Search

Extended-spectrum beta lactamase (ESBL)-producing

Transmission of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) occurs through respiratory droplets passed directly from person to person or indirectly through fomites, such as common use surfaces or objects. The aim of this study was to determine the virucidal efficacy of blue LED (405 nm) and far-UVC (222 nm) light in comparison to standard UVC (254 nm) irradiation for the inactivation of feline infectious peritonitis virus (FIPV) on different matrices as a model for SARS-CoV-2. Wet or dried FIPV on stainless steel, plastic, or paper discs, in the presence or absence of artificial saliva, were exposed to various wavelengths of light for different time periods (1–90 min). Dual activity of blue LED and far-UVC lights were virucidal for most wet and dried FIPV within 4 to 16 min on all matrices. Individual action of blue LED and far-UVC lights were virucidal for wet FIPV but required longer irradiation times (8–90 min) to reach a 4-log reduction. In comparison, LED (265 nm) and germicidal UVC (254 nm) were virucidal on almost all matrices for both wet and dried FIPV within 1 min exposure. UVC was more effective for the disinfection of surfaces as compared to blue LED and far-UVC individually or together. However, dual action of blue LED and far-UVC was virucidal. This combination of lights could be used as a safer alternative to traditional UVC.

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.

The effect of transportation and lairage on the faecal shedding and post-slaughter contamination of carcasses with Escherichia coli O157 and O26 in young calves (4–7-day-old) was assessed in a cohort study at a regional calf-processing plant in the North Island of New Zealand, following 60 calves as cohorts from six dairy farms to slaughter. Multiple samples from each animal at pre-slaughter (recto-anal mucosal swab) and carcass at post-slaughter (sponge swab) were collected and screened using real-time PCR and culture isolation methods for the presence of E. coli O157 and O26 (Shiga toxin-producing E. coli (STEC) and non-STEC). Genotype analysis of E. coli O157 and O26 isolates provided little evidence of faecal–oral transmission of infection between calves during transportation and lairage. Increased cross-contamination of hides and carcasses with E. coli O157 and O26 between co-transported calves was confirmed at pre-hide removal and post-evisceration stages but not at pre-boning (at the end of dressing prior to chilling), indicating that good hygiene practices and application of an approved intervention effectively controlled carcass contamination. This study was the first of its kind to assess the impact of transportation and lairage on the faecal carriage and post-harvest contamination of carcasses with E. coli O157 and O26 in very young calves.

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.

Current culture methods to investigate changes in Escherichia coli community structure are often slow and laborious. Genes such as gnd (6-phosphogluconate dehydrogenase) have a highly variable nucleotide sequence and may provide a target for E. coli microbiome analysis using culture-independent methods. Metabarcoded PCR primers were used to generate separate libraries from calf faecal samples for high throughput sequencing. Although a total of 348 separate gnd sequence types (gSTs) were identified, 188 were likely to be due to sequencing errors. Of the remaining 160 gSTs, 92 did not match those in a database of 319 separate gnd sequences. ‘Animal’ was the main determinant of E. coli diversity with limited impact of sample type or DNA extraction method on intra-host E. coli community variation from faeces and recto-anal mucosal swab samples. This culture-independent study has addressed the difficulties of quantifying bacterial intra-species diversity and revealed that, whilst individual animals may harbour >50 separate E. coli strains, communities are dominated by E. coli in experimental studies designed to assess the impact of interventions on the gut microbiome.

Shiga toxin-producing Escherichia coli (STEC) are a leading cause of foodborne outbreaks of human disease, but they reside harmlessly as an asymptomatic commensal in the ruminant gut. STEC serogroup O145 are difficult to isolate as routine diagnostic methods are unable to distinguish non-O157 serogroups due to their heterogeneous metabolic characteristics, resulting in under-reporting which is likely to conceal their true prevalence. In light of these deficiencies, the purpose of this study was a twofold approach to investigate enhanced STEC O145 diagnostic culture-based methods: firstly, to use a genomic epidemiology approach to understand the genetic diversity and population structure of serogroup O145 at both a local (New Zealand) (n = 47) and global scale (n = 75) and, secondly, to identify metabolic characteristics that will help the development of a differential media for this serogroup. Analysis of a subset of E. coli serogroup O145 strains demonstrated considerable diversity in carbon utilisation, which varied in association with eae subtype and sequence type. Several carbon substrates, such as D-serine and D-malic acid, were utilised by the majority of serogroup O145 strains, which, when coupled with current molecular and culture-based methods, could aid in the identification of presumptive E. coli serogroup O145 isolates. These carbon substrates warrant subsequent testing with additional serogroup O145 strains and non-O145 strains. Serogroup O145 strains displayed extensive genetic heterogeneity that was correlated with sequence type and eae subtype, suggesting these genetic markers are good indicators for distinct E. coli phylogenetic lineages. Pangenome analysis identified a core of 3,036 genes and an open pangenome of >14,000 genes, which is consistent with the identification of distinct phylogenetic lineages. Overall, this study highlighted the phenotypic and genotypic heterogeneity within E. coli serogroup O145, suggesting that the development of a differential media targeting this serogroup will be challenging.

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.

Confinement odour was investigated. Volatiles were extracted directly from the pack, using solid phase microextraction and analysed by gas chromatography-mass spectrometry. Sensory evaluation and microbiological analysis of the meat surface were also performed. Commercial samples of vacuum packed lamb legs (n=85), from two meat processing plants, were kept for 7weeks at -1.5°C then at different regimes of temperature (-1.5 to +4°C) until 11, 12 or 13weeks. Persistent odour was observed in 66% of samples, confinement odour in 24% and no odour in 11%. Volatiles associated with confinement odour (3-methyl-butanal, 3-hydroxy-2-butanone and sulphur dioxide) corresponded with end/sub products of glucose fermentation and catabolism of amino acids by bacteria (all bacteria naturally found in meat and do not represent a risk to health). Confinement odour could indicate a stage at which the environment for bacteria growth is becoming favourable for the production of volatiles with strong odours that are noticed by the consumer.

Shiga toxin-producing Escherichia coli strains (STEC) are food-borne pathogens. While E. coli O157:H7 is commonly associated with cattle, less is known about the prevalence of non-O157 STEC serogroups in bovines. This study evaluated the prevalence and virulence status of O157:H7 and six E. coli O-serogroups (O26, O103, O45, O145, O121, O111) in New Zealand dairy farms using molecular as well as culture-based methods. Fresh farm dairy effluent (FDE) (n = 36) and composite calf faeces (n = 12) were collected over three samplings from 12 dairy farms. All seven target serogroups were detected through molecular techniques. Of the 202 isolates which were serologically confirmed following traditional culturing and immunomagnetic separation (IMS), O103, O26, O45 and O121 were the most common serogroups, being found in 81, 47, 42 and 32% of the FDE and in 17, 33, 25 and 9% of the calf faeces respectively. The majority (157/202) of the isolates were negative for stx and eae virulence genes. The prevalence of the seven target STEC was low, and only nine O26 isolates (4%) were recovered from four of the farms. The study has highlighted the need for improving the isolation of Top 7 STEC from the stx-negative populations present in fresh dairy effluent and calf faeces. SIGNIFICANCE AND IMPACT OF THE STUDY: Shiga toxin-producing Escherichia coli (STEC) are important food-borne pathogens that can cause severe illness in humans. Cattle are asymptomatic reservoirs for STEC, and transmission to humans can be by consumption of food products or water contaminated with cattle faeces. Our study investigated the prevalence of O157:H7 and six E. coli serogroups of STEC (O26, O103, O45, O145, O121, O111) over time in the dairy reservoir and increases the knowledge and understanding of these pathogens on pasture-based farms. Such information is required to develop risk-assessment models aiming at limiting transmission of these STEC to human.

Aims To investigate the transmission route of Carnobacterium from the farm environment to the meat-manufacturing plant and potential risk for meat spoilage. Methods and results A sheep farm-level survey of Carnobacterium, consisting of 150 environmental and animal (no 100) associated samples, was carried out on two farms. A further 20 lamb carcass samples were taken from an abattoir servicing one of the farms. The majority of Carnobacterium maltaromaticum isolates were associated with fleece followed by hard sheep contact surfaces, rectal-anal mucosal swabs and carcasses. Enterobacterial repetitive intergenic consenus PCR (ERIC-PCR) profiling revealed four distinct ERIC types. Each ERIC type was found on both farms, three of which were also found on lamb carcasses. Conclusions Enterobacterial repetitive intergenic consenus PCR was effective at demonstrating within-species variability in C. maltaromaticum. This study provides initial information showing that farm sources maybe an important transmission route of Carnobacterium for contamination of lamb carcasses and subsequently the meat processing environment. Significance and impact of the study Data on distribution, diversity, sources and transmission routes for meat product contamination is limited for spoilage bacteria. This study highlights the importance of good hygienic slaughter practices and cleaning routines to remove accumulated detritus from the handling of animals that may lead to cross-contamination.

Vacuum-packaging and stringent control of storage temperatures enable the export of meat to distant markets, supplying a chilled product that can favourably compete with local fresh meats. To save fuel and reduce emissions, the speed of ships travelling to international markets has decreased resulting in requirement for the shelf-life of chilled lamb to be extended beyond the recognised time of 60-70 days. Growth of microorganisms and ability to cause spoilage of vacuum-packed lamb are dependent on many factors, including the type and initial concentration of spoilage bacteria, meat pH, water activity, availability of substrates, oxygen availability and, most importantly, storage time and temperature of the packaged product. This paper reviews the existing knowledge of the spoilage bacteria affecting vacuum-packed lamb, discusses the impact of these bacteria on product quality, shelf-life and spoilage, and concludes that under specified conditions the shelf-life of chilled lamb can be extended to beyond 70 days.

The spoilage potential of Brochothrix thermosphacta, Serratia proteamaculans and Rahnella aquatilis was investigated in vacuum packaged high (5.9 to 6.4) and low (5.4 to 5.8) pH lamb. Vacuum packaged fore shank (m. extensor carpi radialis) and striploins (m. longissimus dorsi) (n = 306) inoculated with ~ 100 CFU of individual bacteria were stored for twelve weeks at temperatures − 1.5, 0, 2 and 7 °C. Spoilage characteristics and bacterial numbers were recorded and analysed in comparison to un-inoculated control samples. All three bacterial species were shown to grow in vacuum packaged lamb of pH values between 5.4 and 6.4, when stored at chilled temperatures (− 1.5 to 7 °C) for up to 84 days. B. thermosphacta and S. proteamaculans caused spoilage to the meat under these conditions whilst R. aquatilis spoiled high pH meat at 7 °C. These results go against previous beef models stipulating that Brochothrix and Enterobacteriacae species cannot grow on or cause spoilage of low pH meat in the absence of oxygen.

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°C for 5½ 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.

The use of lactic acid bacteria (LAB) as protective cultures in vacuum-packed chill-stored meat has potential application for assuring and improving food quality, safety and market access. In a study to identify candidate strains suitable for evaluation in a meat model, agar-based methods were employed to screen 181 chilled meat and meat process-related LAB for strains inhibitory to pathogens and spoilage organisms of importance to the meat industry. Six meat-derived strains, including Lactobacillus sakei and Lactococcus lactis, were found to be inhibitory to one or more of the target strains Listeria monocytogenes, Brochothrix thermosphacta, Campylobacter jejuni and Clostridium estertheticum. The inhibitory agents appeared to be either cell-associated or molecules released extracellularly with bacteriocin-like properties. Variations detected in the antimicrobial activity of LAB associated with changes to test parameters such as substrate composition underlined the importance of further in situ evaluation of the inhibitory strains in stored meat trials.

A new multiplex PCR and two specific TaqMan assays were developed to target the emerging pathogens A. butzleri and A. cryaerophilus. The assays also included an internal control to verify the presence of bacterial target DNA and amplification integrity. The multiplex assay used a published primer set (CRY1 and CRY2) for detecting A. cryaerophilus DNA (Houf, K., Tutenel, A., De Zutter, L., Van Hoof, J. and Vandamme, P., 2000. Development of a multiplex PCR assay for the simultaneous detection and identification of Arcobacter butzleri, Arcobacter cryaerophilus and Arcobacter skirrowii. FEMS microbiology letters, 193 (1): 89-94.) and a novel A. butzleri primer set designed to target the rpoB/C gene sequences. To improve sample throughput and assay sensitivity a TaqMan assay for each Arcobacter spp. was developed which again utilised the heterogeneity contained in the rpoB/C and 23s rRNA gene sequences. The two TaqMan assays provided >2 log improvement in detection sensitivity for both Arcobacter spp. compared with the multiplex PCR assay and were able to detect

We examined the bacterial community present on an Intralox conveyor belt system in an operating lamb boning room by sequencing the 16S ribosomal DNA (rDNA) of bacteria extracted in the presence or absence of cultivation. RFLP patterns for 16S rDNA clone library and cultures were generated using HaeIII and MspI restriction endonucleases. 16S rDNA amplicons produced 8 distinct RFLP pattern groups. RFLP groups I-IV were represented in the clone library and RFLP groups I and V-VIII were represented amongst the cultured isolates. Partial DNA sequences from each RFLP group revealed that all group I, II and VIII representatives were Pseudomonas spp., group III were Sphingomonas spp., group IV clones were most similar to an uncultured alpha proteobacterium, group V was similar to a Serratia spp., group VI with an Alcaligenes spp., and group VII with Microbacterium spp. Sphingomonads were numerically dominant in the culture-independent clone library and along with the group IV alpha proteobacterium were not represented amongst the cultured isolates. Serratia, Alcaligenes and Microbacterium spp. were only represented with cultured isolates. Pseudomonads were detected by both culture-dependent (84% of isolates) and culture-independent (12.5% of clones) methods and their presence at high frequency does pose the risk of product spoilage if transferred onto meat stored under aerobic conditions. The detection of sphingomonads in large numbers by the culture-independent method demands further analysis because sphingomonads may represent a new source of meat spoilage that has not been previously recognised in the meat processing environment. The 16S rDNA collections generated by both methods were important at representing the diversity of the bacterial population associated with an Intralox conveyor belt system.

PCR assays that can identify the presence of variola virus (VARV) sequences in an unknown DNA sample were developed using principles established for the amplification refractory mutation system (ARMS). The assay's specificity utilised unique single nucleotide polymorphisms (SNP) identified among Orthopoxvirus (OPV) orthologs of the vaccinia virus Copenhagen strain A13L and A36R genes. When a variola virus specific primer was used with a consensus primer in an ARMS assay with different Orthopoxvirus genomes, a PCR product was only amplified from variola virus DNA. Incorporating a second consensus primer into the assay produced a multiplex PCR that provided Orthopoxvirus generic and variola-specific products with variola virus DNA. We tested two single nucleotide polymorphisms with a panel of 43 variola virus strains, collected over 40 years from countries across the world, and have shown that they provide reliable markers for variola virus identification. The variola virus specific primers did not produce amplicons with either assay format when tested with 50 other Orthopoxvirus DNA samples. Our analysis shows that these two polymorphisms were conserved in variola virus genomes and provide a reliable signature of Orthopoxvirus species identification.

The spoilage potential of Brochothrix campestris and Brochothrix thermosphacta was investigated in vacuum-packed lamb. Striploins (n=338) were inoculated and stored for twelve weeks at temperatures -1.5, 0, 2 and 7 °C. Growth around 5-6 log10 CFU/cm(2) was recorded after six weeks at 0, 2 and 7 °C, and ~3 log10 CFU/cm(2) after nine weeks at -1.5 °C. B. campestris was shown to cause spoilage by nine weeks at temperatures above 0 °C by the presence of green drip and unacceptable odours. Molecular based assays for the detection and differentiation of B. thermosphacta and B. campestris were developed and validated. A TaqMan assay was designed to target a unique single-nucleotide polymorphism in the Brochothrix 16s rRNA gene with a sensitivity of

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 2 h to 3 days. Vegetative cells of Clostridium estertheticum subsp. estertheticum survived 7 days 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 53 days. Hot and cold water washing extended the shelf life by 12 to 13 days in comparison to untreated packs.

A new real-time PCR assay was developed targeted to the psychrotolerant spoilage bacteria, Clostridum estertheticum, a causative agent of ‘blown-pack’ spoilage of vacuum packaged meats during chilled storage. Further, a robust validation of the sensitivity and specificity in different meat processing related matrices was carried out. Results show that real-time PCR is a valid method for the detection of C. estertheticum spores as long as consideration is given to the matrix being tested and the sensitivity of detection required. For meat, hide, blood/drip and environmental swabs it was possible to detect low numbers of C. estertheticum spores (approx 3 spores per ml) by direct real-time PCR (without pre-enrichment of the samples). For faeces and soil matrices, a cold temperature enrichment step was required prior to DNA extraction and real-time PCR analysis to increase the ability to detect samples containing C. estertheticum spores; this was particularly important when the samples contained low numbers of spores (less than 3 spores per ml). For matrices with high levels of PCR inhibitors such as soil, it was necessary to dilute the extracted DNA sample 100 fold especially for detection of high levels of contamination (greater than 103 per ml) otherwise a pre-enrichment was required.

Lyophilization was used to concentrate bovine feces prior to DNA extraction and analysis using real-time PCR. Lyophilization significantly improved the sensitivity of detection compared to that in fresh feces and was associated with reliable quantification of both Escherichia coli O157:H7 and Campylobacter jejuni bacteria present in feces at concentrations ranging between 2 log 10 and 6 log 10 CFU g − 1 .

The abilities of five Lactobacillus sakei strains and one Lactococcus lactis strain to retain inhibitory activity against several target organisms in the flora of product during 12 weeks storage of vacuum-packaged lamb and beef was investigated. L. sakei strains were generally found capable of developing dominant populations on both beef and lamb. L. lactis 75 grew poorly on lamb did not inhibit co-inoculated Brochothrix thermosphacta. Lamb inoculated with the Sakacin-A producer L. sakei Lb706 had lower Listeria monocytogenes populations than lamb inoculated with a bacteriocin-negative variant. In beef packs inoculated with Clostridium estertheticum spores and L. sakei strain 27, 44 or 63, the development of blown-pack spoilage was delayed by up to one week. Campylobacter jejuni inoculated onto beef was recovered from fewer packs when it was co-inoculated with 3000 CFU cm(-2) of L. sakei strain 27, 44 or 63. Observed inhibition did not always correlate with inhibition observed in earlier media-based studies, supporting the view that functionality identified using simple media-based screening methods may not be replicated in the complex environment of stored foods, and vice-versa. These findings further define a set of L. sakei strains with potential for the extended bio-preservation of minimally processed fresh beef and lamb.

The diversity of microflora associated with peroxyacetic acid (POAA) treated and untreated beef was investigated by 16S rDNA gene cloning, DGGE analysis and conventional bacterial cultivation. Following vacuum packaging, POAA treated and untreated meat samples were stored for up to 18 weeks at -1.5 degrees C. Each culture independent method showed Carnobacterium spp. to predominate on both POAA treated and untreated meat. However, 16S rDNA gene analysis also detected the presence of psychrotolerant Clostridium spp. in the POAA-treated beef. Culture-dependent analysis did not distinguish Carnobacterium spp. from Lactobacilli. Although culture-dependent analysis showed an increase in the ratio of Enterobacteriaceae to lactic acid bacteria from weeks 6-18 in the POAA treated compared with the untreated meat, the numbers of Enterobacteriaceae were significantly less on POAA treated than on untreated meat. The combination of data collected by culture-dependent and independent techniques provided the most robust approach for elucidating the efficacy of chemical sanitization of chilled vacuum-packaged beef. If conventional cultivation is used for monitoring bacterial spoilage of vacuum-packaged chilled meats it is recommended that culture methods specific for Carnobacterium and Clostridium spp. should be included in order to provide a more complete indication of microbial diversity.

We transformed Aspergillus niger with the full length cDNA gene encoding hen egg-white lysozyme (HEWL) and its secretion signal sequence. Lysozyme levels up to 12 mg/l were secreted when expression was controlled by the A. awamori glucoamylase (GAM) promoter and 1 mg/l when controlled by the A. nidulans glyceraldehyde-3-phosphate dehydrogenase (GPD) promoter. N-terminal sequence analysis of the recombinant protein indicated that the signal peptide was correctly processed by the A. niger secretory apparatus. The specific catalytic activity of the recombinant protein was identical to that of authentic hen lysozyme. The recombinant HEWL was examined by 2D 1H-NMR spectroscopy and shown to have a spectrum identical to that of authentic HEWL indicating that the protein was correctly folded.

Recent investigations of blown pack spoilage in New Zealand chilled vacuum-packaged meats have found moderate to high numbers of Enterobacteriaceae in the spoilage flora, but no clostridia, such as C. estertheticum and C. gasigenes, that are usually associated with blown pack spoilage. This study showed that pyschrotolerant Enterobacteriaceae produced gas in a lamb homogenate model under anaerobic conditions and that these organisms could cause blown pack spoilage of vacuum-packaged chilled meats. Significant gas production was observed with the majority of the psychrotolerant Enterobacteriaceae strains tested including presumptive species of Enterobacter, Serratia, Hafnia and Rahnella. However, no gas was produced in lamb homogenates inoculated with presumptive species of Ewingella americana or Yersinia enterocolitica. Gas production was also confirmed in vacuum-packaged lamb shoulders stored at 4 degrees C for 21 days after being inoculated with individual representative Enterobacteriaceae isolates. Biochemical characterisation proved to be more useful than genotype-based typing of 16S rRNA genes for discriminating different psychrotolerant Enterobacteriaceae from naturally contaminated meat microflora.

This study evaluated the use of PFGE and single enzyme AFLP techniques for the determination of the genetic relationships between Staphyloccocus aureus isolates from human, bovine, ovine and food related sources and reports the prevalence of 'classic' (sea to see) and 'new' (seg, seh, sei, sej, sem, sen and seo) staphylococcal enterotoxin (se) genes in 92 S. aureus strains. A sub-set of the se genotyping results was confirmed by ELISA and the presence of SE toxin determined in isolates from different sources. A 100% correlation was observed, between detection of enterotoxin genes sea-see and expression of corresponding enterotoxin proteins in vitro. The se genotyping data generated from 90 of the S. aureus isolates showed that many of the S. aureus strains producing identical se genotypes correlated with both AFLP and PFGE pattern types. However, single enzyme AFLP technique did not possess the discriminatory power of the PFGE method, but similar clonal relationships were observed by both techniques in many of the isolates tested. Results reported here include the first comprehensive study using a single enzyme AFLP technique to investigate the genetic background of S. aureus isolates from a wide distribution including animal, human and food related sources.

The human genome provides a reference sequence, which is a template for resequencing studies that aim to discover and interpret the record of common ancestry that exists in extant genomes. To understand the nature and pattern of variation and linkage disequilibrium comprising this history, we present a study of approximately 31 kb spanning an approximately 70 kb region of FMR1, sequenced in a sample of 20 humans (worldwide sample) and four great apes (chimp, bonobo, and gorilla). Twenty-five polymorphic sites and two insertion/deletions, distributed in 11 unique haplotypes, were identified among humans. Africans are the only geographic group that do not share any haplotypes with other groups. Parsimony analysis reveals two main clades and suggests that the four major human geographic groups are distributed throughout the phylogenetic tree and within each major clade. An African sample appears to be most closely related to the common ancestor shared with the three other geographic groups. Nucleotide diversity, pi, for this sample is 2.63 +/- 6.28 x 10(-4). The mutation rate, mu is 6.48 x 10(-10) per base pair per year, giving an ancestral population size of approximately 6200 and a time to the most recent common ancestor of approximately 320,000 +/- 72,000 per base pair per year. Linkage disequilibrium (LD) at the FMR1 locus, evaluated by conventional LD analysis and by the length of segment shared between any two chromosomes, is extensive across the region.

The efficacy of a peroxyacetic acid formulation (POAA) at reducing Escherichia coli O157:H7 contamination on external carcass surfaces of hot-boned beef and veal with a commercial spray apparatus was determined. Hot-boned external carcass surfaces were inoculated with either a high dose (10(6) CFU/cm2) in fresh bovine feces or with a low dose (10(3) CFU/cm2) in diluent of laboratory-cultured E. coli O157:H7. Treatments included a water wash, a POAA (180 ppm) wash, or a water plus POAA wash. Samples were extracted from the external carcass surface with a cork borer to determine the numbers of viable E. coli O157:H7 remaining on the carcass surface after treatment. Although a water wash alone resulted in a 1.25 (94.4%) and a 1.31 (95.1%) mean log reduction on veal and beef inoculated with a high dose of E. coli O157:H7, the POAA treatment resulted in a substantially greater mean log reduction of 3.56 and 3.59 (>99.9%). The water wash only resulted in a 33.9% reduction on veal and 62.8% on beef inoculated with a low dose of E. coli O157:H7, whereas POAA treatment greatly improved pathogen reduction to 98.9 and 97.4% on veal and beef, respectively. The combination of a water wash followed by a POAA treatment resulted in a similar E. coli O157:H7 reduction to that achieved by POAA treatment alone. In conclusion, POAA treatment significantly reduced viable E. coli O157:H7 numbers on experimentally contaminated beef and veal carcasses, which justifies its use as a chemical intervention for the removal of this human pathogen.