Sources of psychrophilic and psychrotolerant clostridia causing spoilage of vacuum-packed chilled meats, as determined by PCR amplification procedure.

AIMS: To determine possible preslaughter and processing sources of psychrophilic and psychrotolerant clostridia causing spoilage of vacuum-packed chilled meats. METHODS AND RESULTS: Molecular methods based on the polymerase chain reaction (PCR) amplification of specific 16S rDNA fragments were used to detect the presence of Clostridium gasigenes, Clostridium estertheticum, Clostridium algidicarnis and Clostridium putrefaciens in a total of 357 samples collected from ten slaughter stock supply farms, slaughter stock, two lamb-processing plants, their environments, dressed carcasses and final vacuum-packed meat stored at -0.5 degrees C for 5(1/2) weeks. Clostridium gasigenes, C. estertheticum and C. algidicarnis/C. putrefaciens were commonly detected in farm, faeces, fleece and processing environmental samples collected at the slaughter floor operations prior to fleece removal, but all these micro-organisms were detected in only 4 out of 26 cooling floor and chiller environmental samples. One out of 42 boning room environmental samples tested positive for the presence of C. gasigenes and C. estertheticum, but 25 out of 42 of these samples were positive for C. algidicarnis/C. putrefaciens. Nearly all of the 31 faecal samples tested positive for the presence of C. gasigenes and C. estertheticum; however, only two of these samples were positive for C. algidicarnis and/or C. putrefaciens. Clostridial species that were subject to this investigation were frequently detected on chilled dressed carcasses. CONCLUSIONS: The major qualitative and quantitative differences between the results of PCR detection obtained with the primers specific for 'blown pack' -causing clostridia (C. gasigenes and C. estertheticum) and those obtained with primers specific for C. algidicarnis and C. putrefaciens suggest that the control of meat spoilage caused by different groups of meat clostridia is best approached individually for each group. SIGNIFICANCE AND IMPACT OF THE STUDY: This paper provides information significant for controlling meat spoilage-causing clostridia in the meat-processing plants.

Influence of peroxyacetic acid-based carcass rinse on the onset of "blown pack" spoilage in artificially inoculated vacuum-packed chilled beef.

"Blown pack" spoilage is an increasingly reported spoilage condition of vacuum-packed chilled meats. This spoilage condition is primarily caused by a psychrophilic obligately anaerobic microorganism, Clostridium estertheticum. The present study investigated whether peroxyacetic acid (POAA)-based carcass rinse can delay the onset of gas production in chilled vacuum-packed beef artificially inoculated with C. estertheticum spores. The variables studied were (i) two prepackaging meat rinses (water and POAA-based rinse); (ii) three levels of C. estertheticum spores (0, 4, and 40 spores per cm2); and (iii) three postpackaging storage temperatures (-1.5, 0, and 2 degrees C). Treatment with POAA-based rinse marginally delayed the onset of pack blowing in packs carrying high numbers of C. estertheticum spores but not in packs carrying low levels of inoculum or in uninoculated controls. The presence of as few as 4 spores per cm2 of meat surface effectively decreased by two-thirds the nominal shelf life of vacuum-packed chilled beef. Increasing the inoculum by 10-fold to 40 spores per cm2 resulted in the additional acceleration of the onset of pack blowing. The onset of gas production was significantly delayed by storing the packaged product at -1.5 degrees C rather than at 0 degrees C. The results of this study indicate that the POAA-based rinse tested will not eliminate the spoilage threat posed by clostridial blown pack spoilage spores present on meat surfaces. POAA-based rinse can be used alone to achieve some extension of shelf life of beef cuts heavily contaminated with C. estertheticum spores. Alternatively, the rinse may offer an opportunity for a more substantial extension of shelf life of contaminated cuts when used with additional hurdles.

Efficacy of heat and ethanol spore treatments for the isolation of psychrotrophic Clostridium spp. associated with the spoilage of chilled vacuum-packed meats.

Psychrotrophic Clostridium spp. associated with chilled meat spoilage are difficult to isolate and culture. In this study the kinetics of heat and ethanol spore inactivation was determined as a first step towards optimising the recovery of psychrotrophic clostridial spores from meat and environmental samples. To determine heat inactivation, spores of nine isolates associated with spoiled chilled or frozen meat and a psychrotrophic reference strain Clostridium algidicarnis NCFB 2931, suspended in phosphate buffer, were exposed to temperatures between 75 degrees C and 95 degrees C for 0 to 120 min using a submerged tube procedure. Survivors after various temperature-time combinations were enumerated on Peptone Yeast Extract Glucose Starch (PYGS) agar containing lysozyme. D-values and z-values for each spore suspension were determined from their respective survival curves. To determine ethanol inactivation, similar phosphate buffer spore suspensions were mixed with equal volumes of absolute ethanol, incubated at 20 degrees C and survivors enumerated on lysozyme-containing PYGS agar after 0 to 300 min. Based on spore heat inactivation, the 10 isolates could be grouped as having either heat-sensitive or heat-resistant spores. For heat-sensitive spore types, 60 min ethanol treatment gave maximum spore recovery whereas for heat-resistant spore types, heat treatment at 80 degrees C for 10 min gave the best recovery. When the spore heat-resistance type is unknown, as would be the case when attempting an isolation from spoiled product, both an ethanol treatment and a separate heat treatment should be used, to ensure maximum spore recovery.

Influence of culture media on the recovery of psychrotrophic Clostridium spp. associated with the spoilage of vacuum-packed chilled meats.

This study was undertaken to determine the influence of culture media on the quantitative recovery of vegetative cells and spores of psychrotrophic Clostridium spp. associated with the spoilage of chilled meats. For recovery of vegetative cells and spores (presumptive counts), 24 h or 48 h broth cultures in Peptone Yeast Extract Glucose Starch (PYGS) broth were used; for spore counts, concentrated spore suspensions derived from 35-day cultures were used. For presumptive counts, seven non-selective and eight selective media were tested. Recovery of psychrotrophic clostridia with optimum growth temperatures between 15 degrees C and 20 degrees C was best with non-selective media, such as Peptone Yeast Extract Glucose Starch (PYGS) agar with lysozyme; recovery of clostridia with growth optima between 25 degrees C and 30 degrees C was best with selective media, such as Shahidi Ferguson Perfringens (SFP) agar. For organisms with the lower optimum growth temperature (heat-sensitive group) spore recovery after heat treatment (80 degrees C for 10 min) was best if a 2 M (pH 10) thioglycollate treatment (10 min at 45 degrees C) was used, followed by plating onto a lysozyme- or egg-yolk-containing medium. For organisms with the higher optimum growth temperature (heat-resistant group), spore recovery was best on Glucose Starch agar without added lysozyme. The diversity among psychrotrophic Clostridium spp. associated with chilled meat spoilage precludes the identification of a single 'best' recovery medium or technique. Consequently, a variety of complementary selective techniques and media must be used if comprehensive recovery is to be assured.

Association of psychrotrophic Clostridium spp. with deep tissue spoilage of chilled vacuum-packed lamb.

Early spoilage of commercial vacuum-packed chilled lamb legs was manifested as an objectionable 'cheesy', deep tissue odour that became evident when a cut was made into the stifle joint. Investigation of the probable causative agents led to the isolation of two psychrotrophic strains of clostridia. The isolates could not be identified using traditional identification schemes. One isolate was able to produce strong, objectionable 'cheesy' odours in deep tissues of artificially inoculated beef.

Molecular differentiation of clostridia associated with "blown pack" spoilage of vacuum-packed meats using internal transcribed spacer polymorphism analysis.

The 16S-23S rDNA internal transcribed spacer (ITS) polymorphism analysis was assessed for its suitability in rapid discrimination between species of psychrophilic and psychrotolerant clostridia associated with "blown pack" spoilage of vacuum-packed meats. DNA isolated from 10 reference and 20 meat strains of psychrophilic and psychrotolerant clostridia were used as templates in PCR amplification with primers complementary to conserved regions of the 3' end of the 16S rRNA and 5' end of the 23S rRNA genes directly flanking the spacer. The majority of strains showed multi-band ITS patterns when products of spacer amplification were visualised on an agarose gel. With the majority of meat strains, PCR amplification generated single banding pattern for a single clostridial species. However, meat strains of Cl. algidicarnis produced four different ITS banding patterns. With reference strains of psychrophilic and psychrotolerant clostridia, variation in spacer length was also observed between nonproteolytic Cl. botulinum type B (17B), E (Beluga) and F (202F). On the other hand, the number and size of the ITS amplification products could not be used for a differentiation of Cl. laramiense ATCC 51254(T) from Cl. estertheticum DSM 8809(T), Cl. putrefaciens DSM 1291(T) from Cl. algidicarnis NCFB 2931(T), or Cl. frigidicarnis strains from nonproteolytic Cl. botulinum type B (17B). The presence of interstrain, and lack of interspecies, ITS polymorphism observed in the present study with some clostridial species may preclude the use of 16S-23S rDNA spacer amplification for species-level discrimination and identification, respectively, of psychrophilic and psychrotolerant clostridia associated with meat spoilage. However, where interstrain, intraspecies heterogeneity of ITS amplification products exists, ITS analysis could be useful for tracing back psychrophilic and psychrotolerant clostridia responsible for meat spoilage to their meat plant sources.

Psychrotrophic Clostridium spp. associated with 'blown pack' spoilage of chilled vacuum-packed red meats and dog rolls in gas-impermeable plastic casings.

'Blown pack' spoilage of vacuum-packed chilled beef, lamb and venison, and of a cooked meat product, chilled dog rolls packed in an oxygen-impermeable plastic casing, was characterised by sensory, chemical and microbiological analysis. Investigation of the probable causative agents led to the isolation of eight strains of psychrotrophic clostridia. Three strains have been provisionally identified as C. difficile, C. beijerinckii and C. lituseburense; the other five remain unidentified. In inoculation studies only one isolate produced significant amount of gas on meat, causing pack 'blowing'. It is, therefore, possible that 'blown pack' spoilage involves a synergism with one or more other organisms.

The effect of peroxyacetic acid-based sanitizer, heat and ultrasonic waves on the survival of Clostridium estertheticum spores in vitro.

AIM: To determine the effect of selected physical and chemical treatments on the survival of 'blown pack'-causing Clostridium estertheticum. METHODS AND RESULTS: The study investigated the survival of the spores of 'blown pack'-causing C. estertheticum following the four treatments, which include: heat alone, ultrasound followed by heat treatment, peroxyacetic acid (POAA)-based sanitizer followed by heat treatment and POAA sanitizer followed by heat treatment in the presence of 20% animal fat. No C. estertheticum survivors were recovered in spore preparations that underwent either of the two treatments with the sanitizer, resulting in the inactivation of 4 to 5 log CFU ml(-1) of spores. Similarly, no survivors were detected in spore preparations that were treated with the sanitizer for 5 min at room temperature without further heat treatment. When using heat alone and ultrasound followed by heat treatment, complete spore inactivation did not occur for spores heated at times and temperature combinations other than 240 s at 100 degrees C. CONCLUSIONS: POAA sanitizer used with or without heat is capable of in vitro inactivation of at least 4 log CFU ml(-1)C. estertheticum spores. SIGNIFICANCE AND IMPACT OF THE STUDY: The data generated in the study provide background information for controlling 'blown pack'-causing clostridia on dressed carcasses and in meat plant environment.

Use of restriction fragment length polymorphism analysis to differentiate strains of psychrophilic and psychrotropic clostridia associated with blown pack' spoilage of vacuum-packed meats.

Reference and meat strains of psychrophilic and psychrotrophic clostridia were differentiated using restriction fragment length polymorphism (RFLP) analysis of genomic DNA (DNA-RFLP) and the polymerase chain reaction-amplified 16S rDNA gene (PCR-RFLP). Groupings obtained with PCR-RFLP were confirmed with 16S rDNA gene sequencing. DNA-RFLP resolved 19 of the 22 meat strains into 11 groups. Three meat strains were untypable using this method. All reference strains representing different genotypic species could be distinguished by the restriction patterns of 16S rDNA genes. With PCR-RFLP, the 22 meat strains produced eight distinct genotypes. 16S rDNA gene sequencing confirmed that each genotype was represented by a distinct sequence. PCR-RFLP restriction patterns of 15 meat strains matched those of one of two of the seven reference strains used. Seven meat strains whose RFLP restriction patterns of 16S rDNA genes differed from those of any reference strains probably represent four previously undescribed species. Although RFLP analysis of the amplified 16S rDNA gene allowed differentiation of psychrophilic and psychrotrophic clostridia at the genotypic species level and below, comparison of PCR-RFLP patterns and 16S rDNA sequences of unknown clostridial isolates with patterns and sequences of reference strains may not effect ready identification of these micro-organisms. The results of this study will be useful in diagnosis of the cause of premature spoilage of chilled vacuum-packed meats and in tracing spoilage-causing clostridia to their source(s) in the abattoir.

PCR detection of psychrotolerant clostridia associated with deep tissue spoilage of vacuum-packed chilled meats.

AIMS: To develop a practical molecular procedure that directly (without isolation) and specifically detects the presence of clostridia, which cause the deep tissue spoilage condition. METHODS AND RESULTS: A primer set was designed and a PCR amplification procedure developed to detect the presence of Clostridium algidicarnis and Cl. putrefaciens 16S rDNA gene fragments in meat. The procedure yielded amplicons of the expected size with homologous DNA templates, but failed to give PCR products with DNAs from 47 food clostridia and common meat spoilage micro-organisms. The minimum level of detection was 10(4) cfu g-1 for nonenriched meat samples. Based on the established specificity of these primers, as well as DNA sequencing of amplicons, the presence of Cl. algidicarnis and/or Cl. putrefaciens was confirmed in a swab sample taken from the cartilage of an ovine stifle joint, which on opening exhibited strong offensive odours. CONCLUSIONS: The developed method can be used for rapid detection of clostridia causing deep tissue spoilage in commercial vacuum packs. SIGNIFICANCE AND IMPACT OF THE STUDY: The paper reports practical procedures that can be used for rapid confirmation of the causative agents of deep tissue clostridial spoilage in commercial vacuum-packed chilled meats.

A PCR survey of psychrotrophic Clostridium botulinum-like isolates for the presence of BoNT genes.

Isolates (259) of psychrotrophic Clostridium spp. associated with either blown pack spoilage (five isolates) or slaughter stock (254 isolates) were screened for the presence of botulinum neurotoxin (BoNT) genes using degenerate PCR primers capable of amplifying A, B, E, F and G BoNT genes. No BoNT gene amplification products were detected using DNA templates from the 259 psychrotrophic isolates, including 249 isolates that showed the same 16S rRNA gene Restriction Fragment Length Polymorphism (RFLP) patterns as authentic Cl. botulinum type B. It is concluded that although the growth of such micro-organisms in vacuum-packed chilled meat leads to product spoilage, it does not prejudice product safety.

Abattoir sources of psychrophilic clostridia causing blown pack spoilage of vacuum-packed chilled meats determined by culture-based and molecular detection procedures.

AIMS: To identify the abattoir source(s) of psychrophilic clostridia causing 'blown pack' spoilage of vacuum-packed chilled meats. METHODS AND RESULTS: Molecular procedures were used to detect the presence of specific 16S rRNA gene fragments of blown pack-causing clostridia in samples collected from a commercial abattoir and its environs. Blown pack-causing clostridia were consistently detected in hide, soil and faecal samples, as well as in samples collected at slaughter plant locations associated with handling of animals and animal carcasses prior to pelt removal. CONCLUSIONS: The data indicate that pelts per se or soil particles/faecal material attached thereto are the most probable primary reservoir of blown pack clostridia in the abattoir. SIGNIFICANCE AND IMPACT OF THE STUDY: The paper provides information critical for controlling blown pack spoilage in commercial meat-processing plants.

PCR detection of psychrophilic Clostridium spp. causing 'blown pack' spoilage of vacuum-packed chilled meats.

AIMS: To develop a practical molecular procedure that directly, without isolation, and specifically detects the presence of clostridia which cause 'blown pack' spoilage of vacuum-packed meat. METHODS AND RESULTS: Primer sets and PCR amplification procedures were developed that detect the presence of 16S rDNA gene and/or 16S-23S rDNA internal transcribed spacer fragments of 'blown pack' causing clostridia in meat. The specificity of the developed procedures was evaluated with DNA obtained from close phylogenetic neighbours of 'blown pack' causing clostridia, food clostridia and common meat spoilage microorganisms. The sensitivity of detection was assessed in non-enriched and low-temperature-enriched beef mince inoculated with serially diluted pure cultures of Clostridium estertheticum DSMZ 8809T and Cl. gasigenes DB1AT. The efficacy of detection procedures was evaluated for naturally contaminated vacuum-packed meat samples. Three primer sets, 16SE, 16SDB and EISR, produced amplicons of the expected size with DNA templates from target clostridia, but failed to yield PCR products with DNAs from any other microorganisms tested. With 16SE and 16SDB primers, minimum levels of detection were 104 CFU g(-1) for non-enriched, and 102 CFU g(-1) for enriched meat samples. Based on the established specificity of these primers, as well as DNA sequencing of amplicons, Cl. gasigenes was confirmed as the causative agent of 'blown pack' spoilage in two packs, and Cl. estertheticum as the causative agent in the third. CONCLUSIONS: The developed method can be used for rapid detection of 'blown pack' causing clostridia in commercial blown packs, or following low temperature enrichment, for detection of these microorganisms in meat containing as few as 100 clostridial cells per gram. SIGNIFICANCE AND IMPACT OF THE STUDY: The paper reports practical procedures that can be used for rapid confirmation of the causative agents of clostridial 'blown pack' spoilage in commercial spoiled packs, or for detection of psychrophilic clostridia in epidemiological trace back of 'blown pack' spoilage incidents in meat processing plants.

Clostridium algidixylanolyticum sp. nov., a psychrotolerant, xylan-degrading, spore-forming bacterium.

A psychrotolerant Clostridium species was isolated from vacuum-packed, temperature-abused raw lamb. Colonies of this micro-organism on sheep-blood agar were circular with an entire margin, grey-white, translucent and beta-haemolytic. Cells were single, tapered, motile rods. Elliptical subterminal spores were produced in the late stationary growth phase. Spores did not cause swelling of the maternal cells. The micro-organism was obligately anaerobic. In peptone yeast extract glucose starch (PYGS) broth at pH 7.0, the micro-organism grew optimally between 25.5 and 30.0 degrees C. The temperature range for growth was 2.5-32.2 degrees C. At 26 degrees C, the micro-organism grew optimally at pH 6.8 to 7.0. The pH range for anaerobic growth was 4.7-9.1. The micro-organism was saccharoclastic, hydrolysed starch and degraded xylan. The fermentation products formed in PYGS broth were acetate, formate, lactate, ethanol, butyrate, butanol, hydrogen and carbon dioxide. The G + C content of the DNA was 38.4 mol%. Phylogenetic analyses indicated that the strain belongs to cluster XIVa of the genus Clostridium (sensu Collins et al. 1994). The new strain differed from phylogenetically related clostridia in terms of cellular fatty acid composition, soluble protein profiles and phenotypic properties. On the basis of phenotypic and genotypic characterization data, the strain was assigned to a new species, namely Clostridium algidixylanolyticum. The type strain is strain SPL73T (= DSM 12273T).

The abattoir source of culturable psychrophilic Clostridium spp. causing 'blown pack' spoilage of vacuum-packed chilled venison.

AIMS: To identify the abattoir source(s) of culturable psychrophilic clostridia causing 'blown pack' spoilage of vacuum-packed chilled meats. METHODS AND RESULTS: Psychrophilic and psychrotolerant clostridia were isolated from hides, faeces and tonsils of deer slaughter stock, and from a meat plant environment. The isolates were differentiated using restriction fragment length polymorphism analysis of the 16S rDNA gene (PCR-RFLP) and 16S-23S rDNA internal transcribed spacer (ITS) analysis. PCR-RFLP group I clostridia were found to have restriction patterns indistinguishable from the patterns of 'blown pack'-causing Clostridium gasigenes DB1A(T) and R26. Gas production in packs inoculated with vegetative cells of PCR-RFLP group I clostridia was first evident after 14 days at 2 degrees C. The prevalence of these clostridia was similar in hide and faecal samples from slaughter animals, but these micro-organisms were absent from tonsils and the meat plant environment. Banding patterns of PCR-RFLP group II clostridia showed some cross-similarity with patterns of the 'blown pack'-causing micro-organism Cl. estertheticum DSM 8809(T) and Cl. estertheticum-like meat strains. The majority of clostridia in PCR-RFLP group II were found in the faeces of slaughter animals. Isolates representing PCR-RFLP group II did not, however, produce gas in vacuum packs stored at 2 degrees C for 84 days. CONCLUSIONS: The data suggest that soil particles attached to hide or present in faeces are the most probable primary reservoir from which 'blown pack' clostridia are introduced onto carcasses. Therefore, dressing procedure hygiene remains paramount in order to control the spread of psychrophilic Clostridium spp. in a meat plant. SIGNIFICANCE AND IMPACT OF THE STUDY: The paper provides information critical for controlling 'blown pack' spoilage in meat processing plants. It reports on the use of molecular techniques for determination of abattoir sources of 'blown pack'-causing clostridia.

Clostridium frigidicarnis sp. nov., a psychrotolerant bacterium associated with 'blown pack' spoilage of vacuum-packed meats.

Two strains of a psychrotolerant Clostridium, isolated from vacuum-packed, temperature-abused beef, were characterized using a multiphasic approach. The strains were Gram-positive motile rods producing elliptical subterminal spores during early stationary growth phase. The strains were psychrotolerant. At pH 7.0, they grew between 3.8 and 40.5 degrees C; their optimum growth temperature was 30.0-38.5 degrees C. At 30 degrees C, the pH range for growth was between 4.7 and 9.5; the optimum pH for growth was 6.4-7.2. The organisms were proteolytic and saccharolytic, lecithinase-positive and hydrolysed gelatin. The fermentation products formed in peptone/yeast extract/glucose/starch broth were acetate, ethanol, butyrate, isovalerate, butanol, isobutyrate, oxalacetate, lactate, hydrogen and carbon dioxide. The DNA G + C compositions of the two meat strains were 27.3 and 28.4 mol%. Phylogenetic analyses indicated that the strains belong to Cluster I of the genus Clostridium (sensu Collins et al., 1994). The new strains differed from phylogenetically related clostridia in terms of cellular fatty acid composition, soluble protein profiles and phenotypic properties. On the basis of phenotypic and genotypic characterization data, the strains were assigned to a new species for which the name Clostridium frigidicarnis is proposed; strain SPL77AT (= DSM 12271T) is the type strain.

Clostridium gasigenes sp. nov., a psychrophile causing spoilage of vacuum-packed meat.

Two psychrophilic Clostridium strains, DB1AT and R26, were isolated from incidences of 'blown-pack' spoilage of vacuum-packed chilled lamb. Vacuum packs of meat inoculated with these strains developed gas bubbles and pack distension within 14 d storage at 2 degrees C. The two main gases responsible for pack distension were carbon dioxide and hydrogen. 1-Butanol, butyric and acetic acid and butyl esters were the major volatile compounds produced by the strains in the artificially inoculated packs. The unknown strains were Gram-positive motile rods producing elliptical subterminal spores during the late-stationary growth phase. At pH 7.0, they grew from -1.5 to 26 degrees C, and their optimum growth temperature was 20-22 degrees C. At 20 degrees C, the pH range for growth was 5.4-8.9 and the optimum pH for growth was 6.2-8.6. In peptone/yeast extract broth, the organisms grew little or not at all in the absence of fermentable carbohydrates. Both strains hydrolysed gelatin, aesculin and starch. The fermentation products formed in peptone yeast extract glucose starch broth were ethanol, acetate, butyrate, lactate, butanol, carbon dioxide and hydrogen. The G+C contents of the DNA of strains DB1AT and R26 were 29.4 and 28.3 mol%, respectively. Phylogenetic analyses indicated that the strains belong to cluster I of the genus Clostridium (sensu Collins et al. 1994). The new strains differed from the phylogenetically related clostridia in cellular fatty acid composition, soluble protein profiles and phenotypic properties. On the basis of rDNA analysis and phenotypic and phylogenetic characterization, the strains were assigned to a new species for which the name Clostridium gasigenes is proposed. Strain DB1AT (= DSM 12272T) is designated as the type strain.