The serine proteases CspA and CspC are essential for germination of spores of Clostridium perfringens SM101 through activating SleC and cortex hydrolysis.

Clostridium perfringens SM101 genome encodes three serine proteases (CspA, CspB, and CspC), and genetic evidence indicates that CspB is required for processing of pro-SleC into active SleC, an enzyme essential for degradation of the peptidoglycan cortex during spore germination. In this study, the expression of cspA and cspC, as well as the germination and colony formation by spores of cspAC and cspC mutants of strain SM101, were assessed. We demonstrated that 1) the cspA and cspC genes were expressed as a bicistronic operon only during sporulation in the mother cell compartment of SM101; 2) both cspAC and cspC mutant spores were unable to germinate significantly with either KCl, l-glutamine, brain heart infusion (BHI) broth, or a 1:1 chelate of Ca2+ and dipicolinic acid (DPA); 3) consistent with germination results, both cspAC and cspC mutant spores were defective in normal DPA release; 4) the colony formation by cspAC and cspC mutant spores was ~106-fold lower than that of wild-type spores, although decoated mutant spores yielded wild-type level colony formation on plates containing lysozyme; 5) no processing of inactive pro-SleC into active SleC was observed in cspAC and cspC mutant spores during germination; and finally, 6) the defects in germination, DPA release, colony formation and SleC processing in cspAC and cspC mutant spores were complemented by the wild-type cspA-cspC operon. Collectively, these results indicate that both CspA and CspC are essential for C. perfringens spore germination through activating SleC and inducing cortex hydrolysis.

RelA/DTD-mediated regulation of spore formation and toxin production by Clostridium perfringens type A strain SM101.

RelA is a global regulator for stationary phase development in the model bacterium Bacillus subtilis. The relA gene forms a bicistronic operon with the downstream dtd gene. In this study, we evaluated the significance of RelA and DTD proteins in spore formation and toxin production by an important gastrointestinal pathogen Clostridium perfringens. Our ß-glucuronidase assay showed that in C. perfringens strain SM101, relA forms a bicistronic operon with its downstream dtd gene, and the relA promoter is expressed during both vegetative and sporulation conditions. By constructing double relA dtd and single dtd mutants in C. perfringens SM101, we found that: (1) RelA is required for maintaining the efficient growth capacity of SM101 cells during vegetative conditions; (2) both RelA and DTD are required for spore formation and enterotoxin (CPE) production by SM101; (3) RelA/DTD activate CodY, which is known to activate spore formation and CPE production in SM101 by activating a key sporulation-specific σ factor F; (4) as expected, RelA/DTD activate sporulation-specific σ factors (σE, σF, σG and σK) by positively regulating Spo0A production; and finally (5) RelA, but not DTD, negatively regulates phospholipase C (PLC) production by repressing plc gene expression. Collectively, our results demonstrate that RelA modulates cellular physiology such as growth, spore formation and toxin production by C. perfringens type A strain SM101, although DTD also plays a role in these pleiotropic functions in coordination with RelA during sporulation. These findings have implications for the understanding of the mechanisms involved in the infectious cycle of C. perfringens.

l-lysine (pH 6.0) induces germination of spores of Clostridium perfringens type F isolates carrying chromosomal or plasmid-borne enterotoxin gene.

C. perfringens type F isolates carrying enterotoxin gene (cpe) on the chromosome (C-cpe isolates) are mostly associated with food poisoning, while isolates carrying plasmid-borne cpe (P-cpe isolates) with non-food-borne gastrointestinal diseases. Spore germination is considered the most essential step for initiation of these diseases. Identifying the most effective germinants for spores of C-cpe and P-cpe isolates should help developing novel strategies involving induction of spore germination followed by inactivation of germinated spores with mild treatments. In this study, we showed that (i) l-lysine (pH 6.0) triggered germination of spores of all tested C-cpe and P-cpe isolates; although extremely low concentration of l-lysine (5-10 mM) induced germination of C-cpe spores, 10-fold higher concentration (50 mM) was required for P-cpe spore germination; (ii) P-cpe strain F4969 gerKC spores did not germinate, C-cpe strain SM101 gerKC spores germinated extremely poorly and these gerKC spores released significantly less DPA as compared to wild type spores; and these defects were restored to a nearly wild-type level by complementing gerKC spores with wild-type gerKC; and (iii) F4969 gerAA spores also did not germinate, and released less DPA than wild-type spores in presence of l-lysine (pH 6.0); and these defects were restored partially (germination) and fully (DPA release) by complimenting gerAA spores with wild-type gerAA. Collectively, our current study identified l-lysine as a universal germinant for spores of both C-cpe and P-cpe isolates and provided evidence that GerKC (from SM101 or F4969) and F4969 GerAA play major roles in l-lysine-induced germination.

Bicarbonate and amino acids are co-germinants for spores of Clostridium perfringens type A isolates carrying plasmid-borne enterotoxin gene.

Clostridium perfringens type A isolates carrying a chromosomal enterotoxin (cpe) gene (C-cpe) are generally linked to food poisoning, while isolates carrying cpe on a plasmid (P-cpe) are associated with non-food-borne gastrointestinal diseases. Both C-cpe and P-cpe isolates can form metabolically dormant spores, which through germination process return to actively growing cells to cause diseases. In our previous study, we showed that only 3 out of 20 amino acids (aa) in phosphate buffer (pH 7.0) triggered germination of spores of P-cpe isolates (P-cpe spores). We now found that 14 out of 20 individual aa tested induced germination of P-cpe spores in the presence of bicarbonate buffer (pH 7.0). However, no significant spore germination was observed with bicarbonate (pH 7.0) alone, indicating that aa and bicarbonate are co-germinants for P-cpe spores. P-cpe strain F4969 gerKC spores did not germinate, and gerAA spores germinated extremely poorly as compared to wild-type and gerKA spores with aa-bicarbonate (pH 7.0) co-germinants. The germination defects in gerKC and gerAA spores were partially restored by complementing gerKC or gerAA spores with wild-type gerKC or gerAA, respectively. Collectively, this study identified aa-bicarbonate as a novel nutrient germinant for P-cpe spores and provided evidence that GerKC and GerAA play major roles in aa-bicarbonate induced germination.

The inhibitory effects of essential oil constituents against germination, outgrowth and vegetative growth of spores of Clostridium perfringens type A in laboratory medium and chicken meat.

C. perfringens type A is the causative agent of C. perfringens type A food poisoning (FP) and non-food-borne (NFB) human gastrointestinal diseases. Due to its ability to form highly heat-resistant spores, it is of great interest to develop strategies alternative to thermal processing to inactivate C. perfrinegens. Thus, in this study we evaluated the inhibitory effects of essential oil constituents (EOCs) (cinnamaldehyde, eugenol, allyl isothiocyanate (AITC), and carvacrol) against germination, outgrowth and vegetative growth of spores of C. perfringens FP and NFB disease isolates in laboratory medium and chicken meat. The cinnamaldehyde, eugenol and carvacrol, but not AITC, all at 0.05-0.1%, inhibited the germination of spores of all tested C. perfringens isolates in Tripticase-glucose-yeast extract (TGY) medium. Furthermore, all tested EOCs at 0.05-0.1% arrested the outgrowth and vegetative growth of C. perfringens spores in TGY, with AITC and carvacrol being the most effective. However, among four tested EOCs, only AITC (at 0.5%-2.0%) was able to inhibit the growth of C. perfringens spores in chicken meat and no such inhibitory effect was observed even with a 10-fold higher concentration (5%) of carvacrol. In conclusion, our current work identified AITC as an effective EOC to control spores and vegetative cells of C. perfringens isolates in laboratory medium and chicken meat. Further studies on evaluating the effectiveness of different combination of EOCs against C. perfringens spore growth in different meat products should establish an effective use of EOCs to control the risk of C. perfringens-mediated illnesses.

Expansion of the Clostridium perfringens toxin-based typing scheme.

Clostridium perfringens causes many different histotoxic and enterotoxic diseases in humans and animals as a result of its ability to produce potent protein toxins, many of which are extracellular. The current scheme for the classification of isolates was finalized in the 1960s and is based on their ability to produce a combination of four typing toxins - α-toxin, ß-toxin, ε-toxin and ι-toxin - to divide C. perfringens strains into toxinotypes A to E. However, this scheme is now outdated since it does not take into account the discovery of other toxins that have been shown to be required for specific C. perfringens-mediated diseases. We present a long overdue revision of this toxinotyping scheme. The principles for the expansion of the typing system are described, as is a mechanism by which new toxinotypes can be proposed and subsequently approved. Based on these criteria two new toxinotypes have been established. C. perfringens type F consists of isolates that produce C. perfringens enterotoxin (CPE), but not ß-toxin, ε-toxin or ι-toxin. Type F strains will include strains responsible for C. perfringens-mediated human food poisoning and antibiotic associated diarrhea. C. perfringens type G comprises isolates that produce NetB toxin and thereby cause necrotic enteritis in chickens. There are at least two candidates for future C. perfringens toxinotypes, but further experimental work is required before these toxinotypes can formally be proposed and accepted.

Inactivation Strategies for Clostridium perfringens Spores and Vegetative Cells.

Clostridium perfringens is an important pathogen to human and animals and causes a wide array of diseases, including histotoxic and gastrointestinal illnesses. C. perfringens spores are crucial in terms of the pathogenicity of this bacterium because they can survive in a dormant state in the environment and return to being live bacteria when they come in contact with nutrients in food or the human body. Although the strategies to inactivate C. perfringens vegetative cells are effective, the inactivation of C. perfringens spores is still a great challenge. A number of studies have been conducted in the past decade or so toward developing efficient inactivation strategies for C. perfringens spores and vegetative cells, which include physical approaches and the use of chemical preservatives and naturally derived antimicrobial agents. In this review, different inactivation strategies applied to control C. perfringens cells and spores are summarized, and the potential limitations and challenges of these strategies are discussed.

Chitosan inhibits enterotoxigenic Clostridium perfringens type A in growth medium and chicken meat.

Clostridium perfringens is a spore-forming bacterium and a major cause of bacterial food-borne illness. In this study, we evaluated the inhibitory effects of chitosan against spore germination, spore outgrowth and vegetative growth of C. perfringens food poisoning (FP) isolates. Chitosan of differing molecular weights inhibited germination of spores of all tested FP isolates in a KCl germinant solution containing 0.1 mg/ml chitosan at pH 4.5. However, higher level (0.25 mg/ml) of chitosan was required to effectively arrest outgrowth of the germinated C. perfringens spores in Tripticase-yeast extract-glucose (TGY) medium. Furthermore, chitosan (1.0 mg/ml) was bacteriostatic against vegetative cells of C. perfringens in TGY medium. Although chitosan showed strong inhibitory activities against C. perfringens in laboratory medium, higher levels (2.0 mg/g) were required to achieve similar inhibition of spores inoculated into chicken meat. In summary, the inhibitory effects of chitosan against C. perfringens FP isolates was concentration dependent, and no major difference was observed when using different molecule weight chitosan as an inhibitor. Our results contribute to a better understanding on the potential application of chitosan in cooked meat products to control C. perfringens-associated disease.

Survival of Clostridium difficile spores at low water activity.

Clostridium difficile is frequently found in meat and meat products. Germination efficiency, defined as colony formation, was previously investigated at temperatures found in meat handling and processing for spores of strain M120 (animal isolate), R20291 (human isolate), and DK1 (beef isolate). In this study, germination efficiency of these spore strains was assessed in phosphate buffered saline (PBS, aw ∼1.00), commercial beef jerky (aw ∼0.82/0.72), and aw-adjusted PBS (aw ∼0.82/0.72). Surface hydrophobicity was followed for spores stored in PBS. After three months and for all PBS aw levels tested, M120 and DK1 spores showed a ∼1 decimal reduction in colony formation but this was not the case when kept in beef jerky suggesting a protective food matrix effect. During storage, and with no significant aw effect, an increase in colony formation was observed for R20291 spores kept in PBS (∼2 decimal log increase) and beef jerky (∼1 decimal log increase) suggesting a loss of spore superdormancy. For all strains, no significant changes in spore surface hydrophobicity were observed after storage. Collectively, these results indicate that depending on the germination properties of C. difficile spores and the media properties, their germination efficiency may increase or decrease during long term food storage.

Characterization of germinants and their receptors for spores of non-food-borne Clostridium perfringens strain F4969.

Clostridium perfringens type A can cause both food poisoning (FP) and non-food-borne (NFB) gastrointestinal diseases. Our previous study reported that a mixture of l-asparagine and KCl (AK)-germinated spores of FP and NFB isolates well, but KCl and, to a lesser extent, l-asparagine induced spore germination only in FP isolates. We now report that the germination response of FP and NFB spores differsignificantly in several defined germinants and rich media. Spores of NFB strain F4969 gerAA, gerKA-KC or gerKC mutants lacking specific germinant receptor proteins germinated more slowly than wild-type spores with rich media, did not germinate with AK and germinated poorly compared to wild-type spores with l-cysteine. The germination defects in the gerKA-KC spores were largely due to loss of GerKC as (i) gerKA spores germinated significantly with all tested germinants, while gerKC spores exhibited poor or no germination; and (ii) germination defects in gerKC spores were largely restored by expressing the wild-type gerKA-KC operon in trans. We also found that gerKA-KC, gerAA and gerKC spores, but not gerKA spores, released dipicolinic acid at a slower rate than wild-type spores with AK. The colony-forming efficiency of F4969 gerKC spores was also ~35-fold lower than that of wild-type spores, while gerAA and wild-type spores had similar viability. Collectively, these results suggest that the GerAA and GerKC proteins play roles in normal germination of C. perfringens NFB isolates and that GerKC, but not GerAA, is important in these spores' apparent viability.

Transcriptional Profile during Deoxycholate-Induced Sporulation in a Clostridium perfringens Isolate Causing Foodborne Illness.

UNLABELLED: Clostridium perfringens type A is a common source of foodborne illness (FBI) in humans. Vegetative cells sporulate in the small intestinal tract and produce the major pathogenic factor C. perfringens enterotoxin. Although sporulation plays a critical role in the pathogenesis of FBI, the mechanisms inducing sporulation remain unclear. Bile salts were shown previously to induce sporulation, and we confirmed deoxycholate (DCA)-induced sporulation in C. perfringens strain NCTC8239 cocultured with human intestinal epithelial Caco-2 cells. In the present study, we performed transcriptome analyses of strain NCTC8239 in order to elucidate the mechanism underlying DCA-induced sporulation. Of the 2,761 genes analyzed, 333 were up- or downregulated during DCA-induced sporulation and included genes for cell division, nutrient metabolism, signal transduction, and defense mechanisms. In contrast, the virulence-associated transcriptional regulators (the VirR/VirS system, the agr system, codY, and abrB) were not activated by DCA. DCA markedly increased the expression of signaling molecules controlled by Spo0A, the master regulator of the sporulation process, whereas the expression of spo0A itself was not altered in the presence or absence of DCA. The phosphorylation of Spo0A was enhanced in the presence of DCA. Collectively, these results demonstrated that DCA induced sporulation, at least partially, by facilitating the phosphorylation of Spo0A and activating Spo0A-regulated genes in strain NCTC8239 while altering the expression of various genes. IMPORTANCE: Disease caused by Clostridium perfringens type A consistently ranks among the most common bacterial foodborne illnesses in humans in developed countries. The sporulation of C. perfringens in the small intestinal tract is a key event for its pathogenesis, but the factors and underlying mechanisms by which C. perfringens sporulates in vivo currently remain unclear. Bile salts, major components of bile, which is secreted from the liver for the emulsification of lipids, were shown to induce sporulation. However, the mechanisms underlying bile salt-induced sporulation have not yet been clarified. In the present study, we demonstrate that deoxycholate (one of the bile salts) induces sporulation by facilitating the phosphorylation of Spo0A and activating Spo0A-regulated genes using a transcriptome analysis. Thus, this study enhances our understanding of the mechanisms underlying sporulation, particularly that of bile salt-induced sporulation, in C. perfringens.

Effects of High-Pressure Treatment on Spores of Clostridium Species.

UNLABELLED: This work analyzes the high-pressure (HP) germination of spores of the food-borne pathogen Clostridium perfringens (with inner membrane [IM] germinant receptors [GRs]) and the opportunistic pathogen Clostridium difficile (with no IM GRs), which has growing implications as an emerging food safety threat. In contrast to those of spores of Bacillus species, mechanisms of HP germination of clostridial spores have not been well studied. HP treatments trigger Bacillus spore germination through spores' IM GRs at ∼150 MPa or through SpoVA channels for release of spores' dipicolinic acid (DPA) at ≥400 MPa, and DPA-less spores have lower wet heat resistance than dormant spores. We found that C. difficile spores exhibited no germination events upon 150-MPa treatment and were not heat sensitized. In contrast, 150-MPa-treated unactivated C. perfringens spores released DPA and became heat sensitive, although most spores did not complete germination by fully rehydrating the spore core, but this treatment of heat-activated spores led to almost complete germination and greater heat sensitization. Spores of both clostridial organisms released DPA during 550-MPa treatment, but C. difficile spores did not complete germination and remained heat resistant. Heat-activated 550-MPa-HP-treated C. perfringens spores germinated almost completely and became heat sensitive. However, unactivated 550-MPa-treated C. perfringens spores did not germinate completely and were less heat sensitive than spores that completed germination. Since C. difficile and C. perfringens spores use different mechanisms for sensing germinants, our results may allow refinement of HP methods for their inactivation in foods and other applications and may guide the development of commercially sterile low-acid foods. IMPORTANCE: Spores of various clostridial organisms cause human disease, sometimes due to food contamination by spores. Because of these spores' resistance to normal decontamination regimens, there is continued interest in ways to kill spores without compromising food quality. High hydrostatic pressure (HP) under appropriate conditions can inactivate bacterial spores. With growing use of HP for food pasteurization, advancement of HP for commercial production of sterile low-acid foods requires understanding of mechanisms of spores' interactions with HP. While much is known about HP germination and inactivation of spores of Bacillus species, how HP germinates and inactivates clostridial spores is less well understood. In this work we have tried to remedy this information deficit by examining germination of spores of Clostridium difficile and Clostridium perfringens by several HP and temperature levels. The results may give insight that could facilitate more efficient methods for spore eradication in food sterilization or pasteurization, biodecontamination, and health care.

Antimicrobial resistance in Salmonella enterica serovar typhi and paratyphi in South Asia-current status, issues and prospects.

The human race owes a debt of gratitude to antimicrobial agents, penicillin and its successors that have saved people from tremendous pain and suffering in the last several decades. Unfortunately, this consideration is no more true, as millions of people are prone to the challenging threat of emergence of antimicrobial resistance worldwide and the menace is more distressing in developing countries. Comparable with other bacterial species, Salmonella enterica serovar Typhi (S. typhi) and Paratyphi (S. paratyphi) have been evolving multidrug resistance (MDR) against a wide array of antibiotics, including chloramphenicol, ampicillin and co-trimoxazole, and globally affecting 21 million people with 220,000 deaths each year. S. typhi and S. paratyphi infections are also endemic in South Asia and a series of antibiotics used to treat these infections, have been losing efficacy against enteric fever. Currently, quinolones are regarded as a choice to treat MDR Salmonella in these regions. Travel-related cases of enteric fever, especially from South Asian countries are the harbinger of the magnitude of MDR Salmonella in that region. Conclusively, the MDR will continue to grow and the available antimicrobial agents would become obsolete. Therefore, a radical and aggressive approach in terms of rational use of antibiotics during treating infections is essentially needed.

High hydrostatic pressure-induced inactivation of bacterial spores.

High hydrostatic pressure (HHP) is the most-widely adopted novel non-thermal technology for the commercial pasteurization of foods. However, HHP-induced inactivation of bacterial spores remains a challenge due to spore resistance to the treatment limits of currently available industrial HHP units (i.e. ~650 MPa and 50 °C). Several reports have demonstrated that high pressure can modulate the germination machinery of bacterial spores, rendering them susceptible to subsequent inactivation treatments. Unfortunately, high pressure-induced germination is a unique phenomenon for spores of the genus Bacillus but not of Clostridium. Alternative strategies to inactivate bacterial spores at commercially available pressure and temperature levels include promoting the germination step by inclusion of known germinants into the food formulation to increase the lethality of HHP treatments on bacterial spores. The aim of this review is to provide an overview of the molecular basis involved in pressure-triggered germination of bacterial spores and of novel strategies to inactivate bacterial spores with HHP treatments.

In vitro cytotoxicity induced by Clostridium perfringens isolate carrying a chromosomal cpe gene is exclusively dependent on sporulation and enterotoxin production.

Clostridium perfringens type A is a common source of food poisoning (FP) and non-food-borne (NFB) gastrointestinal diseases in humans. In the intestinal tract, the vegetative cells sporulate and produce a major pathogenic factor, C. perfringens enterotoxin (CPE). Most type A FP isolates carry a chromosomal cpe gene, whereas NFB type A isolates typically carry a plasmid-encoded cpe. In vitro, the purified CPE protein binds to a receptor and forms pores, exerting a cytotoxic activity in epithelial cells. However, it remains unclear if CPE is indispensable for C. perfringens cytotoxicity. In this study, we examined the cytotoxicity of cpe-harboring C. perfringens isolates co-cultured with human intestinal epithelial Caco-2 cells. The FP strains showed severe cytotoxicity during sporulation and CPE production, but not during vegetative cell growth. While Caco-2 cells were intact during co-culturing with cpe-null mutant derivative of strain SM101 (a FP strain carrying a chromosomal cpe gene), the wild-type level cytotoxicity was observed with cpe-complemented strain. In contrast, both wild-type and cpe-null mutant derivative of the NFB strain F4969 induced Caco-2 cell death during both vegetative and sporulation growth. Collectively, the Caco-2 cell cytotoxicity caused by C. perfringens strain SM101 is considered to be exclusively dependent on CPE production, whereas some additional toxins should be involved in F4969-mediated in vitro cytotoxicity.