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Enterococci, which are on the WHO list of priority pathogens, are commonly encountered in hospital acquired infection and are becoming increasing significant due to the development of strains resistant to multiple antibiotics. Enterococci are also important microorganisms in the environment, and their presence is frequently used as an indicator of faecal pollution. Their success is related to their ability to survive within a broad range of habitats and the ease by which they acquire mobile genetic elements, including plasmids, from other bacteria. The enterococci are frequently present within a bacterial biofilm, which provides stability and protection to the bacterial population along with an opportunity for a variety of bacterial interactions. Enterococci can accept extrachromosomal DNA both from within its own species and from other bacterial species, and this is enhanced by the proximity of the donor and recipient strains. It is this exchange of genetic material that makes the role of biofilms such an important aspect of the success of enterococci. There remain many questions regarding the most suitable model systems to study enterococci in biofilms and regarding the transfer of genetic material including antibiotic resistance in these biofilms. This review focuses on some important aspects of biofilm in the context of horizontal gene transfer (HGT) in enterococci. [ABSTRACT FROM AUTHOR]

Aims: Beach water quality is regulated by faecal indicator bacteria levels, sand is not, despite known human health risk from exposure to beach sand. We compared the performance of three methods to extract bacterial DNA from beach sand as a step toward a standard method. Methods and results: The analytical sensitivity of quantitative polymerase chain reaction (qPCR) for Enterococcus was compared for the slurry (suspension, agitation, membrane filtration of supernatant), versus direct extraction using PowerSoil™ or PowerMax Soil™ kits. The slurry method had the lowest limit of detection at 20–80 gene copies g−1, recovered significantly more DNA, and the only method that detected Enterococcus by qPCR in all samples; therefore, the only method used in subsequent experiments. The slurry method reflected the spatial variability of Enterococcus in individual transect samples. Mean recovery efficiency of the microbial source tracking marker HF183 from wastewater spiked marine and freshwater beach sand was 100.8% and 64.1%, respectively, but varied, indicating that the mixing protocol needs improvement. Conclusions: Among the three methods, the slurry method had the best analytical sensitivity and produced extracts that were useful for culture or molecular analysis. Significance and impact of study: Standardization of methods for extraction of bacterial DNA from sand facilitates comparisons among studies, and ultimately contributes to the safety of recreational beaches. [ABSTRACT FROM AUTHOR]

Aims: To determine inactivation of Cryptosporidium parvum oocysts and reduction of Escherichia coli and enterococci in cattle slurry added aqueous ammonia. Methods and Results: Escherichia coli, enterococci and nonviable C. parvum oocysts (DAPI+PI+) were enumerated every second day for 2 weeks in cattle slurry amended with 60 mmol l−1 aq. ammonia and compared with untreated slurry at three temperatures. Regardless of temperature, the proportion of nonviable C. parvum oocysts increased significantly faster over time in slurry with added ammonia than raw slurry (P = 0·021) corresponding to 62·0% higher inactivation (P = 0·001) at day 14. Additionally, 91·8% fewer E. coli and 27·3% fewer enterococci were observed in slurry added ammonia at day 14 compared to raw slurry. Conclusion: The addition of aqueous ammonia to raw slurry significantly reduced the viability of C. parvum oocysts and numbers of bacterial indicators. Hence, ammonia is usable at lower pathogen concentrations in slurry before application to agricultural land. Significance and Impact of the Study: Livestock waste is a valuable source of plant nutrients and organic matter, but may contain high concentrations of pathogens like E. coli and Cryptosporidium sp. that can be spread in the environment, and cause disease outbreaks. However, die‐off rates of pathogens in organic waste can increase following increasing ammonia concentrations. [ABSTRACT FROM AUTHOR]

Aims: The intestinal microbiota of newborn calves is largely unexplored even if it is of great significance for their future health. Therefore, the aim of the study was to gain a better insight into the development dynamics of certain bacterial groups during the first week of life. Methods and Results: Faecal samples of healthy Simmental calves (dual‐purpose breed; n = 80), bottle fed and raised in a dairy farm were taken immediately after birth and at 6/12/24/48/72/168 h (h) after birth. Samples were analysed using cultural, biochemical and molecular–biological methods. The aerobe, anaerobe, Enterobacteriaceae and Enterococcus counts of healthy calves increased significantly between 6 and 24 h postpartum (P <0·05). Apart from the anaerobes, bacterial counts decreased after reaching a plateau at 24–48 h. Enterococcus faecalis was detected in significantly higher counts compared to E. faecium (P <0·05). Lactobacilli developed more slowly and increased until day 7 after birth to a mean value of 6·8 × 107 CFU per g. MALDI‐TOF analysis of 2338 lactobacilli isolates resulted in 36 different species. Conclusions: Lactobacillus reuteri became the most common Lactobacillus sp. during the first week of life. Significance and Impact of the Study: This fact seems to be very important for the calf's intestinal health because L. reuteri is known to show in vitro bactericidal effects against bacterial pathogens and anti‐infective activities against rotaviruses and Cryptosporidium parvum. [ABSTRACT FROM AUTHOR]

Aims The thermal resistance of desiccation-adapted Salmonella Senftenberg 775/W was compared with those of indigenous enterococci and total aerobic bacteria in poultry litter. Methods and Results Aged broiler litter and composted turkey litter with 20, 30, 40 and 50% moisture contents were inoculated with desiccation-adapted Salm. Senftenberg 775/W, and then heat-treated at 75 and 85°C. Compared to total aerobic bacteria, there were better correlations between mean log reductions of desiccation-adapted Salm. Senftenberg 775/W and indigenous enterococci in broiler litter samples with 20, 30, 40 and 50% moisture contents at 75°C ( R2 > 0·91), and 20, 30 and 40% moisture contents at 85°C ( R2 > 0·87). The mean log reductions of Salm. Senftenberg 775/W were better correlated with those of indigenous enterococci in turkey litter samples with 20, 30, 40 and 50% moisture contents at 75°C ( R2 > 0·88), and 20 and 30% moisture contents at 85°C ( R2 = 0·83) than those of total aerobic bacteria, which had a better correlation in turkey litter sample with 40% ( R2 = 0·98) moisture content at 85°C. Conclusion Indigenous enterococci may be used to validate the thermal processing of poultry litter, as it predicts the survival behaviour of Salmonella under some treatment conditions. Significance and the Impact of the Study This study provides some scientific data for poultry litter processors when validating the effectiveness of thermal processing. [ABSTRACT FROM AUTHOR]

Aims The spread of bacteria that are simultaneously resistant to disinfectants and antimicrobials would constitute an unsettling scenario. In order to explore an association between antimicrobial resistance and reduced susceptibility to biocides/microbicides (disinfectants) in agriculture, we investigated Escherichia coli ( n = 438) and enterococci ( n = 120) isolated from six different flocks of the same poultry farm with known history of antimicrobial treatment. Methods and Results Susceptibility to disinfectants (formic acid and a quaternary ammonium compound (QAC), didecyldimethylammoniumchloride-DDAC) was assessed by macrodilution according to guidelines of the German Veterinary Society. Escherichia coli, Enterococcus faecalis and Enterococcus faecium were screened (i) for reduced biocide susceptibility and (ii) for an association of biocide susceptibility and antimicrobial resistance including the production of extended-spectrum beta-lactamases (ESBL) and the hyperproduction of AmpC-type beta-lactamases. DDAC inhibited ESBL/AmpC(hyper)-producing E. coli ( n = 53) from poultry at similar or slightly lower inhibitory concentrations, compared with non-ESBL/AmpC strains (median MIC = 0·36 vs 1·44 mg l−1). In contrast, DDAC-MICs were positively correlated with several other antibiotic MICs (e.g. piperacillin and sulphamethoxazole + trimethoprim in E. coli, chloramphenicol in E. faecalis) and increased DDAC-MICs were statistically linked to high-level aminoglycoside resistance in enterococci (streptomycin high level). DDAC-MICs did not correlate with the presence of the integron marker qacEDelta1. Conclusions This study provides indication that residual disinfectant might be able to select antimicrobial-resistant enterococci, but not ESBL-/AmpC (hyper)producing E. coli from poultry. Significance and Impact of the Study While ESBL-/AmpC- E. coli were inhibited at disinfectant concentrations comparable to or lower than wildtype values, low concentrations of QACs might be able to select other antimicrobial-resistant E. coli or enterococci-a finding with special significance for the food processing industry, where QACs are regularly used. [ABSTRACT FROM AUTHOR]

Aims Development of Taqman MGB real-time PCR (q-PCR) assays for the quantitative detection of virulence factor genes in pure culture and food samples with regard to food safety assessment. Methods and Results New Taqman primers and probes were designed for the ace, esp and gelE genes based on the determinants of virulence profiles of enterococcal strains from GenBank. The high specificity and accuracy of the Taqman probe assay was confirmed. The limit of detection for the different virulence genes was 102 CFU ml−1 or CFU g−1 for pure culture and meat samples, and 103 CFU g−1 for cheese samples. Conclusion This method provides the specific and rapid detection and quantification of ace, esp and gelE genes compared to conventional PCR assays, thus allowing the rapid and direct safety assessment of Enterococcus genus in food samples. Significance and Impact of the Study This study presents efficient methods that can be used directly on food products for the rapid quantification and tracing of virulence genes, regarding food safety assessment. Moreover, this is the first study to quantify these virulence factors using a specific Taqman q-PCR assay in food samples. [ABSTRACT FROM AUTHOR]

Aims To investigate the UVB-independent and exogenous indirect photoinactivation of eight human health-relevant bacterial species in the presence of photosensitizers. Methods and Results Eight bacterial species were exposed to simulated sunlight with greatly reduced UVB light intensity in the presence of three synthetic photosensitizers and two natural photosensitizers. Inactivation curves were fit with shoulder log-linear or first-order kinetic models, from which the presence of a shoulder and magnitude of inactivation rate constants were compared. Eighty-four percent reduction in the UVB light intensity roughly matched a 72-95% reduction in the overall bacterial photoinactivation rate constants in sensitizer-free water. With the UVB light mostly reduced, the exogenous indirect mechanism contribution was evident for most bacteria and photosensitizers tested, although most prominently with the Gram-positive bacteria. Conclusions Results confirm the importance of UVB light in bacterial photoinactivation and, with the reduction of the UVB light intensity, that the Gram-positive bacteria are more vulnerable to the exogenous indirect mechanism than Gram-negative bacteria. Significance and Impact of the Study UVB is the most important range of the sunlight spectrum for bacterial photoinactivation. In aquatic environments where photosensitizers are present and there is high UVB light attenuation, UVA and visible wavelengths can contribute to exogenous indirect photoinactivation. [ABSTRACT FROM AUTHOR]

Aims The goal was to determine the effect of starch source (corn, oats and wheat) and concentration on: (i) total amylolytic bacteria, Group D Gram-positive cocci ( GPC), lactobacilli and lactate-utilizing bacteria, and (ii) fermentation by equine microbiota. Methods and Results When faecal washed cell suspensions were incubated with any substrate amylolytics increased over time. However, at 24 h there were 10 and 1000-fold more amylolytics with corn than wheat or oats respectively. Predominant amylolytics isolated were Enterococcus faecalis (corn, wheat) and Streptococcus bovis (oats). GPC increased with any substrate, but decreased during stationary phase in oats only. Lactobacilli decreased during stationary phase with corn only. By 24 h, oats had more lactate-utilizers and lactobacilli and fewer GPC than corn and wheat. More gas was produced from oats or wheat than from corn. Conclusions These results indicate that the growth of bacteria and fermentative capacity associated with starch metabolism is starch source dependent. Significance and Impact of the Study This study demonstrates a relationship between starch source and microbial changes independent of host digestion. However, future research is needed to evaluate the effect of starch source on the hindgut microbial community in vivo. [ABSTRACT FROM AUTHOR]

Aims To investigate how the growth stage of Enterococcus faecalis affects its photoinactivation in clear water. Methods and Results Enterococcus faecalis were grown in batch cultures to four different growth stages or grown in chemostats set at four different dilution rates, then harvested and exposed to full spectrum or UVB-blocked simulated sunlight. Experiments were conducted in triplicate in clear water with no added sensitizers. Decay curves were shoulder-log linear and were generally not statistically different in experiments conducted under full spectrum light. Shoulders were longer and first order inactivation rates smaller when experiments were seeded with cells grown to stationary as compared to exponential phase, and for slower growing cells when experiments were done under UVB-blocked light. Chemostat-sourced bacteria generally showed less variability among replicates than batch-sourced cells. Conclusions The physiological state of cells and the method via which they are being generated may affect the photoinactivation experimental results. Significance and Impact of the Study Photoinactivation experiments conducted with exponential phase cells may overestimate the photoinactivation kinetics in the environment, particular if UVB-independent mechanisms predominate. Chemostat-sourced cells are likely to provide more consistent experimental results than batch-sourced cells. [ABSTRACT FROM AUTHOR]

Aims This study was conducted to determine antibiotic susceptibility patterns among the faecal indicator bacteria ( FIB), Escherichia coli and enterococci, and to determine the potential for freshwater beaches to serve as reservoirs of resistance genes where transfer of resistant phenotypes takes place or de novo resistance may evolve. Methods and Results One hundred and forty-seven E. coli and 150 enterococci collected from sand and water at recreational beaches along Lake Huron, Michigan, USA were screened against commonly used antibiotics. Resistance was apparent in both E. coli (19% resistant) and enterococci (65% resistant). Antibiotic-resistant E. coli were capable of growing in beach sand microcosms and were able to transfer a plasmid-encoded kanamycin-resistance gene in sand microcosms. Furthermore, resistant phenotypes were stable in the sand environment even in the absence of the corresponding antibiotic. Conclusions Antibiotic-resistant FIB were prevalent and persistent in the beach habitat. Significance and Impact of the Study Active populations of FIB at beaches express antibiotic resistance phenotypes and have the ability to transfer antibiotic resistance. These human-associated bacteria may be intermediaries in the movement of resistance between environmental and clinical reservoirs. [ABSTRACT FROM AUTHOR]

Aims The relationship between land use (undeveloped, cattle grazing, urban), faecal indicator bacteria ( FIB) levels and microbial source tracking ( MST) marker detection was investigated in lakes created following phosphate mining. Methods and Results Faecal coliforms and enterococci were cultured, and MST markers were detected by PCR [ Methanobrevibacter smithii, human polyomaviruses ( HPy Vs), ruminant, human ( HF183) and general Bacteroidales]. FIB levels varied significantly by sampling date and were correlated with antecedent rainfall. FIB levels varied with land use category only in the case of faecal coliform levels in sediments of urban lakes, which were significantly greater than those in undeveloped or cattle-impacted lakes. Ruminant Bacteroidales were detected consistently in cattle-impacted lakes (57%) and rarely in other lakes. HPy Vs was the only human source marker detected. Conclusions Rainfall was more strongly associated with FIB levels than land use category. The detection frequency of only the ruminant MST marker was associated with land use. Significance and Impact of Study Microbial source tracking ( MST) can fine-tune the assessment of human health risk from recreational use of inland waters, particularly when similar FIB levels but different surrounding land use and probable impacts exist. [ABSTRACT FROM AUTHOR]

The development of a microbial source tracking (MST) method is strongly desired to ensure public health and bacteriological safety in coastal recreation areas. We try to specify the source of faecal pollution by applying pulsed-field gel electrophoresis (PFGE) to the study of the aquatic environment on Aoshima Beach, Japan. Enterococcus faecium, an enterococcus, was used as a faecal indicator bacterium in this study. Enterococcus faecium strains were isolated and identified from each water sample collected from Aoshima Beach and five rivers (Oyodo, Kiyotake, Kaeda, Chifuku and Tsukunami Rivers) that might be potential sources of faecal pollution. Enterococcus faecium strains collected from water samples were analysed using PFGE. The similarities of all the PFGE types of the Ent. faecium strains were compared using dendrogram analysis. The PFGE types of the strains isolated from Aoshima Beach showed a high similarity to those of the strains isolated from the Oyodo River at a 0·9 similarity level. It was suggested that the Oyodo River is the source of faecal pollution on Aoshima Beach. The PFGE analysis using enterococci is a potential tool for the MST of faecal indicator bacteria that can be applied to the study of the coastal environment. This is one of the studies that PFGE was applied to the coastal environment. The approach using PFGE could estimate the river that is source of faecal pollution in Aoshima Beach. By applying PFGE as a tool of MST method, detailed information of faecal pollution in coastal area can be provided. [ABSTRACT FROM AUTHOR]

Water quality at two Florida beaches was compared using faecal indicator bacteria measurements, microbial source tracking (MST) methods for detecting human source pollution and the assessment of pathogen presence. These values were also compared before and after remediation of wastewater infrastructure at one beach. Faecal coliforms, Escherichia coli and enterococci were enumerated in estuarine water and sediment samples. PCR assays for the human-associated esp gene of Enterococcus faecium and human polyomaviruses (HPyVs) were used to detect human sewage. Culturable Salmonella and enteric viruses were also analysed. MST identified human sewage contamination at one beach, leading to repair of a sewer main and relocation of portable restrooms. Exceedances of Florida recreational water regulatory standards were significantly reduced after remediation (by 52% for faecal coliforms and 39% for enterococci), and the frequency of detection of MST markers decreased. Coxsackie virus B4 and HPyVs were codetected following a major sewage spill, but Salmonella was not detected during the study. These data indicate that infrastructure remediation significantly reduced pollution from human sewage at the impacted beach. A comprehensive microbial water quality study that can identify contamination sources through the use of MST markers and close collaboration with local/and state agencies can result in tangible actions to improve recreational water quality and safety. [ABSTRACT FROM AUTHOR]

Aims: This study evaluated the applicability of standard faecal indicator bacteria (SFIB) for alpine mountainous water resources monitoring. Methods and Results: Escherichia coli, enterococci (ENTC) and Clostridium perfringens were investigated by standard or frequently applied phenotypic and genotypic methods in a broad range of animal and human faecal sources in a large alpine mountainous area. Clostridium perfringens occurred only in human, livestock and carnivorous source groups in relevant average concentrations (log 4·7-7·0 CFU g−1) but not in herbivorous wildlife sources. Escherichia coli proved to be distributed in all faecal source groups with remarkably balanced average concentrations (log 7·0-8·4 CFU g−1). Except for single faecal samples from the cattle source group, prevalence rates for ENTC source groups were generally >87% with average concentrations of log 5·3-7·7 CFU g−1. To test the faecal indication capacity in the environment, faecal prevalence data were comparatively analysed with results from the concurrently performed multi-parametric microbial source tracking effort on karst spring water quality from the investigated alpine mountainous catchment ( Reischer et al. 2008 ; Environ Microbiol 10:2598-2608). Conclusion: Escherichia coli and enterococci are reliable faecal indicators for alpine mountainous water resources monitoring, although E. coli is the more sensitive one. Clostridium perfringens did not prove to be an indicator of general faecal pollution but is suggested a conservative microbial source tracking marker for anthropogenic faecal influence. Significance and Impact of the Study: Applicability of SFIB is currently hotly debated. This is the first study providing comprehensive information on the applicability of SFIB at alpine mountainous habitats. [ABSTRACT FROM AUTHOR]

Aim: This study investigated the growth potential of Salmonella serotype Typhimurium and faecal indicator organisms in compost materials and the correlation between bacterial growth potential and the physico-chemical composition of the compost substrate and temperature. Methods and Results: Survival of Salm. Typhimurium, Enterococcus spp. and total coliforms at 14, 24 and 37°C was determined in material of different degrees of maturity collected from composting plants for household waste and manure. All three micro-organisms showed the potential for growth in the material from active composts (Solvita index 4) but inactivation generally occurred over time in mature compost material (Solvita index 7–8). Conclusions: Salm. Typhimurium had the potential for growth in psychrophilic/mesophilic (P/M) zones of immature compost material and its growth potential correlated negatively with the maturity of the compost and the temperature within the simulated P/M zone. Significance and Impact of the Study: The risk of pathogen regrowth in P/M zones during organic waste composting further emphasizes the importance of good management practices and of avoiding P/M zones in combination with low compost maturity. [ABSTRACT FROM AUTHOR]

Aims: The role of enterococci in food fermentation and as probiotics counteracts with their increasing importance as human pathogens. Over the years, several virulence factors have been described, mainly in clinical strains but also in food isolates. However, differential expression of such traits may modulate the pathogenic potential of the harbouring enterococci. To further unravel such differential response, this study aims to identify environmental cues responsible for triggering the expression of virulence-related genes. Methods and Results: The differential expression of eight virulence-related genes ( cylMBAI, agg, esp, efaA fs and efaAfm) in 16 enterococci from distinct origins, grown in conditions simulating environmental colonization and infection sites, was analysed by reverse transcriptase PCR. The expression profiles were environmental and strain-dependent, because no constant response was observed neither for clinical nor food enterococci. Conclusions: Virulence expression profiles are strain-specific and unrelated with strain’s origin or species allocation. Significance and Impact of the Study: The current study constitutes the first approach aimed at the evaluation of the differential expression of enterococcal virulence-related genes combining so many growth environments, enterococcal species and origins. So, with this investigation, we intend to contribute to the clarification of enterococcal pathogenicity potential, especially for food strains. [ABSTRACT FROM AUTHOR]

Aims: The aim of this study was to identify and characterize the population of Enterococcus sp. in domestic wastewater as it flows through a constructed wetland. Methods and Results: Four hundred and eighty-four Enterococcus isolates were collected from the inlet, various sites within and from the outlet of a plastic lined constructed wetland in College Station, TX. The wetland treated septic tank effluent that passed sequentially through two 1·89 m3 septic tanks and a 1·89 m3 pump tank allowing 48 l doses at a 24 l min−1 rate. The Enterococcus isolates were identified to species using the commercial Biolog system. The 484 Enterococcus isolates were comprised of ten different species, including Enterococcus faecalis (30·6%), Enterococcus pseudoavium (24·0%), Enterococcus casseliflavus (12·8%), Enterococcus faecium (11·2%), Enterococcus mundtii (7·9%), Enterococcus gallinarum (6·2%), Enterococcus dispar (3·7%), Enterococcus hirae (2·1%), Enterococcus durans and Enterococcus flavescens both 0·8%. Of the 88 isolates collected from the inlet, only 9·1% of the isolates were identified as Ent. faecalis and Ent. pseudoavium (36·4%) was identified as the predominant species. Whereas of the 74 isolates collected from the outlet, the predominant species were identified as Ent. faecalis (29·7%). Species identification varied among sites within the wetland, but often Ent. faecalis was the predominant species. Conclusions: Our data suggest that while Ent. faecalis is the predominant species of Enterococcus found in domestic wastewater, the populations may shift during treatment as the wastewater flows through the constructed wetland. Significance and Impact of the Study: We found that shifts in Enterococcus species composition occurred during domestic wastewater treatment. This has implications for the identification of faecal pollution based on the presence of specific bacterial types associated with domestic wastewater. [ABSTRACT FROM AUTHOR]

Aims: To determine the occurrence of the human pathogen, Vibrio vulnificus, in south Texas coastal waters. Methods and Results: Coastal waters were sampled monthly between August 2006 and July 2007. Water temperature, dissolved oxygen, pH, salinity, conductivity and turbidity were measured during each sampling event. Culture-based techniques utilizing Vibrio vulnificus agar (VVA) and membrane- Enterococcus indoxyl-β-d-glucoside agar (mEI) were used to assess the occurrence and levels of V. vulnificus and the faecal contamination indicator group, enterococci, respectively. Vibrio vulnificus isolates were confirmed using colony-blot hybridization with the species-specific VVAP probe. Vibrio vulnificus was isolated at all sites throughout the year even when the water temperature dropped to 9·71°C. Significant correlations were found between concentrations of V. vulnificus and the abiotic factors, water temperature ( P = 0·002) and dissolved oxygen ( P = 0·028), as well as between concentrations of V. vulnificus and enterococci ( P < 0·001). Conclusions: This study demonstrated the year-round presence of V. vulnificus in coastal waters of south Texas. Significance and Impact of the Study: These findings indicate that the potential for human exposure to the pathogen, V. vulnificus, exists throughout the year. It also suggests that routinely monitored data might be used to predict the occurrence of the pathogen. [ABSTRACT FROM AUTHOR]

Aims: Microbial water quality and possible human sources of faecal pollution were assessed in a Florida estuary that serves shellfishing and recreational activities. Methods and Results: Indicator organisms (IO), including faecal coliforms, Escherichia coli and enterococci, were quantified from marine and river waters, sediments and oysters. Florida recreational water standards were infrequently exceeded (6–10% of samples); however, shellfishing standards were more frequently exceeded (28%). IO concentrations in oysters and overlaying waters were significantly correlated, but oyster and sediment IO concentrations were uncorrelated. The human-associated esp gene of Enterococcus faecium was detected in marine and fresh waters at sites with suspected human sewage contamination. Lagrangian drifters, used to determine the pathways of bacterial transport and deposition, suggested that sediment deposition from the Ochlockonee River contributes to frequent detection of esp at a Gulf of Mexico beach. Conclusions: These data indicate that human faecal pollution affects water quality in Wakulla County and that local topography and hydrology play a role in bacterial transport and deposition. Significance and Impact of the Study: A combination of IO enumeration, microbial source tracking methods and regional hydrological study can reliably inform regulatory agencies of IO sources, improving risk assessment and pollution mitigation in impaired waters. [ABSTRACT FROM AUTHOR]

Aims: The absence of standardized methods for quantifying faecal indicator bacteria (FIB) in sand hinders comparison of results across studies. The purpose of the study was to compare methods for extraction of faecal bacteria from sands and recommend a standardized extraction technique. Methods and Results: Twenty-two methods of extracting enterococci and Escherichia coli from sand were evaluated, including multiple permutations of hand shaking, mechanical shaking, blending, sonication, number of rinses, settling time, eluant-to-sand ratio, eluant composition, prefiltration and type of decantation. Tests were performed on sands from California, Florida and Lake Michigan. Most extraction parameters did not significantly affect bacterial enumeration.anova revealed significant effects of eluant composition and blending; with both sodium metaphosphate buffer and blending producing reduced counts. Conclusions: The simplest extraction method that produced the highest FIB recoveries consisted of 2 min of hand shaking in phosphate-buffered saline or deionized water, a 30-s settling time, one-rinse step and a 10 : 1 eluant volume to sand weight ratio. This result was consistent across the sand compositions tested in this study but could vary for other sand types. Significance and Impact of the Study: Method standardization will improve the understanding of how sands affect surface water quality. [ABSTRACT FROM AUTHOR]

Aims: Micro-organisms and molecular markers for microbial source tracking (MST) in coastal waters are often present at low numbers, and often exhibit significant variability in time and space. In this study, we investigated the uptake, accumulation, and persistence of human associated Enterococcus in the mussel Mytilus edulis. Methods and Results: The human associated molecular markers esp in Enterococcus faecium, and M66 in Enterococcus faecalis were targetted by PCR in seawater and mussel samples from coastal sites affected by sewage contamination. Both native mussels and mussels transplanted from pristine to polluted sites were included. The results showed that the esp and M66 markers were often not detectable in seawater whereas mussels were enriched in the markers. Human associated E. faecalis accumulated rapidly in M. edulis, and reached maximum levels after 4–6 h with concentration 30–300 times greater than in the surrounding seawater. Enterococcus faecalis retained in M. edulis showed a survival comparable to planktonic E. faecalis in seawater with half lives of 30 and 22 h, respectively. Human associated markers remained detectable for 120 h in M. edulis after faecal contamination. Conclusions: The study demonstrated that native and transplanted M. edulis can accumulate and retain human associated molecular markers relevant for MST. Significance and Impact of the Study: Mussels should be considered as additional targets in MST studies in coastal waters. [ABSTRACT FROM AUTHOR]

Aims: The aim of the study was to determine if vancomycin-resistant Enterococcus spp. [VRE] carrying vanA and/or vanB genes were present in public marine beaches and a fishing pier [2001–2003, 2008] from Washington and California [2008]. Methods: PCR assays for the vanA and/or vanB genes with verification by DNA–DNA hybridization of the PCR products were used. Positive isolates were speciated using the BD BBL Crystal™ Identification and/or by sequencing the 16S ribosomal region. Results: Eighteen (8%) of 227 isolates including Enterococcus faecalis, Enterococcus faecium, Enterococcus casseliflavus/gallinarum and a Staphylococcus epidermidis carrying vanA and/or vanB genes, from four of six Washington and one of two California sites, were identified. Selected VRE and the S. epidermidis were able to transfer their van genes to an E. faecalis recipient at frequencies ranging from 1·9 × 10−6 to 6·7 × 10−9. Conclusions: Vancomycin-resistant Enterococcus spp. was isolated from five of the seven sites suggesting that other North America public beaches could be the reservoirs for VRE and should be assessed. Significance & Impact of the Study: This is the first report of isolation and characterization of VRE strains (and a vanB Staphylococcus sp.) from North American environmental sources suggesting that public beaches may be a reservoir for possible transmission of VRE to beach visitors. [ABSTRACT FROM AUTHOR]

Aims: This study sought to evaluate the distribution of the enterococcal surface protein ( esp) gene in Enterococcus faecium in the Pacific coast environment as well as the distribution and diversity of the gene in Northern California animal hosts. Methods and Results: Over 150 environmental samples from the Pacific coast environment (sand, surf zone, fresh/estuarine, groundwater, and storm drain) were screened for the esp gene marker in E. faecium, and the marker was found in 37% of the environmental samples. We examined the host specificity of the gene by screening various avian and mammalian faecal samples, and found the esp gene to be widespread in nonhuman animal faeces. DNA sequence analysis performed on esp polymerase chain reaction amplicons revealed that esp gene sequences were not divergent between hosts. Conclusions: Our data confirm recent findings that the E. faecium variant of the esp gene is not human-specific. Significance and Impact of the Study: Our results suggest that the use of the esp gene for microbial source tracking applications may not be appropriate at all recreational beaches. [ABSTRACT FROM AUTHOR]

Aims: The aim of this study was to examine a rapid method for detecting Escherichia coli and enterococci in recreational water. Methods and Results: Water samples were assayed for E. coli and enterococci by traditional and immunomagnetic separation/adenosine triphosphate (IMS/ATP) methods. Three sample treatments were evaluated for the IMS/ATP method: double filtration, single filtration, and direct analysis. Pearson’s correlation analysis showed strong, significant, linear relations between IMS/ATP and traditional methods for all sample treatments; strongest linear correlations were with the direct analysis ( r = 0·62 and 0·77 for E. coli and enterococci, respectively). Additionally, simple linear regression was used to estimate bacteria concentrations as a function of IMS/ATP results. The correct classification of water-quality criteria was 67% for E. coli and 80% for enterococci. Conclusions: The IMS/ATP method is a viable alternative to traditional methods for faecal-indicator bacteria. Significance and Impact of the Study: The IMS/ATP method addresses critical public health needs for the rapid detection of faecal-indicator contamination and has potential for satisfying US legislative mandates requiring methods to detect bathing water contamination in 2 h or less. Moreover, IMS/ATP equipment is considerably less costly and more portable than that for molecular methods, making the method suitable for field applications. [ABSTRACT FROM AUTHOR]

Aims: The aim of this study was to identify the prevalence of environmentally adapted enterococci strains by analysing biochemical fingerprinting (BF) data of 3952 enterococci isolates collected over 5 years from the six catchments in Southeast Queensland, Australia. Methods and Results: A BF method was used to type 3952 enterococci isolates from six catchments. The environmental isolates were compared with a large existing BF library comprised of 5803 enterococci isolates from 10 host groups. Environmental isolates belonged to 801 biochemical phenotypes (BPTs), of which, an average of 29.2% was specific to each catchment. When compared with the BF library, an average of 79·5% BPTs from each catchment was identical to those in the library (i.e. host-origin BPTs). The remaining 20·5% was regarded as non-host origin BPTs, as they were not in the library and constituted only 5·3% of the total isolates tested for each catchment. Conclusions: Our data suggest that less than 5% of studied environmental strains was not identical to those in the library and seemed to be of environmental origin. From a microbial source tracking context, such low level of environmentally adapted strains can have a minimal impact on the performance of the library-based methods if a large number of isolates were tested from both the host groups and environmental waters. Significance and Impact of the Study: These data shed light on the importance of the size and representativeness of library–based source-tracking methods and their implications for the identification of faecal pollution in environmental waters. [ABSTRACT FROM AUTHOR]

Aims: This study examined vancomycin-susceptible Enterococcus (VSE) from deli salads for streptogramin resistance and presence of cpd, agg and gelE genes. Methods and Results: Fifteen VSE from retail salads were isolated for identification and antimicrobial susceptibility testing by MicroScan, Etest and agar diffusion. Clinical vancomycin-resistant Enterococcus ( n = 32) and animal VSE ( n = 17) were included for comparative purposes. Multiplex PCR was used to detect the following genes: agg, gelE, cpd, vatD, vatE and sodA. Results showed fewer streptogramin-susceptible Enterococcus faecium isolated from salad (1/6, 17%) and animals (6/10, 60%) than from clinical (26/29, 90%) sources. A low level of erythromycin susceptibility was detected among salad (2/6, 33%) and animal (3/10, 30%) Ent. faecium isolates . Food and animal VSE demonstrated similarities in antimicrobial resistance profiles. All Enterococcus faecalis carried one or more of the selected genes cpd (40%), gelE (33%) and agg (27%). The vatD or vatE genes were not detected in any of the isolates. Conclusions: Experiments demonstrated that streptogramin resistance and virulence genes agg, cpd and gelE are present in enterococci isolated from deli salads. Significance and Impact of the Study: This study provides useful information regarding streptogramin resistance and virulence determinants in enterococci from foods associated with multi-component ingredients. [ABSTRACT FROM AUTHOR]

Aim: Three hundred and two enterococci were isolated from food, animal and clinical samples in order to evaluate the incidence of vancomycin-resistant enterococci (VRE) and bacteriocin, cytolysin, haemolysin, gelatinase production. Methods and Results: Among the isolates, 27 (8·9%) were VRE, and 17 (63%) of these showed, by the deferred antagonism method, bacteriocin production against Gram-positive and some Gram-negative indicators. Eight bacteriocin producers displayed by polymerase chain reaction an enterocin structural gene: six Enterococcus faecium the Enterocin A, two Enterococcus faecalis the Enterocin P genes. The enterocins AS-48, 31, L50 and 1071A/B genes were not found. Regarding the virulence factors, two VRE produced gelatinase and seven were haemolytic. Gelatinase gelE gene was found in 19 strains and cytolysin cylL L gene in eight. Among the strains showing the cylL L gene, only two E. faecalis expressed a β-haemolysis. Conclusions: Our results showed the persistence of VRE in food, animal and clinical samples. Many of these strains displayed antibacterial activity and sometimes different components of virulence, which could emphasize their pathogenicity. Significance and Impact of the Study: This work indicates the need of a constant monitoring of enterococci in order to assess their possible pathogenic properties. The strains of interest in the food industry or used as probiotics should be tested for antibiotic resistance and virulence traits. [ABSTRACT FROM AUTHOR]

Aims: Enzyme substrates based on 4-methylumbelliferone are widely used for the detection of Escherichia coli and enterococci in water, by detection of β-glucuronidase and β-glucosidase activity respectively. This study aimed to synthesize and evaluate novel umbelliferone-based substrates with improved sensitivity for these two enzymes. Methods and Results: A novel β-glucuronide derivative based on 6-chloro-4-methylumbelliferone (CMUG) was synthesized and compared with 4-methylumbelliferyl- β-d-glucuronide (MUG) using 42 strains of E. coli in a modified membrane lauryl sulfate broth. Over 7 h of incubation, the fluorescence generated from the hydrolysis of CMUG by E. coli was over twice that from MUG, and all of the 38 glucuronidase-positive strains generated a higher fluorescence with CMUG compared with MUG. Neither substrate caused inhibition of bacterial growth in any of the tested strains. Four β-glucosidase substrates were also synthesized and evaluated in comparison with 4-methylumbelliferyl- β-d-glucoside (MU-GLU) using 42 strains of enterococci in glucose azide broth. The four substrates comprised β-glucoside derivatives of umbelliferone-3-carboxylic acid and its methyl, ethyl and benzyl esters. Glucosides of the methyl, ethyl and benzyl esters of umbelliferone-3-carboxylic acid, were found to be superior to MU-GLU for the detection of enterococci, especially after 18 h of incubation, while umbelliferone-3-carboxylic acid- β-d-glucoside was inferior. However, the variability in detectable β-glucosidase activity among the different strains of enterococci in short-term assays using the three carboxylate esters (7 h incubation) may compromise their use for rapid detection and enumeration of these faecal indicator bacteria. Conclusions: The β-glucuronidase substrate CMUG appears to be a more promising detection system than the various β-glucosidase substrates tested. Significance and Impact of the Study: The novel substrate CMUG showed enhanced sensitivity for the detection of β-glucuronidase-producing bacteria such as E. coli, with a clear potential for application in rapid assays for the detection of this indicator organism in natural water and other environmental samples. [ABSTRACT FROM AUTHOR]

Aims: To compare accuracy of genus and species level identification of presumptive enterococci isolates from the marine environment using conventional biochemical testing, four commercial identification systems and 16S rRNA sequence analysis. Methods and Results: Ninety-seven environmental bacterial isolates identified as presumptive enterococci on mEI media were tested using conventional and Enterococcus genus screen biochemical tests, four commercial testing systems and 16S rRNA sequencing. Conventional and Enterococcus genus screen biochemical testing, 16S rRNA sequencing and two commercial test systems achieved an accuracy of ≥94% for Enterococcus genus confirmation. Conventional biochemical testing and 16S rRNA sequencing achieved an accuracy of ≥90% for species level identification. Conclusions: For confirmation of Enterococcus genus from mEI media, conventional or genus screen biochemical testing, 16S rRNA sequencing and the four commercial systems were correct 79–100% of the time. For speciation to an accuracy of 90% or better, either conventional biochemical testing or 16S rRNA sequencing is required. Significance and Impact of the Study: Accurate identification of presumptive environmental Enterococcus isolates to genus and species level is an integral part of laboratory quality assurance and further characterization of Enterococcus species from pollution incidents. This investigation determines the ability of six different methods to correctly identify environmental isolates. [ABSTRACT FROM AUTHOR]

d.m. ferguson, d f. moore, m.a. getrich and m.h. zhowandai. 2005. To determine the levels and species distribution of enterococci in intertidal and marine sediments and coastal waters at two beaches frequently in violation of bacterial water standards. Faecal indicator bacteria were extracted from sediment and enumerated using membrane filtration. High levels of enterococci were detected in intertidal sediments in a seasonal river and near a storm drain outlet. Low levels were found in marine sediments at 10 m depths and in surf zone sand. Bacterial isolates presumptively identified as Enterococcus on mEI media were speciated. The predominant species found in both water and sediment included Enterococcus faecalis, Enterococcus faecium, Enterococcus hirae, Enterococcus casseliflavus and Enterococcus mundtii. A number of isolates (11–26%) from regulatory water samples presumptively identified as enterococci on mEI media were subsequently identified as species other than Enterococcus. At both study sites, the distribution of species present in water was comparable with those in sediments and the distribution of species was similar in water samples passing and exceeding bacterial indicator standards. High levels of Enterococcus in intertidal sediments indicate retention and possible regrowth in this environment. Resuspension of enterococci that are persistent in sediments may cause beach water quality failures and calls into question the specificity of this indicator for determining recent faecal contamination. [ABSTRACT FROM AUTHOR]

b. martin, m. garriga, m. hugas and t. aymerich. 2005.To determine the biodiversity of enterococci from slightly fermented sausages (chorizo and fuet) at species and strain level by molecular typing, while considering their safety aspects.Species-specific PCR and partial sequencing of 16S rRNA andsodAgenes were used to identify enterococcal population.Enterococcus faeciumwas the most frequently isolated species followed byE. faecalis,E. hiraeandE. durans. Randomly amplified polymorphic DNA (RAPD)-PCR revealed species-specific clusters and allowed strain typing. Sixty strains of 106 isolates exhibited different RAPD profiles indicating a high genetic variability. All theE. faecalisstrains carried virulence genes (efaAfs,esp,aggandgelE) and allE. faeciumisolates carriedefaAfmgene.Enterococcus faecalisshowed higher antibiotic resistance than the other species. Only oneE. faeciumstrain showedvanAgenotype (high-level resistance to glycopeptides) andE. gallinarumandE. casseliflavus/flavescensisolates showedvanC1andvanC2/C3genotypes (low-level resistance only to vancomycin) respectively.E. faecalishas been mainly associated with virulence factors and antimicrobial multi-resistance and, although potential risk for human health is low, the presence of this species in slightly fermented sausages should be avoided to obtain high quality products.The enterococcal population of slightly fermented sausages has been thoroughly characterized. Several relevant safety aspects have been revealed. [ABSTRACT FROM AUTHOR]

w. ahmed, r. neller and m. katouli. 2005.To provide evidence of septic system failure by comparing two faecal indicator bacteria, enterococci andEscherichia coli, from defective septic tanks and adjacent creeks.A biochemical fingerprinting method was used to type and compare enterococci andE. colistrains from 39 septic tanks with creek water samples. Phenotypic diversity of enterococci (0·5 ± 0·3) andE. coli(0·5 ± 0·3) in septic tanks were significantly lower than those found in water samples (0·8 ± 0·1,P < 0·0001 for enterococci and 0·9 ± 0·1,P < 0·0001 forE. coli). Among 1072 enterococci isolates tested from septic tanks, 203 biochemical phenotypes (BPTs) were found of which 98 BPTs from 33 septic tanks were identical to several water samples. Similarly, among 621E. coliisolates tested from septic tanks, 159 BPTs were found of which 53 BPTs from 26 septic tanks were also identical to water samples. The number of the latter bacteria was significantly (P = 0·01) higher in water samples collected from downstream compared with that of upstream in the study area. A high similarity between the populations of both indicator bacteria was also found between defective septic tanks and downstream water samples further indicating the contamination of both creeks by defective septic systems.Biochemical fingerprinting of faecal indicator bacteria is a useful and rapid method to provide direct evidence for septic system failure. Combination of both faecal indicator bacteria (enterococci andE. coli) provides a better judgement of the performance of a septic system.This study is the first to provide direct evidence of septic system failure by identifying the presence of specific bacterial types in septic tanks and surface waters. Based on our findings, we suggest that the performance evaluation of a septic system should be accompanied by direct analysis of faecal indicator bacteria. [ABSTRACT FROM AUTHOR]

n.m.k. yousif, p. dawyndt, h. abriouel, a. wijaya, u. schillinger, m. vancanneyt, j. swings, h.a. dirar, w.h. holzapfel and c.m.a.p. franz. 2004.To identify enterococci from Hussuwa, a Sudanese fermented sorghum product, and determine their technological properties and safety for possible inclusion in a starter culture preparation.Twenty-twoEnterococcusisolates from Hussuwa were identified asEnterococcus faeciumby using phenotypic and genotypic tests such as 16S rDNA gene sequencing, RAPD-PCR and restriction fragment length polymorphism of the 16S/23S intergenic spacer region fingerprinting. Genotyping revealed that strains were not clonally related and exhibited a considerable degree of genomic diversity. Some strains possessed useful technological properties such as production of bacteriocins and H2O2 or utilization of raffinose and stachyose. None producedα-amylase or tannase. A safety investigation revealed that all strains were susceptible to the antibiotics ampicillin, gentamicin, chloramphenicol, tetracycline and streptomycin, but some were resistant to ciprofloxacin, erythromycin, penicillin and vancomycin. Production of biogenic amines or presence of genes encoding virulence determinants occurred in some strains.Enterococcus faeciumstrains are associated with fermentation of Sudanese Hussuwa. Some strains exhibited useful technological properties such as production of antimicrobial agents and fermentation of indigestible sugars, which may aid in stabilizing and improving the digestibility of the product respectively.Enterococci were shown to play a role in the fermentation of African foods. While beneficial properties of these bacteria are indicated, their presence in this food may also imply a hygienic risk as a result of antimicrobial resistances or presence of virulence determinants. [ABSTRACT FROM AUTHOR]

Abstract Aims: The influence of two disinfection techniques on natural biofilm development during drinking water treatment and subsequent distribution is compared with regard to the supply of a high-quality drinking water. Methods and Results: The growth of biofilms was studied using the biofilm device technique in a real public technical drinking water asset. Different pipe materials which are commonly used in drinking water facilities (hardened polyethylene, polyvinyl chloride, steel and copper) were used as substrates for biofilm formation. Apart from young biofilms, several months old biofilms were compared in terms of material dependence, biomass and physiological state. Vital staining of biofilms with 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) and the DNA-specific 4′,6-diamidino-2-phenylindole (DAPI) staining resulted in a significant difference in physiological behaviour of biofilm populations depending on the disinfection technique. Compared with chlorine dioxide disinfection (0·12–0·16 mg l-1 ), the respiratory activities of the micro-organisms were increased on all materials during u.v. disinfection (u.v.254 ; 400 J m-2 ). The biofilm biocoenosis was analysed by in situ hybridization with labelled oligonucleotides specific for some subclasses of Proteobacteria . Using PCR and additional hybridization techniques, the biofilms were also tested for the presence of Legionella spp., atypical mycobacteria and enterococci. The results of the molecular-biological experiments in combination with cultivation tests showed that enterococci were able to pass the u.v. disinfection barrier and persist in biofilms of the distribution system, but not after chlorine dioxide disinfection. Conclusions: The results indicated that bacteria are able to regenerate and proliferate more effectively after u.v. irradiation at the waterworks, and chlorine dioxide disinfection appears to be more applicative to maintain a biological stable drinking water. Significance and Impact of the Study: As far as the application of u.v. disinfection is used for conditioning of critical water sources for drinking water, the efficiency of u.v. irradiation in natural systems should reach a high standard to avoid adverse impacts on human health. [ABSTRACT FROM AUTHOR]

Abstract Aims: To evaluate the numbers and selected phenotypic and genotypic characteristics of the faecal indicator bacteria Escherichia coli and enterococci in gull faeces at representative Great Lakes swimming beaches in the United States. Methods and Results: E. coli and enterococci were enumerated in gull faeces by membrane filtration. E. coli genotypes (rep-PCR genomic profiles) and E. coli (Vitek® GNI+) and enterococci (API® rapid ID 32 Strep and resistance to streptomycin, gentamicin, vancomycin, tetracycline and ampicillin) phenotypes were determined for isolates obtained from gull faeces both early and late in the swimming season. Identical E. coli genotypes were obtained only from single gull faecal samples but most faecal samples yielded more than one genotype (median of eight genotypes for samples with 10 isolates). E. coli isolates from the same site that clustered at ≥85% similarity were from the same sampling date and shared phenotypic characteristics, and at this similarity level there was population overlap between the two geographically isolated beach sites. Enterococcus API® profiles varied with sampling date. Gull enterococci displayed wide variation in antibiotic resistance patterns, and high-level resistance to some antibiotics. Conclusions: Gull faeces could be a major contributor of E. coli (105 –109 CFU g-1 ) and enterococci (104 –108 CFU g-1 ) to Great Lakes recreational waters. E. coli and enterococci in gull faeces are highly variable with respect to their genotypic and phenotypic characteristics and may exhibit temporal or geographic trends in these features. Significance and Impact of the Study: The high degree of variation in genotypic or phenotypic characteristics of E. coli or enterococci populations within gull hosts will require extensive sampling for adequate characterization, and will... [ABSTRACT FROM AUTHOR]

Abstract Aims: Carbon source utilization profiles as a phenotypic fingerprinting methodology for determining sources of faecal pollution in water were evaluated. Methods and Results: Three hundred and sixty-five Enterococcus isolates were collected from known faecal sources in four different geographical regions and were identified to species with the commercial Biolog system. Discriminant analysis (DA) was used to identify the substrate-containing wells that best classified the 365 isolates by source. By using 30 of the 95 wells for the analysis, the average rate of correct classification (ARCC) by source was 92·7% for a human vs non-human two-way classification when isolates from all regions were combined into one library. Corresponding ARCCs for other classification schemes were 81·9% for a four-way classification of human vs livestock vs wildlife vs domestic pets, and 85·7% for a three-way classification without human isolates. When three individual libraries were made based on classification of sources within Enterococcus species, the ARCC was 95·3% for the Ent. faecalis library, 95·8% for the Ent. gallinarum library and 94·7% for the Ent. mundtii library. Thirty Enterococcus isolates (unknown sources) were obtained from each of three stream sites where a specific source of pollution was apparent; 90·0% of the isolates from a human-suspected source were classified as human, 86·6% were classified as livestock from a livestock-suspected site, and 93·3% were classified as wildlife from a wildlife-suspected site. Conclusions: Phenotypic fingerprinting with carbon source utilization profiles provided levels of correct classification by sources from an Enterococcus library that were in the upper range of those reported in the literature. ARCCs for three Enterococcus species-specific libraries were very high and may be the best approach for further developing this concept and methodology. Significance and Impact of the... [ABSTRACT FROM AUTHOR]