Emulsified and Nanoemulsified Essential Oils in the Control of Clostridium botulinum and Clostridium sporogenes in Mortadella.

Clostridium botulinum is a foodborne pathogen responsible for severe neuroparalytic disease associated with the ingestion of pre-formed toxin in food, with processed meats and canned foods being the most affected. Control of this pathogen in meat products is carried out using the preservative sodium nitrite (NaNO2), which in food, under certain conditions, such as thermal processing and storage, can form carcinogenic compounds. Therefore, the objective was to use nanoemulsified essential oils (EOs) as natural antimicrobial agents, with the aim of reducing the dose of NaNO2 applied in mortadella. The antimicrobial activity of nanoemulsions prepared with mixtures of EOs of garlic, clove, pink pepper, and black pepper was evaluated on endospores and vegetative cells of C. botulinum and Clostridium sporogenes (surrogate model) inoculated in mortadella prepared with 50 parts per million NaNO2. The effects on the technological (pH, water activity, and color) and sensory characteristics of the product were also evaluated. The combinations of EOs and their nanoemulsions showed sporicidal effects on the endospores of both tested microorganisms, with no counts observed from the 10th day of analysis. Furthermore, bacteriostatic effects on the studied microorganisms were observed. Regarding the technological and sensorial characteristics of the product, the addition of the combined EOs had a negative impact on the color of the mortadella and on the flavor/aroma. Despite the strong commercial appeal of adding natural preservatives to foods, the effects on flavor and color must be considered. Given the importance of controlling C. botulinum in this type of product, as well as the reduction in the amount of NaNO2 used, this combination of EOs represents a promising antimicrobial alternative to this preservative, encouraging further research in this direction.

The synergic interaction between environmental factors (pH and NaCl) and the physiological state (vegetative cells and spores) provides new possibilities for optimizing processes to manage risk of C. sporogenes spoilage.

Clostridium sporogenes has been widely used as a surrogate for proteolytic C. botulinum for validating thermal processes in low-acid cans. To limit the intensity of heat treatments, industrials must use other ways of control as an association of acidic and saline environment after a low heat treatment. The probability of growth of pH (7-4.4), sodium chloride concentration (0-11%) and heat treatment (80°C-10 min; 100°C-1.5 min and 5.2 min) were studied on C. sporogenes PA 3679 spores and vegetative cells. Vegetative cells or heat-treated spores were inoculated in PYGm broth at 30 °C for 48 days in anaerobic conditions. Vegetative cells growth (pH 4.6-pH 4.5; 7%-8% NaCl) range is larger than the spore one (pH 5.2-pH 5.0; 6%-7% NaCl). Spores germination and outgrowth rage is decreased if the spores are heat-treated at 100 °C for 1.5 min (pH 5.5-5.3; 4%-5% NaCl) and 5.2 min (pH 5.7-5.3; 4%-5% NaCl). The C. sporogenes PA 3679 spores germination and outgrowth is impacted by their physiological state. The synergic interaction between environmental factors (pH and NaCl) and the physiological state (vegetative cells and spores) opening new possibilities for optimizing food formulation processes to manage the risks of C. sporogenes spoilage.

Comparative assessment of the therapeutic drug targets of C. botulinum ATCC 3502 and C. difficile str. 630 using in silico subtractive proteomics approach.

Growing antimicrobial resistance of the pathogens against multiple drugs posed a serious threat to the human health worldwide. This fueled the need of identifying the novel therapeutic targets that can be used for developing new class of the drugs. Recently, there is a substantial rise in the rate of Clostridium infections as well as in the emergence of virulent and antibiotic resistant strains. Hence, there is an urgent need for the identification of potential therapeutic targets and the development of new drugs for the treatment and prevention of Clostridium infections. In the present study, a combinatorial approach involving systems biology and comparative genomics strategy was tested against Clostridium botulinum ATCC 3502 and Clostridium difficile str. 630 pathogens, to render potential therapeutic target at qualitative and quantitative level. This resulted in the identification of five common (present in both the pathogens, 34 in C. botulinum ATCC 3502 and 42 in C. difficile str. 630) drug targets followed by virtual screening-based identification of potential inhibitors employing molecular docking and molecular dynamics simulations. The identified targets will provide a solid platform for the designing of novel wide-spectrum lead compounds capable of inhibiting their catalytic activities against multidrug-resistant Clostridium in the near future.

Antimicrobial Susceptibility of 260 Clostridium botulinum Type A, B, Ba, and Bf Strains and a Neurotoxigenic Clostridium baratii Type F Strain Isolated from California Infant Botulism Patients.

Infant botulism is an infectious intestinal toxemia that results from colonization of the infant large bowel by Clostridium botulinum (or rarely, by neurotoxigenic Clostridium baratii or Clostridium butyricum), with subsequent intraintestinal production and absorption of botulinum neurotoxin that then produces flaccid paralysis. The disease is often initially misdiagnosed as suspected sepsis or meningitis, diagnoses that require prompt empirical antimicrobial therapy. Antibiotics may also be needed to treat infectious complications of infant botulism, such as pneumonia or urinary tract infection. Clinical evidence suggests (see case report below) that broad-spectrum antibiotics that are eliminated by biliary excretion may cause progression of the patient's paralysis by lysing C. botulinum vegetative cells in the large bowel lumen, thereby increasing the amount of botulinum neurotoxin available for absorption. The purpose of this antimicrobial susceptibility study was to identify an antimicrobial agent with little or no activity against C. botulinum that could be used to treat infant botulism patients initially diagnosed with suspected sepsis or meningitis, or who acquired secondary infections, without lysing C. botulinum Testing of 12 antimicrobial agents indicated that almost all California infant botulism patient isolates are susceptible to most clinically utilized antibiotics and are also susceptible to newer antibiotics not previously tested against large numbers of C. botulinum patient isolates. No antibiotic with little or no activity against C. botulinum was identified. These findings reinforce the importance of promptly treating infant botulism patients with human botulism immune globulin (BIG-IV [BabyBIG]).

Mechanism of bio molecule stabilized selenium nanoparticles against oxidation process and Clostridium Botulinum.

The bio molecules from plant leaf extract utilized in the preparation of selenium material at the nano scale. The selenium ion was reduced to selenium nanoparticles in the presence of molecule residue of the plant leaf extract. The bio molecule stabilized selenium nanoparticles were grown gradually in the reaction mixture. The selenium nanoparticles were characterized using atomic absorption spectroscopy, fourier transform inferred spectroscopy, X-ray diffraction, scanning electronic microscope and transmission electronic microscope. The selenium nanoparticles were synthesized successfully as the nano-crystalline pure hexagonal phase and the size range of 26-41 nm with spherical in shape. The activity and mechanism of nanoparticles suggested that the selenium nanoparticles are causes of leakage of reducing sugars and protein of pathogens membrane cell. The selenium nano are responsible for death and fully inhibited the microbial growth of pathogen. The bio molecule stabilized selenium nanoparticles were also investigated for the antioxidant agent. Selenium nanoparticles showed scavenging activity up to 94.48%. These results recommended that the advantages of using this method for synthesis of selenium nanoparticles with excellent antioxidant and antimicrobial mechanism and activity, which can be used as the antioxidant and antibiotic agent.

Small molecule non-peptide inhibitors of botulinum neurotoxin serotype E: Structure-activity relationship and a pharmacophore model.

Botulinum neurotoxins (BoNTs) are the most poisonous biological substance known to humans. They cause flaccid paralysis by blocking the release of acetylcholine at the neuromuscular junction. Here, we report a number of small molecule non-peptide inhibitors of BoNT serotype E. The structure-activity relationship and a pharmacophore model are presented. Although non-peptidic in nature, these inhibitors mimic key features of the uncleavable substrate peptide Arg-Ile-Met-Glu (RIME) of the SNAP-25 protein. Among the compounds tested, most of the potent inhibitors bear a zinc-chelating moiety connected to a hydrophobic and aromatic moiety through a carboxyl or amide linker. All of them show low micromolar IC50 values.

Camelid-derived heavy-chain nanobody against Clostridium botulinum neurotoxin E in Pichia pastoris.

Botulinum neurotoxins (BoNTs) result in severe and often fatal disease, botulism. Common remedial measures such as equine antitoxin and human botulism immunoglobulin in turn are problematic and time-consuming. Therefore, diagnosis and therapy of BoNTs are vital. The variable domain of heavy-chain antibodies (VHH) has unique features, such as the ability to identify and bind specifically to target epitopes and ease of production in bacteria and yeast. The Pichia pastoris is suitable for expression of recombinant antibody fragments. Disulfide bond formation and correct folds of protein with a high yield are some of the advantages of this eukaryotic host. In this study, we have expressed and purified the camelid VHH against BoNT/E in P. pastoris. The final yield of P. pastoris-expressed antibody was estimated to be 16 mg/l, which is higher than that expressed by Escherichia coli. The nanobody expressed in P. pastoris neutralized 4LD50 of the BoNT/E upon i.p. injection in 25% of mice. The nanobody expressed in E. coli extended the mice's survival to 1.5-fold compared to the control. This experiment indicated that the quality of expressed protein in the yeast is superior to that of the bacterial expression. Favorable protein folding by P. pastoris seems to play a role in its better toxin-binding property.

Protective potential of recombinant non-purified botulinum neurotoxin serotypes C and D.

Botulinum neurotoxin (BoNT) serotypes C and D are responsible for cattle botulism, a fatal paralytic disease that results in great economic losses in livestock production. Vaccination is the main approach to prevent cattle botulism. However, production of commercially available vaccines (toxoids) involves high risk and presents variation of BoNT production between batches. Such limitations can be attenuated by the development of novel nontoxic recombinant vaccines through a simple and reproducible process. The aim of this study was to evaluate the protective potential of recombinant non-purified botulinum neurotoxin serotypes C and D. Bivalent vaccines containing 200 µg rHCC and rHCD each were formulated in three different ways: (1) purified antigens; (2) recombinant Escherichia coli bacterins; (3) recombinant E. coli cell lysates (supernatant and inclusion bodies). Guinea pigs immunized subcutaneously with recombinant formulations developed a protective immune response against the respective BoNTs as determined by a mouse neutralization bioassay with pooled sera. Purified recombinant antigens were capable of inducing 13 IU/mL antitoxin C and 21 IU/mL antitoxin D. Similarly, both the recombinant bacterins and the cell lysate formulations were capable of inducing 12 IU/mL antitoxin C and 20 IU/mL antitoxin D. These values are two times as high as compared to values induced by the commercial toxoid used as control, and two to ten times as high as the minimum amount required by the Brazilian Ministry of Agriculture, Livestock and Food Supply (MAPA), respectively. Therefore, we used a practical, industry-friendly, and efficient vaccine production process that resulted in formulations capable of inducing protective immune response (neutralizing antitoxins) against botulism serotypes C and D.

Prioritizing drug targets in Clostridium botulinum with a computational systems biology approach.

A computational and in silico system level framework was developed to identify and prioritize the antibacterial drug targets in Clostridium botulinum (Clb), the causative agent of flaccid paralysis in humans that can be fatal in 5 to 10% of cases. This disease is difficult to control due to the emergence of drug-resistant pathogenic strains and the only available treatment antitoxin which can target the neurotoxin at the extracellular level and cannot reverse the paralysis. This study framework is based on comprehensive systems-scale analysis of genomic sequence homology and phylogenetic relationships among Clostridium, other infectious bacteria, host and human gut flora. First, the entire 2628-annotated genes of this bacterial genome were categorized into essential, non-essential and virulence genes. The results obtained showed that 39% of essential proteins that functionally interact with virulence proteins were identified, which could be a key to new interventions that may kill the bacteria and minimize the host damage caused by the virulence factors. Second, a comprehensive comparative COGs and blast sequence analysis of these proteins and host proteins to minimize the risks of side effects was carried out. This revealed that 47% of a set of C. botulinum proteins were evolutionary related with Homo sapiens proteins to sort out the non-human homologs. Third, orthology analysis with other infectious bacteria to assess broad-spectrum effects was executed and COGs were mostly found in Clostridia, Bacilli (Firmicutes), and in alpha and beta Proteobacteria. Fourth, a comparative phylogenetic analysis was performed with human microbiota to filter out drug targets that may also affect human gut flora. This reduced the list of candidate proteins down to 131. Finally, the role of these putative drug targets in clostridial biological pathways was studied while subcellular localization of these candidate proteins in bacterial cellular system exhibited that 68% of the proteins were located in the cytoplasm, out of which 6% was virulent. Finally, this framework may serve as a general computational strategy for future drug target identification in infectious diseases.

Inhibition of Listeria monocytogenes and Clostridium botulinum in Uncured Shredded Pork and Turkey Packaged Under Reduced Oxygen Conditions.

Cooked, uncured meat products packaged under reduced oxygen packaging conditions require the control of anaerobic and facultative anaerobic pathogens if they are held at temperatures greater than 3°C at retail or consumer level. The objective of this study was to determine the inhibition of Listeria monocytogenes and Clostridium botulinum in cooked, uncured shredded turkey and pork formulated with synthetic or clean-label antimicrobials. Treatments of shredded meat products were prepared with or without antimicrobials using turkey thigh or breast that were cooked to 85°C, shredded, and chilled before inoculation with the target pathogen. L. monocytogenes inoculated samples were stored at 7.2°C, whereas C. botulinum samples were stored at 12.8°C; triplicate samples were assayed every 2 weeks. In the first set of experiments, L. monocytogenes populations increased 2 to 3 logs within 2 weeks of storage at 7.2°C in both meat control treatments without antimicrobials and in pork with 4% lactate-diacetate blend (LD). A 1-log increase was observed in turkey with 4% LD and Pork with 2% cultured dextrose-vinegar-rosemary (CDVR) under the same storage conditions; a 1-log increase was observed in turkey with CDVR at 4 weeks. The second set of experiments tested the effect of pH reduction (to less than 5.5 by the addition of 0.5% citric acid) in combination with 2% CDVR when added to the brine precook or postcook during shredding. Populations of L. monocytogenes increased 4-log within 2 and 4 weeks at 7.2°C for the control turkey and pork formulations, respectively. No growth was observed in 12 weeks for any antimicrobial CDVR-CA treatments regardless of how antimicrobial was added. Similarly, botulinum toxin was detected in both control treatments at week 2 at 12.8°C, but no toxicity was observed in either antimicrobial treatment through 12 weeks. These data suggest that a combination of 2% cultured dextrose-vinegar-rosemary extract plus 0.5% citric acid to reduce pH inhibits the growth of L. monocytogenes and toxin production of C. botulinum in uncured shredded turkey and pork products stored under mild temperature abuse conditions for up to 12 weeks in reduced oxygen packaging.

Glyphosate suppresses the antagonistic effect of Enterococcus spp. on Clostridium botulinum.

During the last 10-15 years, an increase of Clostridium botulinum associated diseases in cattle has been observed in Germany. The reason for this development is currently unknown. The normal intestinal microflora is a critical factor in preventing intestinal colonisation by C. botulinum as shown in the mouse model of infant botulism. Numerous bacteria in the gastro-intestinal tract (GIT) produce bacteriocines directed against C. botulinum and other pathogens: Lactic acid producing bacteria (LAB) such as lactobacilli, lactococci and enterococci, generate bacteriocines that are effective against Clostridium spp. A reduction of LAB in the GIT microbiota by ingestion of strong biocides like glyphosate could be an explanation for the observed increase in levels of C. botulinum associated diseases. In the present paper, we report on the toxicity of glyphosate to the most prevalent Enterococcus spp. in the GIT. Ingestion of this herbicide could be a significant predisposing factor that is associated with the increase in C. botulinum mediated diseases in cattle.

Peptide inhibitors of botulinum neurotoxin serotype A: design, inhibition, cocrystal structures, structure-activity relationship and pharmacophore modeling.

Clostridium botulinum neurotoxins are classified as Category A bioterrorism agents by the Centers for Disease Control and Prevention (CDC). The seven serotypes (A-G) of the botulinum neurotoxin, the causative agent of the disease botulism, block neurotransmitter release by specifically cleaving one of the three SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins and induce flaccid paralysis. Using a structure-based drug-design approach, a number of peptide inhibitors were designed and their inhibitory activity against botulinum serotype A (BoNT/A) protease was determined. The most potent peptide, RRGF, inhibited BoNT/A protease with an IC(50) of 0.9 µM and a K(i) of 358 nM. High-resolution crystal structures of various peptide inhibitors in complex with the BoNT/A protease domain were also determined. Based on the inhibitory activities and the atomic interactions deduced from the cocrystal structures, the structure-activity relationship was analyzed and a pharmacophore model was developed. Unlike the currently available models, this pharmacophore model is based on a number of enzyme-inhibitor peptide cocrystal structures and improved the existing models significantly, incorporating new features.

Functional EL-HN Fragment as a Potent Candidate Vaccine for the Prevention of Botulinum Neurotoxin Serotype E.

Clostridium botulinum produces botulinum neurotoxin (BoNT), which is the most toxic known protein and the causative agent of human botulism. BoNTs have similar structures and functions, comprising three functional domains: catalytic domain (L), translocation domain (HN), and receptor-binding domain (Hc). In the present study, BoNT/E was selected as a model toxin to further explore the immunological significance of each domain. The EL-HN fragment (L and HN domains of BoNT/E) retained the enzymatic activity without in vivo neurotoxicity. Extensive investigations showed EL-HN functional fragment had the highest protective efficacy and contained some functional neutralizing epitopes. Further experiments demonstrated the EL-HN provided a superior protective effect compared with the EHc or EHc and EL-HN combination. Thus, the EL-HN played an important role in immune protection against BoNT/E and could provide an excellent platform for the design of botulinum vaccines and neutralizing antibodies. The EL-HN has the potential to replace EHc or toxoid as the optimal immunogen for the botulinum vaccine.

Important role of class I heat shock genes hrcA and dnaK in the heat shock response and the response to pH and NaCl stress of group I Clostridium botulinum strain ATCC 3502.

Class I heat shock genes (HSGs) code for molecular chaperones which play a major role in the bacterial response to sudden increases of environmental temperature by assisting protein folding. Quantitative reverse transcriptase real-time PCR gene expression analysis of the food-borne pathogen Clostridium botulinum grown at 37°C showed that the class I HSGs grpE, dnaK, dnaJ, groEL, and groES and their repressor, hrcA, were expressed at constant levels in the exponential and transitional growth phases, whereas strong downregulation of all six genes was observed during stationary phase. After heat shock from 37 to 45°C, all HSGs were transiently upregulated. A mutant with insertionally inactivated hrcA expressed higher levels of class I HSGs during exponential growth than the wild type, followed by upregulation of only groES and groES after heat shock. Inactivation of hrcA or of dnaK encoding a major chaperone resulted in lower maximum growth temperatures than for the wild type and reduced growth rates under optimal conditions compared to the wild type. The dnaK mutant showed growth inhibition under all tested temperature, pH, and NaCl stress conditions. In contrast, the growth of an hrcA mutant was unaffected by mild temperature or acid stress compared to the wild-type strain, indicating that induced class I HSGs support growth under moderately nonoptimal conditions. We show that the expression of class I HSGs plays a major role for survival and growth of C. botulinum under the stressful environmental conditions that may be encountered during food processing or growth in food products, in the mammalian intestine, or in wounds.

Disinfection methods for spores of Bacillus atrophaeus, B. anthracis, Clostridium tetani, C. botulinum and C. difficile.

To evaluate disinfection methods for environments contaminated with bioterrorism-associated microorganism (Bacillus anthracis), we performed the following experiments. First, the sporicidal effects of sodium hypochlorite on spores of five bacterial species were evaluated. Bacillus atrophaeus was the most resistant to hypochlorite, followed in order by B. anthracis, Clostridium botulinum and Clostridium tetani, and Clostridium difficile. Subsequently, using B. atrophaeus spores that were the most resistant to hypochlorite, the sporicidal effects of hypochlorite at lower pH by adding vinegar were evaluated. Hypochlorite containing vinegar had far more marked sporicidal effects than hypochlorite alone. Cleaning with 0.5% (5000 ppm) hypochlorite containing vinegar inactivated B. atrophaeus spores attached to vinyl chloride and plywood plates within 15 s, while that not containing vinegar did not inactivate spores attached to cement or plywood plates even after 1 h. Therefore, the surfaces of cement or plywood plates were covered with gauze soaked in 0.5% hypochlorite containing vinegar, and the sporicidal effects were evaluated. B. atrophaeus spores attached to plywood plates were not inactivated even after 6 h, but those attached to cement plates were inactivated within 5 min. On the other hand, covering the surfaces of plywood plates with gauze soaked in 0.3% peracetic acid and gauze soaked in 2% glutaral inactivated B. atrophaeus spores within 5 min and 6 h, respectively. These results suggest that hypochlorite containing vinegar is effective for disinfecting vinyl chloride, tile, and cement plates contaminated with B. anthracis, and peracetic acid is effective for disinfecting plywood plates contaminated with such microorganism.

Partial characterization of bacteriocins produced by two Lactobacilus strains with probiotic properties.

The probiotic characteristics of Lactobacillus brevis BG18 and Lb. plantarum BG33, isolated from traditional Turkish Tulum cheese were assessed. These two bacteriocinproducer strains exhibited good probiotic characteristics such as resistance in media containing 0.3% bile salt, pepsin (3 mg mL⁻¹), and pancreatine (1 mg mL⁻¹) as well as acid resistance at pH 2. They were also adhered to Caco-2 epithelial cells in a manner comparable to Escherichia coli LMG3083 (ETEC) and Salmonella Typhimurium SL1344. The strains produced a heat-stable antimicrobial compound that was shown to be proteinaceous in nature, and therefore, referred to as bacteriocins. The bacteriocins were able to inhibit growth of a number grampositive bacteria such as Listeria monocytogenes, Clostridium botulinum, Staphylococcus aureus and Bacillus cereus. Tricine-SDS-PAGE of the active fraction resulted in single bands with estimated molecular masses of 2.5 kDA and 2.7 kDA for Lb. brevis BG18 and Lb. plantarum BG33 bacteriocins, respectively.

Posttranslational Regulation of Botulinum Neurotoxin Production in Clostridium botulinum Hall A-hyper.

Botulinum neurotoxins (BoNTs) are the most toxic substances known to humankind and are the causative agents of the neuroparalytic disease botulism. Despite the overall importance of BoNTs in public health and safety, as a bioterrorism concern, and in pharmaceutical development, little is known about the molecular mechanisms mediating BoNT stability and degradation in various environments. Previous studies using Clostridium botulinum strain ATCC 3502 revealed that high levels of arginine (20 g/liter) repressed BoNT production approximately 1,000-fold. In the present study, the mechanisms of toxin reduction in arginine-enriched cultures of C. botulinum strain Hall A-hyper, which we have previously genetically manipulated using ClosTron technology, were explored. Cultures were grown in toxin production medium (TPM) and TPM enriched with arginine. Cultures were analyzed for growth (optical density at 600 nm [OD600]), changes in pH, and BoNT formation and stability. Our data indicate that arginine enrichment of C. botulinum strain Hall A-hyper cultures results in a pH shift that induces pH-dependent posttranslational control mechanisms. We further show that independent of arginine, maintenance of an acidic culture pH during growth of C. botulinum strain Hall A-hyper plays a central role in toxin stability and that an extracellular metalloprotease produced by the culture results in BoNT degradation at pH levels between ⁓6.5 and 8.0. IMPORTANCE Botulinum neurotoxin (BoNT) is a public health and bioterrorism concern as well as an important and widely used pharmaceutical, yet the regulation of its synthesis by BoNT-producing clostridia is not well understood. This paper highlights the role of environmentally controlled posttranslational regulatory mechanisms influencing processing and stability of biologically active BoNTs produced by C. botulinum. The results of this work will help enhance public health and safety measures and our ability to evaluate safety risks of novel BoNTs and improve production and quality of BoNTs for pharmaceutical use.

Effects of carbon dioxide on growth of proteolytic Clostridium botulinum, its ability to produce neurotoxin, and its transcriptome.

The antimicrobial gas carbon dioxide is frequently used in modified atmosphere packaging. In the present study, the effects of CO2 (10 to 70%, vol/vol) on gene expression (measured using quantitative reverse transcription-PCR and a whole-genome DNA microarray) and neurotoxin formation (measured using an enzyme-linked immunosorbent assay [ELISA]) by proteolytic Clostridium botulinum type A1 strain ATCC 3502 were studied during the growth cycle. Interestingly, in marked contrast to the situation with nonproteolytic C. botulinum types B and E, CO2 had little effect on any of these parameters. At all CO2 concentrations, relative expression of neurotoxin cluster genes peaked in the transition between exponential and stationary phases, with evidence of a second rise in expression in late stationary phase. Microarray analysis enabled identification of coding sequences whose expression profiles matched those of the neurotoxin cluster. Further research is needed to determine whether these are connected to neurotoxin formation or are merely growth phase associated.

Effect of media, additives, and incubation conditions on the recovery of high pressure and heat-injured Clostridium botulinum spores.

The effect of additives and post-treatment incubation conditions on the recovery of high pressure and heat-injured (i.e., processed at 620 MPa and 95 and 100 degrees C for 5 min) spores of Clostridium botulinum strains, 62-A (proteolytic type A) and 17-B (nonproteolytic type B) was studied. High pressure and heat-injured spores were inoculated into TPGY (Trypticase-Peptone-Glucose-Yeast extract) anaerobic broth media containing additives (lysozyme, L-alanine, L-aspartic acid, dipicolonic acid, sodium bicarbonate, and sodium lactate) at various concentrations (0-10 microg/ml) individually or in combination. The spore counts of high pressure and heat-injured 62-A and 17-B recovered from TPGY broth containing lysozyme (10 microg/ml) incubated for 4 months versus that recovered from peptone-yeast extract-glucose-starch (PYGS) plating agar containing lysozyme (10 microg/ml) incubated under anaerobic conditions for 5 days were also compared. None of the additives either individually or in combination in TPGY broth improved recovery of injured spore enumeration compared to processed controls without additives. Addition of lysozyme at concentrations of 5 and 10 microg/ml in TPGY broth improved initial recovery of injured spores of 17-B during the first 4 days of incubation but did not result in additional recovery at the end of the 4 month incubation compared to the processed control without lysozyme. Adding lysozyme at a concentration of 10 microg/ml to PYGS plating agar resulted in no effect on the recovery of high pressure and heat-injured 62-A and 17-B spores. The recovery counts of high pressure and heat-injured spores of 62-A and 17-B were lower (i.e., <1.0 log units) with PYGS plating agar compared to the MPN method using TPGY broth as the growth medium.