Tolerance of three fungal species to lithium and cobalt: Implications for bioleaching of spent rechargeable Li-ion batteries.

AIMS: This paper aims to quantify the growth and organic acid production of Aspergillus niger, Penicillium chrysogenum and Penicillium simplicissimum when these fungi are exposed to varying levels of lithium (Li) and cobalt (Co). The study also tests whether pre-exposing the fungi to these metals enables the fungi to develop tolerance to Li or Co. METHODS AND RESULTS: When cultures of A. niger, P. chrysogenum or P. simplicissimum were exposed to 250 mg l-1 of Li or Co, biomass production and excretion of organic acids were significantly inhibited after 5 days of growth compared to cultures grown in the absence of these metals. Pre-exposing cultures of A. niger to 250 mg l-1 of Li or Co for 20 days significantly increased biomass production when the fungus was subsequently sub-cultured into 250 or 500 mg l-1 of Li or Co. However, pre-exposure of P. chrysogenum or P. simplicissimum to 250 mg l-1 of Li or Co for 20 days did not increase biomass production. CONCLUSIONS: Aspergillus niger, but not the Penicillium species, developed tolerance to Li and to Co during the 20-day pre-exposure period. Therefore, processes that utilize fungal bioleaching with A. niger to mobilize and recover valuable metals such as Li or Co should consider a pre-exposure step for fungi to improve their tolerance to metal toxicity. SIGNIFICANCE AND IMPACT OF THE STUDY: Fungi may have the ability to extract valuable metals such as Li and Co from spent rechargeable batteries. However, the toxicity of the extracted metals can inhibit fungal growth and organic acid production. Pre-exposure to metals may alleviate toxicity for some fungal species. This knowledge can be used to improve the design of bioleaching protocols, increasing the potential for fungal bioleaching to become an economical and environmentally friendly method of recovering Li and Co from spent batteries.

The Interplay Between Predation, Competition, and Nutrient Levels Influences the Survival of Escherichia coli in Aquatic Environments.

Nutrient levels, competition from autochthonous microorganisms, and protozoan predation may all influence survival of fecal microorganisms as they transition from the gastrointestinal tract to aquatic habitats. Although Escherichia coli is an important indicator of waterborne pathogens, the effects of environmental stressors on its survival in aquatic environments remain poorly understood. We manipulated organic nutrient, predation, and competition levels in outdoor microcosms containing natural river water, sediments, and microbial populations to determine their relative contribution to E. coli survival. The activities of predator (protozoa) and competitor (indigenous bacteria) populations were inhibited by adding cycloheximide or kanamycin. We developed a statistical model of E. coli density over time that fits with the data under all experimental conditions. Predation and competition had significant negative effects on E. coli survival, while higher nutrient levels increased survival. Among the main effects, predation accounted for the greatest variation (40 %) compared with nutrients (25 %) or competition (15 %). The highest nutrient level mitigated the effect of predation on E. coli survival. Thus, elevated organic nutrients may disproportionately enhance the survival of E. coli, and potentially that of other enteric bacteria, in aquatic habitats.

Comparison of concentration methods for quantitative detection of sewage-associated viral markers in environmental waters.

Pathogenic human viruses cause over half of gastroenteritis cases associated with recreational water use worldwide. They are relatively difficult to concentrate from environmental waters due to typically low concentrations and their small size. Although rapid enumeration of viruses by quantitative PCR (qPCR) has the potential to greatly improve water quality analysis and risk assessment, the upstream steps of capturing and recovering viruses from environmental water sources along with removing PCR inhibitors from extracted nucleic acids remain formidable barriers to routine use. Here, we compared the efficiency of virus recovery for three rapid methods of concentrating two microbial source tracking (MST) viral markers human adenoviruses (HAdVs) and polyomaviruses (HPyVs) from one liter tap water and river water samples on HA membranes (90 mm in diameter). Samples were spiked with raw sewage, and viral adsorption to membranes was promoted by acidification (method A) or addition of MgCl2 (methods B and C). Viral nucleic acid was extracted directly from membranes (method A), or viruses were eluted with NaOH and concentrated by centrifugal ultrafiltration (methods B and C). No inhibition of qPCR was observed for samples processed by method A, but inhibition occurred in river samples processed by B and C. Recovery efficiencies of HAdVs and HPyVs were ∼10-fold greater for method A (31 to 78%) than for methods B and C (2.4 to 12%). Further analysis of membranes from method B revealed that the majority of viruses were not eluted from the membrane, resulting in poor recovery. The modification of the originally published method A to include a larger diameter membrane and a nucleic acid extraction kit that could accommodate the membrane resulted in a rapid virus concentration method with good recovery and lack of inhibitory compounds. The frequently used strategy of viral absorption with added cations (Mg(2+)) and elution with acid were inefficient and more prone to inhibition, and will result in underestimation of the prevalence and concentrations of HAdVs and HPyVs markers in environmental waters.

Run-off studies demonstrate parallel transport behaviour for a marker of poultry fecal contamination and Staphylococcus aureus.

AIMS: To determine whether poultry litter marker gene LA35 is correlated with pathogens and fecal indicator bacteria (FIB) in run-off from poultry litter-amended plots. METHODS AND RESULTS: A rainfall simulator with various vegetative filter strip lengths was employed to evaluate the correlation of a microbial source tracking (MST) marker for poultry feces/litter (the 16S rRNA gene of Brevibacterium sp. LA35 [LA35] measured by quantitative PCR) with pathogens and FIB in run-off. LA35 was correlated with Staphylococcus aureus, Escherichia coli, Enterococcus spp. and Bacteroidales levels. Salmonella was present at low concentration in litter, but became undetectable by qPCR in run-off. Escherichia coli, LA35 and Staph. aureus exhibited mass-based first flush behaviour in the run-off. CONCLUSIONS: Correlation of LA35 with FIB and pathogens in run-off from poultry litter-amended fields suggest comparable transport mechanisms and that LA35 is a useful tracer for harmful bacteria in the environment released from poultry litter. SIGNIFICANCE AND IMPACT OF THE STUDY: To protect human health, an effective marker for poultry fecal contamination should exhibit similar fate and transport characteristics compared to pathogens. This study is among the first to demonstrate such a relationship in run-off for a MST marker.

Microbial water quality in freshwater lakes with different land use.

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 (HPyVs), 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. HPyVs 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.

Microbial source tracking to identify human and ruminant sources of faecal pollution in an ephemeral Florida river.

AIMS: Levels and sources of faecal indicator bacteria (FIB) in an ephemeral Florida river were assessed under different rainfall/flow patterns to explore the effects of rainfall on water quality. METHODS AND RESULTS: Quantitative PCR for sewage markers [human-associated Bacteroides HF183 and human polyomaviruses (HPyVs)] and PCR for ruminant faecal markers were used to explore contamination sources. Escherichia coli, faecal coliform and enterococci levels consistently exceeded recreational water quality criteria, and sediment FIB levels were about 100-fold higher compared with water. HPyVs detections cooccurred with HF183, which was frequently detected near septic systems. Ruminant markers were detected only in livestock-grazing areas. Significantly greater faecal coliform and E. coli concentrations were observed under no-flow conditions and the levels of faecal coliforms in water column and sediments were negatively correlated with duration since last rain event. CONCLUSIONS: Septic systems and cattle grazing in this watershed contributed to the formation of FIB reservoirs in sediments, which were persistent following prolonged rainfall. SIGNIFICANCE AND IMPACT OF THE STUDY: Ephemeral water bodies that flow only under the direct influence of recent rainfall are rarely studied. FIB levels in the New River in Florida were greater during dry weather than wet weather, which contrasts with most observations and may be attributed to bacterial reservoirs formed in still pool, sediments and water-saturated soils in this subtropical environment.

Investigation of human sewage pollution and pathogen analysis at Florida Gulf coast beaches.

AIMS: 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. METHODS AND RESULTS: 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. CONCLUSIONS: These data indicate that infrastructure remediation significantly reduced pollution from human sewage at the impacted beach. SIGNIFICANCE AND IMPACT OF THE STUDY: 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.

Indicator microbes correlate with pathogenic bacteria, yeasts and helminthes in sand at a subtropical recreational beach site.

AIMS: Research into the relationship between pathogens, faecal indicator microbes and environmental factors in beach sand has been limited, yet vital to the understanding of the microbial relationship between sand and the water column and to the improvement of criteria for better human health protection at beaches. The objectives of this study were to evaluate the presence and distribution of pathogens in various zones of beach sand (subtidal, intertidal and supratidal) and to assess their relationship with environmental parameters and indicator microbes at a non-point source subtropical marine beach. METHODS AND RESULTS: In this exploratory study in subtropical Miami (Florida, USA), beach sand samples were collected and analysed over the course of 6 days for several pathogens, microbial source tracking markers and indicator microbes. An inverse correlation between moisture content and most indicator microbes was found. Significant associations were identified between some indicator microbes and pathogens (such as nematode larvae and yeasts in the genus Candida), which are from classes of microbes that are rarely evaluated in the context of recreational beach use. CONCLUSIONS: Results indicate that indicator microbes may predict the presence of some of the pathogens, in particular helminthes, yeasts and the bacterial pathogen Staphylococcus aureus including methicillin-resistant forms. Indicator microbes may thus be useful for monitoring beach sand and water quality at non-point source beaches. SIGNIFICANCE AND IMPACT OF THE STUDY: The presence of both indicator microbes and pathogens in beach sand provides one possible explanation for human health effects reported at non-point sources beaches.

Identification of a Brevibacterium marker gene specific to poultry litter and development of a quantitative PCR assay.

AIM: To identify a DNA sequence specific to a bacterium found in poultry litter that was indicative of faecal contamination by poultry sources. METHODS AND RESULTS: Faecally contaminated poultry litter and soils were used as source material for the development of a quantitative polymerase chain reaction (qPCR) method targeting the 16S rRNA gene of a Brevibacterium sp. The identified sequence had 98% nucleotide identity to the 16S rRNA gene of Brevibacterium avium. The qPCR method was tested on 17 soiled litter samples; 40 chicken faecal samples; and 116 nontarget faecal samples from cattle, swine, ducks, geese, and human sewage collected across the United States. The 571-bp product was detected in 76% of poultry-associated samples, but not in 93% of faecal samples from other sources. Marker concentrations were 10(7) -10(9) gene copies per gram in soiled litter, up to 10(5) gene copies per gram in spread-site soils, and 10(7) gene copies per litre in field run-off water. Results were corroborated by a blinded study conducted by a second laboratory. CONCLUSION: The poultry-specific PCR product is a useful marker gene for assessing the impact of faecal contamination as a result of land-applied poultry litter. SIGNIFICANCE AND IMPACT OF THE STUDY: This study describes the first quantitative, sensitive and specific microbial source tracking method for the detection of poultry litter contamination.

Application of microbial source tracking methods in a Gulf of Mexico field setting.

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.

Microbial population dynamics in laboratory-scale solid waste bioreactors in the presence or absence of biosolids.

AIMS: Decomposition of solid waste is microbially mediated, yet little is known about the associated structure and temporal changes in prokaryotic communities. Bioreactors were used to simulate landfill conditions and archaeal and bacterial community development in leachate was examined over 8 months. METHODS AND RESULTS: Municipal solid waste (MSW) was deposited in laboratory bioreactors with or without biosolids and combustion residues (ash). The near-neutral pH fell about half a log by day 25, but recovered to approximately 7.0 by day 50. Cell concentrations in bioreactors containing only MSW were significantly higher than those from co-disposal bioreactors. Archaeal and bacterial community structure was analysed by denaturing gradient gel electrophoresis targeting 16S rRNA genes, showing temporal population shifts for both domains. mcrA sequences retrieved from a co-disposal bioreactor were predominantly affiliated with the orders Methanosarcinales and Methanomicrobiales. CONCLUSION: Regardless of waste composition, microbial communities in bioreactor leachates exhibited high diversity and distinct temporal trends. The solid waste filled bioreactors allowed simulation of solid waste decomposition in landfills while also reducing the variables. SIGNIFICANCE AND IMPACT OF THE STUDY: This study advances the basic understanding of changes in microbial community structure during solid waste decomposition, which may ultimately improve the efficiency of solid waste management.

Pepper mild mottle virus: A plant pathogen with a greater purpose in (waste)water treatment development and public health management.

An enteric virus surrogate and reliable domestic wastewater tracer is needed to manage microbial quality of food and water as (waste)water reuse becomes more prevalent in response to population growth, urbanization, and climate change. Pepper mild mottle virus (PMMoV), a plant pathogen found at high concentrations in domestic wastewater, is a promising surrogate for enteric viruses that has been incorporated into over 29 water- and food-related microbial quality and technology investigations around the world. This review consolidates the available literature from across disciplines to provide guidance on the utility of PMMoV as either an enteric virus surrogate and/or domestic wastewater marker in various situations. Synthesis of the available research supports PMMoV as a useful enteric virus process indicator since its high concentrations in source water allow for identifying the extent of virus log-reductions in field, pilot, and full-scale (waste)water treatment systems. PMMoV reduction levels during many forms of wastewater treatment were less than or equal to the reduction of other viruses, suggesting this virus can serve as an enteric virus surrogate when evaluating new treatment technologies. PMMoV excels as an index virus for enteric viruses in environmental waters exposed to untreated domestic wastewater because it was detected more frequently and in higher concentrations than other human viruses in groundwater (72.2%) and surface waters (freshwater, 94.5% and coastal, 72.2%), with pathogen co-detection rates as high as 72.3%. Additionally, PMMoV is an important microbial source tracking marker, most appropriately associated with untreated domestic wastewater, where its pooled-specificity is 90% and pooled-sensitivity is 100%, as opposed to human feces where its pooled-sensitivity is only 11.3%. A limited number of studies have also suggested that PMMoV may be a useful index virus for enteric viruses in monitoring the microbial quality of fresh produce and shellfish, but further research is needed on these topics. Finally, future work is needed to fill in knowledge gaps regarding PMMoV's global specificity and sensitivity.

Determination of wild animal sources of fecal indicator bacteria by microbial source tracking (MST) influences regulatory decisions.

Fecal indicator bacteria (FIB) are used to assess fecal pollution levels in surface water and are among the criteria used by regulatory agencies to determine water body impairment status. While FIB provide no information about pollution source, microbial source tracking (MST) does, which contributes to more direct and cost effective remediation efforts. We studied a watershed in Florida managed for wildlife conservation that historically exceeded the state regulatory guideline for fecal coliforms. We measured fecal coliforms, enterococci, a marker gene for avian feces (GFD), and a marker gene for human-associated Bacteroides (HF183) in sediment, vegetation, and water samples collected monthly from six sites over two years to: 1) assess the influence of site, temporal factors, and habitat (sediment, vegetation, and water) on FIB and MST marker concentrations, 2) test for correlations among FIB and MST markers, and 3) determine if avian feces and/or human sewage contributed to FIB levels. Sediment and vegetation had significantly higher concentrations of FIB and GFD compared to water and thus may serve as microbial reservoirs, providing unreliable indications of recent contamination. HF183 concentrations were greatest in water samples but were generally near the assay limit of detection. HF183-positive results were attributed to white-tailed deer (Odocoileus virginianus) feces, which provided a false indication of human sewage in this water body. FIB and GFD were positively correlated while FIB and HF183 were negatively correlated. We demonstrated that birds, not sewage, were the main source of FIB, thus avoiding implementation of a total maximum daily load program (TMDL). Our results demonstrate that the concomitant use of FIB and MST can improve decision-making and provide direction when water bodies are impaired, and provides a strategy for natural source exclusion in water bodies impacted by wild animal feces.

Confirmation of putative stormwater impact on water quality at a Florida beach by microbial source tracking methods and structure of indicator organism populations.

The effect of a stormwater conveyance system on indicator bacteria levels at a Florida beach was assessed using microbial source tracking methods, and by investigating indicator bacteria population structure in water and sediments. During a rain event, regulatory standards for both fecal coliforms and Enterococcus spp. were exceeded, contrasting with significantly lower levels under dry conditions. Indicator bacteria levels were high in sediments under all conditions. The involvement of human sewage in the contamination was investigated using polymerase chain reaction (PCR) assays for the esp gene of Enterococcus faecium and for the conserved T antigen of human polyomaviruses, all of which were negative. BOX-PCR subtyping of Escherichia coli and Enterococcus showed higher population diversity during the rain event; and higher population similarity during dry conditions, suggesting that without fresh inputs, only a subset of the population survives the selective pressure of the secondary habitat. These data indicate that high indicator bacteria levels were attributable to a stormwater system that acted as a reservoir and conduit, flushing high levels of indicator bacteria to the beach during a rain event. Such environmental reservoirs of indicator bacteria further complicate the already questionable relationship between indicator organisms and human pathogens, and call for a better understanding of the ecology, fate and persistence of indicator bacteria.

Microbial source tracking in shellfish harvesting waters in the Gulf of Nicoya, Costa Rica.

Current microbial water quality monitoring is generally limited to culture-based measurements of fecal indicator bacteria (FIB). Given the many possible sources of fecal pollution within a watershed and extra-intestinal FIB reservoirs, it is important to determine source(s) of fecal pollution as a means to improve water quality and protect public health. The principal objective of this investigation was to characterize the microbial water quality of shellfish harvesting areas in the Gulf of Nicoya, Costa Rica during 2015. In order to achieve this objective, the specificity and sensitivity of 11 existing microbial source tracking (MST) PCR assays, associated with cows (BacCow), dogs (BacCan, DogBac), domestic wastewater (PMMoV), general avian (GFD), gulls (Gull2), horses (HorseBac, HoF), humans (HF183, HPyV), and pigs (PF), were evaluated using domestic wastewater and animal fecal samples collected from the region. The sensitivity of animal-associated assays ranged from 13 to 100%, while assay specificity ranged from 38 to 100%. The specificity of pepper mild mottle virus (PMMoV) and human polyomavirus (HPyV) was 100% for domestic wastewater, as compared to 94% specificity of the HF183 Bacteroidales marker. PMMoV was identified as a useful domestic wastewater-associated marker, with concentrations as high as 1.1 × 105 copies/ml and 100% sensitivity and specificity. Monthly surface water samples collected from four shellfish harvesting areas were analyzed using culture-based methods for Escherichia coli as well as molecular methods for FIB and a suite of MST markers, which were selected for their specificity in the region. While culturable E. coli results suggested possible fecal pollution during the monitoring period, the absence of human/domestic wastewater-associated markers and low FIB concentrations determined using molecular methods indicated sufficient microbial water quality for shellfish harvesting. This is the first study to our knowledge to test the performance of MST markers in Costa Rica as well as in Central America. Given the lack of wastewater treatment and the presence of secondary sources of FIB, this study highlights the importance of an MST toolbox approach to characterize water quality in tropical regions. Furthermore, it confirms and extends the geographic range of PMMoV as an effective tool for monitoring domestic wastewater pollution.

Utility of Helicobacter spp. associated GFD markers for detecting avian fecal pollution in natural waters of two continents.

Avian fecal droppings may negatively impact environmental water quality due to the presence of high concentrations of fecal indicator bacteria (FIB) and zoonotic pathogens. This study was aimed at evaluating the performance characteristics and utility of a Helicobacter spp. associated GFD marker by screening 265 fecal and wastewater samples from a range of avian and non-avian host groups from two continents (Brisbane, Australia and Florida, USA). The host-prevalence and -specificity of this marker among fecal and wastewater samples tested from Brisbane were 0.58 and 0.94 (maximum value of 1.00). These values for the Florida fecal samples were 0.30 (host-prevalence) and 1.00 (host-specificity). The concentrations of the GFD markers in avian and non-avian fecal nucleic acid samples were measured at a test concentration of 10 ng of nucleic acid at Brisbane and Florida laboratories using the quantitative PCR (qPCR) assay. The mean concentrations of the GFD marker in avian fecal nucleic acid samples (5.2 × 10(3) gene copies) were two orders of magnitude higher than non-avian fecal nucleic acid samples (8.6 × 10(1) gene copies). The utility of this marker was evaluated by testing water samples from the Brisbane River, Brisbane and a freshwater creek in Florida. Among the 18 water samples tested from the Brisbane River, 83% (n = 18) were positive for the GFD marker, and the concentrations ranged from 6.0 × 10(1)-3.2 × 10(2) gene copies per 100 mL water. In all, 92% (n = 25) water samples from the freshwater creek in Florida were also positive for the GFD marker with concentrations ranging from 2.8 × 10(1)-1.3 × 10(4) gene copies per 100 mL water. Based on the results, it can be concluded that the GFD marker is highly specific to avian host groups, and could be used as a reliable marker to detect the presence and amount of avian fecal pollution in environmental waters.

The influence of rainfall on the incidence of microbial faecal indicators and the dominant sources of faecal pollution in a Florida river.

AIMS: An assessment of microbial densities in an urbanized Florida watershed was performed during a period of changing rainfall patterns to investigate the role of climate coupled with urbanization in declining water quality. METHODS AND RESULTS: Concentrations of traditional and alternative faecal indicators were assessed by standard methods over 24 months. Sources of faecal contamination were determined by antibiotic resistance analysis (ARA) of faecal coliform (FC) bacteria. Composite indices of indicator organisms based on a suite of microbial measurements were used to quantify pollution impacts in the river. ARA results found that FC from wild animal sources dominated during the drought, and the relative frequency of FC from human sources increased after cumulative rainfall increased to near-normal levels. CONCLUSIONS: Changes observed in faecal indicator densities and in FC sources during changing rainfall patterns strongly suggest a role of precipitation on the sources and extent of microbial pollution in urbanized coastal watersheds. SIGNIFICANCE AND IMPACT OF THE STUDY: Bacterial source tracking coupled with a composite index of microbial contamination resulted in a more complete picture of microbial pollution within the river, as opposed to the general practice of reliance on one indicator organism. Improved land use decisions in urban areas are necessary to insure maintenance of coastal environmental health under changing climate patterns and population density.

Evaluation of antibiotic resistance analysis and ribotyping for identification of faecal pollution sources in an urban watershed.

AIMS: The accuracy of ribotyping and antibiotic resistance analysis (ARA) for prediction of sources of faecal bacterial pollution in an urban southern California watershed was determined using blinded proficiency samples. METHODS AND RESULTS: Antibiotic resistance patterns and HindIII ribotypes of Escherichia coli (n = 997), and antibiotic resistance patterns of Enterococcus spp. (n = 3657) were used to construct libraries from sewage samples and from faeces of seagulls, dogs, cats, horses and humans within the watershed. The three libraries were analysed to determine the accuracy of host source prediction. The internal accuracy of the libraries (average rate of correct classification, ARCC) with six source categories was 44% for E. coli ARA, 69% for E. coli ribotyping and 48% for Enterococcus ARA. Each library's predictive ability towards isolates that were not part of the library was determined using a blinded proficiency panel of 97 E. coli and 99 Enterococcus isolates. Twenty-eight per cent (by ARA) and 27% (by ribotyping) of the E. coli proficiency isolates were assigned to the correct source category. Sixteen per cent were assigned to the same source category by both methods, and 6% were assigned to the correct category. Addition of 2480 E. coli isolates to the ARA library did not improve the ARCC or proficiency accuracy. In contrast, 45% of Enterococcus proficiency isolates were correctly identified by ARA. CONCLUSIONS: None of the methods performed well enough on the proficiency panel to be judged ready for application to environmental samples. SIGNIFICANCE AND IMPACT OF THE STUDY: Most microbial source tracking (MST) studies published have demonstrated library accuracy solely by the internal ARCC measurement. Low rates of correct classification for E. coli proficiency isolates compared with the ARCCs of the libraries indicate that testing of bacteria from samples that are not represented in the library, such as blinded proficiency samples, is necessary to accurately measure predictive ability. The library-based MST methods used in this study may not be suited for determination of the source(s) of faecal pollution in large, urban watersheds.

Regulation of extracellular copper-binding proteins in copper-resistant and copper-sensitive mutants of Vibrio alginolyticus.

Extracellular proteins of wild-type Vibrio alginolyticus were compared with those of copper-resistant and copper-sensitive mutants. One copper-resistant mutant (Cu40B3) constitutively produced an extracellular protein with the same apparent molecular mass (21 kDa) and chromatographic behavior as copper-binding protein (CuBP), a copper-induced supernatant protein which has been implicated in copper detoxification in wild-type V. alginolyticus. Copper-sensitive V. alginolyticus mutants displayed a range of alterations in supernatant protein profiles. CuBP was not detected in supernatants of one copper-sensitive mutant after cultures had been stressed with 50 microM copper. Increased resistance to copper was not induced by preincubation with subinhibitory levels of copper in the wild type or in the copper-resistant mutant Cu40B3. Copper-resistant mutants maintained the ability to grow on copper-amended agar after 10 or more subcultures on nonselective agar, demonstrating the stability of the phenotype. A derivative of Cu40B3 with wild-type sensitivity to copper which no longer constitutively expressed CuBP was isolated. The simultaneous loss of both constitutive CuBP production and copper resistance in Cu40B3 indicates that constitutive CuBP production is necessary for copper resistance in this mutant. These data support the hypothesis that the extracellular, ca. 20-kDa protein(s) of V. alginolyticus is an important factor in survival and growth of the organism at elevated copper concentrations. The range of phenotypes observed in copper-resistant and copper-sensitive V. alginolyticus indicate that altered sensitivity to copper was mediated by a variety of physiological changes.