Prevalence and genotype diversity of Campylobacter jejuni in hunted reared pheasants (Phasianus colchicus) in Finland.

Campylobacter spp., especially C. jejuni, is the most common zoonotic pathogen in humans worldwide. In Nordic countries, the prevalence of C. jejuni in broilers, which is an important reservoir of human infections, is generally low. Thus, other sources of domestically acquired infections besides chicken meat need to be considered. Game birds are known to carry a variety of zoonotic agents, including Campylobacter. The aim of this study was to investigate Campylobacter spp. carriage in a flock of reared pheasants at hunting in two successive samplings to better understand the dynamics of Campylobacter infections in pheasants. Overall, 72% of the intestinal samples were positive for Campylobacter spp. by direct culture on mCCDA. C. jejuni was the only species identified. The isolates were genotyped using whole genome sequencing (WGS), multilocus sequence typing (MLST), and ad hoc whole genome MLST (wgMLST). Two distinct C. jejuni clones were identified among the 18 isolates studied, representing MLST sequence types (STs) ST-45 and ST-699. The ST-45 isolates were closely related to previous human clinical isolates using core genome MLST (cgMLST). In contrast, the ST-699 isolates forming the dominant clone in the latter sampling were quite distinct from previously described cgMLST profiles from different hosts and sources worldwide. In conclusion, the intestine of reared pheasants is commonly colonized by C. jejuni and may carry genotypes relevant to infections in livestock and humans. Hygienic measures are needed to limit the spread of infection in reared flocks. Especially farmers and hunters having direct contact with pheasant offal need to be aware of the associated zoonosis risk to protect themselves and their working dogs alike. Biosecurity measures to improve the safety and reduce the zoonosis risk associated with pheasant farming should be further investigated.

Vagococcus proximus sp. nov. and Vagococcus intermedius sp. nov., originating from modified atmosphere packaged broiler meat.

During our studies on spoilage microbiomes of modified atmosphere packaged broiler meat, we isolated three strains (PNs007T, STAA11T and STAA25) of unknown identity. In this present polyphasic taxonomy study, including genome-based analyses, we discovered that these isolates represent two novel species belonging to the genus Vagococcus. In all phylogenetic analyses, PNs007T was positioned very close to Vagococcus fessus but both the average nucleotide identity (ANI; 89.5 %) and digital DNA-DNA hybridization (dDDH; 38.3 %) values distinguished it as a novel vagococcal species. STAA11T and STAA25 were genetically highly similar (16S rRNA, ANI and dDDH 100 %). The phylogenetic position of STAA11T was adjacent to but out of the cluster containing V. fessus, Vagococcus coleopterorum and PNs007T. According to the ANI (76.2-76.4 %) and dDDH (<22.6 %) values it also represented a novel vagococcal species. Phenotypic characteristics and chemotaxonomic properties of both novel species were typical for vagococci and they contained C16 : 0 (25.5-30.1 %) and C18 : 1 ω9c (67.3-73.0 %) as the major cellular fatty acids. The streptomycin-resistant genotype of STAA11T and STAA25 allowing the growth on streptomycin thallous acetate actidione medium was considered to result from a modification in codon 104 of the rpsL gene leading to P104A substitution. The ability of STAA11T and STAA25 to produce ammonia from arginine separated them from PNs007T, which did not show arginine deiminase activity. We propose the names Vagococcus proximus sp. nov. (type strain PNs007T=DSM 115185T=CCUG 76696T) and Vagococcus intermedius sp. nov. (type strain STAA11T=DSM 115183T=CCUG 76697T) for these novel species.

Comparative genomics of 40 Weissella paramesenteroides strains.

Weissella strains are often detected in spontaneously fermented foods. Because of their abilities to produce lactic acid and functional exopolysaccharides as well as their probiotic traits, Weissella spp. improve not only the sensorial properties but also nutritional values of the fermented food products. However, some Weissella species have been associated with human and animal diseases. In the era of vast genomic sequencing, new genomic/genome data are becoming available to the public on daily pace. Detailed genomic analyses are due to provide a full understanding of individual Weissella species. In this study, the genomes of six Weissella paramesenteroides strains were de novo sequenced. The genomes of 42 W. paramesenteroides strains were compared to discover their metabolic and functional potentials in food fermentation. Comparative genomics and metabolic pathway reconstructions revealed that W. paramesenteroides is a compact group of heterofermentative bacteria with good capacity of producing secondary metabolites and vitamin Bs. Since the strains rarely harbored plasmid DNA, they did not commonly possess the genes associated with bacteriocin production. All 42 strains were shown to bear vanT gene from the glycopeptide resistance gene cluster vanG. Yet none of the strains carried virulence genes.

Microbiological quality and safety of vacuum-packaged white-tailed deer meat stored at 4 °C.

Vacuum packaging is widely used for extending the shelf life of commercial fresh meat products. It also ensures product hygiene during distribution and storage. However, very little information exists concerning the effects of vacuum packaging on the shelf life of deer meat. One of our aims was to evaluate how storage under vacuum at 4 °C affects the microbial quality and safety of white-tailed deer (Odocoileus virginianus) meat cuts. This was assessed in a longitudinal study based on sensory analyses and measurements of (1) mesophilic aerobic bacteria (MAB), (2) lactic acid bacteria (LAB), (3) enterobacteria (EB), (4) and Escherichia coli (EC) counts, and the presence of foodborne pathogens (Campylobacter, Salmonella, stx-harbouring E. coli (STEC), Yersinia and Listeria). Microbiomes were additionally investigated by 16S rRNA gene amplicon sequencing at the time of spoilage. In total, 50 vacuum-packaged meat cuts from the carcasses of 10 wild white-tailed deer harvested in southern Finland in December 2018 were analysed. A significant (p < 0.001) drop in the odour and appearance scores and a significant increase in MAB (p < 0.001) and LAB (p = 0.001) counts of the vacuum-packaged meat cuts were observed after 3 weeks of storage at 4 °C. A very strong correlation (rs = 0.9444, p < 0.001) between the MAB and LAB counts were found during the 5-week sampling period. Clear spoilage changes, manifested as sour off-odours (odour scores ≤2) and pale colour, were detected in the meat cuts spoilt after 3-week storage. High (≥8 log10 cfu/g) MAB and LAB counts were also detected. According to the 16S rRNA gene amplicon analyses, Lactobacillus was the dominant bacterial genus in these samples, demonstrating that LAB can cause rapid spoilage of vacuum-packaged deer meat cuts stored at 4 °C. The rest of the samples were spoilt after four or five weeks of storage, and a vast number of bacterial genera were identified in them. Listeria and STEC were detected by PCR in 50 % and 18 % of the meat cut samples, respectively, which may indicate a public health problem. Our results demonstrate that it is very challenging to ensure the quality and safety of vacuum-packaged deer meat stored at 4 °C, and freezing is therefore recommended to prolong the shelf life.

Microsporidian Infection in Mosquitoes (Culicidae) Is Associated with Gut Microbiome Composition and Predicted Gut Microbiome Functional Content.

The animal gut microbiota consist of many different microorganisms, mainly bacteria, but archaea, fungi, protozoans, and viruses may also be present. This complex and dynamic community of microorganisms may change during parasitic infection. In the present study, we investigated the effect of the presence of microsporidians on the composition of the mosquito gut microbiota and linked some microbiome taxa and functionalities to infections caused by these parasites. We characterised bacterial communities of 188 mosquito females, of which 108 were positive for microsporidian DNA. To assess how bacterial communities change during microsporidian infection, microbiome structures were identified using 16S rRNA microbial profiling. In total, we identified 46 families and four higher taxa, of which Comamonadaceae, Enterobacteriaceae, Flavobacteriaceae and Pseudomonadaceae were the most abundant mosquito-associated bacterial families. Our data suggest that the mosquito gut microbial composition varies among host species. In addition, we found a correlation between the microbiome composition and the presence of microsporidians. The prediction of metagenome functional content from the 16S rRNA gene sequencing suggests that microsporidian infection is characterised by some bacterial species capable of specific metabolic functions, especially the biosynthesis of ansamycins and vancomycin antibiotics and the pentose phosphate pathway. Moreover, we detected a positive correlation between the presence of microsporidian DNA and bacteria belonging to Spiroplasmataceae and Leuconostocaceae, each represented by a single species, Spiroplasma sp. PL03 and Weissella cf. viridescens, respectively. Additionally, W. cf. viridescens was observed only in microsporidian-infected mosquitoes. More extensive research, including intensive and varied host sampling, as well as determination of metabolic activities based on quantitative methods, should be carried out to confirm our results.

Marination increased tyramine levels in rainbow trout fillet strips packaged under modified atmosphere.

Marinades are increasingly used to manufacture raw fish products. In corresponding meats, marinating is known to have a major effect on the composition of the microbiome, but the effect of marinating on fish is not known as well. This knowledge gap prompted our study of the microbial ecology and amine formation in marinated and unmarinated modified atmosphere commercially packaged rainbow trout fillet strips. According to our findings, marination increased the maximum concentrations (7-8 log CFU/g) of psychrotrophic bacteria by one logarithmic unit and led to 5 times higher average tyramine concentrations than the corresponding unmarinated product. Instead, trimethylamine concentrations were 30 times higher in the unmarinated product than those in the marinated one. According to the 16 S rRNA sequence analyses, lactic acid bacteria (LAB) predominated in the marinated strips one day after the use-by date, whereas in the unmarinated strips Fusobacteriaceae and LAB were the dominating taxa. Based on the culture-dependent analysis, Latilactobacillus fuchuensis was the prevailing LAB in both products. Since the subset of L. fuchuensis strains tested was able to produce tyramine in vitro, we hypothesise that the use of the acidic marinade activated the production of tyrosine-decarboxylating enzymes in L. fuchuensis and led to the increased tyramine concentrations.

Pangenome and genomic taxonomy analyses of Leuconostoc gelidum and Leuconostoc gasicomitatum.

BACKGROUND: Leuconostoc gelidum and Leuconostoc gasicomitatum have dual roles in foods. They may spoil cold-stored packaged foods but can also be beneficial in kimchi fermentation. The impact in food science as well as the limited number of publicly available genomes prompted us to create pangenomes and perform genomic taxonomy analyses starting from de novo sequencing of the genomes of 37 L. gelidum/L. gasicomitatum strains from our culture collection. Our aim was also to evaluate the recently proposed change in taxonomy as well as to study the genomes of strains with different lifestyles in foods. METHODS: We selected as diverse a set of strains as possible in terms of sources, previous genotyping results and geographical distribution, and included also 10 publicly available genomes in our analyses. We studied genomic taxonomy using pairwise average nucleotide identity (ANI) and calculation of digital DNA-DNA hybridisation (dDDH) scores. Phylogeny analyses were done using the core gene set of 1141 single-copy genes and a set of housekeeping genes commonly used for lactic acid bacteria. In addition, the pangenome and core genome sizes as well as some properties, such as acquired antimicrobial resistance (AMR), important due to the growth in foods, were analysed. RESULTS: Genome relatedness indices and phylogenetic analyses supported the recently suggested classification that restores the taxonomic position of L. gelidum subsp. gasicomitatum back to the species level as L. gasicomitatum. Genome properties, such as size and coding potential, revealed limited intraspecies variation and showed no attribution to the source of isolation. The distribution of the unique genes between species and subspecies was not associated with the previously documented lifestyle in foods. None of the strains carried any acquired AMR genes or genes associated with any known form of virulence. CONCLUSION: Genome-wide examination of strains confirms that the proposition to restore the taxonomic position of L. gasicomitatum is justified. It further confirms that the distribution and lifestyle of L. gelidum and L. gasicomitatum in foods have not been driven by the evolution of functional and phylogenetic diversification detectable at the genome level.

Longitudinal Metatranscriptomic Analysis of a Meat Spoilage Microbiome Detects Abundant Continued Fermentation and Environmental Stress Responses during Shelf Life and Beyond.

Microbial food spoilage is a complex phenomenon associated with the succession of the specific spoilage organisms (SSO) over the course of time. We performed a longitudinal metatranscriptomic study on one modified-atmosphere-packaged (MAP) beef product to increase understanding of the longitudinal behavior of a spoilage microbiome during shelf life and beyond. Based on the annotation of the mRNA reads, we recognized three stages related to the active microbiome that were descriptive of the sensory quality of the beef: acceptable product (AP), early spoilage (ES), and late spoilage (LS). Both the 16S RNA taxonomic assignments from the total RNA and functional annotations of the active genes showed that these stages were significantly different from each other. However, the functional gene annotations showed more pronounced differences than the taxonomy assignments. Psychrotrophic lactic acid bacteria (LAB) formed the core of the SSO, according to the transcribed reads. Leuconostoc species were the most abundant active LAB throughout the study period, whereas the transcription activity of Streptococcaceae (mainly Lactococcus) increased after the product had spoiled. In the beginning of the experiment, the community managed environmental stress by cold-shock responses, which were followed by expression of the genes involved in managing oxidative stress. Glycolysis, the pentose phosphate pathway, and pyruvate metabolism were active throughout the study at a relatively stable level. However, the proportional transcription activities of the enzymes in these pathways changed over time.IMPORTANCE It is generally known which organisms are the typical SSO in foods, whereas the actively transcribed genes and pathways during microbial succession are poorly understood. This knowledge is important, since better approaches to food quality evaluation and shelf life determination are needed. Therefore, we conducted this study to find longitudinal markers that are connected to quality deterioration in a MAP beef product. This kind of RNA marker could be used to develop novel types of rapid quality analysis tools in the future. New tools are needed, since even though SSO can be detected and their concentrations determined using the current microbiological methods, results from these analyses cannot predict how close in time a spoilage community is to the production of clear sensory defects. The main reason for this is that the species composition of a spoilage community does not change dramatically during late shelf life, whereas the ongoing metabolic activities lead to the development of notable sensory deterioration.

Two copies of the ail gene found in Yersinia enterocolitica and Yersinia kristensenii.

Yersinia enterocolitica is the most common Yersinia species causing foodborne infections in humans. Pathogenic strains carry the chromosomal ail gene, which is essential for bacterial attachment to and invasion into host cells and for serum resistance. This gene is commonly amplified in several PCR assays detecting pathogenic Y. enterocolitica in food samples and discriminating pathogenic isolates from non-pathogenic ones. We have isolated several non-pathogenic ail-positive Yersinia strains from various sources in Finland. For this study, we selected 16 ail-positive Yersinia strains, which were phenotypically and genotypically characterised. Eleven strains were confirmed to belong to Y. enterocolitica and five strains to Yersinia kristensenii using whole-genome alignment, Parsnp and the SNP phylogenetic tree. All Y. enterocolitica strains belonged to non-pathogenic biotype 1A. We found two copies of the ail gene (ail1 and ail2) in all five Y. kristensenii strains and in one Y. enterocolitica biotype 1A strain. All 16 Yersinia strains carried the ail1 gene consisting of three different sequence patterns (A6-A8), which were highly similar with the ail gene found in high-pathogenic Y. enterocolitica biotype 1B strains (A2). The Ail protein encoded by the ail1 gene was highly conserved compared to the Ail protein encoded by the ail2 gene. Multiple sequence alignment of the ail gene and Ail protein were conducted with MAFF. In total, 10 ail sequence variations have been identified, of which 8 conserved ones belonged to the ail1 gene. According to our results, the detection of ail alone is not sufficient to predict the pathogenicity of Yersinia isolates.

Growth and metabolic characteristics of fastidious meat-derived Lactobacillus algidus strains.

Lactobacillus algidus is a meat spoilage bacterium often dominating the bacterial communities on chilled, packaged meat. Yet, L. algidus strains are rarely recovered from meat, and only few studies have focused on this species. The main reason limiting detailed studies on L. algidus is related to its poor growth on the media routinely used for culturing food spoilage bacteria. Thus, our study sought to develop reliable culture media for L. algidus to enable its recovery from meat, and to allow subculturing and phenotypic analyses of the strains. We assessed the growth of meat-derived L. algidus strains on common culture media and their modifications, and explored the suitability of potential media for the recovery of L. algidus from meat. Moreover, we determined whether 12 meat-derived L. algidus strains selected from our culture collection produce biogenic amines that may compromise safety or quality of meat, and finally, sequenced de novo and annotated the genomes of two meat-derived L. algidus strains to uncover genes and metabolic pathways relevant for phenotypic traits observed. MRS agar supplemented with complex substances (peptone, meat and yeast extract, liver digest) supported the growth of L. algidus, and allowed the recovery of new L. algidus isolates from meat. However, most strains grew poorly on standard MRS agar and on general-purpose media. In MRS broth, most strains grew well but a subset of strains required supplementation of MRS broth with additional cysteine. Supplementation of MRS broth with catalase allowed growth in aerated cultures suggesting that the strains produced hydrogen peroxide when grown aerobically. The strains tested (n = 12) produced ornithine from arginine and putrescine from agmatine, and two strains produced tyramine from tyrosine. Our findings reveal that L. algidus populations are underestimated if routine culture protocols are applied, and prompt concerns that L. algidus may generate tyramine or putrescine in meat or fermented meat products.

Metabolomics and bacterial diversity of packaged yellowfin tuna (Thunnus albacares) and salmon (Salmo salar) show fish species-specific spoilage development during chilled storage.

Microbial (colony counts, 16S rRNA gene amplification), chemical (pH, 1H NMR spectroscopy) and sensory changes in raw Atlantic Salmon (Salmo salar) and tuna (Thunnus albacares) fillets stored under vacuum at 3 °C were evaluated over a period of 12 days. Both species of fish are globally important and among the ten most consumed fishes in the world. Although the sensory analyses showed a decrease in the quality of both fish species, only the salmon fillets were considered spoiled at the end of the storage period. In salmon, trimethylamine was the main spoilage product and bacterial colony counts reached an average of 7.3 log10 cfu/g. The concentration of glucose decreased and the concentration of organic acids increased during storage revealing glucose fermentation. Photobacterium was the dominating genus in the salmon studied. In the tuna studied, the bacterial colony counts reached only an average of 4.6 log10 cfu/g. The dominating bacteria in tuna were Pseudomonas spp. Glucose levels did not decrease, suggesting that amino acids and lactate most likely acted as carbon sources for bacteria in tuna. In conclusion, the study revealed that salmon was clearly a more perishable fish than tuna.

Food Spoilage-Associated Leuconostoc, Lactococcus, and Lactobacillus Species Display Different Survival Strategies in Response to Competition.

Psychrotrophic lactic acid bacteria (LAB) are the prevailing spoilage organisms in packaged cold-stored meat products. Species composition and metabolic activities of such LAB spoilage communities are determined by the nature of the meat product, storage conditions, and interspecies interactions. Our knowledge of system level responses of LAB during such interactions is very limited. To expand it, we studied interactions between three common psychrotrophic spoilage LAB (Leuconostoc gelidum, Lactococcus piscium, and Lactobacillus oligofermentans) by comparing their time course transcriptome profiles obtained during their growth in individual, pairwise, and triple cultures. The study revealed how these LAB employed different strategies to cope with the consequences of interspecies competition. The fastest-growing bacterium, Le. gelidum, attempted to enhance its nutrient-scavenging and growth capabilities in the presence of other LAB through upregulation of carbohydrate catabolic pathways, pyruvate fermentation enzymes, and ribosomal proteins, whereas the slower-growing Lc. piscium and Lb. oligofermentans downregulated these functions. These findings may explain the competitive success and predominance of Le. gelidum in a variety of spoiled foods. Peculiarly, interspecies interactions induced overexpression of prophage genes and restriction modification systems (mechanisms of DNA exchange and protection against it) in Lc. piscium and Lb. oligofermentans but not in Le. gelidum Cocultivation induced also overexpression of the numerous putative adhesins in Lb. oligofermentans These adhesins might contribute to the survival of this slowly growing bacterium in actively growing meat spoilage communities.IMPORTANCE Despite the apparent relevance of LAB for biotechnology and human health, interactions between members of LAB communities are not well known. Knowledge of such interactions is crucial for understanding how these communities function and, consequently, whether there is any possibility to develop new strategies to interfere with their growth and to postpone spoilage of packaged and refrigerated foods. With the help of controlled experiments, detailed regulation events can be observed. This study gives an insight into the system level interactions and the different competition-induced survival strategies related to enhanced uptake and catabolism of carbon sources, overexpression of adhesins and putative bacteriocins, and the induction of exchange of genetic material. Even though this experiment dealt with only three LAB strains in vitro, these findings agreed well with the relative abundance patterns typically reported for these species in natural food microbial communities.

Exploring lot-to-lot variation in spoilage bacterial communities on commercial modified atmosphere packaged beef.

Understanding the factors influencing meat bacterial communities is important as these communities are largely responsible for meat spoilage. The composition and structure of a bacterial community on a high-O2 modified-atmosphere packaged beef product were examined after packaging, on the use-by date and two days after, to determine whether the communities at each stage were similar to those in samples taken from different production lots. Furthermore, we examined whether the taxa associated with product spoilage were distributed across production lots. Results from 16S rRNA amplicon sequencing showed that while the early samples harbored distinct bacterial communities, after 8-12 days storage at 6 °C the communities were similar to those in samples from different lots, comprising mainly of common meat spoilage bacteria Carnobacterium spp., Brochothrix spp., Leuconostoc spp. and Lactococcus spp. Interestingly, abundant operational taxonomic units associated with product spoilage were shared between the production lots, suggesting that the bacteria enable to spoil the product were constant contaminants in the production chain. A characteristic succession pattern and the distribution of common spoilage bacteria between lots suggest that both the packaging type and the initial community structure influenced the development of the spoilage bacterial community.

Lactobacillus oligofermentans glucose, ribose and xylose transcriptomes show higher similarity between glucose and xylose catabolism-induced responses in the early exponential growth phase.

BACKGROUND: Lactobacillus oligofermentans has been mostly isolated from cold-stored packaged meat products in connection with their spoilage, but its precise role in meat spoilage is unknown. It belongs to the L. vaccinostercus group of obligate heterofermentative lactobacilli that generally ferment pentoses (e.g. xylose and ribose) more efficiently than hexoses (e.g. glucose). However, more efficient hexose utilization can be induced. The regulation mechanisms of the carbohydrate catabolism in such bacteria have been scarcely studied. To address this question, we provided the complete genome sequence of L. oligofermentans LMG 22743(T) and generated time course transcriptomes during its growth on glucose, ribose and xylose. RESULTS: The genome was manually annotated and its main functional features were examined. L. oligofermentans was confirmed to be able to efficiently utilize several hexoses and maltose, which is, presumably, induced by its repeated cultivation with glucose in vitro. Unexpectedly, in the beginning of the exponential growth phase, glucose- and xylose-induced transcriptome responses were more similar, whereas toward the end of the growth phase xylose and ribose transcriptomes became more alike. The promoter regions of genes simultaneously upregulated both on glucose and xylose in comparison with ribose (particularly, hexose and xylose utilization genes) were found to be enriched in the CcpA- binding site. Transcriptionally, no glucose-induced carbon catabolite repression was detected. The catabolism of glucose, which requires initial oxidation, led to significant overexpression of the NAD(P)H re-oxidation genes, the upstream regions of which were found to contain a motif, which was highly similar to a Rex repressor binding site. CONCLUSIONS: This paper presents the second complete genome and the first study of carbohydrate catabolism-dependent transcriptome response for a member of the L. vaccinostercus group. The transcriptomic changes detected in L. oligofermentans for growth with different carbohydrates differ significantly from those of facultative heterofermentative lactobacilli. The mechanism of CcpA regulation, putatively contributing to the observed similarities between glucose- and xylose-induced transcriptome responses and the absence of stringent carbon catabolite control, requires further studies. Finally, the cell redox balance maintenance, in terms of the NAD(P)+/NAD(P)H ratio, was predicted to be regulated by the Rex transcriptional regulator, supporting the previously made inference of Rex-regulons for members of the Lactobacillaceae family.

Complete genome sequence of Leuconostoc gelidum subsp. gasicomitatum KG16-1, isolated from vacuum-packaged vegetable sausages.

Leuconostoc gelidum subsp. gasicomitatum is a predominant lactic acid bacterium (LAB) in spoilage microbial communities of different kinds of modified-atmosphere packaged (MAP) food products. So far, only one genome sequence of a poultry-originating type strain of this bacterium (LMG 18811(T)) has been available. In the current study, we present the completely sequenced and functionally annotated genome of strain KG16-1 isolated from a vegetable-based product. In addition, six other vegetable-associated strains were sequenced to study possible "niche" specificity suggested by recent multilocus sequence typing. The genome of strain KG16-1 consisted of one circular chromosome and three plasmids, which together contained 2,035 CDSs. The chromosome carried at least three prophage regions and one of the plasmids encoded a galactan degradation cluster, which might provide a survival advantage in plant-related environments. The genome comparison with LMG 18811(T) and six other vegetable strains suggests no major differences between the meat- and vegetable-associated strains that would explain their "niche" specificity. Finally, the comparison with the genomes of other leuconostocs highlights the distribution of functionally interesting genes across the L. gelidum strains and the genus Leuconostoc.

Development of spoilage bacterial community and volatile compounds in chilled beef under vacuum or high oxygen atmospheres.

Research into microbial community development and metabolism is essential to understand meat spoilage. Recent years have seen the emergence of powerful molecular techniques that are being used alongside conventional microbiology approaches. This enables more accurate studies on meat spoilage. The aim of this study was to investigate the influence of packaging (under vacuum and in high oxygen atmosphere) on the development of microbial communities and metabolic activities at 6 °C by using culture-dependent (cultivation, ribotyping) and culture-independent (amplicon sequencing) methods. At the beginning of shelf life, the microbial community mostly consisted of Carnobacterium and Lactobacillus. After two weeks of storage, Lactococcus and Lactobacillus were the dominant genera under vacuum and Leuconostoc in high oxygen meat packages. This indicates that oxygen favoured the genus Leuconostoc comprising only heterofermentative species and hence potential producers of undesirable compounds. Also the number of volatile compounds, such as diacetyl, 1-octen-3-ol and hexanoic acids, was higher in high oxygen packages than under vacuum packages. The beef in high oxygen atmosphere packaging was detected as spoiled in sensory evaluation over 10 days earlier than beef under vacuum packaging. Leuconostoc gelidum, Lactococcus piscium, Lactobacillus sakei and Lactobacillus algidus were the most common species of bacteria. The results obtained from identification of the isolates using ribotyping and amplicon sequencing correlated, except for L. algidus, which was detected in both types of packaging by amplicon sequencing, but only in vacuum packaged samples using the culture-based technique. This indicates that L. algidus grew, but was not cultivable in high oxygen beef using the Nordic Committee on Food Analysis standard method.

Volatile organic compounds and Photobacterium phosphoreum associated with spoilage of modified-atmosphere-packaged raw pork.

Accumulation of volatile organic compounds was monitored in association with sensory quality, bacterial concentrations and culture-independent microbial community analyses in raw pork loin and pork collar during storage under high-oxygen modified atmosphere at +4 °C. Of the 48 volatile compounds detected in the pork samples, the levels of acetoin, diacetyl and 3-methyl-1-butanol had the highest correlations with the sensory scores and bacterial concentrations. These compounds accumulated in all of the four monitored lots of non-sterile pork but not in the sterilized pork during chilled storage. According to the culture-dependent and culture-independent characterization of bacterial communities, Brochothrix thermosphacta, lactic acid bacteria (Carnobacterium, Lactobacillus, Lactococcus, Leuconostoc, Weissella) and Photobacterium spp. predominated in pork samples. Photobacterium spp., typically not associated with spoilage of meat, were detected also in 8 of the 11 retail packages of pork investigated subsequently. Eleven isolates from the pork samples were shown to belong to Photobacterium phosphoreum by phenotypic tests and sequencing of the 16S rRNA and gyrB gene fragments. Off-odors in pork samples with high proportion of Photobacterium spp. were associated with accumulation of acetoin, diacetyl and 3-methyl-1-butanol in meat, but these compounds did not explain all the off-odors reported in sensory analyses.

Lactic acid bacteria in dried vegetables and spices.

Spices and dried vegetable seasonings are potential sources of bacterial contamination for foods. However, little is known about lactic acid bacteria (LAB) in spices and dried vegetables, even though certain LAB may cause food spoilage. In this study, we enumerated LAB in 104 spices and dried vegetables products aimed for the food manufacturing industry. The products were obtained from a spice wholesaler operating in Finland, and were sampled during a one-year period. We picked isolates (n = 343) for species identification based on numerical analysis of their ribotyping patterns and comparing them with the corresponding patterns of LAB type strains. We found LAB at levels >2 log CFU/g in 68 (65%) of the samples, with the highest counts detected from dried onion products and garlic powder with counts ranging from 4.24 to 6.64 log CFU/g. The LAB identified were predominantly Weissella spp. (61%) and Pediococcus spp. (15%) with Weissella confusa, Weissella cibaria, Weissella paramesenteroides, Pediococcus acidilactici and Pediococcus pentosaceus being the species identified. Other species identified belonged to the genera of Enterococcus spp. (8%), Leuconostoc spp. (6%) and Lactobacillus spp. (2%). Among the LAB identified, Leuconostoc citreum, Leuconostoc mesenteroides and W. confusa have been associated with food spoilage. Our findings suggest that spices and dried vegetables are potential sources of LAB contamination in the food industry.

Oral tylosin administration is associated with an increase of faecal enterococci and lactic acid bacteria in dogs with tylosin-responsive diarrhoea.

The term tylosin-responsive diarrhoea (TRD) is used for canine recurrent diarrhoea cases for which no underlying cause can be found after extensive diagnostic investigations, but which show a response to the antibiotic tylosin in a few days. The objective of this prospective, one-arm longitudinal trial was to assess the effects of oral tylosin administration on the faecal levels of potentially probiotic bacteria, such as Enterococcus spp. and lactic acid bacteria (LAB), in dogs with TRD. This trial included 14 client-owned suspected TRD dogs that were on tylosin treatment and had firm faeces. Treatment was then terminated and dogs were followed up for up to 2 months to determine the recurrence of diarrhoea. Once diarrhoea started, dogs received tylosin (orally, 25 mg/kg, once daily for 7 days). At the end of the treatment period, stools were firm again in 11 dogs (TRD dogs); three dogs continued having diarrhoea and were excluded from the study. Faecal samples were collected at all three time-points for culture of LAB and enterococci. In TRD dogs, the colony counts of Enterococcus spp. (P = 0.003), LAB (P = 0.037), tylosin-resistant Enterococcus spp. (P <0.001) and LAB (P <0.001) were significantly higher when the dogs were on tylosin treatment and had normal faecal consistency compared to when they had diarrhoea following discontinuation of tylosin. In conclusion, cessation of diarrhoea in TRD dogs with tylosin treatment could be mediated by selection of a specific lactic acid population, the Enterococcus spp., due to their potential probiotic properties.

Lactic acid bacteria and their controversial role in fresh meat spoilage.

Lactic acid bacteria (LAB) constitute a heterogeneous group that has been widely associated with fresh meat and cooked meat products. They represent a controversial cohort of microbial species that either contribute to spoilage through generation of offensive metabolites and the subsequent organoleptic downgrading of meat or serve as bioprotective agents with strains of certain species causing unperceivable or no alterations. Therefore, significant distinction among biotypes is substantiated by studies determining spoilage potential as a strain-specific trait corroborating the need to revisit the concept of spoilage.