Clostridium Perfringens Spores Research Articles

Use of calcium, potassium, and sodium lactates to control germination and outgrowth of Clostridium perfringens spores during chilling of injected pork

Inhibition of Clostridium perfringens spore germination and outgrowth during abusive chilling regimes was investigated by the incorporation of lactates of calcium (CaL), potassium (KL) and sodium (NaL) in injected pork. Lactates (Ca, K, or Na) were incorporated into injected pork samples at four different concentrations (1.0%, 2.0%, 3.0%, and 4.8%), along with a no-lactate control. A three-strain cocktail of C. perfringens spores was inoculated into the product (injected pork) to obtain a final spore population of ca. 2.0–2.5 log 10 CFU/g. Chilling of injected pork (control) from 54.4 to 7.2 °C within 6.5, 9, 12, 15, 18, and 21 h exponential chill rates resulted in C. perfringens population increases of 0.49, 2.40, 4.02, 5.03, 6.24, and 6.30 log 10 CFU/g, respectively. Addition of CaL at 1.0% or KL and NaL ⩾2.0% to injected pork was able to control C. perfringens germination and outgrowth to <1 log CFU/g, meeting the USDA-FSIS performance standard. However, extension of chilling rates beyond 9.0 h (up to 21 h) required addition of CaL (⩾2.0%), KL or NaL (⩾3.0%) to meet the stabilization performance standard. In general, CaL was more effective compared to KL or NaL for all the chilling regimes, in reducing the potential risk of C. perfringens germination and outgrowth.

Multistate Evaluation of an Ultrafiltration-Based Procedure for Simultaneous Recovery of Enteric Microbes in 100-Liter Tap Water Samples

Ultrafiltration (UF) is increasingly being recognized as a potentially effective procedure for concentrating and recovering microbes from large volumes of water and treated wastewater. Because of their very small pore sizes, UF membranes are capable of simultaneously concentrating viruses, bacteria, and parasites based on size exclusion. In this study, a UF-based water sampling procedure was used to simultaneously recover representatives of these three microbial classes seeded into 100-liter samples of tap water collected from eight cities covering six hydrologic areas of the United States. The UF-based procedure included hollow-fiber UF as the primary step for concentrating microbes and then used membrane filtration for bacterial culture assays, immunomagnetic separation for parasite recovery and quantification, and centrifugal UF for secondary concentration of viruses. Water samples were tested for nine water quality parameters to investigate whether water quality data correlated with measured recovery efficiencies and molecular detection levels. Average total method recovery efficiencies were 71, 97, 120, 110, and 91% for phiX174 bacteriophage, MS2 bacteriophage, Enterococcus faecalis, Clostridium perfringens spores, and Cryptosporidium parvum oocysts, respectively. Real-time PCR and reverse transcription-PCR (RT-PCR) for seeded microbes and controls indicated that tap water quality could affect the analytical performance of molecular amplification assays, although no specific water quality parameter was found to correlate with reduced PCR or RT-PCR performance.

Mode of Action of a Germination-Specific Cortex-Lytic Enzyme, SleC, ofClostridium perfringensS40

The hydrolysis of the bacterial spore peptidoglycan (cortex) is a crucial event in spore germination. It has been suggested that SleC and SleM, which are conserved among clostridia, are to be considered putative cortex-lytic enzymes in Clostridium perfringens. However, little is known about the details of the hydrolytic process by these enzymes during germination, except that SleM functions as a muramidase. Muropeptides derived from SleC-digested decoated spores of a Bacillus subtilis mutant that lacks the enzymes, SleB, YaaH and CwlJ, related to cortex hydrolysis were identified by amino acid analysis and mass spectrometry. The results suggest that SleC is most likely a bifunctional enzyme possessing lytic transglycosylase activity and N-acetylmuramoyl-L-alanine amidase activity confined to cross-linked tetrapeptide-tetrapeptide moieties of the cortex structure. Furthermore, it appears that during germination of Clostridium perfringens spores, SleC causes merely small and local changes in the cortex structure, which are necessary before SleM can function.

Removal and fate of Cryptosporidium parvum, Clostridium perfringens and small-sized centric diatoms ( Stephanodiscus hantzschii) in slow sand filters

The decimal elimination capacity (DEC) of slow sand filtration (SSF) for Cryptosporidium parvum was assessed to enable quantitative microbial risk analysis of a drinking water production plant. A mature pilot plant filter of 2.56 m 2 was loaded with C. parvum oocysts and two other persistent organisms as potential surrogates; spores of Clostridium perfringens (SCP) and the small-sized (4–7 μm) centric diatom (SSCD) Stephanodiscus hantzschii. Highly persistent micro-organisms that are retained in slow sand filters are expected to accumulate and eventually break through the filter bed. To investigate this phenomenon, a dosing period of 100 days was applied with an extended filtrate monitoring period of 150 days using large-volume sampling. Based on the breakthrough curves the DEC of the filter bed for oocysts was high and calculated to be 4.7 log. During the extended filtrate monitoring period the spatial distribution of the retained organisms in the filter bed was determined. These data showed little risk of accumulation of oocysts in mature filters most likely due to predation by zooplankton. The DEC for the two surrogates, SCP and SSCD, was 3.6 and 1.8 log, respectively. On basis of differences in transport behaviour, but mainly because of the high persistence compared to the persistence of oocysts, it was concluded that both spores of sulphite-reducing clostridia (incl. SCP) and SSCD are unsuited for use as surrogates for oocyst removal by slow sand filters. Further research is necessary to elucidate the role of predation in Cryptosporidium removal and the fate of consumed oocysts.

Inhibition of Germination and Outgrowth of Clostridium perfringens Spores by Lactic Acid Salts during Cooling of Injected Turkey

Inhibition of Clostridium perfringens spore germination and outgrowth by lactic acid salts (calcium, potassium, and sodium) during exponential cooling of injected turkey product was evaluated. Injected turkey samples containing calcium lactate, potassium lactate, or sodium lactate (1.0, 2.0, 3.0, or 4.8% [w/w]), along with a control (product without lactate), were inoculated with a three-strain cocktail of C. perfringens spores to achieve a final spore population of 2.5 to 3.0 log CFU/g. The inoculated product was heat treated and exponentially cooled from 54.5 to 7.2°C within 21, 18, 15, 12, 9, or 6.5 h. Cooling of injected turkey (containing no antimicrobials) resulted in C. perfringens germination and an outgrowth of 0.5, 2.4, 3.4, 5.1, 5.8, and 5.8 log CFU/g when exponentially cooled from 54.4 to 7.2°C in 6.5, 12, 15, 18, and 21 h, respectively. The incorporation of antimicrobials (lactates), regardless of the type (Ca, Na, or K salts), inhibited the germination and outgrowth of C. perfringens spores at all the concentrations evaluated (1.0, 2.0, 3.0, and 4.8%) compared to the injected turkey without acetate (control). Increasing the concentrations of the antimicrobials resulted in a greater inhibition of the spore germination and outgrowth in the products. In general, calcium lactate was more effective in inhibiting the germination and outgrowth of C. perfringens spores at ≥1.0% concentration than were sodium and potassium lactates. Incorporation of these antimicrobials in cooked, ready-to-eat turkey products can provide additional protection in controlling the germination and outgrowth of C. perfringens spores during cooling (stabilization).

Carvacrol, Cinnamaldehyde, Oregano Oil, and Thymol Inhibit Clostridium perfringens Spore Germination and Outgrowth in Ground Turkey during Chilling†

Inhibition of Clostridium perfringens by plant-derived carvacrol, cinnamaldehyde, thymol, and oregano oil was evaluated during abusive chilling of cooked ground turkey. Test substances were mixed into thawed turkey product at concentrations of 0.1, 0.5, 1.0, or 2.0% (wt/wt) along with a heat-activated three-strain C. perfringens spore cocktail to obtain final spore concentrations of ca. 2.2 to 2.8 log CFU spores per g of turkey meat. Aliquots (5 g) of the ground turkey mixtures were vacuum packaged and then cooked in a water bath, where the temperature was raised to 60°C in 1 h. The products were cooled from 54.4 to 7.2°C in 12, 15, 18, or 21 h, resulting in 2.9-, 5.5-, 4.9-, and 4.2-log CFU/g increases, respectively, in C. perfringens populations in samples without antimicrobials. Incorporation of test compounds (0.1 to 0.5%) into the turkey completely inhibited C. perfringens spore germination and outgrowth (P ≤ 0.05) during exponential cooling in 12 h. Longer chilling times (15, 18, and 21 h) required greater concentrations (0.5 to 2.0%) to inhibit spore germination and outgrowth. Cinnamaldehyde was significantly (P < 0.05) more effective (<1.0-log CFU/g growth) than the other compounds at a lower concentration (0.5%) at the most abusive chilling rate evaluated (21 h). These findings establish the value of the plant-derived antimicrobials for inhibiting C. perfringens in commercial ground turkey products.

Microbiological quality of groundwater sources used by rural communities in Limpopo Province, South Africa

A survey of the microbiological quality of water from 194 boreholes (97 privately owned and 97 communal boreholes) in the rural Thitale-Hlanganani area of the Limpopo Province, South Africa was carried out between August 2002 and August 2003. Very little information on the microbiological quality of privately-owned boreholes in rural communities is available raising concerns about the safety of these groundwater supplies. In this study, levels of total coliforms, thermotolerant (faecal) coliforms, faecal enterococci, Clostridium perfringens (vegetative cells and spores) and somatic coliphages were determined for community and privately-owned borehole water. The average counts for total coliforms, faecal coliforms, faecal enterococci and Clostridium perfringens exceeded the South African recommended guideline limits of 0-10 counts.100 ml(-1) for total coliforms and 0 counts. 100 ml(-1) for faecal coliforms, faecal enterococci and Clostridium perfringens respectively. Comparisons between the levels of indicator bacteria present in private and communal boreholes during dry seasons indicated a statistical difference for faecal enterococci bacteria (p = 0.005) and Clostridium perfringens (p = 0.08). Comparisons between the levels of indicator bacteria present in private and communal boreholes during rainy seasons indicated statistical differences between total coliforms (p = 0.05), faecal coliforms (p = 0.03) and Clostridium perfringens (p = 0.009) bacteria. No significant differences in the presence of somatic coliphages in both private and communal borehole water were see. The results indicated the need for environmental impact assessment studies to monitor the microbiological quality of groundwater sources in rural communities.

Stress modifies the vaginal flora in cyclic female rats*

The understanding of the pathogenesis of vaginitis is directly dependent upon the factors which promote the colonization by bacterial genital microflora. Disruption of the bacterial barrier function afforded by the predominant microflora can lead to colonization by pathogenic or opportunistic pathogenic subdominant species. The present study focused on the effects of stress on the bacteria of the vaginal flora. For this purpose two types of psychological stress were employed: acute and subacute mild unpredictable stress. Estimation of the vaginal flora of 22 Wistar rats was performed according to the distinct phases of the estrous cycle: PE (proestrous), E (estrous), ME (metestrous), and DE (diestrous). Bacteria isolated from the vaginal flora were identified following Bergey’s Manual criteria. Escherichia coli, Clostridium , and Clostridium perfringens (vegetative and spore forms), showed increased numbers at PE-E after acute and subacute stress. Enterococcus was increased after acute stress, in all stages, while S treptococcus was increased after acute stress only at ME-DE. Staphylococcus aureus was detected at ME-DE only after stress. Staphylococcus epidermidis seemed to be predominant, but not influenced by stress at PE-E. However, it was detected in increased numbers at ME-DE after stress. Fusobacterium was detected at low levels in all phases and remained unaffected after exposure to stress. Both types of stress increased the numbers of Bacteroides at PE-E. Finally, the absence of Lactobacillus was noticed at PE-E 24 h after acute and subacute mild unpredictable stress, while Lactobacillus was decreased at ME-DE 72 h after stress. Hormonal fluctuations during the different phases of the estrous cycle may be associated with alterations in the vaginal flora. The data showed that stress acting on the vaginal flora and its immunity potential may contribute to disturbances of the natural balance in the area by altering the numbers of several bacteria that belong to the vaginal microflora. Continuous research on the factors that regulate the vaginal microflora is considered as essential, to prevent complications in infections that may be caused by indigenous microorganisms. Key words: stress, vaginal flora, clostridium, lactobacillus

Survival of faecal indicators and enteroviruses in soil after land-spreading of municipal sewage sludge

One concern in regard to the spreading of sewage sludge is the potential for contamination of soil and water by pathogens present in sludge. We studied the survival of enteric micro-organisms in sewage sludge following direct land-spreading. The sludge produced by a wastewater treatment plant (capacity equivalent to 2000 inhabitants; sludge storage tank of 700 m 3) was spread on a soil, at a rate of 80 m 3/ha. The tested micro-organisms included three of specific sanitary interest: faecal indicators, spores of Clostridium perfringens and enteroviruses. The results highlighted three types of behaviour associated with these three groups of micro-organisms. The enteroviruses were not detected 2 weeks after spreading on the soil whereas the concentration of faecal indicators fell slowly with an observed decrease of between 1.2 and 1.8 logarithmic units over 2 months (but without the initial level of the soil being reached). Lastly, the concentrations of C. perfringens remained stable. The different survival times of the studied micro-organisms in soil, confirm the necessity to include several indicators (bacteria and viruses) to estimate reliably the sanitary risk related to sludge spreading.

Chitosan Protects Cooked Ground Beef and Turkey Against Clostridium perfringens Spores During Chilling

ABSTRACT: We investigated the inhibition of Clostridium perfringens spore germination and outgrowth by the biopolymer chitosan during abusive chilling of cooked ground beef (25% fat) and turkey (7% fat) obtained from a retail store. Chitosan was mixed into the thawed beef or turkey at concentrations of 0.5%, 1.0%, 2.0%, or 3.0% (w/w) along with a heat‐activated 3‐strain spore cocktail to obtain a final spore concentration of 2 to 3 log10 CFU/g. Samples (5 g) of the ground beef or turkey mixtures were then vacuum‐packaged and cooked to 60 °C in 1 h in a temperature‐controlled water bath. Thereafter, the products were cooled from 54.4 to 7.2 °C in 12, 15, 18, or 21 h, resulting in 4.21, 4.51, 5.03, and 4.70 log10 CFU/g increases, respectively, in C. perfringens populations in the ground beef control samples without chitosan. The corresponding increases for ground turkey were 5.27, 4.52, 5.11, and 5.38 log10 CFU/g. Addition of chitosan to beef or turkey resulted in concentration‐ and time‐dependent inhibition in the C. perfringens spore germination and outgrowth. At 3%, chitosan reduced by 4 to 5 log10 CFU/g C. perfringens spore germination and outgrowth (P≤ 0.05) during exponential cooling of the cooked beef or turkey in 12, 15, or 18 h. The reduction was significantly lower (P &lt; 0.05) at a chilling time of 21 h, about 2 log10 CFU/g, that is, 7.56 log10 CFU/g (unsupplemented) compared with 5.59 log10 CFU/g (3% chitosan). The results suggest that incorporation of 3% chitosan into ground beef or turkey may reduce the potential risk of C. perfringens spore germination and outgrowth during abusive cooling from 54.4 to 7.2 °C in 12, 15, or 18 h.

Control of Clostridium perfringens in Cooked Ground Beef by Carvacrol, Cinnamaldehyde, Thymol, or Oregano Oil during Chilling

Inhibition of Clostridium perfringens spore germination and outgrowth by carvacrol, cinnamaldehyde, thymol, and oregano oil was evaluated during abusive chilling of cooked ground beef (75% lean) obtained from a local grocery store. Test substances were mixed into thawed ground beef at concentrations of 0.1, 0.5, 1.0, or 2.0% (wt/wt) along with a heat-activated three-strain C. perfringens spore cocktail to obtain final spore concentrations of ca. 2.8 log spores per g. Aliquots (5 g) of the ground beef mixtures were vacuum-packaged and then cooked in a water bath, the temperature of which was raised to 60°C in 1 h. The products were cooled from 54.4 to 7.2°C in 12, 15, 18, or 21 h, resulting in 3.18, 4.64, 4.76, and 5.04 log CFU/g increases, respectively, in C. perfringens populations. Incorporation of test compounds (≥0.1%) into the beef completely inhibited C. perfringens spore germination and outgrowth (P ≤ 0.05) during exponential cooling of the cooked beef in 12 h. Longer chilling times (15, 18, and 21 h) required greater concentrations to inhibit spore germination and outgrowth. Cinnamaldehyde was significantly (P < 0.05) more effective (<1.0 log CFU/g growth) at a lower concentration (0.5%) at the most abusive chilling rate evaluated (21 h) than the other compounds. Incorporation of lower levels of these test compounds with other antimicrobials used in meat product formulations may reduce the potential risk of C. perfringens germination and outgrowth during abusive cooling regimes.

Thermal inactivation of Bacillus cereus and Clostridium perfringens vegetative cells and spores in pork luncheon roll

The aim of this study was to design a thermal treatment(s) for pork luncheon roll, which would destroy Bacillus cereus and Clostridium perfringens vegetative cells and spores. B. cereus and C. perfringens vegetative and spore cocktails were used to inoculate luncheon meat. Samples were subjected to different temperatures and removal times. The decimal-reduction times ( D-values) were calculated by linear regression analysis ( D=−1/slope of a plot of log surviving cells versus time). The log 10 of the resulting D-values were plotted against their corresponding temperatures to calculate (−1/slope of the curve) the thermal resistance ( z-values) of each cocktail. The D-values for vegetative cells ranged from 1 min (60 °C) to 33.2 min (50 °C) for B. cereus and from 0.9 min (65 °C) to 16.3 min (55 °C) for C. perfringens. The D-values for B. cereus spores ranged from 2.0 min (95 °C) to 32.1 min (85 °C) and from 2.2 min (100 °C) to 34.2 min (90 °C) for C. perfringens. The z-values were calculated to be 6.6 and 8.5 °C for B. cereus vegetative and spores, respectively, and 7.8 and 8.4 °C for C. perfringens vegetative cells and spores, respectively. The D-values of B. cereus and C. perfringens suggest that a mild cook of 70 °C for 12 s and 1.3 min would achieve a 6 log reduction of B. cereus and C. perfringens vegetative cells, respectively. The equivalent reduction of B. cereus and C. perfringens spores would require the pork luncheon meat to be heated for 36 s at 105 and 110 °C, respectively. The results of this study provide the thermal inactivation data necessary to design a cooking protocol for pork luncheon roll that would inactivate B. cereus and C. perfringens vegetative cells and spores. The data may also be used in future risk assessment studies.

Fecal Contamination of Agricultural Soils Before and After Hurricane-Associated Flooding in North Carolina

Hurricane Floyd and other storms in 1999 caused widespread and extensive flooding of eastern North Carolina and environmental contamination with fecal wastes from municipal wastewater and livestock operations. Because wastewater contains high levels of pathogenic micro-organisms, principal health risks to humans from flooding are consumption of crops grown in fecally contaminated soil and ingestion of contaminated water. Flood waters polluted with microbial and other contaminants also may be detrimental to the health of livestock and plant crops. In the present study, agricultural soils impacted by flood waters were analyzed for bacterial and viral indicators of fecal contamination. Total coliforms, fecal coliforms, Escherichia coli, spores of Clostridium perfringens, and both male specific (F+) and somatic coliphages were recovered from soil and assayed in liquid culture media. A number of samples were positive for the presence of fecal coliforms, E. coli, and coliphages, indicating the presence of human or animal feces. Most samples were positive for total coliforms, and almost all samples contained high levels of Cl. perfringens spores. The levels of Cl. perfringens spores were significantly (P < 0.001) higher in flooded soil (post-Hurricane Floyd) compared to pre-flood soil. Persistent fecal contamination of soil, as demonstrated by the high levels of Cl. perfringens spores, suggests the need for additional or alternative measures to protect crop-growing areas, including prospective microbiological monitoring and improved protection of watersheds from incidents capable of releasing fecal material.

Laboratory Evaluation of Thermophilic‐Anaerobic Digestion to Produce Class A Biosolids. 2. Inactivation of Pathogens and Indicator Organisms in a Continuous‐Flow Reactor Followed by Batch Treatment

Thermophilic-anaerobic digestion in a single-stage, mixed, continuous-flow reactor is not approved in the United States as a process capable of producing Class A biosolids for land application. This study was designed to evaluate the inactivation of pathogens and indicator organisms in such a reactor followed by batch treatment in a smaller reactor. The combined process was evaluated at 53 degrees C with sludges from three different sources and at 51 and 55 degrees C with sludge from one of the sources. Feed sludge to the continuous-flow reactor was spiked with the pathogen surrogates Ascaris suum and vaccine-strain poliovirus. Feed and effluent were analyzed for these organisms and for indigenous Salmonella spp., fecal coliforms, Clostridium perfringens spores, and somatic and male-specific coliphages. No viable Ascaris eggs were observed in the effluent from the continuous reactor at 53 or 55 degrees C, with greater than 2-log removals across the digester in all cases. Approximately 2-log removal was observed at 51 degrees C, but all samples of effluent biosolids contained at least one viable Ascaris egg at 51 degrees C. No viable poliovirus was found in the digester effluent at any of the operating conditions, and viable Salmonella spp. were measured in the digester effluent in only one sample throughout the study. The ability of the continuous reactor to remove fecal coliforms to below the Class A monitoring limit depended on the concentration in the feed sludge. There was no significant removal of Clostridium perfringens across the continuous reactor under any condition, and there also was limited removal of somatic coliphages. The removal of male-specific coliphages across the continuous reactor appeared to be related to temperature. Overall, at least one of the Class A pathogen criteria or the fecal coliform limit was exceeded in at least one sample in the continuous-reactor effluent at each temperature. Over the range of temperatures evaluated, the maximum time required to meet the Class A criteria by batch treatment of the continuous-reactor effluent was 1 hour for Ascaris suum and Salmonella spp. and 2 hours for fecal coliforms.

Inhibition of Clostridium perfringens sporulation by Bacteroides fragilis and short-chain fatty acids

The effect a common fecal organism, Bacteroides fragilis, has on the sporulation of Clostridium perfringens, an organism linked to some cases of antibiotic associated diarrhea, was examined. Established B. fragilis cultures significantly decreased the number of heat resistant spores formed by C. perfringens ATCC 12915 and increased the number of vegetative cells. To determine if short-chain fatty acids (SCFA), fermentation products of B. fragilis, inhibited sporulation, the SCFA were added to sporulation broth. Sporulation decreased in the presence of acetate, isobutyrate, isovalerate, and succinate. Vegetative cell number for C. perfringens decreased in the cultures with isobutyrate. Propionate did not affect sporulation or vegetative cell number. The data support the hypothesis that the decrease in short-chain fatty acid concentration following antibiotic therapy predisposes patients to diarrheas caused by C. perfringens.

Inactivation of enteric microbes in water by electro-chemical oxidant from brine (NaCl) and free chlorine

Oxidant solutions of mostly free chlorine can be electrochemically produced on-site from brine (NaCl) solution and used to disinfect water at the household or community level. In this study electrochemical oxidant (ECO) from brine and free chlorine were evaluated under laboratory conditions for inactivation of test microbes. Purified suspensions of Escherichia coli, the rugose strain of Vibrio cholerae, Clostridium perfringens spores, MS2 coliphage and Cryptosporidium parvum oocysts were treated with 2 mg/L or 5 mg/L solutions of ECO or free chlorine at 5 degrees C and 25 degrees C and pH 6, 8, and 10 (pH 7 and 25 degrees C only for C. parvum oocysts) for contact times <60 min. Under nearly all conditions, inactivation kinetics were more rapid for E. coli, V. cholerae, C. perfringens spores and MS2 coliphage with ECO than with free chlorine. ECO reduced E. coli, V. cholerae and MS2 by >4 log10 within 30 min and C. perfringens spores by >2 log10 within 10 min at pH 8 and 25 degrees C. Contrary to previous results, however, C. parvum oocysts were not inactivated by ECO, and the reasons for this difference are uncertain. The on-site electrolytic generation of oxidants from brine provided a convenient and inexpensive disinfectant containing free chlorine that was effective against many enteric microbes, for the treatment of household and community drinking-water supplies worldwide. However, the effectiveness of such oxidants for inactivating C. parvum oocysts was variable and sometimes ineffective.

Production of class A biosolids with anoxic low dose alkaline treatment and odor management

The feasibility of full-scale anoxic disinfection of dewatered and digested sludge from Winnipeg, Manitoba with low lime doses and lagoon fly ash was investigated to determine if a class A product could be produced. Lime doses of 50 g, 100 g, and 200 g per kg of biosolids (dry) were used along with fly ash doses of 500 g, 1,000 g, and 1,500 g per kg of biosolids (dry). The mixed product was buried in eight-10 cubic metre trenches at the West End Water Pollution Control Center in Winnipeg. The trenches were backfilled with dirt and trapped to simulate anoxic conditions. Sampling cages were packed with the mixed product and pathogens non-indigenous to Winnipeg's biosolids. The cages were buried amongst the mixed biosolids in the trench. The non-indigenous pathogens spiked in the laboratory were the helminth Ascaris suum and the enteric virus reovirus. Samples were removed at days 12, 40, 69, 291, and 356 and were tested for the presence of fecal Coliform, Clostridium perfringens spores, Ascaris suum eggs, and reovirus. The pH, total solids, and free ammonia content of the mixed product were also determined for each sample. Odor was quantified for samples at both 291 and 356 days. Fecal Coliform bacteria and reovirus were completely inactivated for doses as low as 100 g lime per kg biosolids (dry) and 50 g lime + 500 g fly ash per kg biosolids (dry). Spores of the bacteria C. perfringens experienced a 4-log reduction when treated with 100 g lime per kg biosolids and a 5-log reduction when treated with doses as low as 50 g lime + 500 g fly ash per kg biosolids (dry) after 69 days. Ascaris eggs were completely inactivated in 5 gram packets for all treatments involving 100 g lime per kg biosolids (dry) after 69 days. Class A pathogen requirements were met for all treatments involving a lime dose of at least 100 g per kg biosolids. The odor potential from the produced biosolids is also assessed.

NUMERICAL ANALYSIS OF THE GROWTH OF CLOSTRIDIUM PERFRINGENS IN COOKED BEEF UNDER ISOTHERMAL AND DYNAMIC CONDITIONS1

ABSTRACT The main objective of this study was to develop a numerical technique to solve a set of biologically‐based differential equations used to describe the growth behaviors of bacteria under isothermal conditions in food systems. A 4th‐order Runge‐Kutta method was incorporated to a computer program to solve these equations covering the entire range of bacterial growth, including lag, exponential, and stationary phases. The differential growth models were tested using the spores of Clostridium perfringens inoculated to ground beef and incubated under various isothermal conditions between 17–50C. Results of numerical analysis showed that the differential equations could accurately describe the growth of C. perfringens in cooked ground beef under isothermal conditions. The differential equations were also used to estimate the growth of C. perfringens in cooked ground beef under four different dynamic temperature profiles: 2‐square waves, exponential and linear cooling. In combination with a secondary kinetic model, the growth of C. perfringens in cooked ground beef under dynamic conditions was accurately estimated. Results showed the differences between the estimated and experimentally observed growth curves under dynamic conditions were generally less than 0.5 log (CFU/g). The methodology developed in this study can be a new approach for the food industry, food retailers and consumers, and regulatory agencies to predict and estimate the bacterial growth in cooked meat products under dynamic temperature abuse and deviation conditions.

Increased Inactivation of Ozone-Treated Clostridium perfringens Vegetative Cells and Spores on Fabricated Beef Surfaces Using Mild Heat

Ozone treatment of beef surfaces enhanced the effectiveness of cooking temperatures ranging from 45 to 75°C against enterotoxin-producing strains of Clostridium perfringens. Vegetative cells on beef surfaces at an initial concentration of 5.59±0.17 log CFU/g were reduced significantly (P < 0.05) to 4.09±0.72 log CFU/g and 3.50±0.90 log CFU/g after combined treatments with aqueous ozone (5 ppm) and subsequent heating at 45 and 55°C, respectively. Spores on the beef surface were likewise significantly reduced from an initial concentration of 2.94±0.37 log spores per g to 2.07±0.38 log spores per g and 1.70±0.37 log spores per g after the combined treatment with aqueous ozone (5 ppm) and subsequent heating at 55 and 75°C, respectively. Fluorescent nucleic acid stains were used with confocal fluorescence microscopy to show that spores remaining attached to the meat were protected from treatment-specific injury. This study provides evidence for the decreased resistance of both vegetative cells and spores of C. perfringens with ozone treatment that is followed by heat treatment at temperatures that would not otherwise be as effective, thus lowering the requirements for cooking beef while maintaining a margin of safety.

Photocatalytic inactivation of Clostridium perfringens and coliphages in water

This study presents results of the photocatalytic inactivation of two groups of microorganisms: spores of the anaerobic bacterium Clostridium perfringens and coliphage. A cylindrical reactor impregnated with titanium dioxide and irradiated with ultraviolet light (15 W) was used. Parameters such as color, turbidity, hydraulic detention time (HDT) and initial concentration of microorganisms were evaluated in relation to the efficiency of the inactivation process. According to the experiments with the bacterium C. perfringens, the reduction in number of microorganisms was higher than 98% after an irradiation time of 152 seconds, independent of color and turbidity. For solutions with low turbidities efficiency of the coliphage inactivation reached approximately 100% between 89 and 104 HDT, while this value was 98% for solutions with higher turbidities.