Occurrence of phages infecting Escherichia coli O157:H7 carrying the Stx 2 gene in sewage from different countries

Shiga toxin-converting bacteriophages are involved in the pathogenicity of some enteric bacteria, such as Escherichia coli O157:H7. Recent studies have demonstrated a relatively high presence of Shiga toxin 2 phages in sewage from Spain, but no data on sewage from other areas were available. In order to evaluate the presence of such phages in sewage from diverse geographical origins, 33 sewage samples, including samples from eight different European countries as well as from New Zealand and South Africa were analysed. Using an experimental approach based on the detection of Stx 2 gene by a phage enrichment culture followed by PCR, bacteriophages infecting E. coli O157:H7 carrying the Shiga toxin 2 gene were detected in 15 of the samples studied. Results presented here show that the presence of phages carrying the Stx 2 gene is common in sewage from developed countries.     

Abundance in Sewage of Bacteriophages That Infect Escherichia coli O157:H7 and That Carry the Shiga Toxin 2 Gene

Shiga toxin-converting bacteriophages are involved in the pathogenicity of some enteric bacteria, such as Escherichia coli O157:H7, but data on the occurrence and distribution of such phages as free particles in nature were not available. An experimental approach has been developed to detect the presence of the Shiga toxin 2 (Stx 2)-encoding bacteriophages in sewage. The Stx 2 gene was amplified by PCR from phages concentrated from 10-ml samples of sewage. Moreover, the phages carrying the Stx 2 gene were detected in supernatants from bacteriophage enrichment cultures by using an Stx 2-negative E. coli O157:H7 strain infected with phages purified from volumes of sewage as small as 0.02 ml. Additionally, the A subunit of Stx 2 was detected in the supernatants of the bacteriophage enrichment cultures, which also showed cytotoxic activity for Vero cells. By enrichment of phages concentrated from different volumes of sewage and applying the most-probable-number technique, it was estimated that the number of phages infectious for E. coli O157:H7 and carrying the Stx 2 gene was in the range of 1 to 10 per ml of sewage from two different origins. These values were approximately 1% of all phages infecting E. coli O157:H7.     

Immunomagnetic assay,classic culture method and fermentation tube test in the recovery of Escherichia coli O157 from sewage

The diffusion of E. coli O157 was studied in the effluents of a large sewage treatment plant. Three methods were used: the Culture Method, Immunomagnetic Separation (IMS) and Fermentation Tube Test. Vero cells assay and PCR were used to assess toxin production and the presence of virulence genes. E. coli O157 were only detected using IMS and the Fermentation Tubes Test: one strain in raw sewage and 2 in a sample of disinfected effluent. The latter resulted positive for the production of verocytotoxin and possessed genes coding for vt2 and type gamma intimin. The best results were obtained with SMAC-CT-BCIG medium. The use of various different recovery techniques confirmed the difficulty involved in isolating E. coli O157 from water samples.     

Selection and biochemical activity of a microbial biocenosis in the process of concentrated sewage treatment

A bacterial cenosis responsible for the destruction of organic wastes having a complex composition was selected under the conditions of continuous cultivation on highly concentrated industrial sewage. Most of the bacterial species and all of the yeasts and fungi were eliminated from the original microflora when the concentration of the purified sewage was increased from 2 to 12 g O2 per litre (in terms of the overall chemical oxygen uptake, COU) and the dilution rate (D) varied from 0.08 to 0.25 h-1. The number of species was reduced to 10 in the cenosis. The degree of oxidation of organic compounds in the sewage was 94 to 97% at the maximal parameters of the process. The specific rate of oxidation as well as the respiration and dehydrogenase activities increased with a rise in both COU and D. The highest biochemical activity was observed at D=0.25 h-1:     

Prevalence of verotoxin-producing Escherichia coli (VTEC) in slurry,farmyard manure and sewage sludge in France

AIMS: The aims of the present study were to determine VTEC prevalence in manure, slurry and sewage sludge in France and to characterize the VTEC strains isolated (virulence genes and serotype). METHODS AND RESULTS: Seven hundred and fifty-two samples from 55 farmyard manures, 136 bovine and porcine faeces, 114 slurries, 10 composts, and 437 samples from outflows of sewage wastewater treatment plants were analysed. Twenty-four percent contained isolates which were PCR positive for stx gene. Twenty-one VTEC strains were recovered from positive samples by colony hybridization: 76% of them were positive for stx(2) gene, 33% for stx(1) gene,and 19% for eae gene. One strain belonged to serotype O157:H7 and two others to serogroups O26 and O55, respectively. CONCLUSIONS: Some of the VTEC strains isolated from environments in France should be considered as potentially pathogenic for humans. SIGNIFICANCE AND IMPACT OF THE STUDY: Appropriate handling or use of manure, slurry and sewage sludge is necessary so that contamination of the environment and food by VTEC can be prevented.     

Characterization of Shiga toxin-producing Escherichia coli isolated from aquatic environments

This study reports the phenotypic and genotypic characterization of 144 Shiga toxin-producing Escherichia coli (STEC) strains isolated from urban sewage and animal wastewaters using a Shiga toxin 2 gene variant (stx(2))-specific DNA colony hybridization method. All the strains were classified as E. coli and belonged to 34 different serotypes, some of which had not been previously reported to carry the stx(2) genes (O8:H31, O89:H19, O166:H21 and O181:H20). Five stx(2) subtypes (stx(2), stx(2c), stx(2d), stx(2e) and stx(2g)) were detected. The stx(2), stx(2c), stx(2d) and stx(2e) subtypes were present in urban sewage and stx(2e) was the only stx(2) subtype found in pig wastewater samples. The stx(2c) and stx(2g) were more associated with cattle wastewater. One strain was positive for the intimin gene (eae) and five strains of serotypes were positive for the adhesin encoded by the saa gene. A total of 41 different seropathotypes were found. On the basis of occurrence of virulence genes, most non-O157 STEC strains are assumed to be low-virulence serotypes.     

Evaluation of pilot-scale modified A2O processes for the removal of nitrogen compounds from sewage

In this study, pilot-scale experiments for the removal of nitrogen from sewage obtained from a county Y sewer system were performed using modified A₂O processes. Using this approach, the total amount of nitrogen discharged during denitrification of the influent was average 38.6 mg/L and a level of average 10.8 mg/L was maintained throughout the denitrification process, which resulted in an average removal efficiency that was greater than 72%. The nitrogen components in the effluent water consisted of 22% ammonia nitrogen, 6% nitrite nitrogen and 72% nitrate nitrogen, reaching a nitrification efficiency of 94%. In conclusion, since these advanced treatment methods, which involve modified A₂O processes, were successfully employed to remove nitrogen from sewage discharge, they hold promise for wide spread use by treatment plants.     

Biological elimination of nitrous oxide under oxidative conditions

The process of biological N2O elimination under oxidative conditions has been discribed. It was performed by samples of soil, litter, water, ooze, sewage and compost. The process differs from denitrification, is not accompanied with dinitrogen formation and its rate is maximal at 15% of oxygen in the gaseous phase. Acetylene, an inhibitor of denitrification has no effect on elimination of nitrous oxide under oxidative conditions.     

Photolysis primes biodegradation of benzo[a]pyrene

14C-labeled benzo[a]pyrene (BaP) was used as a model-compound for polycyclic aromatic hydrocarbons (PAH) in order to assess the effect of photolytic pretreatment on the subsequent fate of BaP in sewage sludge and soil test systems. Photolysis was performed in methanolic solution with or without 0.1 M H2O2, under either UV light (300 nm) or natural sunlight. The presence of H2O2 greatly enhanced the rate of photolysis both with UV and with natural sunlight. Intact BaP resisted biodegradation in both test systems. Photolysis transformed BaP to polar materials that were subject to increased mineralization and binding in both biological test systems. As shown by the Ames assay, photolysis decreased the mutagenicity of BaP to test strains TA98 and TA104 only moderately. The photolysate had an increased acute toxicity and lost its need for activation by S-9 enzymes. However, during subsequent incubation in soil or sewage sludge, mutagenicity decreased rapidly by one to two orders of magnitude and acute toxicity disappeared due to the mineralization and binding of photoproducts to humic materials. Photolysis of BaP and similar PAH compounds represents a useful treatment option that could be applied to certain PAH-containing petroleum refinery sludge and to coal tar residues in order to facilitate their detoxification and environmentally safe disposal.     

Photolysis primes biodegradation of benzo[a]pyrene.

14C-labeled benzo[a]pyrene (BaP) was used as a model-compound for polycyclic aromatic hydrocarbons (PAH) in order to assess the effect of photolytic pretreatment on the subsequent fate of BaP in sewage sludge and soil test systems. Photolysis was performed in methanolic solution with or without 0.1 M H2O2, under either UV light (300 nm) or natural sunlight. The presence of H2O2 greatly enhanced the rate of photolysis both with UV and with natural sunlight. Intact BaP resisted biodegradation in both test systems. Photolysis transformed BaP to polar materials that were subject to increased mineralization and binding in both biological test systems. As shown by the Ames assay, photolysis decreased the mutagenicity of BaP to test strains TA98 and TA104 only moderately. The photolysate had an increased acute toxicity and lost its need for activation by S-9 enzymes. However, during subsequent incubation in soil or sewage sludge, mutagenicity decreased rapidly by one to two orders of magnitude and acute toxicity disappeared due to the mineralization and binding of photoproducts to humic materials. Photolysis of BaP and similar PAH compounds represents a useful treatment option that could be applied to certain PAH-containing petroleum refinery sludge and to coal tar residues in order to facilitate their detoxification and environmentally safe disposal.     

Occurrence of virulence genes associated with enterohemorrhagic Escherichia coli in raw municipal sewage

Municipal sewage influent was screened for the presence of the virulence genes encoding Shiga-like toxins SLT-I and SLT-II (slt-I and slt-II) and intimin (eaeA) and those involved in biosynthesis of O157 (rfbE) and H7 (fliC) antigens by multiplex PCR to simultaneously identify the enterohemorrhagic Escherichia coli (EHEC) O157:H7 and its virulence factors in a single reaction. The screening was carried out monthly from October 2004 to September 2005. Direct PCR analysis using total DNA from sewage concentrate showed the presence of at least one virulence gene in 100% samples (n = 12). Sixty six percent of these samples were also positive for rfbE (O157) gene and fliC (H7) gene. The PCR amplification of these genes was possible when the concentration was above 20 cells ml⁻¹. From the multiplex PCR of the isolates following plating on Cefixime-Tellurite Sorbitol MacConkey (CT-SMAC) agar to detect non-sorbitol fermenting (NSF) colonies (n = 600), one E. coli strain carrying slt-II gene and two strains of E. coli O157:H7 carrying slt-I were detected. The results show that municipal sewage represents a potential reservoir of EHEC. CT-SMAC agar was proved to have limited E. coli O157:H7 selectivity and only 0.005% (3/600) sensitivity for sewage samples due to the high frequency (43%) of NSF strains in sewage. The enrichment of sewage sample in modified E. coli broth (mEC) increased the sensitivity of PCR resulting in the clearer amplification of five genes. Amplification of target cell type in mEC broth implied that EHEC were present in sewage in a culturable and hence potentially infectious state. However, pre-enrichment did not affect the selectivity of CT-SMAC because frequency of NSF colonies remained the same as that obtained without enrichment. The study, therefore, underscores the need for more sensitive screening techniques that can be routinely employed for the regular monitoring of sewage influent.     

Molecular characterization of Vibrio parahaemolyticus of similar serovars isolated from sewage and clinical cases of diarrhoea in Calcutta,India

Vibrio parahaemolyticus is a seafood-borne halophilic pathogen that causes acute gastroenteritis in humans. During the course of an investigation on the incidence of V. parahaemolyticus in sewage water samples of Calcutta, India, we isolated eight (26.7%) strains of V. parahaemolyticus from 30 samples. Among these strains, five (62.5%) carried the thermostable direct hemolysin (tdh) gene, a major virulence marker of V. parahaemolyticus. Two strains belonged to serovar O5:K3 and the remaining three to O5:KUT, which is common among clinical strains of V. parahaemolyticus isolated from hospitalized patients of Calcutta with acute diarrhoea. The tdh positive sewage strains of V. parahaemolyticus were compared by randomly amplified polymorphic DNA (RAPD)-PCR and pulsed-field gel electrophoresis (PFGE) with strains of similar serovars selected from our culture collection to determine the genetic relatedness. Our results showed that except for sharing the similar serovar, sewage and clinical strains of V. parahaemolyticus were genetically different. In addition, toxRS-targeted group-specific (GS) PCR and open reading frame 8 (ORF-8) PCR showed that the sewage strains did not belong to the pandemic genotype. Since the sewage in Calcutta is directly used for cultivation of vegetables and for pisciculture, the presence of tdh positive V. parahaemolyticus in the sewage highlights the need for constant monitoring of the environment.     

Prevalence of the stx2 gene in coliform populations from aquatic environments

Shiga toxin-producing Escherichia coli strains are human pathogens linked to hemorrhagic colitis and hemolytic uremic syndrome. The major virulence factors of these strains are Shiga toxins Stx1 and Stx2. The majority of the genes coding for these toxins are borne by bacteriophages. Free Stx2-encoding bacteriophages have been found in aquatic environments, but there is limited information about the lysogenic strains and bacteria present in the environment that are susceptible to phage infection. The aim of this work was to study the prevalence and the distribution of the stx(2) gene in coliform bacteria in sewage samples of different origins. The presence of the stx(2) gene was monitored every 2 weeks over a 1-year period in a municipal sewage treatment plant. A mean value of 10(2) genes/ml was observed without significant variation during the study period. This concentration was of the same order of magnitude in raw municipal sewage of various origins and in animal wastewater from several slaughterhouses. A total of 138 strains carrying the stx(2) gene were isolated by colony hybridization. This procedure detected approximately 1 gene-carrying colony per 1,000 fecal coliform colonies in municipal sewage and around 1 gene-carrying colony per 100 fecal coliform colonies in animal wastewaters. Most of the isolates belonged to E. coli serotypes other than E. coli O157, suggesting a low prevalence of strains of this serotype carrying the stx(2) gene in the wastewater studied.     

Sewage workers: risk of acquiring enteric virus infections including hepatitis A

To determine if sewage workers have an increased risk of acquiring viral infections, 66 workers at a small wastewater plant in north-eastern Italy and 72 control subjects recruited from blood donors were enrolled in a seroprevalence study to determine whether sewage workers are at increased risk of acquiring viral infections. In order to evaluate various risk factors, a questionnaire was filled out by each worker whereas seropositivity to Hepatitis A virus, Coxsackievirus B2 - B3 - B4 - B5, and Echovirus types 1 and 9 was determined in the laboratory. Anti-HAV antibodies were present in 37.8% of sewage workers and 36.1% of subjects in the control group. The difference was not statistically significant in the two groups, whereas a significant association was observed regarding age (P < 0.3). No association was observed with the occupational age, or with number and duration of contacts per day. The lack of evident occupational risk for hepatitis A among sewage workers may be explained by the adult age of the workers (mean age 41.3 years, range 22-58 years), and thus the antibody titre against different enteroviruses was determined. No statistically significant differences were evident with the raw values, but considering the 90 degrees percentile as a dichotomic value for the antibody levels a strong and significant association was present with Coxsackievirus B3 (O.R. 22.85, C.I. 95% 2.93-178.08) and Coxsackievirus B2 (O.R. 14.25, C.I. 95% 1.78-113.87). Analysis of the data confirms a limited risk of acquiring infection and/or disease but also the evident possibility of silent exposure to the viruses. The shift in HAV epidemiology and increased morbidity and mortality in adult age suggest that active immunization against hepatitis A should be considered.     

底物种类和浓度对污泥重金属生物淋滤效果的影响

以生污泥为材料,研究单质硫(浓度分别为5、10、20 g/L)、FeSO4?7H2O (浓度分别为10、20 g/L）和硫代硫酸钠（浓度分别为10、20 g/L）三种底物的不同浓度对污泥中Zn、Cu、Pb、Cd、Cr和Ni六种重金属生物淋滤效果的影响。结果表明：单质硫的致酸性最好,第4天pH值就达到2.0左右,Zn、Cu、Pb、Cr和Ni的滤出量最大（原污泥Cd未检出）,并以底物投放浓度在10～20 g/L为佳。FeSO4?7H2O的致酸性和重金属滤出量较单质硫弱。硫代硫酸钠在浓度10 g/L时,致酸性和重金属的滤出量介于单质硫和FeSO4?7H2O之间,但浓度20 g/L时滤液呈碱性,重金属难于滤出甚至不滤出。     

Strategies to mitigate N2O emissions from biological nitrogen removal systems

N2O emissions from the biological treatment of sewage, manure, landfill leachates and industrial effluents have gained considerable interest among policy makers and environmental scientists. Estimated global emission rates from these sources can contribute up to 10% of the anthropogenic N2O emissions. Particularly at the level of a treatment plant, the N2O impact can be very significant and reach up to 80% of the operational CO2 footprint. Imperfect nitritation by an imbalance in the two-step nitritation metabolism of ammonia-oxidizing bacteria is considered as the main contributor to N2O production with hydroxylamine and particularly nitrite as key precursors. Monitoring of these compounds is warranted to understand and abate N2O emissions. Mitigation strategies should also comprise optimizations of the process parameters as well as bio-augmentative approaches empowered to restore the functional capacity and to deal with unwanted accumulation of intermediates. These strategies require validation for their effectiveness and costs at full-scale.     

Development of a simple and rapid fluorogenic procedure for identification of vibrionaceae family members

We describe a simple colony overlay procedure for peptidases (COPP) for the rapid fluorogenic detection and quantification of Vibrionaceae from seawater, shellfish, sewage, and clinical samples. The assay detects phosphoglucose isomerase with a lysyl aminopeptidase activity that is produced by Vibrionaceae family members. Overnight cultures are overlaid for 10 min with membranes containing a synthetic substrate, and the membranes are examined for fluorescent foci under UV illumination. Fluorescent foci were produced by all the Vibrionaceae tested, including Vibrio spp., Aeromonas spp., and Plesiomonas spp. Fluorescence was not produced by non-Vibrionaceae pathogens. Vibrio cholerae strains O1, O139, O22, and O155 were strongly positive. Seawater and oysters were assayed, and 87 of 93 (93.5%) of the positive isolates were identified biochemically as Vibrionaceae, principally Vibrio vulnificus, Vibrio parahaemolyticus, Aeromonas hydrophila, Photobacterium damselae, and Shewanella putrefaciens. None of 50 nonfluorescent isolates were Vibrionaceae. No Vibrionaceae were detected in soil, and only A. hydrophila was detected in sewage. The COPP technique may be particularly valuable in environmental and food-testing laboratories and for monitoring water quality in the aquaculture industry.     

Method for Detection of Microorganisms That Produce Gaseous Nitrogen Oxides

A method was developed to detect NO- or N₂O-producing bacteria in solid or liquid medium by their ability to oxidize the redox indicator resazurin from its reduced colorless form to its oxidized pink form. The method was sensitive to as little as 35 nM N₂O or 0.5 nM NO. Ninety-one percent of the colonies that oxidized resazurin on plates also produced N₂O in slant cultures. Forty-four percent of the colonies that did not oxidize resazurin did produce N₂O. This percentage was reduced to 15% when colonies in which the coloration was difficult to discern were picked to slants to determine whether they oxidized the slant. The production of N₂O preceded the oxidation of resazurin by liquid cultures of Escherichia coli and a sludge isolate. With the denitrifying sewage isolate, the disappearance of N₂O was followed by the return of resazurin to its reduced state. Wolinella succinogenes was found to produce small amounts of N₂O from NO₃⁻, which resulted in a transient oxidation of resazurin.     

污泥土地利用对农作物及土壤的影响研究

以沈阳北部污水处理厂污泥为研究对象 ,开展了污泥土地利用对农作物及土壤环境影响的研究 .结果表明 ,污泥土地利用可提高土壤中N、P及有机质含量 .污泥用量为 2 2 .5t·hm-2 和 45t·hm-2 时有利于作物生长发育 ,使水稻生物量与对照相比分别增加了 11.48%、11.83%.污泥施用量只要控制在 45t·hm-2以内 ,污泥中的重金属不会对土壤、农产品质量及地下水产生不良的影响 .     

Nitrous oxide emissions in the Shanghai river network: implications for the effects of urban sewage and IPCC methodology

Global nitrogen (N) enrichment has resulted in increased nitrous oxide (N₂O) emission that greatly contributes to climate change and stratospheric ozone destruction, but little is known about the N₂O emissions from urban river networks receiving anthropogenic N inputs. We examined N₂O saturation and emission in the Shanghai city river network, covering 6300 km², over 27 months. The overall mean saturation and emission from 87 locations was 770% and 1.91 mg N₂O‐N m^?2 d^?1, respectively. Nitrous oxide (N₂O) saturation did not exhibit a clear seasonality, but the temporal pattern was co‐regulated by both water temperature and N loadings. Rivers draining through urban and suburban areas receiving more sewage N inputs had higher N₂O saturation and emission than those in rural areas. Regression analysis indicated that water ammonium (NH₄⁺) and dissolved oxygen (DO) level had great control on N₂O production and were better predictors of N₂O emission in urban watershed. About 0.29 Gg N₂O‐N yr^?1 N₂O was emitted from the Shanghai river network annually, which was about 131% of IPCC's prediction using default emission values. Given the rapid progress of global urbanization, more study efforts, particularly on nitrification and its N₂O yielding, are needed to better quantify the role of urban rivers in global riverine N₂O emission.