Complete sequencing and diversity analysis of the enterotoxin-encoding plasmids in Clostridium perfringens type A non-food-borne human gastrointestinal disease isolates

Enterotoxin-producing Clostridium perfringens type A isolates are an important cause of food poisoning and non-food-borne human gastrointestinal diseases, e.g., sporadic diarrhea (SPOR) and antibiotic-associated diarrhea (AAD). The enterotoxin gene (cpe) is usually chromosomal in food poisoning isolates but plasmid-borne in AAD/SPOR isolates. Previous studies determined that type A SPOR isolate F5603 has a plasmid (pCPF5603) carrying cpe, IS1151, and the beta2 toxin gene (cpb2), while type A SPOR isolate F4969 has a plasmid (pCPF4969) lacking cpb2 and IS1151 but carrying cpe and IS1470-like sequences. By completely sequencing these two cpe plasmids, the current study identified pCPF5603 as a 75.3-kb plasmid carrying 73 open reading frames (ORFs) and pCPF4969 as a 70.5-kb plasmid carrying 62 ORFs. These plasmids share an approximately 35-kb conserved region that potentially encodes virulence factors and carries ORFs found on the conjugative transposon Tn916. The 34.5-kb pCPF4969 variable region contains ORFs that putatively encode two bacteriocins and a two-component regulator similar to VirR/VirS, while the approximately 43.6-kb pCPF5603 variable region contains a functional cpb2 gene and several metabolic genes. Diversity studies indicated that other type A plasmid cpe+/IS1151 SPOR/AAD isolates carry a pCPF5603-like plasmid, while other type A plasmid cpe+/IS1470-like SPOR/AAD isolates carry a pCPF4969-like plasmid. Tn916-related ORFs similar to those in pCPF4969 (known to transfer conjugatively) were detected in the cpe plasmids of other type A SPOR/AAD isolates, as well as in representative C. perfringens type B to D isolates carrying other virulence plasmids, possibly suggesting that most or all C. perfringens virulence plasmids transfer conjugatively.     

Epsilon-toxin plasmids of Clostridium perfringens type D are conjugative

Isolates of Clostridium perfringens type D produce the potent epsilon-toxin (a CDC/U.S. Department of Agriculture overlap class B select agent) and are responsible for several economically significant enterotoxemias of domestic livestock. It is well established that the epsilon-toxin structural gene, etx, occurs on large plasmids. We show here that at least two of these plasmids are conjugative. The etx gene on these plasmids was insertionally inactivated using a chloramphenicol resistance cassette to phenotypically tag the plasmid. High-frequency conjugative transfer of the tagged plasmids into the C. perfringens type A strain JIR325 was demonstrated, and the resultant transconjugants were shown to act as donors in subsequent mating experiments. We also demonstrated the transfer of "unmarked" native epsilon-toxin plasmids into strain JIR325 by exploiting the high transfer frequency. The transconjugants isolated in these experiments expressed functional epsilon-toxin since their supernatants had cytopathic effects on MDCK cells and were toxic in mice. Using the widely accepted multiplex PCR approach for toxin genotyping, these type A-derived transconjugants were genotypically type D. These findings have significant implications for the C. perfringens typing system since it is based on the toxin profile of each strain. Our study demonstrated the fluid nature of the toxinotypes and their dependence upon the presence or absence of toxin plasmids, some of which have for the first time been shown to be conjugative.     

Detection of the etx gene (ε-toxin inducer) in plasmids of high molecular weight in Clostridium perfringens type D

The purpose of this work is to correlate the production of epsilon-toxin in a set of strains of Clostridium perfringens type D with the presence of the etx gene, either genomic or in plasmids. Total DNA obtained from strains with a different level of toxin production was explored by PCR and all the strains showed the amplification signal. Different methods were used to obtain plasmid profiles and all of the bands were assayed by PCR. The detection of the etx gene was only shown in several high molecular plasmids. These results were confirmed by a Southern blot. We suggest that the localization of the etx gene in different plasmids could be associated with the epsilon-toxin production level.     

Genotyping of Clostridium perfringens isolated from calves with neonatal diarrhea

Neonatal diarrhea is one of the main causes of losses in cattle herds. Clostridium perfringens is a widespread enteropathogen, and is responsible for many animal diseases such as bovine neonatal diarrhea. Fecal samples from 141 diarrheic calves and 129 healthy calves, aged up to 28 days and belonging to three herds were examined. Rates of culture positivity were 36.2% and 30.2% for diarrheic and nondiarrheic calves, respectively. Multiple isolates from primary isolation plates were subjected to simultaneous genotyping by multiplex PCR, with primers amplifying fragments of alpha (cpa), beta (cpb), epsilon (etx), iota (itxA), enterotoxin (cpe) and beta2 (cpb2) toxin-encoding genes. Only 17/51 (33.3%) and 17/39 (43.6%) of these mixtures from diarrheic and nondiarrheic calves, respectively, yielded genotype information, suggesting that this may not be a viable approach to genotyping of isolates. Fourteen isolate mixtures from animals with diarrhea had only cpa (type A), one had cpa and cpb2 (type A beta2 positive), one with cpa, itxA, and cpb2 (type E, beta2 positive), and one with cpa, etx, itxA, and cpb2 toxin producing strains. Among 17 isolate mixtures from healthy calves, 10 were exclusively type A, one was type A cpb2 positive, two were type E, three were type E cpb2 positive, and one was types D and E cpb2 positive. There was no correlation between isolation of a given toxin type and the presence of diarrhea.     

Molecular typing of Clostridium perfringens isolated from turkey meat by multiplex PCR

Aims:  To determine the presence of toxin genes in 22 Clostridium perfringens isolated from turkey meat samples by molecular typing.
Methods and Results:  For this purpose, alpha ( cpa ), beta ( cpb ), beta 2 ( cpb2 ), epsilon ( etx ), iota ( iA ) and enterotoxin ( cpe ) toxin genes were analysed by multiplex PCR. All 22 turkey meat Cl. perfringens isolates were found to carry the cpa , gene but in none of the isolates cpb , etx, iap or cpe genes were detected. Results showed that all isolates represented type A and were cpe negative.
Conclusions:  Our results indicate that Cl. perfringens type A is the most common type in turkey meat. Also multiplex PCR is effective and rapid method for typing of Cl. perfringens .
Significance and Impact of the Study:  It is the first study about molecular typing of Cl. perfringens using multiplex PCR in turkey meat samples in Turkey.     

Complete DNA sequence and gene analysis of the virulence plasmid pCP301 of Shigella flexneri 2a

The complete nucleotide sequence and organization of the large virulence plasmid pCP301 (termed by us) of Shigella flexneri 2a strain 301 were determined and analyzed. The result showed that the entire DNA sequence of pCP301 is composed of 221618 bp which form a circular plasmid. Sequence analysis identified 272 open reading frames (ORFs), among which, 194 correspond to the proteins described previously, 61 have low identity (<60%) to known proteins and the rest 17 have no regions of significant homology with proteins in database. The genes of pCP301 mainly include the genes associated with bacterial virulence, the genes associated with regulation and the genes relating to plasmid maintenance, stability and DNA metabolism. Insertion sequence (IS) elements are 68 kb in length and account for 30 percent of complete sequence of the plasmid which indicates that gene multiple rearrangements of the pCP301 have taken place in Shigella flexneri evolution history. The research result is helpful for interpreting the pathogenesis of Shigella, as well as the genetics and evolution of the plasmid.     

Prevalence and Characterization of nim Genes of Bacteroides spp. Isolated in Hungary

Members of the Bacteroides spp. are the anaerobic pathogens most commonly isolated from human clinical material. Metronidazole has been the drug of choice for the prevention and treatment of Bacteroides and other such anaerobic infections for nearly 40 years. Although the emergence of metronidazole-resistance in Bacteroides spp. was reported recently, the published rate remains low, at <5%. Isolates of Bacteroides spp. originating from different wards at the University Hospital of Szeged in Hungary were investigated for metronidazole resistance genes by PCR and characterized by other molecular methods. We report here the first strains of Bacteroides spp. with elevated MICs to metronidazole from Hungary. Of 167 isolates, four had metronidazole MICs >2 μg/mL, and two were positive fornim genes with metronidazole MICs of 4 and 12 μg/mL. The nim genes were determined to be nim A and nim B, and proved to be activated by upstream copies of the IS1186 element. The nim A gene was localized in a pIP417-like plasmid, and the nim B gene on the chromosome.     

Comparative Experiments To Examine the Effects of Heating on Vegetative Cells and Spores of Clostridium perfringens Isolates Carrying Plasmid Genes versus Chromosomal Enterotoxin Genes

Clostridium perfringens enterotoxin (CPE) is an important virulence factor for both C. perfringens type A food poisoning and several non-food-borne human gastrointestinal diseases. Recent studies have indicated that C. perfringens isolates associated with food poisoning carry a chromosomal cpe gene, while non-food-borne human gastrointestinal disease isolates carry a plasmid cpe gene. However, no explanation has been provided for the strong associations between certain cpe genotypes and particular CPE-associated diseases. Since C. perfringens food poisoning usually involves cooked meat products, we hypothesized that chromosomal cpe isolates are so strongly associated with food poisoning because (i) they are more heat resistant than plasmid cpe isolates, (ii) heating induces loss of the cpe plasmid, or (iii) heating induces migration of the plasmid cpe gene to the chromosome. When we tested these hypotheses, vegetative cells of chromosomal cpe isolates were found to exhibit, on average approximately twofold-higher decimal reduction values (D values) at 55°C than vegetative cells of plasmid cpe isolates exhibited. Furthermore, the spores of chromosomal cpe isolates had, on average, approximately 60-fold-higher D values at 100°C than the spores of plasmid cpe isolates had. Southern hybridization and CPE Western blot analyses demonstrated that all survivors of heating retained their cpe gene in its original plasmid or chromosomal location and could still express CPE. These results suggest that chromosomal cpe isolates are strongly associated with food poisoning, at least in part, because their cells and spores possess a high degree of heat resistance, which should enhance their survival in incompletely cooked or inadequately warmed foods.     

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.     

Emergence of OXA-48-Producing Klebsiella pneumoniae in Taiwan

The isolation of OXA-48-producing Enterobacteriaceae has increased dramatically in Mediterranean countries in the past 10 years, and has recently emerged in Asia. Between January 2012 and May 2014, a total of 760 carbapenem non-susceptible Klebsiella pneumoniae (CnSKP) isolates were collected during a Taiwan national surveillance. Carbapenemases were detected in 210 CnSKP isolates (27.6%), including 162 KPC-2 (n = 1), KPC-3, KPC-17, and NDM-1 (n = 1 each), OXA-48 (n = 4), IMP-8 (n = 18), and VIM-1 (n = 24). The four bla _OXA-48 CnSKP isolates were detected in late 2013. Herein we report the emergence OXA-48-producing K. pneumoniae isolates in Taiwan. PFGE analysis revealed that the four isolates belonged to three different pulsotypes. Three isolates harboured bla _CTX-M genes and belonged to MLST type ST11. In addition, the plasmids belonged to the incompatibility group, IncA/C. One isolate belonged to ST116 and the plasmid incompatibility group was non-typeable. The sequence upstream of the bla _OXA-48 gene in all four isolates was identical to pKP_OXA-48N1, a bla _OXA-48-carrying plasmid. This is the first report of OXA-48-producing Enterobacteriaceae in Taiwan and the second report to identify bla _OXA-48 on an IncA/C plasmid in K. pneumoniae. Given that three isolates belong to the same pandemic clone (ST11) and possess the IncA/C plasmid and similar plasmid digestion profile that indicated the role of clonal spread or plasmid for dissemination of bla _OXA-48 gene, the emergence of OXA-48-producing K. pneumoniae in Taiwan is of great concern.     

Complete DNA sequence and gene analysis of the virulence plasmid pCP301 of Shigella flexneri 2a

Bacteria Shigella spp. are highly contagious, severely harmful and gram-negative facultative intracellular pathogens. They may cause shigellosis characterized by fever, dehydration and hematochezia in clinic, and shigellosis has been remaining a leading cause of infant mortality in the world. Shigella belongs to the family Enterobacteriaceae and the group Escherichiaeae, which are divided into four species and at least 47 serotypes: Shigella dysenteriae (13 serotypes), Shigella flexneri (15 …     

Further Comparison of Temperature Effects on Growth and Survival of Clostridium perfringens Type A Isolates Carrying a Chromosomal or Plasmid-Borne Enterotoxin Gene

Clostridium perfringens type A isolates can carry the enterotoxin gene (cpe) on either their chromosome or a plasmid, but food poisoning isolates usually have a chromosomal cpe gene. This linkage between chromosomal cpe isolates and food poisoning has previously been attributed, at least in part, to better high-temperature survival of chromosomal cpe isolates than of plasmid cpe isolates. In the current study we assessed whether vegetative cells and spores of chromosomal cpe isolates also survive better than vegetative cells and spores of plasmid cpe isolates survive when the vegetative cells and spores are subjected to low temperatures. Vegetative cells of chromosomal cpe isolates exhibited about eightfold-higher decimal reduction values (D values) at 4°C and threefold-higher D values at −20°C than vegetative cells of plasmid cpe isolates exhibited. After 6 months of incubation at 4°C and −20°C, the average log reductions in viability for spores of plasmid cpe isolates were about fourfold and about threefold greater, respectively, than the average log reductions in viability for spores from chromosomal cpe isolates. C. perfringens type A isolates carrying a chromosomal cpe gene also grew significantly faster than plasmid cpe isolates grew at 25°C, 37°C, or 43°C. In addition, chromosomal cpe isolates grew at higher maximum and lower minimum temperatures than plasmid cpe isolates grew. Collectively, these results suggest that chromosomal cpe isolates are commonly involved in food poisoning because of their greater resistance to low (as well as high) temperatures for both survival and growth. They also indicate the importance of proper low-temperature storage conditions, as well as heating, for prevention of C. perfringens type A food poisoning.     

Prevalence of enterotoxigenic Clostridium perfringens Isolates in Pittsburgh (Pennsylvania) area soils and home kitchens

In the United States and Europe, food poisoning due to Clostridium perfringens type A is predominantly caused by C. perfringens isolates carrying a chromosomal enterotoxin gene (cpe). Neither the reservoir for these isolates nor the point in the food chain where these bacteria contaminate foods is currently understood. Therefore, the current study investigated whether type A isolates carrying a chromosomal cpe gene are present in two potential reservoirs, i.e., soil and home kitchen surfaces. No C. perfringens isolates were recovered from home kitchen surfaces, but most surveyed soil samples contained C. perfringens. The recovered soil isolates were predominantly type A, but some type C, D, and E soil isolates were also identified. All cpe-positive isolates recovered from soil were genotyped as type A, with their cpe genes on cpe plasmids rather than the chromosome. However, two cpe-positive soil isolates did not carry a classical cpe plasmid. Both of those atypical cpe-positive soil isolates were sporulation capable yet failed to produce C. perfringens enterotoxin, possibly because of differences in their upstream promoter regions. Collectively these results suggest that neither soil nor home kitchen surfaces represent major reservoirs for type A isolates with chromosomal cpe that cause food poisoning, although soil does appear to be a reservoir for cpe-positive isolates causing non-food-borne gastrointestinal diseases.     

Complete DNA sequence and gene analysis of the virulence plasmid pCP301 of <Emphasis Type="Italic">Shigella flexneri</Emphasis> 2a

The complete nucleotide sequence and organization of the large virulence plasmid pCP301 (termed by us) of Shigella flexneri 2a strain 301 were determined and analyzed. The result showed that the entire DNA sequence of pCP301 is composed of 221618 bp which form a circular plasmid. Sequence analysis identified 272 open reading frames (ORFs), among which, 194 correspond to the proteins described previously, 61 have low identity (<60%) to known proteins and the rest 17 have no regions of significant homology with proteins in database. The genes of pCP301 mainly include the genes associated with bacterial virulence, the genes associated with regulation and the genes relating to plasmid maintenance, stability and DNA metabolism. Insertion sequence (IS) elements are 68 kb in length and account for 30 percent of complete sequence of the plasmid which indicates that gene multiple rearrangements of the pCP301 have taken place in Shigella flexneri evolution history. The research result is helpful for interpreting the pathogenesis of Shigella, as well as the genetics and evolution of the plasmid.     

Comparative Effects of Osmotic, Sodium Nitrite-Induced, and pH-Induced Stress on Growth and Survival of Clostridium perfringens Type A Isolates Carrying Chromosomal or Plasmid-Borne Enterotoxin Genes

About 1 to 2% of Clostridium perfringens isolates carry the enterotoxin gene (cpe) necessary for causing C. perfringens type A food poisoning. While the cpe gene can be either chromosomal or plasmid borne, food poisoning isolates usually carry a chromosomal cpe gene. Previous studies have linked this association between chromosomal cpe isolates (i.e., C-cpe isolates) and food poisoning, at least in part, to both the spores and vegetative cells of C-cpe isolates being particularly resistant to high and low temperatures. The current study now reveals that the resistance phenotype of C-cpe isolates extends beyond temperature resistance to also include, for both vegetative cells and spores, enhanced resistance to osmotic stress (from NaCl) and nitrites. However, by omitting one outlier isolate, no significant differences in pH sensitivity were detected between the spores or vegetative cells of C-cpe isolates versus isolates carrying a plasmid-borne cpe gene. These results indicate that both vegetative cells and spores of C-cpe isolates are unusually resistant to several food preservation approaches in addition to temperature extremes. The broad-spectrum nature of the C-cpe resistance phenotype suggests these bacteria may employ multiple mechanisms to persist and grow in foods prior to their transmission to humans.     

Comparative genomics and phylogeny of the IncI1 plasmids: a common plasmid type among porcine enterotoxigenic Escherichia coli

Increasing reports of multidrug resistance conferred by conjugative plasmids of Enterobacteriaceae necessitate a better understanding of their evolution. One such group is the narrow-host-range IncI1 plasmid type, known for their ability to carry genes encoding resistance to extended-spectrum beta lactamases. The focus of this study was to perform comparative sequencing of IncI1 plasmids from porcine enterotoxigenic Escherichia coli (ETEC), isolated irrespective of antimicrobial susceptibility phenotype. Five IncI1 plasmids of porcine ETEC origin and one IncI1 plasmid from a Salmonella enterica serovar Kentucky isolate from a healthy broiler chicken were sequenced and compared to existing IncI1 plasmid sequences in an effort to better understand the overall genetic composition of the IncI1 plasmid lineages. Overall, the sequenced porcine ETEC IncI1 plasmids were divergent from other sequenced IncI1 plasmids based upon multiple means of inferred phylogeny. High occurrences of IncI1 and IncA/C plasmid-associated genes and the bla_TEM and bla_CMY-2 beta lactamase genes were observed among porcine ETEC. However, the presence of bla_TEM and bla_CMY-2 did not strongly correlate with IncI1 plasmid possession, suggesting that these plasmids in porcine ETEC are not primarily associated with the carriage of such resistance genes. Overall, this work suggests a conservation of the IncI1 plasmid backbone among sequenced plasmids with a single locus for the acquisition of accessory genes, such as those associated with antimicrobial resistance. Furthermore, the high occurrence of IncI1 and IncA/C plasmids among clinical E. coli from commercial swine facilities is indicative of extensive horizontal gene transfer among porcine ETEC.     

Multiplex PCR assay for toxinotyping Clostridium perfringens isolates obtained from Finnish broiler chickens

AIMS: The aim of the study was to determine the presence of genes coding for alpha (cpa), beta (cpb), epsilon (etx), iota (iA) and enterotoxin (cpe) from Clostridium perfringens broiler chicken isolates, using multiplex PCR assay established in the study. METHODS AND RESULTS: The multiplex PCR assay was shown to be specific when tested with 10 C. perfringens strains representing different toxin types, and 15 strains of other bacterial species. All 118 broiler chicken C. perfringens isolates were shown to carry the cpa gene but not cpb, etx, iap or cpe genes, signifying that all isolates represented type A and were cpe-negative. CONCLUSIONS: The assay established in the study enables the simultaneous detection of the major toxin genes and the cpe gene from C. perfringens isolates. SIGNIFICANCE AND IMPACT OF THE STUDY: The present study offers a new primer pair for detecting cpa, combined with a multiplex PCR assay. In addition, the study provides data of the presence of different toxin genes in C. perfringens isolates obtained from broiler chickens.     

Cloning of the determinants for microcin D93 production and analysis of three different D-type microcin plasmids

Three different microcin plasmids coding for D-type microcins were analyzed. Two of the plasmids (pMccD93 and pCP101) were small, multicopy plasmids and were closely related. The third plasmid (pCP106) was a conjugative, antibiotic multiresistance plasmid. Although plasmids pCP101 and pCP106 were previously classified as A-type microcin plasmids, we have determined that they are, in fact, D type. Furthermore, the determinants for microcin D93 production were cloned from plasmid pMccD93, and a DNA probe for the region implicated in the synthesis of microcin was obtained. This probe hybridized to plasmid C from Escherichia coli strain V517, indicating that this plasmid might be involved in the synthesis of a D-type microcin. The characteristics of replication of plasmid pCP106 were analyzed and appeared to be similar to those of ColEl plasmids, although pCP106 is a conjugative single-copy plasmid.