Clostridium Perfringens Spores Research Articles

Retraction for Sarker et al., "Comparative Experiments To Examine the Effects of Heating on Vegetative Cells and Spores of Clostridium perfringens Isolates Carrying Plasmid Genes versus Chromosomal Enterotoxin Genes".

Retraction for Sarker et al., "Comparative Experiments To Examine the Effects of Heating on Vegetative Cells and Spores of Clostridium perfringens Isolates Carrying Plasmid Genes versus Chromosomal Enterotoxin Genes".

Effects of spore purity on the wet heat resistance of Clostridium perfringens, Bacillus cereus and Bacillus subtilis spores

Heat resistance of spores of Clostridium perfringens 8238 (Hobbs Serotype 2), Bacillus cereus NCTC 11143 (4810/72), and Bacillus subtilis PS533, an isogenic derivative of strain PS832 (a 168 strain) was determined in ground beef at 95 °C. Spore purification was by centrifugation and washing with sterile distilled water (dH2O), followed by sonication and then Histodenz centrifugation for B. subtilis and C. perfringens, and centrifugation and washing with sterile dH2O followed by Histodenz centrifugation for B. cereus. Bags containing inoculated beef samples were submerged in a temperature-controlled water bath and held at 95 °C for predetermined lengths of time. Surviving spore populations were enumerated by plating on mannitol egg yolk polymyxin agar (MYP) agar plates for B. cereus and B. subtilis, and on tryptose-sulfite-cycloserine agar (TSC) agar plates for C. perfringens. Survivor curves were fitted to linear, linear with tail, and Weibull models using the USDA Integrated Pathogen Modeling Program (IPMP) 2013 software. The Weibull model provided a relatively better fit to the data since the root mean square error (RMSE), mean square error (MSE), sum of squared errors (SSE), and Akaike information criterion (AIC) values were lower than the values obtained using the linear or the linear with tail models. Additionally, the Weibull model accurately predicted the observed D-values at 95 °C for the three spore-formers since the accuracy factor (Af) values ranged from 1.03 to 1.08 and the bias factor (Bf) values were either 1.00 or 1.01. Times at 95 °C to achieve a 3-log reduction decreased from 206 min for C. perfringens spores purified with water washes alone to 191 min with water washes followed by sonication and Histodenz centrifugation, from 7.9 min for B. cereus spores purified with water washes alone to 1.4 min with water washes followed by Histodenz centrifugation, and from 20.6 min for B. subtilis spores purified with water washes alone to 6.7 min for water washes followed by sonication and Histodenz centrifugation. Thermal-death-time values reported in this study will assist food processors to design thermal processes to guard against bacterial spores in cooked foods. In addition, clearly spore purity is an additional factor in spore wet heat resistance, although the cause of this effect is not clear.

Evaluation of the additive effects of volatile fatty acids and moderate heat treatment for enhancing the inactivation of vegetative cells and spores of Clostridium perfringens by flow cytometry

ObjectivesClostridium perfringens is a well-known spore-forming bacterium that can resist the environment. A mixture of volatile fatty acids or thermal treatments can interact with these bacteria. The aim of this study was to evaluate the effects of different volatile fatty acid concentrations and moderate heat treatment on Clostridium perfringens sporulation. MethodsA pure culture of Clostridium perfringens type A in Duncan Strong medium was treated with a mixture of volatile fatty acids at several concentrations. A thermal treatment was also tested. To evaluate the effects, a double staining method was employed, and treatments on Clostridium perfringens were analysed by flow cytometry. ResultsModerate heat treatment destroyed vegetative forms but had no effect on sporulating forms. Volatile fatty acids combined with moderate heat treatment inhibited Clostridium perfringens sporulation. ConclusionsThe use of flow cytometry as an original method for evaluating the treatment of Clostridium perfringens is of interest because of its simplicity, short time to obtain results, and the level of information provided on the microbial population (impact on metabolism). A combination of mild treatments (moderate heat treatment + volatile fatty acids) to decrease the Clostridium perfringens concentration when these bacteria sporulate is a very promising finding for inhibiting Clostridium perfringens propagation.

Fluence requirements in existing UV disinfection facilities to comply with EU validation performance targets for reclaimed water: a case study.

Since 2020, there is a new European Regulation (EU, 2020/741) on minimum requirements for water reuse, where routine and validation monitoring requirements (log reductions of indicator microorganisms and reference pathogens) have been established. Many reclamation facilities that are already in operation might have difficulties to comply with these performance targets. Existing disinfection systems must be expanded and upgraded. In the case of UV disinfection systems, fluence requirements must be determined to properly design with a focus on the safety and economic-environmental viability of reclaimed water. This study can be used as a reference to develop fluence-response curves for Clostridium perfringens spores, Escherichia coli, and total and F-specific coliphages, indicator microorganisms referred to in the new European Regulation. Eight UV-LED collimated beam tests were performed. Samples were obtained from filtered effluent of secondary treatment from two wastewater treatment plants (WWTPs) which ranged between 30 and 54%. Results showed UV sensitivity of 33.46 mJ/cm2 log I for C. perfringens spores and 2.86 mJ/cm2 log I for E. coli, both from environmental origin. Coliphages were inactivated below the limit of quantitation. The non-dominance of MS2 phages in environmental F-specific coliphages was observed.

Effects of Different Bacteriostats on the Dynamic Germination of Clostridium perfringens Spores.

Bacteriostats, as chemical substances that inhibit bacterial growth, are widely used in the sterilization process; however, their effects on spindle spores are unclear. In this study, the effects of bacteriostats, including nine commonly used food additives and four detergents, on the growth of Clostridium perfringens spores were investigated. The results showed that 0.07‱ ethylenediaminetetraacetate had a good inhibitory effect on C. perfringens spore growth, and the spore turbidity decreased by 4.8% after incubation for 60 min. Furthermore, 0.3‱ tea polyphenols, 0.8‱ D-isoascorbic acid, and 0.75‱ potassium sorbate promoted leakage of contents during spore germination. Among the four detergents, 5‱ glutaraldehyde solution presented the best inhibitory effect on the growth of C. perfringens spores, and the spore turbidity decreased by 5.6% after incubation for 60 min. Further analysis of the inactivation mechanism of spores by the bacteriostats was performed by comparing the leakage of UV-absorbing substances during germination. The results revealed that bacteriostats could not directly kill the spores, but could inactivate them by inhibiting germination or damaging the spore structure during germination, thus preventing the formation of bacterial vegetative bodies. These findings provide important information and reference for the mechanism underlying the effects of different bacteriostatic agents on spore growth.

Effects of sporulation conditions on the growth, germination, and resistance of Clostridium perfringens spores

Clostridium perfringens can form metabolically dormant spores that can survive in meat preservation processes and cause food spoilage and human disease upon germination and outgrowth. The characteristics of spores in food products are closely related to the sporulation environment. To control or inactivate C. perfringens spores in food industry, the effects of sporulation conditions on the spores characteristics should be examined. This study aimed to investigate the effects of temperature (T), pH, and water activity (aw) on the growth, germination, and wet-heat resistance of C. perfringens C1 spores isolated from food product. The results showed that C. perfringens C1 spores produced at T = 37 °C, pH = 8, and aw = 0.997 had the highest sporulation rate and germination efficiency and lowest wet-heat resistance. A further increase in pH and sporulation temperature reduced the spore counts and germination efficiency, but enhanced spores' wet-heat resistance. By using air-drying method and Raman spectroscopy analysis, the water content, composition, and levels of calcium dipicolinic acid, proteins, and nucleic acids in spores produced under different sporulation conditions were determined. The results obtained revealed that sporulation conditions should be carefully considered during food production and processing, thus providing a novel insight into prevention and control of spores in food industry.

Divalent Cation Signaling in Clostridium perfringens Spore Germination

Spore germination plays an essential role in the pathogenesis of Clostridium perfringens-associated food poisoning. Germination is initiated when bacterial spores sense various stimuli, including chemicals and enzymes. A previous study showed that dipicolinic acid (DPA) chelated with calcium (Ca-DPA) significantly stimulated spore germination in C. perfringens. However, whether Ca2+ or DPA alone can induce germination is unknown. Therefore, we aimed to evaluate the possible roles of Ca2+ and other divalent cations present in the spore core, such as Mn2+ and Mg2+, in C. perfringens spore germination. Our study demonstrated that (i) Ca-DPA, but not DPA alone, induced C. perfringens spore germination, suggesting that Ca2+ might play a signaling role; (ii) all tested calcium salts induced spore germination, indicating that Ca2+ is critical for germination; (iii) the spore-specific divalent cations Mn2+ and Mg2+, but not Zn2+, induced spore germination, suggesting that spore core-specific divalent cations are involved in C. perfringens spore germination; and (iv) endogenous Ca2+ and Mg2+ are not required for induction of C. perfringens spore germination, whereas exogenous and partly endogenous Mn2+ are required. Collectively, our results suggest that exogenous spore core-specific divalent cation signals are more important than endogenous signals for the induction of spore germination.

Inactivation of biohazards in healthcare wastewater by E-Beam and Gamma irradiation: a comparative study.

The main objective of this study is to evaluate the effect of irradiation by Gamma rays and Electron Beam (E-Beam) on naturally occurring microorganisms shed in healthcare wastewater issued from multi-specialties hospital. We examined the susceptibility of naturally occurring total indicator bacteriophages towards Gamma rays and E-Beam irradiation to evaluate their appropriateness as viral indicators for healthcare wastewater quality control. Results showed that healthcare wastewater is a rich matrix containing bacteriophages surrogates of pathogenic waterborne viruses (4.5 Log10 PFU/100mL for SOMCPH and 2.3 Log10 PFU/100mL for FRNAPH), antibiotic resistant bacteria (Mean concentrations from 2.3 to 5.5 Log10 CFU/100mL), molds and yeasts (2.7 Log10 CFU/100mL), and spores of Clostridium perfringens (Mean concentration of 3.3 Log10 CFU/100mL). After E-Beam irradiation, naturally occurring bacteria in healthcare wastewater showed lower resistance patterns (D10 values ranging between 0.21 ± 0.005 and 0.59 ± 0.005) compared to those obtained after Gamma irradiation (D10 values ranging between 0.25 ± 0.015 and 0.70 ± 0.0001). Spores of Clostridium perfringens were the most resistant assayed microbes either after E-Beam (D10 values of 3.74 ± 0.005) or Gamma irradiation (D10 values of 4.77 ± 0.025) of collected samples. According to inactivation patterns, a dose of 10kGy was sufficient for a complete inactivation of spores. Bacteriophages isolated from healthcare wastewater showed the same resistance patterns as those previously obtained in urban treated sewage and were inactivated using higher doses than waterborne bacteria (D10 values of SOMCPH 1.46 ± 0.057; D10 values of FRNAPH 1.03 ± 0.057). Their resistance to irradiation treatment in such complex matrix corroborates their use to survey the viral quality of healthcare wastewater before their discharge in the urban sanitation network. D10 value analysis showed that bacteria and bacteriophages inactivation by E-Beam irradiation required lower doses than those required for their inactivation using Gamma rays. According to inactivation patterns, a dose of 7kGy was sufficient for total inactivation of both pathogenic bacteria and viruses. Thus, E-Beam irradiation seems to be an efficient physical pre-treatment process for healthcare wastewater treatment prior to its discharge in urban sanitation system to ensure compliance with environmental standards and protect public health.

Occurrence of Clostridium perfringens vegetative cells and spores throughout an industrial production process of black soldier fly larvae (Hermetia illucens)

The main use of black soldier fly larvae (Hermetia illucens) is currently as an animal feed ingredient. While the bacterial community of the larvae has been characterised repeatedly via sequencing, microbiological safety assessment based on culture-dependent techniques is still scarce. This study focused on the occurrence of the spore-forming foodborne pathogen Clostridium perfringens during rearing and consecutive processing of the larvae, based on observations in a single rearing facility. C. perfringens vegetative cells and spores were determined, in addition to total viable counts, total aerobic spore counts and intrinsic parameters including pH, water activity and moisture content. All samples were obtained from an industrial production plant. In a preliminary experiment, substrate ingredients and dried larvae were analysed, but the larvae were produced with a previous batch of the substrate mixture. A second, more detailed, experiment was performed where all samples were collected sequentially from the same production run (substrate ingredients, substrate mixture, starting larvae, harvested larvae, residue, dried larvae and stored dried larvae). In the two experiments, (presumptive) C. perfringens, as determined on tryptose sulphite cycloserine agar, was found at low numbers in the ingredients and in the second experiment it was also found in the substrate mixture. Over the two experiments, total C. perfringens counts (i.e. vegetative cells plus spores) ranged between 3.0±0.1 and <1.2±0.5 log cfu/g and C. perfringens spores ranged between 2.5±0.1 and <1.0±0.0 log cfu/g. Interestingly, vegetative cells and spores of C. perfringens were below the detection limit in all larvae samples. Therefore, it appears that at this production site and based on the samples investigated, the pathogen did not colonise the larvae. However, these results indicate that insect producers should monitor this pathogen among others, and install good hygiene practices to avoid contamination.

Microbial faecal pollution of river water in a watershed of tropical Ethiopian highlands is driven by diffuse pollution sources

Tropical communities in the developing world depend heavily on riverine systems for their socioeconomic development. However, these resources are poorly protected from diffuse pollution, and there is a lack of quantitative information regarding the microbial pollution characteristics of riverine water, despite frequently reported gastrointestinal diseases. The aim of our study was to apply faecal taxation (i.e., faecal pellet counting in representative test areas to estimate the potential availability of diffuse pollution sources) in combination with a detailed microbiological faecal pollution analysis in a riverine environment to elucidate the importance of diffuse pollution. To realize this approach, ambient faecal pellets, a multiparametric data set for standard faecal indicator bacteria (SFIB), including Escherichia coli, Clostridium perfringens spores and enterococci from catchment soil and river water, and a number of riverine water physicochemical variables were analysed during a one-year cycle. We demonstrated that the abundance of ambient faecal pellets, which were consistently counted at reference sites in the catchment, was associated with faecal pollution in the river water. Water SFIB, dissolved oxygen, nutrients, conductivity and total suspended solids were strongly linked with the abundance of ambient faecal pellets in the river catchment, as demonstrated by principal component analysis (PCA). Elevated concentrations of SFIB in the riverine water in the absence of rainfall also suggested the direct input of faecal bacteria into the riverine water by livestock (e.g., during watering) and humans (e.g., during bathing). Statistical analyses further revealed that the microbiological water quality of the investigated riverine water was not influenced by SFIB potentially occurring in the soil. This study demonstrates the importance of diffuse faecal pollution sources as major drivers of the microbiological quality of riverine water in the Ethiopian highlands. In addition, the new successfully applied integrated approach could be very useful for developing predictive models, which would aid in forecasting riverine microbiological quality in tropical developing countries.

Effect of vacuum cooling followed by ozone repressurization on Clostridium perfringens germination and outgrowth in cooked pork meat under temperature-abuse conditions

The effect of combining vacuum cooling with an ozone-based inhibition process (InhVac) on Clostridium perfringens spore germination and outgrowth in cooked pork meat after exponential chilling (from 54.4 to 7.2 °C in 12, 15, 18, or 21 h) and isothermal storage (20, 25, 30, 36, or 45 °C) was evaluated. Ice cooling (IC) and vacuum cooling (VC) were used to compare the effects with InhVac. The samples were inoculated with a three-strain mixture of C. perfringens spores to obtain concentration of ca. 3 log10 CFU/g. C. perfringens growth in samples treated by InhVac were 0.1, 0.37 and 0.9 log10 CFU/g after 15, 18 and 21 h of cooling from 54.4 to 7.2 °C respectively, significantly lower (P<0.05) than those in samples subjected to IC (1.01, 2.10 and 2.8 log10 CFU/g) and VC (0.56, 1.01 and 2.13 log10 CFU/g). Compared to VC and IC, InhVac treatment increased the lag phase (λ), decreased the growth rates (μmax), and extended the sample shelf-life (the time until a 1 log10 CFU/g increase in C. perfringes from the initial concentration value) at all storage temperatures. InhVac-treated samples not only had a longer shelf-life than those treated by VC, but also exhibited almost two times longer shelf-life compared to those subjected to IC regardless of storage temperatures. Additionally, statistical indexes showed that a primary modified Gompertz model and a secondary Square Root model could fit the data well. Industrial relevanceIn this study, an innovative inhibition approach (InhVac) was found to show a better antimicrobial effect on C. perfringens germination and outgrowth in cooked pork meat compared to ice cooling and vacuum cooling under temperature-abuse conditions. A primary modified Gompertz model and a secondary Square Root model could be used to predict the C. perfringens growth in samples subjected to InhVac treatment.

Control of Clostridium perfringens spore germination and outgrowth by potassium lactate and sodium diacetate in ham containing reduced sodium chloride

The effect of potassium lactate and sodium diacetate mixture (Opti.Form PD4®) on Clostridium perfringens spore germination and outgrowth during abusive cooling of reduced sodium ham was evaluated. A ham formulation containing NaNO2 (0 or 100 ppm), NaCl (1 or 2%) and Opti.Form PD4® (0, 1.5 or 2.5%) was prepared. Meat (10 g) was vacuum-packaged, stored under refrigeration (5 °C) for 3 or 24 h and inoculated with a three-strain C. perfringens spore cocktail. Samples were vacuum-packaged, heat treated (75 °C, 20 min) and cooled from 54.4 °C to 7.2 °C within 9, 15 or 21 h. C. perfringens population increases of 5.18 and 4.26 log CFU/g were observed in non-cured ham (control; stored for 3 h) formulated with 1 or 2% of NaCl, respectively and cooled exponentially within 21 h. Incorporation of Opti.Form PD4® (1.5%) inhibited C. perfringens spore germination and outgrowth regardless of the cooling rate (9, 15 or 21 h). Addition of NaNO2 (100 ppm) enhanced antimicrobial activity of Opti.Form PD4®. In general, abusive cooling (≥15 h) of ham after 24 h of storage resulted in greater populations of C. perfringens. Addition of NaNO2, Opti.Form PD4® and processing immediately resulted in greater inhibition of C. perfringens spore germination and outgrowth.

Quantitative microbial risk assessment of occupational and public risks associated with bioaerosols generated during the application of dairy cattle wastewater as biofertilizer

The reuse or recycling of wastewater provides environmental and economic benefits, representing a sustainable and circular alternative for the management of liquid waste. However, the application of effluents to agricultural crops via spraying creates a potentially dangerous situation for individuals exposed to airborne pathogens. This study used Quantitative Microbial Risk Assessment (QMRA) tools to quantitatively assess the microbial risks of occupational and public exposures to bioaerosols in fertigation scenarios by spraying untreated and treated dairy cattle wastewater. Analyses of Escherichia coli (EC) and spores of Clostridium perfringens (CpSP) in raw and treated effluents as well as pathogen / indicator ratios from the literature were used to estimate the concentrations of Escherichia coli O157:H7 (EC O157:H7) and Cryptospodirium spp. (Crypto) in the air, and the results were applied to an atmospheric microbiological dispersion model. From the concentrations of pathogens in the air, infectious risks for downwind receptors were calculated. The risks of infection by EC O157:H7 to workers at 10 m and 50 m away from the emission source ranged between 3.81 × 10 1 and 2.68 × 10 3 pppy (per person per year), whereas to residents at 100 m and 500 m ranged from 4.59 × 10 1 to 1.51 × 10 4 pppy. Peak values (95th percentile) of occupational and public risks associated with the exposure to Crypto were 3.41 × 10 3 and 6.84 × 10 4 pppy at 10 m and 50 m from the source, respectively, and were lower than 1.48 × 10 6 pppy regarding exposures to CpSP. Anaerobic digestion reduced risks by approximately one order of magnitude. The distance from the source was inversely proportional to the risk of exposure. It is recommended that wastewater is treated prior to its reuse and the adoption of application methods with low aerosolization potential. In addition, the need for workers to use personal protective equipment (PPE) is highlighted.

Inhibition of germination and outgrowth of Clostridium perfringens spores by buffered calcium, potassium and sodium citrates in cured and non-cured injected pork during cooling

Inhibition of Clostridium perfringens spore germination and outgrowth by buffered calcium, potassium, and sodium citrates (Ca-Cit, K-Cit, and Na-Cit, respectively) during exponential cooling of non-cured and cured pork was evaluated. Antimicrobials were added to a base formulation of a pork product at a final concentration of 0.5, 1.0 or 1.5% w/w along with a control (without citrates). A three strain cocktail of C. perfringens spores was added to achieve a final spore population of 2.5–3.0 log CFU/g. Heat treatment and exponential cooling of inoculated, non-cured and cured control pork from 54.4 °C to 7.2 °C in 6.5, 9, 12, 15, 18, and 21 h resulted in C. perfringens spore germination and outgrowth of 0.52, 1.53, 4.45, 4.81, 5.83, 6.98 log CFU/g and 0.52, 1.55, 3.45, 4.59, 5.65 and 6.04 log CFU/g, respectively. Incorporation of K- or Na-citrates (>1.0%) inhibited germination and outgrowth of C. perfringens spores (<1.0 log CFU/g) compared to the control product. Ca-Cit was more effective in inhibiting C. perfringens spore germination and outgrowth at 0.5% compared to sodium and potassium citrates. Incorporation of Ca, K or Na citrates into pork product formulations can inhibit C. perfringens spores during extended cooling or during cooling process deviations.

Growth of Clostridium perfringens in roasted chicken and braised beef during cooling – One-step dynamic analysis and modeling

Roasted chicken (RC) and braised beef (BB) are two important commercial products widely consumed in China. Prepared with a mixture of different spices and other ingredients, these products may be contaminated with Clostridium perfringens spores. The spores may germinate, outgrow, and multiply to a dangerous level during cooling after cooking if the cooling process is not sufficiently fast, causing outbreaks of food poisoning due to production of C. perfringens enterotoxin.This study was conducted to investigate the growth of C. perfringens from spores in these products under various cooling conditions and to develop predictive models. Inoculated samples were exposed to different cooling profiles to observe the bacterial growth. Analyzed with the one-step dynamic kinetic analysis method, various kinetic parameters were determined. The estimated Tmin, Topt, and Tmax were 8.8, 46.4, and 51.5 °C in RC, and 10.3, 43.5, and 53.4 °C in BB, all agreeing well with the cardinal temperatures of this microorganism in meats. The optimum specific growth rate (μopt) was 6.06 ln CFU/g per h in RC, and 5.64 ln CFU/g per h in BB.The validation of the growth models showed that the RMSE of prediction was relatively low (0.2–0.3 log CFU/g). The majority (94%) of the residual errors of prediction was within ±0.5 log CFU/g, and 76% within ±0.3 log CFU/g of the experimental observations. While accurate, the predictions are mostly fail-safe. The estimated kinetic parameters and predictive models developed in this study can be used to predict the growth of C. perfringens in RC and BB during cooling after cooking to prevent the outbreaks caused by the growth of this pathogen.

Microbial contamination of poultry litter during fattening period

The results of the research into the microbiological contamination of litter used by broiler chickens are presented. Litter samples were taken prior to the introduction of chicks (day 0) and in 7-day intervals until the end of the fattening period. The total numbers of aerobic mesophilic microorganisms, yeasts, moulds and Clostridium perfringens spores, and the presence of bacteria of the Salmonella genus were determined. The total microbial count in newly laid litter was 7 log10 CFU/g, which increased to 9 log10 CFU/g by the 4th week. However, at the end of the 5th week, it was at the same level as in newly laid litter. C. perfringens spores, presumably originating from chicks? faeces, were first detected on day 7. In the next 7 days their number increased, reaching 3-4 log10 CFU/g, and remained at approximately same levels until the end of the research. The initial mould contamination was 5-6 log10 CFU/g. However, from day 21 moulds were not isolated, but only yeasts of the Saccharomyces genus. It is supposed that these were deposited with chicks? faeces, due to their presence in complete broiler feed. No bacteria of the Salmonella genus were ever isolated from the litter. In conclusion, the total numbers of microorganisms in deep litter reach their peak in approximately a month, which is followed by their decrease. Deep litter is a favourable environment for probiotic yeast cultures. Added to feed intended for broilers, they can positively influence the microbial composition of litter, providing healthier environment to fattening broilers.

Hybrid electrochemical-granular activated carbon system for the treatment of greywater

Three-dimensional (3D) flow-through electrochemical reactor filled with granular activated carbon (GAC) was employed for the treatment of greywater. Polarization of GAC by the electric field, without the direct contact with the terminal current feeders, resulted in oxidant production and degradation of the adsorbed/electrosorbed contaminants. This in turn enabled electrochemical regeneration of GAC and an improved performance compared to a conventional two-dimensional (2D) electrochemical system. The synergy between electrooxidation and GAC adsorption enhanced the performance of the 3D system by up to 21% and 23% in terms of COD and TOC removal, respectively, compared to the summed up performances of GAC and 2D electrochemical systems operating separately. In long-term operation of the 3D system with a previously saturated GAC, the regeneration efficiency of GAC was increased from 42 to 65% over 31 consecutive runs. Disinfection efficiency was evaluated using Escherichia coli (E. coli), spores of Clostridium perfringens (SCP) and somatic coliphages (SOMCPH) as model organisms for pathogenic bacteria, protozoan surrogate and viruses, respectively. GAC could not remove any of the abovementioned microbial indicators. 2D and 3D electrochemical systems achieved similar log removals of E. coli (4.6–5.1 log), SCP (0.1–0.6 log) and SOMCPH (3.2–3.3 log). However, the proportions of viable cells from the total cells adsorbed onto the GAC was reduced from 72% to 22% when an electric field was applied, further confirming that electrochemical polarization of GAC caused the killing of the adsorbed microorganisms. Simultaneous adsorption/electrosorption and electrocatalysis in the GAC packed bed lowered the energy consumption from 46 kWh kg COD−1 removed in conventional 2D system, to 30 kWh kg COD−1 removed in the 3D electrochemical system, thus enabling significant savings in the operational costs.

Use of bacterial spores in monitoring water quality and treatment.

Because Clostridium perfringens spores are both specific to sewage contamination and environmentally stable, they are considered as possible conservative indicators of human fecal contamination and possible surrogates for environmentally stable pathogens. This review discusses the reasons and summarizes methods for monitoring spores in water. Cultural methods are still preferred over qPCR for routine water quality monitoring because of their low costs. Membrane filter (MF) methods are preferred over the more laborious and less accurate most probable number methods. The most commonly used MF media are TSC medium and mCP medium. TSC normally allows higher recoveries than mCP. TSC produces fewer false-positive results than mCP; however, it does produce more false-negatives. Two newer methods have substantial potential, CP Chromo Select agar, which allows better recoveries and greater specificity than mCP, and the Fung double tube method, which creates anaerobic conditions and allows enumeration of colonies in tubes in 5-6 hours. Aerobic spores are not associated with fecal contamination but they can be surrogates for environmentally stable pathogens in monitoring water for treatment efficacy; Bacillus cereus spores are normally measured on nutrient agar by the MF method.

Distribution of Enterotoxin- and Epsilon-Positive Clostridium perfringens Spores in U.S. Retail Spices

The role of spices as vehicles of foodborne illness prompted an examination of bacterial spores in these products. Here, we report on the levels and characteristics of spores of Clostridium perfringens associated with 247 U.S. retail spices. Forty-three confirmed isolates from 17% of samples were obtained, present at levels ranging from 3.6 to 2,400/g. Twenty-seven (63%) of C. perfringens isolates were positive for the enterotoxin gene ( cpe). Seven random spice isolates produced enterotoxin at levels of between 4 and 16 ng/mL, compared with three outbreak (control) strains that each produced enterotoxin levels of >1,024 ng/mL. D95°C levels (1.0 to 3.3 min) of spores of four randomly selected spice isolates suggests a plasmid-localized cpe, while one had D95°C (>45 min) consistent with chromosomally located cpe. Five of the 43 isolates possessed the epsilon toxin gene ( etx, as well as cpe). Foods could easily become contaminated with spores of cpe-positive C. perfringens by the addition of spices. Because of its spore-forming ability, its rapid generation times at elevated temperatures, improper heating, cooling, and holding conditions could lead to elevated levels of C. perfringens in foods, a requirement for its implication in foodborne outbreaks.

Inactivation of microbiota from urban wastewater by single and sequential electrocoagulation and electro-Fenton treatments

This work aims at comparing the ability of two kinds of electrochemical technologies, namely electrocoagulation (EC) and electro-Fenton (EF), to disinfect primary and secondary effluents from municipal wastewater treatment plants. Heterotrophic bacteria, Escherichia coli, enterococci, Clostridium perfringens spores, somatic coliphages and eukaryotes (amoebae, flagellates, ciliates and metazoa) were tested as indicator microorganisms. EC with an Fe/Fe cell at 200 A m−2 and natural pH allowed >5 log unit removal of E. coli and final concentration below 1 bacteria mL−1 of coliphages and eukaryotes from both effluents in ca. 60 min, whereas heterotrophic bacteria, enterococci and spores were more resistant. A larger removal was obtained for the primary effluent, probably because the flocs remove higher amount of total organic carbon (TOC), entrapping more easily the microbiota. EF with a boron-doped diamond (BDD) anode and an air-diffusion cathode that produces H2O2 on site was first performed at pH 3.0, with large or even total inactivation of microorganisms within 30 min. A more effective microorganism removal was attained as compared to EC thanks to •OH formed from Fenton's reaction. A quicker disinfection was observed for the secondary effluent owing to its lower TOC content, allowing the attack of greater quantities of electrogenerated oxidants on microorganisms. Wastewater disinfection by EF was also feasible at natural pH (∼7), showing similar abatement of active microorganisms as a result of the synergistic action of generated oxidants like active chlorine and coagulation with iron hydroxides. A sequential EC/EF treatment (30 min each) was more effective for a combined decontamination and disinfection of urban wastewater.