Analytical comparison of nine PCR primer sets designed to detect the presence of Escherichia coli/Shigella in water samples

The analytical performance of 9 different PCR primer sets designed to detect Escherichia coli and Shigella in water has been evaluated in terms of ubiquity, specificity, and analytical detection limit. Of the 9 PCR primer sets tested, only 3 of the 5 primer sets targeting uidA gene and the primer set targeting tuf gene amplified DNA from all E. coli strains tested. However, of those 4 primer sets, only the primer set targeting the tuf gene also amplified DNA from all Shigella strains tested. For the specificity, only the primer sets targeting the uidA gene were 100% specific although the primer sets targeting 16S rRNA, phoE, and tuf genes only amplified Escherichia fergusonii as non-specific target. Finally, the primer set targeting the 16S-ITS-23S gene region, was not specific as it amplified DNA from many other Enterobacteriaceae species. In summary, only the assay targeting the tuf gene detected all E. coli/Shigella strains tested in this study. However, if it becomes important to discriminate between E. coli and E. fergusonii, assays targeting the uidA gene would represent a good choice although none of them were totally ubiquitous to detect of the presence of Shigella strains.     

Metabolic turnover and catalase activity of biofilms of Pseudomonas fluorescens (ATCC 17552) as related to copper corrosion

In this work we report the results of a combined biochemical and electrochemical study aimed to analyze both the growth of biofilms of Pseudomonas fluorescens on copper samples and its possible role in the instability of the metal/electrolyte interface.DNA and RNA were quantified along the time for biofilms grown on copper and glass to estimate both the growth of the bacterial population and its metabolic state (through the RNA/DNA ratio). The expression and specific activity of catalase were also determined to gain insight into their possible role in corrosion acceleration. The electrochemical behavior of the biofilm/copper interface was monitored by Linear Polarization Resistance (Rp) and electrochemical impedance spectroscopy (EIS) along the experiments.Results showed a longer lag phase for biofilms developing on copper that included a period of high metabolic activity (as measured by the RNA/DNA ratio) without biomass growth. Biological activity introduced a new time constant at intermediate frequencies in EIS spectra whose capacitive behavior increased with the biofilm development. The increment in this biofilm-related signal was accompanied by a strong limitation to charge transfer through a diffusion controlled process probably due to oxygen exhaustion by cells respiration, while the resistance of the interface decreased presumably due to oxide dissolution by local acidification under the colonies. In addition, catalase activity was found to be high in mature copper-tolerant biofilms, which differentially express a catalase isoform not present in biofilms growing on glass.     

Survey of pathogenic free-living amoebae and Legionella spp. in mud spring recreation area

Acanthamoeba, Hartmannella, and Naegleria are free-living amoebae, ubiquitous in aquatic environments. Several species within these genera are recognized as potential human pathogens. These free-living amoebae may facilitate the proliferation of their parasitical bacteria, such as Legionella. In this study, we identified Acanthamoeba, Hartmannella, Naegleria, and Legionella using various analytical procedures and investigated their occurrence at a mud spring recreation area in Taiwan. We investigated factors potentially associated with the prevalence of the pathogens, including various water types, and physical and microbiological water quality parameters. Spring water was collected from 34 sites and Acanthamoeba, Hartmannella, Naegleria, and Legionella were detected in 8.8%, 35.3%, 14.7%, and 47.1%, respectively. The identified species of Acanthamoeba included Acanthamoeba castellanii and Acanthamoeba polyphaga. Nearly all the Hartmannella isolates are identified as Hartmannella vermiformis. The Naegleria species included Naegleria australiensis and its sister groups, and two other isolates referred to a new clade of Naegleria genotypes. The Legionella species identified included unnamed Legionella genotypes, Legionella pneumophila serotype 6, uncultured Legionella spp., Legionella lytica, Legionella drancourtii, and Legionella waltersii. Significant differences (Mann-Whitney U test, P < 0.05) were observed between the presence/absence of Hartmannella and total coliforms, between the presence/absence of Naegleria and heterotrophic plate counts, and between the presence/absence of Legionella and heterotrophic plate counts. This survey confirms that pathogenic free-living amoebae and Legionella are prevalent in this Taiwanese mud spring recreation area. The presence of pathogens should be considered a potential health threat when associated with human activities in spring water.     

Imposex and butyltin body burden in Nassarius nitidus (Jeffreys, 1867), in coastal waters within the Basque Country (northern Spain)

Levels of imposex (superimposition of male characters, upon females) and the presence of sterile females are assessed in the gastropod Nassarius nitidus (Jeffreys, 1867), at 22 locations in the Basque Country (northern Spain). At 18 of these localities, butyltin bioaccumulation (tributyltin (TBT); dibutyltin; monobutyltin) was analysed using isotope dilution and GC-ICP-MS. Higher imposex levels and TBT body burden were found in confined harbours, with a large vessel traffic or the presence of a fishing fleet or a shipyard. For the first time, four apparently sequential types of aborted capsules are described in this species. Another novelty is the interspecific comparison between imposex intensities in sympatrically living populations of N. nitidus and N. reticulatus. This showed that sensitivity to TBT pollution of both species is relatively similar (in terms of Relative Penis Length Index). Since N. nitidus has a restricted habitat distribution due to its low presence in wave-exposed habitats, its complementary use with other species is recommended for its use in TBT monitoring programmes.     

Possible interaction between a rodenticide treatment and a pathogen in common vole (Microtus arvalis) during a population peak

A common vole (Microtus arvalis) population peak in Northern Spain in 2007 was treated with large scale application of chlorophacinone, an anticoagulant rodenticide of the indandione family. Voles found dead and trapped alive were collected in treated and untreated areas. Residues of chlorophacinone were analyzed in liver of voles by HPLC-UV. Also, the presence of the pathogen Francisella tularensis was analyzed by PCR in samples of vole spleen. Chlorophacinone (82-3800 ng/g; wet weight liver) was only detected in voles found dead in treated areas (55.5%). The prevalence of F. tularensis in voles found dead in treated areas was also particularly high (66.7%). Moreover, chlorophacinone levels were lower in voles that were PCR-positive for F. tularensis (geometric mean [95% CI], 418 [143-1219] ng/g) than in those that were PCR-negative (1084 [581-2121] ng/g). Interactions between pathogens and rodenticides might be considered to reduce the doses used in baits or to avoid the use of the more toxic 2nd generation anticoagulant rodenticides.     

Molecular characterization of antimicrobial resistance in enterococci and Escherichia coli isolates from European wild rabbit (Oryctolagus cuniculus)

A total of 44 Escherichia coli and 64 enterococci recovered from 77 intestinal samples of wild European rabbits in Portugal were analyzed for resistance to antimicrobial agents. Resistance in E. coli isolates was observed for ampicillin, tetracycline, sulfamethoxazole/trimethoprim, streptomycin, gentamicin, tobramycin, nalidixic acid, ciprofloxacin and chloramphenicol. None of the E. coli isolates produced extended-spectrum beta-lactamases (ESBLs). The bla_TEM, aadA, aac(3)-II, tet(A) and/or tet(B), and the catA genes were demonstrated in all ampicillin, streptomycin, gentamicin, tetracycline, and chloramphenicol-resistant isolates respectively, and the sul1 and/or sul2 and/or sul3 genes in 4 of 5 sulfamethoxazole/trimethoprim resistant isolates. Of the enterococcal isolates, Enterococcus faecalis was the most prevalent detected species (39 isolates), followed by E. faecium (21 isolates) and E. hirae (4 isolates). More than one-fourth (29.7%) of the isolates were resistant to tetracycline; 20.3% were resistant to erythromycin, 14.1% were resistant to ciprofloxacin and 10.9% were resistant to high-level-kanamycin. Lower level of resistance (< 10%) was detected for ampicillin, quinupristin/dalfopristin and high-level-gentamicin, -streptomycin. No vancomycin-resistance was detected in the enterococci isolates. Resistance genes detected included aac(6′)-aph(2″), ant(6)-Ia, tet(M) and/or tet(L) in all gentamicin, streptomycin and tetracycline-resistant isolates respectively. The aph(3′)-IIIa gene was detected in 6 of 7 kanamycin-resistant isolates, the erm(B) gene in 11 of 13 erythromycin-resistant isolates and the vat(D) gene in the quinupristin/dalfopristin-resistant E. faecium isolate. This survey showed that faecal bacteria such as E. coli and enterococci of wild rabbits could be a reservoir of antimicrobial resistance genes.     

Biomass and nutrient content of sessile oak (Quercus petraea (Matt.) Liebl.) and beech (Fagus sylvatica L.) stem and branches in a mixed stand in southern Belgium

Accurate estimates of the amounts of nutrients immobilised in the organs and tissues of different tree species are of prime importance to make appropriate tree species selection and determine the harvesting regime that will ensure forest sustainability. Sixteen sessile oaks (Quercus petraea (Matt.) Liebl.) (64-129 years; stem diameters: 17-57 cm) and twelve beeches (Fagus sylvatica L.) (43-86 years; stem diameters: 9-50 cm) were destructively sampled from a mixed stand located on an acid brown soil in southern Belgium. Statistical models were developed to investigate the differences in nutrient concentrations between tree species, between aboveground tree compartments of the same species, and between tissues of the same compartment. For stem tissues, vertical concentration profiles were described using a versatile equation. Allometric equations were used to predict biomass and nutrient content of tree compartments based on tree dimensions. Broadly speaking, nutrient concentrations tended to be somewhat higher for oak compared with beech, but the amplitude and the direction of inter-species differences varied greatly, depending on the nutrient and the tree compartment. For both species, living branch nutrient concentrations tended to decrease with increasing branch diameter, except for Ca (oak) and Mg (beech). Nutrient concentrations were consistently higher in bark than in wood; this difference between tissues was quite pronounced for Ca, particularly in the case of oak. The biomass and nutrient content equations were used to investigate the effects of tree species and harvesting regime on nutrient exports at harvesting. For equivalent harvesting scenarios, beech was found to induce higher Mg exports than oak, and inversely for Ca. Assuming stand clear cutting, complete tree harvesting would increase average nutrient exports from 65% (Ca) to 162% (P) compared with a stem-only harvesting scenario. These results provide valuable information in the current context of the more intensive utilization of forest products.     

Protection against UV disinfection of E. coli bacteria and B. subtilis spores ingested by C. elegans nematodes

Nematodes, which occur abundantly in granular media filters of drinking water treatment plants and in distribution systems, can ingest and transport pathogenic bacteria and provide them protection against chemical disinfectants. However, protection against UV disinfection had not been investigated to date.In this study, Caenorhabditis elegans nematodes (wild-type strain N2) were allowed to feed on Escherichia coli OP50 and Bacillus subtilis spores before being exposed to 5 and 40 mJ/cm² UV fluences, using a collimated beam apparatus (LP, 254 nm). Sonication (15 W, 60 s) was used to extract bacteria from nematode guts following UV exposure in order to assess the amount of ingested bacteria that resisted the UV treatment using a standard culture method. Bacteria located inside the gut of C. elegans were shown to benefit from a significant protection against UV. Approximately 15% of the applied UV fluence of 40 mJ/cm² (as typically used in WTP) was found to reach the bacteria located inside nematode guts based on the inactivation of recovered bacteria (2.7 log reduction of E. coli bacteria and 0.7 log reduction of B. subtilis spores at 40 mJ/cm²). To our knowledge, this study is the first demonstration of the protection effect of bacterial internalization by higher organisms against UV treatment, using the specific case of E. coli and B. subtilis spores ingested by C. elegans.     

Influence of physicochemical and operational parameters on Nitrobacter and Nitrospira communities in an aerobic activated sludge bioreactor

To understand how to optimize performance of a partially nitrifying plant, the dynamics of Nitrospira and Nitrobacter abundance were studied over a 1 year period using quantitative polymerase chain reaction (qPCR) and their relative contributions to nitrite oxidation assessed including the affects of temperature and dissolved oxygen (DO). Correlation coefficients linking shifts in the community composition of nitrite-oxidizing bacteria (NOB) to operational or environmental variables indicated Nitrospira was significantly and negatively correlated to nitrite concentrations (r = −0.45, P < 0.01) and DO (r = −0.46, P < 0.01), while temperature showed a strong positive correlation (r = 0.59, P < 0.0001). However, the Nitrobacter portion of the total NOB populations showed a positive correlations with DO (r = 0.38, P < 0.01) and hydraulic retention time (HRT) (r = 0.33, P < 0.05), as well as being negatively correlated with temperature (r = −0.49, P < 0.001) suggesting specific niche adaptations within the NOB community. Nitrospira was dominant being better adapted to the low DO and shorter sludge retention times (SRT) of this plant, while Nitrobacter increased in abundance during the winter months, when temperatures were lower and DO concentrations higher. Principal component analysis (PCA) results supported these findings by the close proximity of Nitrospira and temperature biplots of PC1 and PC2 as well as grouping Nitrobacter, NO₂⁻-N, HRT, and DO in the loadings together. The clustering of samples from specific dates also exhibited a strong seasonality.     

Feeding characteristics of a golden alga (Poterioochromonas sp.) grazing on toxic cyanobacterium Microcystis aeruginosa

Microcystis aeruginosa has quickly risen in infamy as one of the most universal and toxic bloom-forming cyanobacteria. Here we presented a species of golden alga (Poterioochromonas sp. strain ZX1), which can feed on toxic M. aeruginosa without any adverse effects from the cyanotoxins. Using flow cytometry, the ingestion and maximal digestion rates were estimated to be 0.2∼1.2 and 0.2 M. aeruginosa cells (ZX1 cell)⁻¹ h⁻¹, respectively. M. aeruginosa in densities below 10⁷ cells mL⁻¹ could be grazed down by ZX1, but no significant decrease was observed when the initial density was 3.2 × 10⁷ cells mL⁻¹. ZX1 grazing was a little influenced by the light intensity (0.5∼2500 lx) and initial pH of the medium (pH = 5.0∼9.5). ZX1 could not survive in continuous darkness for longer than 10 days. The pH value was adjusted to 8 by ZX1 while to 10 by M. aeruginosa. This study may shed light on understanding the ecological interactions between M. aeruginosa and mixotrophic Poterioochromonas sp. in aquatic ecosystems.     

Metal specific partitioning in a parasite-host assemblage of the cestode Ligula intestinalis and the cyprinid fish Rastrineobola argentea

When evaluating metal accumulation patterns in parasite-host assemblages species specific metal requirements should be taken into account. The aim of the present study was therefore to determine the metal specific partitioning in a parasite-host assemblage of the cestode Ligula intestinalis and the cyprinid fish Rastrineobola argentea and to determine the effect of the parasites on the metal balance of the fish. To this purpose the host-parasite assemblage was analysed for several metals at sites in the coastal zone of Lake Victoria differing in metal contamination. Our results showed that some elements (Ca, Sr, and Mg) reflected the physiological differences of bone formation and ionic balance and pointed to physiological disturbances of infested R. argentea. Other essential metals including Cu and Co were subject of element competition between fish and parasite, while only a micro-element (Cr) and a non-essential metal (Cd) displayed a partitioning with high concentration in the parasite. The present study clearly demonstrated the impact of the large cestodes on their small fish hosts and it is concluded that the partitioning of metals in the assemblage of R. argentea and L. intestinalis is subject to metal specific mechanisms for essential and non-essential elements.     

Differentiation and identification of Shigella spp. and enteroinvasive Escherichia coli in environmental waters by a molecular method and biochemical test

Both Shigella spp. and enteroinvasive Escherichia coli (EIEC) are important human pathogens that are responsible for the majority of cases of endemic bacillary dysentery. However, they are difficult to identify and differentiate by biochemical tests or molecular methods alone. In this study, we developed a procedure to detect Shigella spp. and EIEC from environmental water samples using membrane filtration followed by nutrient broth enrichment, isolation using selective culture plates, and identification of the invasion plasmid antigen H (ipaH) gene by PCR amplification and DNA sequencing. Finally, we used a biochemical test and a serological assay to differentiate between Shigella and EIEC. Among the 93 water samples from nine reservoirs and one watershed, 76 (81.7%) water samples of culture plates had candidate colonies of Shigella and EIEC and 5 water samples were positive (5.4%) for a Shigella- and EIEC-specific polymerase chain reaction targeting the ipaH gene. Guided by the molecular method, the biochemical test, and the serological assay, 11 ipaH gene-positive isolates from 5 water samples were all identified as EIEC.     

Zebra mussel (Dreissena polymorpha) parasites: Potentially useful bioindicators of freshwater quality?

In environmental quality bioassessment studies, analysis of host-parasite interactions may well be a valuable alternative to classical macroinvertebrate sampling approaches. Herein, we investigated whether zebra mussel (Dreissena polymorpha) parasites could be useful biomonitoring tools. Mussel populations were sampled twice at two sites in northeastern France representing different levels of contamination and were characterized for parasite infection following standard histological methods. Our results indicated that sites of different environmental quality (i.e. chemical contamination) exhibited different parasite communities characterized by different trematode species and parasite associations. An additional significant finding was the positive correlation established between the prevalence of Rickettsiales-like organisms and metal contamination. Multivariate analyses were valuable in examining parasite communities.     

In situ biomonitoring of freshwater quality using the New Zealand mudsnail Potamopyrgus antipodarum (Gray) exposed to waste water treatment plant (WWTP) effluent discharges

Mollusk species have been shown to be sensitive to various endocrine disrupting compounds (EDC) at environmentally relevant concentrations. Waste water treatment plant (WWTP) effluents are a major source of potential or known EDC in the aquatic environment. The aim of this study was to develop an in situ exposure method using the New Zealand mudsnail Potamopyrgus antipodarum (Molluska, Hydrobiidea) to assess the impact of water quality on the life traits of this species, by focusing on its reproduction. The impact of three WWTP discharges on three different receiving rivers was studied. The effects of WWTP effluent on adult survival, weight, reproduction and vertebrate-like sex-steroid levels in snails were monitored for three to four weeks. Although the physicochemical and hydrological parameters varied greatly between the rivers, the caging experiments allowed us to detect significant impairment of the life traits of snails when exposed downstream of the WWTPs discharge. While adult survival was not affected by exposure, reproduction was significantly impacted downstream from the WWTP effluent discharges (60-70% decrease of embryos without shells after three to four weeks exposure) independently of the river. Modulations of steroid levels proved to be an informative parameter with an increase of testosterone downstream of the discharges, and increases and decreases of 17β-estradiol levels according to site. The endpoints used proved to be an adapted method for field exposures and allowed the discrimination between upstream and downstream sites.     

EnergyPlus vs. DOE-2.1e: The effect of ground-coupling on energy use of a code house with basement in a hot-humid climate

For low-rise buildings, the heat losses or gains through the ground coupled building envelope can be a significant load component. Studies have shown that current simulation tools give dissimilar results for the ground coupled heat transfer (GCHT) with basements. This paper quantifies and explains the differences between EnergyPlus and DOE-2.1e (DOE-2) basement GCHTs based on their results for an all electric code house in a hot and humid climate zone. The code house was modeled with two basement configurations i.e. a conditioned basement and an unconditioned crawlspace. DOE-2 was used with Winkelmann's basement model and EnergyPlus was used with its GCHT calculator utility, Basement. The results revealed that the ground isolated EnergyPlus houses used 3-23% more cooling, 12-29% less heating and 3-7% lower overall HVAC electricity use when compared to the ground isolated DOE-2 houses. Ground coupling added up to three times more heat loss in EnergyPlus than in DOE-2. This increased the overall energy use difference between these two programs from 3-7% (ground isolated) to 14-25% (ground coupled). These results showed that a truth standard is required for basement heat transfer calculations of low-rise buildings.     

Assessing the ecotoxicity of vinyl chloride using green alga P. subcapitata, nematode C. elegans, and the SOS chromotest in a closed system without headspace

The ecotoxicity of vinyl chloride (VC) was evaluated using green alga, nematode, and the SOS chromotest. The green alga and nematode tested were Pseudokirchneriella subcapitata, and Caenorhabditis elegans, respectively. Because of the tendency of VC to escape from an aqueous exposure medium to the air phase, all tests in the present study were performed in a closed system without headspace to minimize the losses of VC. Previous studies on VC toxicity were performed in an open system or closed system with headspace. VC inhibits the growth of P. subcapitata. The 48-h IC50 value for P. subcapitata exposed to VC was calculated to be 5.15 mg/L. The survival of C. elegans was not influenced at concentrations of up to 60 mg/L; however, VC has an adverse effect on the reproduction of C. elegans. In a stress-related gene expression test using C. elegans, a significant and concentration-dependent expression of heat shock protein 16.2 was observed, indicating that VC induces the stress of C. elegans at the genetic level. The results of the SOS chromotest using Escherichia coli PQ37 showed an IF_max value of 1.11, indicating that VC is not genotoxic. The present study demonstrated that VC has an adverse effect on the algal growth and reproductive and genetic levels of C. elegans. A closed system without headspace is an effective method of testing the aquatic toxicity of volatile organic compounds such as VC.     

Sublethal toxicity and biotransformation of pyrene in Lumbriculus variegatus (Oligochaeta)

The aim of this work was to study the toxicity and biotransformation of polyaromatic hydrocarbon (PAH) pyrene in the oligochaete aquatic worm, Lumbriculus variegatus. PAHs are ubiquitous environmental pollutants that pose a hazard to aquatic organisms, and metabolizing capability is poorly known in the case of many invertebrate species. To study the toxicity and biotransformation of pyrene, the worm was exposed for 15 days to various concentrations of water-borne pyrene. The dorsal blood vessel pulse rate was used as a sublethal endpoint. Pyrene biotransformation by L. variegatus was studied and the critical body residues (CBR) were estimated for pyrene toxicity. The toxicokinetics of pyrene uptake was evaluated. A combination of radiolabeled (¹⁴C) and nonlabeled pyrene was used in the exposures, and liquid scintillation counting (LSC) and high-pressure liquid chromatography were employed in both water and tissue residue analyses. The results showed that L. variegatus was moderately able to metabolize pyrene to 1-hydroxypyrene (1-HP), thus demonstrating that the phase-I-like oxidizing enzyme system metabolizes pyrene in L. variegatus. The amount of the 1-HP was 1-2% of the amount of pyrene in the worm tissues. The exposure to pyrene reduced the blood vessel pulse rate significantly (p < 0.05), showing that pyrene had a narcotic effect. The estimated CBRs remained constant during the exposure time, varying from 0.120 to 0.174 mmol pyrene/kg worm wet weight. The bioconcentration factors (BCF) decreased as exposure concentration increased. It was suggested that the increased toxicity of pyrene accounted for the decrease in BCFs by lowering the activity of the organism.     

Haematological status of wintering great tits (Parus major) along a metal pollution gradient

In the long-term biomonitoring of wild populations inhabiting polluted areas, the use of non-destructive biomarkers as markers of condition is very important. We examined the possible effects of metal pollution on the haematological status of adult great tits (Parus major) along a well-established pollution gradient near a non-ferrous smelter in Belgium. We measured blood and feather metal concentrations and assessed the haematological status (amount of red blood cells, haemoglobin concentration, haematocrit, mean corpuscular volume and mean corpuscular haemoglobin) of adult great tits during winter at four study sites. Metal concentrations in blood and feathers indicated that cadmium and lead were the most important metals in the pollution gradient under study. Measurements of haematological parameters revealed that haemoglobin concentration, haematocrit, mean corpuscular volume and mean corpuscular haemoglobin were lower in great tits from the more polluted sites. These parameters were significantly negatively correlated with blood lead concentration. The amount of red blood cells, however, did not significantly differ among study sites. Our results indicate that the haematological status of great tits is negatively affected by metal pollution and may therefore be used as a successful biomarker for monitoring the negative impact of metal exposure in the wild.