Sulfite-reducing clostridia in the sediment of a high mountain lake (Laguna Grande, Gredos, Spain) as indicators of fecal pollution.

We studied the vertical distribution of sulfite-reducing clostridia in the sediment of a Spanish high-mountain lagoon (Laguna Grande de Gredos, central Spain), with optimal sediment characteristics (temperature < 20 degrees C) to maintain spores without growing. This allowed us to assess the original numbers of sulfite-reducing clostridia endospores settled, without postdepositional growing. Sulfite-reducing clostridia are normal inhabitants of the intestinal microbiota of humans and other mammals. These microorganisms may form endospores, which allow the bacteria to survive in almost any habitat, either terrestrial or aquatic, waiting for favorable conditions for growth. Sulfite-reducing clostridia could be suitable indicators of past human pollution because they have a great longevity in natural habitats and they cannot multiply at temperatures below 20 degrees C or in the presence of O2. We found a great increase in the numbers of clostridia (expressed as colony-forming units per gram [CFU/g] of dry weight of sediment) since the 1970s, which reflects the rise of human pressure caused by the practice of outdoor activities. Clostridia CFU/g rose dramatically after the faulty operation of the depuration system of a mountain refuge built close to the lagoon. We compared the vertical distribution of clostridia CFU/g from Laguna Grande sediments with those from a neighbor lagoon (Laguna Cimera), which showed less tourist pressure and no direct disposal of sewage. Finally, we agree with the usefulness of the numbers of sulfite-reducing clostridia as indicators of past pollution.     

青岛市秋季空气微生物群落多样性

采用KC-6120空气综合采样器采集空气微生物样品,通过构建16S/18SrDNA克隆文库方法分析青岛市市区街道秋季空气微生物群落结构特征.结果表明： 空气细菌分布在6大类,分别为变形菌门(78.8%)、厚壁菌门(14.6%)、放线菌门(4.0%)、浮霉菌门(1.3%)、蓝藻门(0.7%)和栖热菌门(0.6%),优势菌属为不动杆菌属(39.7%)、葡萄球菌属(11.3%)、鞘脂单胞菌属(8.6%)和副球菌属(6.0%).空气真菌分布在子囊菌门(97.5%)和担子菌门(2.5%),优势菌属为核腔菌属(76.5%)、炭角菌属(13.6%)和外瓶霉属(2.5%).空气微生物中存在不动杆菌属、鞘脂单胞菌、葡萄球菌等致病菌或条件致病菌,以及引发多种农作物枯萎死亡的麦类核腔菌、团炭角菌和角状平脐疣孢等真菌.     

青岛市秋季空气微生物群落多样性

采用KC-6120空气综合采样器采集空气微生物样品,通过构建16S/18SrDNA克隆文库方法分析青岛市市区街道秋季空气微生物群落结构特征.结果表明： 空气细菌分布在6大类,分别为变形菌门(78.8%)、厚壁菌门(14.6%)、放线菌门(4.0%)、浮霉菌门(1.3%)、蓝藻门(0.7%)和栖热菌门(0.6%),优势菌属为不动杆菌属(39.7%)、葡萄球菌属(11.3%)、鞘脂单胞菌属(8.6%)和副球菌属(6.0%).空气真菌分布在子囊菌门(97.5%)和担子菌门(2.5%),优势菌属为核腔菌属(76.5%)、炭角菌属(13.6%)和外瓶霉属(2.5%).空气微生物中存在不动杆菌属、鞘脂单胞菌、葡萄球菌等致病菌或条件致病菌,以及引发多种农作物枯萎死亡的麦类核腔菌、团炭角菌和角状平脐疣孢等真菌.     

Coliform aerosols generated from the surface of dewatered sewage applied to a forest clearcut.

Concentrations of airborne coliform bacteria as high as 1.5 X 10(4) m-3 were observed 8 cm above anaerobically digested sewage sludge applied to a forest clearcut. Dry conditions and high wind speeds tended to favor aerosol generation.     

Bacteriological indicators of faecal contamination: result of a loading experiment with untreated urban wastewater

Some observations were made on the behaviour of total coliforms, faecal coliforms, enterococci, numbers of aerobic bacteria, salmonellas and sulphur-reducing clostridia as bacterial indicators of faecal contamination of groundwater. A controlled irrigation experiment was carried out with untreated residual water in the alluvial aquifer of the Vega of Granada (Spain). The results obtained confirm the value of these parameters as useful indicators of very recent faecal contamination; and changes were detected as the level of the freatic layer increased and the chemical composition of the groundwater changed. These groups of micro-organisms persisted for about 200 h, with the exception of the aerobes which survived for much longer. Salmonellas were present at levels too low to calculate the extent of faecal contamination and sulphur-reducing clostridia were not detected. The results obtained show that irrigation with untreated wastewater offers a lower risk of microbiological contamination of groundwater compared with the direct addition of waters decanted and/or previously filtered.     

Coliform aerosols generated from the surface of dewatered sewage applied to a forest clearcut.

Concentrations of airborne coliform bacteria as high as 1.5 X 10(4) m-3 were observed 8 cm above anaerobically digested sewage sludge applied to a forest clearcut. Dry conditions and high wind speeds tended to favor aerosol generation.     

Occurrence and densities of bacteriophages proposed as indicators and bacterial indicators in river waters from Europe and South America

AIMS: To evaluate the feasibility of bacteriophages as a complementary tool for water quality assessment in surface waters from different parts of the globe. METHODS AND RESULTS: Faecal coliform bacteria, enterococci, spores of sulphite-reducing clostridia, somatic coliphages, F-specific RNA bacteriophages and bacteriophages infecting Bacteroides fragilis were determined by standardized methods in raw sewage and in 392 samples of river water from 22 sampling sites in 10 rivers in Argentina, Colombia, France and Spain, which represent very different climatic and socio-economic conditions. The results showed that the indicators studied maintained the same relative densities in the raw sewage from the different areas. Classifying the river water samples according to the content of faecal coliform bacteria, it can be observed that the relative densities of the different bacterial indicators and bacteriophages changed according to the concentration of faecal coliform bacteria. There was a relative increase in the densities of all groups of bacteriophages and sulphite-reducing clostridia with respect to faecal coliforms and enterococci in the samples with low counts of faecal coliform bacteria. CONCLUSIONS: The numbers of bacterial indicators and bacteriophages were similar in the different geographical areas studied. Once released in rivers, the persistence of the different micro-organisms differed significantly. Bacteriophages and spores of sulphite-reducing clostridia persisted longer than faecal coliforms and enterococci. SIGNIFICANCE AND IMPACT OF THE STUDY: Bacteriophages in river water samples provide additional information to that provided by bacteria about the fate of faecal micro-organisms in river water. The easy, fast and cheap methods for phage determination are feasible both in industrialized and developing countries.     

Occurrence and levels of indicators and selected pathogens in different sludges and biosolids

AIMS: Determine the occurrence and levels of pathogens and indicators in raw and treated sludges and compare their persistence after two different treatments. METHODS AND RESULTS: Helminth ova, Cryptosporidium spp., Salmonella spp., enteroviruses, and bacterial and viral indicators were determined in raw sludges and biosolids produced after mesophilic and thermophilic treatments. Except Salmonella, all of the parameters were quantified. Helminth ova were found at very low concentrations even in raw sludges. Viable Cryptosporidium oocysts were still present in most samples of treated sludges. Faecal coliforms, spores of sulphite-reducing clostridia (SSRC), and somatic coliphages were the only indicators with values above their detection limits in most of the samples. CONCLUSIONS: Pathogens were still detected in some treated sludge samples. SSRC were the most resistant micro-organisms to treatments and hence may be an indicator for the reduction of protozoan oocysts. Somatic coliphages constitute an alternative as viral indicators due to their detection in sludges before and after treatment. SIGNIFICANCE AND IMPACT OF THE STUDY: Because of the persistence of some pathogens after sludge treatments, additional indicators are needed. SSRC and somatic coliphages are good candidates. Easy and inexpensive methods for the determination of these indicators are feasible both in industrialized and developing countries.     

Distribution and persistence of fecal bacterial populations in liquid and dewatered sludge from a biological treatment plant

The changes in composition and structure of fecal coliforms (FC) and enterococci (ENT) populations, as well as the elimination of spores of sulphite-reducing bacteria (SRB), were compared between municipal sewage and their derived sludge in a biological treatment plant in order to determine any selective reduction or adsorption to sludge during the treatment process. Additionally, the persistence of antibiotic-resistant enterococcal populations in two kinds of sludge was also considered to evaluate their potential elimination in the treatment process. Microbial indicators, vancomycin-resistant and erythromycin-resistant enterococci were enumerated. The structure and composition of FC and ENT populations were determined by biochemical fingerprinting and clustering analyses. Raw and treated sewage showed a concentration of FC 1 log unit higher than ENT and nearly 2 log units higher than spores of SRB. However, the three studied indicators showed similar concentrations in both types of sludge. Consequently, FC were eliminated in higher proportion than ENT and spores of SRB in sludge. FC and ENT populations showed high diversity and similarity population indexes for all kinds of samples. Antibiotic-resistant enterococci persisted in a similar proportion in respect to total enterococci not only in treated sewage but also in sludge. The persistence of antibiotic-resistant strains in sludge as well as in treated sewage should be considered if they are used for land disposal or for water reutilization, respectively.     

Survival of bacterial indicator species and bacteriophages after thermal treatment of sludge and sewage

The inactivation of naturally occurring bacterial indicators and bacteriophages by thermal treatment of a dewatered sludge and raw sewage was studied. The sludge was heated at 80 degrees C, and the sewage was heated at 60 degrees C. In both cases phages were significantly more resistant to thermal inactivation than bacterial indicators, with the exception of spores of sulfite-reducing clostridia. Somatic coliphages and phages infecting Bacteroides fragilis were significantly more resistant than F-specific RNA phages. Similar trends were observed in sludge and sewage. The effects of thermal treatment on various phages belonging to the three groups mentioned above and on various enteroviruses added to sewage were also studied. The results revealed that the variability in the resistance of phages agreed with the data obtained with the naturally occurring populations and that the phages that were studied were more resistant to heat treatment than the enteroviruses that were studied. The phages survived significantly better than Salmonella choleraesuis, and the extents of inactivation indicated that naturally occurring bacteriophages can be used to monitor the inactivation of Escherichia coli and Salmonella.     

Development of an obligate anaerobe specific biocide

Summary Anaerobic bacteria, such as sulfate-reducing bacteria and clostridia, are capable of generating H₂S and organic acids which corrode metallurgy resulting in millions of dollars of damage to industry annually. The bacteria are obligate anaerobes which grow typically on equipment surfaces under deposits such as biofilms. A successful method of penetrating biofilm and killing the anaerobic bacteria specifically has not been previously presented. We have investigated whether a blend of 1-(2-hydroxyethyl)-2-methyl-5-nitroimidazole (metronidazole) and a biodispersant would killDesulfovibrio, Desulfotomaculum, andClostridium species grown in the laboratory and in field applications. We found the blend significantly reduced the anaerobes in laboratory cultures. However, in a bioreactor designed to induced a high level of biofilm production and enhance underdeposit growth of anaerobic bacteria, a 40–58% increase in the antibiotic-biodispersant blend concentration was required. The metronidazole blend killed obligate anaerobic bacteria specifically but was non-toxic to aerobic bacteria and fungi. These results were confirmed in cooling tower field trial studies.     

Isolation and characterization of a new Clostridium sp. that performs effective cellulosic waste digestion in a thermophilic methanogenic bioreactor

A methanogenic bioreactor that utilized wastepaper was developed and operated at 55 degrees C. Microbial community structure analysis showed the presence of a group of clostridia that specifically occurred during the period of high fermentation efficiency. To isolate the effective cellulose digester, the sludge that exhibited high fermentation efficiency was inoculated into a synthetic medium that contained cellulose powder as the sole carbon source and was successively cultivated. A comprehensive 16S rRNA gene sequencing study revealed that the enriched culture contained various clostridia that had diverse phylogenetic positions. The microorganisms were further enriched by successive cultivation with filter paper as the substrate, as well as the bait carrier. A resultant isolate, strain EBR45 (= Clostridium sp. strain NBRC101661), was a new member of the order Clostridiales phylogenetically and physiologically related to Clostridium thermocellum and Clostridium straminisolvens. Specific PCR-based monitoring demonstrated that strain EBR45 specifically occurred during the high fermentation efficiency period in the original methanogenic sludge. Strain EBR45 effectively digested office paper in its pure cultivation system with a synthetic medium.     

Potential Health Hazards from Microbial Aerosols in Densely Populated Urban Regions

Aerosolized bacteria were recovered up to 930 m downwind of three sewage treatment plants in Jefferson County, Ky. This distance includes homes in the proximity of several hundred such plants in that county. Bacterial counts were elevated on foliage near activated sludge tanks; although these counts decreased rapidly, at 48 h after exposure they were significantly higher than the counts on unexposed leaves. The 50% lethal dose of aerosolized Klebsiella pneumoniae was comparable to the 50% lethal dose of a virulent clinical isolate, and enteric bacteria were recovered from the respiratory organs of mice after forced inhalation adjacent to an aerated sludge tank. The coliform density in the effluents of the plants tested was inversely related to the airborne bacterial load at those plants. This relationship was attributed to the correlation between effluent quality and extent of aeration of activated sludge. Wind direction and distance influenced the airborne counts, but the extreme variation in counts indicates that it is not possible to predict emission rates accurately in an open ecosystem. Airborne enteric bacteria also were isolated near a decorative fountain used by humans for wading. The discovery of these sources of aerosolized microorganisms from polluted waters in densely populated areas suggests that a potential health hazard may be created by the increased probability of inhaling and ingesting microorganisms of fecal origin.     

The intestinal microflora of infants: composition of fecal flora in breast-fed and bottle-fed infants

The fecal flora of 35 breast-fed and 35 bottle-fed babies was determined. Bifidobacteria were the predominant fecal bacteria in both groups. Conversely, the counts of most of the other bacteria, such as bacteroides, eubacteria, peptococci, veillonella, clostridia, enterobacteria, streptococci, and bacilli in the bottle-fed group were significantly higher than those in the breast-fed group. The frequencies of occurrence of lecithinase positive clostridia, clostridia-others, pseudomonas and bacilli in the bottle-fed group were significantly higher than those in the breast-fed group. Twenty-one genera and 103 species or biovars of microorganisms were isolated from the feces of the breast-fed group and 20 genera and 97 species or biovars from the bottle-fed group. The organism that showed the highest number and the highest frequency of occurrence in both groups was Bifidobacterium breve. Bifidobacterium infantis, which was formerly the most prevalent Bifidobacterium species in baby feces, was never isolated in this study. Further, the counts and incidences of Clostridium paraputrificum, C. perfringens, and Bacillus subtilis, the counts of C. clostridiiforme, Bacteroides vulgatus, Veillonella parvula, Lactobacillus acidophilus, Escherichia coli, Streptococcus bovis, S. faecalis, and S. faecium and the incidences of C. difficile, C. tertium, and Pseudomonas aeruginosa in the bottle-fed infants were significantly higher than those in the breast-fed infants.     

Coaggregation among nonflocculating bacteria isolated from activated sludge

Thirty-two strains of nonflocculating bacteria isolated from sewage-activated sludge were tested by a spectrophotometric assay for their ability to coaggregate with one other in two-membered systems. Among these strains, eight showed significant (74 to 99%) coaggregation with Acinetobacter johnsonii S35 while only four strains coaggregated, to a lesser extent (43 to 65%), with Acinetobacter junii S33. The extent and pattern of coaggregation as well as the aggregate size showed good correlation with cellular characteristics of the coaggregating partners. These strains were identified by sequencing of full-length 16S rRNA genes. A. johnsonii S35 could coaggregate with strains of several genera, such as Oligotropha carboxidovorans, Microbacterium esteraromaticum, and Xanthomonas spp. The role of Acinetobacter isolates as bridging organisms in multigeneric coaggregates is indicated. This investigation revealed the role of much-neglected nonflocculating bacteria in floc formation in activated sludge.     

Occurrence of Listeria sp and L monocytogenes in sewage sludge used for land application: effect of dewatering,liming and storage in tank on survival of Listeria species

The application of sewage sludge to agricultural land is widely used in France. To determine the impact of sludge treatments, concentrations of Listeria sp., Listeria monocytogenes and faecal indicators were monitored in five types of sludge from three sewage treatment plants in Angers (France) and its suburbs over a 1-year period. On the whole, bacteria were reduced in numbers through sludge treatments. Apart from liming, which leads to reduced levels of bacteria below detection limits, other sludge treatments did not eliminate Listeria sp. and faecal indicators. Listeria sp. and L. monocytogenes were found respectively in 87% and 73% of dewatered sludges and in 96% and 80% of sludges stored in tanks. Concentrations of L. monocytogenes, ranging from 0.15 to 20 MPN g(-1) dry matter in dewatered sludge and from 1 to 240 MPN g(-1) dry matter in sludge stored in tanks, did not show seasonal variations. Spreading of sanitised sludge onto agricultural land results in the addition of 10(6)-10(8) L. monocytogenes per hectare per year, which may contribute to the increase in the dissemination of this pathogenic species in the environment.     

Involvement of Acinetobacter sp. in the floc-formation in activated sludge process

The coaggregation behavior of Acinetobacter johnsonii S35 isolate with sewage bacteria was assessed by a spectrophotometric assay using different samples from a municipal wastewater treatment plant and a community plant. A. johnsonii S35 coaggregated well with other free bacteria and microflocs at the mixing ratios of 0.2:1-0.6:1 of A. johnsonii S35 and sewage samples. In addition, the size of coaggregates became larger (100 μm or more) under the same conditions. A. johnsonii S35 cells were highly adsorbed (adsorption=93-99%) onto sludge samples. Microbial adhesion to hydrocarbon (MATH) test and adsorption to octyl-Sepharose CL-4B showed that A. johnsonii S35 cells and sludge samples had a hydrophobic character. The population of Acinetobacter spp. in sewage treatment plants was 2-7% and its role in bioflocculation was discussed. The present study revealed that A. johnsonii S35 isolate can play as a bridging organism and contribute in floc-formation in activated sludge process.     

Isolation and Characterization of a New Clostridium sp. That Performs Effective Cellulosic Waste Digestion in a Thermophilic Methanogenic Bioreactor

A methanogenic bioreactor that utilized wastepaper was developed and operated at 55°C. Microbial community structure analysis showed the presence of a group of clostridia that specifically occurred during the period of high fermentation efficiency. To isolate the effective cellulose digester, the sludge that exhibited high fermentation efficiency was inoculated into a synthetic medium that contained cellulose powder as the sole carbon source and was successively cultivated. A comprehensive 16S rRNA gene sequencing study revealed that the enriched culture contained various clostridia that had diverse phylogenetic positions. The microorganisms were further enriched by successive cultivation with filter paper as the substrate, as well as the bait carrier. A resultant isolate, strain EBR45 (= Clostridium sp. strain NBRC101661), was a new member of the order Clostridiales phylogenetically and physiologically related to Clostridium thermocellum and Clostridium straminisolvens. Specific PCR-based monitoring demonstrated that strain EBR45 specifically occurred during the high fermentation efficiency period in the original methanogenic sludge. Strain EBR45 effectively digested office paper in its pure cultivation system with a synthetic medium.     

A multiple logistic model for predicting the occurrence of Campylobacter jejuni and Campylobacter coli in water

A multiple logistic regression model was established to predict the occurrence of Campylobacter jejuni/coli , related to index bacteria such as faecal coliforms, faecal streptococci, and sulphite-reducing clostridia, in a water source in southern Norway. The fitted model indicated that faecal coliforms were strong predictors for C. jejuni/coli , although the water temperature also had a strong influence on results. Sulphite-reducing clostridia, faecal streptococci, and season of the year had no significant influence on the results, in spite of their apparent predictor value as separate variables. The model employed offers a new approach to the relationship between index bacteria and the occurrence of pathogenic bacteria in water. Similar models can also be established in general food microbiology.     

Evaluation of broiler litter with reference to the microbial composition as assessed by using 16S rRNA and functional gene markers

Very little is known about the microbial composition of animal bedding wastes, including poultry litter, and what is known has been deduced from standard culture methods, by which some fastidious organisms that exist in the environment may not be detected. We evaluated the bacterial composition of poultry litter by using a combination of culture and molecular detection. Total aerobic bacteria in poultry litter were detected by culture at 10(9) CFU/g of material. Enteric bacteria such as Enterococcus spp. and coliforms composed 0.1 and 0.01%, respectively, of the total aerobic cultivatable bacteria in poultry litter; no Salmonella strains were detected by culture. In order to characterize the most abundant bacterial groups, we sequenced 16S ribosomal DNA (rDNA) genes amplified by PCR with microbial community DNA isolated from poultry litter as the template. From the 16S rDNA library, 31 genera were identified. Twelve families or groups were identified with lactobacilli and Salinococcus spp. forming the most abundant groups. In fact, 82% of the total sequences were identified as gram-positive bacteria with 62% of total belonging to low G+C gram-positive groups. In addition to detection of 16S rDNA sequences associated with the expected fecal bacteria present in manure, we detected many bacterial sequences for organisms, such as Globicatella sulfidofaciens, Corynebacterium ammoniagenes, Corynebacterium urealyticum, Clostridium aminovalericum, Arthrobacter sp., and Denitrobacter permanens, that may be involved in the degradation of wood and cycling of nitrogen and sulfur. Several sequences were identified in the library for bacteria associated with disease in humans and poultry such as clostridia, staphylococci, and Bordetella spp. However, specific PCR targeting other human and veterinary pathogens did not detect the presence of Salmonella, pathogenic Escherichia coli, Campylobacter spp., Yersinia spp., Listeria spp., or toxigenic staphylococci. PCR and DNA hybridization revealed the presence of class 1 integrons with gene cassettes that specify resistance to aminoglycosides and chloramphenicol. Only from understanding the microbial community of animal wastes such as poultry litter can we manage animal disease and limit the impact of animal waste on the environment and human and animal health.