Elina Säde

Comparison of microbial communities in marinated and unmarinated broiler meat by metagenomics

Most raw poultry sold in Finland at the retail level is mixed with marinades containing oil, sugar, spices and acetic acid and packaged under modified atmosphere. Premature spoilage of marinated poultry preparations has been observed and associated with high levels of Leuconostoc spp. in meat. In this study we investigated whether marination of broiler fillet strips increased the proportion of Leuconostoc spp. in the microbial communities. To obtain a comprehensive view of the microbiota, we sequenced total DNA and 16S rRNA gene amplicons from the microbial communities. The lactic acid bacterial communities were characterized also by identification of colonies. The results showed that marinade increased the proportions of the spoilage-associated Leuconostoc gasicomitatum in the communities as well as the proportions of Leuconostoc gelidum and Lactobacillus spp. The proportions of Carnobacterium, Vagococcus, Brochothrix thrermosphacta, Clostridium, Enterobacteriaceae and Vibrio were diminished in marinated meat. Analysis of 16S rRNA gene amplicons resulted in 312 and 284 operational taxonomical units (dissimilarity 0.03) in unmarinated and marinated meat, respectively, indicating that the meat communities were more diverse than hitherto shown. Metagenomic analysis revealed a number of bacterial taxa that have not been associated with late shelf-life meat before, including Vagococcus and Vibrio that belonged to the predominating part of the microbial community in unmarinated meat. According to the functional analysis of the metagenomes, the communities in both marinated and unmarinated poultry were characterized by high proportions (15.6% or 17.9%) of genes involved in carbohydrate metabolism.

Genome Sequence of a Food Spoilage Lactic Acid Bacterium, Leuconostoc gasicomitatum LMG 18811T, in Association with Specific Spoilage Reactions

ABSTRACT Leuconostoc gasicomitatum is a psychrotrophic lactic acid bacterium causing spoilage of cold-stored, modified-atmosphere-packaged (MAP), nutrient-rich foods. Its role has been verified by challenge tests in gas and slime formation, development of pungent acidic and buttery off odors, and greening of beef. MAP meats have especially been prone to L. gasicomitatum spoilage. In addition, spoilage of vacuum-packaged vegetable sausages and marinated herring has been reported. The genomic sequencing project of L. gasicomitatum LMG 18811T was prompted by a need to understand the growth and spoilage potentials of L. gasicomitatum, to study its phylogeny, and to be able to knock out and overexpress the genes. Comparative genomic analysis was done within L. gasicomitatum LMG 18811T and the three fully assembled Leuconostoc genomes (those of Leuconostoc mesenteroides, Leuconostoc citreum, and Leuconostoc kimchii) available. The genome of L. gasicomitatum LMG 18811T is plasmid-free and contains a 1,954,080-bp circular chromosome with an average GC content of 36.7%. It includes genes for the phosphoketolase pathway and alternative pathways for pyruvate utilization. As interesting features associated with the growth and spoilage potential, LMG 18811T possesses utilization strategies for ribose, external nucleotides, nucleosides, and nucleobases and it has a functional electron transport chain requiring only externally supplied heme for respiration. In respect of the documented specific spoilage reactions, the pathways/genes associated with a buttery off odor, meat greening, and slime formation were recognized. Unexpectedly, genes associated with platelet binding and collagen adhesion were detected, but their functionality and role in food spoilage and processing environment contamination need further study.

Characterization and evaluation of the spoilage potential of Lactococcus piscium isolates from modified atmosphere packaged meat

A total of 222 psychrotrophic lactococci isolated from use-by day, modified atmosphere packaged (MAP) meat were identified to the species level by numerical analyses of EcoRI and ClaI ribopatterns and phylogenetic sequence analyses of 16S, rpoA and pheS genes. In addition, their meat spoilage potential was studied. The majority of the isolates (n=215) were identified as Lactococcus piscium, while seven isolates belonged to Lactococcus raffinolactis. L. piscium was shown to be adapted to growing in a variety of MAP meat products including broiler, turkey, pork, and minced meat from beef and pork, where they belonged to the predominating microbiota at the end of the storage. Numerical analyses of EcoRI and ClaI ribopatterns, and phylogenetic sequence analyses of rpoA and pheS genes were shown to be reliable tools in species level identification of meat lactococci. The spoilage potential of L. piscium was evaluated by inoculating representative isolates to MAP pork stored at 6 °C for 22 days. Development of spoilage population was monitored using a culture-independent T-RFLP approach. The sensory shelf life of pork inoculated with L. piscium was shortened compared to the uninoculated control. Alongside with the inoculated L. piscium isolates, Leuconostoc spp. present as initial contaminants in the samples thrived. This shows that even though lactococci were inoculated at higher levels compared to the natural microbiota, they did not occupy the niche and prevent the growth of other lactic acid bacteria.

Identification of Enterococci from Broiler Products and a Broiler Processing Plant and Description of Enterococcus viikkiensis sp. nov.

ABSTRACT In two previous studies dealing with lactic acid bacteria (LAB) from modified-atmosphere-packaged (MAP) broiler products and a broiler processing plant, several isolates remained unidentified. According to 16S rRNA gene sequence analysis, 36 isolates were assigned to the genus Enterococcus. Numerical analysis of combined HindIII and EcoRI ribopatterns of these isolates resulted in species-specific clusters that were congruent with the clusters obtained by both DNA-directed RNA polymerase subunit A (rpoA) and phenylalanyl-tRNA synthetase α chain (pheS) housekeeping gene analyses. In the analyses, a group of five isolates distinct from any known enterococcal species clustered together. The five isolates were positioned in the Enterococcus avium group, with E. devriesei being the closest phylogenetic neighbor. The DNA-DNA hybridization levels with E. devriesei ranged from 28.8 to 54.3% and indicated that these strains represented a novel species. The name Enterococcus viikkiensis sp. nov. is proposed, with strain DSM 24043T (LMG 26075T) being the type strain. Our study demonstrated that the identification of enterococci within the E. avium phylogenetic group demands polyphasic taxonomic approaches. The rpoA and pheS gene similarities (99.0 to 99.2% and 94.3 to 95.4%, respectively) between E. viikkiensis and its closest phylogenetic neighbor, E. devriesei, were higher than those previously reported within the enterococci. In addition, the phenotypic profiles of the species in the E. avium group were also highly similar, and some traits were found to be misleading for enterococci, such as E. viikkiensis does not grow at 45°C. The numerical analysis of combined HindIII and EcoRI ribopatterns was of considerable assistance in distinguishing enterococcal species within the E. avium group.

High number of Yersinia enterocolitica 4/O:3 in cold-stored modified atmosphere-packed pig cheek meat.

Yersinia enterocolitica is a psychrotrophic, facultative anaerobic zoonotic bacterium belonging to family Enterobacteriaceae and it can be transmitted from pigs to humans through pork. The growth of bacteria belonging to Enterobacteriaceae and aerobic spoilage bacteria is usually effectively restricted by 20% or more CO(2) enriched atmosphere at refrigerated temperatures. In this study, 40 samples of meat strips from pig cheek (musculus masseter) and 40 samples from hind leg (m. semimembranosus) muscles were packaged in modified atmosphere (MA) (30% CO(2)/70% O(2)) and stored at 6°C for 12d. Twenty naturally contaminated samples per muscle type were studied on days 1 and 13. Violet red bile glucose (VRBG) and de Man Rogosa Sharpe (MRS) agar plates were used for enumeration of Enterobacteriaceae including Y. enterocolitica and lactic acid bacteria, respectively. During the 12-d storage at 6°C in MA, the mean number of bacteria on pork strips of cheek meat was increasing from 1.6 to 4.5 log cfu/g and from 3.1 to 7.2 log cfu/g on VRBG and MRS agar plates, respectively. Most of the oxidase-negative isolates on VRBG plates, which were isolated from the cheek meat samples after 12-d cold storage in MA, were identified as Y. enterocolitica 4/O:3. The mean number of this pathogen was 4.1 log cfu/g varying between 2.3 and 5.4 log cfu/g. The pH of the cheek meat and leg meat was measured on days 1 and 13, and it remained high (pH>6) in most cheek meat samples during the storage. No Y. enterocolitica 4/O:3 was isolated from meat strips of hind leg. This study shows that cheek meat of slaughter pigs is contaminated with Y. enterocolitica 4/O:3 and that this pathogen can grow well on raw pork packaged in MA at 6°C even in the presence of high number of lactic acid bacteria.

The effect of marination on lactic acid bacteria communities in raw broiler fillet strips.

Marination with marinade containing salt, sugar, and acetic acid is commonly used in Finland to enhance the value of raw broiler meat. In this study, we investigated the effect of marination, marinade components and storage time on composition of bacterial communities in modified atmosphere-packaged (MAP) broiler fillet strips. The communities were characterized using two culture-independent methods: 16S rRNA gene fragment sequencing and terminal restriction fragment length polymorphism. In unmarinated broiler fillet strips, Lactococcus spp. and Carnobacterium spp. predominated at the early storage phase but were partially replaced by Lactobacillus spp. and Leuconostoc spp. when the chilled storage time was extended. In the marinated fillet strips, Lactobacillus spp. and Leuconostoc spp. predominated independent from the storage time. By mixing the different marinade components with broiler meat, we showed that marination changed the community composition and favored Leuconostoc spp. and Lactobacillus spp. by the combined effect of carbohydrates and acetic acid in marinade. Marination increased the maximum level of lactic acid bacteria in broiler meat and enhanced CO2 production and acidification of meat during the chilled storage. Accumulation of CO2 in package head-space due to the enhanced growth of Leuconostoc spp. in marinated meat may lead to bulging of packages, which is a spoilage defect frequently associated with marinated and MAP raw broiler preparations in Finland.

Multilocus Sequence Typing of Leuconostoc gelidum subsp. gasicomitatum, a Psychrotrophic Lactic Acid Bacterium Causing Spoilage of Packaged Perishable Foods

ABSTRACT Leuconostoc gelidum subsp. gasicomitatum is a psychrotrophic lactic acid bacterium (LAB) that causes spoilage of a variety of modified-atmosphere-packaged (MAP) cold-stored food products. During the past 10 years, this spoilage organism has been increasingly reported in MAP meat and vegetable products in northern Europe. In the present study, the population structure within 252 L. gelidum subsp. gasicomitatum strains was determined based on a novel multilocus sequence-typing (MLST) scheme employing seven housekeeping genes. These strains had been isolated from meat and vegetable sources over a time span of 15 years, and all 68 previously detected pulsed-field gel electrophoresis (PFGE) genotypes were represented. A total of 46 sequence types (STs) were identified, with a majority of the strains (>60%) belonging to three major STs, which were grouped into three clonal complexes (CCs) and 17 singletons by Global Optimal eBURST (goeBURST). The results by Bayesian analysis of population structure (BAPS) mostly correlated with the grouping by goeBURST. Admixture analysis by BAPS indicated a very low level of exchange of genetic material between the subpopulations. Niche specificity was observed within the subpopulations: CC1 and BAPS cluster 1 consisted mostly of strains from a variety of MAP meats, whereas vegetable strains grouped together with strains from MAP poultry within CC2 and BAPS cluster 2. The MLST scheme presented in this study provides a shareable and continuously growing sequence database enabling global comparison of strains associated with spoilage cases. This will further advance our understanding of the microbial ecology of this industrially important LAB.

Characterization of European Yersinia enterocolitica 1A strains using restriction fragment length polymorphism and multilocus sequence analysis

Yersinia enterocolitica is currently divided into two subspecies: subsp. enterocolitica including highly pathogenic strains of biotype 1B and subsp. palearctica including nonpathogenic strains of biotype 1A and moderately pathogenic strains of biotypes 2–5. In this work, we characterized 162 Y. enterocolitica strains of biotype 1A and 50 strains of biotypes 2–4 isolated from human, animal and food samples by restriction fragment length polymorphism using the HindIII restriction enzyme. Phylogenetic relatedness of 20 representative Y. enterocolitica strains including 15 biotype 1A strains was further studied by the multilocus sequence analysis of four housekeeping genes (glnA, gyrB, recA and HSP60). In all the analyses, biotype 1A strains formed a separate genomic group, which differed from Y. enterocolitica subsp. enterocolitica and from the strains of biotypes 2–4 of Y. enterocolitica subsp. palearctica. Based on these results, biotype 1A strains considered nonpathogenic should not be included in subspecies palearctica containing pathogenic strains of biotypes 2–5.