Emergence of genetic diversity and multi-drug resistant Clostridium perfringens from wild birds.

BACKGROUND: Clostridium perfringens (C. perfringens) is an important zoonotic microorganism that can cause animal and human infections, however information about the prevalence status in wild birds of this pathogenic bacterium is currently limited. RESULT: In this study, 57 strains of C. perfringens were isolated from 328 fecal samples of wild birds. All the isolates were identified as type A and 70.18% of the isolates carried the cpb2 gene. Antimicrobial susceptibility testing showed that and 22.80% of the isolates were classified as multidrug-resistant strains. The MLST analysis of the 57 isolates from wild birds was categorized into 55 different sequence types (STs) and clustered into eight clonal complexes (CCs) with an average of 20.1 alleles and the Simpson Diversity index (Ds) of 0.9812, and revealed a high level of genetic diversity within the C. perfringens populations. Interestingly, the isolates from swan goose were clustered in the same CC while isolates from other bird species were more scattered suggesting that a potential difference in genetic diversity among the C. perfringens populations associated with different bird species. CONCLUSION: C. perfringens exhibits a wide range of host adaptations, varying degrees of antimicrobial resistance, and a high degree of genetic diversity in wild birds. Understanding the prevalence, toxin type, antimicrobial resistance, and genetic diversity of C. perfringens in wildlife populations is essential for developing effective strategies for disease control and management.

The bacterial profile and antibiotic susceptibility in skin and soft tissue infections at a tertiary care hospital of Quetta, Pakistan.

Objectives: To determine the bacterial profile and antibiotic susceptibility in skin and soft tissue infections among patients in a tertiary care setting. METHODS: The cross-sectional cohort study was conducted at the Centre for Advanced Studies in Vaccinology and Biotechnology, University of Balochistan, Quetta, Pakistan, from June 2021 to May 2022, and comprised bacteriainfected skin samples that were collected from the Bolan Medical Complex Hospital, Quetta, and the Sandeman Provincial Hospital, Quetta. The swab samples were immediately cultured, and positive samples were evaluated for biochemical tests, antibiotic susceptibility test and polymerase chain reaction. Data was analysed using SPSS 22. RESULTS: Of the 800 samples, 598(74.7%) tested positive for pathogenic bacteria. Staphylococcus aureus accounted for 316(39.5%) infections, followed by clostridium perfringens 18.96(2.37%), escherichia coli 120(15.12%), pseudomonas aeruginosa 98(12.25%) and klebsiella pneumoniae 44(5.5%). Among all the infected samples, 380(47.5%) belonged to males, 218(27.25%) to patients aged 5-20 years, 448(56%) to the uneducated subjects, and 462(57.87%) to patients having lower socioeconomic status. Pseudomonas aeruginosa showed the highest level of resistance against all antibiotics. Conclusion: Regular surveillance and proper use of antibiotics should be encouraged in hospitals to limit the spread of antibiotic resistance against pathogenic bacteria.

A case of Crimean-Congo hemorrhagic fever with the bacteremia of Clostridium perfringens.

Crimean-Congo hemorrhagic fever (CCHF) is a worldwide tick-borne viral infection in humans. The aim of the study is to report a case of a female patient with severe CCHF with the bacteremia of Clostridium perfringens. An 18-year-old woman admitted to the emergency department with sudden onset of fever, nausea and vomiting, myalgia, headache, generalized abdominal pain. It was learned that the patient was living in a rural area and had a history of tick bite 3 days before the admission. At laboratory examination, bicytopenia, abnormal liver function tests, and abnormal coagulation parameters were observed. The diagnosis of the case was confirmed with a positive real-time polymerase chain reaction. On the third day of hospitalization, she had an increase in abdominal pain, confusion, and respiratory distress. She was transferred to the intensive care unit for close monitoring. On the fifth day of hospitalization, she developed fever again. Catheter and peripheral anaerobic blood cultures grew C. perfringens. No evidence of perforation was observed on abdominal tomography. It has been successfully treated with a multidisciplinary approach. CCHF demonstrates different types of clinical presentations, except for common symptoms of fever and hemorrhage. A case of CCHF with C. perfringens bacteremia has not been previously reported before.

Bone and joint infections caused by Clostridium perfringens: a case series.

The objective of this study was to evaluate antimicrobial therapy outcomes of bone and joint infections (BJI) caused by Clostridium perfringens. We investigated remission of symptoms and the absence of relapse or reinfection during follow-up. Among the 8 patients with C. perfringens BJI, the type of infection was early prosthesis infection (n = 2), osteosynthetic device infection (n = 4), and chronic osteomyeletis (n = 2). Clindamycin-rifampicin combination was given in 4 cases and metronidazole in 4 cases. The overall success rate was 87.5%. Among the 7 patients who completed antibiotic treatment, the success rate was 100%. The clindamycin-rifampicin combination appeared to be effective in patients with C. perfringens BJI.

Activity of ethanolic extract of Eucalyptus globulus leaves against multi drug resistant poultry pathogens in broiler chicks.

The present study was designed to evaluate the antimicrobial activity of E. globulus leaves in broiler chicks. Total (n=255) day-old chicks were segregated into five groups i.e. Pathogenic E. coli, S. pullorum, S. gallinarum and C. perfringens type A and control negative group. Each bacterial challenged (1x 107 CFU) group was divided into control positive, antibiotic, probiotic and E. globulus group. Experimental birds were exposed to E. coli, S. pullorum, S. gallinarum and C. perfringens type A at different ages. At 35th day of experiment the log reduction for each group was determined. The highest log reduction in E. coli and C. perfringens Type A colonies count were found in E. globulus (3.26) (2.33) treated group followed by antibiotic (2.85) (1.59) and probiotic (2.84) (1.50) respectively. The log reduction in S. pullorum colonies count was highest in E. globulus (2.50) followed by probiotic (2.24) and antibiotic (2.16). The S. gallinarum colonies count log reduction was found highest for antibiotic (2.84) followed by probiotic (2.48) and E. globulus group. The results of in-vivo experiment revealed that ethanolic extract of E. globulus has antibacterial activity and it can be used as a replacement to low level of antibiotics added in poultry feed.

Phytochemical composition and In-vitro activity of ethanolic extract of Eucalyptus globulus leaves against multidrug resistant poultry pathogens.

Aim of the present study was to determine the In-vitro antibacterial activity of ethanolic extract of E. globulus leaves against common multidrug resistant poultry pathogens. Phytochemical analysis through HPLC revealed that kaempeferol (7.315min) followed by querecetin (6.655min) and myrecetin (3.655min). Percent area of kaempeferol (6826.88%) was highest, followed by myrecetin (5516.22%) and querecetin (163.748%). Phytochemical investigation of ethanolic extract of E. globulus leaves through GCMS showed highest retention time (min) α-pinene (20.43) and α-terpineol (20.15) accompanied by spathulenol (11.97), piperitone (11.04). The ethanolic extracts of E. globulus leaves showed a highest zone of inhibition against S. pullorum SP6; 20.64± 2.08, E. coli SE 12; 19.75± 2.83, C. perfringens type A (CPM38-01); 19.46± 2.02. The highest level of MIC of E. globulus noted were against S. gallinarum S22; 133.37±53.294, S. gallinarum S1; 130.20±45.10, S. gallinarum S4; 129.47±24.182, S. gallinarum S3; 126.83±72.392. In conclusion, the study confirmed that the ethanolic extract of E. globulus is composed of active ingredients having antibacterial activity and can be referred as an alternate to antibiotics.

Essential oil combinations against Clostridium perfringens and Clostridium septicum - the causative agents of gas gangrene.

AIMS: The inhibitory and bactericidal effect of a wide range of essential oils, and their selected combinations against two pathogens (Clostridium perfringens and Clostridium septicum), the causative pathogens of gas gangrenous infections were investigated. Fractional inhibitory indices were also calculated to determine the interactions. METHODS AND RESULTS: The Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) assays were used to determine the efficacy of the essential oils. Santalum austrocaledonicum demonstrated the highest activity inhibiting both Clostridial pathogens at the lowest concentration of 0·02 mg ml-1 . Santalum austrocaledonicum combined with Cymbopogon martinii had the strongest inhibition against C. perfringens (MIC 0·02 mg ml-1 ) and C. septicum (MIC 0·01 mg ml-1 ). Selected combinations demonstrated synergy (ΣFIC ≤ 0·50) in combination against both pathogens tested. Antagonism was also observed in many combinations. CONCLUSIONS: Selected essential oils, when studied either individually or in combination, have high inhibitory and bactericidal effects against both Clostridial strains. Nine combinations have proven to be synergistic with 23 combinations additive; 96 indifferent and 77 having an antagonistic effect against the pathogenic strains. Some combinations demonstrated extreme antagonism and as such, careful consideration needs to be given to essential oil selection against these pathogens. SIGNIFICANT IMPACT OF THE STUDY: Very few essential oils have been antimicrobially screened (MIC and MBC) against Clostridial strains and furthermore, the efficacies in combination are not known.

Insights on toxin genotyping, virulence, antibiogram profiling, biofilm formation and efficacy of disinfectants on biofilms of Clostridium perfringens isolated from poultry, animals and humans.

AIMS: This study aimed to determine the toxin genotypes, virulence determinants and antibiogram of Clostridium perfringens isolated from poultry, animals and humans. Biofilm formation and the efficacy of disinfectants on C. perfringens biofilms were studied. METHODS AND RESULTS: Thirty C. perfringens isolates (20 clinical and 10 from chicken carcasses) were genotyped by PCR and all isolates were genotype A (cpa+). The overall prevalence of cpe, cpb2, netB and tpeL virulence genes was 6·7, 56·7, 56·7 and 36·7% respectively. Twenty-one isolates (70%) were multidrug-resistant, 8 (26·7%) were extensive drug-resistant and one isolate (3·3%) was pan drug-resistant. The average multiple antibiotic resistance index was 0·7. Biofilms were produced by 63·3% of C. perfringens isolates and categorized as weak (36·7%), moderate (16·7%) and strong (10%). Sodium hypochlorite caused significant reduction in C. perfringens biofilms (P < 0·0001). CONCLUSIONS: All C. perfringens strains in this study were type A, resistant to multiple antibiotics and most of them were biofilm producers. Sodium hypochlorite showed higher efficacy in reducing C. perfringens biofilms. SIGNIFICANCE AND IMPACT OF THE STUDY: This study reported the efficacy of disinfectants in reducing C. perfringens biofilms of economic and public health concern and recommends application on surfaces in farms, food processing plants and slaughterhouses.

A study on fungal defensin against multidrug-resistant Clostridium perfringens and its treatment on infected poultry.

In the present study, we aimed to investigate the antibacterial activity and mechanisms of plectasin-derived peptide NZ2114 in vitro and its therapeutic effects in vivo on broilers challenged with Clostridium perfringens. In vitro assay showed that NZ2114 had potent (minimal inhibitory concentration, 0.91 µM) and rapid antibacterial activity (99.9% reduction within 2 h), and the dual antibacterial mechanisms (including interfering with the cell membrane and intracellular DNA) against C. perfringens CVCC 2030. In vivo study, NZ2114 tended to increase linearly and quadratically the average daily gain as NZ2114 level increased and was the highest at 20 mg/L. NZ2114 at 10 ~ 40 mg/L dramatically reduced jejunal lesion score. Besides, the levels of IL-6, TNF-α, and IL-1ß tended to downregulate linearly and quadratically as the NZ2114 level increased and were all the lowest at the dose of 20 mg/L. NZ2114 significantly upregulated those levels of IgA, IgG, IgM, and sIgA with a linear and quadratic dose effect, with the highest IgA, IgG, IgM, and sIgA at 20 mg/L. Finally, NZ2114 tended to linearly and quadratically increase the numerical value of crypt depth, with the lowest value at 40 mg/L. Lincomycin only dramatically reduced the jejunal lesion score and increased the numerical value of crypt depth. These results indicate that NZ2114 has the potential as a new alternative to antibiotics for the treatment of C. perfringens-induced necrotic enteritis infection.Key points• NZ2114 could kill C. perfringens by dual antibacterial mechanisms• Broiler necrotic enteritis model induced by C. perfringens was established• NZ2114 treatment could ameliorate C. perfringens-induced necrotic enteritis.

Evaluation of the expression and immunogenicity of four versions of recombinant Clostridium perfringens beta toxin designed by bioinformatics tools.

Beta toxins (CPB) produced by Clostridium perfringens type B and C cause various diseases in animals, and the use of toxoids is an important prophylactic measure against such diseases. Promising recombinant toxoids have been developed recently. However, both soluble and insoluble proteins expressed in Escherichia coli can interfere with the production and immunogenicity of these antigens. In this context, bioinformatics tools have been used to design new versions of the beta toxin, and levels of expression and solubility were evaluated in different strains of E. coli. The immunogenicity in sheep was assessed using the molecule with the greatest potential that was selected on analyzing these results. In silico analyzes, greater mRNA stability (-169.70 kcal/mol), solubility (-0.755), and better tertiary structure (-0.12) were shown by rCPB-C. None of the strains of E. coli expressed rFH8-CPB, but a high level of expression and solubility was shown by rCPB-C. Higher levels of total and neutralizing anti-CPB antibodies were observed in sheep inoculated with bacterins containing rCPB-C. Thus, this study suggests that due to higher productivity of rCPB-C in E. coli and immunogenicity, it is considered as the most promising molecule for the production of a recombinant vaccine against diseases caused by the beta toxin produced by C. perfringens type B and C.

Prevalence and characterization of Clostridium perfringens isolated from different chicken farms in China.

Clostridium perfringens (C. perfringens) is a common pathogenic microorganism present in nature, which can cause animal and human diseases, such as necrotizing enteritis (NE) in poultry. Little is known about the current prevalence status of C. perfringens from poultry farms of different types and regions in China. From December 2018 to August 2019, we investigated the prevalence, genotype distribution and drug resistance of C. perfringens from Guangdong, Pingyin, Tai'an and Weifang. A total of 622 samples were collected and processed for C. perfringens isolation, among which 239 (38.42%) samples were determined to be positive for C. perfringens. A total of 312 isolates of C. perfringens were recovered (1-5 strains were isolated for each positive sample), and 98.72% of the isolates were identified as type A, while the others were type F. Antimicrobial susceptibility testing revealed that 47.71% of the isolates were resistant to at least five classes of commonly used antibiotics. Multilocus sequence typing (MLST) showed that 74 representative isolates were divided into 63 sequence types (STs), and the Simpson's diversity index (Ds) of the STs for the five farms was 0.9799. 37.84% of the isolates were classified into seven clonal complexes (CC1-CC7), and the isolates from the same farm were more concentrated in the minimum spanning tree. In addition, some cloaca isolates and feed isolates were distributed in the same ST or CC; this result indicates that the C. perfringens in chicken can come from the environment (feed etc.).

Toxinotyping and molecular characterization of antimicrobial resistance in Clostridium perfringens isolated from different sources of livestock and poultry.

The present study was designed to understand the presence of antimicrobial resistance among the prevalent toxinotypes of Clostridium perfringens recovered from different animals of Tamil Nadu, India. A total of 75 (10.76%) C. perfringens were isolated from 697 multi-species fecal and intestinal content samples. C. perfringens type A (90.67%), type C (2.67%), type D (4%) and type F (2.67%) were recovered. Maximum number of isolates were recovered from dog (n = 20, 24.10%) followed by chicken (n = 19, 5.88%). Recovered isolates were resistant to gentamicin (44.00%), erythromycin (40.00%), bacitracin (40.00%), and tetracycline (26.67%), phenotypically and most of the isolates were found to be resistant to multiple antimicrobials. Genotypic characterization revealed that tetracycline (41.33%), erythromycin (34.66%) and bacitracin (17.33%) resistant genes were present individually or in combination among the isolates. Combined results of phenotypic and genotypic characterization showed the highest percentage of erythromycin resistance (26.66%) among the isolates. None of the isolates showed amplification for lincomycin resistance genes. The correlation matrix analysis of genotypic resistance showed a weak positive relationship between the tetracycline and bacitracin resistance while a weak negative relationship between the tetracycline and erythromycin resistance. The present study thus reports the presence of multiple-resistance genes among C. perfringens isolates that may be involved in the dissemination of resistance to other bacteria present across species. Further insights into the genome can help to understand the mechanism involved in gene transfer so that measures can be taken to prevent the AMR spread.

The Multifunctional Sactipeptide Ruminococcin C1 Displays Potent Antibacterial Activity In Vivo as Well as Other Beneficial Properties for Human Health.

The world is on the verge of a major antibiotic crisis as the emergence of resistant bacteria is increasing, and very few novel molecules have been discovered since the 1960s. In this context, scientists have been exploring alternatives to conventional antibiotics, such as ribosomally synthesized and post-translationally modified peptides (RiPPs). Interestingly, the highly potent in vitro antibacterial activity and safety of ruminococcin C1, a recently discovered RiPP belonging to the sactipeptide subclass, has been demonstrated. The present results show that ruminococcin C1 is efficient at curing infection and at protecting challenged mice from Clostridium perfringens with a lower dose than the conventional antibiotic vancomycin. Moreover, antimicrobial peptide (AMP) is also effective against this pathogen in the complex microbial community of the gut environment, with a selective impact on a few bacterial genera, while maintaining a global homeostasis of the microbiome. In addition, ruminococcin C1 exhibits other biological activities that could be beneficial for human health, as well as other fields of applications. Overall, this study, by using an in vivo infection approach, confirms the antimicrobial clinical potential and highlights the multiple functional properties of ruminococcin C1, thus extending its therapeutic interest.

Assessment of Immune Response and Efficacy of Essential Oils Application on Controlling Necrotic Enteritis Induced by Clostridium perfringens in Broiler Chickens.

Necrotic enteritis (NE) caused by Clostridium perfringens is one of the most important enteric diseases in poultry. The antibacterial activity of two different essential oil (EO) blends against C. perfringens was investigated both in vitro and in vivo. Additionally, the immunological response to EO treatment was assessed. In the in vitro study, the antibacterial activity of EO formulas and commonly used antibiotics was evaluated against C. perfringens using disk diffusion assay, minimum inhibitory concentration (MIC) assay, and minimum bactericidal concentration (MBC) assay. In the in vivo study, NE experimental infection was performed on 440 Ross broiler chicks at 19 days of age for 4 continuous days. The chicks were treated with either EOs or amoxicillin at 22 days of age for 5 continuous days. One day after the end of treatment, the birds' performance was evaluated by calculating the feed conversion ratio. Serum samples from 120 birds were collected to measure the levels of IL-1ß, IFN-γ, IL-8, IL-10, and IL-17. After that, all birds were slaughtered, and their small intestines were subjected to gross and histopathological evaluation. In addition, bacterial counts in the small intestines were evaluated. In the in vitro study, EOs showed higher antimicrobial activities in comparison with antibiotics against C. perfringens. In the in vivo study, birds treated with EOs showed a significant decrease in bacterial counts, a significant decrease in intestinal lesions, and a significant improvement in performance compared with untreated birds (p < 0.05). Moreover, treating birds with EOs directed the immune system toward an anti-inflammatory pathway. None of the treated birds died due to NE compared with the 10% mortality rate in untreated birds. In conclusion, EOs might be an effective and safe alternative to antibiotics in the treatment of chicken NE.

The Biological Activity of Monarda didyma L. Essential Oil and Its Effect as a Diet Supplement in Mice and Broiler Chicken.

The use of growth-promoting antibiotics in livestock faces increasing scrutiny and opposition due to concerns about the increased occurrence of antibiotic-resistant bacteria. Alternative solutions are being sought, and plants of Lamiaceae may provide an alternative to synthetic antibiotics in animal nutrition. In this study, we extracted essential oil from Monarda didyma, a member of the Lamiaceae family. We examined the chemical composition of the essential oil and then evaluated the antibacterial, antioxidant, and anti-inflammatory activities of M. didyma essential oil and its main compounds in vitro. We then evaluated the effectiveness of M. didyma essential oil in regard to growth performance, feed efficiency, and mortality in both mice and broilers. Carvacrol (49.03%) was the dominant compound in the essential oil extracts. M. didyma essential oil demonstrated antibacterial properties against Escherichia coli (MIC = 87 µg·mL-1), Staphylococcus aureus (MIC = 47 µg·mL-1), and Clostridium perfringens (MIC = 35 µg·mL-1). Supplementing the diet of mice with essential oil at a concentration of 0.1% significantly increased body weight (+5.4%) and feed efficiency (+18.85%). In broilers, M. didyma essential oil significantly improved body weight gain (2.64%). Our results suggest that adding M. didyma essential oil to the diet of broilers offers a potential substitute for antibiotic growth promoters.

Antibacterial activity of cyadox against Clostridium perfringens in broilers and a dosage regimen design based on pharmacokinetic-pharmacodynamic modeling.

Necrotic enteritis is an intestinal disease caused by Clostridium perfringens (C. perfringens) that results in high economic losses to the poultry industry. The purpose of this study was to investigate the antibacterial activity of cyadox against C. perfringens and to formulate its dosage regimen based on pharmacokinetics/pharmacodynamics (PK/PD) modeling in broilers. The PK parameters of cyadox in ileum of healthy and infected broilers following oral administration at 30 mg/kg body weight (BW) were investigated and PD study the MIC, MBC, MPC, and PAE were determined. The time-killing curves were established in vitro and ex vivo to evaluate the antibacterial activity of cyadox against C. perfringens. The results revealed that the MIC of cyadox against C. perfringens was 1-16 µg/mL. After oral administration of cyadox, the peak concentration (Cmax), maximum concentration time (Tmax), and area under the concentration-time curve (AUC) in ileum content of broilers were 143.55-161.48 µg/mL, 1.08-1.25 h, and 359.51-405.69 µg h/mL respectively. After Integrating the in vivo PK and ex vivo PD data the AUC24h/MIC values needed for bacteriostatic, bactericidal and bacterial eradication were 27.71 h, 78.93 h, and 165.14 h, respectively. By model validation, the cure rate was 85.71%. In conclusion, a dosage regimen of 14.02 mg/kg repeated after every 12 h for 3-5days was expected to be therapeutically effective in broilers against C. perfringens with MIC ≤2 µg/mL.

The Tcp plasmids of Clostridium perfringens require the resP gene to ensure stable inheritance.

Many of the disease-causing toxins of the pathogenic bacterium Clostridium perfringens are harboured on large, highly stable, conjugative plasmids. Previous work has established the requirement of a ParMRC-like partitioning system for plasmid maintenance, but little is known about other mechanisms used to ensure stable plasmid inheritance. The archetypal 47 kb Tcp plasmid, pCW3, encodes a gene, resP, whose putative product has sequence similarity to members of the serine recombinase family of site-specific recombinases. ResP is therefore likely to function to resolve plasmid multimers. Sequence analysis identified that resP genes are present on all C. perfringens plasmid families, suggesting a conserved function in these plasmids. To assess the requirement of resP for the stability of pCW3, deletion mutants were constructed. Deletion of resP from pCW3 resulted in a marked instability phenotype that was rescued upon complementation with the wild-type resP gene. Complementation with resP genes from two different C. perfringens plasmids demonstrated that only closely related resP genes can complement the mutation on pCW3. The function of ResP in vivo was examined using an Escherichia coli model system, which determined that two directly repeated res sites were required for the resolution of DNA and that ResP could resolve multimeric plasmid forms into monomeric units. Based on these findings we concluded that ResP could catalyse the resolution of plasmid multimers and was required for the maintenance of Tcp plasmids within C. perfringens. Overall, the results of this study have significant implications for our understanding of the maintenance of toxin-encoding plasmids within C. perfringens.

Effect of combination of Oxyrase and sodium thioglycolate on growth of Clostridium perfringens from spores under aerobic incubation.

Clostridium perfringens is a strictly anaerobic pathogen that requires absence of oxygen for its growth in laboratory experiments, which is usually attained by using an anaerobic chamber or anaerobic jars. However, it has been demonstrated that C. perfringens may survive for short periods of times due to its adaptive response to O2. Therefore, the objective of this study was to explore the application of Oxyrase (OX) and sodium thioglycolate (ST) as oxygen scavengers, used alone or in combination, for observation of the growth of C. perfringens under aerobic incubation. The growth of C. perfringens from spores in Schaedler Anaerobe Agar containing different levels and combinations of OX and ST was observed at temperatures between 20 and 50 °C. The kinetic parameters, including lag time, specific growth rate, and maximum cell concentrations in the stationary phase, were determined. The results indicated that ST at concentrations of 0.025 and 0.05% (w/w), although allowing eventual growth of C. perfringens, prolonged its lag times, while OX at 1.5% only allowed growth at a lower growth rate in comparison to anaerobic incubation. OX at 3% enhanced the growth of C. perfringens at temperatures between 30 and 50 °C, while higher levels of OX were needed in the medium to support the growth of C. perfringens during storage at 25 °C (>6% OX) and 20 °C (>9% OX), due to the effect of temperature on enzyme activity. No significant difference was found in the kinetic parameters of C. perfringens incubated aerobically with OX and the control (without OX or ST) in an anaerobic chamber. Therefore, OX at appropriate concentrations may allow the observation of the growth of C. perfringens under aerobic incubation conditions without the need of an anaerobic device.

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.

Effects of Chitosan on Clostridium perfringens and Application in the Preservation of Pork Sausage.

The effects of chitosan with 95% deacetylation degree (DD95) on the spore germination, cell proliferation, and heat resistance of Clostridium perfringens CCRC 10,648 and CCRC 13,019 were investigated, and its application on pork sausage with sodium nitrite reduction was also evaluated. DD95 chitosan can strongly reduce the heat resistance of both strains. The D80 and D100 values for strain CCRC 13,019 decreased from 40.98 and 4.64 min to 39.21 and 3.26 min, respectively, as a result of adding 250 ppm DD95; meanwhile, addition of chitosan decreased the D80 and D100 values for CCRC 10,648 from 41.15 and 6.46 min to 39.52 and 3.78 min, respectively. In pork sausage, addition of 3000 ppm DD95 chitosan considerably slowed down the bacterial proliferation and volatile basic nitrogen production. There were no significant differences in color (L* and b* values), shearing force, and hardness in the pork sausages with or without DD95 chitosan during storage at 4 and 25 °C. However, the addition of DD95 chitosan in pork sausage significantly retarded the decrease of the a* value. Therefore, DD95 chitosan could reduce the concentration of sodium nitrite required in pork sausages for color retention.