Identification of Germinants and Expression of Germination Genes in Clostridium perfringens Strains Isolated from Diarrheic Animals.

In this study, we investigated the spore germination phenotype of Clostridium perfringens strains isolated from diarrheic animals (animal strains). The transcripts of germination-specific genes and their protein products were also measured. Our study found the following results: (i) animal strains spores germinated at a slower rate with AK (mixture of L-asparagine and KCl), L-cysteine, or L-lysine, but the extent of germination varied based on strains and germinants used; (ii) none of the amino acids (excluding L-cysteine and L-lysine) were identified as a universal germinant for spores of animal strains; (iii) animal strain spores germinated better at a pH range of 6.0-7.0; (iv) all tested germination-specific genes were expressed in animal strains; the levels of expression of major germinant receptor gene (gerKC) were higher and the cortex hydrolysis machinery genes (cspB and sleC) were lower in animal strains, compared to the food poisoning strain SM101; and (v) the levels of CspB and SleC were significantly lower in spores of animal strains compared to strain SM101, suggesting that these animal strains lack an efficient spore cortex hydrolysis machinery. In summary, our findings suggest that the poor or slow spore germination in C. perfringens animal strains might be due to incomplete spore cortex hydrolysis.

Divalent Cation Signaling in Clostridium perfringens Spore Germination.

Spore germination plays an essential role in the pathogenesis of Clostridium perfringens-associated food poisoning. Germination is initiated when bacterial spores sense various stimuli, including chemicals and enzymes. A previous study showed that dipicolinic acid (DPA) chelated with calcium (Ca-DPA) significantly stimulated spore germination in C. perfringens. However, whether Ca2+ or DPA alone can induce germination is unknown. Therefore, we aimed to evaluate the possible roles of Ca2+ and other divalent cations present in the spore core, such as Mn2+ and Mg2+, in C. perfringens spore germination. Our study demonstrated that (i) Ca-DPA, but not DPA alone, induced C. perfringens spore germination, suggesting that Ca2+ might play a signaling role; (ii) all tested calcium salts induced spore germination, indicating that Ca2+ is critical for germination; (iii) the spore-specific divalent cations Mn2+ and Mg2+, but not Zn2+, induced spore germination, suggesting that spore core-specific divalent cations are involved in C. perfringens spore germination; and (iv) endogenous Ca2+ and Mg2+ are not required for induction of C. perfringens spore germination, whereas exogenous and partly endogenous Mn2+ are required. Collectively, our results suggest that exogenous spore core-specific divalent cation signals are more important than endogenous signals for the induction of spore germination.

Characterization of Putative Sporulation and Germination Genes in Clostridium perfringens Food-Poisoning Strain SM101.

Bacterial sporulation and spore germination are two intriguing processes that involve the expression of many genes coherently. Phylogenetic analyses revealed gene conservation among spore-forming Firmicutes, especially in Bacilli and Clostridia. In this study, by homology search, we found Bacillus subtilis sporulation gene homologs of bkdR, ylmC, ylxY, ylzA, ytaF, ytxC, yyaC1, and yyaC2 in Clostridium perfringenes food-poisoning Type F strain SM101. The ß-glucuronidase reporter assay revealed that promoters of six out of eight tested genes (i.e., bkdR, ylmC, ytaF, ytxC, yyaC1, and yyaC2) were expressed only during sporulation, but not vegetative growth, suggesting that these genes are sporulation-specific. Gene knock-out studies demonstrated that C. perfringens ΔbkdR, ΔylmC, ΔytxC, and ΔyyaC1 mutant strains produced a significantly lower number of spores compared to the wild-type strain. When the spores of these six mutant strains were examined for their germination abilities in presence of known germinants, an almost wild-type level germination was observed with spores of ΔytaF or ΔyyaC1 mutants; and a slightly lower level with spores of ΔbkdR or ΔylmC mutants. In contrast, almost no germination was observed with spores of ΔytxC or ΔyyaC2 mutants. Consistent with germination defects, ΔytxC or ΔyyaC2 spores were also defective in spore outgrowth and colony formation. The germination, outgrowth, and colony formation defects of ΔytxC or ΔyyaC2 spores were restored when ΔytxC or ΔyyaC2 mutant was complemented with wild-type ytxC or yyaC2, respectively. Collectively, our current study identified new sporulation and germination genes in C. perfringens.

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.

Sporulation and Germination in Clostridial Pathogens.

As obligate anaerobes, clostridial pathogens depend on their metabolically dormant, oxygen-tolerant spore form to transmit disease. However, the molecular mechanisms by which those spores germinate to initiate infection and then form new spores to transmit infection remain poorly understood. While sporulation and germination have been well characterized in Bacillus subtilis and Bacillus anthracis, striking differences in the regulation of these processes have been observed between the bacilli and the clostridia, with even some conserved proteins exhibiting differences in their requirements and functions. Here, we review our current understanding of how clostridial pathogens, specifically Clostridium perfringens, Clostridium botulinum, and Clostridioides difficile, induce sporulation in response to environmental cues, assemble resistant spores, and germinate metabolically dormant spores in response to environmental cues. We also discuss the direct relationship between toxin production and spore formation in these pathogens.

An enhanced green fluorescence protein (EGFP)-based reporter assay for quantitative detection of sporulation in Clostridium perfringens SM101.

Clostridium perfringens type F is a spore-forming anaerobe that causes bacterial food-borne illness in humans. The disease develops when ingested vegetative cells reach the intestinal tract and begin to form spores that produce the diarrheagenic C. perfringens enterotoxin (CPE). Given that CPE production is regulated by the master regulator of sporulation (transcription factor Spo0A), the identification of sporulation-inducing factors in the intestine is relevant to better understanding of the disease. To examine these factors, we established assays to quantify C. perfringens sporulation stage under microscopy by using two fluorescent reporters, namely, Evoglow-Bs2 and CpEGFP. When the reporter genes were placed under control of the cpe promoter, both protein products were expressed specifically during sporulation. However, the intensity of the anaerobic reporter Evoglow-Bs2 was weak and rapidly photobleached during microscopic observation. Alternatively, CpEGFP, a canonical green fluorescence protein with optimized codon usage for Clostridium species, was readily detectable in the mother-cell compartment of most bacteria at early stages of sporulation. Additionally, CpEGFP expression predicted final spore yield and was quantifiable in 96-well plates using fluorescence plate reader. These results indicate that CpEGFP can be used to analyze the sporulation of C. perfringens and has a potential application in the large-scale screening of sporulation-regulating biomolecules.

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.

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.

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.

Inactivation of Clostridium perfringens spores adhered onto stainless steel surface by agents used in a clean-in-place procedure.

Enterotoxigenic Clostridium perfringens, a leading foodborne pathogen can be cross-contaminated from food processing stainless steel (SS) surfaces to the finished food products. This is mostly due to the high resistance of C. perfringens spores adhered onto SS surfaces to various disinfectants commonly used in food industries. In this study, we aimed to investigate the survivability and adherence of C. perfringens spores onto SS surfaces and then validate the effectiveness of a simulated Clean-in-Place (CIP) regime on inactivation of spores adhered onto SS surfaces. Our results demonstrated that, 1) C. perfringens spores adhered firmly onto SS surfaces and survived for at-least 48 h, unlike their vegetative cells who died within 30 min, after aerobic incubation at refrigerated and ambient temperatures; 2) Spores exhibited higher levels of hydrophobicity than vegetative cells, suggesting a correlation between cell surface hydrophobicity and adhesion to solid surfaces; 3) Intact spores were more hydrophobic than the decoated spores, suggesting a positive role of spore coat components on spores' hydrophobicity and thus adhesion onto SS surfaces; and finally 4) The CIP regime (NaOH + HNO3) successfully inactivated C. perfringens spores adhered onto SS surfaces, and most of the effect of CIP regime appeared to be due to the NaOH. Collectively, our current findings may well contribute towards developing a strategy to control cross-contamination of C. perfringens spores into food products, which should help reducing the risk of C. perfringens-associated food poisoning outbreaks.

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.

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.

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.

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.