Comparative Proteomic Analysis of Extracellular Proteins of Clostridium perfringens Type A and Type C Strains

Clostridium perfringens is a medically important clostridial pathogen and an etiological agent causing several diseases in humans and animals. C. perfringens and its toxins have been listed as potential biological and toxin warfare (BTW) agents; thus, efforts to develop strategies for detection and protection are warranted. Forty-eight extracellular proteins of C. perfringens type A and type C strains have been identified here using a 2-dimensional gel electrophoresis-mass spectrometry (2-DE-MS) technique. The SagA protein, the DnaK-type molecular chaperone hsp70, endo-beta-N-acetylglucosaminidase, and hypothetical protein CPF_0656 were among the most abundant proteins secreted by C. perfringens ATCC 13124. The antigenic component of the exoproteome of this strain has also been identified. Most of the extracellular proteins were predicted to be involved in carbohydrate transport and metabolism (16%) or cell envelope biogenesis or to be outer surface protein constituents (13%). More than 50% of the proteins were predictably secreted by either classical or nonclassical pathways. LipoP and TMHMM indicated that nine proteins were extracytoplasmic but cell associated. Immunization with recombinant ornithine carbamoyltransferase (cOTC) clearly resulted in protection against a direct challenge with C. perfringens organisms. A significant rise in IgG titers in response to recombinant cOTC was observed in mice, and IgG2a titers predominated over IgG1 titers (IgG2a/IgG1 ratio, 2). The proliferation of spleen lymphocytes in cOTC-immunized animals suggested a cellular immune response. There were significant increases in the levels of gamma interferon (IFN-γ) and interleukin 2 (IL-2), suggesting a Th1 type immune response.Clostridium perfringens is a medically important clostridial pathogen and an etiological agent causing several diseases in humans and animals; the former include gas gangrene, food poisoning, necrotizing enterocolitis of infants, and enteritis necroticans (28, 37, 45). C. perfringens is an obligately anaerobic rod-shaped bacterium commonly found in the gastrointestinal tracts of both animals and humans and widely distributed in soil and sewage. The ability of C. perfringens to cause disease is associated with the production of a variety of extracellular toxins (13 different toxins have been reported so far). On the basis of differential production of toxins, the strains of C. perfringens can be divided into five types, A through E (35), of which type A and type C strains are implicated in human diseases while other types are of veterinary importance. Type A strains cause gas gangrene, the most destructive of all clostridial diseases, which is characterized by rapid destruction of tissue with production of gas (4, 42). The incidence of disease ranged from 1% or less of wounded personnel during World War II to 10% of wounded personnel during World War I (27). Hundreds of thousands of soldiers died of gas gangrene as a result of battlefield injuries, and C. perfringens was widely recognized as the most important causal organism of the disease. Besides gas gangrene, type A strains also cause gastrointestinal diseases in humans (food poisoning, antibiotic-associated diarrhea, sporadic diarrhea, sudden infant death syndrome) and animals (diarrhea in foals and pigs, etc.). C. perfringens type C strains cause necrotic enteritis in humans and animals, in addition to enterotoxemia in sheep. Moreover, C. perfringens and its toxins have been listed as potential biological and toxin warfare (BTW) agents; therefore, efforts to develop strategies for detection and protection are warranted.Interest in a vaccine against gas gangrene has been intermittent; most efforts were made during World Wars I and II and were devoted to the therapeutic use of antisera. Such antisera, raised against toxoids of all five species of clostridia associated with gas gangrene, were shown to have benefits if they were given soon after trauma (20). Active immunization against the disease has received little attention until a few years ago (32, 43, 44). A number of clinical studies of other pathogenic bacteria, including Clostridium difficile, have highlighted the importance of nontoxin protein antigens in disease expression, especially in colonization by the bacterium (9, 12, 16, 26).Despite a sudden spurt of activity in the proteomic characterization of bacterial pathogens, for reasons unknown, clostridia have been largely ignored. Clostridium difficile is the only clostridial species whose proteome has been analyzed to some extent (34). Proteomic methodology has been used to elucidate proteins regulated by the VirR/VirS system in Clostridium perfringens (40).To invade, multiply in, and colonize host tissues, a pathogen must be able to evade the host immune system and obtain nutrients essential for growth. The factors involved in these complex processes are largely unknown and of crucial importance for the understanding of microbial pathogenesis. The exoproteins of Gram-positive bacteria are likely to contain some of these key factors. The term “secretome” refers to and takes into account both the protein secretion systems and the secreted proteins; in monoderm bacteria (Gram-positive cell envelope architecture), these proteins can also be found in the membrane and/or cell wall. The proteins found in the extracellular milieu of Gram-positive bacteria are hence extracellular proteins, or exoproteins, which form the exoproteome; these exoproteins are not necessarily secreted by known secretion systems (15). This terminology is used for the subproteome described in this study.Further, the correct identification of C. perfringens pathovars is critical for epidemiological studies and for the development of effective preventive measures, including vaccination. It is not well understood why C. perfringens produces illnesses that differ so greatly in severity. Specific illnesses may reflect the particular type of tissue that high numbers of the organism are able to invade. Clearly, the presence of certain toxins is one explanation, but it falls short in many instances. For example, strains that cause food poisoning may differ from those that cause gas gangrene only by the presence of an enterotoxin gene in the former, yet food-poisoning strains have never been found to cause gas gangrene. Elucidation of the exoproteins of this bacterium is likely to reveal factors responsible for the host specificity of the different C. perfringens pathovars.The present investigation was carried out with the following objectives: (i) identification of dominant exoproteins from the C. perfringens type A strain ATCC 13124 (a gas gangrene isolate and the species type strain) and comparison with those of type C strains, (ii) elucidation of immunogenic components of the exoproteome, (iii) in silico analysis of the identified proteins with respect to localization, predicted function, and likely potential as surface markers for detection and as vaccine candidates, and (iv) validation of selected candidates with respect to vaccine potential in an experimental mouse model of gas gangrene. To the best of our knowledge, ours is the first proteomic elucidation of the C. perfringens exoproteome. The identification of extracellular proteins of C. perfringens type A and type C strains in the present investigation advances our understanding of the pathogenesis of the disease and opens significant opportunities for the identification of diagnostic markers and the development of vaccines against gas gangrene in humans.     

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.     

Evidence That the Enterotoxin Gene Can Be Episomal in Clostridium perfringens Isolates Associated with Non-Food-Borne Human Gastrointestinal Diseases

Clostridium perfringens enterotoxin (CPE) is responsible for the diarrheal and cramping symptoms of human C. perfringens type A food poisoning. CPE-producing C. perfringens isolates have also recently been associated with several non-food-borne human gastrointestinal (GI) illnesses, including antibiotic-associated diarrhea and sporadic diarrhea. The current study has used restriction fragment length polymorphism (RFLP) and pulsed-field gel electrophoresis (PFGE) analyses to compare the genotypes of 43 cpe-positive C. perfringens isolates obtained from diverse sources. All North American and European food-poisoning isolates examined in this study were found to carry a chromosomal cpe, while all non-food-borne human GI disease isolates characterized in this study were determined to carry their cpe on an episome. Collectively, these results provide the first evidence that distinct subpopulations of cpe-positive C. perfringens isolates may be responsible for C. perfringens type A food poisoning versus CPE-associated non-food-borne human GI diseases. If these putative associations are confirmed in additional surveys, cpe RFLP and PFGE genotyping assays may facilitate the differential diagnosis of food-borne versus non-food-borne CPE-associated human GI illnesses and may also be useful epidemiologic tools for identifying reservoirs or transmission mechanisms for the subpopulations of cpe-positive isolates specifically responsible for CPE-associated food-borne versus non-food-borne human GI diseases.     

Genotypic and Phenotypic Characterization of Clostridium perfringens Isolates from Darmbrand Cases in Post-World War II Germany

Clostridium perfringens type C strains are the only non-type-A isolates that cause human disease. They are responsible for enteritis necroticans, which was termed Darmbrand when occurring in post-World War II Germany. Darmbrand strains were initially classified as type F because of their exceptional heat resistance but later identified as type C strains. Since only limited information exists regarding Darmbrand strains, this study genetically and phenotypically characterized seven 1940s era Darmbrand-associated strains. Results obtained indicated the following. (i) Five of these Darmbrand isolates belong to type C, carry beta-toxin (cpb) and enterotoxin (cpe) genes on large plasmids, and express both beta-toxin and enterotoxin. The other two isolates are cpe-negative type A. (ii) All seven isolates produce highly heat-resistant spores with D₁₀₀ values (the time that a culture must be kept at 100°C to reduce its viability by 90%) of 7 to 40 min. (iii) All of the isolates surveyed produce the same variant small acid-soluble protein 4 (Ssp4) made by type A food poisoning isolates with a chromosomal cpe gene that also produce extremely heat-resistant spores. (iv) The Darmbrand isolates share a genetic background with type A chromosomal-cpe-bearing isolates. Finally, it was shown that both the cpe and cpb genes can be mobilized in Darmbrand isolates. These results suggest that C. perfringens type A and C strains that cause human food-borne illness share a spore heat resistance mechanism that likely favors their survival in temperature-abused food. They also suggest possible evolutionary relationships between Darmbrand strains and type A strains carrying a chromosomal cpe gene.     

Contributions of NanI Sialidase to Caco-2 Cell Adherence by Clostridium perfringens Type A and C Strains Causing Human Intestinal Disease

Previous studies showed that Clostridium perfringens type D animal disease strain CN3718 uses NanI sialidase for adhering to enterocyte-like Caco-2 cells. The current study analyzed whether NanI is similarly important when type A and C human intestinal disease strains attach to Caco-2 cells. A PCR survey determined that the nanI gene was absent from typical type A food poisoning (FP) strains carrying a chromosomal enterotoxin (CPE) gene or the genetically related type C Darmbrand (Db) strains. However, the nanI gene was present in type A strains from healthy humans, type A strains causing CPE-associated antibiotic-associated diarrhea (AAD) or sporadic diarrhea (SD), and type C Pig-Bel strains. Consistent with NanI sialidase being the major C. perfringens sialidase when produced, FP and Db strains had little supernatant sialidase activity compared to other type A or C human intestinal strains. All type A and C human intestinal strains bound to Caco-2 cells, but NanI-producing strains had higher attachment levels. When produced, NanI can contribute to host cell attachment of human intestinal disease strains, since a nanI null mutant constructed in type A SD strain F4969 had lower Caco-2 cell adhesion than wild-type F4969 or a complemented strain. Further supporting a role for NanI in host cell attachment, sialidase inhibitors reduced F4969 adhesion to Caco-2 cells. Collectively, these results suggest that NanI may contribute to the intestinal attachment and colonization needed for the chronic diarrhea of CPE-associated AAD and SD, but this sialidase appears to be dispensable for the acute pathogenesis of type A FP or type C enteritis necroticans.     

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.     

Type IV pili and the CcpA protein are needed for maximal biofilm formation by the gram-positive anaerobic pathogen Clostridium perfringens

The predominant organizational state of bacteria in nature is biofilms. Biofilms have been shown to increase bacterial resistance to a variety of stresses. We demonstrate for the first time that the anaerobic gram-positive pathogen Clostridium perfringens forms biofilms. At the same concentration of glucose in the medium, optimal biofilm formation depended on a functional CcpA protein. While the ratio of biofilm to planktonic growth was higher in the wild type than in a ccpA mutant strain in middle to late stages of biofilm development, the bacteria shifted from a predominantly biofilm state to planktonic growth as the concentration of glucose in the medium increased in a CcpA-independent manner. As is the case in some gram-negative bacteria, type IV pilus (TFP)-dependent gliding motility was necessary for efficient biofilm formation, as demonstrated by laser confocal and electron microscopy. However, TFP were not associated with the bacteria in the biofilm but with the extracellular matrix. Biofilms afforded C. perfringens protection from environmental stress, including exposure to atmospheric oxygen for 6 h and 24 h and to 10 mM H₂O₂ for 5 min. Biofilm cells also showed 5- to 15-fold-increased survival over planktonic cells after exposure to 20 μg/ml (27 times the MIC) of penicillin G for 6 h and 24 h, respectively. These results indicate C. perfringens biofilms play an important role in the persistence of the bacteria in response to environmental stress and that they may be a factor in diseases, such as antibiotic-associated diarrhea and gas gangrene, that are caused by C. perfringens.     

Complete genome sequence of ΦCP51, a temperate bacteriophage of Clostridium perfringens

During sequencing of the genome of Clostridium perfringens strain 5147-97, a putative prophage was identified, located within a gene for a proposed flavodoxin oxidoreductase. Mitomycin C induction of this strain released a bacteriophage whose morphological features examined by electron microscopy indicated it belonged to the family Siphoviridae. This phage was hence designated as vB_CpeS-CP51. The 39,108-bp genome includes 50 predicted open reading frames (ORFs), including two that may affect sporulation, and two predicted tRNAs. To determine the ends of the prophage, PCR was performed using primers facing outwards from the proposed end genes. This confirmed the presence of a circularised genome in PEG-precipitated bacteriophage particles.     

Development and preliminary evaluation of a slide latex agglutination assay for detection of Clostridium perfringens type A enterotoxin

A slide latex agglutination (SLA) assay was developed for rapid screening for Clostridium perfringens type A enterotoxin (CPE). SLA specifically detected CPE added to buffer or normal feces (sensitivity limit of 1 μg CPE/g feces). Using clinical fecal samples from C. perfringens food poisoning cases, a strong correlation was shown between (1) SLA results and results from other CPE assays and (2) between SLA results and illness status.     

Multiplex PCR genotyping assay that distinguishes between isolates of Clostridium perfringens type A carrying a chromosomal enterotoxin gene (cpe) locus, a plasmid cpe locus with an IS1470-like sequence, or a plasmid cpe locus with an IS1151 sequence

Clostridium perfringens type A isolates carrying the enterotoxin (cpe) gene are important causes of both food poisoning and non-food-borne diarrheas in humans. In North America and Europe, food poisoning isolates were previously shown to carry a chromosomal cpe gene, while non-food-borne gastrointestinal (GI) disease isolates from those two geographic locations were found to have a plasmid cpe gene. In this report, we describe the development of an economical multiplex PCR cpe genotyping assay that works with culture lysates to distinguish among type A isolates carrying a chromosomal cpe gene, a plasmid cpe gene with a downstream IS1470-like sequence, or a plasmid cpe gene with a downstream IS1151 sequence. When this multiplex PCR assay was applied in molecular epidemiologic studies, it was found that (i) all 57 examined type A isolates with a plasmid cpe gene have either IS1470-like or IS1151 sequences downstream of the plasmid cpe gene; (ii) an IS1470-like sequence, rather than an IS1151 sequence, is more commonly present downstream of the plasmid cpe gene (particularly in North American non-food-borne human GI disease isolates); and (iii) as previously shown in the United States and Europe, isolates carrying the chromosomal cpe gene also appear to be the major cause of C. perfringens food poisoning in Japan. The superiority of this new multiplex PCR assay over existing cpe genotyping approaches should facilitate further molecular epidemiologic investigations of C. perfringens enterotoxin-associated GI illnesses and their associated cpe-positive type A isolates.     

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.     

The NanI and NanJ Sialidases of Clostridium perfringens Are Not Essential for Virulence

The essential toxin in Clostridium perfringens-mediated gas gangrene or clostridial myonecrosis is alpha-toxin, although other toxins and extracellular enzymes may also be involved. In many bacterial pathogens extracellular sialidases are important virulence factors, and it has been suggested that sialidases may play a role in gas gangrene. C. perfringens strains have combinations of three different sialidase genes, two of which, nanI and nanJ, encode secreted sialidases. The nanI and nanJ genes were insertionally inactivated by homologous recombination in derivatives of sequenced strain 13 and were shown to encode two functional secreted sialidases, NanI and NanJ. Analysis of these derivatives showed that NanI was the major sialidase in this organism. Mutation of nanI resulted in loss of most of the secreted sialidase activity, and the residual activity was eliminated by subsequent mutation of the nanJ gene. Only a slight reduction in the total sialidase activity was observed in a nanJ mutant. Cytotoxicity assays using the B16 melanoma cell line showed that supernatants containing NanI or overexpressing NanJ enhanced alpha-toxin-mediated cytotoxicity. Finally, the ability of nanI, nanJ, and nanIJ mutants to cause disease was assessed in a mouse myonecrosis model. No attenuation of virulence was observed for any of these strains, providing evidence that neither the NanI sialidase nor the NanJ sialidase is essential for virulence.Clostridium perfringens type A is the causative agent of human gas gangrene, or clostridial myonecrosis, and human food poisoning (25, 27). It produces many secreted hydrolytic enzymes and toxins, including alpha-toxin and perfringolysin O. C. perfringens strains can also encode up to three sialidases, but the three sialidase genes, nanH, nanI, and nanJ, are not present in all of the strains that have been completely sequenced. Strain ATCC 13124 encodes all three sialidases (18), while strain 13 encodes both of the large sialidases, NanI and NanJ, but not the smaller NanH enzyme (32). The food poisoning isolate SM101 encodes NanH but not NanI or NanJ (18).Sialidases have been implicated in the virulence of several bacterial pathogens. They have been shown to enhance the pathogenesis of disease through synergistic effects with other bacterial factors. For example, Vibrio cholerae sialidase enhances the activity of cholera toxin (10), Pseudomonas aeruginosa sialidase increases the binding of this organism to the cells of susceptible patients (6), and the two sialidases of Streptococcus pneumoniae contribute to the progression of infection in several animal models (16, 23, 37). More recently, a surface-exposed sialidase was shown to be required for persistence of the canine pathogen Capnophagia canimorsus (15).Alpha-toxin is an essential virulence factor in gas gangrene (2), and perfringolysin O, although not essential, has been found to have a synergistic role with alpha-toxin, enhancing the disease process (3). Synergy between alpha-toxin and the NanI sialidase was also observed in experiments that showed that purified alpha-toxin had greater pathological effects on cultured cell lines that had been pretreated with NanI (8). Inoculation of mice with both purified alpha-toxin and NanI resulted in increased levels of plasma creatine kinase, a marker for muscle necrosis, compared to the levels after inoculation of alpha-toxin alone (8).The sialidases of C. perfringens have different cellular locations. NanH (43 kDa) lacks a signal peptide and is located in the cytoplasm (12, 24). It has been proposed that NanH is involved in the cleavage of short oligosaccharides that enter the cell and are subsequently broken down for nutritional purposes (41). By contrast, NanI (77 kDa) contains a signal peptide, is secreted from the cell, and is readily isolated from cell-free supernatants (19, 38). A high-resolution structure of the catalytic domain of NanI in a complex with its sialic acid substrate has recently been described (20). NanI may also play a role in nutrition, releasing sialic acid from higher-order gangliosides for subsequent transport into the cell (41). As a result of its location, NanI may also interact with the extracellular environment of the host tissue during infection. In addition to its synergy with alpha-toxin (8), NanI has also been shown to have synergistic effects with ɛ-toxin (31), which is required for C. perfringens type B- and D-mediated diseases (34, 39, 40). Very limited information is available for the 129-kDa NanJ enzyme, but recent studies have shown that in addition to sialidase motifs this enzyme contains an additional galactose binding domain (5).The objective of our study was to determine the role of NanI and NanJ in the pathogenesis of gas gangrene. Mutagenesis of the genes (nanI and nanJ) encoding each of the secreted sialidases was carried out using the strain 13 derivative JIR325. Then the relative contribution of each sialidase to total sialidase production was determined, and virulence was assessed using the mouse myonecrosis model. The results showed that NanI is the major sialidase in this strain but that neither enzyme is essential for virulence.     

Comparative genomic analysis using microarray demonstrates a strong correlation between the presence of the 80-kilobase pathogenicity island and pathogenicity in Kanagawa phenomenon-positive Vibrio parahaemolyticus strains

Vibrio parahaemolyticus is a gram-negative marine bacterium. A limited population of the organisms causes acute gastroenteritis in humans. Almost all of the clinical V. parahaemolyticus isolates exhibit beta-type hemolysis on Wagatsuma agar, known as the Kanagawa phenomenon (KP). KP is induced by the thermostable direct hemolysin produced by the organism and has been considered a crucial marker to distinguish pathogenic strains from nonpathogenic ones. Since 1996, so-called “pandemic clones,” the majority of which belong to serotype O3:K6, have caused worldwide outbreaks of gastroenteritis. In this study, we used a DNA microarray constructed based on the genome sequence of a pandemic V. parahaemolyticus strain, RIMD2210633, to examine the genomic composition of 22 strains of V. parahaemolyticus, including both pathogenic (pandemic and nonpandemic) and nonpathogenic strains. More than 86% of the RIMD2210633 genes were conserved in all of the strains tested. Many variably present genes formed gene clusters on the genome of RIMD2210633 and were probably acquired through lateral gene transfer. At least 65 genes over 11 loci were specifically present in the pandemic strains compared with any of the nonpandemic strains, suggesting that the difference between pandemic and nonpandemic strains is not due to a simple genetic event. Only the genes in the 80-kb pathogenicity island (Vp-PAI) on chromosome II, including two tdh genes and a set of genes for the type III secretion system, were detected only in the KP-positive pathogenic strains. These results strongly suggest that acquisition of this Vp-PAI was crucial for the emergence of V. parahaemolyticus strains that are pathogenic for humans.     

A new genome of Acidithiobacillus thiooxidans provides insights into adaptation to a bioleaching environment

Acidithiobacillus thiooxidans is a sulfur oxidizing acidophilic bacterium found in many sulfur-rich environments. It is particularly interesting due to its role in bioleaching of sulphide minerals. In this work, we report the genome sequence of At. thiooxidans Licanantay, the first strain from a copper mine to be sequenced and currently used in bioleaching industrial processes. Through comparative genomic analysis with two other At. thiooxidans non-metal mining strains (ATCC 19377 and A01) we determined that these strains share a large core genome of 2109 coding sequences and a high average nucleotide identity over 98%. Nevertheless, the presence of 841 strain-specific genes (absent in other At. thiooxidans strains) suggests a particular adaptation of Licanantay to its specific biomining environment. Among this group, we highlight genes encoding for proteins involved in heavy metal tolerance, mineral cell attachment and cysteine biosynthesis. Several of these genes were located near genetic motility genes (e.g. transposases and integrases) in genomic regions of over 10 kbp absent in the other strains, suggesting the presence of genomic islands in the Licanantay genome probably produced by horizontal gene transfer in mining environments.     

Synergistic Effects of Clostridium perfringens Enterotoxin and Beta Toxin in Rabbit Small Intestinal Loops

The ability of Clostridium perfringens type C to cause human enteritis necroticans (EN) is attributed to beta toxin (CPB). However, many EN strains also express C. perfringens enterotoxin (CPE), suggesting that CPE could be another contributor to EN. Supporting this possibility, lysate supernatants from modified Duncan-Strong sporulation (MDS) medium cultures of three CPE-positive type C EN strains caused enteropathogenic effects in rabbit small intestinal loops, which is significant since CPE is produced only during sporulation and since C. perfringens can sporulate in the intestines. Consequently, CPE and CPB contributions to the enteropathogenic effects of MDS lysate supernatants of CPE-positive type C EN strain CN3758 were evaluated using isogenic cpb and cpe null mutants. While supernatants of wild-type CN3758 MDS lysates induced significant hemorrhagic lesions and luminal fluid accumulation, MDS lysate supernatants of the cpb and cpe mutants caused neither significant damage nor fluid accumulation. This attenuation was attributable to inactivating these toxin genes since complementing the cpe mutant or reversing the cpb mutation restored the enteropathogenic effects of MDS lysate supernatants. Confirming that both CPB and CPE are needed for the enteropathogenic effects of CN3758 MDS lysate supernatants, purified CPB and CPE at the same concentrations found in CN3758 MDS lysates also acted together synergistically in rabbit small intestinal loops; however, only higher doses of either purified toxin independently caused enteropathogenic effects. These findings provide the first evidence for potential synergistic toxin interactions during C. perfringens intestinal infections and support a possible role for CPE, as well as CPB, in some EN cases.