Toxinotyping of necrotic enteritis-producing and commensal isolates of Clostridium perfringens from chickens fed organic diets

The present study determined the effect of Clostridium perfringens isolates taken from necrotic enteritis (NE) outbreaks on organic farms in a NE virulence testing model. Thirteen strains were isolated in the course of the study. Six C. perfringens field isolates were taken from a naturally occurring NE outbreak on an organic farm. Polymerase chain reaction toxinotyping was used to establish C. perfringens strains, as well as to create a toxin profile. All field isolates were found to be type A and positive for alpha, beta-2 and netB toxin genes. During the NE virulence model, digesta samples were collected before oral inoculation to define the C. perfringens found as part of the natural flora. Three of the five natural flora isolates were found to be C. perfringens type E while the other two isolates were type A; only four of five isolates were positive for either netB or beta-2 toxin genes. Two isolates collected after inoculation were C. perfringens type A positive for cpb2 and netB. All isolates were tested positive for the quorum-sensing-related gene luxS, regardless of the strain source. The presence of luxS, alpha, netB and beta-2 toxin genes seems not to be a determinant of the disease as they were present in isolates from both outbreak birds as well as healthy and pre-inoculated birds. The C. perfringens field isolates induced mild NE lesions in one-half of the birds during the challenge study. Other mechanisms must play a role in the development of the disease beyond toxinotype, potentially including intestinal ecology and health, which would account for acute disease as seen in the field outbreak.     

NetB-producing and beta2-producing Clostridium perfringens associated with subclinical necrotic enteritis in laying hens in the Netherlands

Since 2006 increasing numbers of laying hen flocks with decreased production have been reported in the Netherlands. At necropsy, birds from affected flocks showed multifocal areas of necrosis in the duodenum. Histologically the duodenum had moderate to marked villus atrophy and fusion with crypt hyperplasia and a mixed inflammatory infiltrate within the lamina propria underlying focal areas of degenerative epithelium. Multifocally, free within the intestinal lumen and associated with epithelial necrosis, were marked numbers of large rod-shaped bacteria. Anaerobic culturing and subsequent toxin typing revealed, in 19 out of 73 affected birds, the presence of Clostridium perfringens strains, either type A or type C harbouring the atypical allele of cpb2 and netB. Eighteen out of these 19 birds carried C. perfringens strains capable of producing beta2 toxin in vitro and all of these birds harboured C. perfringens strains capable of producing NetB toxin in vitro. In contrast, specific pathogen free (SPF) birds lacked gross or histological lesions in their duodenum, and C. perfringens type C was isolated from four out of 15 SPF birds tested. One of these isolates harboured the consensus three allele of cpb2 that produced beta2 toxin in vitro. None of the C. perfringens isolates originating from SPF birds harboured netB. These findings might indicate that the NetB toxin produced by C. perfringens is associated with subclinical necrotic enteritis in layers, whereas the involvement of beta2 toxin in subclinical necrotic enteritis, if any, might be variant dependent.     

Prevalence of β2-Toxigenic Clostridium perfringens in Horses with Intestinal Disorders

The incidence of a new, yet unassigned toxin type of Clostridium perfringens containing the genes for the α-toxin and the recently described β2-toxin in horses with intestinal disorders is reported. The study included 18 horses suffering from typical typhlocolitis, 7 horses with atypical typhlocolitis, 16 horses with other intestinal disorders, and 58 horses without intestinal disease. In total, 20 samples of ingesta of the small and large intestines, five biopsy specimens of the intestinal wall, and 74 fecal samples were analyzed bacteriologically. C. perfringens isolates were typed for the presence of the α-, β-, β2-, and -toxin and enterotoxin genes by PCR, including a newly developed PCR for the detection of the β2-toxin gene cpb2. β2-Toxigenic C. perfringens was detected in samples from 13 of 25 (52%) horses with typical or atypical typhlocolitis, with a particularly high incidence in specimens of ingesta and biopsy specimens (75%), whereas only 6 of 16 specimens from horses with other intestinal diseases yielded β2-toxigenic C. perfringens. No β2-toxigenic C. perfringens was found in the samples from the 58 control horses, of which only one fecal sample contained C. perfringens type A. Among the samples from the 15 horses with fatal cases of typical and atypical typhlocolitis 9 (60%) were positive for β2-toxigenic C. perfringens, whereas samples from only 4 of the 10 (40%) animals with nonfatal cases of infection were positive. We found an interesting correlation between the antibiotic-treated horses which were positive for β2-toxigenic C. perfringens and lethal progression of the disease. No C. perfringens strains isolated in this study contained genes for the β- and -toxins and enterotoxin. The high incidence of β2-toxigenic C. perfringens in samples of ingesta, biopsy specimens of the intestinal wall, and feces from horses suffering or dying from typhlocolitis together with the absence of this organism in healthy horses provides strong evidence that β2-toxigenic C. perfringens play an important role in the pathogenesis of typhlocolitis.     

Characterization of Clostridium perfringens TpeL Toxin Gene Carriage,Production, Cytotoxic Contributions,and Trypsin Sensitivity

Large clostridial toxins (LCTs) are produced by at least four pathogenic clostridial species, and several LCTs are proven pivotal virulence factors for both human and veterinary diseases. TpeL is a recently identified LCT produced by Clostridium perfringens that has received relatively limited study. In response, the current study surveyed carriage of the tpeL gene among different C. perfringens strains, detecting this toxin gene in some type A, B, and C strains but not in any type D or E strains. This study also determined that all tested strains maximally produce, and extracellularly release, TpeL at the late-log or early-stationary growth stage during in vitro culture, which is different from the maximal late-stationary-phase production reported previously for other LCTs and for TpeL production by C. perfringens strain JIR12688. In addition, the present study found that TpeL levels in culture supernatants can be repressed by either glucose or sucrose. It was also shown that, at natural production levels, TpeL is a significant contributor to the cytotoxic activity of supernatants from cultures of tpeL-positive strain CN3685. Lastly, this study identified TpeL, which presumably is produced in the intestines during diseases caused by TpeL-positive type B and C strains, as a toxin whose cytotoxicity decreases after treatment with trypsin; this finding may have pathophysiologic relevance by suggesting that, like beta toxin, TpeL contributes to type B and C infections in hosts with decreased trypsin levels due to disease, diet, or age.     

Four Foodborne Disease Outbreaks Caused by a New Type of Enterotoxin-Producing Clostridium perfringens

The epidemiological and bacteriological investigations on four foodborne outbreaks caused by a new type of enterotoxin-producing Clostridium perfringens are described. C. perfringens isolated from patients of these outbreaks did not produce any known enterotoxin and did not carry the C. perfringens enterotoxin gene. However, the culture filtrates of these isolates induced the accumulation of fluid in rabbit ileal loop tests. The molecular weight of the new enterotoxin may be between 50,000 and 100,000, although the known C. perfringens enterotoxin is ca. 35,000. This new enterotoxin was heat labile, and its biological activities were inactivated by heating for 5 min at 60°C. The new enterotoxin was sensitive to pH values higher than 11.0 and protease treatment but was resistant to trypsin treatment. These results suggest that the new enterotoxin may be a protein. Although C. perfringens enterotoxin induced morphological changes in Vero cells, the changes induced by the new enterotoxin differed from those by the known C. perfringens enterotoxin. The new enterotoxin also induced morphological changes in L929 cells, whereas the known C. perfringens enterotoxin did not, because L929 cells lacked an appropriate enterotoxin receptor. Although C. perfringens enterotoxin is recognized as the only diarrheagenic toxin responsible for C. perfringens foodborne outbreaks, the results of the present study indicate that C. perfringens isolated from these four outbreaks produced a new type of enterotoxin.     

A chicken intestinal ligated loop model to study the virulence of Clostridium perfringens isolates recovered from antibiotic-free chicken flocks

Necrotic enteritis (NE) is a major problem in antibiotic-free (ABF) chicken flocks and specific strains of Clostridium perfringens are known to induce NE. The objective of this study was to develop a chicken intestinal ligated loop model in order to compare the virulence of various C. perfringens strains recovered from consecutive ABF flocks with and without NE. Intestinal loops were surgically prepared in 10 anaesthetized specific-pathogen-free chickens and alternately inoculated with C. perfringens isolates or brain heart infusion (BHI) media. Histological lesion scoring was performed for each loop. All strains from NE-affected flocks induced histological lesions compatible with NE whereas inoculation of loops with a commensal C. perfringens strain or BHI did not. Among inoculated strains, CP0994 (netB-positive and cpb2-positive) and CP-2003-1256 (netB-positive) demonstrated mean histological lesion scores significantly higher (P?netB-negative and cpb2-positive) induced intestinal lesions without significantly higher scores. In loops where villi were colonized by Gram-positive rods, significantly higher (P?C. perfringens is a critical step in the pathogenesis of NE. Finally, we demonstrated the importance of controlling virulent C. perfringens strains in ABF chicken flocks as a highly virulent strain can be present in consecutive flocks with NE and possibly affect multiple flocks.     

Clostridium perfringens tpeL is expressed during sporulation

Clostridium perfringens TpeL belongs to the family of large clostridial toxins. Although recent studies demonstrated the expression of tpeL during vegetative growth nothing is known about tpeL expression during sporulation. Our current study demonstrated that tpeL: (i) is also expressed during sporulation; (ii) expression is dependent on the master regulator of sporulation, Spo0A, and the sporulation-specific sigma factor, SigE; and (iii) is localized to the mother cell compartment of the sporulating cell. In summary, these results indicate that TpeL is a sporulation-regulated C. perfringens toxin.     

Characterization of Toxin Plasmids in Clostridium perfringens Type C Isolates

Clostridium perfringens type C isolates cause enteritis necroticans in humans or necrotizing enteritis and enterotoxemia in domestic animals. Type C isolates always produce alpha toxin and beta toxin but often produce additional toxins, e.g., beta2 toxin or enterotoxin. Since plasmid carriage of toxin-encoding genes has not been systematically investigated for type C isolates, the current study used Southern blot hybridization of pulsed-field gels to test whether several toxin genes are plasmid borne among a collection of type C isolates. Those analyses revealed that the surveyed type C isolates carry their beta toxin-encoding gene (cpb) on plasmids ranging in size from ∼65 to ∼110 kb. When present in these type C isolates, the beta2 toxin gene localized to plasmids distinct from the cpb plasmid. However, some enterotoxin-positive type C isolates appeared to carry their enterotoxin-encoding cpe gene on a cpb plasmid. The tpeL gene encoding the large clostridial cytotoxin was localized to the cpb plasmids of some cpe-negative type C isolates. The cpb plasmids in most surveyed isolates were found to carry both IS1151 sequences and the tcp genes, which can mediate conjugative C. perfringens plasmid transfer. A dcm gene, which is often present near C. perfringens plasmid-borne toxin genes, was identified upstream of the cpb gene in many type C isolates. Overlapping PCR analyses suggested that the toxin-encoding plasmids of the surveyed type C isolates differ from the cpe plasmids of type A isolates. These findings provide new insight into plasmids of proven or potential importance for type C virulence.Clostridium perfringens isolates are classified into five toxinotypes (A to E) based upon the production of four (α, β, ɛ, and ι) typing toxins (29). Each toxinotype is associated with different diseases affecting humans or animals (25). In livestock species, C. perfringens type C isolates cause fatal necrotizing enteritis and enterotoxemia, where toxins produced in the intestines absorb into the circulation to damage internal organs. Type C-mediated animal diseases result in serious economic losses for agriculture (25). In humans, type C isolates cause enteritis necroticans, which is also known as pigbel or Darmbrand (15, 17), an often fatal disease that involves vomiting, diarrhea, severe abdominal pain, intestinal necrosis, and bloody stools. Acute cases of pigbel, resulting in rapid death, may also involve enterotoxemia (15).By definition, type C isolates must produce alpha and beta toxins (24, 29). Alpha toxin, a 43-kDa protein encoded by the chromosomal plc gene, has phospholipase C, sphingomyelinase, and lethal properties (36). Beta toxin, a 35-kDa polypeptide, forms pores that lyse susceptible cells (28, 35). Recent studies demonstrated that beta toxin is necessary for both the necrotizing enteritis and lethal enterotoxemia caused by type C isolates (33, 37). Besides alpha and beta toxins, type C isolates also commonly express beta2 toxin, perfringolysin O, or enterotoxin (11).There is growing appreciation that naturally occurring plasmids contribute to both C. perfringens virulence and antibiotic resistance. For example, all typing toxins, except alpha toxin, can be encoded by genes carried on large plasmids (9, 19, 26, 30-32). Other C. perfringens toxins, such as the enterotoxin or beta2 toxin, can also be plasmid encoded (6, 8, 12, 34). Furthermore, conjugative transfer of several C. perfringens antibiotic resistance plasmids or toxin plasmids has been demonstrated, supporting a key role for plasmids in the dissemination of virulence or antibiotic resistance traits in this bacterium (2).Despite their pathogenic importance, the toxin-encoding plasmids of C. perfringens only recently came under intensive study (19, 26, 27, 31, 32). The first carefully analyzed C. perfringens toxin plasmids were two plasmid families carrying the enterotoxin gene (cpe) in type A isolates (6, 8, 12, 26). One of those cpe plasmid families, represented by the ∼75-kb prototype pCPF5603, has an IS1151 sequence present downstream of the cpe gene and also carries the cpb2 gene, encoding beta2 toxin. A second cpe plasmid family of type A isolates, represented by the ∼70-kb prototype pCPF4969, lacks the cpb2 gene and carries an IS1470-like sequence, rather than an IS1151 sequence, downstream of the cpe gene. The pCPF5603 and pCPF4969 plasmid families share an ∼35-kb region that includes transfer of a clostridial plasmid (tcp) locus (26). The presence of this tcp locus likely explains the demonstrated conjugative transfer of some cpe plasmids (5) since a similar tcp locus was shown to mediate conjugative transfer of the C. perfringens tetracycline resistance plasmid pCW3 (2).The iota toxin-encoding plasmids of type E isolates are typically larger (up to ∼135 kb) than cpe plasmids of type A isolates (19). Plasmids carrying iota toxin genes often encode other potential virulence factors, such as lambda toxin and urease, as well as a tcp locus (19). Many iota toxin plasmids of type E isolates share, sometimes extensively, sequences with cpe plasmids of type A isolates (19). It has been suggested that many iota toxin plasmids evolved from the insertion of a mobile genetic element carrying the iota toxin genes near the plasmid-borne cpe gene in a type A isolate, an effect that silenced the cpe gene in many type E isolates (3, 19).Plasmids carrying the epsilon toxin gene (etx) vary from ∼48 kb to ∼110 kb among type D isolates (32). In part, these etx plasmid size variations in type D isolates reflect differences in toxin gene carriage. For example, the small ∼48-kb etx plasmids present in some type D isolates lack both the cpe gene and the cpb2 gene. In contrast, larger etx plasmids present in other type D isolates often carry the cpe gene, the cpb2 gene, or both the cpe and cpb2 genes. Thus, the virulence plasmid diversity of type D isolates spans from carriage of a single toxin plasmid, possessing from one to three distinct toxin genes, to carriage of three different toxin plasmids.In contrast to the variety of etx plasmids found among type D isolates, type B isolates often or always share the same ∼65-kb etx plasmid, which is related to pCPF5603 but lacks the cpe gene (27). This common etx plasmid of type B isolates, which carries a cpb2 gene and the tcp locus, is also present in a few type D isolates. Most type B isolates surveyed to date carry their cpb gene, encoding beta toxin, on an ∼90-kb plasmid, although a few of those type B isolates possess an ∼65-kb cpb plasmid distinct from their ∼65-kb etx plasmid (31).To our knowledge, the cpb gene has been mapped to a plasmid (uncharacterized) in only a single type C strain (16). Furthermore, except for the recent localization of the cpe gene to plasmids in type C strains (20), plasmid carriage of other potential toxin genes in type C isolates has not been investigated. Considering the limited information available regarding the toxin plasmids of type C isolates, our study sought to systematically characterize the size, diversity, and toxin gene carriage of toxin plasmids in a collection of type C isolates. Also, to gain insight into possible mobilization of the cpb gene by insertion sequences or conjugative transfer, the presence of IS1151 sequences or the tcp locus on type C toxin plasmids was investigated.     

A Live Oral Recombinant Salmonella enterica Serovar Typhimurium Vaccine Expressing Clostridium perfringens Antigens Confers Protection against Necrotic Enteritis in Broiler Chickens

Necrotic enteritis (NE) in broiler chickens is caused by Clostridium perfringens, and there is currently no effective vaccine for NE. We previously showed that in broiler chickens protection against NE can be achieved through intramuscular immunization with alpha toxin (AT) and hypothetical protein (HP), and we subsequently identified B-cell epitopes in HP. In the present study, we identified B-cell epitopes in AT recognized by chickens immune to NE. The gene fragments encoding immunodominant epitopes of AT as well as those of HP were codon optimized for Salmonella and cloned into pYA3493, and the resultant plasmid constructs were introduced into an attenuated Salmonella enterica serovar Typhimurium χ9352 vaccine vehicle. The expression of these Clostridium perfringens proteins, alpha toxoid (ATd) and truncated HP (HPt), was confirmed by immunoblotting. The protection of broiler chickens against experimentally induced NE was assessed at both the moderate and the severe levels of challenge. Birds immunized orally with Salmonella expressing ATd were significantly protected against moderate NE, and there was a nonsignificant trend for protection against severe challenge, whereas HPt-immunized birds were significantly protected against both severities of challenge. Immunized birds developed serum IgY and mucosal IgA and IgY antibody responses against Clostridium and Salmonella antigens. In conclusion, this study identified, for the first time, the B-cell epitopes in AT from an NE isolate recognized by chickens and showed the partial protective ability of codon-optimized ATd and HPt against NE in broiler chickens when they were delivered orally by using a Salmonella vaccine vehicle.Necrotic enteritis (NE) in broiler chickens is caused by Clostridium perfringens. This disease poses a resurging threat to the poultry industry in jurisdictions where there has been a ban on the use of antimicrobials for growth promotion, since broiler chickens reared under intensive conditions in the absence of antimicrobial prophylaxis are at risk of NE (12). There has consequently been an increased effort in recent years to understand the pathogenesis of and immunity to NE. Emerging evidence is that there are distinct NE strains in chickens capable of producing disease characterized by the presence of a novel toxin, NetB (14), and possibly by other features, including intraspecies growth inhibition by C. perfringens (29, 33). The toxin historically implicated in the pathogenesis of NE was alpha toxin (AT), a phospholipase C that was also identified as an important immunogen in protection against NE (2, 3, 22). A recent study has shown that AT is not essential in pathogenesis (15). However, the pathogenesis of NE may be more complex than the involvement of NetB rather than AT, since some non-NE isolates can be netB positive and some NE isolates can be netB negative (5, 23).Although immunization appears to offer the best alternative to antimicrobials for the control of NE, no effective active vaccine is available for broiler chickens and understanding of the basis of immunity to NE is incomplete. Recent findings reported from our laboratory have indicated that different selected secreted proteins, including AT, have roles in providing protection against NE, with their efficacies varying with the severity of the challenge (17, 18). Two important immunogens, namely, AT and a hypothetical protein (HP), protected birds immunized intramuscularly against severe experimentally induced NE. Subsequently, a region of HP that contained immunoreactive B-cell epitopes was cloned into an attenuated Salmonella vehicle, and birds immunized orally with this vaccine were shown to be significantly but not completely protected against NE (19), and among the clostridial genes expressed in Salmonella, HP gave the best protection. In that study, use of the plasmid expression vector pYA3342 resulted in the relatively poor expression of the cloned genes. There is evidence that AT is an important protective immunogen in immunity to NE (11, 18, 22), although its effect may be indirect (35), when it is delivered as a toxoid subcutaneously (6) or through an attenuated Salmonella oral vaccine expressing the carboxy-terminal domain (35); the choice of the C-terminal domain was based on its successful use as a protective immunogen in mouse model of gas gangrene (31, 35). Although the immunogenic B-cell epitopes of AT for mice have been identified (21), the B-cell epitopes that are recognized by chickens immune to NE and that may be important for protection have not been characterized.The current study was carried out with two main objectives: (i) to identify the B-cell epitopes in AT by using serum from chickens immune to NE to obtain information about the possible antibody recognition sites and (ii) to clone and express the immunodominant epitopes of AT and of HP (identified in a previous study) in an improved Salmonella antigen expression system and to compare the abilities of these oral vaccine constructs to provide immunity against infection in an experimental NE challenge model at both moderate and severe levels of challenge.     

BEC,a Novel Enterotoxin of Clostridium perfringens Found in Human Clinical Isolates from Acute Gastroenteritis Outbreaks

Clostridium perfringens is a causative agent of food-borne gastroenteritis for which C. perfringens enterotoxin (CPE) has been considered an essential factor. Recently, we experienced two outbreaks of food-borne gastroenteritis in which non-CPE producers of C. perfringens were strongly suspected to be the cause. Here, we report a novel enterotoxin produced by C. perfringens isolates, BEC (binary enterotoxin of C. perfringens). Culture supernatants of the C. perfringens strains showed fluid-accumulating activity in rabbit ileal loop and suckling mouse assays. Purification of the enterotoxic substance in the supernatants and high-throughput sequencing of genomic DNA of the strains revealed BEC, composed of BECa and BECb. BECa and BECb displayed limited amino acid sequence similarity to other binary toxin family members, such as the C. perfringens iota toxin. The becAB genes were located on 54.5-kb pCP13-like plasmids. Recombinant BECb (rBECb) alone had fluid-accumulating activity in the suckling mouse assay. Although rBECa alone did not show enterotoxic activity, rBECa enhanced the enterotoxicity of rBECb when simultaneously administered in suckling mice. The entertoxicity of the mutant in which the becB gene was disrupted was dramatically decreased compared to that of the parental strain. rBECa showed an ADP-ribosylating activity on purified actin. Although we have not directly evaluated whether BECb delivers BECa into cells, rounding of Vero cells occurred only when cells were treated with both rBECa and rBECb. These results suggest that BEC is a novel enterotoxin of C. perfringens distinct from CPE, and that BEC-producing C. perfringens strains can be causative agents of acute gastroenteritis in humans. Additionally, the presence of becAB on nearly identical plasmids in distinct lineages of C. perfringens isolates suggests the involvement of horizontal gene transfer in the acquisition of the toxin genes.     

Characterization of Virulence Plasmid Diversity among Clostridium perfringens Type B Isolates

The important veterinary pathogen Clostridium perfringens type B is unique for producing the two most lethal C. perfringens toxins, i.e., epsilon-toxin and beta-toxin. Our recent study (K. Miyamoto, J. Li, S. Sayeed, S. Akimoto, and B. A. McClane, J. Bacteriol. 190:7178-7188, 2008) showed that most, if not all, type B isolates carry a 65-kb epsilon-toxin-encoding plasmid. However, this epsilon-toxin plasmid did not possess the cpb gene encoding beta-toxin, suggesting that type B isolates carry at least one additional virulence plasmid. Therefore, the current study used Southern blotting of pulsed-field gels to localize the cpb gene to ∼90-kb plasmids in most type B isolates, although a few isolates carried a ∼65-kb cpb plasmid distinct from their etx plasmid. Overlapping PCR analysis then showed that the gene encoding the recently discovered TpeL toxin is located ∼3 kb downstream of the plasmid-borne cpb gene. As shown earlier for their epsilon-toxin-encoding plasmids, the beta-toxin-encoding plasmids of type B isolates were found to carry a tcp locus, suggesting that they are conjugative. Additionally, IS1151-like sequences were identified upstream of the cpb gene in type B isolates. These IS1151-like sequences may mobilize the cpb gene based upon detection of possible cpb-containing circular transposition intermediates. Most type B isolates also possessed a third virulence plasmid that carries genes encoding urease and lambda-toxin. Collectively, these findings suggest that type B isolates are among the most plasmid dependent of all C. perfringens isolates for virulence, as they usually carry three potential virulence plasmids.Isolates of the Gram-positive, spore-forming anaerobe Clostridium perfringens are classified (31) into five different types (A to E), depending upon their production of four (alpha, beta, epsilon, and iota) lethal typing toxins. All C. perfringens types produce alpha-toxin; in addition, type B isolates produce both beta- and epsilon-toxins, type C isolates produce beta-toxin, type D isolates produce epsilon-toxin and type E isolates produce iota-toxin. Except for the chromosomal alpha-toxin gene (plc), all C. perfringens typing toxins are encoded by genes resident on large plasmids (11, 22, 23, 32, 33). Large plasmids can also encode other C. perfringens toxins, such as the enterotoxin (CPE) or beta2-toxin (8, 9, 14, 35), as well as other potential virulence factors such as urease (12, 23).The large virulence plasmids of C. perfringens are only now being characterized (23, 28, 29, 33). The first analyzed, and still most studied, C. perfringens toxin plasmids are the CPE-encoding plasmids of type A isolates (14, 28). In type A isolates, most plasmids carrying the enterotoxin gene (cpe) belong to one of two families: (i) a 75.3-kb plasmid with a cpe locus containing an IS1151 element and the cpb2 gene encoding beta2-toxin or (ii) a 70.5-kb plasmid that lacks the cpb2 gene and carries a cpe locus with an IS1470-like sequence instead of an IS1151 element. Sequence comparisons (28) revealed that these two cpe plasmid families of type A isolates share a conserved region of ∼35 kb that includes the transfer of clostridial plasmid (tcp) locus, which is related to the conjugative transposon Tn916. Confirming that cpe plasmids can be conjugative, mixed mating studies have directly demonstrated transfer of the cpe plasmid from type A isolate F4969 to other C. perfringens isolates (5). A similar tcp locus is also shared by the tetracycline resistance plasmid pCW3 and several other toxin plasmids (2, 23, 28, 29, 33), as discussed below. Mutagenesis analyses demonstrated the importance of several genes in the tcp locus for conjugative transfer of pCW3 (2) and, by extension, presumably the tcp-carrying, conjugative toxin plasmids, such as the cpe plasmid of isolate F4969 (5) and some etx plasmids of type D isolates (19).Although the sequence of an iota-toxin-encoding plasmid has not yet been published, pulsed-field gel electrophoresis (PFGE) and PCR analyses determined that these plasmids are typically larger than the cpe plasmids of type A isolates (23). Specifically, iota-toxin plasmids are often ≥100 kb in size, reaching up to a size of ∼135 kb. These plasmids of type E isolates often encode, in addition to the iota-toxin, other potential virulence factors such as lambda-toxin and urease. These plasmids also carry a tcp locus, suggesting that they may be capable of conjugative transfer. Interestingly, many iota-toxin plasmids appear to be related, sometimes extensively, to the cpe plasmids of type A isolates. Consequently, it has been suggested (3, 23) that many iota-toxin plasmids arose from insertion of an iota-toxin gene-carrying mobile genetic element near the cpe gene on a tcp-carrying type A plasmid. This insertional event apparently inactivated the cpe gene, so most or all type E isolates now carry silent cpe genes (3, 23).The epsilon-toxin-encoding plasmids of type D isolates show considerable size variations (33), ranging from ∼48 kb to ∼110 kb. These size variations in type D etx plasmids reflect, in part, differences among their toxin gene carriage. The small 48-kb etx plasmids present in some type D isolates typically lack either the cpe gene or the cpb2 gene (encoding beta2-toxin), while the larger (>75-kb) etx plasmids found in other type D isolates can also carry the cpe gene, the cpb2 gene, or both the cpe and cpb2 genes. Consequently, some type D isolates carry a toxin plasmid encoding only etx, other type D isolates carry a toxin plasmid with up to three different functional toxin genes (etx, cpb2, and cpe), and the remaining type D isolates carry their etx, cpe, and cpb2 genes on up to three distinct plasmids.C. perfringens type B isolates uniquely produce both beta- and epsilon-toxins, the two most lethal C. perfringens toxins (13). These bacteria are important pathogens of sheep but also cause disease in goats, calves, and foals (26). For unknown reasons, diseases caused by C. perfringens type B isolates apparently are restricted to certain geographic regions (24, 25, 26). C. perfringens type B enterotoxemias initiate when these bacteria proliferate in the gut, accompanied by toxin production. Those toxins initially affect the intestines but later are absorbed and act systemically. Studies from our group (13) showed that beta- and epsilon-toxins each contribute to lethality in a mouse model involving intravenous injection of type B culture supernatants.There has been characterization of only one type B virulence plasmid to date. Our recent study (29) showed that most, if not all, type B isolates carry a common etx plasmid of ∼65 kb that also possesses a tcp locus and a cpb2 gene, although not the cpb gene encoding beta-toxin. Interestingly, the type B etx plasmid is highly (80%) related to the ∼75-kb cpe- and cpb2-carrying plasmid found in some type A isolates (28). The ∼65-kb etx plasmid present in most, if not all, type B isolates is also carried by a minority of type D isolates (29).The absence of the cpb gene from their etx plasmids suggested that most type B isolates might carry additional virulence plasmids. Therefore, the current study was performed to better address virulence plasmid carriage and diversity among type B disease isolates.     

Cross-Sectional and Longitudinal Multilocus Sequence Typing of Pseudomonas aeruginosa in Cystic Fibrosis Sputum Samples

Multilocus sequence typing (MLST) is a genetic typing tool designed to provide information about the relatedness of isolates at the core genome level. The utility of MLST in regard to cystic fibrosis (CF)-related infection with Pseudomonas aeruginosa is unknown. The molecular clock speed of the MLST genes was studied using 219 colonies isolated longitudinally from 49 patients with CF. A cross-sectional study examining 27 to 46 colonies per sputum sample for samples from 16 patients was also undertaken. The molecular clock speed was estimated to be 2.05 × 10⁻⁵ (upper 95% confidence limit) or 4.75 × 10⁻⁶ (50% confidence limit) point mutations per nucleotide per year. In the cross-sectional study, 50% of patients were infected with more than one sequence type. There was evidence of point mutations, recombination events, and coinfection with epidemic and unique strains. A clonal complex that was highly genetically distinct from the rest of the P. aeruginosa population was identified. The MLST scheme uses genes with an appropriate clock speed and provides useful information about the genetic variation of P. aeruginosa within and between patients with CF.Multilocus sequence typing (MLST) is a typing tool based on the DNA sequences of several housekeeping genes. Housekeeping genes are chosen because they are assumed to be under low selection pressure and therefore to have a low molecular clock speed, i.e., to undergo mutations at a low rate. This allows MLST to provide data about the core evolutionary genome of a bacterium which allow deductions about population structure and the relatedness of different strains. Where populations are rapidly diversifying by recombination, as may occur in a clinical outbreak, MLST is able to identify closely related isolates (22) which may have undergone changes in one or two loci. MLST has recently been used to estimate the molecular clock speed of the housekeeping genes in cystic fibrosis (CF)-related Burkholderia cepacia complex infection and to identify presumed recombination events in infected patients (24). We hypothesized that the clock speed of the housekeeping genes of the Pseudomonas aeruginosa MLST scheme in patients with CF would be of the same order and that point mutations and recombination events would be identified. These hypotheses were tested by examining sequential isolates from chronically infected patients with CF (clock speed study) and by examining multiple isolates from single sputum samples (cross-sectional study).     

Evaluation of molecular typing methods in characterizing a European collection of epidemic methicillin-resistant Staphylococcus aureus strains: the HARMONY collection

We analyzed a representative sample of methicillin-resistant Staphylococcus aureus (MRSA) from 11 European countries (referred to as the HARMONY collection) using three molecular typing methods used within the HARMONY group to examine their usefulness for large, multicenter MRSA surveillance networks that use these different laboratory methodologies. MRSA isolates were collected based on their prevalence in each center and their genetic diversity, assessed by pulsed-field gel electrophoresis (PFGE). PFGE groupings (≤3 bands difference between patterns) were compared to those made by sequencing of the variable repeats in the protein A gene spa and clonal designations based on multilocus sequence typing (MLST), combined with PCR analysis of the staphylococcal chromosome cassette containing the mec genes involved in methicillin resistance (SCCmec). A high level of discrimination was achieved using each of the three methodologies, with discriminatory indices between 89.5% and 91.9% with overlapping 95% confidence intervals. There was also a high level of concordance of groupings made using each method. MLST/SCCmec typing distinguished 10 groups containing at least two isolates, and these correspond to the majority of nosocomial MRSA clones described in the literature. PFGE and spa typing resolved 34 and 31 subtypes, respectively, within these 10 MRSA clones, with each subtype differing only slightly from the most common pattern using each method. The HARMONY group has found that the methods used in this study differ in their availability and affordability to European centers involved in MRSA surveillance. Here, we demonstrate that the integration of such technologies is achievable, although common protocols (such as we have developed for PFGE) may also be important, as is the use of centralized Internet sites to facilitate data analysis. PFGE and spa-typing data from analysis of MRSA isolates from the many centers that have access to the relevant equipment can be compared to reference patterns/sequences, and clonal designations can be made. In the majority of cases, these will correspond to those made by the (more expensive) method of choice—MLST/SCCmec typing—and these alternative methods can therefore be used as frontline typing systems for multicenter surveillance of MRSA.     

Clostridium perfringens type A strains carrying a plasmid-borne enterotoxin gene (genotype IS1151-cpe or IS1470-like-cpe) as a common cause of food poisoning

The prevalences of various genotypes of enterotoxin gene-carrying (cpe-positive) Clostridium perfringens type A in 24 different food poisoning outbreaks were 75% (chromosomal IS1470-cpe), 21% (plasmid-borne IS1470-like-cpe), and 4% (plasmid-borne IS1151-cpe). These results show that C. perfringens type A carrying the plasmid-borne cpe is a common cause of food poisoning.     

Comparative population structure analysis of Campylobacter jejuni from human and poultry origin in Bangladesh

Campylobacter jejuni is the most important cause of antecedent infections leading to Guillain–Barré syndrome (GBS) and Miller Fisher syndrome (MFS). The objective of the present study was to define the genetic diversity, population structure, and potential role of poultry in the transmission of Campylobacter to humans in Bangladesh. We determined the population structure of C. jejuni isolated from poultry (n?=?66) and patients with enteritis (n?=?39) or GBS (n?=?10). Lipooligosaccharide (LOS) typing showed that 50/66 (76 %) C. jejuni strains isolated from poultry could be assigned to one of five LOS locus classes (A–E). The distribution of neuropathy-associated LOS locus classes A, B, and C were 30/50 (60 %) among the typable strains isolated from poultry. The LOS locus classes A, B, and C were significantly associated with GBS and enteritis-related C. jejuni strains more than for the poultry strains [(31/38 (82 %) vs. 30/50 (60 %), p?p?C. jejuni isolated from humans and poultry. There seems to be a lack of overlap between the major human and chicken clones, which suggests that there may be additional sources for campylobacteriosis other than poultry in Bangladesh.     

Necrotic enteritis in broilers: an updated review on the pathogenesis

Clostridium perfringens-induced necrotic enteritis and related subclinical disease have become economically significant problems for the broiler industry. Fortunately, scientific interest in this topic has grown: new C. perfringens virulence factors have been discovered and new insight gained about the pathogenesis of necrotic enteritis. It has been shown that alpha toxin, for a long time thought to be the key virulence factor, is not essential for the development of the disease. Moreover, it is now clearly established that only certain C. perfringens strains are capable of inducing necrotic enteritis under specific conditions that predispose to the disease and they constitute only a minority in the intestinal tract of healthy chickens. A novel pore-forming toxin, NetB, has been identified in these virulent avian C. perfringens strains. Using a gene knockout mutant, it has been shown that NetB is a critical virulence factor in the pathogenesis of necrotic enteritis in broilers. In addition to toxin production, other factors have been described that contribute to the ability of certain C. perfringens strains to cause necrotic enteritis in broilers. It has been suggested that proteolytic enzymes play an important role in the initial stages of necrotic enteritis since the villi are first affected at the level of the basement membrane and the lateral domain of the enterocytes. In field outbreaks of necrotic enteritis, a single clone of C. perfringens is dominant in intestines of all affected birds, as opposed to the mixture of different C. perfringens strains that can be isolated from healthy bird intestines. It has been proposed that bacteriocin production is responsible for the dominance of a single strain in necrotic enteritis cases. Furthermore, it has been shown that virulent strains are more able to adhere to extracellular matrix molecules than non-virulent strains. The current knowledge on the pathogenesis of the disease has been summarized in this short review.     

Clostridium perfringens Type E Animal Enteritis Isolates with Highly Conserved, Silent Enterotoxin Gene Sequences

Several Clostridium perfringens genotype E isolates, all associated with hemorrhagic enteritis of neonatal calves, were identified by multiplex PCR. These genotype E isolates were demonstrated to express α and ι toxins, but, despite carrying sequences for the gene (cpe) encoding C. perfringens enterotoxin (CPE), were unable to express CPE. These silent cpe sequences were shown to be highly conserved among type E isolates. However, relative to the functional cpe gene of type A isolates, these silent type E cpe sequences were found to contain nine nonsense and two frameshift mutations and to lack the initiation codon, promoters, and ribosome binding site. The type E animal enteritis isolates carrying these silent cpe sequences do not appear to be clonally related, and their silent type E cpe sequences are always located, near the ι toxin genes, on episomal DNA. These findings suggest that the highly conserved, silent cpe sequences present in most or all type E isolates may have resulted from the recent horizontal transfer of an episome, which also carries ι toxin genes, to several different type A C. perfringens isolates.     

A correlative analysis of epidemiologic and molecular characteristics of methicillin-resistant Staphylococcus aureus clones from diverse geographic locations with virulence measured by a Caenorhabditis elegans host model

Methicillin-resistant Staphylococcus aureus (MRSA) strains from different geographic areas have different genetic backgrounds, suggesting independent clonal evolutions. To better understand the virulence of MRSA strains and the relationship to their clonal and geographic origins, we undertook an analysis of epidemiologic, molecular, and virulence characteristics of a large number of MRSA isolates from geographically diverse origins, in a Caenorhabditis elegans infection model. A total of 99 MRSA isolates collected between 1993 and 2010 at the Geneva University Hospitals from diverse global origins were characterized with Panton–Valentine leukocidin (PVL), toxic shock syndrome toxin (TSST), accessory gene regulator (agr) group, staphylococcal cassette chromosome mec (SCCmec), S. aureus protein A (spa), multilocus sequence typing (MLST), and pulsed-field gel electrophoresis (PFGE) typing. Epidemiologic data were provided from clinical records. The bacterial virulence was tested in a C. elegans host model. The inter-relationships of epidemiological/molecular characteristics in association with nematocidal activities were analyzed with univariate and two-factor analysis of variance (ANOVA). Community-associated MRSA (CA-MRSA) strains were more virulent than hospital-associated MRSA (HA-MRSA), with higher nematocidal activities in CA-MRSA strains (0.776 vs. 0.506, p?=?0.0005). All molecular characteristics (PVL, TSST, spa, SCCmec, MLST, and PFGE types) showed a significant association with nematocidal activities on univariate analysis (p?<?0.005). PVL was not a significant predictor after adjusting for genomic backgrounds using spa, MLST, or PFGE typing. The dominant CA-MRSA strains in North America showed higher nematocidal activities than strains from other regions (p?<?0.0001). Strains with global origins containing distinct genetic backgrounds have different virulence in the C. elegans model. Nematocidal activities were most highly correlated with SCCmec, spa, MLST, and PFGE typing, suggesting that genomic background rather than a single exotoxin characteristic was the most discriminating predictor of virulence.     

An abbreviated MLVA identifies Escherichia coli ST131 as the major extended-spectrum β-lactamase-producing lineage in the Copenhagen area

Rapid bacterial typing is a valuable and necessary tool in the prevention and detection of outbreaks. The purpose of this study was to adapt a multilocus variable number of tandem repeats analysis (MLVA) for analysis on a benchtop capillary electrophoresis instrument and compare the modified assay with multilocus sequence typing (MLST) for typing cefpodoxime-resistant Escherichia coli (E. coli). Further, we identified the causative resistance mechanisms and epidemiological type of infection for isolates producing extended-spectrum β-lactamases (ESBLs). A collection of E. coli resistant to cefpodoxime was typed by MLST and a modified MLVA assay using a benchtop capillary electrophoresis instrument. Resistance mechanisms were identified by polymerase chain reaction (PCR) and sequencing. Patient history was examined to establish the epidemiological type of infection for ESBL-producing E. coli. MLVA yielded typing results homologous with MLST and it correctly identified E. coli sequence type (ST) 131 that was accounting for 45 % of all ESBL-producing isolates in the sample collection. The majority (76.7 %) of ESBL-producing isolates was healthcare-related and only 23.3 % of the ESBL-producing isolates were community-onset infections (COI), regardless of the ST. Patients with COI were significantly more often of female gender and younger age compared to healthcare-associated infections (HCAI) and hospital-onset infections (HOI). In conclusion, the modified MLVA is a useful tool for the rapid typing of E. coli and it identified ST131 as the predominating ESBL-producing lineage in Copenhagen. Healthcare-related infections were the predominant infection setting of ESBL-producing E. coli and the demographic characteristics differed between patients with COI and healthcare-related infections.