Antimicrobial resistance in Enterococcus spp. isolated in inflow, effluent and sludge from municipal sewage water treatment plants

Antimicrobial resistance of enterococci was investigated in 42 samples of crude inflow, treated effluent and sludge collected in 14 municipal sewage treatment plants of Portugal. A total of 983 enterococci were recovered and tested, using the diffusion agar method, regarding their sensitivity to 10 different antimicrobial drugs. Multidrug resistance was present in 49.4% of the isolates. Only 3.3% and 0.6% of the investigated strains were resistant to ampicillin and vancomycin, respectively. Resistances found against rifampicin (51.5%), tetracycline (34.6%), erythromycin (24.8%) and nitrofurantoin (22.5%), are causes for substantial concern. Almost 14% of isolates were resistant to ciprofloxacin. Wastewater treatment resulted in enterococci decrease between 0.5 and 4log; nevertheless, more than 4.4 x 10(5)CFU/100ml were present in the outflow of the plants. Our data indicate that the use of antimicrobials had created a large pool of resistance genes and that sewage treatment processes are unable to avoid the dissemination of resistant enterococci into the environment.     

Comparison of Enterococcus measurements in freshwater at two recreational beaches by quantitative polymerase chain reaction and membrane filter culture analysis

Cell densities of the fecal pollution indicator genus, Enterococcus, were determined by a rapid (3 h or less) quantitative polymerase chain reaction (QPCR) analysis method in 100 ml water samples collected from recreational beaches on Lake Michigan and Lake Erie during the summer of 2003. Measurements by this method were compared with counts of Enterococcus colony-forming units (CFU) determined by Method 1600 membrane filter (MF) analysis using mEI agar. The QPCR method had an estimated 95% confidence, minimum detection limit of 27 Enterococcus cells per sample in analyses of undiluted DNA extracts and quantitative analyses of multiple lake water samples, spiked with known numbers of these organisms, gave geometric mean results that were highly consistent with the spike levels. At both beaches, the geometric means of ambient Enterococcus concentrations in water samples, determined from multiple collection points during each sampling visit, showed approximately lognormal distributions over the study period using both QPCR and MF analyses. These geometric means ranged from 10 to 8548 cells by QPCR analysis and 1-2499 CFU by MF culture analysis in Lake Michigan (N=56) and from 8 to 8695 cells by QPCR and 3-1941 CFU by MF culture in Lake Erie (N=47). Regression analysis of these results showed a significant positive correlation between the two methods with an overall correlation coefficient (r) of 0.68.     

Water quality prediction of marine recreational beaches receiving watershed baseflow and stormwater runoff in southern California, USA

Beach advisories are issued to the public in California when the concentration of fecal indicator bacteria (FIB), including total coliform, fecal coliform (or Escherichia coli), and Enterococcus, exceed their recreational water health standards, or when the amount of a rainfall event is above the pre-determined threshold. However, it is not fully understood about how and to what degree stormwater runoff or baseflow exerts impacts on beach water quality. Furthermore, current laboratory methods used to determine the FIB levels take 18-96 h, which is too slow to keep pace with changes in FIB levels in water. Thus, a beach may not be posted when it is contaminated, and may be posted under advisory when bacterial levels have already decreased to within water quality standards. The study was designed to address the above critical issues. There were large temporal and spatial variations in FIB concentrations along two popular State Beaches in San Diego, CA, USA. The rainstorm-induced runoff from the watersheds exerts significant impacts on the marine recreational water quality of the beaches adjacent to lagoons during the first 24-48 h after a rain event. The large volume of stormwater runoff discharging to beaches caused high FIB concentrations in beach water not only at the lagoon outlet channel and the mixing zone, but also at the locations 90 m away from the channel northward or southward along the shoreline. The geomorphology of beach shoreline, distance from the outlet channel, wind strength, wind direction, tide height, wave height, rainfall, time lapse after a rainstorm, or channel flow rate played a role in affecting the distribution of FIB concentrations in beach water. Despite the great temporal and spatial variability of FIB concentrations along a shoreline, the artificial neural network-based models developed in this study are capable of successfully predicting FIB concentrations at different beaches, different locations, and different times under baseflow or rainstorm conditions. The models are based on readily measurable variables including temperature, conductivity, pH, turbidity, channel water flow, rainfall, and/or time lapse after a rainstorm. The established models will help fill the current gap between beach posting and actual water quality and make more meaningful and effective decisions on beach closures and advisories.     

Traditional and molecular analyses for fecal indicator bacteria in non-point source subtropical recreational marine waters

The use of enterococci as the primary fecal indicator bacteria (FIB) for the determination of recreational water safety has been questioned, particularly in sub/tropical marine waters without known point sources of sewage. Alternative FIB (such as the Bacteroidales group) and alternative measurement methods (such as rapid molecular testing) have been proposed to supplement or replace current marine water quality testing methods which require culturing enterococci. Moreover, environmental parameters have also been proposed to supplement current monitoring programs. The objective of this study was to evaluate the health risks to humans from exposure to subtropical recreational marine waters with no known point source. The study reported symptoms between one set of human subjects randomly assigned to marine water exposure with intensive environmental monitoring compared with other subjects who did not have exposure. In addition, illness outcomes among the exposed bathers were compared to levels of traditional and alternative FIB (as measured by culture-based and molecular-based methods), and compared to easily measured environmental parameters. Results demonstrated an increase in self-reported gastrointestinal, respiratory and skin illnesses among bathers vs. non-bathers. Among the bathers, a dose–response relationship by logistic regression modeling was observed for skin illness, where illness was positively related to enterococci enumeration by membrane filtration (odds ratio = 1.46 [95% confidence interval = 0.97–2.21] per increasing log10 unit of enterococci exposure) and positively related to 24 h antecedent rain fall (1.04 [1.01–1.07] per increasing millimeters of rain). Acute febrile respiratory illness was inversely related to water temperature (0.74 [0.56–0.98] per increasing degree of water temperature). There were no significant dose–response relationships between report of human illness and any of the other FIB or environmental measures. Therefore, for non-point source subtropical recreational marine waters, this study suggests that humans may be at increased risk of reported illness, and that the currently recommended and investigational FIB may not track gastrointestinal illness under these conditions; the relationship between other human illness and environmental measures is less clear.     

Antimicrobial resistance of Listeria monocytogenes isolated from dairy-based food products

In this study Listeria monocytogenes (L. monocytogenes) was isolated from three traditionally consumed Lebanese dairy-based food products. One hundred and sixty four samples (45 samples of Baladi cheese, 36 samples of Shankleesh and 83 of Kishk) were collected from the Bekaa Valley in the Northeast region of Lebanon. Suspected Listeria colonies were selected and initially identified by using standard biochemical tests. Initial identification of the positive L. monocytogenes colonies was confirmed at the molecular level by Polymerase Chain Reaction (n = 30) and the confirmed isolates were evaluated for their susceptibility to 10 commonly used antimicrobials. All of the 30 isolates were confirmed to be L. monocytogenes yielding a PCR product of ∼ 660 base pairs (bp). L. monocytogenes was detected in 26.67%, 13.89% and 7.23% of the Baladi cheese, Shankleesh and Kishk samples, respectively. The highest resistance in L. monocytogenes isolates was noted against oxacillin (93.33%) followed by penicillin (90%). The results provide an indication of the contamination levels of dairy-based foods in Lebanon and highlight the emergence of multi-drug resistant Listeria in the environment.     

Rapid QPCR-based assay for fecal Bacteroides spp. as a tool for assessing fecal contamination in recreational waters

Concentrations of fecal indicator bacteria (FIB; e.g. Escherichia coli, and Enterococcus sp.) can only be used in limited ways for determining the source of fecal contamination in recreational waters because they cannot distinguish human from non-human fecal contamination. Several Bacteroides spp. have been suggested as potential alternative indicators. We have developed a rapid, culture-independent method for quantifying fecal Bacteroides spp. using quantitative PCR (QPCR) targeting the 16S rRNA gene. The assay specifically targets and quantifies the most common human Bacteroides spp. The details of the method are presented, including analyses of a wide range of fecal samples from different organisms. Specificity and performance of the QPCR assay were also tested via a laboratory experiment where human sewage and gull guano were inoculated into a range of environmental water samples. Concentrations of fecal Bacteroides spp., total Enterococcus sp., Enterococcus faecium, Enterococcus faecalis, and Enterococcus casseliflavus were measured using QPCR, and total Enterococcus sp. and E. coli were quantified by membrane filtration (MF). Samples spiked with gull guano were highly concentrated with total Enterococcus sp., E. coli, E. faecalis, and E. casseliflavus, demonstrating that these indicators are prominent in animal feces. On the other hand, fecal Bacteroides spp. concentrations were high in samples containing sewage and were relatively low in samples spiked with gull guano. Sensitivity and specificity results suggest that the rapid fecal Bacteroides spp. QPCR assay may be a useful tool to effectively predict the presence and concentration of human-specific fecal pollution.     

Correlation between cone penetration resistance and standard penetration in parts of the coastal plain sands,Southeastern Nigeria

Test results using the cone and standard penetrometers carried out in parts of the Coastal Plain Sands were correlated by graphical means. Results indicate that the cone resistance q_c, increases with increasing standard penetration, N, giving a positive slope on a normal arithmetic plot. The least square equation relating both parameters is in the form y=mx. The q_c and N values are then used as the basis for rating the engineering characteristics of the Coastal Plain Sands.     

Surveillance of adenoviruses and noroviruses in European recreational waters

Exposure to human pathogenic viruses in recreational waters has been shown to cause disease outbreaks. In the context of Article 14 of the revised European Bathing Waters Directive 2006/7/EC (rBWD, CEU, 2006) a Europe-wide surveillance study was carried out to determine the frequency of occurrence of two human enteric viruses in recreational waters. Adenoviruses were selected based on their near-universal shedding and environmental survival, and noroviruses (NoV) selected as being the most prevalent gastroenteritis agent worldwide. Concentration of marine and freshwater samples was done by adsorption/elution followed by molecular detection by (RT)-PCR. Out of 1410 samples, 553 (39.2%) were positive for one or more of the target viruses. Adenoviruses, detected in 36.4% of samples, were more prevalent than noroviruses (9.4%), with 3.5% GI and 6.2% GII, some samples being positive for both GI and GII. Of 513 human adenovirus-positive samples, 63 (12.3%) were also norovirus-positive, whereas 69 (7.7%) norovirus-positive samples were adenovirus-negative. More freshwater samples than marine water samples were virus-positive. Out of a small selection of samples tested for adenovirus infectivity, approximately one-quarter were positive. Sixty percent of 132 nested-PCR adenovirus-positive samples analysed by quantitative PCR gave a mean value of over 3000 genome copies per L of water. The simultaneous detection of infectious adenovirus and of adenovirus and NoV by (RT)PCR suggests that the presence of infectious viruses in recreational waters may constitute a public health risk upon exposure. These studies support the case for considering adenoviruses as an indicator of bathing water quality.     

Differential patterns of antimicrobial resistance in population subsets of Escherichia coli isolated from waste- and surface waters

The species Escherichia coli comprises different subgroups with distinct phylogeny, physiology and ecology and, thus, presumably, with different roles in antimicrobial resistance dissemination. E. coli strains isolated from raw and treated municipal wastewater and from urban water streams were characterized in terms of phylogenetic groups, antimicrobial resistance patterns and the presence of class 1 and class 2 integrons. Our main objective was to investigate the contribution of the different phylo-groups in antimicrobial resistance dissemination in urban waters. Groups A and B1 were predominant in all types of water, evidencing, respectively, the lowest and the highest resistance prevalence. Municipal wastewater treatment was accompanied by significant increases of ciprofloxacin and streptomycin resistance (p < 0.01). Antimicrobial resistance prevalence differed significantly between the different phylo-groups and within the same group, mainly in group A. Such differences contributed to explain the higher ciprofloxacin and streptomycin resistance rates observed in treated effluent in comparison with the raw wastewater. We conclude that the dynamics of the bacterial populations has a major role on the dissemination of antimicrobial resistance in the environment.     

Diversity and antifungal susceptibility of yeasts from tropical freshwater environments in Southeastern Brazil

Yeast communities were isolated from water and sediment samples of two unpolluted natural lakes, located inside Rio Doce State Park, and two rivers located outside of this Park in Southeastern Brazil. A total of 134 yeast isolates were obtained and identified as belonging to 36 species. The numbers of fecal coliforms and yeast species were higher in rivers than in lakes. The genus Candida had the highest number of species with the presence of opportunistic pathogens such as Candida krusei, C. tropicalis, C. guilliermondii and C. parapsilosis. Yeasts able to grow at 37 degrees C were tested in relation to their susceptibility to common used antifungal drugs. Yeast isolates (13%) were susceptible to ketoconazole, 79% to fluconazole, 31% to terbinafine and 78% of the strains were susceptible to amphotericin B. Seven isolates from different Candida species were resistant to all antifungals tested. The high number of fecal coliforms found in these aquatic environments and the presence of resistant yeast strains to common used antifungal drugs suggest that these environments can pose potential health risks for people utilizing the contaminated waters.     

Antimicrobial resistance of fecal bacteria in waters of the Seine river watershed (France)

We studied the prevalences of antimicrobial resistance (AR) and multiple antimicrobial resistance (MAR) among the fecal bacteria found in the rivers of a large watershed under strong anthropogenic pressures, the Seine river watershed (France). Two groups of fecal indicator bacteria, Escherichia coli and intestinal enterococci, were tested for their susceptibility to 16 and 10 antimicrobials respectively, using the disk diffusion method. We found that 42% of the 214 E. coli river isolates were AR (resistant to at least one antimicrobial) and 35% were MAR (resistant to at least two antimicrobials). Among the 148 intestinal enterococci isolates from rivers, 83% were AR and 49% were MAR. We also investigated the sources of AR fecal bacteria found in the rivers of the watershed. A total of 715 E. coli isolates and 476 intestinal enterococci isolates were collected in point sources (municipal and hospital wastewaters) and non-point sources (surface runoff and soil leaching waters from agricultural or forest areas). For E. coli, the prevalence of AR differed widely from source to source and ranked in this order: hospital wastewaters (71%) > municipal wastewaters (44%) > agricultural non-point sources (16%) > forest non-point sources (2%). The prevalence of MAR ranked similarly, and the same trend was observed for intestinal enterococci. The AR level of fecal bacteria in the sources was related to their expected exposure level to antimicrobials before their release into the environment. A MAR index was calculated for every source and a good discrimination between them was thus obtained. At the global scale of the Seine river watershed, domestic wastewaters seemed more likely to be the predominant source of the AR fecal bacteria found in the rivers. This was corroborated by the similarity of the MAR indices from river and municipal wastewater isolates for both fecal indicators.     

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The chemical composition of highway drainage waters IV. Alkyllead compounds in runoff waters

Alkyllead compounds have been analysed in road drainage water samples collected during seven storm events using a sensitive gas chromatography-atomic absorption technique. The most commonly observed alkyllead species were tetraethyllead, triethyllead and trimethyllead. Similar compounds were also observed in rainwater collected at the site, but not in road surface dust or bottom sediment from the drainage channel. In all samples analysed the concentration of alkyllead was < 5% of the dissolved (< 0.45 μm) inorganic lead. It is postulated that washout of alkyllead from the atmosphere is the major source of alkyllead compounds in the drainage waters analysed. Alkyllead species were also identified in rainwater collected at other sites.     

Occurrence of rotaviruses and enteroviruses in recreational waters of Oak Creek, Arizona

Recent epidemiological studies have shown a relationship between swimming in recreational waters meeting bacteriological standards and gastroenteritis with a suggested viral etiology. No previous studies have been conducted in the United States on the occurrence of human pathogenic enteric viruses in freshwater recreational areas. The presence of enteroviruses and rotaviruses was investigated in Oak Creek, Arizona, a heavily used recreational area. Water samples were filtered through positively charged filters (168–1555 I.), eluted with beef extract, and assayed for human enteroviruses and rotaviruses. Eighteen of the 41 recreational water samples were positive for enterovirus or rotavirus. Of these, nine samples exceeded the Arizona State recommended limit of 1 PFU 40 l⁻¹ for full body contact in effluent dominated recreational waters. Several virus positive samples met the recommended fecal coliform standards (200 CFU 100 ml⁻¹) for recreational waters indicating the inadequacy of bacterial standards for monitoring viral water quality. The isolation of the pathogenic enteric viruses (i.e., poliovirus 1, echovirus 1, coxsackievirus B1 and B6 and rotavirus) from this popular recreational water demonstrates the potential for transmission of viral disease.     

Estimated human health risks from exposure to recreational waters impacted by human and non-human sources of faecal contamination

This work was conducted to determine whether estimated risks following exposure to recreational waters impacted by gull, chicken, pig, or cattle faecal contamination are substantially different than those associated with waters impacted by human sources such as treated wastewater. Previously published Quantitative Microbial Risk Assessment (QMRA) methods were employed and extended to meet these objectives. Health outcomes used in the analyses were infection from reference waterborne pathogens via ingestion during recreation and subsequent gastrointestinal (GI) illness. Illness risks from these pathogens were calculated for exposure to faecally contaminated recreational water at the U.S. regulatory limits of 35 cfu 100 mL⁻¹ enterococci and 126 cfu 100 mL⁻¹Escherichia coli. The probabilities of GI illness were calculated using pathogen dose-response relationships from the literature and Monte Carlo simulations. Three scenarios were simulated, representing a range of feasible interpretations of the available data. The primary findings are that: 1) GI illness risks associated with exposure to recreational waters impacted by fresh cattle faeces may not be substantially different from waters impacted by human sources; and 2) the risks associated with exposure to recreational waters impacted by fresh gull, chicken, or pig faeces appear substantially lower than waters impacted by human sources. These results suggest that careful consideration may be needed in the future for the management of recreational waters not impacted by human sources.     

Contribution of treated wastewater to the contamination of recreational river areas with Cryptosporidium spp. and Giardia duodenalis

Samples of the influent and final effluent from 12 wastewater treatment plants from Galicia (NW, Spain) were analyzed for the presence of Cryptosporidium spp. oocysts and Giardia duodenalis cysts. All of the plants discharge effluent to a hydrographic basin in which there are numerous recreational areas and fluvial beaches. The samples (25-50 liters) were collected in spring, summer, autumn and winter of 2007. A total of 96 samples were analyzed using techniques included in the US Environmental Protection Agency Method 1623. To identify the genotypes present, the following genes were amplified and sequenced: 18S SSU rRNA (Cryptosporidium spp.) and beta-giardina (G. duodenalis). Both parasites were detected in influent and effluent samples from all treatment plants (100%) throughout the year, and G. duodenalis always outnumbered Cryptosporidium spp. The mean concentration of G. duodenalis per liter of influent was significantly higher (P<0.05) than the mean concentration of Cryptosporidium spp. per liter of influent. The mean concentrations of parasites in influent samples ranged from 6 to 350 Cryptosporidium spp. oocysts per liter and from 89 to 8305 G. duodenalis cysts per liter. In final treated effluent, the mean concentration of parasites ranged from 2 to 390 Cryptosporidium spp. oocysts per liter and from 79 to 2469 G. duodenalis cysts per liter. The distribution of results per season revealed that in all plants, the highest number of (oo)cysts were detected in spring and summer. Cryptosporidium parvum, Cryptosporidium andersoni, Cryptosporidium hominis and assemblages A-I, A-II, E of G. duodenalis were detected. The risk of contamination of water courses by Cryptosporidium spp. and G. duodenalis is therefore considerable. It is important that wastewater treatment authorities reconsider the relevance of the levels of contamination by both parasites in wastewater, and develop adequate countermeasures.     

Relationships between microbial water quality and environmental conditions in coastal recreational waters: the Fylde coast, UK.

This paper explores ways in which the analysis of microbial data from routine compliance monitoring, in combination with basic environmental data, can provide insight into the factors affecting faecal-indicator organism concentrations in coastal waters. In the case study presented, eight designated bathing waters on the Fylde coast are continuing to exhibit unreliable compliance with the Imperative standards for total coliform (TC) and faecal coliform (FC) concentrations specified in the EU Bathing Water Directive (76/160/EEC), despite significant reductions in geometric mean concentrations following recent major investment in the sewerage infrastructure. Faecal streptococci (FS) concentrations have remained high and have not been improved by the new sewerage schemes. The results suggest that, prior to the schemes, higher bacterial concentrations were strongly associated with rainfall; and sewage sources were important for TC and FC, but less important for FS, which may have been more strongly affected by diffuse catchment sources. In the post-schemes period, catchment sources appear to be of greater significance; rainfall remains as a significant, though less important, predictor; and tide height at time of sampling, together with variables such as sunshine and the proportion of onshore winds (which affect the survival and movement of bacteria that have already entered the coastal waters), assume greater significance. The approach used here provides a cost-effective management tool for the exploratory investigation of any monitoring point that is failing to meet recreational water quality standards.     

Accumulation and trophic transfer of organotins in a marine food web from the Danish coastal waters

The presence of organotin compounds, e.g., tributyltin (TBT) and triphenyltin (TPhT) including the di- and monosubstituted breakdown products, was studied in a representative marine food web in order to assess the accumulation potential at different trophic levels in Danish coastal waters. This included samples of two species of seaweed, four species of invertebrates, four species of fish, five species of birds and two species of mammals. All organisms were sampled away from harbour areas and the organotin concentrations found in this study can therefore be considered to reflect a general level in organisms living in Danish coastal waters. All the samples analysed contained organotin compounds. The highest hepatic concentrations of butyltins were found in flounder (60-259 ng g-1 wet weight [ww], as Sn), eider duck (12-202 ng g-1 ww) and harbour porpoise (134-2283 ng g-1 ww). The lowest concentrations were found in seaweed and a plant-feeding bird. TPhT or its degradation products were also found in most of the samples with the highest concentrations in flounder (9.8-74 ng g-1 ww), cod (23-28 ng g-1 ww) and great black-backed gull (19-24 ng g-1 ww). This indicates an input of TPhT in the region, probably from the use as antifouling agent. A high variance in accumulation potential was found between the species, even between species at the same trophic level, which probably reflects the species-specific differences in exposure routes and the capabilities to metabolise and eliminate the organotin compounds. This study gives evidence of the importance of biomagnification of butyltin in harbour porpoises and, to a lesser extent, in fish and birds.     

Evaluation of public health risks at recreational beaches in Lake Michigan via detection of enteric viruses and a human-specific bacteriological marker

Each year the National Resource Defense Council addresses the quality of US beaches by routine bacterial indicators. In the Great Lakes region the indicator used is Escherichia coli and for 2007 more beaches were closed and impacted than ever before. In this study, water quality was addressed at two Lake Michigan Beaches over the 2004 swimming season by monitoring infectious enteric viruses by cell culture and integrated PCR and for a human sewage marker based on the Enterococcal Surface Protein (esp). Our goals for this study were to 1) examine the occurrence and variety of human enteric viruses present during peak usage of the beaches 2) determine key variables for development of predictive models for viruses; and 3) use quantitative risk assessment to estimate the potential health impact. Our results demonstrate that for both beaches predictive models of virus pollution were best described utilizing physical parameters like wind speed, wind direction and water temperature. The esp marker was not predictive of human viruses. The daily risk of acquiring a viral infection at either of the beaches ranged from 0.2 to 2.4/1000 swimmers using a quantitative microbial risk assessment model, with three swims during a day at the beach for children and over the season, the risk was 9-15/1000 swimmers using adenovirus as the model.Conclusions: Lake Michigan recreational beaches are being adversely impacted by human fecal pollution. Monitoring for the traditional indicators of water quality does not address viral risks and models can be developed and potentially used as real-time water quality forecasting tools.     

The marine corrosion of structural steels in brackish and fresh waters

High-quality models are required to describe the corrosion of structural steels in marine, brackish and fresh waters and these models should, preferably, be based on a proper understanding of the corrosion processes involved. The models should then be used to interpret field data, rather than attempting to derive models empirically from field data. On this basis a multi-phase corrosion–time model has been proposed previously for mild and low alloy steels subject to immersion in ‘at-sea’ conditions. This model is reviewed and extended to brackish and fresh waters in this paper. The scientific principles show that salinity is not the critical issue for the corrosiveness of water. Instead, the critical parameters are the temperature, pH and calcium carbonate solubility when corrosion is governed by oxidation. For later corrosion, governed by anaerobic bacterial activity, the available nutrient level and rate of supply of nutrients control the observed rate of corrosion. The data available at the present time for estimating all the parameters of the new model are somewhat preliminary and new corrosion monitoring programmes will be required to augment the information and to fully characterize the corrosivity of coastal and harbour waters. However, indicative trends for the parameters, based on currently available data, are given and permit the construction of corrosion loss curves for practical applications.