Differentiation and identification of Shigella spp. and enteroinvasive Escherichia coli in environmental waters by a molecular method and biochemical test

Both Shigella spp. and enteroinvasive Escherichia coli (EIEC) are important human pathogens that are responsible for the majority of cases of endemic bacillary dysentery. However, they are difficult to identify and differentiate by biochemical tests or molecular methods alone. In this study, we developed a procedure to detect Shigella spp. and EIEC from environmental water samples using membrane filtration followed by nutrient broth enrichment, isolation using selective culture plates, and identification of the invasion plasmid antigen H (ipaH) gene by PCR amplification and DNA sequencing. Finally, we used a biochemical test and a serological assay to differentiate between Shigella and EIEC. Among the 93 water samples from nine reservoirs and one watershed, 76 (81.7%) water samples of culture plates had candidate colonies of Shigella and EIEC and 5 water samples were positive (5.4%) for a Shigella- and EIEC-specific polymerase chain reaction targeting the ipaH gene. Guided by the molecular method, the biochemical test, and the serological assay, 11 ipaH gene-positive isolates from 5 water samples were all identified as EIEC.     

Method for rapid detection of viable Escherichia coli in water using real-time NASBA

A rapid real-time NASBA method was developed for detection of Escherichia coli in water samples. In this method, a fragment of the clpB-mRNA is amplified and a specific molecular beacon probe is used to detect the amplified mRNA fragment during the NASBA reaction. The method was shown to be specific and sensitive (1 viable E. coli in 100 ml) and can be performed within 3-4 h. Different inactivation processes (starvation, heat, UV-irradiation and chlorine) were employed to study the relationship between culturability and the ability to detect E. coli using NASBA. Detection of clpB-mRNA correlated with culturability after starvation or chlorine treatment. After UV-irradiation or heat-inactivation, detection of the increase in production of clpB-mRNA in viable E. coli cells after heat-shock induction correlated with culturability. Application of the NASBA method on tap water, treated sewage and surface water samples showed that culture and NASBA yielded comparable results in these different matrices. This study demonstrates that the NASBA method has high potential as a rapid test for microbiological water quality monitoring.     

Mechanisms of Escherichia coli inactivation by several disinfectants

The objective of this study was to elucidate dominant mechanisms of inactivation, i.e. surface attack versus intracellular attack, during application of common water disinfectants such as ozone, chlorine dioxide, free chlorine and UV irradiation. Escherichia coli was used as a representative microorganism. During cell inactivation, protein release, lipid peroxidation, cell permeability change, damage in intracellular enzyme and morphological change were comparatively examined. For the same level of cell inactivation by chemical disinfectants, cell surface damage was more pronounced with strong oxidant such as ozone while damage in inner cell components was more apparent with weaker oxidant such as free chlorine. Chlorine dioxide showed the inactivation mechanism between these two disinfectants. The results suggest that the mechanism of cell inactivation is primarily related to the reactivity of chemical disinfectant. In contrast to chemical disinfectants, cell inactivation by UV occurred without any changes measurable with the methods employed. Understanding the differences in inactivation mechanisms presented herein is critical to identify rate-limiting steps involved in the inactivation process as well as to develop more effective disinfection strategies.     

ERIC-PCR identification of the spread of airborne Escherichia coli in pig houses

To understand the spread of microbial aerosols in pig houses, with Escherichia coli (E. coli) as indicator, the airborne E. coli in 4 pig houses and their surroundings at different points 10, 50 m upwind and 10, 50, 100, 200 and 400 m downwind respectively from the pig houses were collected, and the concentrations were calculated at each sampling point. Furthermore, the feces of pigs were collected to separate E. coli. The ERIC-PCR (Enterobacterial Repetitive Intergenic Consensus-Polymerase Chain Reaction) technology was used to amplify the isolated E. coli DNA samples, then the amplified results were analyzed by NTSYS-pc (Version 2.10) to identify the similarity of isolated E. coli. The results showed that the airborne E. coli concentrations in indoor air of the 4 pig houses (21-35 CFU m⁻³ air) were much higher than those in upwind and downwind air (P < 0.05), but there were no significant differences (P > 0.05) at downwind distances. The ERIC-PCR results also showed that 52.4% of the fecal E. coli (four houses being respectively 2/4, 50%; 2/4, 50%; 3/6, 50%; 4/7, 57.1%) were identical to the indoor airborne E. coli isolates, and there was more than 90% similarity between the majority of E. coli (50%, 21/42) isolated from downwind air at 10, 50, 100 and 200 m and those from indoor air or feces. It could be concluded that the aerosols in pig houses can spread to the surroundings, and thus effective measures should be taken to control and minimize the spread of microbial aerosols.     

Molecular characterization of antimicrobial resistance in enterococci and Escherichia coli isolates from European wild rabbit (Oryctolagus cuniculus)

A total of 44 Escherichia coli and 64 enterococci recovered from 77 intestinal samples of wild European rabbits in Portugal were analyzed for resistance to antimicrobial agents. Resistance in E. coli isolates was observed for ampicillin, tetracycline, sulfamethoxazole/trimethoprim, streptomycin, gentamicin, tobramycin, nalidixic acid, ciprofloxacin and chloramphenicol. None of the E. coli isolates produced extended-spectrum beta-lactamases (ESBLs). The bla_TEM, aadA, aac(3)-II, tet(A) and/or tet(B), and the catA genes were demonstrated in all ampicillin, streptomycin, gentamicin, tetracycline, and chloramphenicol-resistant isolates respectively, and the sul1 and/or sul2 and/or sul3 genes in 4 of 5 sulfamethoxazole/trimethoprim resistant isolates. Of the enterococcal isolates, Enterococcus faecalis was the most prevalent detected species (39 isolates), followed by E. faecium (21 isolates) and E. hirae (4 isolates). More than one-fourth (29.7%) of the isolates were resistant to tetracycline; 20.3% were resistant to erythromycin, 14.1% were resistant to ciprofloxacin and 10.9% were resistant to high-level-kanamycin. Lower level of resistance (< 10%) was detected for ampicillin, quinupristin/dalfopristin and high-level-gentamicin, -streptomycin. No vancomycin-resistance was detected in the enterococci isolates. Resistance genes detected included aac(6′)-aph(2″), ant(6)-Ia, tet(M) and/or tet(L) in all gentamicin, streptomycin and tetracycline-resistant isolates respectively. The aph(3′)-IIIa gene was detected in 6 of 7 kanamycin-resistant isolates, the erm(B) gene in 11 of 13 erythromycin-resistant isolates and the vat(D) gene in the quinupristin/dalfopristin-resistant E. faecium isolate. This survey showed that faecal bacteria such as E. coli and enterococci of wild rabbits could be a reservoir of antimicrobial resistance genes.     

Towards understanding inter-strain attachment variations of Escherichia coli during transport in saturated quartz sand

Although Escherichia coli is an indicator of fecal contamination in aquifers, limited research has been devoted to understanding the biological processes involved in the initial attachment of E. coli transported in abiotic porous media. The roles of the various surface structures of E. coli, like lipopolysaccharides (LPS), autotransporter proteins, and fimbriae are unknown. The objective of this research was to establish the effects of variations in surface characteristics of the outer membrane of E. coli on the attachment efficiency of 54 E. coli strains upon transport in saturated quartz sand under identical flow conditions. We used column experiments to assess retention of the E. coli strains, and we determined sphericity, motility, zeta-potential, and aggregation of all strains. LPS composition was determined based on known serotypes, and the presence/absence of 22 genes encoding surface characteristics was determined with qualitative PCR. The results indicated that under identical flow conditions, there was a variation of two orders of magnitude in the maximum breakthrough concentrations of the 54 E. coli strains. Of all factors we investigated, no single factor was able to explain attachment efficiency variations statistically significantly. However, low attachment efficiencies were associated (p = 0.13) with LPS containing saccharides with phosphate and/or carboxyl groups. These saccharide groups are acidic and likely charged with a negative O-atom, which reduced attachment to the negatively charged quartz surface. In addition, of the 22 genes tested, Afa was most associated (p = 0.21) with attachment efficiency. The work presented here bridges knowledge on colloid transport and molecular microbiology, and tries to offer a more holistic view on the attachment of planktonic E. coli bacteria to (abiotic) quartz grain surfaces. Future research should involve the use of microbiological techniques in order to be able to map the unique or grouped characteristics of E. coli in aquifers, and to assess the usefulness of E. coli as a fecal indicator in aquifers.     

Evaluation of statistical models for predicting Escherichia coli particle attachment in fluvial systems

Modeling surface water Escherichia coli fate and transport requires partitioning E. coli into particle-attached and unattached fractions. Attachment is often assumed to be a constant fraction or is estimated using simple linear models. The objectives of this study were to: (i) develop statistical models for predicting E. coli attachment and virulence marker presence in fluvial systems, and (ii) relate E. coli attachment to a variety of environmental parameters. Stream water samples (n = 60) were collected at four locations in a rural, mixed-use watershed between June and October 2012, with four storm events (>20 mm rainfall) being captured. The percentage of E. coli attached to particles (>5 μm) and the occurrences of virulence markers were modeled using water quality, particle concentration, particle size distribution, hydrology and land use factors as explanatory variables. Three types of statistical models appropriate for highly collinear, multidimensional data were compared: least angle shrinkage and selection operator (LASSO), classification and regression trees using the general, unbiased, interaction detection and estimation (GUIDE) algorithm, and multivariate adaptive regression splines (MARS). All models showed that E. coli particle attachment and the presence of E. coli virulence markers in the attached and unattached states were influenced by a combination of water quality, hydrology, land-use and particle properties. Model performance statistics indicate that MARS models outperform LASSO and GUIDE models for predicting E. coli particle attachment and virulence marker occurrence. Validating the MARS modeling approach in multiple watersheds may allow for the development of a parameterizing model to be included in watershed simulation models.     

Survival of Escherichia coli in two sewage treatment plants using UV irradiation and chlorination for disinfection

We investigated the survival of Escherichia coli in two STPs utilising UV irradiation (STP-A) or chlorination (STP-B) for disinfection. In all, 370 E. coli strains isolated from raw influent sewage (IS), secondary treated effluent (STE) and effluent after the disinfection processes of both STPs were typed using a high resolution biochemical fingerprinting method and were grouped into common (C-) and single (S-) biochemical phenotypes (BPTs). In STP-A, 83 BPTs comprising 123 isolates were found in IS and STE, of which 7 BPTs survived UV irradiation. Isolates tested from the same sites of STP-B (n = 220) comprised 122 BPTs, however, only two BPTs were found post-chlorination. A representative isolate from each BPT from both STPs was tested for the presence of 11 virulence genes (VGs) associated with uropathogenic (UPEC) or intestinal pathogenic (IPEC) E. coli strains. Strains surviving UV irradiation were distributed among seven phylogenetic groups with five BPTs carrying VGs associated with either UPEC (4 BPTs) or IPEC (1 BPT). In contrast, E. coli strains found in STP-B carried no VGs. Strains from both STPs were resistant to up to 12 out of the 21 antibiotics tested but there was no significant difference between the numbers of antibiotics to which surviving strains were resistant to in these STPs. Our data suggests that some E. coli strains have a better ability to survive STPs utilising chlorination and UV irradiation for disinfection. However, strains that survive UV irradiation are more diverse and may carry more VGs than those surviving SPTs using chlorination.     

A cross comparison of QPCR to agar-based or defined substrate test methods for the determination of Escherichia coli and enterococci in municipal water quality monitoring programs

Molecular methods such as quantitative, real-time polymerase chain reaction (QPCR) are intended to shorten the period between sampling and publicly available results. Cross comparison studies in Racine, WI, USA evaluated QPCR against agar-based (US EPA Method 1600) and defined substrate (IDEXX Colilert-18^®) methods for the detection and quantification of Escherichia coli and enterococci in a variety of aqueous environments (wastewater, stormwater, and surface water). Regulatory outcomes were also compared based on choice of indicator and method. Positive correlation was seen between QPCR cell equivalents and viable cells through the wastewater treatment process and in all surface water samples (river or freshwater bathing beach) but not in direct stormwater discharge. For surface water samples, correlation improved with the application of a site-specific corrective factor, with regulatory action correctly predicted 98% of the time at bathing beaches. This study suggests the potential utility of QPCR for certain water quality monitoring applications.     

High diversity and abundance of antibiotic-resistant Escherichia coli isolated from humans and farm animal hosts in Jeonnam Province, South Korea

The spread of antibiotics resistance among bacteria is a threat to human health. Since South Korea uses approximately 1.5 times more antibiotics than do other OECD countries, this is likely to impact the numbers and types of antibiotic-resistant bacteria found in the environment. In this study we examined feces from domesticated animals and humans for the diversity and abundance of antibiotic-resistant Escherichia coli. Abundant antibiotic-resistant E. coli were isolated from all the tested animals and humans and were examined by horizontal, fluorophore-enhanced, rep-PCR (HFERP) DNA fingerprint analysis. A total of 793 unique, non-clonal, E. coli isolates were obtained from the 513 human and animal hosts examined. Antibiotic resistance analysis, done using 14 antibiotics, indicated that 72.3% of the isolates (573 of 793) were found resistant to more than one antibiotic. The E. coli isolated from swine were resistant to the greatest number of antibiotics. Tetracycline resistant E. coli were routinely isolated from all animal hosts (36 to 77% per host), except for dairy cattle (9.3%). Twenty nine E. coli isolates from all hosts, except for duck, were resistant to more than 10 antibiotics. Gene transfer and southern hybridization studies revealed that resistance to 13 of the antibiotics was self-transmissible, and likely mediated by plasmids and integrons. Since genetically diverse and numerically abundant antibiotic-resistant E. coli were consistently recovered from chicken, swine and other domesticated animals in South Korea, our results suggest that the use of sub-therapeutic levels of antibiotics for disease prophylaxis and growth promotion should be curtailed.     

Production of Shiga-like toxins in viable but nonculturable Escherichia coli O157:H7

Escherichia coli O157:H7, a causative agent of hemolytic uremic syndrome, can enter into a viable but nonculturable (VBNC) state when under stress. To date, it is unknown whether VBNC cells produce Shiga-like toxins (Stx). To address this question, we confirmed the expression of the stx1 and stx2 genes and the production of Stx in VBNC E. coli O157:H7 cells. To quantitatively assess the production of Stx in VBNC cells, we developed a Vero-cell microplate cytotoxicity assay based on the correspondence of the cytotoxicity of VBNC cells on Vero cells to the number of inoculated VBNC cells. Using this method, we found that all VBNC cells induced by river water, PBS buffer, deionized water, or chloraminated water retained the ability to produce Stx, and that they had differing levels of Stx. Both aged (19-month-old) VBNC cells induced by river water and fresh VBNC cells induced by chloraminated water showed very low half maximal inhibitory concentration (IC₅₀; 6.6 × 10⁴ and 7.1 × 10⁴ respectively), corresponding to higher levels of toxins produced than VBNC cells induced by deionized water and PBS buffer. VBNC cells originating from different isolates may vary in Stx production, and the VBNC cells from bovine isolates produced higher levels of Stx than those from clinical isolates. These results demonstrate a potential health risk of VBNC E. coli O157:H7 in environmental water and the importance of monitoring VBNC E. coli O157:H7.     

Multi-scale temporal and spatial variation in genotypic composition of Cladophora-borne Escherichia coli populations in Lake Michigan

High concentrations of Escherichia coli in mats of Cladophora in the Great Lakes have raised concern over the continued use of this bacterium as an indicator of microbial water quality. Determining the impacts of these environmentally abundant E. coli, however, necessitates a better understanding of their ecology. In this study, the population structure of 4285 Cladophora-borne E. coli isolates, obtained over multiple three day periods from Lake Michigan Cladophora mats in 2007-2009, was examined by using DNA fingerprint analyses. In contrast to previous studies that have been done using isolates from attached Cladophora obtained over large time scales and distances, the extensive sampling done here on free-floating mats over successive days at multiple sites provided a large dataset that allowed for a detailed examination of changes in population structure over a wide range of spatial and temporal scales. While Cladophora-borne E. coli populations were highly diverse and consisted of many unique isolates, multiple clonal groups were also present and accounted for approximately 33% of all isolates examined. Patterns in population structure were also evident. At the broadest scales, E. coli populations showed some temporal clustering when examined by year, but did not show good spatial distinction among sites. E. coli population structure also showed significant patterns at much finer temporal scales. Populations were distinct on an individual mat basis at a given site, and on individual days within a single mat. Results of these studies indicate that Cladophora-borne E. coli populations consist of a mixture of stable, and possibly naturalized, strains that persist during the life of the mat, and more unique, transient strains that can change over rapid time scales. It is clear that further study of microbial processes at fine spatial and temporal scales is needed, and that caution must be taken when interpolating short term microbial dynamics from results obtained from weekly or monthly samples.     

Large scale analysis of virulence genes in Escherichia coli strains isolated from Avalon Bay, CA

Contamination of recreational waters with Escherichia coli and Enterococcus sp. is a widespread problem resulting in beach closures and loss of recreational activity. While E. coli is frequently used as an indicator of fecal contamination, and has been extensively measured in waterways, few studies have examined the presence of potentially pathogenic E. coli strains in beach waters. In this study, a combination of high-throughput, robot-assisted colony hybridization and PCR-based analyses were used to determine the genomic composition and frequency of virulence genes present in E. coli isolated from beach water in Avalon Bay, Santa Catalina Island, CA. A total of 24,493 E. coli isolates were collected from two sites at a popular swimming beach between August through September 2007 and from July through August 2008. All isolates were examined for the presence of shiga-like toxins (stx1/stx2), intimin (eaeA), and enterotoxins (ST/LT). Of the 24,493 isolates examined, 3.6% contained the eaeA gene, indicating that these isolates were potential EPEC strains. On five dates, however, greater than 10% of the strains were potential EPEC, suggesting that incidence of virulence genes at this beach has a strong temporal component. No STEC or ETEC isolates were detected, and only eight (<1.0%) of the potential EPEC isolates were found to carry the EAF plasmid. The potential EPEC isolates mainly belonged to E. coli phylogenetic groups B1 or B2, and carried the β intimin subtype. DNA fingerprint analyses of the potential EPEC strains indicated that the isolates belonged to several genetically diverse groups, although clonal isolates were frequently detected. While the presence of virulence genes alone cannot be used to determine the pathogenicity of strains, results from this study show that potential EPEC strains can be found in marine beach water and their presence needs to be considered as one of the factors used in decisions concerning beach closures.     

Macromolecule mediated transport and retention of Escherichia coli O157:H7 in saturated porous media

The role of extracellular macromolecules on Escherichia coli O157:H7 transport and retention was investigated in saturated porous media. To compare the relative transport and retention of E. coli cells that are macromolecule rich and deficient, macromolecules were partially cleaved using a proteolytic enzyme. Characterization of bacterial cell surfaces, cell aggregation, and experiments in a packed sand column were conducted over a range of ionic strength (IS). The results showed that macromolecule-related interactions contribute to retention of E. coli O157:H7 and are strongly linked to solution IS. Under low IS conditions (IS ≤ 0.1 mM), partial removal of the macromolecules resulted in a more negative electrophoretic mobility of cells and created more unfavorable conditions for cell-quartz and cell-cell interactions as suggested by Derjaguin-Landau-Verwey-Overbeek (DLVO) interaction energy profiles and cell aggregation kinetics. Consequently, less retention was observed for enzyme treated cells in the corresponding column experiments. In addition, a time-dependent deposition process (i.e., ripening) was observed for untreated cells, but not for treated cells, supporting the fact that the macromolecules enhanced cell-cell interactions. Additional column experiments for untreated cells under favorable conditions (IS ≥ 1 mM) showed that a significant amount of the cells were reversibly retained in the column, which contradicts predictions of DLVO theory. Furthermore, a non-monotonic cell retention profile was observed under favorable attachment conditions. These observations indicated that the presence of macromolecules hindered irreversible interactions between the cells and the quartz surface.     

Survival of manure-borne E. coli in streambed sediment: Effects of temperature and sediment properties

Escherichia coli bacteria are commonly used as indicator organisms to designate of impaired surface waters and to guide the design of management practices to prevent fecal contamination of water. Stream sediments are known to serve as a reservoir and potential source of fecal bacteria (E. coli) for stream water. In agricultural watersheds, substantial numbers of E. coli may reach surface waters, and subsequently be deposited into sediments, along with fecal material in runoff from land-applied manures, grazing lands, or wildlife excreta. The objectives of this work were (a) to test the hypothesis that E. coli survival in streambed sediment in the presence of manure material will be affected by sediment texture and organic carbon content and (b) to evaluate applicability of the exponential die-off equation to the E. coli survival data in the presence of manure material. Experiments were conducted at three temperatures (4 °C, 14 °C, and 24 °C) in flow-through chambers using sediment from three locations at the Beaverdam Creek Tributary in Beltsville, Maryland mixed with dairy manure slurry in the proportion of 1000:1. Indigenous E. coli populations in sediments ranged from ca. 10¹ to 10³ MPN g⁻¹ while approx 10³ manure-borne E. coli MPN g⁻¹ were added. E. coli survived in sediments much longer than in the overlaying water. The exponential inactivation model gave an excellent approximation of data after 6-16 days from the beginning of the experiment. Slower inactivation was observed with the increase in organic carbon content in sediments with identical granulometric composition. The increase in the content of fine particles and organic carbon in sediments led not only to the slower inactivation but also to lower sensitivity of the inactivation to temperature. Streambed sediment properties have to be documented to better evaluate the role of sediments as reservoirs of E. coli that can affect microbiological stream water quality during high flow events.     

Protection against UV disinfection of E. coli bacteria and B. subtilis spores ingested by C. elegans nematodes

Nematodes, which occur abundantly in granular media filters of drinking water treatment plants and in distribution systems, can ingest and transport pathogenic bacteria and provide them protection against chemical disinfectants. However, protection against UV disinfection had not been investigated to date.In this study, Caenorhabditis elegans nematodes (wild-type strain N2) were allowed to feed on Escherichia coli OP50 and Bacillus subtilis spores before being exposed to 5 and 40 mJ/cm² UV fluences, using a collimated beam apparatus (LP, 254 nm). Sonication (15 W, 60 s) was used to extract bacteria from nematode guts following UV exposure in order to assess the amount of ingested bacteria that resisted the UV treatment using a standard culture method. Bacteria located inside the gut of C. elegans were shown to benefit from a significant protection against UV. Approximately 15% of the applied UV fluence of 40 mJ/cm² (as typically used in WTP) was found to reach the bacteria located inside nematode guts based on the inactivation of recovered bacteria (2.7 log reduction of E. coli bacteria and 0.7 log reduction of B. subtilis spores at 40 mJ/cm²). To our knowledge, this study is the first demonstration of the protection effect of bacterial internalization by higher organisms against UV treatment, using the specific case of E. coli and B. subtilis spores ingested by C. elegans.     

DNA extraction and Escherichia coli quantification of anaerobically digested biosolids using the competitive touchdown PCR method

Accurate enumeration of indicator organisms such as Escherichia coli is important for assessing the safety of water and wastewater samples. Recent research has shown that E. coli can enter a viable but non-culturable state; therefore, traditional cultivation methods could potentially underestimate the quantities of the organisms. The goals of the research were to develop and verify a DNA extraction protocol and a quantitative polymerase chained reaction (PCR) method for E. coli enumeration in digested biosolids. A solvent-based DNA extraction protocol with extensive cell lysis recovered approximately 78-84% of spiked DNA. In comparison, a commercial kit only recovered 28-42% of DNA, likely from inefficient cell lysis. The developed competitive touchdown PCR (cPCR) method for E. coli enumeration was comparable to both real-time PCR (rt-PCR) and cultivation methods with sensitivity of approximately 50,000-500,000 E. coli per gram dry solids (DS), which is suitable for Class B biosolids monitoring in the US and "conventional" biosolids in the European Union. The cPCR protocol provides a less expensive alternative than the rt-PCR as a culturing independent method for enumerating E. coli.     

Photocatalytic inactivation of E. coli in surface water using immobilised nanoparticle TiO2 films

Photocatalysis is a promising method for the disinfection of potable water in developing countries where solar irradiation can be employed, thus reducing the cost of treatment. In addition to microbial contamination, water normally contains suspended solids, dissolved inorganic ions and organic compounds (mainly humic substances) which may affect the efficacy of solar photocatalysis. In this work the photocatalytic and photolytic inactivation rates of Escherichia coli using immobilised nanoparticle TiO₂ films were found to be significantly lower in surface water samples in comparison to distilled water. The presence of nitrate and sulphate anions spiked into distilled water resulted in a decrease in the rate of photocatalytic disinfection. The presence of humic acid, at the concentration found in the surface water, was found to have a more pronounced affect, significantly decreasing the rate of disinfection. Adjusting the initial pH of the water did not markedly affect the photocatalytic disinfection rate, within the narrow range studied.     

Isolation and characterization of Bacteroides host strain HB-73 used to detect sewage specific phages in Hawaii

Previous studies have shown that Escherichia coli and enterococci are unreliable indicators of fecal contamination in Hawaii because of their ability to multiply in environmental soils. In this study, the method of detecting Bacteroides phages as specific markers of sewage contamination in Hawaii’s recreational waters was evaluated because these sewage specific phages cannot multiply under environmental conditions. Bacteroides hosts (GB-124, GA-17), were recovered from sewage samples in Europe and were reported to be effective in detecting phages from sewage samples obtained in certain geographical areas. However, GB-124 and GA-17 hosts were ineffective in detecting phages from sewage samples obtained in Hawaii. Bacteroides host HB-73 was isolated from a sewage sample in Hawaii, confirmed as a Bacteroides sp. and shown to recover phages from multiple sources of sewage produced in Hawaii at high concentrations (5.2-7.3 × 10⁵ PFU/100 mL). These Bacteroides phages were considered as potential markers of sewage because they also survived for three days in fresh stream water and two days in marine water. Water samples from Hawaii’s coastal swimming beaches and harbors, which were known to be contaminated with discharges from streams, were shown to contain moderate (20-187 CFU/100 mL) to elevated (173-816 CFU/100 mL) concentrations of enterococci. These same samples contained undetectable levels (<10 PFU/100 mL) of F+ coliphage and Bacteroides phages and provided evidence to suggest that these enterococci may not necessarily be associated with the presence of raw sewage. These results support previous conclusions that discharges from streams are the major sources of enterococci in coastal waters of Hawaii and the most likely source of these enterococci is from environmental soil rather than from sewage.