An investigation of the environmental niches of blown pack spoilage causing Clostridium estertheticum and Clostridium gasigenes on New Zealand beef and sheep farms.

The transfer of blown pack spoilage causing Clostridium spores from the farm to the meat plant is of growing concern to the meat industry. This study investigated the environmental niches of these Clostridium spp., specifically Clostridium estertheticum and Clostridium gasigenes in the beef and sheep farm environments in New Zealand. Faecal, soil, grass, drinking water, puddle water and feed (fodder beet, hay, bailage and silage, where available) samples were collected on five beef and sheep farms during Winter and Spring in 2018, in North and South Island, respectively. Beef and sheep farm samples were tested for C. estertheticum and C. gasigenes using enrichment plus PCR, qPCR and direct plating. C. estertheticum was detected in bovine faecal (4%), soil (2-18%) and grass (0-12%) samples at concentration of up to 2.0 log10 cfu/g. C. gasigenes were found in 18-46% of faecal, 16-82% of soil, 12-44% of grass, 0-44.4% of drinking water and 0-58.3% of puddle water samples tested and the direct counts ranged from 2.4 log10 cfu/ml in puddle water to 3.4 log10 cfu/g in soil. C. estertheticum were detected by qPCR in sheep farms in ovine feces (2.3%), soil (2.3%) and fodder beet (10%). All other sample types (grass, drinking water, puddle water, baleage, hay, silage and fodder beet) were negative using direct and enrichment plus PCR methods. In contrast C. gasigenes was detected in of faecal (22.7-38.6%), soil (22.7-84.1%), grass (17.5-34.1%) drinking water (35.7-78.6%), puddle water (33.3-40%), hay baleage (57%), silage (2%) and fodder beet (10%) at concentrations of up to 3.7 log10 cfu/g/ml. It was concluded that C. estertheticum and C. gasigenes were common on beef and sheep farms with the latter having higher incidence and mean concentration.

An investigation into the ecological niches and seasonal nature of Clostridium estertheticum and Clostridium gasigenes in the Irish beef farm environment.

Blown pack spoilage (BPS) of vacuum packaged beef is caused by psychrotolerant and psychrophilic Clostridium species, primarily Clostridium estertheticum and Clostridium gasigenes. The aim of this study was to investigate the environmental niches and impact of season on these BPS Clostridium spp. on Irish beef farms. On each of five different beef farms, faecal (10), soil (5), silage (5), air (5), bedding straw (5), drinking water (5) and puddle/ditch water (5) samples were collected during Spring, Summer, Autumn and Winter and tested for C. estertheticum and C. gasigenes using culture (direct plating and enrichment) and molecular (conventional PCR and quantitative PCR (qPCR)) based techniques. C. estertheticum and C. gasigenes were detected in all sample types, with qPCR detection rates ranging from 4% to 50% and at concentrations of up to 1·5 log10 CFU per g and 3·5 log10 CFU per g, respectively. The impact of season was not clear as the results were mixed depending on the detection method used. It was concluded that BPS-causing C. estertheticum and C. gasigenes are widely distributed in the beef farm environment.

An investigation of the effect of rapid slurry chilling on blown pack spoilage of vacuum-packaged beef primals.

The aim of this study was to investigate if rapid slurry chilling would retard or prevent blown pack spoilage (BPS) of vacuum-packaged beef primals. Beef primals were inoculated with Clostridium estertheticum subspp. estertheticum (DSMZ 8809), C. estertheticum subspp. laramenise (DSMZ 14864) and C. gasigenes (DSMZ 12272), and vacuum-packaged with and without heat shrinkage (90°C for 3 s). These packs were then subjected to immediate chilling in an ice slurry or using conventional blast chilling systems and stored at 2°C for up to 100 days. The onset and progress of BPS was monitored using the following scale; 0-no gas bubbles in drip; 1-gas bubbles in drip; 2-loss of vacuum; 3-'blown'; 4-presence of sufficient gas inside the packs to produce pack distension and 5-tightly stretched, 'overblown' packs/packs leaking. Rapid slurry chilling (as compared to conventional chilling) did not significantly affect (P > 0.05) the time to the onset or progress of BPS. It was therefore concluded that rapid chilling of vacuum-packaged beef primals, using an ice slurry system, may not be used as a control intervention to prevent or retard blown pack spoilage. SIGNIFICANCE AND IMPACT OF THE STUDY: This study adds to our growing understanding of blown pack spoilage of vacuum-packaged beef primals and suggests that rapid chilling of vacuum-packaged beef primals is not a control option for the beef industry. The results suggest that neither eliminating the heat shrinkage step nor rapid chilling of vacuum-packaged beef retard the time to blown pack spoilage.

A four-year survey of blown pack spoilage Clostridium estertheticum and Clostridium gasigenes on beef primal cuts.

UNLABELLED: The objective of this study was to determine the incidence of Clostridium estertheticum and Clostridium gasigenes on beef primals taking sample type and season into account. Molecular methods using direct extraction of DNA without enrichment and based on the polymerase chain reaction (PCR) amplification of 16S rDNA fragments were used to test for the presence of Cl. estertheticum and Cl. gasigenes in 4826 beef primal samples (1967 drip, 1896 wet swab and 963 dry swab) provided by 10 commercial beef abattoirs over a 4-year period. Overall 1·5% of samples were PCR positive with the incidence of Cl. estertheticum and Cl. gasigenes being 0·8 and 0·7%, respectively. Although the highest incidence of Cl. estertheticum (4·0%) and Cl. gasigenes (5·1%) was observed in June and November, respectively, seasonal differences were not significant (P < 0·05). Drip samples yielded more positive results than swab samples. It was concluded that a low but persistent percentage of beef primal cuts are contaminated with blown pack spoilage Clostridia. There was no seasonal effect and drip may be a more effective test sample than swabs. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides data on blown pack spoilage contamination rates of beef primal cuts (pieces of meat initially cut from the carcass during butchering) over an extended period of time. The results show the risk of contamination is low but persistent throughout the year necessitating continuous sporicidal treatment of plant and equipment. Moreover, the higher prevalence of positive meat drip samples suggests sampling regimes should be based on this sample type.

Effects of meat pH on growth of 11 species of psychrotolerant clostridia on vacuum packaged beef and blown pack spoilage of the product.

The aim of the study was to determine the effects of meat pH on the abilities of 11 psychrotolerant Clostridium spp. to grow on, and to possibly cause blown pack spoilage of vacuum packaged beef. Beef steaks of pH 5.4-5.6, 5.7-5.9 or ≥6.0, i.e. of normal, intermediate or high pH were prepared and vacuum packaged. Groups of 3 steaks of the same pH range were inoculated with log phase cultures of Clostridium algoriphilum, Clostridium algidixylanolyticum, Clostridium bowmanii, Clostridium estertheticum, Clostridium frigoris, Clostridium frigidicarnis, Clostridium gasigenes, Clostridium lacusfryxellense, Clostridium psychrophilum, Clostridium tagluense or Clostridium vincentii. Each pack was resealed immediately after the steak was inoculated, and pack volumes were determined by water displacement, immediately after resealing and at intervals during storage at 2 °C for 56 days. All of the clostridia grew in packs of high pH beef but none caused pack swelling. Packs of intermediate pH beef inoculated with C. estertheticum began to swell after 14 days, with a mean rate of increase of pack volumes of 6.80 ml/day. One pack of intermediate pH beef inoculated with C. frigoris was swollen after 37 days. Packs of normal pH beef that had been inoculated with C. estertheticum began swelling after 14 days with a mean rate of increase of pack volumes of 7.70 ml/day. Packs of normal or intermediate pH beef inoculated with other clostridia did not swell. After storage, the numbers of most Clostridium spp., as determined by real-time PCR were greater on beef of high pH than of lower pH values, but the numbers of C. frigidicarnis and C. lacusfryxellense were highest on intermediate pH meat, the numbers of C. estertheticum were higher on meat of lower than of high pH, and the numbers of C. tagluense were the same on meat of all pH values. With high pH meat, glucose was reduced to very low level in rinse fluids from packs that had been inoculated with any Clostridium sp. With intermediate and normal pH meat, glucose was reduced to very low concentrations in only rinse fluids from beef that had been inoculated with C. estertheticum.

Phylogeny and Physiological Diversity of Cold-adapted Anaerobic Bacteria Isolated from Rice Field Soil in Japan.

Cold-adapted or psychrotrophic fermentative anaerobic bacteria were isolated from rice field soil in a temperate area in Japan using anaerobic enrichment cultures incubated at 5°C. Most isolates were obligately anaerobic, spore-forming rods and affiliated with different lineages of the genus Clostridium based on 16S rRNA gene sequences. The growth temperature ranges and physiological properties of three representative clostridial isolates (C5S7, C5S11T, and C5S18) were examined. Strain C5S7 grew at 0°C, but not at 20°C, and was identified as Clostridium estertheticum, a psychrophile isolated from spoiled, vacuum-packed, chilled meat (blown pack spoilage, BPS). Strain C5S7 produced butyrate, n-butanol, and abundant gases (H2 and CO2) as major fermentation products from the carbohydrates utilized. Strain C5S11T, which was recently described as Clostridium gelidum sp. nov., possessed psychrotrophic properties and grew at temperatures between 0 and 25°C. Strain C5S11T was saccharolytic, decomposed polysaccharides, such as inulin, pectin, and xylan, and produced acetate, butyrate, and gases. Strain C5S18 also grew at 0°C and the optimum growth temperature was 15°C. Strain C5S18 did not ferment carbohydrates and grew in a manner that was dependent on proteinaceous substrates. This strain was identified as the psychrotolerant species, Clostridium tagluense, originally isolated from a permafrost sample. Collectively, the present results indicate that psychrotrophic anaerobic bacteria with different physiological properties actively degrade organic matter in rice field soil, even in midwinter, in a cooperative manner using different substrates. Furthermore, different psychrotrophic species of the genus Clostridium with the ability to cause BPS inhabit cultivated soil in Japan.

Gases and volatile compounds associated with micro-organisms in blown pack spoilage of Brazilian vacuum-packed beef.

UNLABELLED: This study correlated the composition of the spoilage bacterial flora with the main gaseous and volatile organic compounds (VOCs) found in the package headspace of spoiled, chilled, vacuum-packed meat. Fifteen chilled, vacuum-packed beef samples, suffering from blown pack spoilage, were studied using 16S rRNA clone sequencing. More than 50% of the bacteria were identified as lactic acid bacteria (LAB), followed by clostridia and enterobacteria. Fifty-one volatile compounds were detected in the spoiled samples. Although the major spoilage compounds were identified as alcohols and aldehydes, CO2 was identified as the major gas in the spoiled samples by headspace technique. Different species of bacteria contribute to different volatile compounds during meat spoilage. LAB played an important role in blown pack deterioration of the Brazilian beef studied. SIGNIFICANCE AND IMPACT OF THE STUDY: The data generated by this study provided useful information to correlate the microbial contamination of Enterobacteriaceae, lactic acid bacteria and Clostridium with the VOC and gaseous compound production to define, in a faster manner, not only the type of contamination, but also to prevent it.

Synergistic Effect of Static Magnetic Field and Modified Atmosphere Packaging in Controlling Blown Pack Spoilage in Meatballs.

This study aimed to compare the microbial diversity in meatballs with or without blown pack spoilage (BPS) to determine the cause of BPS and to assess the synergistic effect of static magnetic field (SMF) and modified atmosphere packaging (MAP) to reduce the phenomenon of BPS. Results showed that the BPS group with a 2.26-fold larger volume and packaging containing 71.85% CO2 had Klebsiella spp. (46.05%) and Escherichia spp. (39.96%) as the dominant bacteria, which was different from the spoilage group. The results of isolation and identification of strains from the BPS group and their inoculation test confirmed that Klebsiella pneumoniae was the major strain-inducing BPS in meatballs due to its pack-swelling ability. SMF (5 mT) treatment combined with MAP (40%CO2 + 60%N2), which did not influence the sensory quality of meatballs, had a significant synergistic effect on preventing the increase in pack volume. Compared with the control group, this synergistic treatment effectively delayed bacterial growth, drop in pH, and the increase of TBARS. The findings of this study will provide further guidance for meatball manufacturers to adopt effective strategies to reduce the BPS of meatballs.

The survival of blown pack spoilage associated Clostridium estertheticum and Clostridium gasigenes spores during the ensiling of grass.

Blown pack spoilage (BPS) of vacuum packaged primals, caused by Clostridium estertheticum and Clostridium gasigenes, is a serious issue for the beef industry. There are multiple sources of these bacteria on beef farms, including grass and associated feed preparations. The aim of this study was to investigate the survival of C. estertheticum and C. gasigenes spores during the ensiling of grass and the subsequent opening of the silos. Grass, harvested from fields, with and without cattle slurry amendment, was inoculated with approximately 100 spores/g and ensiled using a laboratory (silo) model system at 20°C in the dark. Adding formic acid or sucrose resulted in six treatment combination as follows: no slurry (NS), no slurry plus formic acid (NSFA), no slurry plus sucrose (NSS), slurry (S), slurry plus formic acid (SFA) and slurry plus sucrose (SS). During the silage fermentation, samples were removed periodically and tested for C. estertheticum, C. gasigenes, total viable, Escherichia coli, Enterobacteriaceae and lactic acid bacteria (LAB) counts. The pH, ethanol, volatile fatty acids (VFA), lactic acid and ammonia concentrations were also monitored throughout the experiment. C. estertheticum did not survive the ensiling process, regardless of treatment. In contrast, C. gasigenes grew in the early stages and was detected during the entirety of the fermentation for all treatments. Based on these observations, it was concluded that the silage fermentation process described would not remove C. gasigenes and contaminated grass may result in contaminated feed for animals.

Whole Genome Sequence-Based Identification of Clostridium estertheticum Complex Strains Supports the Need for Taxonomic Reclassification Within the Species Clostridium estertheticum.

Isolates within the Clostridium estertheticum complex (CEC) have routinely been identified through the 16S rRNA sequence, but the high interspecies sequence similarity reduces the resolution necessary for species level identification and often results in ambiguous taxonomic classification. The current study identified CEC isolates from meat juice (MJS) and bovine fecal samples (BFS) and determined the phylogeny of species within the CEC through whole genome sequence (WGS)-based analyses. About 1,054 MJS were screened for CEC using quantitative real-time PCR (qPCR). Strains were isolated from 33 MJS and 34 BFS qPCR-positive samples, respectively. Pan- and core-genome phylogenomics were used to determine the species identity of the isolates. Average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) were used to validate the species identity. The phylogeny of species within the CEC was determined through a combination of these methods. Twenty-eight clostridia strains were isolated from MJS and BFS samples out of which 13 belonged to CEC. At 95% ANI and 70% dDDH thresholds for speciation, six CEC isolates were identified as genomospecies2 (n=3), Clostridium tagluense (n=2) and genomospecies3 (n=1). Lower thresholds of 94% ANI and 58% dDDH were required for the classification of seven CEC isolates into species C. estertheticum and prevent an overlap between species C. estertheticum and Clostridium frigoriphilum. Combination of the two species and abolishment of current subspecies classification within the species C. estertheticum are proposed. These data demonstrate the suitability of phylogenomics to identify CEC isolates and determine the phylogeny within CEC.

Detection of Psychrophilic Clostridium spp. Causing "Blown Pack" Spoilage in Meat Juice Samples from Chilled Vacuum-Packed Beef and Lamb Meat Imported from Different Countries to Switzerland.

"Blown pack" spoilage (BPS) of chilled vacuum-packed meat is mainly caused by anaerobic and psychrophilic Clostridium spp., including C. estertheticum, C. gasigenes, C. frigoriphilum, and C. frigidicarnis. Recently, its occurrence has been reported in several countries, especially in internationally traded meat. Therefore, this study aimed at detecting the occurrence of psychrophilic Clostridium spp. causing BPS in meat juice samples (MJS) from chilled vacuum-packed beef and lamb meat imported from other countries to Switzerland. One hundred fifty-four MJS (n = 78 from beef; n = 76 from lamb meat) were screened for psychrophilic Clostridium spp. by quantitative PCR, whereby MJS with a crossing point PCR cycle value <35 and >35 were considered positive and negative, respectively. Psychrophilic Clostridium spp. were detected in 10 MJS, of which 2 were from beef and 8 were from lamb meat. The two beef MJS originated from Spain and Lithuania, whereas the lamb MJS originated from New Zealand (six) and Australia (two). This is the first report of psychrophilic Clostridium spp. in MJS from chilled vacuum-packed beef and lamb meat imported from other countries to Switzerland and provides further evidence that the risk of BPS in lamb meat is higher than in beef.

Comparative Genome Analysis and Phenotypic Characterization of Clostridium gasigenes CGAS001 Isolated From Chilled Vacuum-Packed Lamb Meat.

Genomic data for psychrophilic bacteria causing blown pack spoilage (BPS) are limited. This study characterizes the genome of a novel Clostridium gasigenes strain CGAS001 isolated from meat juice sample (MJS) of vacuum-packed lamb meat by comparing it with the type strain C. gasigenes DSM 12272 and five strains representing four other BPS-causing Clostridium sensu stricto species. Phenotypic characteristics of the strain, which include biochemical characteristics, antimicrobial resistance and production of putative polyketide, have been determined. The size of its draft genome is 4.1 Mb with 3,845 coding sequences, 28.7% GC content and 95 RNA genes that include 75 tRNAs, 17 rRNAs, and 3 ncRNAs. Average Nucleotide Identity (ANI) and digital DNA-DNA Hybridization (dDDH) predict that C. gasigenes CGAS001 and DSM 12272 constitute a single species (ANI and dDDH = 98.3% for speciation) but two distinct subspecies (dDDH = 73.3% for subspeciation). The genome is characterized by saccharolytic, lipolytic and proteolytic genes as well as hemolysins and phospholipases, which are consistent with its phenotype. The genome also reveals the ability of C. gasigenes to synthesize polyketides which is demonstrated by the antimicrobial activity of a crude polyketide extract against Listeria monocytogenes and Enterococcus devriesei. The strain is resistant to polymyxin B and streptomycin. The genetic and phenotypic analyses suggest that CGAS001 constitutes a novel subspecies of C. gasigenes adapted to a saprophytic lifestyle and can synthesize narrow spectrum antimicrobial compounds.

Relevant Aspects of Clostridium estertheticum as a Specific Spoilage Organism of Vacuum-Packed Meat.

Clostridium estertheticum is a psychrotolerant, gram-positive, motile, anaerobic, spore-forming, rod-shaped bacteria that causes blown pack spoilage (BPS). Spoilage occurs in vacuum-packed meat without temperature abuse. Having been reported in the last 30 years in several countries, BPS by Cl. estertheticum is a major issue around the world and presents a huge economic impact on the meat industry. Despite being an important spoilage microorganism, studies on Cl. estertheticum are challenged by numerous aspects. These include, lack or poor growth in laboratory media, long culturing periods, and unpredictable isolation on the media. These factors hamper the detection of Cl. estertheticum before occurrence of BPS, which further undermines efforts to prevent the occurrence of BPS. Nevertheless, considerable developments have taken place with regard to culture-independent methods. Although information on Cl. estertheticum is available, it is limited and remains highly fragmented. Therefore, this review collates the available information and discusses relevant aspects of Cl. estertheticum as a specific spoilage organism of BPS in vacuum-packed meat.

Antimicrobial effect of different peroxyacetic acid and hydrogen peroxide formats against spores of Clostridium estertheticum.

"Blown pack" spoilage is primarily caused by Clostridium estertheticum. The primary source of contamination is probably pelts, faeces and soil during opening cuts and de-hiding. Peroxyacetic acid (POAA) based fogs are commonly included in an abattoir's routine cleaning process. Hydrogen peroxide (H2O2) is a powerful oxidizing agent that penetrates microbe cell walls causing cell death. In this study, we compared the ability of H2O2 and OXYSAN ZS (POAA containing 1-hydroxyethylidine-1,1-diphosphonic acid as a stabilizer) in different formats to inactivate C. estertheticum spores. Hydrogen peroxide treatment using Phytagel™ gel as carrier was effective on fleece against both naturally contaminating microflora and C. estertheticum spores. This is the first time an antimicrobial treatment has been shown to inactivate C. estertheticum spores on such a complex and highly contaminated matrix. Both H2O2 and OXYSAN ZS treatments inactivated C. estertheticum spores on stainless steel indicating their potential use as an in-plant decontamination procedure or inclusion in routine in-process cleaning.

Development of a multiplex qPCR for the species identification of Clostridium estertheticum, C. frigoriphilum, C. bowmanii and C. tagluense-like from blown pack spoilage (BPS) meats and from wild boars.

Psychrophilic and psychrotolerant clostridia (n = 110) were isolated from vacuum-packed meat (beef and lamb), fresh venison and from skin and fecal samples of wild boars. They were identified to species level using MALDI-TOF MS, sequence and phylogeny analysis of the 16S rRNA and species specific multiplex qPCR. The results of all three methods were concordant. The majority of isolates were identified as C. tagluense-like Group I (n = 34) and Group II (n = 42). Thirty-five isolates could be identified to species level as follows: C. estertheticum (n = 15), C. frigoriphilum (n = 13), C. frigidicarnis (n = 1) and C. bowmanii (n = 5). This is the first report of detection and identification of C. frigoriphilum and C. tagluense-like Group II as causative agents of blown pack spoilage of beef. The species specific multiplex qPCR developed in this study could be applied to identify and to quantify the Clostridium species described above in suspicious meat juice samples.

Comparison of hot versus cold boning of beef carcasses on bacterial growth and the risk of blown pack spoilage.

Primals were prepared from beef Longissimus thoracis et lumborum (LTL), psoas major (PM), quadriceps femoris (QF) and semitendinosus (S) muscles from cold and hot boned carcasses, vacuum-packaged and stored for 42 or 100days at 2°C and 7°C. Storage temperature, carcass or primal surface temperature, pH and aw were monitored. Samples were taken periodically and tested for total viable count mesophilic (TVCm), TVC psychrophilic (TVCp), total Enterobacteriaceae count (TEC), presumptive Pseudomonas spp., lactic acid bacteria (LAB), Clostridium spp. and Brochothrix thermosphacta. A fifth muscle, biceps femoris (BF), was used to examine the impact of hot boning on blown pack spoilage (BPS). Primal counts increased to 6-7log10cfucm-2 after 6weeks. Significantly (P<0.05) higher TEC, Pseudomonas spp. and Br. thermosphacta counts were observed on cold versus hot boned primals. In contrast, significantly (P<0.05) higher TVC, LAB and Clostridium spp. concentrations were obtained on hot boned beef. Moreover, BPS pack distension/bursting occurred considerably sooner in hot boned product.

Real-time PCR methods for the detection of blown pack spoilage causing Clostridium species; C. estertheticum, C. gasigenes and C. ruminantium.

A set of real-time PCR methods for the detection of C. estertheticum, C. gasigenes and C. ruminantium, the causative agents of blown pack spoilage (BPS) in vacuum packaged beef, was developed. Robust validation of the sensitivity and specificity was carried out in the three matrices (beef meat drip, wet environmental swabs and dry environmental swabs) as encountered in our testing laboratory and against Clostridium strains (n=76) and non-Clostridium strains (n=36). It was possible to detect 4-5 spores per ml for C. estertheticum, 2 spores per ml for C. gasigenes and 8 spores per ml for C. ruminantium, without the need for enrichment of the samples. This high sensitivity is particularly important for the beef sector, not just for testing spoiled product but also in the early detection of contaminated beef and in validation of sporicidal cleaning procedures for critical pieces of equipment such as the vacuum packaging machine, which have the potential to contaminate large volumes of product.

Controlling Blown Pack Spoilage Using Anti-Microbial Packaging.

Active (anti-microbial) packaging was prepared using three different formulations; Auranta FV; Inbac-MDA and sodium octanoate at two concentrations (2.5 and 3.5 times their minimum inhibitory concentration (MIC, the lowest concentration that will inhibit the visible growth of the organisms) against Clostridium estertheticum, DSMZ 8809). Inoculated beef samples were packaged using the active packaging and monitored for 100 days storage at 2 °C for blown pack spoilage. The time to the onset of blown pack spoilage was significantly (p < 0.01) increased using Auranta FV and sodium octanoate (caprylic acid sodium salt) at both concentrations. Moreover, sodium octanoate packs had significantly (p < 0.01) delayed blown pack spoilage as compared to Auranta FV. It was therefore concluded that Auranta FV or sodium octanoate, incorporated into the packaging materials used for vacuum packaged beef, would inhibit blown pack spoilage and in the case of the latter, well beyond the 42 days storage period currently required for beef primals.

Characterization of germination of spores of Clostridium estertheticum, the primary causative agent of blown pack spoilage of vacuum packaged beef.

The aim of this study was to investigate the effect of various factors on the germination of Clostridium estertheticum endospores (spores) in relation to beef. The effect of heat on germination was determined by recovering C. estertheticum on Columbia agar from spore suspensions not heated or heated at 63, 70 or 80°C for various times. The effects of pH, temperature and oxygen were determined, by enumeration of remaining ungerminated spores during incubation in Meat Juice medium (MJM). Amino acids and lactate were tested for their ability to trigger germination of C. estertheticum spores by monitoring dipicolinic acid (DPA) release. Heat treatment of spores at 80°C for ≤20min significantly (p<0.05) increased the numbers of spores recovered on blood agar. Neither incubation temperature nor oxygen affected germination in MJM. The optimal pH for germination was 7.0 to 7.5. Incubation with leucine or aspartic acid caused a 1.3% release of DPA, the highest among all amino acids tested. Incubation with lactate resulted in a 4.1% release of DPA, which was significantly (p<0.05) higher than those from incubation with amino acids. The DPA release from incubation with lactate, lactate with amino acids, or MJM was similar (p>0.05).

Detection of psychrophilic and psychrotolerant Clostridium spp. in chilled fresh vacuum-packed meat using different PCR methods.

Since 1989, blown pack spoilage has been recognized as a special form of spoilage in vacuum-packed raw and cooked beef. However, only limited information concerning the occurrences of bacteria causing blown pack spoilage on chilled fresh meat is available. In this study, a total of 63 beef and 33 lamb commercially available samples from different countries and without any signs of spoilage were examined for contamination with psychrophilic and psychrotolerant Clostridium spp. using different PCR systems. In total, 34.4% of the chilled fresh vacuum-packed meats were PCR positive. A higher number of lamb samples were identified as PCR positive compared with beef. A geographical relationship between positive results and the origin of the samples could not be determined. PCR system described by Brightwell and Clemens (Development and validation of a real-time PCR assay specific for Clostridium estertheticum and C. estertheticum-like psychrotolerant bacteria. Meat Sci 2012;92:697-703) gave the highest number of positive detections compared with the Broda, Boerema and Bell PCR system (PCR detection of psychrophilic Clostridium spp. causing 'blown pack' spoilage of vacuum-packed chilled meats. J Appl Microbiol 2003;94:515-22). Eight clostridia isolates from two German beef and four Welsh lamb samples were isolated overall. Three of these clostridia isolates were identified as Clostridium estertheticum whereas five clostridia isolates remain unidentified. The study shows that psychrophilic and psychrotolerant Clostridium spp. are more prevalent in retail samples than previously suspected.