestive tract was assessed. Three RTEFs (chicken salad, chicken burger, and carrot cake) were sampled from five fast-food restaurants five times in summer (SU) and winter (WI). The prevalence of enterococci was significantly higher in SU (92.0% of salad samples and 64.0% of burger samples) than in WI (64.0% of salad samples and 24.0% of burger samples). The overall concentrations of enterococci during the two seasons were similar (~10³ CFU/g); the most prevalent were Enterococcus casseliflavus (41.5% of isolates) and Enterococcus hirae (41.5%) in WI and Enterococcus faecium (36.8%), E. casseliflavus (27.6%), and Enterococcus faecalis (22.4%) in SU. Resistance in WI was detected primarily to tetracycline (50.8%), ciprofloxacin (13.8%), and erythromycin (4.6%). SU isolates were resistant mainly to tetracycline (22.8%), erythromycin (22.1%), and kanamycin (13.0%). The most common tet gene was tet(M) (35.4% of WI isolates and 11.9% of SU isolates). The prevalence of virulence genes (gelE, asa1, cylA, and esp) and marker genes for clinical isolates (EF_0573, EF_0592, EF_0605, EF_1420, EF_2144, and pathogenicity island EF_0050) was low (≤12.3%). Genotyping of E. faecalis and E. faecium using pulsed-field gel electrophoresis revealed that the food contamination likely originated from various sources and that it was not clonal. Our conservative estimate (single AR gene copy per cell) for the influx of tet genes alone to the human digestive tract is 3.8 × 10⁵ per meal (chicken salad). This AR gene influx is frequent because RTEFs are commonly consumed and that may play a role in the acquisition of AR determinants in the human digestive tract.     

Prevalence of Campylobacter spp., Escherichia coli, and Salmonella Serovars in Retail Chicken, Turkey, Pork, and Beef from the Greater Washington, D.C., Area

A total of 825 samples of retail raw meats (chicken, turkey, pork, and beef) were examined for the presence of Escherichia coli and Salmonella serovars, and 719 of these samples were also tested for Campylobacter spp. The samples were randomly obtained from 59 stores of four supermarket chains during 107 sampling visits in the Greater Washington, D.C., area from June 1999 to July 2000. The majority (70.7%) of chicken samples (n = 184) were contaminated with Campylobacter, and a large percentage of the stores visited (91%) had Campylobacter-contaminated chickens. Approximately 14% of the 172 turkey samples yielded Campylobacter, whereas fewer pork (1.7%) and beef (0.5%) samples were positive for this pathogen. A total of 722 Campylobacter isolates were obtained from 159 meat samples; 53.6% of these isolates were Campylobacter jejuni, 41.3% were Campylobacter coli, and 5.1% were other species. Of the 212 chicken samples, 82 (38.7%) yielded E. coli, while 19.0% of the beef samples, 16.3% of the pork samples, and 11.9% of the turkey samples were positive for E. coli. However, only 25 (3.0%) of the retail meat samples tested were positive for Salmonella. Significant differences in the bacterial contamination rates were observed for the four supermarket chains. This study revealed that retail raw meats are often contaminated with food-borne pathogens; however, there are marked differences in the prevalence of such pathogens in different meats. Raw retail meats are potential vehicles for transmitting food-borne diseases, and our findings stress the need for increased implementation of hazard analysis of critical control point (HACCP) and consumer food safety education efforts.     

Bifidobacteria in Feces and Environmental Waters

Bifidobacteria have been recommended as potential indicators of human fecal pollution in surface waters even though very little is known about their presence in nonhuman fecal sources. The objective of this research was to shed light on the occurrence and molecular diversity of this fecal indicator group in different animals and environmental waters. Genus- and species-specific 16S rRNA gene PCR assays were used to study the presence of bifidobacteria among 269 fecal DNA extracts from 32 different animals. Twelve samples from three wastewater treatment plants and 34 water samples from two fecally impacted watersheds were also tested. The species-specific assays showed that Bifidobacterium adolescentis, B. bifidum, B. dentium, and B. catenulatum had the broadest host distribution (11.9 to 17.4%), whereas B. breve, B. infantis, and B. longum were detected in fewer than 3% of all fecal samples. Phylogenetic analysis of 356 bifidobacterial clones obtained from different animal feces showed that ca. 67% of all of the sequences clustered with cultured bifidobacteria, while the rest formed a supercluster with low sequence identity (i.e., <94%) to previously described Bifidobacterium spp. The B. pseudolongum subcluster (>97% similarity) contained 53 fecal sequences from seven different animal hosts, suggesting the cosmopolitan distribution of members of this clade. In contrast, two clades containing B. thermophilum and B. boum clustered exclusively with 37 and 18 pig fecal clones, respectively, suggesting host specificity. Using species-specific assays, bifidobacteria were detected in only two of the surface water DNA extracts, although other fecal anaerobic bacteria were detected in these waters. Overall, the results suggest that the use of bifidobacterial species as potential markers to monitor human fecal pollution in natural waters may be questionable.     

Evidence for Extensive Resistance Gene Transfer among Bacteroides spp. and among Bacteroides and Other Genera in the Human Colon

Transfer of antibiotic resistance genes by conjugation is thought to play an important role in the spread of resistance. Yet virtually no information is available about the extent to which such horizontal transfers occur in natural settings. In this paper, we show that conjugal gene transfer has made a major contribution to increased antibiotic resistance in Bacteroides species, a numerically predominant group of human colonic bacteria. Over the past 3 decades, carriage of the tetracycline resistance gene, tetQ, has increased from about 30% to more than 80% of strains. Alleles of tetQ in different Bacteroides species, with one exception, were 96 to 100% identical at the DNA sequence level, as expected if horizontal gene transfer was responsible for their spread. Southern blot analyses showed further that transfer of tetQ was mediated by a conjugative transposon (CTn) of the CTnDOT type. Carriage of two erythromycin resistance genes, ermF and ermG, rose from <2 to 23% and accounted for about 70% of the total erythromycin resistances observed. Carriage of tetQ and the erm genes was the same in isolates taken from healthy people with no recent history of antibiotic use as in isolates obtained from patients with Bacteroides infections. This finding indicates that resistance transfer is occurring in the community and not just in clinical environments. The high percentage of strains that are carrying these resistance genes in people who are not taking antibiotics is consistent with the hypothesis that once acquired, these resistance genes are stably maintained in the absence of antibiotic selection. Six recently isolated strains carried ermB genes. Two were identical to erm(B)-P from Clostridium perfringens, and the other four had only one to three mismatches. The nine strains with ermG genes had DNA sequences that were more than 99% identical to the ermG of Bacillus sphaericus. Evidently, there is a genetic conduit open between gram-positive bacteria, including bacteria that only pass through the human colon, and the gram-negative Bacteroides species. Our results support the hypothesis that extensive gene transfer occurs among bacteria in the human colon, both within the genus Bacteroides and among Bacteroides species and gram-positive bacteria.     

Campylobacter spp., Giardia spp., Cryptosporidium spp., Noroviruses, and Indicator Organisms in Surface Water in Southwestern Finland, 2000-2001

A total of 139 surface water samples from seven lakes and 15 rivers in southwestern Finland were analyzed during five consecutive seasons from autumn 2000 to autumn 2001 for the presence of various enteropathogens (Campylobacter spp., Giardia spp., Cryptosporidium spp., and noroviruses) and fecal indicators (thermotolerant coliforms, Escherichia coli, Clostridium perfringens, and F-RNA bacteriophages) and for physicochemical parameters (turbidity and temperature); this was the first such systematic study. Altogether, 41.0% (57 of 139) of the samples were positive for at least one of the pathogens; 17.3% were positive for Campylobacter spp. (45.8% of the positive samples contained Campylobacter jejuni, 25.0% contained Campylobacter lari, 4.2% contained Campylobacter coli, and 25.0% contained Campylobacter isolates that were not identified), 13.7% were positive for Giardia spp., 10.1% were positive for Cryptosporidium spp., and 9.4% were positive for noroviruses (23.0% of the positive samples contained genogroup I and 77.0% contained genogroup II). The samples were positive for enteropathogens significantly (P < 0.05) less frequently during the winter season than during the other sampling seasons. No significant differences in the prevalence of enteropathogens were found when rivers and lakes were compared. The presence of thermotolerant coliforms, E. coli, and C. perfringens had significant bivariate nonparametric Spearman's rank order correlation coefficients (P < 0.001) with samples that were positive for one or more of the pathogens analyzed. The absence of these indicators in a logistic regression model was found to have significant predictive value (odds ratios, 1.15 × 10⁸, 7.57, and 2.74, respectively; P < 0.05) for a sample that was negative for the pathogens analyzed. There were no significant correlations between counts or count levels for thermotolerant coliforms or E. coli or the presence of F-RNA phages and pathogens in the samples analyzed.     

Methanobrevibacter ruminantium as an Indicator of Domesticated-Ruminant Fecal Pollution in Surface Waters

A PCR-based assay (Mrnif) targeting the nifH gene of Methanobrevibacter ruminantium was developed to detect fecal pollution from domesticated ruminants in environmental water samples. The assay produced the expected amplification product only when the reaction mixture contained DNA extracted from M. ruminantium culture, bovine (80%), sheep (100%), and goat (75%) feces, and water samples from a bovine waste lagoon (100%) and a creek contaminated with bovine lagoon waste (100%). The assay appears to be specific and sensitive and can distinguish between domesticated- and nondomesticated-ruminant fecal pollution in environmental samples.     

Phylogenetic analysis of the fecal microbial community in herbivorous land and marine iguanas of the Galápagos Islands using 16S rRNA-based pyrosequencing

Herbivorous reptiles depend on complex gut microbial communities to effectively degrade dietary polysaccharides. The composition of these fermentative communities may vary based on dietary differences. To explore the role of diet in shaping gut microbial communities, we evaluated the fecal samples from two related host species—the algae-consuming marine iguana (Amblyrhynchus cristatus) and land iguanas (LI) (genus Conolophus) that consume terrestrial vegetation. Marine and LI fecal samples were collected from different islands in the Galápagos archipelago. High-throughput 16S rRNA-based pyrosequencing was used to provide a comparative analysis of fecal microbial diversity. At the phylum level, the fecal microbial community in iguanas was predominated by Firmicutes (69.5±7.9%) and Bacteroidetes (6.2±2.8%), as well as unclassified Bacteria (20.6±8.6%), suggesting that a large portion of iguana fecal microbiota is novel and could be involved in currently unknown functions. Host species differed in the abundance of specific bacterial groups. Bacteroides spp., Lachnospiraceae and Clostridiaceae were significantly more abundant in the marine iguanas (MI) (P-value>1E−9). In contrast, Ruminococcaceae were present at >5-fold higher abundance in the LI than MI (P-value>6E−14). Archaea were only detected in the LI. The number of operational taxonomic units (OTUs) in the LI (356–896 OTUs) was >2-fold higher than in the MI (112–567 OTUs), and this increase in OTU diversity could be related to the complexity of the resident bacterial population and their gene repertoire required to breakdown the recalcitrant polysaccharides prevalent in terrestrial plants. Our findings suggest that dietary differences contribute to gut microbial community differentiation in herbivorous lizards. Most importantly, this study provides a better understanding of the microbial diversity in the iguana gut; therefore facilitating future efforts to discover novel bacterial-associated enzymes that can effectively breakdown a wide variety of complex polysaccharides.     

Antibiotic Resistance of Enterococci in American Bison (Bison bison) from a Nature Preserve Compared to That of Enterococci in Pastured Cattle

Enterococci isolated from a bison population on a native tall-grass prairie preserve in Kansas were characterized and compared to enterococci isolated from pastured cattle. The species diversity was dominated by Enterococcus casseliflavus in bison (62.4%), while Enterococcus hirae was the most common isolate from cattle (39.7%). Enterococcus faecalis was the second most common species isolated from bison (16%). In cattle, E. faecalis and Enterococcus faecium were isolated at lower percentages (3.2% and 1.6%, respectively). No resistance to ampicillin, chloramphenicol, gentamicin, or high levels of vancomycin was detected from either source. Tetracycline and erythromycin resistance phenotypes, encoded by tetO and ermB, respectively, were common in cattle isolates (42.9% and 12.7%, respectively). A significant percentage of bison isolates (8% and 4%, respectively) were also resistant to these two antibiotics. The tetracycline resistance genes from both bison and cattle isolates resided on mobile genetic elements and showed a transfer frequency of 10⁻⁶ per donor, whereas erythromycin resistance was not transferable. Resistance to ciprofloxacin was found to be higher in enterococci from bison (14.4%) than in enterococci isolated from cattle (9.5%). The bison population can serve as a sentinel population for studying the spread and origin of antibiotic resistance.     

Quantification of Bacterial Groups within Human Fecal Flora by Oligonucleotide Probe Hybridization

To investigate the population structure of the predominant phylogenetic groups within the human adult fecal microbiota, a new oligonucleotide probe designated S-G-Clept-1240-a-A-18 was designed, validated, and used with a set of five 16S rRNA-targeted oligonucleotide probes. Application of the six probes to fecal samples from 27 human adults showed additivity of 70% of the total 16S rRNA detected by the bacterial domain probe. The Bacteroides group-specific probe accounted for 37% ± 16% of the total rRNA, while the enteric group probe accounted for less than 1%. Clostridium leptum subgroup and Clostridium coccoides group-specific probes accounted for 16% ± 7% and 14% ± 6%, respectively, while Bifidobacterium and Lactobacillus groups made up less than 2%.     

Detection of Human-Derived Fecal Pollution in Environmental Waters by Use of a PCR-Based Human Polyomavirus Assay

Regulatory agencies mandate the use of fecal coliforms, Escherichia coli or Enterococcus spp., as microbial indicators of recreational water quality. These indicators of fecal pollution do not identify the specific sources of pollution and at times underestimate health risks associated with recreational water use. This study proposes the use of human polyomaviruses (HPyVs), which are widespread among human populations, as indicators of human fecal pollution. A method was developed to concentrate and extract HPyV DNA from environmental water samples and then to amplify it by nested PCR. HPyVs were detected in as little as 1 μl of sewage and were not amplified from dairy cow or pig wastes. Environmental water samples were screened for the presence of HPyVs and two additional markers of human fecal pollution: the Enterococcus faecium esp gene and the 16S rRNA gene of human-associated Bacteroides. The presence of human-specific indicators of fecal pollution was compared to fecal coliform and Enterococcus concentrations. HPyVs were detected in 19 of 20 (95%) samples containing the E. faecium esp gene and Bacteroides human markers. Weak or no correlation was observed between the presence/absence of human-associated indicators and counts of indicator bacteria. The sensitivity, specificity, and correlation with other human-associated markers suggest that the HPyV assay could be a useful predictor of human fecal pollution in environmental waters and an important component of the microbial-source-tracking “toolbox.”     

Occurrence of Microbial Indicators and Clostridium perfringens in Wastewater, Water Column Samples, Sediments, Drinking Water, and Weddell Seal Feces Collected at McMurdo Station, Antarctica

McMurdo Station, Antarctica, has discharged untreated sewage into McMurdo Sound for decades. Previous studies delineated the impacted area, which included the drinking water intake, by using total coliform and Clostridium perfringens concentrations. The estimation of risk to humans in contact with the impacted and potable waters may be greater than presumed, as these microbial indicators may not be the most appropriate for this environment. To address these concerns, concentrations of these and additional indicators (fecal coliforms, Escherichia coli, enterococci, coliphage, and enteroviruses) in the untreated wastewater, water column, and sediments of the impacted area and drinking water treatment facility and distribution system at McMurdo Station were determined. Fecal samples from Weddell seals in this area were also collected and analyzed for indicators. All drinking water samples were negative for indicators except for a single total coliform-positive sample. Total coliforms were present in water column samples at higher concentrations than other indicators. Fecal coliform and enterococcus concentrations were similar to each other and greater than those of other indicators in sediment samples closer to the discharge site. C. perfringens concentrations were higher in sediments at greater distances from the discharge site. Seal fecal samples contained concentrations of fecal coliforms, E. coli, enterococci, and C. perfringens similar to those found in untreated sewage. All samples were negative for enteroviruses. A wastewater treatment facility at McMurdo Station has started operation, and these data provide a baseline data set for monitoring the recovery of the impacted area. The contribution of seal feces to indicator concentrations in this area should be considered.     

Sunlight Inactivation of Fecal Indicator Bacteria and Bacteriophages from Waste Stabilization Pond Effluent in Fresh and Saline Waters

Sunlight inactivation in fresh (river) water of fecal coliforms, enterococci, Escherichia coli, somatic coliphages, and F-RNA phages from waste stabilization pond (WSP) effluent was compared. Ten experiments were conducted outdoors in 300-liter chambers, held at 14°C (mean river water temperature). Sunlight inactivation (k_S) rates, as a function of cumulative global solar radiation (insolation), were all more than 10 times higher than the corresponding dark inactivation (k_D) rates in enclosed (control) chambers. The overall k_S ranking (from greatest to least inactivation) was as follows: enterococci > fecal coliforms ≥ E. coli > somatic coliphages > F-RNA phages. In winter, fecal coliform and enterococci inactivation rates were similar but, in summer, enterococci were inactivated far more rapidly. In four experiments that included freshwater-raw sewage mixtures, enterococci survived longer than fecal coliforms (a pattern opposite to that observed with the WSP effluent), but there was little difference in phage inactivation between effluents. In two experiments which included simulated estuarine water and seawater, sunlight inactivation of all of the indicators increased with increasing salinity. Inactivation rates in freshwater, as seen under different optical filters, decreased with the increase in the spectral cutoff (50% light transmission) wavelength. The enterococci and F-RNA phages were inactivated by a wide range of wavelengths, suggesting photooxidative damage. Inactivation of fecal coliforms and somatic coliphages was mainly by shorter (UV-B) wavelengths, a result consistent with photobiological damage. Fecal coliform repair mechanisms appear to be activated in WSPs, and the surviving cells exhibit greater sunlight resistance in natural waters than those from raw sewage. In contrast, enterococci appear to suffer photooxidative damage in WSPs, rendering them susceptible to further photooxidative damage after discharge. This suggests that they are unsuitable as indicators of WSP effluent discharges to natural waters. Although somatic coliphages are more sunlight resistant than the other indicators in seawater, F-RNA phages are the most resistant in freshwater, where they may thus better represent enteric virus survival.     

Fecal indicators and zoonotic pathogens in household drinking water taps fed from rainwater tanks in Southeast Queensland, Australia

In this study, the microbiological quality of household tap water samples fed from rainwater tanks was assessed by monitoring the numbers of Escherichia coli bacteria and enterococci from 24 households in Southeast Queensland (SEQ), Australia. Quantitative PCR (qPCR) was also used for the quantitative detection of zoonotic pathogens in water samples from rainwater tanks and connected household taps. The numbers of zoonotic pathogens were also estimated in fecal samples from possums and various species of birds by using qPCR, as possums and birds are considered to be the potential sources of fecal contamination in roof-harvested rainwater (RHRW). Among the 24 households, 63% of rainwater tank and 58% of connected household tap water (CHTW) samples contained E. coli and exceeded Australian drinking water guidelines of <1 CFU E. coli per 100 ml water. Similarly, 92% of rainwater tanks and 83% of CHTW samples also contained enterococci. In all, 21%, 4%, and 13% of rainwater tank samples contained Campylobacter spp., Salmonella spp., and Giardia lamblia, respectively. Similarly, 21% of rainwater tank and 13% of CHTW samples contained Campylobacter spp. and G. lamblia, respectively. The number of E. coli (P = 0.78), Enterococcus (P = 0.64), Campylobacter (P = 0.44), and G. lamblia (P = 0.50) cells in rainwater tanks did not differ significantly from the numbers observed in the CHTW samples. Among the 40 possum fecal samples tested, Campylobacter spp., Cryptosporidium parvum, and G. lamblia were detected in 60%, 13%, and 30% of samples, respectively. Among the 38 bird fecal samples tested, Campylobacter spp., Salmonella spp., C. parvum, and G. lamblia were detected in 24%, 11%, 5%, and 13% of the samples, respectively. Household tap water samples fed from rainwater tanks tested in the study appeared to be highly variable. Regular cleaning of roofs and gutters, along with pruning of overhanging tree branches, might also prove effective in reducing animal fecal contamination of rainwater tanks.     

A Rapid,Specific Membrane Filtration Procedure for Enumeration of Enterococci in Recreational Water

A two-step membrane filter (MF) method with mE medium, upon which the membrane must be incubated for 48 h and then transferred to a substrate medium to differentiate enterococci, is recommended by the U.S. Environmental Protection Agency to measure enterococci in fresh and marine recreational waters. The original mE medium was modified by reducing the triphenyltetrazolium chloride from 0.15 to 0.02 g/liter and adding 0.75 g of indoxyl β-d-glucoside per liter. The new MF medium, mEI medium, detected levels of enterococci in 24 h comparable to those detected by the original mE medium in 48 h, with the same level of statistical confidence. In addition, the use of mEI medium eliminated the need to transfer the membrane to a substrate medium to differentiate enterococci from other genera of the fecal streptococcal group. Colonies from mEI medium were examined to determine the rates of false-positive and false-negative occurrences. mEI medium had a false-positive rate of 6.0% and a false-negative rate of 6.5%. Interlaboratory testing of the MF method with mEI medium demonstrated that the relative reproducibility standard deviations among laboratories ranged from 2.2% for marine water to 18.9% for freshwater. The comparative recovery studies, specificity determinations, and multilaboratory evaluation indicated that mEI medium has analytical performance characteristics equivalent to those of mE medium. The simplicity of use and decreased incubation time with mEI medium will facilitate the detection and quantification of enterococci in fresh and marine recreational waters.The use of enterococci as an indicator of fecal contamination of recreational water was recommended by the U.S. Environmental Protection Agency (13) in 1986. The recommendation was based on studies which demonstrated that enterococci had a strong direct relationship to swimming-associated illness in both marine water (3) and freshwater (7) environments. A two-step membrane filter (MF) procedure described by Levin et al. (11) was used to quantify enterococci in these studies and is the procedure recommended for measuring the quality of recreational water by the U.S. Environmental Protection Agency Ambient Water Quality Criteria for Bacteria—1986 (13).The two-step MF procedure for enterococci requires 48 h of incubation of the MF at 41°C on a selective primary isolation medium (mE agar) followed by transfer of the MF to an in situ esculin-iron agar (EIA) substrate medium, which is incubated for 20 min at 41°C. Pink to red colonies on the MF that produce a brownish black precipitate on EIA are identified as enterococci. The brownish black precipitate formed on EIA is the result of the hydrolysis of esculin to glucose and coumarin by the enzyme β-glucosidase. Coumarin forms a black precipitate in the presence of ferric citrate. The selective characteristics of the primary isolation medium (mE agar) result from the addition of nalidixic acid, cycloheximide (Acti-Dione), and triphenyltetrazolium chloride (TTC) to the medium and the elevated incubation temperature of 41°C. Nalidixic acid inhibits gram-negative bacteria, cycloheximide inhibits fungi, and TTC (0.15 g/liter) differentiates enterococci from other gram-positive cocci and inhibits background organisms. The specificity of the medium was reported to be 10% false-positive and 11.7% false-negative (11).In 1980, Dufour (6) described a medium, similar to that of Levin et al. (11), for use in a single-step, 24-h MF procedure to enumerate enterococci in marine water and freshwater. The medium contained nalidixic acid, cycloheximide, a reduced concentration of TTC, and indoxyl β-d-glucoside, a chromogenic cellobiose analog used in place of esculin in the primary medium of Levin et al. (11) to differentiate enterococci from fecal streptococci. The addition of indoxyl β-d-glucoside into microbiological media results in β-glucosidase-positive enterococci producing an insoluble indigo blue complex which diffuses into the surrounding media, forming a blue halo around the colony.The present study was undertaken to (i) evaluate modifications to the commercially available base medium mE agar which would produce recovery of enterococci equivalent to that in the two-step, 48-h procedure in a single-step, 24-h procedure; (ii) determine the specificity of the modified medium (mEI medium) for enterococci; and (iii) determine, through collaborative study, the variability among laboratories using mEI medium for samples from various aquatic environments.     

Prevalence and Genetic Characterization of Shiga Toxin-Producing Escherichia coli Isolates from Slaughtered Animals in Bangladesh

To determine the prevalence of Shiga toxin (Stx)-producing Escherichia coli (STEC) in slaughter animals in Dhaka, Bangladesh, we collected rectal contents immediately after animals were slaughtered. Of the samples collected from buffalo (n = 174), cows (n = 139), and goats (n = 110), 82.2%, 72.7%, and 11.8% tested positive for stx₁ and/or stx₂, respectively. STEC could be isolated from 37.9%, 20.1%, and 10.0% of the buffalo, cows, and goats, respectively. STEC O157 samples were isolated from 14.4% of the buffalo, 7.2% of the cows, and 9.1% of the goats. More than 93% (n = 42) of the STEC O157 isolates were positive for the stx₂, eae, katP, etpD, and enterohemorrhagic E. coli hly (hly_EHEC) virulence genes. STEC O157 isolates were characterized by seven recognized phage types, of which types 14 (24.4%) and 31 (24.4%) were predominant. Subtyping of the 45 STEC O157 isolates by pulsed-field gel electrophoresis showed 37 distinct restriction patterns, suggesting a heterogeneous clonal diversity. In addition to STEC O157, 71 STEC non-O157 strains were isolated from 60 stx-positive samples from 23.6% of the buffalo, 12.9% of the cows, and 0.9% of the goats. The STEC non-O157 isolates belonged to 36 different O groups and 52 O:H serotypes. Unlike STEC O157, most of the STEC non-O157 isolates (78.9%) were positive for stx₁. Only 7.0% (n = 5) of the isolates were positive for hly_EHEC, and none was positive for eae, katP, and etpD. None of the isolates was positive for the iha, toxB, and efa1 putative adhesion genes. However, 35.2% (n = 25), 11.3% (n = 8), 12.7% (n = 9), and 12.7% (n = 9) of the isolates were positive for the lpf_O113, saa, lpfA_O157/01-141, and lpfA_O157/OI-154 genes, respectively. The results of this study provide the first evidence that slaughtered animals like buffalo, cows, and goats in Bangladesh are reservoirs for STEC, including the potentially virulent STEC strain O157.     

Prevalence, Persistence, and Molecular Characterization of Glycopeptide-Resistant Enterococci in Norwegian Poultry and Poultry Farmers 3 to 8 Years after the Ban on Avoparcin

Environmental reservoirs of glycopeptide-resistant enterococci (GRE) in Norway have been linked to former growth promoting use of the glycopeptide avoparcin in poultry production. We have examined the prevalence of fecal GRE in poultry and poultry farmers 3 to 8 years after the Norwegian avoparcin ban in 1995 and performed molecular analyses of the GRE population. Fecal samples from poultry farmers and their flocks on 29 previously avoparcin-exposed farms were collected on five occasions during the study period (1998 to 2003). All flocks (100%) were GRE positive in 1998. Throughout the study period, 78.5% of the poultry samples were GRE positive. Glycopeptide-resistant Enterococcus faecium (GREF) was isolated from 27.6% of the farmer samples in 1998 and from 27.8% of the samples collected between 1998 and 2003. The prevalence of fecal GRE in poultry declined significantly during the study period, but prevalence in samples from the farmers did not decline. PCR analysis revealed a specific Tn1546-plasmid junction fragment in 93.9% of E. faecium isolates. A putative postsegregation killing (PSK) system linked to Tn1546 was detected in 97.1% of the isolates examined. Multilocus sequence typing of glycopeptide-susceptible (n = 10) and -resistant (n = 10) E. faecium isolates from humans (n = 10) and poultry (n = 10) on two farms displayed 17 different sequence types. The study confirms the continuing persistence of a widespread common plasmid-mediated vanA-pRE25-PSK element within a heterogeneous GRE population on Norwegian poultry farms 8 years after the avoparcin ban. Moreover, it suggests an important role of PSK systems in the maintenance of antimicrobial resistance determinants in reservoirs without apparent antimicrobial selection.     

Coxiella burnetii Shedding Routes and Antibody Response after Outbreaks of Q Fever-Induced Abortion in Dairy Goat Herds

Q fever is a zoonosis caused by Coxiella burnetii, a bacterium largely carried by ruminants and shed into milk, vaginal mucus, and feces. The main potential hazard to humans and animals is due to shedding of bacteria that can then persist in the environment and be aerosolized. The purpose of this study was to evaluate shedding after an outbreak of Q fever abortion in goat herds and to assess the relationship with the occurrence of abortions and antibody responses. Aborting and nonaborting goats were monitored by PCR for C. burnetii shedding 15 and 30 days after the abortion episodes. PCR analysis of all samples showed that 70% (n = 50) of the aborting and 53% (n = 70) of the nonaborting goats were positive. C. burnetii was shed into vaginal mucus, feces, and milk of 44%, 21%, and 38%, respectively, of goats that aborted and 27%, 20%, and 31%, respectively, of goats that delivered normally. Statistical comparison of these shedding results did not reveal any difference between these two groups. PCR results obtained for the vaginal and fecal routes were concordant in 81% of cases, whereas those for milk correlated with only 49% of cases with either vaginal or fecal shedding status. Serological analysis, using enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescence assay (IFA), and complement fixation tests, showed that at least 24% of the seronegative goats shed bacteria. Positive vaginal and fecal shedding, unlike positive milk shedding, was observed more often in animals that were weakly positive or negative by ELISA or IFA. Two opposite shedding trends were thus apparent for the milk and vaginal-fecal routes. Moreover, this study showed that a nonnegligible proportion of seronegative animals that delivered normally could excrete C. burnetii.