Inhibitory effects of sucrose palmitic acid ester on the germination-to-outgrowth process of Clostridium perfringens SM101 spores.

As a commercially available esterified compound derived from sucrose and palmitoyl acids, sucrose ester palmitic acid (SEPA) has been used as an emulsifier in food processing. It possesses antibacterial activity against vegetative and spore-forming bacteria, including Clostridium, Moorella, Bacillus, and Geobacillus species, prompting the food industry to use it as a food additive to achieve a desirable shelf life; however, the precise mechanism by which the compound affects the physiological processes of bacteria and how it inhibits bacterial growth remains unclear. In this study, we focused on the inhibitory effect of SEPA on the germination-to-outgrowth process of Clostridium perfringens SM101 spores, a strain widely used as a model of C. perfringens. When the isolated spores were exposed to ≧ 20 µg/ml of SEPA on brain heart infusion agar, bacterial colony formation was completely inhibited. Time-resolved phase-contrast microscopy was employed to visualize the effect of SEPA on the entire regrowth process of SM101 spores. SEPA did not affect the "germination stage," where each spore changes its optical density from phase-bright to phase-dark. In contrast, the presence of SEPA completely blocked the "outgrowth stage," in which the newly synthesized vegetative cell body emerges from the cracked spore shell. The results demonstrate that SEPA inhibits the revival process of the spores of a pathogenic strain of C. perfringens and that the site of its action is the "outgrowth stage" and not the "germination stage," as evidenced by single- cell analysis.

The presence of differentiated C2C12 muscle cells enhances toxin production and growth by Clostridium perfringens type A strain ATCC3624.

Clostridium perfringens type A causes gas gangrene, which involves muscle infection. Both alpha toxin (PLC), encoded by the plc gene, and perfringolysin O (PFO), encoded by the pfoA gene, are important when type A strains cause gas gangrene in a mouse model. This study used the differentiated C2C12 muscle cell line to test the hypothesis that one or both of those toxins contributes to gas gangrene pathogenesis by releasing growth nutrients from muscle cells. RT-qPCR analyses showed that the presence of differentiated C2C12 cells induces C. perfringens type A strain ATCC3624 to upregulate plc and pfoA expression, as well as increase expression of several regulatory genes, including virS/R, agrB/D, and eutV/W. The VirS/R two component regulatory system (TCRS) and its coupled Agr-like quorum sensing system, along with the EutV/W TCRS (which regulates expression of genes involved in ethanolamine [EA] utilization), were shown to mediate the C2C12 cell-induced increase in plc and pfoA expression. EA was demonstrated to increase toxin gene expression. ATCC3624 growth increased in the presence of differentiated C2C12 muscle cells and this effect was shown to involve both PFO and PLC. Those membrane-active toxins were each cytotoxic for differentiated C2C12 cells, suggesting they support ATCC3624 growth by releasing nutrients from differentiated C2C12 cells. These findings support a model where, during gas gangrene, increased production of PFO and PLC in the presence of muscle cells causes more damage to those host cells, which release nutrients like EA that are then used to support C. perfringens growth in muscle.

Growth and no-growth boundary of Clostridium perfringens in cooked cured meats - A logistic analysis and development of critical control surfaces using a solid growth medium.

This study was conducted to evaluate the effect of sodium nitrite (NaNO2, 100-200 ppm), sodium erythorbate (SE, 0-547 ppm), sodium tripolyphosphate (STPP, 0-0.5 %), and sodium chloride (NaCl, 2-3 %) on growth of C. perfringens using a solid growth medium and to develop a growth/no-growth boundary (critical control surface, or CCS) to prevent its growth in cooked cured meat under the optimal temperature condition. Melted Shahidi Ferguson Perfringens (SFP) agar, inoculated with a 3-strain spore cocktail and mixed with NaNO2, SE, STPP, and NaCl according to a Box-Behnken response surface experimental design, was dispersed in 96-well microplates and incubated anaerobically in an incubator programmed to remain at 4 °C for 24 h, heat to 80 °C in 1.75 h, quickly (0.5 h) cool to 46 °C (optimum temperature), and then maintain at 46 °C overnight. The plates were examined optically and visually for colony formation. Any well free of growth was designated as no-growth. Logistic regression was used to analyze the growth probability (P) as affected by NaNO2, SE, STPP, and NaCl and define a CSS as meeting the criterion of P < 1/96. The results showed that STPP and the interactions of SE with NaNO2 and NaCl could reduce the growth probability of C. perfringens in SFP agar. The validation of CCS with ground beef showed an accuracy of 96.3 % for no growth of C. perfringens in the inoculated samples. The results of this study proved that cured meat can be formulated with proper combinations of NaNO2, SE, STPP, and NaCl to prevent the growth of C. perfringens even under the optimum temperature condition, thus preventing food poisoning caused by the growth of this microorganism.

Survival of Clostridium perfringens, Staphylococcus aureus and Salmonella enterica in alternatively cured bacon during cooking and process deviations.

Pork bellies were injected with four different alternative curing brines. The bellies were inoculated on the surface and at a depth of 1 cm with multiple strains of Clostridium perfringens, Staphylococcus aureus and Salmonella enterica. The bellies were processed using either a standard process cycle or an interrupted process cycle to simulate a process deviation. Additionally, laboratory simulation of the same cycles was conducted where surface inoculated pork belly samples (22 ± 1 g) were processed in a circulating water bath. Microbiological populations were determined at the beginning, mid-point and end of the cycles, and the change in population was calculated for each bacterium at each time point, by comparing the population to the initial inoculated population. Irrespective of the brine or process cycle, the populations of all of the inoculated bacteria on both the surface and interior samples had decreased by the end of the process. There was no difference in the reductions in bacterial populations for all of the inoculated bacteria by brine type or by sample location (P > 0.30). There were differences in the microbial population reductions for C. perfringens attributable to the processing cycle (P < 0.001), with less population reductions associated with the standard cycle when compared to the interrupted cycle. However, no differences (P > 0.10) were observed in the population reductions between the two processing cycles for either S. aureus or S. enterica.

NanI Sialidase Enhances the Action of Clostridium perfringens Enterotoxin in the Presence of Mucus.

Clostridium perfringens enterotoxin (CPE) is the main virulence factor for C. perfringens type F strains to cause human gastrointestinal diseases, which can involve lethal enterotoxemia. During type F disease, CPE encounters an adherent mucus layer overlying the intestines, so the current study evaluated if NanI potentiates CPE activity in the presence of adherent mucus. CPE alone caused more cytotoxicity transepithelial electrical resistance (TEER) and permeability to fluorescent dextran (FD) for minimal mucus-producing HT29 cells versus that in their derivative HT29-MTX-E12 cells, which produce abundant adherent mucus. However, for HT29-MTX-E12 cells, the presence of NanI significantly increased CPE binding and pore formation, which enhanced their sensitivity to CPE effects on cytotoxicity, TEER, and FD permeability. When the ability of NanI to potentiate CPE-induced enterotoxemia was then tested in a mouse small intestinal loop enterotoxemia model, a pathophysiologically relevant 50 µg/mL dose of CPE did not kill mice. However, the copresence of purified NanI resulted in significant CPE-induced lethality. More CPE was detected in the sera of mice challenged with 50 µg/mL of CPE when NanI was copresent during challenge. The copresence of NanI and CPE during challenge also significantly increased intestinal histologic damage compared to that after challenge with CPE alone, suggesting that NanI enhancement of CPE-induced intestinal damage may increase CPE absorption into blood. Overall, these results indicate that (i) mucus inhibits CPE action and (ii) NanI can potentiate CPE action in the presence of mucus, which may help explain why type F strains that produce relatively low levels of CPE are still pathogenic. IMPORTANCE NanI is a sialidase produced by some Clostridium perfringens type F strains. Here, we found that NanI can significantly increase the action of C. perfringens enterotoxin (CPE), which is the main toxin responsible for severe human enteric disease caused by type F strains. This effect likely helps to explain why even some type F strains that produce small amounts of CPE are pathogenic.

Stress-Related Herpesvirus Reactivation in Badgers Can Result in Clostridium Proliferation.

Clostridium perfringens is an important food-borne zoonotic pathogen and a member of the commensal gut microbiome of many mammals. Predisposing factors such as coinfection with other pathogens or diet change can, however, cause overgrowth and subsequent disease development. Here we investigated the occurrence of C. perfringens in a free-ranging badger population with up to 100% prevalence of herpesvirus infection. Herpesvirus reactivation is known to be associated with increased susceptibility bacterial infections. PCR screening of rectal swabs from 69 free-ranging badgers revealed 15.9% (11/69, 95% CI = 9.1-26.3%) prevalence of detectable C. perfringens (Type A) DNA in the digestive tracts of assymptomatic animals. The results of Fisher's exact test revealed C. perfringens detection was not biased by age, sex and seasons. However, badgers with genital tract gammaherpesvirus (MusGHV-1) reactivation (p = 0.007) and infection with a specific MusGHV-1 genotype (p = 0.019) were more prone to of C. perfringens proliferation, indicating coinfection biased dynamics of intestinal C. perfringens. An inclusion pattern analysis further indicated that, causally, MusGHV-1 reactivation potentiated C. perfringens detection. Whether or not specific MusGHV-1 genotype infection or reactivation plays a role in C. perfringens overgrowth or disease development in badgers will require further investigation. Nevertheless, a postmortem examination of a single badger that died of fatal disease, likely associated with C. perfringens, revealed MusGHV-1 detection in the small intestine.

Protective Effects of Lactobacillus plantarum Lac16 on Clostridium perfringens Infection-Associated Injury in IPEC-J2 Cells.

Clostridium perfringens (C. perfringens) causes intestinal injury through overgrowth and the secretion of multiple toxins, leading to diarrhea and necrotic enteritis in animals, including pigs, chickens, and sheep. This study aimed to investigate the protective effects of Lactobacillus plantarum (L. plantarum) Lac16 on C. perfringens infection-associated injury in intestinal porcine epithelial cell line (IPEC-J2). The results showed that L. plantarum Lac16 significantly inhibited the growth of C. perfringens, which was accompanied by a decrease in pH levels. In addition, L. plantarum Lac16 significantly elevated the mRNA expression levels of host defense peptides (HDPs) in IPEC-J2 cells, decreased the adhesion of C. perfringens to IPEC-J2 cells, and attenuated C. perfringens-induced cellular cytotoxicity and intestinal barrier damage. Furthermore, L. plantarum Lac16 significantly suppressed C. perfringens-induced gene expressions of proinflammatory cytokines and pattern recognition receptors (PRRs) in IPEC-J2 cells. Moreover, L. plantarum Lac16 preincubation effectively inhibited the phosphorylation of p65 caused by C. perfringens infection. Collectively, probiotic L. plantarum Lac16 exerts protective effects against C. perfringens infection-associated injury in IPEC-J2 cells.

NanI Sialidase Contributes to the Growth and Adherence of Clostridium perfringens Type F Strain F4969 in the Presence of Adherent Mucus.

Clostridium perfringens type F strains causing nonfoodborne human gastrointestinal diseases (NFD) typically produce NanI sialidase as their major secreted sialidase. Type F NFDs can persist for several weeks, indicating their pathogenesis involves intestinal colonization, including vegetative cell growth and adherence, with subsequent sporulation that fosters enterotoxin production and release. We previously reported that NanI contributes to type F NFD strain adherence and growth using Caco-2 cells. However, Caco-2 cells make minimal amounts of mucus, which is significant because the intestines are coated with adherent mucus. Therefore, it was important to assess if NanI contributes to the growth and adherence of type F NFD strains in the presence of adherent mucus. Consequently, the current study first demonstrated greater growth of nanI-carrying versus non-nanI-carrying type F strains in the presence of HT29-MTX-E12 cells, which produce an adherent mucus layer, versus their parental HT29 cells, which make minimal mucus. Demonstrating the specific importance of NanI for this effect, type F NFD strain F4969 or a complementing strain grew and adhered better than an isogenic nanI null mutant in the presence of HT29-MTX-E12 cells versus HT29 cells. Those effects involved mucus production by HT29-MTX-E12 cells since mucus reduction using N-acetyl cysteine reduced F4969 growth and adherence. Consistent with those in vitro results, NanI contributed to growth of F4969 in the mouse small intestine. By demonstrating a growth and adherence role for NanI in the presence of adherent mucus, these results further support NanI as a potential virulence factor during type F NFDs.

Bacteriocin-Like Inhibitory Substance (BLIS) Activity of Enterococcus faecium DB1 Against Biofilm Formation by Clostridium perfringens.

The antibiofilm effect of bacteriocin-like inhibitory substance (BLIS) from Enterococcus faecium DB1 against Clostridium perfringens was investigated in the present study. BLIS of E. faecium DB1 significantly reduced biofilm formation by C. perfringens in a dose-dependent manner for 24 and 48 h. In particular, treatment with BLIS of E. faecium DB1 significantly inhibited biofilm formation by C. perfringens on chicken meat and stainless steel coupon surfaces. Moreover, BLIS of E. faecium DB1 decreased the viability of C. perfringens biofilm and planktonic cells, indicating that the reduction of biofilm formation by C. perfringens might be achieved by killing the bacterial cells. Taken together, the present results suggest that BLIS of E. faecium DB1 can be a promising antibiofilm agent to eradicate C. perfringens.

Predictive model for growth of Clostridium perfringens during cooling of cooked pork supplemented with sodium chloride and sodium pyrophosphate.

A dynamic model was developed to predict growth of Clostridium perfringens in cooked ground pork supplemented with salt (0-3% wt/wt) and sodium pyrophosphate (0-0.3% wt/wt) under varying temperatures. C. perfringens (NCTC 8238, NCTC 8239, and NCTC 10240) spores were heat shocked, cooled, and inoculated into ground pork. Isothermal bacterial growth was quantified with variable salt and phosphate concentrations at temperatures ranging from 15 to 51 °C. The primary Baranyi model was fitted to all C. perfringens growth profiles and gave a satisfactory fit (R2 ≥ 0.85). A quadratic polynomial secondary model was developed (P < 0.0001) to predict the maximum specific growth rate as a function of temperature, salt, and phosphate concentrations (R2 = 0.93). A dynamic model was developed and validated using growth data retrieved from 7 published studies. Thirty three out of 44 predictions were within the acceptable prediction zone (-0.5 ≤ prediction error ≤ 1.0). The developed predictive model can be used to minimize the risk of C. perfringens in pork products supplemented with additives during cooling.

Selection and application of natural antimicrobials to control Clostridium perfringens in sous-vide chicken breasts inhibition of C. perfringens in sous-vide chicken.

Current consumer preferences for both clean label food ingredients and convenience-based foods has provided a unique opportunity to explore the application of novel natural food preservatives in sous vide products. The anaerobic environment and relatively low thermal processing of the sous vide process creates a favorable environment for the survival, germination, and outgrowth of spore-forming bacterium Clostridium perfringens. The aim of this study was to identify effective novel natural ingredient formulations against C. perfringens and apply them within a vacuum-sealed sous vide chicken model exposed to abusive storage and chilling conditions. Among six commercial vinegar-based formulations, liquid vinegar with citrus extract (CE; 1.0%) and with lemon juice concentrate (LJC; 1.5%) were identified as the most effective at inhibiting three individual C. perfringens strains. Both reduced viable cell counts by 5 log CFU/mL (P < 0.05), whereas reductions in spore counts ranged from 2 to 4 log CFU/mL depending on formulation and concentration used. Once incorporated to chicken meat 1.0% CE and 1.5% LJC before sous-vide cooking, completely inhibited the growth of mixed C. perfringens strains (P < 0.05) during storage for 16 days at 12 and 16 °C. Exponential cooling from 54 to 4 °C was performed for 18 h to imitate abusive storage conditions. CE and LJC at 3.0% inhibited growth and reduced counts by 3.4 and 2.9 log CFU/g compared to respective controls. Treatments CE and LJC could be implemented within the formulation of a sous vide chicken product to provide an effective protection against C. perfringens meeting clean label expectations.

NanH Is Produced by Sporulating Cultures of Clostridium perfringens Type F Food Poisoning Strains and Enhances the Cytotoxicity of C. perfringens Enterotoxin.

Clostridium perfringens type F food poisoning (FP) strains cause one of the most common foodborne illnesses. This FP develops when type F FP strains sporulate in the intestines and produce C. perfringens enterotoxin (CPE), which is responsible for the diarrhea and abdominal cramps of this disease. While C. perfringens can produce up to three different sialidases, the current study surveyed FP strains, which confirmed the results of a previous study that they consistently carry the nanH sialidase gene, often as their only sialidase gene. NanH production was found to be associated with sporulating cultures of the surveyed type F FP strains, including SM101 (a transformable derivative of a FP strain). The sporulation-associated regulation of NanH production by strain SM101 growing in modified Duncan-Strong medium (MDS) was shown to involve Spo0A, but it did not require the completion of sporulation. NanH production was not necessary for either the growth or sporulation of SM101 when cultured in MDS. In those MDS cultures, NanH accumulated in the sporulating mother cell until it was released coincidently with CPE. Since CPE becomes extracellular when mother cells lyse to release their mature spores, this indicates that mother cell lysis is also important for NanH release. The copresence of NanH and CPE in supernatants from lysed sporulating cultures was shown to enhance CPE cytotoxicity for Caco-2 cells. This enhancement was attributable to NanH increasing CPE binding and could be replicated with purified recombinant NanH. These in vitro findings suggest that NanH may be an accessory virulence factor during type F FP.IMPORTANCEClostridium perfringens type F strains cause the second most common bacterial foodborne illness in the United States. C. perfringens enterotoxin (CPE) is responsible for the diarrhea and cramping symptoms of this food poisoning (FP). Previous studies showed that NanI sialidase can enhance CPE activity in vitro While many type F FP strains do not produce NanI, they do consistently make NanH sialidase. This study shows that, like CPE, NanH is produced by sporulating type F FP strains and then released extracellularly when their sporulating cells lyse to release their mature spore. NanH was shown to enhance CPE cytotoxicity in vitro by increasing CPE binding to cultured Caco-2 cells. This enhancement could be important because many type F FP strains produce less CPE than necessary (in a purified form) to cause intestinal pathology in animal models. Therefore, NanH represents a potential accessory virulence factor for type F FP.

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.

Evidence That VirS Is a Receptor for the Signaling Peptide of the Clostridium perfringens Agr-like Quorum Sensing System.

Since both the Agr (accessory gene regulator)-like quorum sensing (QS) system and VirS/VirR (VirS/R) two-component regulatory system of Clostridium perfringens positively regulate production of several toxins, including C. perfringens beta toxin (CPB), it has been hypothesized the VirS membrane sensor protein is an Agr-like QS signaling peptide (SP) receptor. To begin evaluating whether VirS is an SP receptor, this study sequenced the virS gene in C. perfringens strains CN3685 and CN1795 because it was reported that agrB mutants of both strains increase CPB production in response to the pentapeptide 5R, likely the natural SP, but only the CN3685 agrB mutant responds to 8R, which is 5R plus a 3-amino-acid tail. This sequencing identified differences between the predicted VirS extracellular loop 2 (ECL2) of CN3685 versus that of CN1795. To explore if those ECL2 differences explain strain-related variations in SP sensitivity and support VirS as an SP receptor, virS agrB double-null mutants of each strain were complemented to swap which VirS protein they produce. CPB Western blotting showed that this complementation changed the natural responsiveness of each strain to 8R. A pulldown experiment using biotin-5R demonstrated that VirS can bind SP. To further support VirS:SP binding and to identify a VirS binding site for SP, a 14-mer peptide corresponding to VirS ECL2 was synthesized. This ECL2 peptide inhibited 5R signaling to agrB mutant and wild-type strains. This inhibition was specific, since a single N to D substitution in the ECL2 peptide abrogated these effects. Collectively, these results support VirS as an important SP receptor and may assist development of therapeutics.IMPORTANCEC. perfringens beta toxin (CPB) is essential for the virulence of type C strains, a common cause of fatal necrotizing enteritis and enterotoxemia in humans and domestic animals. Production of CPB, as well as several other C. perfringens toxins, is positively regulated by both the Agr-like QS system and the VirS/R two-component regulatory system. This study presents evidence that the VirS membrane sensor protein is a receptor for the AgrD-derived SP and that the second extracellular loop of VirS is important for SP binding. Understanding interactions between SP and VirS improves knowledge of C. perfringens pathogenicity and may provide insights for designing novel strategies to reduce C. perfringens toxin production during infections.

Dietary Mannan-oligosaccharides potentiate the beneficial effects of Bifidobacterium bifidum in broiler chicken.

This study investigated the effects of dietary Bifidobacterium bifidum (BFD) and mannan-oligosaccharide (MOS), as a synbiotic, on the production performance, gut microbiology, serum biochemistry, antioxidant profile and health indices of broiler chicken. Six dietary treatments were T1 (negative control), T2 (positive control-20 mg antibiotic BMD kg-1 diet; BMD: bacitracin methylene disalicylate), T3 (0·1% MOS + 106  CFU BFD per g feed), T4 (0·1% MOS + 107  CFU BFD per g feed), T5 (0·2% MOS + 106  CFU BFD per g feed) and T6 (0·2% MOS + 107  CFU BFD per g feed). Significantly (P < 0·01) better growth performance and efficiency was observed in birds supplemented with 0·2% MOS along with 106  CFU BFD per g of feed compared to BMD and control birds. Supplementation with 0·2% MOS along with either 106 or 107  CFU BFD per g feed reduced (P < 0·01) the gut coliform, Escherichia coli, total plate count, and Clostridium perfringens count and increased the Lactobacillus and Bifidobacterium count. Significantly (P < 0·01) higher serum and liver antioxidant enzyme pool, serum HDL cholesterol and lower serum glucose, triglyceride, total cholesterol, cardiac risk ratio, atherogenic coefficient and atherogenic index of plasma were observed in birds supplemented with 0·2% MOS along with 106  CFU BFD per g of feed compared to control or BMD supplemented birds. Better production performance, gut microbial composition, serum biochemistry, antioxidant profile and health indices were depicted by broiler chicken supplemented with 0·2% MOS and 106  CFU BFD per g of feed.

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.

Near-infrared spectroscopy coupled with chemometrics algorithms for the quantitative determination of the germinability of Clostridium perfringens in four different matrices.

Clostridium perfringens (C. perfringens) has the ability to form metabolically-dormant spores that can survive food preservation processes and cause food spoilage and foodborne safety risks upon germination outgrowth. This study was conducted to investigate the effects of different AGFK concentrations (0, 50, 100, 200 mM/mL) on the spore germination of C. perfringens in four matrices, including Tris-HCl, FTG, milk, and chicken soup. C. perfringens spore germinability was investigated using near infrared spectroscopy (NIRS) combined with chemometrics. The spore germination rate (S), the OD600%, and the Ca2+-DPA% were measured using traditional spore germination methods. The results of spore germination assays showed that the optimum germination rate was obtained using 100 mM/L concentrations of AGFK in the FTG medium, and the S, OD600% and Ca2+-DPA% were 98.6%, 59.3% and 95%, respectively. The best prediction models for the S, OD600% and Ca2+-DPA% were obtained using SNV as the preprocessing method for the original spectra, with the competitive adaptive weighted resampling method (CARS) as the characteristic variables related to the selected spore germination methods from NIRS data. The results of the S showed that the optimum model was built by CARS-PLSR (RMSEV = 0.745, Rc = 0.897, RMSEP = 0.769, Rp = 0.883). For the OD600%, interval partial least squares regression (CARS-siPLS) was performed to optimize the models. The calibration yielded acceptable results (RMSEV = 0.218, Rc = 0.879, RMSEP = 0.257, Rp = 0.845). For the Ca2+-DPA%, the optimum model with CARS-siPLS yielded acceptable results (RMSEV = 44.7, Rc = 0.883, RMSEP = 50.2, Rp = 0.872). This indicated that quantitative determinations of the germinability of C. perfringens spores using NIR technology is feasible. A new method based on NIR was provided for rapid, automatic, and non-destructive determination of the germinability of C. perfringens spores.

Growth of Clostridium perfringens in cooked chicken during cooling: One-step dynamic inverse analysis, sensitivity analysis, and Markov Chain Monte Carlo simulation.

The objective of this study was to determine the kinetic parameters and apply Markov Chain Monte Carlo (MCMC) simulation to predict the growth of Clostridium perfringens from spores in cooked ground chicken meat during dynamic cooling. Inoculated samples were exposed to various cooling conditions to observe dynamic growth. A combination of 4 cooling profiles was used in one-step inverse analysis with the Baranyi model as the primary model and the cardinal parameters model as the secondary model. Six kinetic parameters of the Baranyi model and the cardinal parameters model, including Q0, Ymax, µopt, Tmin, Topt, and Tmax, were estimated. The estimated Tmin, Topt, and Tmax were 14.8, 42.9, and 50.5 °C, respectively, with a µopt of 5.25 h-1 and maximum cell density of 8.4 log CFU/g. Correlation analysis showed that both Q0 and Ymax are weakly correlated to other parameters, while the remaining parameters are mostly mildly to strongly correlated with each other. Although it may be difficult to estimate highly correlated parameters using a single temperature profile, one-step analysis with multiple different temperature profiles helped estimate them successfully. The estimated parameters were used as the prior information to construct the posterior distribution for Bayesian analysis. MCMC simulation was used to predict the bacterial growth using different dynamic temperature profiles for validation of the accuracy of the predictive models. The MCMC simulation results showed that the Bayesian analysis produced more accurate predictions of bacterial growth during cooling than the deterministic method. With Bayesian analysis, the root-mean-square-error (RMSE) of prediction was only 0.1 log CFU/g with all residual errors within ±0.25 log CFU/g. Therefore, Bayesian analysis is recommended for predicting the growth of C. perfringens in cooked meat during cooling.

Lactobacillus rhamnosus Granules Dose-Dependently Balance Intestinal Microbiome Disorders and Ameliorate Chronic Alcohol-Induced Liver Injury.

As the functions of Lactobacilli become better understood, there are increasing numbers of applications for Lactobacillus products. Previously, we have demonstrated that Lactobacillus rhamnosus GG (LGG) can prevent alcoholic liver injury. LGG granules were produced by fluid bed granulation with a media composed of starch, skimmed milk powder, whey powder, microcrystalline cellulose and maltose, and LGG fermented liquid that comprised 30-50% of the total weight. We found LGG granules dose-dependently protected against chronic alcoholic liver disease. When alcohol was consumed for 8 weeks with LGG treatment during the last 2 weeks, we demonstrated that the dose dependence of LGG granules can improve alcohol-induced liver injury through decreasing the levels of lipopolysaccharide and tumor necrosis factor-α in serum and prevent liver steatosis by suppressing triglyceride, free fatty acid, and malondialdehyde production in liver. Alcohol feeding caused a decline in the number of both Lactobacillus and Bifidobacterium, with a proportional increase in the number of Clostridium perfringens in ileum, and expansion of the Gram-negative bacteria Proteobacteria, Campylobacterales, and Helicobacter in cecum. However, LGG granule treatment restored the content of these microorganisms. In conclusion, LGG granule supplementation can improve the intestinal microbiota, reduce the number of gram-negative bacteria, and ameliorate alcoholic liver injury.

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.