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.     

Genotypic characterization of hospital Enterococcus faecalis strains using multiple-locus variable-number tandem-repeat analysis

Aims:  The level of genetic diversity and relationships between the specific genotypes and the distribution of virulence determinants among Enterococcus faecalis strains isolated from patients hospitalized in different wards of two hospitals were investigated.
Methods and Results:  Fifty-six clinical strains of E. faecalis , isolated from patients hospitalized in the period of 1999–2004 in several wards in Wrocław (Poland), were analysed by multiple-locus variable-number tandem-repeat analysis (MLVA). Analysis of seven genomic loci identified 40 novel genotypes among the analysed E. faecalis strains, with two major genomic groups, designated I and II, distinguished at a cut-off of 35%. With a similarity cut-off of 85·7%, the genotypes could be combined into 12 clusters (C1–C12), containing at least two isolates. The remaining 18 MLVA types were represented by a single isolate.
Conclusions:  Based on the data obtained by MLVA, it was found that (i) many E. faecalis isolates recovered from patients from the wards whose location allowed the potential transmission of micro-organisms, belonged to closely related MLVA types and (ii) possible relationships between specific E. faecalis genotype and the virulence factors lipase, haemolysin and esp gene can exist.
Significance and Impact of the Study:  Our study confirms that MLVA is a suitable method for the epidemiological study of E. faecalis and for the first time shows possible relationships between specific genotypes and such virulence determinants, i.e. lipase, haemolysin and esp gene.     

Genomic reticulation indicates mixed ancestry in Southern-Hemisphere Mytilus spp. mussels

Previous surveys of allozyme variation in smooth-shell Mytilus spp. mussels have reported the presence in the Southern Hemisphere of both Mytilus edulis and Mytilus galloprovincialis mussels. In the present study, nuclear DNA markers mac-1 and Glu-5 '/ Glu-3 ', both diagnostic for Northern-Hemisphere M. edulis and M. galloprovincialis , were used to further characterize the nuclear genomes of M. edulis from Kerguelen and M. galloprovincialis from Tasmania. Genomic reticulation was observed, with typical M. edulis allelomorphs fixed in both populations at locus mac-1 whereas, at locus Glu-5 '/ Glu-3 ', allelomorphs characteristic of M. galloprovincialis were present in Kerguelen and nearly fixed in Tasmania. Kerguelen mussels had a genome of mixed M. edulis and M. galloprovincialis ancestry without evidence of barriers to merging as shown by Hardy–Weinberg and linkage equilibrium. Tasmanian mussels possessed a predominantly M. galloprovincialis genomic background introgressed by M. edulis allelomorphs at locus mac-1 . Genetic drift superimposed on ancient hybridization and introgression may explain the genomic reticulation observed in both Kerguelen and Tasmanian mussels. There was no evidence of a recent introduction of Northern-Hemisphere M. galloprovincialis or M. edulis to Kerguelen or Tasmania.  © 2007 The Linnean Society of London, Biological Journal of the Linnean Society , 2007, 92 , 747–754.     

Mortality of zebra mussel, Dreissena polymorpha, veligers during downstream transport

1. Streams flowing from lakes which contain zebra mussels, Dreissena polymorpha , provide apparently suitable habitats for mussel colonization and downstream range expansion, yet most such streams contain few adult mussels. We postulated that mussel veligers experience high mortality during dispersal via downstream transport. They tested this hypothesis in Christiana Creek, a lake-outlet stream in south-western Michigan, U.S.A., in which adult mussel density declined exponentially with distance downstream.
2. A staining technique using neutral red was developed and tested to distinguish quickly live and dead veligers. Live and dead veligers were distinguishable after an exposure of fresh samples to 13.3 mg L⁻⁻¹ of neutral red for 3 h.
3. Neutral red was used to determine the proportion of live veligers in samples taken longitudinally along Christiana Creek. The proportion of live veligers (mean ± SE) declined from 90 ± 3% at the lake outlet to 40 ± 8% 18 km downstream.
4. Veligers appear to be highly susceptible to damage by physical forces (e.g. shear), and therefore, mortality in turbulent streams could be an important mechanism limiting zebra mussel dispersal to downstream reaches. Predictions of zebra mussel spread and population growth should consider lake-stream linkages and high mortality in running waters.     

The mediterranean mussel Mytilus galloprovincialis Lmk. in Japan

Mussels ( Mytilus sp.) from Sanriku Bay, NE Honshu, Japan were examined using morphological characters and electrophoretically detectable enzyme polymorphisms. Using both sets of criteria, the mussels were identified as M. galloprovincialis , the mediterranean mussel. This confirms an earlier opinion, which was based on morphological criteria alone, that the mediterranean mussel occurs on the mainland coast of Japan. Investigation of some early Japanese literature suggests that mussels did not occur in this area earlier this century, and M. galloprovincialis may have been introduced to the region of Kobe, around 1930–1935. The present-day distribution of M. edulis and M. galloprovincialis in the Japanese archipelago may be explained by sea-surface temperatures in the region.     

Assessment of environmental pollution at Balearic Islands applying oxidative stress biomarkers in the mussel Mytilus galloprovincialis

The antioxidant enzyme response of the mussel Mytilus galloprovincialis to different degree of pollution was investigated. Antioxidant enzyme activities - catalase (CAT), glutathione peroxidase (GSH-PX), glutathione reductase (GR), superoxide dismutase (SOD) - and malondialdehyde (MDA) concentration were measured in gills and digestive glands of mussels. Mussels from the same origin were transplanted along the Balearic coastal waters in eight stations characterized by a different degree of contamination and human impacts. Antioxidant enzyme activities showed an adaptive response to increase the activities in the more polluted areas. CAT, GR and SOD in gills and CAT and GR in digestive gland presented significant differences between polluted and non-polluted stations. No significant differences were observed in MDA concentration indicating that the antioxidant response is capable to avoid the lipid peroxidation. The use of biomarkers such as CAT and GR in gills and digestive glands of the mussel M. galloprovincialis is a good tool to categorize differences between polluted and non-polluted areas.     

Settlement and growth of the green mussel Perna viridis (L.) in coastal waters: influence of water velocity

Green mussels Perna viridis were observed to be a major foulant in the seawater intake tunnel of a coastal power station. Field experiments were carried out to ascertain what factors were responsible for the successful colonisation by mussels. Two adjacent stations (25 m apart) were selected, one representing the coastal waters and the other representing the intake screens (with higher water velocity). Gonadal activity, larval abundance, spat settlement and growth rate of the mussels were monitored at monthly intervals for a total period of two years. The results showed that the breeding activity of the mussels at the study area is influenced largely by temporal distribution of seawater temperature. However, ensuing larval availability in the coastal waters is more dependent on food availability. On the other hand, spat settlement and growth rate are predominantly influenced by water flow, probably as a result of increased propagule and food flux rate at higher water velocities. Higher water velocity at the intake screens also contributed to mussel dominance by preventing settlement of many potential competitors.     

Vancomycin-resistant Enterococcus spp. in marine environments from the West Coast of the USA

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.     

Predicting zebra mussel fouling on native mussels from physicochemical variables

1. Predictive models of impact are needed for the risk assessment of invasive species. One such species is the Eurasian zebra mussel ( Dreissena polymorpha ), a fouling bivalve that overgrows and kills native mussels (Unionidae) in many North American lakes and rivers. The level of mortality in a native mussel population increases with the fouling intensity – i.e. the mean number of zebra mussels attached to each native mussel.
2. We conducted a multi-site survey within the St Lawrence and Richelieu rivers (Quebec, Canada) to determine whether zebra mussel fouling intensity can be predicted from environmental variables. We found fouling intensity to be positively correlated with calcium concentration [Ca²⁺] and negatively correlated with sediment size, but not affected by local macrophyte cover.
3. A multiple regression model that includes calcium concentration and sediment size explains 86% of variation in fouling intensity across all sites.
4. Analysis of data from invaded sites in North America and Europe revealed a nonlinear relationship in which fouling intensity increases with calcium concentration up to an asymptotic threshold of 24 mg L⁻¹ Ca²⁺.
5. Our results suggest that the community-level impacts of zebra mussels are mediated by abiotic environmental variables and gradients in these variables may provide local habitat refugia for native mussels.     

Tracing of norovirus outbreak strains in mussels collected near sewage effluents

Noroviruses from mussels collected near sewage effluents were compared with local patient outbreak strains. Sequence analyses of RNA polymerase-capsid-poly(A)-3' (3.1-kilobase) regions confirmed the 99.9% similarity between genotype I.1 strains from mussels and patient strains from recreational-bathing outbreaks, indicating the potential usefulness of sentinel norovirus mussel studies in tracing human norovirus contamination of coastal waters.     

Effects of an invasive bivalve on the zooplankton community of the Hudson River

1. Previous studies documented that zebra mussels became abundant in the Hudson River during 1992 causing an 80–90% reduction in phytoplankton biomass. This study used intervention time series analysis of abundance, biomass and reproduction over the period 1987–95 to assess changes in zooplankton in response to the invasion.
2. Zebra mussels caused a size-dependent decline in zooplankton. Microzooplankton, including tintinnid ciliates, rotifers and copepod nauplii all declined in 1992 and were scarce thereafter. Mean abundances of post-naupliar copepods and of cladocerans were also lower following the invasion but these changes were not statistically significant ( P > 0.05). Egg ratios and clutch sizes for the dominant cladoceran, Bosmina freyi , were not significantly related to zebra mussels, even though relatively low egg ratios were observed after the invasion.
3. The strong declines in microzooplankton were probably caused by direct zebra mussel predation. Estimated consumption rates by mussels were roughly equivalent to maximum microzooplankton growth rates.
4. The total biomass of zooplankton in the Hudson River declined by more than 70% following the invasion. Annual average zooplankton biomass was correlated with chlorophyll, but biomass per unit chlorophyll in the Hudson River was much lower than in lakes. The present study hypothesizes that this lower biomass reflects limitations by riverine flow and by predation during summer.     

Escherichia coli detection using mTEC agar and fluorescent antibody direct viable counting on coastal recreational water samples

Aims:  Escherichia coli is the faecal indicator species recommended by the US Environmental Protection Agency (USEPA) for monitoring fresh recreational water. Viable but nonculturable (VBNC) E. coli are living cells that are dormant and not culturable using standard microbiological cultivation methods. This study reports a comparison between the mTEC culture method recommended by USEPA for E. coli enumeration and a fluorescent antibody-direct viable count (FA-DVC) method to visualize living E. coli cells with a microscope.
Methods and Results:  Escherichia coli , faecal coliforms and Enterococcus were detected using standard methods recommended by the USEPA. VBNC E. coli was visualized with FA-DVC. Results were analysed with standard statistical methods (Pearson correlation; paired-sample t -test). Significantly higher numbers of E. coli were detected using the FA-DVC method than using the mTEC method. Escherichia coli results were also compared with faecal coliform (mFC broth) and Enterococcus (mEI agar) counts in the same samples.
Conclusions:  The results of this comparative study demonstrate that E. coli can be present in higher numbers than what are detected with standard culture methods.
Significance and Impact of the Study:  This study re-emphasizes the need for a rapid, accurate and precise method for detecting health risks to humans who use recreational waters.     

Factors influencing the upper temperature tolerances of three mussel species in a brackish water canal: size, season and laboratory protocols

Mussels are the most problematic organisms encountered in the water intake systems of electrical power plants. Various fouling control measures are adopted, among which heat treatment is considered the relatively more attractive from economic and ecological points of view. Thermal tolerance experiments were carried out to determine the effects of mussel size (2-20 mm shell length), season (breeding vs non-breeding), nutritional status (fed vs non-fed), acclimation temperature (5-25 degrees C) and acclimation salinity (1-35%o) on the mortality pattern of three important mussel species, viz. a freshwater mussel Dreissena polymorpha, a brackish water mussel Mytilopsis leucophaeata and a marine mussel Mytilus edulis under different temperatures (36-41 degrees C). The mussels in the 10 mm size group exposed to 36 degrees C showed 100% mortality after 38 min (D. polymorpha), 84 min (M. edulis) and 213 min (M. leucophaeata). The effect of mussel size on M. edulis and M. leucophaeata mortality at different temperatures was significant, with the largest size group of mussels showing greater resistance, while no significant size-dependence was observed in the case of D. polymorpha. All the three mussel species collected during the non-breeding season (June-October). Nutritional status had no significant influence on the thermal tolerance of the three mussels; fed and non-fed mussels showed 100% mortality at comparable rates. Acclimation temperature had a significant effect on the mortality of all three species. Survival time at any given target temperature increased with increasing acclimation temperature. The acclimation salinity showed no significant effect on the thermal tolerance of the three mussel species. In comparison, M. leucophaeata was more tolerant to high temperature stress than the other two species. The present studies clearly show that various factors can influence the mortality of D. polymorpha, M. edulis and M. leucophaeata to elevated temperatures. The results, therefore, suggest that if heat treatment were to be used as a control measure for these mussels, it has to be employed judiciously, depending on the mussel species, mussel size, breeding season, water temperature and salinity.     

Application of PFGE to source tracking of faecal pollution in coastal recreation area: a case study in Aoshima Beach, Japan

Aims: 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. Methods and Results: 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. Conclusions: 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. Significance and Impact of the Study: 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.     

The incidence of the Pea crab, Pinnotheres pisum in the two types of Mytilus (Mollusca: Bivalvia) from Padstow, south-west England

Significant differences in the infection of M. edulis and the "Padstow type" mussel with P. pisum are recorded, and some possible explanations for these differences are discussed.
Both types of Mytilus from the mid and lower regions of the mussel bed showed heavier infections than mussels higher on the shore. Even so, the differences between the two types were still maintained.
A relationship exists between crab and mussel size, larger crabs being found only in larger hosts. The smallest mussel found to be infected with Pinnotheres measured 3·35 cm in length.
Infection in M. edulis was found to increase with increased size of host, the largest occurring mussels having from 80 to 100% infection. Larger mussels occurred in greater numbers in the low shore. It is assumed that infection in the "Padstow type" would show a similar relationship if sufficient recordings had been available.
The presence of the crab causes gill damage, and infected mussels show considerably lower tissue weights and slightly greater shell weights than uninfected mussels of similar size.
The presence of the crab does not appear to influence the reproductive capacity of the mussel.     

The effect of seasonal variation in selective feeding by zebra mussels (Dreissena polymorpha) on phytoplankton community composition

1. To investigate the impact of zebra mussels ( Dreissena polymorpha ) on phytoplankton community composition, temporal variability in selective feeding by the mussels was determined from April to November 2005 in a natural lake using Delayed Fluorescence (DF) excitation spectroscopy.
2. Selective grazing by zebra mussels varied in relation to seasonal phytoplankton dynamics; mussels showed a consistent preference for cryptophytes and avoidance of chlorophytes and cyanobacteria. Diatoms, chrysophytes and dinoflagellates responded differentially to zebra mussel grazing depending on their size. Analysis of excreted products of the zebra mussels revealed that in addition to chlorophytes and cyanobacteria, phytoplankton >50  μ m and very small phytoplankton (≤7  μ m) were largely expelled in pseudofaeces.
3. The zebra mussel is a selective filter-feeder that alters its feeding behaviour in relation to phytoplankton composition to capture and ingest high quality phytoplankton, especially when phytoplankton occur in preferred size ranges. Flexibility of zebra mussel feeding behaviour and variation in susceptibility among phytoplankton groups to mussel ingestion indicate that invading zebra mussels could alter phytoplankton community composition of lakes and have important ecosystem consequences.     

Enumeration and speciation of enterococci found in marine and intertidal sediments and coastal water in southern California

AIMS: 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. METHODS AND RESULTS: 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. CONCLUSIONS: High levels of Enterococcus in intertidal sediments indicate retention and possible regrowth in this environment. SIGNIFICANCE AND IMPACT OF THE STUDY: 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.     

Identification and tracing of Enterococcus spp. by RAPD-PCR in traditional fermented sausages and meat environment

Aims:  Four local small-scale factories were studied to determine the sources of enterococci in traditional fermented sausages.
Methods and Results:  Different points during the production of a traditional fermented sausage type ( fuet ) were evaluated. Randomly amplified polymorphic DNA (RAPD)-PCR was used to type 596 Enterococcus isolates from the final products, the initial meat batter, the casing, the workers' hands and the equipment. Species-specific PCR-multiplex and the partial sequencing of atpA gene and 16S rRNA gene sequencing allowed the identification of the isolates: Enterococcus faecalis (31·4%), Enterococcus faecium (30·7%), Enterococcus sanguinicola (14·9%), Enterococcus devriesei (9·7%), Enterococcus malodoratus (7·2%), Enterococcus gilvus (1·0%), Enterococcus gallinarum (1·3%), Enterococcus casseliflavus (3·4%), Enterococcus hermanniensis (0·2%), and Enterococcus durans (0·2%) . A total of 92 different RAPD-PCR profiles were distributed among the different factories and samples evaluated. Most of the genotypes found in fuet samples were traced back to their source.
Conclusions:  The major sources of enterococci in the traditional fermented sausages studied were mainly the equipment followed by the raw ingredients, although a low proportion was traced back to human origin.
Significance and Impact of the Study:  This work contributes to determine the source of enterococcal contamination in fermented sausages and also to the knowledge of the meat environment.     

Mytilid mussels: global habitat engineers in coastal sediments

Dense beds of mussels of the family Mytilidae occur worldwide on soft-bottoms in cold and warm temperate coastal waters and have usually been considered hot spots of biodiversity. We examined intertidal mussel beds at four distant locations around the globe with the same sampling method, to find out whether this “hot spot” designation holds universally. We studied species assemblages within the matrices of byssally interconnected mussels engineered by Mytilus edulis in the North Sea, by mixed Perumytilus purpuratus and Mytilus chilensis at the southern Chilean coast, by Musculista senhousia in the Yellow Sea and by Xenostrobus inconstans at the coast of southern Australia. In all cases, species assemblages inside mussel beds were significantly different from those outside with many species being restricted to one habitat type. However, species richness and diversity were not generally higher in mussel beds than in ambient sediments without mussels. In the North Sea (M. edulis) and at the Chilean coast (P. purpuratus, M. chilensis), mussel beds have markedly higher species numbers and diversities than surrounding sediments, but this was not the case for mussel beds in Australia (X. inconstans) and the Yellow Sea (M. senhousia) where numbers of associated species were only slightly higher and somewhat lower than in adjacent sediments, respectively. In conclusion, although soft bottom mytilid mussels generally enhance habitat heterogeneity and species diversity at the ecosystem level, mussel beds themselves are not universal centres of biodiversity, but the effects on associated species are site specific.     

Zebra mussels mediate benthic–pelagic coupling by biodeposition and changing detrital stoichiometry

1. The zebra mussel ( Dreissena polymorpha ) is one of the most successful invasive species; it has colonised many aquatic systems in Europe and North America with strong impacts on various ecosystem processes. The effect of D. polymorpha filtration on pelagic seston concentrations has been quantified in several studies, but the magnitude and stoichiometry of the transfer of sestonic biomass into benthic detritus by D. polymorpha and the accompanying enrichment of the benthic habitat is still under-investigated.
2. We studied biodeposition by zebra mussels in two series of laboratory experiments with the food algae Cryptomonas erosa and Scenedesmus obliquus . We also measured the year-round biodeposition rate under natural conditions in the oligotrophic Lake Constance.
3. In all experiments, zebra mussel biodeposition was linearly related to seston concentration. In the field, the relationship changed with a seasonal shift in algal composition and lower biodeposition rates during the spring algal bloom.
4. For both algal species in laboratory experiments, biodeposited material was depleted in phosphorous at an algal concentration ≤0.6 mg ash-free dry mass L⁻¹, but not at higher concentrations. This effect was not observed in the field, probably because of high variation in C : N : P stoichiometry.
5. By mediating the transfer of pelagic resources into the benthos zebra mussels provide a sufficient amount of detritus for benthic invertebrates, especially during summer. Thus, material biodeposited by the mussels might increase benthic secondary production from pelagic resources, and zebra mussels are important mediators of this flux of organic matter from the pelagic zone into the benthos.