qPCR quantification and genetic characterization of Clostridium perfringens populations in biosolids composted for 2 years

Aim: The ability of Clostridium perfringens to survive for a long time in the environment makes it a suitable indicator of faecal pollution, but its use as a routine indicator organism in biosolids and composted biosolids has not yet been adopted. This study was performed to improve our understanding of C. perfringens persistence in composted biosolids by monitoring its presence and studying its genetic diversity. Methods and Results: A culture‐independent TaqMan qPCR assay targeting the cpn60 gene was adapted to enumerate C. perfringens in composted biosolid samples varying in age from 1 to 24 months. The pathogen was detected in all compost samples under study, but no correlation between composting time and number of cpn60 copies was observed. Rep‐PCR detected 14 different C. perfringens genotypes, all belonging to toxinotype A, which is the most common biotype found in human and animal gastrointestinal tracts. Conclusions: Composting did not significantly decrease the number of C. perfringens cells. High genetic diversity of C. perfringens isolates present in composted biosolids is reported for the first time. Significance and Impact of Study: This study evaluated tools for surveillance of composting processes, source tracking and risk assessment of composted biosolids.     

Abilities of the mCP Agar Method and CRENAME Alpha Toxin-Specific Real-Time PCR Assay To Detect Clostridium perfringens Spores in Drinking Water

We first determined the analytical specificity and ubiquity (i.e., the ability to detect all or most strains) of a Clostridium perfringens-specific real-time PCR (rtPCR) assay based on the cpa gene (cpa rtPCR) by using a bacterial strain panel composed of C. perfringens and non-C. perfringens Clostridium strains. All non-C. perfringens Clostridium strains tested negative, whereas all C. perfringens strains tested positive with the cpa rtPCR, for an analytical specificity and ubiquity of 100%. The cpa rtPCR assay was then used to confirm the identity of 116 putative C. perfringens isolates recovered after filtration of water samples and culture on mCP agar. Colonies presenting discordant results between the phenotype on mCP agar and cpa rtPCR were identified by sequencing the 16S rRNA and cpa genes. Four mCP⁻/rtPCR⁺ colonies were identified as C. perfringens, whereas 3 mCP⁺/rtPCR⁻ colonies were identified as non-C. perfringens. The cpa rtPCR was negative with all 51 non-C. perfringens strains and positive with 64 of 65 C. perfringens strains. Finally, we compared mCP agar and a CRENAME (concentration and recovery of microbial particles, extraction of nucleic acids, and molecular enrichment) procedure plus cpa rtPCR (CRENAME + cpa rtPCR) for their abilities to detect C. perfringens spores in drinking water. CRENAME + cpa rtPCR detected as few as one C. perfringens CFU per 100 ml of drinking water sample in less than 5 h, whereas mCP agar took at least 25 h to deliver results. CRENAME + cpa rtPCR also allows the simultaneous and sensitive detection of Escherichia coli and C. perfringens from the same potable water sample. In itself, it could be used to assess the public health risk posed by drinking water potentially contaminated with pathogens more resistant to disinfection.     

Investigating the role of small, acid-soluble spore proteins (SASPs) in the resistance of Clostridium perfringens spores to heat

Background  

Clostridium perfringens type A food poisoning is caused by enterotoxigenic C. perfringens type A isolates that typically possess high spore heat-resistance. The molecular basis for C. perfringens spore heat-resistance remains unknown. In the current study, we investigated the role of small, acid-soluble spore proteins (SASPs) in heat-resistance of spores produced by C. perfringens food poisoning isolates.     

Sewage reflects the distribution of human faecal Lachnospiraceae

Faecal pollution contains a rich and diverse community of bacteria derived from animals and humans, many of which might serve as alternatives to the traditional enterococci and Escherichia coli faecal indicators. We used massively parallel sequencing (MPS) of the 16S rRNA gene to characterize microbial communities from wastewater treatment plant (WWTP) influent sewage from 12 cities geographically distributed across the USA. We examined members of the Clostridiales, which included the families Clostridiaceae, Lachnospiraceae and Ruminococcaceae for their potential as sewage indicators. Lachnospiraceae was one of the most abundant groups of faecal bacteria in sewage, and several Lachnospiraceae high‐abundance sewage pyrotags occurred in at least 46 of 48 human faecal samples. Clone libraries targeting Clostridium coccoides (C. coccoides) in sewage samples demonstrated that Lachnospiraceae‐annotated V6 pyrotags encompassed the previously reported C. coccoides group. We used oligotyping to profile the genus Blautia within Lachnospiraceae and found oligotypes comprised of 24 entropy components that showed patterns of host specificity. These findings suggest that indicators based on Blautia might have the capacity to discriminate between different faecal pollution sources. Development of source‐specific alternative indicators would enhance water quality assessments, which leads to improved ecosystem health and reduced human health risk due to waterborne disease.     

Nosocomial Diarrhoea in the Elderly Due to Enterotoxigenic Clostridium perfringens

To diagnose sporadic diarrhoea due to Clostridium perfringens infection, faecal specimens from elderly patients were examined directly for C. perfringens enterotoxin using reverse passive latex agglutination assay, and then cultured for this organism. C. perfringens isolates from those samples were grouped by slide agglutination and by pulsed-field gel electrophoresis (PFGE). Fifty of the 60 isolates agglutinated with newly raised antiserum WX2 and 38 shared the same genomic PFGE pattern. Characteristics of the epidemics and experimental data suggest that the diarrhoea was caused by a nosocomial spread of C. perfringens, and not by a food-borne outbreak.     

Escherichia coli and enterococci are sensitive and reliable indicators for human,livestock and wildlife faecal pollution in alpine mountainous water resources

Aims: This study evaluated the applicability of standard faecal indicator bacteria (SFIB) for alpine mountainous water resources monitoring. Methods and Results: Escherichia coli, enterococci (ENTC) and Clostridium perfringens were investigated by standard or frequently applied phenotypic and genotypic methods in a broad range of animal and human faecal sources in a large alpine mountainous area. Clostridium perfringens occurred only in human, livestock and carnivorous source groups in relevant average concentrations (log 4·7–7·0 CFU g^?1) but not in herbivorous wildlife sources. Escherichia coli proved to be distributed in all faecal source groups with remarkably balanced average concentrations (log 7·0–8·4 CFU g^?1). Except for single faecal samples from the cattle source group, prevalence rates for ENTC source groups were generally >87% with average concentrations of log 5·3–7·7 CFU g^?1. To test the faecal indication capacity in the environment, faecal prevalence data were comparatively analysed with results from the concurrently performed multi‐parametric microbial source tracking effort on karst spring water quality from the investigated alpine mountainous catchment ( Reischer et al. 2008 ; Environ Microbiol 10:2598–2608). Conclusion: Escherichia coli and enterococci are reliable faecal indicators for alpine mountainous water resources monitoring, although E. coli is the more sensitive one. Clostridium perfringens did not prove to be an indicator of general faecal pollution but is suggested a conservative microbial source tracking marker for anthropogenic faecal influence. Significance and Impact of the Study: Applicability of SFIB is currently hotly debated. This is the first study providing comprehensive information on the applicability of SFIB at alpine mountainous habitats.     

Genetic diversity of Clostridium perfringens type A isolates from animals, food poisoning outbreaks and sludge

Background  

Clostridium perfringens, a serious pathogen, causes enteric diseases in domestic animals and food poisoning in humans. The epidemiological relationship between C. perfringens isolates from the same source has previously been investigated chiefly by pulsed-field gel electrophoresis (PFGE). In this study the genetic diversity of C. perfringens isolated from various animals, from food poisoning outbreaks and from sludge was investigated.     

Detection and typing of Clostridium perfringens from retail chicken meat parts

The objectives of the present work were to investigate the presence of Clostridium perfringens in chicken meat parts (breast, wing, drumstick and leg quarter) by culture methods and to detect the cpa, cpb, etx, iA, cpe and cpb2 toxin genes by multiplex PCR. A total of 200 samples, the raw chicken breasts (n: 50), wings (n: 50), drumsticks (n: 50) and leg quarters (n: 50), were collected from various retail stores. Our results demonstrated that 47 of 50 wing samples (94%), 40 of 50 leg quarter samples (80%), 34 of 50 drumstick samples (66%) and 33 of 50 breast samples (66%) were found to be contaminated with Cl. perfringens. 558 positive isolates obtained from these samples were identified as Cl. perfringens based on the microscopic examination and biochemical tests. It was detected that 545 (97·6%) of 558 Cl. perfringens isolates carried only cpa toxin gene (type A), 12 (2·1%) of them carried both cpa and cpb2 toxin gene (type A‐cpb2), one (0·1%) of them carried both cpa and cpe toxin genes (type A‐cpe), according to the multiplex PCR results, targeted cpa, cpb, cpb2, cpe, etx and iA genes.

Significance and Impact of the Study

This study is the first report of detection of cpe and cpb2 toxin genes in Clostridium perfringens isolated from chicken meats in Turkey. The multiplex PCR protocol described in this study is useful for rapid detection of Clostridium perfringens toxin genes simultaneously in one‐step PCR.     

Diversity and population structure of sewage‐derived microorganisms in wastewater treatment plant influent

The release of untreated sewage introduces non‐indigenous microbial populations of uncertain composition into surface waters. We used massively parallel 454 pyrosequencing of hypervariable regions in rRNA genes to profile microbial communities from eight untreated sewage influent samples of two wastewater treatment plants (WWTPs) in metropolitan Milwaukee. The sewage profiles included a discernible human faecal signature made up of several taxonomic groups including multiple Bifidobacteriaceae, Coriobacteriaceae, Bacteroidaceae, Lachnospiraceae and Ruminococcaceae genera. The faecal signature made up a small fraction of the taxa present in sewage but the relative abundance of these sequence tags mirrored the population structures of human faecal samples. These genera were much more prevalent in the sewage influent than standard indicators species. High‐abundance sequences from taxonomic groups within the Beta‐ and Gammaproteobacteria dominated the sewage samples but occurred at very low levels in faecal and surface water samples, suggesting that these organisms proliferate within the sewer system. Samples from Jones Island (JI – servicing residential plus a combined sewer system) and South Shore (SS – servicing a residential area) WWTPs had very consistent community profiles, with greater similarity between WWTPs on a given collection day than the same plant collected on different days. Rainfall increased influent flows at SS and JI WWTPs, and this corresponded to greater diversity in the community at both plants. Overall, the sewer system appears to be a defined environment with both infiltration of rainwater and stormwater inputs modulating community composition. Microbial sewage communities represent a combination of inputs from human faecal microbes and enrichment of specific microbes from the environment to form a unique population structure.     

Giardia cysts in sewage influent in Bergen, Norway 15-23 months after an extensive waterborne outbreak of giardiasis

Aims: In autumn/winter 2004, a large outbreak of waterborne giardiasis occurred in Bergen, Norway. Over 1 year later, the concentrations and genotypes of Giardia cysts occurring in sewage influent were studied to investigate the impact of the outbreak event on Giardia infections in the community. Methods and Results: Sewage influent samples from four sewage treatment works (STW) serving Bergen were analysed for Giardia cysts on four occasions between 15 and 23 months after the outbreak. Cysts genotypes were investigated at one to three genes. Data from influent analysis from one of the STW before the outbreak, and from patient faecal samples analysed during the outbreak, provided baseline comparative data. Relatively high concentrations of Giardia cysts of diverse genotypes, both from Assemblages A and B, were detected at all STW. Conclusions: Comparison of data suggests that although Giardia cyst concentrations in sewage influent returned to pre‐outbreak levels within 18 months after the outbreak peak, the genetic composition of the isolates remained significantly influenced by the Assemblage B isolate associated with the outbreak. Significance and Impact of the Study: Genotypes associated with an extensive outbreak of giardiasis continued to occur in Giardia infections in Bergen’s population many months after the outbreak was considered to be over.     

Detection and characterization of Clostridium difficile in retail chicken

Aims: This study was designed to evaluate the prevalence of Clostridium difficile contamination of retail chicken. Methods and Results: Chicken legs, thighs and wings were purchased using a standardized method from retail outlets across Ontario, Canada. Selective culture was used for qualitative and quantitative detection of C. difficile. Clostridium difficile was isolated from 26/203 (12·8%) chicken samples; 10/111 (9·0%) thighs, 13/72 (18%) wings and 3/20 (15%) legs (P = 0·19). All isolates were ribotype 078, a strain that has been associated with food animals and potentially community‐associated disease in humans. All positive samples were positive only on enrichment culture. Conclusions: Clostridium difficile could be found relatively commonly in retail chicken meat, albeit at low levels. Significance and Impact of the Study: This is the first study to report C. difficile in chicken meat. Contamination of meat with C. difficile strains implicated in human infections raises concerns about food as a source of C. difficile infection. The relevance of food contamination is completely unclear at this point but food should be investigated as a source of infection.     

Clostridium perfringens in retail chicken

Clostridium perfringens isolates were recovered by enrichment from retail grocery chicken samples (n = 88) in Ontario, Canada, with one sample per site. The gene associated with necrotic enteritis in chickens, netB, was found in 21% of the isolates. The tpeL gene was found in 2% and the cpb2 gene in 68% (95% “atypical” genes) of isolates. This study suggests that netB-positive C. perfringens can reach people through retail chicken.     

Penicillin-binding proteins of clostridia

The penicillin-binding protein (PBP) profiles of 33Clostridium perfringens and sixClostridium species isolated from clinically significant infections were analyzed. Three new PBPs—PBPs 2B, 4B, and 5B (84, 70, and 49 kDa respectively)—and a high-molecular-weight PBP 6 (45 kDa) were demonstrated in theC. perfringens isolates. In addition to PBPs 1 and 2, PBPs 2B and 4B were seen to show low binding affinities for penicillin, although further studies are required to determine their possible roles in the development of penicillin resistance. The PBP profiles of theC. perfringens isolates were complex. Variations in apparent molecular weights (M _r s) of all PBPs, with the exception of PBP 5 and the presence or absence of PBPs 2, 3, and 4B, gave rise to nine different PBP patterns. The high-M _rPBPs 5 and 6, which exhibited high-penicillin-binding affinities, were with only one exception consistent within theC. perfringens isolates. These PBPs 5 and 6 of theC. perfringens isolates and independent PBPs found in the otherClostridium species studied indicate that PBP analysis may assist in the differentiation ofClostridium spacies.     

Comparison of alternatives to in‐feed antimicrobials for the prevention of clinical necrotic enteritis

Aims: The capacity for Lactobacillus johnsonii and an organic acid (OA) blend to prevent Clostridium perfringens‐induced clinical necrotic enteritis (NE) in chickens was studied. Methods and Results: Cobb 500 birds were allocated into six groups (n = 25 birds/pen, eight pens/treatment); Unchallenged, Challenged, Antimicrobial (zinc bacitracin (ZnB)/monensin), OA, probiotic Lact. johnsonii and probiotic sham (Phosphate–buffered saline). All birds were challenged with Eimeria spp. and Cl. perfringens except for unchallenged controls. Birds fed antimicrobials were protected from NE development as indicated by maintenance of body weight, low mortality and clostridium levels, and decreased intestinal macroscopic lesion scores compared to challenged controls (P < 0·05). Lactobacillus johnsonii‐fed birds had reduced lesion scores, whilst OA‐fed birds had decreased Cl. perfringens levels. Both Lact. johnsonii and OA‐fed birds had improved feed efficiency between days 0 and 28 compared to challenged controls; however, mortality and body weights were not improved by either treatment. Microbial profiling indicated that the challenge procedure significantly altered the jejunal microbiota. The microbiota of antimicrobial‐fed birds was significantly different from all other groups. Conclusions: Whilst Lact. johnsonii and OA altered specific intestinal parameters, significant protection against NE was not observed. Significance and Impact of the Study: Lactobacillus johnsonii and OA did not prevent NE; however, some improvements were evident. Other related treatments, or combinations of these two treatments, may provide greater protection.     

Survival of <Emphasis Type="Italic">Clostridium perfringens</Emphasis>During Simulated Transport and Stability of Some Plasmid-borne Toxin Genes under Aerobic Conditions

Clostridium perfringens is a pathogen of great concern in veterinary medicine, because it causes enteric diseases and different types of toxaemias in domesticated animals. It is important that bacteria in tissue samples, which have been collected in the field, survive and for the classification of C. perfringens into the correct toxin group, it is crucial that plasmid-borne genes are not lost during transportation or in the diagnostic laboratory. The objectives of this study were to investigate the survival of C. perfringens in a simulated transport of field samples and to determine the stability of the plasmid-borne toxin genes cpb1 and etx after storage at room temperature and at 4°C. Stability of the plasmid-borne genes cpb1 and etx of C. perfringens CCUG 2035, and cpb2 from C. perfringens CIP 106526, JF 2255 and 6 field isolates in aerobic atmosphere was also studied. Survival of C. perfringens was similar in all experiments. The cpb1 and etx genes were detected in all isolates from samples stored either at room temperature or at 4°C for 24–44 h. Repeated aerobic treatment of C. perfringens CCUG 2035 and CIP 106526 did not result in the loss of the plasmid-borne genes cpb1, cpb2 or etx. Plasmid-borne genes in C. perfringens were found to be more stable than generally reported. Therefore, C. perfringens toxinotyping by PCR can be performed reliably, as the risk of plasmid loss seems to be a minor problem.     

Detection of Clostridium perfringens in tsunami deposits after the Great East Japan Earthquake

The Great East Japan Earthquake struck off the Tohoku and caused a tsunami in 2011. Most of the microbial characteristics of tsunami‐affected soil remain unknown and no published study has shown how a tsunami affects the risk of infection by Clostridium perfringens living in soil. In 2011 and 2015, C. perfringens was assessed in deposits in soil from tsunami‐damaged areas and undamaged areas of Miyagi. It was found that the number of C. perfringens was overwhelmingly greater in 2011 than in 2015 in the tsunami‐damaged areas. According to real‐time PCR, the prevalence C. perfringens organisms (%) was 10³ fold greater in the damaged than in the undamaged areas.     

Occurrence of Clostridium perfringens from different cultivated soils

The occurrence of Clostridium perfringens was estimated in 750 samples originated from a variety of soils bearing various bulb crops: Brawnica oderacea (vegetable), Olea europaea, Daucus carota (carote), Solanum tuberosum (potato), Phaseolus vulgaris (green haricot), Beta vulgaris var. rapaceum (beetroot), Cucurbita pepo (squash), Allium cepa (onion), Cucumis sativus (cucumber) and Capsicum annum (pepper). All isolated strains were tested for their antimicrobial activities to amoxicillin, penicillin G, kanamycin, tetracycline, streptomycin, erythromycin, chloramphenicol and metronidazole.When considering the type of the bulb production, it was observed increased number of C. perfringens spore densities in the most undersurface bulb soils. Moreover, C. perfringens spore are likely to occur in particularly large numbers in soil contaminated by fecal matter. Additionally, there is a close relationship between the spore amount and nature of organic content. Presence of C. perfringens was associated with acidic soil. Most of our strains showed resistance to the studied antibiotics applied usually for human and veterinary care. A systematic monitoring of the cultivated soil ecosystems must include bacteriological parameters together with chemical indices of organic pollution in order to obtain information adequate for assessing their overall quality.     

Changes in the cell wall of Clostridium species following passage in animals

Morphological changes in clostridial isolates after animal passage with other flora in mixed infections were studied by utilizing a subcutaneous abscess model in mice. We used 26 isolates of 7 clostridial species, and one isolate each of Bacteroides fragilis and Klebsiella pneumoniae. Abscesses were induced by all 7 Clostridium perfringens and 3 Clostridium butyricum isolates and by some of the other isolates. A thick granular wall prior to animal inoculation was shown only in C. perfringens, C. butyricum, and C. difficile. This structure was observed in other clostridia only following their animal passage alone or when co-inoculated with K. pneumoniae or B. fragilis.     

Impact of beta-glucan on the faecal microbiota of polypectomized patients: a pilot study

Beta-glucans are polysaccharides present in the cell walls of higher plants, in the seeds of some cereals, and certain yeasts and fungi also produce them. It is suggested that they exhibit, among many other health benefits, protective effects against carcinogenesis in the colon, but there is not enough human data to support this. The aim of the study was to determine the effect of barley-derived beta-glucan in the gut microbiota of polypectomized patients. Subjects were randomly assigned to consume 125 g of bread per day with beta-glucan (3 g/d), or without (placebo group), for 3 months. Thirty-three polypectomized men and women (mean age 57.6 years) were recruited into the study, but only 20 completed. Subjects did not consume any probiotics, prebiotics or antibiotics 2 months prior the intervention, or during the study. Stool samples were collected at baseline, on days 30 and 90 of intervention, as well as 2 weeks after the intervention, for enumeration of total aerobes and anaerobes, coliforms, E. coli, enterococci, Bacteroides spp., Clostridium perfringens, bifidobacteria, lactobacilli and Candida spp. Faecal bacterial enzyme activity (beta-glucuronidase and beta-glucosidase), pH, faecal moisture and the concentration of volatile fatty acids in the faeces were measured. Gastrointestinal symptoms were also recorded. Overall, no significant differences were observed in bacterial viable counts between the two feeding groups. Group specific analysis for β-glucan group revealed significantly decreased total coliform counts on the 30th day of the trial compared to the baseline (p = 0.041). Clostridium perfringens concentration increased without reaching statistical significance, on the 30th day, while it decreased significantly on the 90th day of the intervention compared to the 30th day (p = 0.016). An increase was noted in the molar ratio of acetate on the 90th day of the trial compared to placebo (p = 0.018). The molar ratio of butyrate presented a trend to increase on the 30th day, which decreased (p = 0.013) on the 90th day and then increase 2 weeks after the intervention (p = 0.017) compared to placebo. A decrease was recorded in the β-glucan group in the bloating and abdominal pain score after the 30th day of the intervention (Day 30–37) compared to placebo. During β-glucan administration we did not observe any changes on beta-glucuronidase or beta-glucosidase activity, faecal pH, or on faecal moisture.     

Oral Administration of <Emphasis Type="Italic">Clostridium butyricum</Emphasis> for Modulating Gastrointestinal Microflora in Mice

This study aimed to evaluate the safety of Clostridium butyricum and to investigate the effect of C. butyricum on mice ecosystem in the intestinal tract by way of examining the population of different microorganisms isolated from caecal contents. We firstly evaluated the safety of C. butyricum using acute toxicity test and Ames test. Then forty male BALB/c mice were divided into the following four treatment groups, each consisting of ten mice: normal group, low-dose group, medium-dose group and high-dose group. Caecal contents were removed aseptically, immediately placed into an anaerobic chamber, and dissolved in sterile pre-reduced PBS. The determination of Enterococcus spp., Enterobacter spp., Lactobacillus spp., Bifidobacterium spp. and Clostridium perfringens was analyzed by the spread plate method, cell morphologies and biochemical profiles. The results showed the oral maximum tolerated dose of C. butyricum was more than 10 g/kg body weight in mice and no mutagenicity judged by negative experimental results of Ames test. And in medium- and high-dose groups, the populations of Bifidobacterium spp. and Lactobacillus spp. increased in caecum, as well as the ratios of Bifidobacterium spp. and Lactobacillus spp. to Clostridium perfringens (P < 0.01) as compared with the normal group. This research showed the intake of C. butyricum significantly improved the ecosystem of the intestinal tract in BALB/c mice by increasing the amount of probiotics and reducing the populations of unwanted bacteria.