Human-specific phages infecting Enterococcus host strain MW47: are they reliable microbial source tracking markers?

AIM: The aim of this study was to determine the morphological diversity and environmental survival of human-specific phages infecting Enterococcus faecium host strain MW47, to support their use as microbial source tracking (MST) markers. METHODS AND RESULTS: Twenty phages capable of infecting strain MW47 were propagated and their morphologies were determined using transmission electron microscopy (TEM), which revealed that a heterogeneous group of phages was able to infect strain MW47. Three distinct morphologies from two different families (Myoviridae and Siphoviridae) were observed. In situ inactivation experiments were subsequently conducted to determine their environmental persistence. CONCLUSION: The findings revealed a statistically significant link between morphology and the rate of inactivation, with phages belonging to the Myoviridae family demonstrating more rapid inactivation in comparison to those belonging to the Siphoviridae family. SIGNIFICANCE AND IMPACT OF STUDY: The results suggest that while Enterococcus MW47 phages appear to be a potentially valuable MST tools, significant variations in the persistence of the different phages mean that the approach should be used with caution, as this may adversely affect the reliability of the approach, especially when comparing MW47 phage levels or the presence across different matrices (e.g. levels in sediments or shellfish). This highlights the importance of elucidating the ecological characteristics of newly proposed MST markers before they are used in full-scale MST investigations.

The effect of UV-C radiation (254 nm) on candidate microbial source tracking phages infecting a human-specific strain of Bacteroides fragilis (GB-124).

The enumeration of phages infecting host-specific strains of Bacteroides has been widely recognised as an effective and low-cost method of microbial source tracking (MST). A recently described human-specific Bacteroides host strain (GB-124) has been shown to detect bacteriophages exclusively in human-impacted waters and is emerging as a useful MST tool. However, a better understanding of the morphology and ecological behaviour of the phages, especially in wastewater disinfection processes, is now required in order to validate their role as MST markers. Bacteriophages infecting Bacteroides fragilis GB-124 (n = 21) were isolated from wastewater effluent and irradiated using laboratory-based UV-C (254 nm) collimated beam experiments. Bacteriophages were found to be both a morphologically and ecologically homogeneous group, with all specimens showing highly similar first order log-linear inactivation profiles (mean fluence required to inactivate phages by 4-log(10) was 36 mJ/cm(2)). These findings present the first evidence that phages infecting GB-124 are inactivated by the levels of UV-C radiation routinely delivered during tertiary wastewater treatment processes. More importantly, comparison with previously published inactivation data suggests that their response to UV-C radiation makes GB-124 phages more suitable surrogates for selected enteric viruses in UV disinfection processes than traditional faecal indicator bacteria or human-specific molecular markers.

Virulence traits associated with verocytotoxigenic Escherichia coli O157 recovered from freshwater biofilms.

AIM: To investigate whether epilithic biofilms in freshwater streams in a mixed UK agricultural river catchment harbour Escherichia coli O157, and if so, whether they demonstrate an association with those excreted by grazing farm animals. METHODS AND RESULTS: Flint shingle, native to the study site, was used as a surface for biofilm development within cages of metal lath set into a stream bed at four locations on a chalkland farm. Shingle was collected from all sites once a month, as were pooled faecal samples from five farm animal populations. Subpopulations of E. coli, including E. coli O157 that demonstrated significant phenotypic and genotypic similarity with animal faecal isolates (t-test, P = 0.05) were isolated. Of 1002 E. coli isolates from biofilms and animal faeces, 48 were confirmed as the O157 strain by latex agglutination. The presence of five virulence traits associated with incidence of human disease was tested using PCR. Stx(2) was the most frequently isolated single gene (30 isolates), while stx(1) was the least frequently recovered (four isolates). CONCLUSION: Escherichia coli O157, expressing up to four virulence factors associated with human disease, reside within freshwater biofilms in this agricultural environment. SIGNIFICANCE AND IMPACT OF THE STUDY: Aquatic biofilms may potentially act as a reservoir for these pathogens, and the implications of the findings for the protection of drinking water resources should be further investigated.

Geographical stability of enterococcal antibiotic resistance profiles in Europe and its implications for the identification of fecal sources.

This manuscript describes the use of a recently developed microbial source tracking (MST) technique to determine sources of fecal bacteria (enterococci) from four separate European countries. The investigation aimed to evaluate whether the origin of bacterial populations from France, Spain, and Sweden (n = 456) could be predicted using a library of antibiotic resistance patterns of enterococci of known origin assembled in the United Kingdom (n = 2739). Bacterial isolates were exposed to a battery of 31 antibiotic tests and classified into source groups using discriminant function analysis (DFA). Results showed that while on average 72% of the U.K. isolates could be correctly classified as originating from either municipal wastewater (MW), livestock, or wild birds, only 43% of non-U.K. isolates could be successfully classified into the same source categories. The results suggested that patterns of resistance amongst isolates contained in the U.K. library were not representative of those found in the other locations and that it may not be possible to share libraries over large distances, such as those in this study. Future MST studies using antibiotic resistance analysis (ARA) in Europe may therefore require the assembly of watershed specific libraries, increasing the cost of such studies.

A simplified low-cost approach to antibiotic resistance profiling for faecal source tracking.

Antibiotic resistance profiling (ARP) is a potentially useful method for distinguishing faecal bacteria according to host source. This phenotypic approach has cost benefits over genotypic methods, but existing protocols are time-consuming and manual data handling is open to human error. A simplified, low-cost approach to the ARP technique was developed that used automated data recording techniques combined with simple statistical analyses to compare isolates of the genus Enterococcus from various faecal sources. An initial battery of 21 antibiotics (at up to four concentrations) was chosen for source discrimination. Images of growth or non-growth in microplate wells were stored as bitmaps and converted to binary data to form a database of known antibiotic resistance profiles. Discriminant function analysis (DFA) showed that the average rate of isolates correctly classified by the database (wastewater vs non-wastewater) was 86%. Once the more discriminating antibiotics and their concentrations had been identified, it was possible to reduce the number of tests from 80 to 18 whilst increasing the number of correctly classified human isolates. ARP could offer a low-cost and rapid means of identifying sources of faecal pollution. As such, the technique may be of particular benefit to developing countries, where water quality may have a significant impact on health and where cost is a major factor when choosing environmental management technology.

Comparison of enterococcal populations related to urban and hospital wastewater in various climatic and geographic European regions.

AIMS: Scarce knowledge about the distribution of enterococci species in wastewaters limits any statement on their reliability as faecal indicators or the implications of antibiotic resistance transmission by these organisms through the water cycle. Enterococci have been involved in nosocomial infections and the spreading of antibiotic resistance through the food chain. The species distribution of enterococci and the presence of resistant strains to vancomycin and erythromycin were analysed in more than 400 raw and treated urban wastewaters, surface waters receiving these treated wastewaters and hospital wastewaters from three European countries. METHODS AND RESULTS: A total of 9296 strains were isolated and biochemically phenotyped. The species identification was based on the comparison of biochemical profiles with those of more than 20000 enterococci isolates from an international study. The prevalence of enterococcal isolates resistant to erythromycin (ERE) and vancomycin (VRE) was also analysed. ERE strains were present in a high proportion in all the studied samples. VRE strains were also isolated in all studied countries despite the time elapsed since the use of antimicrobial glycopeptides in animal production was banned in the European Union. CONCLUSIONS: Enterococcus faecalis and Ent. faecium were the most abundant species in all the studied wastewaters. All the studied wastewaters demonstrated high diversity and similar population structure and composition. ERE and VRE isolates were detected in most of the wastewaters. SIGNIFICANCE AND IMPACT OF THE STUDY: Urban and hospital wastewaters are useful targets for the evaluation of the prevalence of ERE and VRE isolates in the environment. It appears that these bacteria could pass through wastewater treatment plants and be transferred to surface waters.

Phenotypic population characteristics of the enterococci in wastewater and animal faeces: implications for the new European directive on the quality of bathing waters.

Faecal pollution of recreational bathing waters may derive from point sources of various wastewaters or from more diffuse sources such as run-off of agricultural wastes. The paper describes the application of population similarity studies to the enterococcal flora of various animal faeces and municipal wastewaters as a means of distinguishing human from animal faecal material. A simplified phenotypic testing technique (PhenePlate, PhP) was used to study the fermentation kinetics of eleven carbohydrates by all bacterial isolates. Enterococcal isolates (1,766) from six sources were investigated. Enterococcal population diversity (measured as Simpson's Diversity Index) in wastewater samples was high (mean D(i) = 0.95) compared with those of non-human faeces. The mean diversity of isolates in seabird faeces was 0.72, in sheep and donkey faeces 0.44, in dog faeces 0.42 and in cattle faeces 0.32. Analysis of population similarity coefficients demonstrated that faeces from sheep and cattle showed the greatest similarity (S(p) = 0.72). Sheep and cattle faeces demonstrated a low similarity to municipal wastewater samples. This would suggest that population similarity studies might be a useful tool for distinguishing the relative contributions of municipal wastewater and agricultural run-off to bathing water pollution. The PhP procedure identified a specific PhP type that appears to have high specificity to non-human faeces. It may, therefore, represent an important tool in source tracking. Additional phenotypic and genotypic analysis of PhP types that demonstrate a high degree of source specificity is required. The benefits and limitations of the use of population similarity studies to distinguish pollution sources are discussed in comparison with other source tracking approaches and the implications of these developments for future European Union legislation on the quality of bathing waters are discussed.

In situ measurement and statistical modelling of Escherichia coli decay in small rivers.

Numerous studies have been carried out on the environmental factors associated with the decay of faecal bacteria in open (fresh or marine) waters. The present study aimed at understanding the fate of bacteria in small streams (flow <20 m3 s(-1)) for which there is a lack of knowledge. An original in situ protocol was developed for measuring the die-off of Escherichia coli (E. coli) from wastewater treatment plants. Based upon 80 values of the decay first-order parameter (K or its inverse T90), collected from five rivers in Normandy (France), a median T90 of 10 h and a minimal T90 of 1.3 h were obtained. K was then modelled as a linear function of variables made up from flow, water temperature and suspended particulate matter (SPM). The set of significant co-variables did not include light indicators. E. coli decay is inversely related to the river flow and it becomes highly significant below 0.3 m3 s(-1). The positive effect of small flows on die-off is increased by water temperature over 15 degrees C, whereas it could be reduced by SPM. The major co-variable of the model (p < 10(-9)) is an empiric composite variable integrating the effect of flow and temperature that explains more than 40% of the variance of K. We interpreted this as an expression of predation by benthic micro-grazers which could be the main cause of E. coli die-off in small streams in temperate countries.

Relationship between mortality rates and hospital patient volume for Medicare patients undergoing major orthopaedic surgery of the hip, knee, spine, and femur.

This study examines the relationship between mortality rates and hospital patient volume for major orthopaedic surgery. All Medicare patients from fiscal years 1993 and 1994 in diagnosis-related groups (DRGs) 209, 210, and 214 were included, covering hip and knee arthroplasty, other hip and femur procedures, and spine procedures. Within DRG 209, five procedures were studied in more detail: total hip arthroplasty, partial hip arthroplasty, revision total hip arthroplasty, total knee arthroplasty, and revision total knee arthroplasty. Higher-volume hospitals had lower mortality rates, both in-house and in-house plus 30-day, for each of the DRGs studied and for each of the individual procedures within DRG 209. Age and sex were examined as potential causes of the differences in mortality rates, but no attempt was made to adjust for comorbidities or orthopaedic degree of difficulty. Results for 1995 are included in an appendix.

Antibiotics derived from a mutant of Bacillus circulans.

A mutant of Bacillus circulans, which produces butirosins only when 2-deoxystreptamine is added to the fermentation medium, was employed in the biosynthesis of antibiotics containing modified aminocyclitols. The blocked mutant converted 2,5-dideoxystreptamine into 5-deoxybutirosamine. Streptamine was incorporated into a complex differing from butirosin by an additional hydroryl at C-2.