Sporulation, Heat Resistance, and Biological Properties of Clostridium perfringens

A sporulation medium for 134 Clostridium perfringens strains, including types A, B, C, D, E, and F, was devised according to Grelet's observation that sporulation occurred when cultural environment became limited in any nutritional requirement indispensable for the growth of the organism. Sporulation took place most prominently when 10% cooked-meat broth (pH 7.2) containing 3% Proteose Peptone and 1% glucose was used for the preculture and 2% Poli Peptone medium (pH 7.8) was used for the subculture medium. Sometimes, terminal spores could be observed. A correlation between sporulation and heat resistance was examined by use of C. perfringens strains isolated from samples heated at different temperatures. Almost all strains isolated from unheated samples and from those heated at lower temperatures gave rise to spores in our sporulation medium, but the spores were weakly heat-resistant, whereas strains isolated from samples heated at 100 C for 60 min were highly heat-resistant but sporulated poorly. A majority of these heat-resistant strains were non-gelatinolytic and definitely salicin-fermenting.     

Persistent High Numbers of Clostridium perfringens in the Intestines of Japanese Aged Adults

TSN agar was applicable for enumeration of Clostridium perfringens in fecal samples of adults but not in those of infants. It was demonstrated using TSN agar that some healthy aged adults had persistently carried C. perfringens at levels ranging from 10⁷ to 10⁹, while some others ranged from 10³ to 10⁶ per ml volume of fecal sample although all of these adults had the same diets. In the test for agglutinability of isolates of C. perfringens collected from two elderly adults, a younger adult and a baby, it was demonstrated that most of the isolates obtained from an aged adult of high levels for 19 months belonged the same serotype, while rapid alteration of serotypes could be observed in three other persons with high or low levels. In spite of as many as 10⁹ C. perfringens per ml of feces, no trace of α-toxin could be detected in the fecal samples. In in vitro tests, fecal suspension suppressed the production of α-toxin although it allowed the organism to grow sufficiently.     

The Ability of Saprotrophic Bacteria Isolated from Natural Habitats to Lyse Yeasts

More than 600 bacterial strains isolated from different horizons of steppe biogeocenoses and zoogenous loci (diplopod intestines and feces) were tested for the ability to lyse yeast cell walls. About half of the strains that were isolated from biotopes with active degradation of plant debris (steppe litters and diplopod intestines and feces) were found to possess yeast-lytic activity. Most of the yeast-lytic strains belonged to the genera Streptomyces, Promicromonospora, Oerskovia, and Agromyces. The yeast-lytic activity of actinobacteria from the genera Agromyces, Mycobacterium, and Micrococcus has not previously been reported.     

Prevalence of Enterotoxigenic Clostridium perfringens in Meats in San Luis,Argentina

Clostridium perfringens is an important pathogen agent causing, among other diseases, enteritis in humans and enterotoxemia in domestic animals. This bacterium can produce more than 15 toxins, one of which is its enterotoxin (CPE), that causes human food poisoning. The aim of this work was (i) to determine the prevalence of C. perfringens in some non-industrial meat foods in San Luis, Argentina, (ii) to characterize the C. perfringens enterotoxigenic strains by PCR, RPLA and the slide reverse passive latex agglutination test, (iii) to type the C. perfringens strains isolated and identification by PCR and (iv) to develop a slide RPLA test. A total of 515 samples of meat food (315 fresh sausages, 100 hamburgers and 100 samples of minced meat) were studied. A 126 C. perfringens strains (24.46%) were isolated and characterized. Of these C. perfringens -positive samples, 48 contained counts higher than 2 log/g. No significant differences were observed between counts performed in iron–milk medium and tryptose–sulfite–cycloserine agar (r= 0.99). Twelve samples (9.52%) exhibited counts with MPN >5log bacteria/g. Modified Tórtora medium (Tm) with thiotone replaced by proteose peptone turned out to be the most useful medium for both sporulation and enterotoxin production. Of the 126 samples tested by PCR and RPLA, nine strains (7.14%) were enterotoxigenic. Similar results were obtained by Slide RPLA, which exhibited a sensitivity of 8 ng/mL. Of the 126 C. perfringens strains , 123 were of type A (97.20%), two were of type C (1.59%) and one of type E (0.79%). All enterotoxigenic strains were classified as type A.     

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.     

Comparative Genomic Hybridization Analysis Shows Different Epidemiology of Chromosomal and Plasmid-Borne cpe-Carrying Clostridium perfringens Type A

Clostridium perfringens, one of the most common causes of food poisonings, can carry the enterotoxin gene, cpe, in its chromosome or on a plasmid. C. perfringens food poisonings are more frequently caused by the chromosomal cpe-carrying strains, while the plasmid-borne cpe-positive genotypes are more commonly found in the human feces and environmental samples. Different tolerance to food processing conditions by the plasmid-borne and chromosomal cpe-carrying strains has been reported, but the reservoirs and contamination routes of enterotoxin-producing C. perfringens remain unknown. A comparative genomic hybridization (CGH) analysis with a DNA microarray based on three C. perfringens type A genomes was conducted to shed light on the epidemiology of C. perfringens food poisonings caused by plasmid-borne and chromosomal cpe-carrying strains by comparing chromosomal and plasmid-borne cpe-positive and cpe-negative C. perfringens isolates from human, animal, environmental, and food samples. The chromosomal and plasmid-borne cpe-positive C. perfringens genotypes formed two distinct clusters. Variable genes were involved with myo-inositol, ethanolamine and cellobiose metabolism, suggesting a new epidemiological model for C. perfringens food poisonings. The CGH results were complemented with growth studies, which demonstrated different myo-inositol, ethanolamine, and cellobiose metabolism between the chromosomal and plasmid-borne cpe-carrying strains. These findings support a ubiquitous occurrence of the plasmid-borne cpe-positive strains and their adaptation to the mammalian intestine, whereas the chromosomal cpe-positive strains appear to have a narrow niche in environments containing degrading plant material. Thus the epidemiology of the food poisonings caused by two populations appears different, the plasmid-borne cpe-positive strains probably contaminating foods via humans and the chromosomal strains being connected to plant material.     

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.     

Transmission identification of Escherichia coli aerosol in chicken houses to their environments using ERIC-PCR

In order to study E. coli aerosol spreading from chicken houses to their surrounding air, air samples, including indoor and outdoor air (upwind 10 and 50 m as well as downwind 10, 50, 100, 200 and 400 m away) of 5 chicken houses were collected using six-stage Andersen microbial samplers and Reuter-Centrifugal samplers (RCS). E. coli concentrations (CFU/m3 air) collected from different sampling sites were calculated. E. coli strains from chicken feces samples were also isolated. Furthermore, the enterobacterial repetitive intergenic consensus (ERIC)-PCR method was applied to amplify the isolated E. coli strain DNA samples. Through the genetic similarity analyses of the E. coli obtained from different sampling sites, the spreading of bioaerosol from animal houses to the ambient air was characterized. The results showed that the isolated E. coli concentrations in indoor air (9―63 CFU/m3) in 5 chicken houses were higher than those in upwind and downwind air, but there were no significant differences between the indoor and downwind sites 10 m away from all the 5 houses (P>0.05). The phylogenetic tree indicated that a part of the E. coli (34.1%) isolated from indoor air had 100% similarity with those isolated from feces, and that most of E. coli isolated (54.5%) from downwind at 10, 50, 100 or even 200 m had 100% similarity with those isolated from indoor air or feces too. But those isolated from upwind air had a lower similarity (73%―92%) with corresponding strains isolated from indoor air or feces. Our results suggested that some strains isolated from downwind air and indoor air originated in the chicken feces, but most of isolates obtained from upwind air samples did not come from the chicken feces or indoor air. Effective hygienic measures should be taken in animal farms to prevent or minimize downwind spreading of microorganism aerosol.     

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.     

Cloning and sequencing of the Clostridium perfringens enterotoxin gene

Several gene banks of Clostridium perfringens in E. coli were constructed. Using a mixture of synthetic 29-mer DNA probes clones were selected containing inserts from the C. perfringens gene coding for the enterotoxin. This has allowed sequencing of the complete gene and its flanking regions. The decuded amino acid sequence (320 a.a.) was found to differ at several sites from the sequence published previously by others. Two 40-mer DNA-probes were used to detect the toxin gene in C. perfringens strains isolated from the faeces of different non-symptomatic animals. Only 6% of the strains were found to possess the gene.     

Noninvasive Analysis of Microbiome Dynamics in the Fruit Fly Drosophila melanogaster

The diversity and structure of the intestinal microbial community has a strong influence on life history. To understand how hosts and microbes interact, model organisms with comparatively simple microbial communities, such as the fruit fly (Drosophila melanogaster), offer key advantages. However, studies of the Drosophila microbiome are limited to a single point in time, because flies are typically sacrificed for DNA extraction. In order to test whether noninvasive approaches, such as sampling of fly feces, could be a means to assess fly-associated communities over time on the same cohort of flies, we compared the microbial communities of fly feces, dissected fly intestines, and whole flies across three different Drosophila strains. Bacterial species identified in either whole flies or isolated intestines were reproducibly found in feces samples. Although the bacterial communities of feces and intestinal samples were not identical, they shared similarities and obviously the same origin. In contrast to material from whole flies and intestines, feces samples were not compromised by Wolbachia spp. infections, which are widespread in laboratory and wild strains. In a proof-of-principle experiment, we showed that simple nutritional interventions, such as a high-fat diet or short-term starvation, had drastic and long-lasting effects on the micobiome. Thus, the analysis of feces can supplement the toolbox for microbiome studies in Drosophila, unleashing the full potential of such studies in time course experiments where multiple samples from single populations are obtained during aging, development, or experimental manipulations.     

A survey of zoo aviaries for the presence of Histoplasma capsulatum and Cryptococcus neoformans

A zoo's aviaries were surveyed for the presence ofH. capsulatum andC. neoformans. Dried feces samples were collected in the 14 aviaries and tested forC. neoformans by using direct Cultural methods. This pathogen was isolated from an indoor aviary housing one White-breasted Toucan, and from another indoor aviary housing two Concave-casqued Hornbills. Soil samples were collected in 12 outdoor aviaries and two other areas of the zoo premises and tested forH. capsulatum andC. neoformans by direct and indirect cultural methods. Neither fungal pathogen was isolated from the soil samples. Fresh feces samples were collected from the two aviaries whereC. neoformans had been isolated in dried feces. Tests for the presence ofC. neoformans were made by direct culture methods, however the organism was not isolated.Contribution No. 154, Department of Infectious Diseases, College of Veterinary Medicine, Agricultural Experiment Station, Manhattan, 66502.     

Clostridium perfringens type A cytotoxic-enterotoxin(s) as triggers for death in the sudden infant death syndrome: Development of a toxico-infection hypothesis

In our studies with the pathogenic bacteriumClostridium perfringens type A and its cytotoxic-enterotoxins (CTEs), we have obtained results that imply an involvement of this organism in the sudden infant death syndrome (SIDS). In fecal samples obtained from SIDS infants (n=164) and non-SIDS infants (n=57),C. perfringens type A was present in high numbers in >80% of SIDS and <2% of control non-SIDS cases respectively. Fecal samples from SIDS infants analyzed by ELISA forC. perfringens type A CTEs showed a very strong positive correlation with the presence of the organism. Histopathological examination of ileal tissue from SIDS infants showed remarkable similarity to tissue from animal models affected byC. perfringens type A CTEs, where the patterns of damage were positively correlated with the age of the animal. We propose that systemic distribution of the CTEs acts parasympathomimetically to trigger a biochemical cascade that alters cardiorespiratory control. Death may subsequently ensue in an immunologically vulnerable infant.Florida Agricultural Experiment Station Journal Series No. R-02419.     

Inhibition of Clostridium botulinum by Strains of Clostridium perfringens Isolated from Soil

Thirty-one soil samples were examined for the presence of organisms capable of inhibiting growth and toxin production of strains of Clostridium botulinum type A. Such organisms were found in eight samples of soil. Inhibiting strains of C. perfringens were found in five samples, of C. sporogenes in three and of Bacillus cereus in three. Three of the C. perfringens strains produced an inhibitor effective on all 11 strains of C. botulinum type A against which they were tested, seven of eight proteolytic type B strains, one nonproteolytic type B strain, five of nine type E strains and all seven type F strains, whether proteolytic or nonproteolytic. They did not inhibit any of 26 type C strains, 6 type D strains, 4 type E strains, or 24 C. sporogenes strains. In mixed culture, an inhibitor strain of C. perfringens repressed growth and toxin production by a C. botulinum type A strain even though it was outnumbered by the latter about 40 times. It also repressed growth and toxin production of C. botulinum in mixed culture of soils in which this latter organism naturally occurred when cooked meat medium but not when trypticase medium was used.     

Clostridium tetani in a Metropolitan Area: A Limited Survey Incorporating a Simplified in Vitro Identification Test

In a limited survey, three toxigenic and one nontoxigenic strains of Clostridium tetani were isolated from 18 environmental samples from metropolitan Boston. No C. tetani was found in 100 samples of human feces, 20 samples of dog feces, and two samples of horse feces. A simple modification of the halo precipitin test was studied in conjunction with the mouse lethality test for tetanus toxigenicity and was found to be a useful, although not a wholly definitive, technique.     

Polymerase chain reaction-denaturing gradient gel electrophoresis analysis of bacterial community structure in the food,intestines, and feces of earthworms

The bacterial communities in the food, intestines, and feces of earthworms were investigated by PCR-denaturing Gradient gel electrophoresis (DGGE). In this study, PCR-DGGE was optimized by testing 6 universal primer sets for microbial 16S rRNA in 6 pure culture strains of intestinal microbes in earthworms. One primer set effectively amplified 16S rRNA from bacterial populations that were found in the food, intestines, and feces of earthworms. Compared with the reference markers from the pure culture strains, the resulting DGGE profiles contained 28 unique DNA fragments. The dominant microorganisms in the food, intestines, and feces of earthworms included Rhodobacterales bacterium, Fusobacteria, Ferrimonas marina, Aeromonas popoffii, and soil bacteria. Other straisn, such as Acinetobacter, Clostridium, and Veillonella, as well as rumen bacteria and uncultured bacteria also were present. These results demonstrated that PCR-DGGE analysis can be used to elucidate bacterial diversity and identify unculturable microorganisms.     

Identification and Characteristics of Nisin Z-Producing <Emphasis Type="Italic">Lactococcus lactis</Emphasis> subsp. <Emphasis Type="Italic">lactis</Emphasis> Isolated from Kimchi

We isolated bacteriocin-producing Lactococcus lactis subsp. lactis from Kimchi. The bacteriocin inhibited strains of Clostridium perfringens, C. difficile, Listeria monocytogenes, vancomycin-resistant Enterococcus, and one out of four methicillin-resistant Staphylococcus aureus strains, as well as some closely related lactic acid bacteria. In tricine-SDS-PAGE, the bacteriocin migrated with an apparent molecular weight of about 4 kDa to the same location as nisin A and crude nisin Z. The gene encoding this bacteriocin was found to be identical to that of nisin Z with direct PCR sequence methods. The inhibitory activity was stable against heat and pH, but it was lost at 100°C for 1 h and at 121°C for 15 min. The bacteriocin was inactivated by proteolytic enzymes, but was not affected by lysozyme, lipase, catalase, or β-glucosidase. There were some differences in characteristics from those of nisins described previously. Received: 21 June 2002 / Accepted: 22 July 2002     

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.     

Genotyping of isolates of Clostridium perfringens from vaccinated and unvaccinated sheep

Enterotoxaemia vaccine is a polyvalence vaccine from different types of Clostridium perfringens, and C. septicum toxins that prevents various diseases, the most important one being enterotoxaemia in sheep. The aim of the present study was to evaluate the enterotoxaemia vaccine effects in reducing isolates of intestinal clostridia genus specifically C. perfringens. Sheep dung samples were randomly collected from 10 places in Kerman, Iran. The samples were taken from 90 vaccinated Kermani sheep against enterotoxaemia and 50 unvaccinated sheep of same age from flocks with similar management. Following processing and culture of the samples, colonies were identified applying morphological, gram stain and biochemical tests. Using these tests C. perfringens were isolated from 27 out of 50 unvaccinated sheep (54.0%) and from 2 out of 90 vaccinated sheep (2.2%). All of the clostridia isolates were analyzed by multiplex PCR. Genotyping of 2 strains isolated from the vaccinated sheep indicated that these strains were type D, while the strains isolated from the unvaccinated sheep were types A, B, C and D; 14.8% (4 out of 27), 22.2% (6 out of 27), 40.7% (11 out of 27) and 22.2% (6 out of 27), respectively. However, no isolate containing the iota gene (type E) was detected. Vaccination against enterotoxaemia had a significant effect (P < 0.01) on reducing C. perfringens isolates. Occurrence of the disease in the vaccinated and unvaccinated groups was 3.3% and 64.0% (P < 0.01), respectively.     

Plasmids in Vibrio parahemolyticus Strains Isolated in Japan and Bangladesh with Special Reference to Different Distributions

We surveyed plasmids in naturally occurring Vibrio parahemolyticus strains isolated in Japan and Bangladesh. Among the strains isolated in Japan, about half of the strains isolated from stools of patients of domestic diarrhea outbreaks as well as of travelers returning from East Asia were found to have plasmids, but no strains from foods had plasmids. In contrast, among the strains isolated in Bangladesh, none of the four strains isolated from patients had plasmids, but two out of eight strains isolated from water had plasmids, suggesting that plasmids are common in strains from the water in Bangladesh. All plasmids so far reported in V. parahemolyticus were detected in strains isolated from stools of patients. Incidences of plasmids in this organism were not so high in either area. In Japan, all plasmids were detected in strains from human intestines at 37 C, but in Bangladesh, where the temperature is around 30–40 C, the plasmids were detected in strains from the natural environment. These results suggested the possibility that these plasmids can come from different bacteria under rather high temperatures and that incidences of plasmids are influenced by the incidences of plasmids in bacteria present in the vicinity of V. parahemolyticus strains. None of these plasmids were found to have any relation to the biological characters tested.