Clonal populations of thermotolerant Enterobacteriaceae in recreational water and their potential interference with fecal Escherichia coli counts

Bacterial strains were isolated from beach water samples using the original Environmental Protection Agency method for Escherichia coli enumeration and analyzed by pulsed-field gel electrophoresis (PFGE). Identical PFGE patterns were found for numerous isolates from 4 of the 9 days sampled, suggesting environmental replication. 16S rRNA gene sequencing, API 20E biochemical testing, and the absence of beta-glucuronidase activity revealed that these clonal isolates were Klebsiella, Citrobacter, and Enterobacter spp. In contrast, 82% of the nonclonal isolates from water samples were confirmed to be E. coli, and 16% were identified as other fecal coliforms. These nonclonal isolates produced a diverse range of PFGE patterns similar to those of isolates obtained directly from untreated sewage and gull droppings. beta-Glucuronidase activity was critical in distinguishing E. coli from other fecal coliforms, particularly for the clonal isolates. These findings demonstrate that E. coli is a better indicator of fecal pollution than fecal coliforms, which may replicate in the environment and falsely elevate indicator organism levels.     

Comparison of gull feces-specific assays targeting the 16S rRNA genes of Catellicoccus marimammalium and Streptococcus spp

Two novel gull-specific quantitative PCR (qPCR) assays were developed using 16S rRNA gene sequences from gull fecal clone libraries: a SYBR green assay targeting Streptococcus spp. (gull3) and a hydrolysis TaqMan assay targeting Catellicoccus marimammalium (gull4). The objectives of this study were to compare the host specificity of a previous C. marimammalium qPCR assay (gull2) with that of the new markers and to examine the presence of the three gull markers in environmental water samples from different geographic locations. Most of the gull fecal samples tested (n = 255) generated positive signals with the gull2 and gull4 assays (i.e., >86%), whereas only 28% were positive with gull3. Low prevalence and abundance of tested gull markers (0.6 to 15%) were observed in fecal samples from six nonavian species (n = 180 fecal samples), whereas the assays cross-reacted to some extent (13 to 31%) with other (nongull) avian fecal samples. The gull3 assay was positive against fecal samples from 11 of 15 avian species, including gull. Of the presumed gull-impacted water samples (n = 349), 86%, 59%, and 91% were positive with the gull2, the gull3, and the gull4 assays, respectively. Approximately 5% of 239 non-gull-impacted water samples were positive with the gull2 and the gull4 assays, whereas 21% were positive witg the gull3 assay. While the relatively high occurrence of gull2 and gull4 markers in waters impacted by gull feces suggests that these assays could be used in environmental monitoring studies, the data also suggest that multiple avian-specific assays will be needed to accurately assess the contribution of different avian sources in recreational waters.     

Clonal Populations of Thermotolerant Enterobacteriaceae in Recreational Water and Their Potential Interference with Fecal Escherichia coli Counts

Bacterial strains were isolated from beach water samples using the original Environmental Protection Agency method for Escherichia coli enumeration and analyzed by pulsed-field gel electrophoresis (PFGE). Identical PFGE patterns were found for numerous isolates from 4 of the 9 days sampled, suggesting environmental replication. 16S rRNA gene sequencing, API 20E biochemical testing, and the absence of β-glucuronidase activity revealed that these clonal isolates were Klebsiella, Citrobacter, and Enterobacter spp. In contrast, 82% of the nonclonal isolates from water samples were confirmed to be E. coli, and 16% were identified as other fecal coliforms. These nonclonal isolates produced a diverse range of PFGE patterns similar to those of isolates obtained directly from untreated sewage and gull droppings. β-Glucuronidase activity was critical in distinguishing E. coli from other fecal coliforms, particularly for the clonal isolates. These findings demonstrate that E. coli is a better indicator of fecal pollution than fecal coliforms, which may replicate in the environment and falsely elevate indicator organism levels.     

Analysis of the Gull Fecal Microbial Community Reveals the Dominance of Catellicoccus marimammalium in Relation to Culturable Enterococci

Gulls are prevalent in beach environments and can be a major source of fecal contamination. Gulls have been shown to harbor a high abundance of fecal indicator bacteria (FIB), such as Escherichia coli and enterococci, which can be readily detected as part of routine beach monitoring. Despite the ubiquitous presence of gull fecal material in beach environments, the associated microbial community is relatively poorly characterized. We generated comprehensive microbial community profiles of gull fecal samples using Roche 454 and Illumina MiSeq platforms to investigate the composition and variability of the gull fecal microbial community and to measure the proportion of FIB. Enterococcaceae and Enterobacteriaceae were the two most abundant families in our gull samples. Sequence comparisons between short-read data and nearly full-length 16S rRNA gene clones generated from the same samples revealed Catellicoccus marimammalium as the most numerous taxon among all samples. The identification of bacteria from gull fecal pellets cultured on membrane-Enterococcus indoxyl-β-d-glucoside (mEI) plates showed that the dominant sequences recovered in our sequence libraries did not represent organisms culturable on mEI. Based on 16S rRNA gene sequencing of gull fecal isolates cultured on mEI plates, 98.8% were identified as Enterococcus spp., 1.2% were identified as Streptococcus spp., and none were identified as C. marimammalium. Illumina deep sequencing indicated that gull fecal samples harbor significantly higher proportions of C. marimammalium 16S rRNA gene sequences (>50-fold) relative to typical mEI culturable Enterococcus spp. C. marimammalium therefore can be confidently utilized as a genetic marker to identify gull fecal pollution in the beach environment.     

Relationship among fecal coliforms, Escherichia coli, and Salmonella spp. in shellfish.

The relationship of fecal coliforms, Escherichia coli, and Salmonella spp. was examined in freshly harvested and stored shellfish. In 16 of 40 freshly collected oyster samples, fecal coliform levels were above the recommended wholesale level suggested by the National Shellfish Sanitation Program (less than or equal to 230/100 g), and Salmonella spp. were present in three of these samples. Salmonella spp. were not, however, present in any sample containing less than 230 fecal coliforms per 100 g. Analysis of the data suggests that low fecal coliform levels in both fresh and stored oysters are good indicators of the absence of Salmonella spp., but that high levels of fecal coliforms are somewhat limited in predicting the presence of Salmonella spp. E. coli levels correlated very strongly with fecal coliform levels in both fresh and stored oysters and clams, suggesting that there is no advantage in replacing fecal coliforms with E. coli as an indicator of shellfish quality.     

Relationship among fecal coliforms, Escherichia coli, and Salmonella spp. in shellfish

The relationship of fecal coliforms, Escherichia coli, and Salmonella spp. was examined in freshly harvested and stored shellfish. In 16 of 40 freshly collected oyster samples, fecal coliform levels were above the recommended wholesale level suggested by the National Shellfish Sanitation Program (less than or equal to 230/100 g), and Salmonella spp. were present in three of these samples. Salmonella spp. were not, however, present in any sample containing less than 230 fecal coliforms per 100 g. Analysis of the data suggests that low fecal coliform levels in both fresh and stored oysters are good indicators of the absence of Salmonella spp., but that high levels of fecal coliforms are somewhat limited in predicting the presence of Salmonella spp. E. coli levels correlated very strongly with fecal coliform levels in both fresh and stored oysters and clams, suggesting that there is no advantage in replacing fecal coliforms with E. coli as an indicator of shellfish quality.     

Detection of Acinetobacter spp. in rural drinking water supplies.

A bacteriological survey was conducted of untreated, individual groundwater supplies in Preston County, W.Va. Nearly 60% of the water supplies contained total coliforms in excess of the U.S. Environmental Protection Agency maximum contaminant level of 1 CFU/100 ml. Approximately one-third of the water systems contained fecal coliforms and/or fecal streptococci. Acinetobacter spp. were detected in 38% of the groundwater supplies at an arithmetic mean density of 8 CFU/100 ml and were present in 16% of the water supplies in the absence of total coliforms, posing some concern about the usefulness of total coliforms as indicators of the presence of this opportunistic pathogen. Slime production, a virulence factor for A. calcoaceticus, was not significantly different between well water isolates and clinical strains, suggesting some degree of pathogenic potential for strains isolated from groundwater. In addition, several Acinetobacter isolates were able to interfere with sheen production by some coliform bacteria on M-Endo medium, adding further to the possible significance of Acinetobacter spp. in groundwater supplies.     

Detection of Acinetobacter spp. in rural drinking water supplies

A bacteriological survey was conducted of untreated, individual groundwater supplies in Preston County, W.Va. Nearly 60% of the water supplies contained total coliforms in excess of the U.S. Environmental Protection Agency maximum contaminant level of 1 CFU/100 ml. Approximately one-third of the water systems contained fecal coliforms and/or fecal streptococci. Acinetobacter spp. were detected in 38% of the groundwater supplies at an arithmetic mean density of 8 CFU/100 ml and were present in 16% of the water supplies in the absence of total coliforms, posing some concern about the usefulness of total coliforms as indicators of the presence of this opportunistic pathogen. Slime production, a virulence factor for A. calcoaceticus, was not significantly different between well water isolates and clinical strains, suggesting some degree of pathogenic potential for strains isolated from groundwater. In addition, several Acinetobacter isolates were able to interfere with sheen production by some coliform bacteria on M-Endo medium, adding further to the possible significance of Acinetobacter spp. in groundwater supplies.     

Microbial impact of Canada geese (Branta canadensis) and whistling swans (Cygnus columbianus columbianus) on aquatic ecosystems.

Quantitative and qualitative analyses of the intestinal bacterial flora of Canada geese and whistling swans were carried out with the finding that wild birds harbor significantly more fecal coliforms than fecal streptococci. The reverse was typical of captive and fasting birds. Neither Salmonella spp. nor Shigella spp. were isolated from 44 migratory waterfowl that were wintering in the Chesapeake Bay region. Enteropathogenic Escherichia coli were detected in seven birds. Geese eliminated 10(7) and swans 10(9) fecal coliforms per day. Results of in situ studies showed that large flocks of waterfowl can cause elevated fecal coliform densities in the water column. From the data obtained in this study, it is possible to predict the microbial impact of migratory waterfowl upon aquatic roosting sites.     

Microbial impact of Canada geese (Branta canadensis) and whistling swans (Cygnus columbianus columbianus) on aquatic ecosystems.

Quantitative and qualitative analyses of the intestinal bacterial flora of Canada geese and whistling swans were carried out with the finding that wild birds harbor significantly more fecal coliforms than fecal streptococci. The reverse was typical of captive and fasting birds. Neither Salmonella spp. nor Shigella spp. were isolated from 44 migratory waterfowl that were wintering in the Chesapeake Bay region. Enteropathogenic Escherichia coli were detected in seven birds. Geese eliminated 10(7) and swans 10(9) fecal coliforms per day. Results of in situ studies showed that large flocks of waterfowl can cause elevated fecal coliform densities in the water column. From the data obtained in this study, it is possible to predict the microbial impact of migratory waterfowl upon aquatic roosting sites.     

Distribution and survival of motile Aeromonas spp. in brackish water receiving sewage treatment effluent.

The spatiotemporal distributions of Aeromonas spp. and fecal coliforms in a cove receiving sewage treatment effluent and draining into a brackish lagoon were studied for 34 months with sampling at six stations. A total of 452 strains of Aeromonas spp. were isolated and identified at the outflow of the treatment system and at stations in the cove. Hemolytic activity of 289 Aeromonas strains was determined. The Aeromonas spp. and fecal coliform distributions showed seasonal cycles in the pond effluent. These seasonal bacterial cycles were persistent in effluent, at the discharge point, and in the cove. However, the abundance levels of these bacterial distributions decreased gradually from the coast to the open lagoon. A dilution model showed that the Aeromonas spp. and fecal coliform distributions in the cove water were subject not only to dilution effect but also to other environmental factors, such as salinity. A. sobria is the most common species identified in the Aeromonas population present in the cove water. Survival studies confirmed that Aeromonas spp., especially A. sobria, were more sensitive to saline and/or marine stress than fecal coliforms. Among the Aeromonas hydrophila and A. sobria strains, 96 and 97%, respectively, produced hemolysin, whereas among the Aeromonas caviae strains, 95% were nonhemolytic.     

Distribution and survival of motile Aeromonas spp. in brackish water receiving sewage treatment effluent.

The spatiotemporal distributions of Aeromonas spp. and fecal coliforms in a cove receiving sewage treatment effluent and draining into a brackish lagoon were studied for 34 months with sampling at six stations. A total of 452 strains of Aeromonas spp. were isolated and identified at the outflow of the treatment system and at stations in the cove. Hemolytic activity of 289 Aeromonas strains was determined. The Aeromonas spp. and fecal coliform distributions showed seasonal cycles in the pond effluent. These seasonal bacterial cycles were persistent in effluent, at the discharge point, and in the cove. However, the abundance levels of these bacterial distributions decreased gradually from the coast to the open lagoon. A dilution model showed that the Aeromonas spp. and fecal coliform distributions in the cove water were subject not only to dilution effect but also to other environmental factors, such as salinity. A. sobria is the most common species identified in the Aeromonas population present in the cove water. Survival studies confirmed that Aeromonas spp., especially A. sobria, were more sensitive to saline and/or marine stress than fecal coliforms. Among the Aeromonas hydrophila and A. sobria strains, 96 and 97%, respectively, produced hemolysin, whereas among the Aeromonas caviae strains, 95% were nonhemolytic.     

在饮用水中协同检测“粪型大肠菌群”的试验方法

阐述了在饮用水中检测"粪型大肠菌群"的意义,强调了大肠菌群与粪型大肠菌群的关系,总结出"粪型大肠菌群"的检测条件。     

Isolation of Aeromonas spp. from an unchlorinated domestic water supply.

The recovery of Aeromonas spp. from the unchlorinated water supply for a Western Australian city of 21,000 people was monitored at several sampling points during a period of 1 year. Membrane filtration techniques were used to count colonies of Aeromonas spp., coliforms, and Escherichia coli in water sampled before entry to service reservoirs, during storage in service reservoirs, and in distribution systems. Aeromonas spp. were identified by subculture on blood agar with ampicillin, oxidase tests, and the use of Kaper medium and then were tested for production of enterotoxins and hemolysins. During the same period, two-thirds of all fecal specimens sent for microbiological examination were cultured on ampicillin-blood agar for Aeromonas spp. Recovery of Aeromonas spp. from water supplies at distribution points correlated with fecal isolations and continued during autumn and winter. Coliforms and E. coli were found most commonly in late summer to autumn. This pattern differs from the summer peak of Aeromonas isolations both from water and from patients with Aeromonas spp.-associated gastroenteritis in Perth, Western Australia, a city with a chlorinated domestic water supply. Of the Aeromonas strains from water, 61% were enterotoxigenic, and 64% produced hemolysins.     

Incidence of coliphage in potable water supplies

Samples of drinking water from different sources in greater Cairo, Egypt, and bottled drinking water were tested for total coliform, fecal coliform, and coliphage populations. Of the 147 samples tested, 4 samples were positive for both total coliforms and coliphage, 65 samples were negative for total coliforms, fecal coliforms, and coliphage, and 78 samples were positive for coliphage and negative for total coliforms and fecal coliforms. The incidence of coliphage in these potable water supplies reflects the probability of human pathogenic virus survival in these waters also.     

Incidence of coliphage in potable water supplies.

Samples of drinking water from different sources in greater Cairo, Egypt, and bottled drinking water were tested for total coliform, fecal coliform, and coliphage populations. Of the 147 samples tested, 4 samples were positive for both total coliforms and coliphage, 65 samples were negative for total coliforms, fecal coliforms, and coliphage, and 78 samples were positive for coliphage and negative for total coliforms and fecal coliforms. The incidence of coliphage in these potable water supplies reflects the probability of human pathogenic virus survival in these waters also.     

Comparison of selected methods for the enumeration of fecal coliforms and Escherichia coli in shellfish.

In a comparison of five selected methods for the enumeration of fecal coliforms and Escherichia coli in naturally contaminated and sewage-seeded mussels (Choromytilus spp.) and oysters (Ostrea spp.), a spread-plate procedure with mFC agar without rosolic acid and preincubation proved the method of choice for routine quality assessment.     

Significance and suitability of <Emphasis Type="Italic">Aeromonas hydrophila</Emphasis> vs. fecal coliforms in assessing microbiological water quality

We examined the significance and suitability of Aeromonas hydrophila versus fecal coliforms in assessing microbiological water quality. For this, we used the membrane filtration method to simultaneously estimate the abundance level of A. hydrophila and fecal coliforms in waters from the Mfoundi river watershed at Yaoundé, and compared how fluctuations in A. hydrophila abundance matched those observed with fecal coliforms index as an indicator of water quality in the system under study. Our results revealed that waters were not safe according to the standards for water quality established by the Word Health Organization (WHO). They also indicated the prevalence of A. hydrophila as compared to fecal coliforms, and suggested that water from the Mfoundi River and its tributaries could be classified as hypereutrophic based on the density of Aeromonas. Moreover, the spatial distribution of fecal coliforms and A. hydrophila exhibited similar trends within the different water bodies investigated, suggesting that A. hydrophila can be used as indicator of water quality in highly polluted waters. We concluded that waters from the Mfoundi River watershed at Yaoundé represent a great potential risk of infection for users, and foresee that the next challenge will be to determine, among other factors, the physico-chemical factors influencing the observed spatial distribution.     

Microbiological implications of periurban agriculture and water reuse in Mexico City

Background

Recycled treated or untreated wastewater represents an important health challenge in developing countries due to potential water related microbiological exposure. Our aim was to assess water quality and health implications in a Mexico City periurban agricultural area.

Methodology/Principal Findings

A longitudinal study in the Xochimilco wetland area was conducted, and 42 sites were randomly selected from 211, including irrigation water canals and effluents of treatment plants. Sample collection took place during rainy and dry seasons (2000–2001). Microbiological parameters (total coliforms, fecal coliforms, streptococci/enterococci, and bacteria other than Vibrio grown on TCBS), Helicobacter pylori, and physicochemical parameters including trihalomethanes (THM) were determined. Fecal coliforms and fecal streptococci are appropriate indicators of human or animal fecal contamination. Fecal coliform counts surpass Mexican and World Health Organization irrigation water guidelines. Identified microorganisms associated with various pathologies in humans and domestic animals comprise Escherichia coli, Klebsiella spp., Salmonella spp., Enterobacter spp., Enterococcus spp., and Pseudomonas spp; H. pylori was also present in the water. An environmental characteristic of the canal system showed high Total Organic Carbon content and relatively low dissolved oxygen concentration; residual chlorine as a disinfection control is not efficient, but THMs do not represent a problem. During the rainy season, temperature and conductivity were higher; in contrast, pH, dissolved oxygen, ammonia, and residual chlorine were lower. This is related with the continuous load of feces from human and animal sources, and to the aquatic systems, which vary seasonally and exhibit evidence of lower water quality in effluents from treatment plants.

Conclusions/Significance

There is a need for improvement of wastewater treatment systems, as well as more efficient monitoring, regulation, and enforcement procedures for wastewater disposal into bodies of water.