Enhancement of the thermostability and activity of mesophilic Clostridium cellulovorans EngD by in vitro DNA recombination with Clostridium thermocellum CelE

The thermal stability and catalytic activity of endoglucanase (EngD) from mesophilic Clostridium cellulovorans were improved by evolutionary molecular engineering. Thermostable mutants were isolated after staggered extension process (StEP) with celE from thermophilic Clostridium thermocellum performed to conduct family shuffling and overlay screening of the resultant mutant library. The relative activity of the best-evolved clone has been improved of about 2 times higher at 50 °C and showed a higher k_cat/K_m value than its engD parental clone. We determined that these variants had two amino acid substitutions (L157N, Q158E) and confirmed their effects by substituting these amino acids in the parental gene by site-directed mutagenesis. These substitutions resulted in an increase in hydrophilic or charged residues. Our results demonstrate that in vitro recombination is an effective approach to improve the thermostability and enzymatic activity of a mesophilic enzyme.     

Strategy to inactivate Clostridium perfringens spores in meat products

The current study aimed to develop an inactivation strategy for Clostridium perfringens spores in meat through a combination of spore activation at low pressure (100–200 MPa, 7 min) and elevated temperature (80 °C, 10 min); spore germination at high temperatures (55, 60 or 65 °C); and inactivation of germinated spores with elevated temperatures (80 and 90 °C, 10 and 20 min) and high pressure (586 MPa, at 23 and 73 °C, 10 min). Low pressures (100–200 MPa) were insufficient to efficiently activate C. perfringens spores for germination. However, C. perfringens spores were efficiently activated with elevated temperature (80 °C, 10 min), and germinated at temperatures lethal for vegetative cells (≥55 °C) when incubated for 60 min with a mixture of l-asparagine and KCl (AK) in phosphate buffer (pH 7) and in poultry meat. Inactivation of spores (∼4 decimal reduction) in meat by elevated temperatures (80–90 °C for 20 min) required a long germination period (55 °C for 60 min). However, similar inactivation level was reached with shorter germination period (55 °C for 15 min) when spore contaminated-meat was treated with pressure-assisted thermal processing (568 MPa, 73 °C, 10 min). Therefore, the most efficient strategy to inactivate C. perfringens spores in poultry meat containing 50 mM AK consisted: (i) a primary heat treatment (80 °C, 10 min) to pasteurize and denature the meat proteins and to activate C. perfringens spores for germination; (ii) cooling of the product to 55 °C in about 20 min and further incubation at 55 °C for about 15 min for spore germination; and (iii) inactivation of germinated spores by pressure-assisted thermal processing (586 MPa at 73 °C for 10 min). Collectively, this study demonstrates the feasibility of an alternative and novel strategy to inactivate C. perfringens spores in meat products formulated with germinants specific for C. perfringens.     

Enterotoxin formation by Clostridium perfringens during sporulation and vegetative growth

Growth, sporulation and enterotoxin production have been followed for three different strains of Clostridium perfringens on a vegetative (FTG) medium and a sporulation (DS) medium. Enterotoxin production was followed by use of enzyme-linked immunosorbent assay (ELISA). All strains produced enterotoxin under both vegetative growth and sporulation in spite of the fact that one strain is known to be enterotoxin negative. The presumed negative strain produced about the same amount of enterotoxin on both media (1 ng/ml). The two other strains produced about 1000–2000 times more enterotoxin during sporulation than during vegetative growth.     

Elevation of heat resistance of Clostridium sporogenes following heat shock

Heat shock of strains of spores of Clostridium sporogenes at 80 or 100°C increased their apparent resistance to heating at 121·1°C, with the exception of NCIB 10696. The presence of glucose (0·5 – 1·0%) in the sporulation medium reduced the percentage of spores which could be activated by heat. Within individual spore populations, some spores required more heat than others to induce activation. Spores of PA 3679 remained activated for 18 months at 4°C and heat shock did not alter the z value. Experiments using dimethylglutaric acid/NaOH buffer suggest that heat shock in acid conditions has maximum effect on heat resistance.     

Inactivation strategy for Clostridium perfringens spores adhered to food contact surfaces

The contamination of enterotoxigenic Clostridium perfringens spores on food contact surfaces posses a serious concern to food industry due to their high resistance to various preservation methods typically applied to control foodborne pathogens. In this study, we aimed to develop an strategy to inactivate C. perfringens spores on stainless steel (SS) surfaces by inducing spore germination and killing of germinated spores with commonly used disinfectants. The mixture of l-Asparagine and KCl (AK) induced maximum spore germination for all tested C. perfringens food poisoning (FP) and non-foodborne (NFB) isolates. Incubation temperature had a major impact on C. perfringens spore germination, with 40 °C induced higher germination than room temperature (RT) (20 ± 2 °C). In spore suspension, the implementation of AK-induced germination step prior to treatment with disinfectants significantly (p < 0.05) enhanced the inactivation of spores of FP strain SM101. However, under similar conditions, no significant spore inactivation was observed with NFB strain NB16. Interestingly, while the spores of FP isolates were able to germinate with AK upon their adhesion to SS chips, no significant germination was observed with spores of NFB isolates. Consequently, the incorporation of AK-induced germination step prior to decontamination of SS chips with disinfectants significantly (p < 0.05) inactivated the spores of FP isolates. Collectively, our current results showed that triggering spore germination considerably increased sporicidal activity of the commonly used disinfectants against C. perfringens FP spores attached to SS chips. These findings should help in developing an effective strategy to inactivate C. perfringens spores adhered to food contact surfaces.     

Short communication: Attempts to identify Clostridium botulinum toxin in milk from three experimentally intoxicated Holstein cows

Three adult lactating Holstein cows were injected in the subcutaneous abdominal vein with 175 ng/kg of body weight of Clostridium botulinum type C toxin (451 cow median toxic doses) to determine if this botulinum toxin crosses the blood-milk barrier. Whole blood (in sodium heparin) and clotted blood serum samples were taken at 0 min, 10 min, and 3, 6, 9, and 12 h postinoculation. Milk samples were taken at 0 min and at 3, 6, 9 and 12 h postinoculation. All samples were tested for the presence of the toxin using the mouse bioassay and immunostick ELISA test. The immunostick ELISA identified the toxin in whole blood and the mouse bioassay identified the toxin in serum at all times examined in all 3 animals. Toxin was not identified by either detection method in milk samples collected from the 3 animals. From these results, it appears that Clostridium botulinum type C toxin does not cross from the blood to the milk in detectable concentrations.     

Effects of copper on germination,growth and sporulation of Clostridium tyrobutyricum

The effects of copper (Cu²⁺) on spore germination, vegetative growth and sporulation of Clostridium tyrobutyricum, which is capable to causing texture and flavour defects in Emmental cheese, were studied. Spore suspensions of three different strains were used as starting material for two experimental set-ups. The first studied the effects of supplemented (0–30 ppm) copper in RCM medium during spore germination and vegetative growth of C. tyrobutyricum measured by plating. The second set-up studied the effects of copper (0–30 ppm) in RCM medium during growth and sporulation of C. tyrobutyricum as measured by optical density at 550 nm and by platings after heat treatment of the samples respectively. Inhibition of germination, vegetative growth and sporulation processes by copper was strain-dependent. Both sporulation and germination were more sensitive than vegetative growth of C. tyrobutyricum to the inhibitory effects of copper. Copper, at the concentrations investigated in this study, inhibits spore germination of C. tyrobutyricum strains. Consequently copper may reduce the risk of late blowing spoilage from in the germination of C. tyrobutyricum spores during the ripening period of Emmental cheese.     

Isolation and partial purification of a proteolytic enzyme preparation from Clostridium perfringens

Although two strains of Clostridium perfringens (ATCC 12915 and 13124) exhibited excellent growth on amino acid and peptone media, only one (ATCC 13124) produced measurable proteolytic enzyme activity. Thus, subsequent purification steps concentrated on isolation of a proteolytic enzyme preparation produced by this strain. Purification and concentration were carried out by precipitating the crude enzyme fraction from the culture filtrate with ZnCl₂, extracting with saturated disodium phosphate and reprecipitating by 60% saturation with (NH₄)₂SO₄. The precipitate was then redissolved in borate buffered saline solution and further purified by successively passing it through a Bio-Gel P-100 column, a DEAE-cellulose column and a Bio-Gel P-200 column. The final step resulted in a 159-fold purification with 12% recovery and a final specific activity of 79 azocoll units/milligramme protein. Although each successive purification step eliminated some of the impurities, the final fraction still showed considerable heterogeneity upon disc-gel electrophoresis.     

Effect of a proteolytic enzyme produced by Clostridium perfringens upon porcine muscle

Purified proteolytic enzyme preparations isolated from actively growing cultures of Clostridium perfringens (ATCC 13124—Type A) were added to aseptic porcine muscle and incubated at 37°C. Samples were removed after one, two and four days incubation. The sarcoplasmic and myofibrillar proteins were extracted and subjected to both conventional and SDS-polyacrylamide disc gel electrophoresis to assess the changes due to the enzyme treatment. The enzyme preparation caused little change in the sarcoplasmic protein fraction but caused some proteolysis in the myofibrillar extract. Comparison of the protein profile from muscle incubated with the purified enzyme preparation and that incubated with the actively growing organisms per se showed that the organisms caused more degradation, suggesting that they produced additional enzymes causing proteolysis of porcine muscle.     

Evaluation of CP Chromo Select Agar for the enumeration of Clostridium perfringens from water

The European Directive on drinking water quality has included mCP agar as the reference method for recovering Clostridium perfringens from drinking waters. In the present study, three media (mCP, TSCF and CP Chromo Select Agar) were evaluated for recovery of C. perfringens in different surface water samples. Out of 139 water samples, using a membrane filtration technique, 131 samples (94.2%) were found to be presumptively positive for C. perfringens in at least one of the culture media. Green colored colonies on CP Chromo Select Agar (CCP agar) were counted as presumptive C. perfringens isolates.     

The effect of temperature abuse on Clostridium perfringens and Bacillus cereus in raw beef and chicken substrates during frozen storage

Raw beef and chicken substrates inoculated with Clostridium perfringens and Bacillus cereus were subjected to severe temperature abuse at intervals during 6 months frozen storage. Clostridium perfringens grew in the thawing substrates at 20°C after one month but not after two months frozen storage. Substantial growth of Cl. perfringens and B. cereus occurred during 24 h of thawing at 27°C in chicken and beef substrates and this was maintained throughout frozen storage. Refreezing thawed samples reduced viable counts of Cl. perfringens and B. cereus by up to 99 and 97% respectively; non-growing cells of Cl. perfringens were much more resistant. Spores of Cl. perfringens and B. cereus were more resistant to prolonged frozen storage than vegetative cells.     

Inactivation of Clostridium sporogenes spores on stainless-steel using heat and an organic acidic chemical agent

The efficacy of a food grade acidic chemical agent for the reduction of Clostridium sporogenes spores on a stainless steel surface was investigated. The chemical agent was a combination of selected fatty acids and lactate esters. Distilled deionized water and 35% hydrogen peroxide were used as negative and positive controls, respectively. Approximately 3 log cfu reductions in viable spore numbers were detected on the steel surfaces for all treatments at room temperature, except the controls. Reductions in the viable spore numbers significantly increased with increasing exposure times and concentrations of the acidic agent. Five log reduction of viable spore number was achieved after 10 min treatment with the 10% agent solution at 68 °C. No viable spores were observed on the 10% agent treated sample after a 60 min exposure time at 75 °C. This research showed that the acidic sanitizer tested in this study could be used to reduce the number of C. sporogenes spores on stainless steel surfaces.     

High-pressure destruction kinetics of Clostridium sporogenes ATCC 11437 spores in milk at elevated quasi-isothermal conditions

The high-pressure sterilization establishment requires data on isobaric and isothermal destruction kinetics of baro-resistant pathogenic and spoilage bacterial spores. In this study, Clostridium sporogenes 11437 spores (10⁷ CFU/ml) inoculated in milk were subjected to different pressure, temperature and time (P, T, t) combination treatments (700–900 MPa; 80–100 °C; 0–32 min). An insulated chamber was used to enclose the test samples during the treatment for maintaining isobaric and quasi-isothermal processing conditions. Decimal reduction times (D values) and pressure and temperature sensitivity parameters, Z_T (pressure constant) and Z_P (temperature constant) were evaluated using a 3 × 3 full factorial experimental design. HP treatments generally demonstrated a minor pressure pulse effect (PE) (no holding time) and the pressure hold time effect was well described by the first order model (R² > 0.90). Higher pressures and higher temperatures resulted in a higher destruction rate and a higher microbial count reduction. At 900 MPa, the temperature corrected D values were 9.1, 3.8, 0.73 min at 80, 90, 100 °C, respectively. The thermal treatment at 0.1 MPa resulted in D values 833, 65.8, 26.3, 6.0 min at 80, 90, 95, 100 °C respectively. By comparison, HP processing resulted in a strong enhancement of spore destruction at all temperatures. Temperature corrected Z_T values were 16.5, 16.9, 18.2 °C at 700, 800, 900 MPa, respectively, which were higher than the thermal z value 9.6 °C. Hence, the spores had lower temperature sensitivity at elevated pressures. Similarly, corrected Z_P values were 714, 588, 1250 MPa at 80, 90, 100 °C, respectively, which illustrated lower pressure sensitivity at higher temperatures. By general comparison, it was concluded that within the range operating conditions employed, the spores were relatively more sensitive to temperature than to pressure.     

The two-component system CBO2306/CBO2307 is important for cold adaptation of Clostridium botulinum ATCC 3502

Clostridium botulinum is a notorious foodborne pathogen. Its ability to adapt to and grow at low temperatures is of interest for food safety. Two-component systems (TCSs) have been reported to be involved in cold-shock and growth at low temperatures. Here we show the importance of TCS CBO2306/CBO2307 in the cold-shock response of C. botulinum ATCC 3502. The relative expression levels of the cbo2306 and cbo2307 were up to 4.4-fold induced in the cold-shocked cultures but negatively regulated in the late-log and stationary growth phase in relation to early logarithmic growth phase in non-shocked cultures. Importance of the CBO2306/CBO2307 in the cold stress was further demonstrated by impaired growth of insertional cbo2306 or cbo2307 knockout mutants in relation to the wild-type strain ATCC 3502. The results suggest that the TCS CBO2306/CBO2307 is important for cold-shock response and adaptation of C. botulinum ATCC 3502 to low temperature.     

Fluorescent in situ hybridization in combination with filter cultivation (FISHFC) method for specific detection and enumeration of viable Clostridium perfringens

To reduce time for enumeration of viable Clostridium perfringens, fluorescence in situ hybridization in combination with filter cultivation (FISHFC) was employed. The method utilized a CLP-180 probe, based on the 16S rRNA region of C. perfringens, and FISHFC fluorescence microscopy to detect C. perfringens, but not organisms from other species. Optimal cultivation requirements for micro-colony formation were TSC medium, anaerobic conditions, 37 °C, and incubation for 6 h. Under these conditions, micro-colony diameters reached 100 μm, a size sufficient for hybridization. Enumeration of C. perfringens using the CLP-180-aided FISHFC method was realized in 9 h as compared to 3–5 days required by the conventional plate count method. Moreover, viable C. perfringens counts of food samples from the two methods were not significantly different.