Psychrotolerant Clostridia Research Articles

Comparative genomics of Clostridium species associated with vacuum-packed meat spoilage

Bacterial species belonging to the genus Clostridium have been recognized as causative agents of blown pack spoilage (BPS) in vacuum packed meat products. Whole-genome sequencing of six New Zealand psychrotolerant clostridia isolates derived from three meat production animal types and their environments was performed to examine their roles in BPS. Comparative genome analyses have provided insight into the genomic diversity and physiology of these bacteria and divides clostridia into two separate species clusters. BPS-associated clostridia encode a large and diverse spectrum of degradative carbohydrate-active enzymes (CAZymes) that enable them to utilize the intramuscular carbohydrate stores and facilitate sporulation. In total, 516 glycoside hydrolases (GHs), 93 carbohydrate esterases (CEs), 21 polysaccharide lyases (PLs), 434 glycosyl transferases (GTs) and 211 carbohydrate-binding protein modules (CBM) with predicted activities involved in the breakdown and transport of carbohydrates were identified. Clostridia genomes have different patterns of CAZyme families and vary greatly in the number of genes within each CAZy category, suggesting some level of functional redundancy. These results suggest that BPS-associated clostridia occupy similar environmental niches but apply different carbohydrate metabolism strategies to be able to co-exist and cause meat spoilage.

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.

Molecular discrimination of New Zealand sourced meat spoilage associated psychrotolerant Clostridium species by ARDRA and its comparison with 16s RNA gene sequencing

ARDRA analysis was carried out on 90 New Zealand psychrotolerant Clostridium isolates derived from three meat production animal types and their environments. The isolates included species associated with spoilage: C. gasigenes, C. algidicarnis, C. tagluense, C. frigidicarnis and C. estertheticum. The isolates fell into 14 distinct ARDRA Groups, with 13 previously characterised meat spoilage-associated isolates shared between 6 of the 14 groups. The accuracy of ARDRA profiling analysis was supported by sequencing the 16s rRNA gene from isolates, including the representative spoilage associated Clostridium species and was consistent with previous phylogenetic relationships and classical cultural characterisation. The ARDRA methodology described in this study successfully discriminated between the different spoilage-associated species of clostridia as well as other pyschrotolerant Clostridium species associated with meat production. This discriminatory molecular screen will aid future source attribution studies as well as enable meat processors to identify and validate control measures for clostridia contamination, thus gaining greater efficacy in controlling meat spoilage caused by psychrotolerant clostridia.

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.

Substrate utilization during incubation in meat juice medium of psychrotolerant clostridia associated with blown pack spoilage

Several new species of psychrophilic or psychrotolerant clostridia have been identified in recent years. Some of these may be involved in ‘blown pack’ spoilage (BPS) of vacuum packaged beef. Organisms that cause BPS must produce large volumes of gas while utilizing substrates available in raw meat. Therefore, Clostridium algoriphilum, Clostridium algidixylanolyticum, Clostridium bowmanii, Clostridium frigoris, Clostridium frigidicarnis, Clostridium gasigenes, Clostridium lacusfryxellense, Clostridium psychrophilum, Clostridium tagluense and Clostridium vincentii were grown in meat juice medium (MJM), and changes in substrate concentrations were monitored to assess the potential for gas production by each organism. All 10 species were able to grow exponentially on glucose with simultaneous hydrolysis of glycogen, reaching maximum values for absorbance at 600 nm of 0.3–1.90. All ceased growing when glucose and glycogen were still detectable in the growth medium. C. frigidicarnis utilized most of the amino acids available in MJM and reduced the concentration of total amino acids by 10 mM. The other 9 species caused little or no reduction in amino acid concentrations. C. algidixylanolyticum and C. frigidicarnis utilized glucose, glycogen and lactate simultaneously during growth and after growth ceased. C. algoriphilum and C. frigoris commenced utilization of lactate, while continuing utilization of glucose and glycogen, only after growth ceased, but utilization of lactate by C. algoriphilum was weak. C. psychrophilum ceased utilization of glucose and glycogen but initiated weak utilization of lactate after growth ceased. The other 5 species did not utilize any substrate after growth ceased. The utilization of glucose, glycogen and relatively large amounts of lactate by C. algidixylanolyticum, C. frigoris, and C. frigidicarnis after growth ceased indicates that these organisms have the potential to cause BPS. The other 7 species appear to lack such potential.

Reduction of spoilage of chilled vacuum-packed lamb by psychrotolerant clostridia

Methods for the reduction of spoilage, of lamb, by psychrotolerant clostridia were investigated including exposure to air, hot and cold water spray washing and tyndallisation. Initially vegetative cells of psychrotolerant clostridia associated with spoilage of chilled vacuum-packed meat were exposed to aerobic cooked meat medium at room temperature (21°C) to determine how long they remained viable. Survival of strains varied from 2h to 3days. Vegetative cells of Clostridium estertheticum subsp. estertheticum survived 7days at 10°C with little reduction in viable numbers. This ruled out exposure to air as a practical method for reducing spoilage. Trials were also carried out on chilled vacuum-packed lamb inoculated with spores of Cl. estertheticum subsp. estertheticum. The time until inoculated packs reached the loss of vacuum stage varied from 38 to 53days. Hot and cold water washing extended the shelf life by 12 to 13days in comparison to untreated packs.

Psychrophilic and psychrotrophic clostridia: sporulation and germination processes and their role in the spoilage of chilled, vacuum‐packaged beef, lamb and venison

SummarySpoilage of beef, lamb and venison by psychrophilic and psychrotrophic clostridial species renders meat unacceptable resulting in financial losses and reduced consumer confidence. A number of clostridial strains, including Clostridium algidicarnis, Clostridium algidixylanolyticum, Clostridium estertheticum, Clostridium frigidicarnis and Clostridium gasigenes, have been implicated in red meat spoilage. Unlike other spoilers, these clostridia are able to grow in anaerobic conditions and at chilled temperatures (some at −1.5 °C the optimal storage temperature for chilled red meat). The spoilage they cause is characterised by softening of the meat, production of large amounts of drip (exudates), offensive odours and in the case of C. estertheticum and C. gasigenes production of gas. Spoilage occurs following the introduction of clostridial spores into vacuum packages during processing. Germination of spores is necessary for the growth of vegetative cells, which cause spoilage. Current mitigation strategies focus on good management practice within meat processing plants. However, this is not always sufficient to prevent spoilage. This review summarises the issues associated with meat spoilage because of psychrotolerant clostridia and discusses areas that require further study.

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

To determine possible preslaughter and processing sources of psychrophilic and psychrotolerant clostridia causing spoilage of vacuum-packed chilled meats. 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. 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. This paper provides information significant for controlling meat spoilage-causing clostridia in the meat-processing plants.

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.

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

To identify the abattoir source(s) of culturable psychrophilic clostridia causing 'blown pack' spoilage of vacuum-packed chilled meats. 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. 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. 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.