Use of power ultrasound to enhance the thermal inactivation of Clostridium perfringens spores in beef slurry.

Clostridium perfringens is a pathogen of concern in pasteurised foods. The main objective of this study was to use power ultrasound to enhance the thermal inactivation of C. perfringens spores in beef slurry. The effect of simultaneous ultrasound and heat (TS, thermosonication) on the spore inactivation in beef slurry was first investigated. At 75 °C, a 60 min TS process (24 kHz, 0.33 W/g) resulted in a less than 1.5 log reduction for both C. perfringens NZRM 898 and NZRM 2621 spores. Then, the thermal inactivation first order kinetic parameters of C. perfringens spores in beef slurry were estimated for the two strains. The D105 °C- and z-values were 2.5 min and 10.6 °C for NZRM 898 and 1.8 min and 10.9 °C for NZRM 2621. After, the effect of a spore heat shock followed by ultrasound on its thermal inactivation in beef slurry was investigated. This heat shock+ultrasound pretreatment was able to double the spore thermal inactivation rate in beef slurry. For example at 95 °C D-value of 20.2 min decreased to 9.8 min, demonstrating that spore exposure to heat shock followed by ultrasonication enhanced its thermal inactivation.

A sporulation factor is involved in the morphological change of Clostridium perfringens biofilms in response to temperature.

Biofilm formation has been associated with bacterial pathogenesis, such as nosocomial and chronic infections, as the resistance of biofilms to environmental stresses has increased. Clostridium perfringens is a Gram-positive spore-forming anaerobic pathogen. This organism survives antibiotic treatment through the formation of biofilms or spores, but the environmental and regulatory factors involved in the biofilm formation remain unclear. Here, we observed that temperature regulates C. perfringens biofilm morphology. At 37°C, C. perfringens adhered to the substrate surface and formed a flat, thin biofilm, herein referred to as adhered biofilm. However, at 25°C, this bacterium did not adhere and produced a threadlike extracellular matrix, forming a viscous, thick biofilm, herein referred to as pellicle biofilm. Pellicle biofilm formation requires the sporulation master regulator, Spo0A, and the toxin regulator, CtrAB, and is enhanced in the absence of the global repressor, AbrB. These transcriptional regulator genes are regulated by each other and temperature. Adhered-biofilm formation requires AbrB and pilA2, which encodes a component of type IV pili (TFP). TFP expression was activated at 37°C and regulated through Spo0A, AbrB, and CtrAB. These results indicate that the morphology of C. perfringens biofilm is dependent on temperature through the differential production of extracellular matrix and the activity of TFP. Moreover, pellicle biofilm formation is involved in sporulation and toxin production. Here, we demonstrated that clostridial biofilm formation is closely associated with sporulation and that the morphological change of the biofilms could play an important role in the pathogenesis of this organism.

The Sialidases of Clostridium perfringens type D strain CN3718 differ in their properties and sensitivities to inhibitors.

Clostridium perfringens causes histotoxic infections and diseases originating in animal or human intestines. A prolific toxin producer, this bacterium also produces numerous enzymes, including sialidases, that may facilitate infection. C. perfringens type D strain CN3718 carries genes encoding three sialidases, including two large secreted sialidases (named NanI and NanJ) and one small sialidase (named NanH) that has an intracellular location in log-phase cultures but is present in supernatants of death phase cultures. Using isogenic mutants of CN3718 that are capable of expressing only NanJ, NanI, or NanH, the current study characterized the properties and activities of each sialidase. The optimal temperature determined for NanJ or NanH enzymatic activity was 37°C or 43°C, respectively, while NanI activity increased until temperature reached 48°C. NanI activity was also the most resistant against higher temperatures. All three sialidases showed optimal activities at pH 5.5. Compared to NanJ or NanH, NanI contributed most to the sialidase activity in CN3718 culture supernatants, regardless of the substrate sialic acid linkage; NanI also released the most sialic acid from Caco-2 cells. Only NanI activity was enhanced by trypsin pretreatment and then only for substrates with an α-2,3- or α-2,6-sialic acid linkage. NanJ and NanI activities were more sensitive than NanH activity to two sialidase inhibitors (N-acetyl-2,3-dehydro-2-deoxyneuraminic acid and siastatin B). The activities of the three sialidases were affected differently by several metal ions. These results indicated that each C. perfringens sialidase has distinct properties, which may allow these enzymes to play different roles depending upon environmental conditions.

Nitrergic response to Clostridium perfringens infection in the rat brain regions: effect of red light irradiation.

A single intraperitoneal injection of a gram-positive pathogen Clostridium perfringens (Cp) causes a remarkable down-regulation the constitutive nitric oxide synthase (cNOS) with a simultaneous increase in the activity of inducible NOS (iNOS) and the level of reactive nitrogen species in the rat brain major regions (cortex, striatum, hippocampus and hypothalamus) at 48 h post-administration of Cp. Treatment by both a semiconductor laser (SCL) and/or a light-emitting diode (LED) with same wavelength, energy density and time exposure (continuous wave, λ=654 nm, fluence=1.27 J/cm(2), time exposure=600 s) could modulate brain nitrergic response following Cp-infection. Besides, unlike the LED, the SCL-irradiation prevents the cNOS inhibition in all the studied brain regions and might be useful in restoring its function in neurotransmission and cerebral blood flow, along with providing a protective effect against nitrosative stress-induced iNOS-mediated injury in the brain regions.

Effect of daylight on regrowth of bacteria in anaerobically digested sludge.

This study investigated the regrowth of total coliform, Salmonella, and Clostridium perfringens in anaerobically digested sludge after centrifuge dewatering in the presence and absence of daylight. Sludge cake and centrate samples were collected from a treatment plant, and half of the samples was stored in daylight and the other half was stored in dark for three weeks. The bacteria levels in the cake and centrate samples were measured periodically throughout the storage period, and all three bacteria showed substantial regrowth. Presence of daylight increased the regrowth of Salmonella both in sludge cake and centrate, and increased the regrowth of total coliform in centrate. Salmonella exhibited the highest regrowth rate in cake among the three bacteria tested both in the presence and absence of light. Daylight did not appear to have a significant impact on the regrowth of Clostridium perfringens in cake and centrate, and on the regrowth of total coliform in cake. This might, however, be caused by the masking effect of the higher initial numbers of these bacteria in the samples. There is need for more research to thoroughly understand the effect of daylight on the regrowth of sludge bacteria.

Using irradiation treatment for reduction of anaerobic bacteria from a wastewater treatment plant.

This study was done to investigate the reduction of anaerobic bacteria (Clostridium perfringens) by ultrasound irradiation technology (USI). C. perfringens is the most important of the sulfite-reducing clostridia and is normally present in human and animal feces. Clostridial spores survive longer than coliforms, Escherichia coli or enterococci, and are consequently used as an indicator of past fecal pollution. The spores are not always inactivated by chlorination, but are not a hazard to health in potable water. C. perfringens are very resistant to chlorine and other drinking-water disinfectants. Conventional water treatment practices including chlorination are sometimes inadequate for inactivation of C. perfringens. Ultrasound irradiation is able to inactive C. perfringens through a number of physical and chemical effects arising from acoustic cavitation. The phenomenon of cavitation, which involves formation, growth, and violent collapse of vapour bubbles in a liquid media, is known to generate a high-intensity pressure, which affects the cell and C. perfringens' viability. Cavitations disrupt C. perfringens. Ultrasound irradiation at a frequency of 42 kHz was used to expose aqueous suspension of C. perfringens to evaluate the disinfection efficacy of ultrasound irradiation. Results indicated a significant increase in reduction of C. perfringens with increasing ultrasound time. C. perfringens was inactivated by 99.98% after 90 min.

Roles of DacB and spm proteins in clostridium perfringens spore resistance to moist heat, chemicals, and UV radiation.

Clostridium perfringens food poisoning is caused mainly by enterotoxigenic type A isolates that typically possess high spore heat resistance. Previous studies have shown that alpha/beta-type small, acid-soluble proteins (SASP) play a major role in the resistance of Bacillus subtilis and C. perfringens spores to moist heat, UV radiation, and some chemicals. Additional major factors in B. subtilis spore resistance are the spore's core water content and cortex peptidoglycan (PG) structure, with the latter properties modulated by the spm and dacB gene products and the sporulation temperature. In the current work, we have shown that the spm and dacB genes are expressed only during C. perfringens sporulation and have examined the effects of spm and dacB mutations and sporulation temperature on spore core water content and spore resistance to moist heat, UV radiation, and a number of chemicals. The results of these analyses indicate that for C. perfringens SM101 (i) core water content and, probably, cortex PG structure have little if any role in spore resistance to UV and formaldehyde, presumably because these spores' DNA is saturated with alpha/beta-type SASP; (ii) spore resistance to moist heat and nitrous acid is determined to a large extent by core water content and, probably, cortex structure; (iii) core water content and cortex PG cross-linking play little or no role in spore resistance to hydrogen peroxide; (iv) spore core water content decreases with higher sporulation temperatures, resulting in spores that are more resistant to moist heat; and (v) factors in addition to SpmAB, DacB, and sporulation temperature play roles in determining spore core water content and thus, spore resistance to moist heat.

Role of small, acid-soluble spore proteins in the resistance of Clostridium perfringens spores to chemicals.

Previous work showed that C. perfringens spores lacking the majority of alpha/beta-type small, acid-soluble spore proteins (SASPs) (termed alpha(-) beta(-) spores) exhibit greatly decreased resistance to moist heat and UV radiation. The current study demonstrated that these alpha(-) beta(-) spores had reduced resistance to hydrogen peroxide, hydrochloric acid, nitrous acid and formaldehyde. These results clearly demonstrate the important role of alpha/beta-type SASPs in the resistance of C. perfringens spores to chemicals.

Antisense-RNA-mediated decreased synthesis of small, acid-soluble spore proteins leads to decreased resistance of clostridium perfringens spores to moist heat and UV radiation.

Previous work has suggested that a group of alpha/beta-type small, acid-soluble spore proteins (SASP) is involved in the resistance of Clostridium perfringens spores to moist heat. However, this suggestion is based on the analysis of C. perfringens spores lacking only one of the three genes encoding alpha/beta-type SASP in this organism. We have now used antisense RNA to decrease levels of alpha/beta-type SASP in C. perfringens spores by approximately 90%. These spores had significantly reduced resistance to both moist heat and UV radiation but not to dry heat. These results clearly demonstrate the important role of alpha/beta-type SASP in the resistance of C. perfringens spores.

Transcription activation of a UV-inducible Clostridium perfringens bacteriocin gene by a novel sigma factor.

Expression of the plasmid-encoded Clostridium perfringens gene for bacteriocin BCN5 was shown to depend in vivo and in vitro on the activity of UviA protein. UviA, also plasmid-encoded, proved to be an RNA polymerase sigma factor and was also partly autoregulatory. The uviA gene has two promoters; one provided a UviA-independent, basal level of gene expression while the stronger, UviA-dependent promoter was only utilized after the cell experienced DNA damage. As a result, BCN5 synthesis is induced by treatment with UV light or mitomycin C. UviA is related to a special class of sigma factors found to date only in Clostridium species and responsible for activating transcription of toxin genes in Clostridium difficile, Clostridium tetani, and Clostridium botulinum.

Reduction of enteric microbes in flushed swine wastewater treated by a biological aerated filter and UV irradiation.

An aerobic biofilter system was studied to assess its effectiveness for reducing enteric microbial indicators in flushed swine wastewater under different seasonal conditions. A laboratory-scale, low-pressure UV collimated beam apparatus was used to investigate the effectiveness of UV irradiation for inactivating enteric bacteria, coliphages, and bacterial spores in treated and untreated swine wastewater having unfiltered absorbances of 5 to 11 cm(-1) and total suspended solids concentrations of 500 to 1200 mg/L. Fecal coliforms, Escherichia coli, enterococci, somatic coliphages, and male-specific coliphages were reduced by 97 to 99% in the biofilter system when reactor water temperatures were between 23 and 32 degrees C. Salmonella were reduced by 95 to 97% when water temperatures were 17 to 32 degrees C. Of the six microbial indicators studied. Clostridium perfringens spores were typically reduced the least by the biofilter system. At an average absorbed UV irradiation dose of 13 mJ/cm2, maximum reductions of fecal coliforms, E. coli, enterococci, C. perfringens spores, and somatic coliphages in biofilter system effluent were 2.2, 2.1, 1.3, 0.2, and 2.3 log10, respectively. The results of this study show that the aerobic biofilter system can be an effective alternative for treatment of flushed swine waste. Ultraviolet irradiation can be effective for further reducing enteric microbe concentrations in biologically-treated swine waste, as well as in lower quality wastewaters, indicating its general potential for pathogen reductions in low-quality wastewaters intended for beneficial reuse.

Irradiation of diets fed to captive exotic felids: microbial destruction, consumption, and fecal consistency.

Two frozen, raw horse meat-based diets fed to captive exotic felids at Brookfield Zoo were irradiated to determine the extent of microbial destruction and whether radiation treatment would affect consumption and/or fecal consistency in exotic cats. Fifteen cats, two African lions (Panthera leo), two Amur tigers (Panthera tigris altaica), one Amur leopard (Panthera pardus orientalis), two clouded leopards (Neofelis nebulosa), two caracals (Felis caracal), one bobcat (Felis rufus), and five fishing cats (Felis viverrinus), housed at Brookfield Zoo were fed nonirradiated and irradiated raw diets containing horse meat with cereal products and fortified with nutrients: Nebraska Brand Feline and/or Canine Diet (Animal Spectrum, North Platte, Nebraska 69103, USA). Baseline data were obtained during a 2-wk control period (nonirradiated diets), which was followed by a 4-wk period of feeding comparable irradiated diets. Feed intake and fecal consistency data were collected. An estimated radiation dose range of 0.5-3.9 kilograys reduced most microbial populations, depending on specific diet and microbe type. Irradiation had no overall effect on either feed consumption or fecal consistency in captive exotic cats, regardless of species, age, sex, or body mass. Data indicate that irradiation of frozen horse meat-based diets (packaged in 2.2-kg portions) result in microbial destruction in these products but that product storage time between irradiation and sampling may also affect microbial reduction. However, irradiation would be an appropriate method for reducing potentially pathologic bacteria in raw meat fed to exotic cats.

[Microwave effect on survival of sporulated bacteria inoculated in minced meat]. / Efecto de las microondas sobre la sobrevivencia de bacterias esporuladas inoculadas en carne molida.

Due to the current tendency of cooking and heating meat prepared foods in microwave ovens and the possibility that they transmit bacterial diseases, the survival rate of spore-forming bacteria was evaluated in minced meat samples. Meat was innoculated with a known number of Bacillus cereus and Clostridium perfringens spores, and laterly thawed and cooked in an Amana microwave oven (2450 Hz). Survival rate was determined according to the methodology described by Vanderzant & Splittstoesser, and the activity of the enzyme acid phosphatase was determined as cooking parameter. B. cereus spore showed a decrease in its number as the time of exposition increased, but without fully disappearing. C. perfringens spores also decreased in number, but showed a later increase, associated with the germination of survival spores.

Efecto de las microondas sobre la sobrevivencia de bacterias esporuladas inoculadas en carne molida / Microwave effect on the survival of sporulated bacteria inoculated in minced meat

Debido a la creciente tendencia de cocinar y recalentar en horno de microondas platillos a base de carne y a la posibilidad de que estos transmitan enfermedades de origen bacteriano, se evaluó la capacidad de sobrevivencia de esporas de bacterias inoculadas en carne molida y tratadas en horno de microonda. Las tortas de carne fueron inoculadas con un número conocido de esporas de Bacillus cereus o Clostridium perfringens y posteriormente cocinadas en horno de microondas Amana de 2450 Hz. Se utilizó la técnica descrita por Vanderzant y Splittstoesser para determinar la tase de sobrevivencia y la actividad de la enxima fosfatasa ácida como parámetro de conocimiento. Las esporas de B. cereus mostraron una tendencia a disminuir en número, conforme aumenta el tiempo de exposición a la radiación, pero si llegar a desaparecer totalmente. Las esporas de C. perfringens disminuyen en número al aumentar el tiempo de exposición, pero presentan un segundo aumento asociado a la germinación de esporas sobrevivientes.

Effect of gamma irradiation on shelf life and bacterial and viral loads in hard-shelled clams (Mercenaria mercenaria).

The feasibility of using 60Co gamma irradiation to inactivate total coliforms, fecal coliforms, Escherichia coli, Clostridium perfringens, and F-coliphage in hard-shelled clams, Mercenaria mercenaria, was investigated. The results of three trials indicated average D10 values of 1.32 kGy for total coliforms, 1.39 kGy for fecal coliforms, 1.54 kGy for E. coli, 2.71 kGy for C. perfringens, and 13.50 kGy for F-coliphage. Irradiation doses of > 0.5 kGy were significantly lethal to the shellfish.

Influence of radiotherapy on intestinal microflora in cancer patients.

We investigated in 15 patients with carcinoma of the uterine cervix or endometrium, who were undergoing postoperative radiation therapy, the effects of different fractionated radiation exposures on counts of fecal bacteria, on the growth of Clostridium difficile and Clostridium perfringens enterotoxin production. We observed a generally significant decrease in intestinal microflora after the first radiation exposure, whereas at the end of radiotherapy all bacteria increased and reached basal values except Enterococcus faecium 1, lactobacilli and total anaerobes. In some patients we observed an overgrowth of some Clostridium spp. which were potential pathogens associated with clinical symptoms. We did not observe an influence of multiple radiations on C. perfringens enterotoxin fecal contents. We conclude that patients receiving radiotherapy may benefit from the intake of oral bacteriotherapy, i.e. live beneficial bacteria such as Bacillus subtilis at the beginning of the irradiation exposure.

Reversal of radiation-dependent heat sensitization of Clostridium perfringens spores.

The effect of solute concentration on the sensitization of Clostridium perfringens spores to heat by ionizing radiation was investigated. As we have shown previously, spores of C. perfringens treated with gamma radiation are now sensitive to subsequent heat treatments than are spores that receive no radiation treatment. When gamma-irradiated spores were heated in the presence of increasing concentrations of glycerol or sucrose, the heat sensitivity induced by irradiation was progressively decreased. The magnitude of the increase in heat resistance induced by extracellular solutes was greater in gamma-irradiated spores than in nonirradiated spores. Based on these observations, it is proposed that the induction of heat sensitivity in spores by radiation is related to the loss of osmoregulatory or dehydrating mechanisms in irradiated spores.

Sensitization of Clostridium perfringens spores to heat by gamma radiation.

Spores of Clostridium perfringens, type A, were given separate or sequential treatments of gamma radiation (0 to 0.7 Mrad) and/or high temperature (93 to 103 degrees C). Prior heating, sufficient to inactivate 40 to 99% of the viable spores, had no effect on the subsequent radiation inactivation rate. Prior irradiation had a sensitizing effect on subsequently heated spores. The degree of sensitization to heat, as measured by thermal inactivation rate, increased with increased radiation pretreatment dose.

Radiation resistance of spores of some Clostridium perfringens strains.

Clostridium perfringens spores (eight strains) were irradiated in a model system with 60Co gamma rays at -30 C. The quantal response data obtained were analyzed with extreme value statistics. It was found (at the 95% confidence level) that all eight strains followed the same rate of death and that this rate was probably (at the 95% level) not exponential. The statistics did not exclude, however, a normal, lognormal, Weibull, or related rate of spore kill. A more definitive study would be required to distinguish between the latter distributions.