Transcriptomic analysis of the response of Photobacterium phosphoreum and Photobacterium carnosum to co-contaminants on chicken meat.

This study investigated the impact of Brochothrix (B.) thermosphacta and Pseudomonas (Ps.) fragi on the transcriptomes of Photobacterium (P.) phosphoreum and P. carnosum on chicken meat under modified atmosphere (MA) and air atmosphere (AA). P. phosphoreum TMW2.2103 responded to MA with a reduced transcript number related to cell division and an enhanced number related to oxidative stress. Concomitantly, the analysis revealed upregulation of fermentation and downregulation of respiration. It predicts enhanced substrate competition in presence of co-contaminants/MA. In contrast, the strain upregulated the respiration in AA, supposably due to improved substrate accessibility in this situation. For P. carnosum TMW2.2149 the respiration was downregulated, and the pyruvate metabolism upregulated under MA. MA/co-contaminant resulted in multiple upregulated metabolic routes. Conversely, AA/co-contaminant resulted only in minor regulations, showing inability to cope with fast growing competitors. Observations reveal different strategies of photobacteria to react to co-contaminants on meat.

Bombella favorum sp. nov. and Bombella mellum sp. nov., two novel species isolated from the honeycombs of Apis mellifera.

As part of a study investigating the microbiome of bee hives and honey, two novel strains (TMW 2.1880T and TMW 2.1889T) of acetic acid bacteria were isolated and subsequently taxonomically characterized by a polyphasic approach, which revealed that they cannot be assigned to known species. The isolates are Gram-stain-negative, aerobic, pellicle-forming, catalase-positive and oxidase-negative. Cells of TMW 2.1880T are non-motile, thin/short rods, and cells of TMW 2.1889T are motile and occur as rods and long filaments. Morphological, physiological and phylogenetic analyses revealed a distinct lineage within the genus Bombella. Strain TMW 2.1880T is most closely related to the type strain of Bombella intestini with a 16S rRNA gene sequence similarity of 99.5 %, and ANIb and in silico DDH values of 94.16 and 56.3 %, respectively. The genome of TMW 2.1880T has a size of 1.98 Mb and a G+C content of 55.3 mol%. Strain TMW 2.1889T is most closely related to the type strain of Bombella apis with a 16S rRNA gene sequence similarity of 99.5 %, and ANIb and in silico DDH values of 85.12 and 29.5 %, respectively. The genome of TMW 2.1889T has a size of 2.07 Mb and a G+C content of 60.4 mol%. Ubiquinone analysis revealed that both strains contained Q-10 as the main respiratory quinone. Major fatty acids for both strains were C16 : 0, C19 : 0 cyclo ω8c and summed feature 8, respectively, and additionally C14 : 0 2-OH only for TMW 2.1880T and C14 : 0 only for TMW 2.1889T. Based on polyphasic evidence, the two isolates from honeycombs of Apis mellifera represent two novel species of the genus Bombella, for which the names Bombella favorum sp. nov and Bombella mellum sp. nov. are proposed. The designated respective type strains are TMW 2.1880T (=LMG 31882T=CECT 30114T) and TMW 2.1889T (=LMG 31883T=CECT 30113T).

Hydrostatic pressure- and halotolerance of Photobacterium phosphoreum and P. carnosum isolated from spoiled meat and salmon.

Photobacterium spp. occur frequently in marine environments but have been recently also found as common spoilers on chilled meats. The environmental conditions in these ecological niches differ especially regarding salinity and ambient pressure. Linking the occurrence of photobacteria in different niches may elucidate its ecology and bring insights for the food industry. We investigated tolerance of Photobacterium (P.) phosphoreum and P. carnosum strains to high hydrostatic pressure and salinity and aligned our observations with presence of relevant genes. The strains were isolated from packaged meats and salmon (or the sea) to identify adaptations to marine and terrestrial habitats. Growth of all P. carnosum strains was reduced by 40 MPa hydrostatic pressure and >3% sodium chloride, suggesting loss of traits associated with marine habitats. In contrast, P. phosphoreum strains were only slightly affected, suggesting general adaptation to marine habitats. In accordance, these strains had gene clusters associated with marine niches, e.g. flagellar and lux-operons, being incomplete in P. carnosum. Occurrence of P. carnosum strains on packaged salmon and P. phosphoreum strains on meats therefore likely results from cross-contamination in meat and fish processing. Still, these strains showed intermediate traits regarding pressure- and halotolerance, suggesting developing adaptation to their respective environment.

Metatranscriptomic analysis of modified atmosphere packaged poultry meat enables prediction of Brochothrix thermosphacta and Carnobacterium divergens in situ metabolism.

In this study, in situ-expressed metabolic routes of Brochothrix (B.) thermosphacta and Carnobacterium (C.) divergens were evaluated based on a metatranscriptomic dataset from bacteria growing on MAP chicken meat (O2/CO2; N2/CO2). Both species exhibited no (C. divergens) or minor transcription regulation (B. thermosphacta) within their main metabolic routes in response to different atmospheres. Both employ pathways related to glucose and ribose. Gluconeogenesis from lipid-borne glycerol is active in the progressing lack of carbohydrates. Pyruvate fates in both species comprise lactate, ethanol, acetate, CO2, formate, C4-compounds and H2O2 (only B. thermosphacta). Both species express genes for a minimal aerobic respiratory chain, but do not possess the genetic setting for a functional citric acid cycle. While products of carbohydrate and glycerol metabolism display mild to medium sensorial off-characteristics, predicted end products of their amino acid metabolism comprise, e.g., isobutyrate and isovalerate (B. thermosphacta) or cadaverine and tyramine (C. divergens) as potent spoilage compounds.

Lactococcus carnosus sp. nov. and Lactococcus paracarnosus sp. nov., two novel species isolated from modified-atmosphere packaged beef steaks.

As part of a study investigating the spoilage microbiome of modified-atmosphere packaged beef from Germany, four novel strains of lactic acid bacteria were isolated and subsequently taxonomically characterized by a polyphasic approach, which revealed that they could not be assigned to known species. The isolates were Gram-staining-positive, coccoid, facultatively anaerobic, non-motile, catalase-negative and oxidase-negative. Morphological, physiological and phylogenetic analysis revealed a distinct lineage within the genus Lactococcus, with Lactococcus piscium and Lactococcus plantarum as closest relatives. Results indicated that they represented two different novel species with two strains, (TMW 2.1612T/TMW 2.1613 and TMW 2.1615T/TMW 2.1614), respectively. The two strains of both novel species shared identical 16S rRNA gene sequences but a MLSA allowed their intraspecies differentiation. The 16S rRNA gene sequences of TMW 2.1612T and TMW 2.1615T had a similarity of 99.85 % to each other and a similarity of 99.85 and 99.78 % the most closely related type strain of Lactococcus piscium, respectively. However, the ANIb value between the respective type strains TMW 2.1612T and TMW 2.1615T, and the type strain of Lactococcus piscium was only 94.3 and 92.0 %, respectively, and 92.9 % between TMW 2.1612T and TMW 2.1615T. The in silico DDH estimate value between the respective type strain TMW 2.1612T and TMW 2.1615T and the most closely related type strain of Lactococcus piscium was only 59.9 and 48.9 %, respectively, and 51.1 % between TMW 2.1612T and TMW 2.1615T. Peptidoglycan type of strain TMW 2.1612T is Lys-Thr-Ala and major fatty acids are summed feature 8 and C16 : 0. Peptidoglycan type of strain TMW 2.1615T is Lys-Ala and major fatty acids are C16 : 0, C19 : 0cyclo ω8c and summed feature 8. On the basis of polyphasic evidence, the meat isolates represent two novel species of the genus Lactococcus, for which the names Lactococcus carnosus sp. nov and Lactococcus paracarnosus sp. nov are proposed. The designated respective type strains are TMW 2.1612T (=DSM 111016T =CECT 30115T) and TWM 2.1615T (=DSM 111017T =CECT 30116T).

Comparative Genomics of Acetic Acid Bacteria within the Genus Bombella in Light of Beehive Habitat Adaptation.

It is known that the bacterial microbiota in beehives is essential for keeping bees healthy. Acetic acid bacteria of the genus Bombella colonize several niches in beehives and are associated with larvae protection against microbial pathogens. We have analyzed the genomes of 22 Bombella strains of different species isolated in eight different countries for taxonomic affiliation, central metabolism, prophages, bacteriocins and tetracycline resistance to further elucidate the symbiotic lifestyle and to identify typical traits of acetic acid bacteria. The genomes can be assigned to four different species. Three genomes show ANIb values and DDH values below species demarcation values to any validly described species, which identifies them as two potentially new species. All Bombella spp. lack genes in the Embden-Meyerhof-Parnas pathway and the tricarboxylic acid cycle, indicating a focus of intracellular carbohydrate metabolism on the pentose phosphate pathway or the Entner-Doudoroff pathway for which all genes were identified within the genomes. Five membrane-bound dehydrogenases were identified that catalyze oxidative fermentation reactions in the periplasm, yielding oxidative energy. Several complete prophages, but no bacteriocins, were identified. Resistance to tetracycline, used to prevent bacterial infections in beehives, was only found in Bombella apis MRM1T. Bombella strains exhibit increased osmotolerance in high glucose concentrations compared to Gluconobacter oxydans, indicating adaption to high sugar environments such as beehives.

Chromohalobacter moromii sp. nov., a moderately halophilic bacterium isolated from lupine-based moromi fermentation.

Three moderately halophilic strains, TMW 2.2308T, TMW 2.2299 and TMW 2.2304, were isolated from a lupine-based moromi fermentation. Initial identification based on their low molecular sub-proteome using mass spectrometry showed relation to the genus Halomonas, however, low score values indicated novelty. The comparison of 16S rRNA gene sequences placed these strains within the genus Chromohalobacter with C. japonicus CECT 7219T (99.67% 16S rRNA sequence similarity to strain TMW2.2308T), C. canadensis DSM 6769T (99.54%) and C. beijerinckii LMG 2148T (99.32%) being their closest relatives. However, average nucleotide highest identity values of TMW 2.2308T to C. beijerinckii LMG 2148T of 93.12% and 92.88% to C. japonicus CECT 7219T demonstrate that it represents a novel species within the genus Chromohalobacter with additional strains TMW 2.2299 (96.91%) and TMW 2.2304 (96.98%). The isolated strains were non-spore-forming, motile and able to grow at temperatures from 5 to 45 °C with an optimum at 37 °C. Growth of TMW 2.2308T occurs at 5 to 25% (w/v) NaCl with optimum growth between 10and 12.5%. The genome of TMW 2.2308T has a size of 3.47 Mb and a G + C content of 61.0 mol%. The polyphasic evidence lead to the classification of TMW 2.2308T, TMW 2.2299 and TMW 2.2304 as members of a novel species of the genus Chromohalobacter. We propose a novel species as Chromohalobacter moromii sp. nov., with TMW 2.2308T (=DSM113153T =CECT30422T) as the type strain.

Impact of Modified Atmospheres on Growth and Metabolism of Meat-Spoilage Relevant Photobacterium spp. as Predicted by Comparative Proteomics.

Modified atmosphere packaging (MAP) is a common strategy to selectively prevent the growth of certain species of meat spoiling bacteria. This study aimed to determine the impact of high oxygen MAP (70% O2, 30% CO2, red and white meats) and oxygen-free MAP (70% N2, 30% CO2, also white meat and seafood) on preventing the growth of spoiling photobacteria on meat. Growth of Photobacterium carnosum and P. phosphoreum was monitored in a meat simulation media under different gas mixtures of nitrogen, oxygen, and carbon dioxide, and samples were taken during exponential growth for a comparative proteomic analysis. Growth under air atmosphere appears optimal, particularly for P. carnosum. Enhanced protein accumulation affected energy metabolism, respiration, oxygen consuming reactions, and lipid usage. However, all the other atmospheres show some degree of growth reduction. An increase in oxygen concentration leads to an increase in enzymes counteracting oxidative stress for both species and enhancement of heme utilization and iron-sulfur cluster assembly proteins for P. phosphoreum. Absence of oxygen appears to switch the metabolism toward fermentative pathways where either ribose (P. phosphoreum) or glycogen (P. carnosum) appear to be the preferred substrates. Additionally, it promotes the use of alternative electron donors/acceptors, mainly formate and nitrate/nitrite. Stress response is manifested as an enhanced accumulation of enzymes that is able to produce ammonia (e.g., carbonic anhydrase, hydroxylamine reductase) and regulate osmotic stress. Our results suggest that photobacteria do not sense the environmental levels of carbon dioxide, but rather adapt to their own anaerobic metabolism. The regulation in presence of carbon dioxide is limited and strain-specific under anaerobic conditions. However, when oxygen at air-like concentration (21%) is present together with carbon dioxide (30%), the oxidative stress appears enhanced compared to air conditions (very low carbon dioxide), as explained if both gases have a synergistic effect. This is further supported by the increase in oxygen concentration in the presence of carbon dioxide. The atmosphere is able to fully inhibit P. carnosum, heavily reduce P. phosphoreum growth in vitro, and trigger diversification of energy production with higher energetic cost, highlighting the importance of concomitant bacteria for their growth on raw meat under said atmosphere.

Genome Sequence of the Diploid Yeast Debaryomyces hansenii TMW 3.1188.

Debaryomyces hansenii TMW 3.1188 is a halotolerant diploid yeast that was isolated from lupine moromi fermentation. Here, we report on the 24.77-Mbp genome of a diploid strain of the species D. hansenii.

Competing formate- and carbon dioxide-utilizing prokaryotes in an anoxic methane-emitting fen soil.

Methanogenesis in wetlands is dependent on intermediary substrates derived from the degradation of biopolymers. Formate is one such substrate and is stimulatory to methanogenesis and acetogenesis in anoxic microcosms of soil from the fen Schlöppnerbrunnen. Formate dissimilation also yields CO(2) as a potential secondary substrate. The objective of this study was to resolve potential differences between anaerobic formate- and CO(2)-utilizing prokaryotes of this fen by stable isotope probing. Anoxic soil microcosms were pulsed daily with low concentrations of [(13)C]formate or (13)CO(2) (i.e., [(13)C]bicarbonate). Taxa were evaluated by assessment of 16S rRNA genes, mcrA (encoding the alpha-subunit of methyl-coenzyme M reductase), and fhs (encoding formyltetrahydrofolate synthetase). Methanogens, acetogens, and formate-hydrogen lyase-containing taxa appeared to compete for formate. Genes affiliated with Methanocellaceae, Methanobacteriaceae, Acetobacteraceae, and Rhodospirillaceae were (13)C enriched (i.e., labeled) in [(13)C]formate treatments, whereas genes affiliated with Methanosarcinaceae, Conexibacteraceae, and Solirubrobacteraceae were labeled in (13)CO(2) treatments. [(13)C]acetate was enriched in [(13)C]formate treatments, but labeling of known acetogenic taxa was not detected. However, several phylotypes were affiliated with acetogen-containing taxa (e.g., Sporomusa). Methanosaetaceae-affiliated methanogens appeared to participate in the consumption of acetate. Twelve and 58 family-level archaeal and bacterial 16S rRNA phylotypes, respectively, were detected, approximately half of which had no isolated representatives. Crenarchaeota constituted half of the detected archaeal 16S rRNA phylotypes. The results highlight the unresolved microbial diversity of the fen Schlöppnerbrunnen, suggest that differing taxa competed for the same substrate, and indicate that Methanocellaceae, Methanobacteriaceae, Methanosarcinaceae, and Methanosaetaceae were linked to the production of methane, but they do not clearly resolve the taxa responsible for the apparent conversion of formate to acetate.

Influence of the packaging atmosphere and presence of co-contaminants on the growth of photobacteria on chicken meat.

Fresh meat is commonly packaged in modified atmosphere to decelerate spoilage processes. The applied gas mixture affects the growth of spoilage organisms and selectively shapes the spoilage community. In this study, we investigated the impact of O2 and CO2 on the growth of Photobacterium (P.) phosphoreum and P. carnosum strains in situ on chicken meat by packaging under different modified atmospheres (air, 70% O2/30% CO2, 70% N2/30% CO2, 100% N2). Combination of 70% O2 and 30% CO2 resulted in significant growth reduction of the analyzed strains, suggesting inhibitory effects of both gases in combination. In contrast, 30% CO2 alone had only a minor effect and photobacteria are supposed to have a growth advantage over other meat spoilers in this atmosphere. Additionally, single growth of the strains in the different atmospheres was compared when challenged with the presence of Pseudomonas (Ps.) fragi or Brochothrix (B.) thermosphacta as prominent co-contaminants in different ratios (10:1, 1:1, 1:10). Presence of co-contaminants resulted in increased cell numbers of P. carnosum TMW2.2149 but reduced or unchanged cell numbers of P. phosphoreum TMW2.2103 in most packaging atmospheres. The initial ratio of photobacteria and co-contaminants defined the relative abundance during storage but did not change the type of the interaction. Our results suggest either a commensalistic (P. carnosum) or competitive interaction (P. phosphoreum) of photobacteria and co-contaminants on modified atmosphere packaged chicken, respectively. Furthermore, in a mix comprising seven prominent spoilers, strains of both Photobacterium species prevailed as a constant part of the spoilage microbiome during 7 days of refrigerated storage on chicken meat packaged under O2/CO2 atmosphere.

Comparative genomics of Photobacterium species from terrestrial and marine habitats.

Photobacterium (P.) is a genus widely studied in regards to its association with and ubiquitous presence in marine environments. However, certain species (P. phosphoreum, P. carnosum, P. iliopiscarium) have been recently described to colonize and spoil raw meats without a marine link. We have studied 27 strains from meat as well as 26 strains from marine environments in order to probe for intraspecies marine/terrestrial subpopulations and identify distinct genomic features acquired by environmental adaptation. We have conducted phylogenetic analysis (MLSA, ANI, fur, codon usage), search of plasmids (plasmidSPADES), phages (PHASTER), CRISPR-cas operons (CRISPR-finder) and secondary metabolites gene clusters (antiSMASH, BAGEL), in addition to a targeted gene search for specific pathways (e.g. TCA cycle, pentose phosphate, respiratory chain) and elements relevant for growth, adaptation and competition (substrate utilization, motility, bioluminescence, sodium and iron transport). P. carnosum appears as a conserved single clade, with one isolate from MAP fish clustering apart that doesn't, however, show distinct features that could indicate different adaptation. The species harbors genes for a wide carbon source utilization (glycogen/starch, maltose, pullulan, fucose) for colonization of diverse niches in its genome. P. phosphoreum is represented by two different clades on the phylogenetic analyses not correlating to their origin or distribution of other features analyzed that can be divided into two novel subspecies based on genome-wide values. A more diverse antimicrobial activity (sactipeptides, microcins), production of secondary metabolites (siderophores and arylpolyenes), stress response and adaptation (bioluminescence, sodium transporters, catalase, high affinity for oxygen cytochrome cbb3 oxidase, DMSO reductase and proton translocating NADH dehydrogenase) is predicted compared to the other species. P. iliopiscarium was divided into two clades based on source of isolation correlating with phylogeny and distribution of several traits. The species shows traits common to the other two species, similar carbon utilization/transport gene conservation as P. carnosum for the meat-isolated strains, and predicted utilization of marine-common DMSO and flagellar cluster for the sea-isolated strains. Results additionally suggest that photobacteria are highly prone to horizontal acquisition/loss of genetic material and genetic transduction, and that it might be a strategy for increasing the frequency of strain- or species-specific features that offers a growth/competition advantage.

Comparative Proteomics Reveals the Anaerobic Lifestyle of Meat-Spoiling Pseudomonas Species.

The ability of certain Pseudomonas (P.) species to grow or persist in anoxic habitats by either denitrification, acetate fermentation, or arginine fermentation has been described in several studies as a special property. Previously, we had isolated strains belonging to the species P. lundensis, P. weihenstephanensis, and P. fragi from anoxic modified atmosphere packaged (MAP) minced beef and further proved their anaerobic growth in vitro on agar plates. This follow-up study investigated the anaerobic growth of two strains per respective species in situ on inoculated chicken breast filet under 100% N2 modified atmosphere. We were able to prove anaerobic growth of all six strains on chicken breast filet with cell division rates of 0.2-0.8/day. Furthermore, we characterized the anaerobic metabolic lifestyle of these Pseudomonas strains by comparative proteomics, upon their cultivation in meat simulation media, which were constantly gassed with either air or 100% N2 atmospheres. From these proteomic predictions, and respective complementation by physiological experiments, we conclude that the Pseudomonas strains P. fragi, P. weihenstephanensis, P. lundensis exhibit a similar anaerobic lifestyle and employ arginine fermentation via the arginine deiminase (ADI) pathway to grow anaerobically also on MAP meats. Furthermore, glucose fermentation to ethanol via the ED-pathway is predicted to enable long term survival but no true growth, while respiratory growth with nitrate as alternative electron acceptor or glucose fermentation to acetate could be excluded due to absence of essential genes. The citric acid cycle is partially bypassed by the glyoxylate shunt, functioning as the gluconeogenetic route without production of NADH2 under carbon limiting conditions as e.g., in packaged meats. Triggered by an altered redox balance, we also detected upregulation of enzymes involved in protein folding as well as disulfide bonds isomerization under anoxic conditions as a counteracting mechanism to reduce protein misfolding. Hence, this study reveals the mechanisms enabling anaerobic grow and persistence of common meat-spoiling Pseudomonas species, and further complements the hitherto limited knowledge of the anaerobic lifestyle of Pseudomonas species in general.

Effect of earthworm feeding guilds on ingested dissimilatory nitrate reducers and denitrifiers in the alimentary canal of the earthworm.

The earthworm gut is an anoxic nitrous oxide (N(2)O)-emitting microzone in aerated soils. In situ conditions of the gut might stimulate ingested nitrate-reducing soil bacteria linked to this emission. The objective of this study was to determine if dissimilatory nitrate reducers and denitrifiers in the alimentary canal were affected by feeding guilds (epigeic [Lumbricus rubellus], anecic [Lumbricus terrestris], and endogeic [Aporrectodea caliginosa]). Genes and gene transcripts of narG (encodes a subunit of nitrate reductase and targets both dissimilatory nitrate reducers and denitrifiers) and nosZ (encodes a subunit of N(2)O reductase and targets denitrifiers) were detected in guts and soils. Gut-derived sequences were similar to those of cultured and uncultured soil bacteria and to soil-derived sequences obtained in this study. Gut-derived narG sequences and narG terminal restriction fragments (TRFs) were affiliated mainly with Gram-positive organisms (Actinobacteria). The majority of gut- and uppermost-soil-derived narG transcripts were affiliated with Mycobacterium (Actinobacteria). In contrast, narG sequences indicative of Gram-negative organisms (Proteobacteria) were dominant in mineral soil. Most nosZ sequences and nosZ TRFs were affiliated with Bradyrhizobium (Alphaproteobacteria) and uncultured soil bacteria. TRF profiles indicated that nosZ transcripts were more affected by earthworm feeding guilds than were nosZ genes, whereas narG transcripts were less affected by earthworm feeding guilds than were narG genes. narG and nosZ transcripts were different and less diverse in the earthworm gut than in mineral soil. The collective results indicate that dissimilatory nitrate reducers and denitrifiers in the earthworm gut are soil derived and that ingested narG- and nosZ-containing taxa were not uniformly stimulated in the guts of worms from different feeding guilds.

Development of a rapid detection method for Photobacterium spp. using Loop-mediated isothermal amplification (LAMP).

While the abundance of photobacteria has previously been exclusively associated with marine environments and spoilage of seafood, several recent studies have demonstrated their status as pervasive constituents of the microbiota on packaged meats. Since their ubiquitous nature has been revealed, detection of their presence on meat, their entry route into meat processing environments and prevention of their growth is a novel emerging challenge for the food industry. In this study, we have developed a highly sensitive and specific loop-mediated isothermal amplification (LAMP) assay for the detection of relevant species of photobacteria on foods, and tested its efficacy on meats. The gene encoding trimethylamine-N-oxide reductase (torA) was chosen as the target for this assay. Designed primers based on the gene sequence proved their specificity by testing 67 isolates of 5 species of photobacteria (positive) as well as 63 strains of 16 species of other common meat spoilers (negative). The optimized assay takes 2 h including sample preparation and has a detection limit of only 10-11 copies (50 fg/reaction) of the average Photobacterium (P.) genome per reaction. Its applicability could be successfully demonstrated on naturally and artificially contaminated chicken, beef and pork samples and evaluated by comparison with a culture-dependent approach using selective media and MALDI-TOF MS for identification. The developed LAMP assay revealed presence of photobacteria on one naturally contaminated chicken sample stored at 4 °C long before (3 days) confirmation by the culture-dependent approach. This study demonstrates that the developed LAMP assay represents a reliable and sensitive method for rapid detection of photobacteria on meats. However, its specificity would allow the applicability of the methodology to be extended to other foods, e.g. fish and seafood where presence of photobacteria is directly linked to their shelf life. The method has no requirement for specialized equipment or specially trained personal allowing an easy implementation within the quality control of the food industry. Considering the lot-to-lot variations observed on meats regarding the presence of photobacteria and the impracticality of implementing quantitative methods within the routine control, the LAMP method can simplify and reduce the workload for detection of photobacteria on high sample numbers. Consequently, producers can identify batches/plants that need more stringent control, and are provided with a tool to determine the entry route of photobacteria into the processing and distribution chain of raw meats.

Comparative Proteomics of Meat Spoilage Bacteria Predicts Drivers for Their Coexistence on Modified Atmosphere Packaged Meat.

Besides intrinsic and extrinsic factors such as antagonism for organic substrates or temperature, the storage atmosphere of meat has a high influence on the development of its initial microbiota. Specific modified atmospheres (MAs) selectively suppress growth of aerobic and anaerobic bacteria, thus reshaping the initial microbiota. As some microorganisms are more tolerant to MA, they overgrow competitors and produce metabolites that cause rejection of the product. In order to elucidate responses to different MA by means of metabolic adaptation and competition for organic substrates on meat, the typical representative meat spoilage bacteria Brochothrix (B.) thermosphacta TMW2.2101 and four lactic acid bacteria Carnobacterium (C.) divergens TMW2.1577, C. maltaromaticum TMW2.1581, Leuconostoc (L.) gelidum subsp. gelidum TMW2.1618 and L. gelidum subsp. gasicomitatum TMW2.1619 were chosen. Bacteria were grown in sterile glass bottles filled with a meat simulation medium, which was aerated constantly with either air, 100%_N2, 30%_CO2/70%_O2 or 30%_CO2/70%_N2. Growth of bacteria during incubation at 25°C and stirring at 120 rpm was monitored over 48 h and a label-free quantitative mass spectrometric approach was employed to determine changes within the bacterial proteomes in response to oxygen and carbon dioxide. Both Leuconostoc subsp. were intrinsically tolerant to MA, exhibiting no proteomic regulation of enzymes, whereas the other species provide a set of metabolic adaptation mechanism, enabling higher resistance to the detrimental effects of MA. Those mechanisms comprise: enhanced oxidative stress reduction, adjustment of the pyruvate metabolism and catabolic oxygen consumption in response to oxygen and intracellular pH homeostasis, maintenance of osmotic balance and alteration of the fatty acid composition in response to carbon dioxide. We further evaluated the potential of industrial used MA to inhibit specific bacterial spoilage. No bacterial inhibition is predicted for 30%_CO2/70%_O2 for the analyzed species, whereas 30%_CO2/70%_N2 predictively inhibits C. divergens TMW21577 and B. thermosphacta TMW2.2101. Furthermore, species-specific metabolic pathways enabling different and preferential carbon source utilization were identified, which enable non-competitive coexistence of respective bacteria on meat, resulting in synergistic spoilage. In conclusion, this study gives mechanistically explanations of their acknowledged status as typical spoilage organisms on MAP meats.

Bap and Cell Surface Hydrophobicity Are Important Factors in Staphylococcus xylosus Biofilm Formation.

Staphylococcus (S.) xylosus is a coagulase-negative Staphylococcus species naturally present in food of animal origin with a previously described potential for biofilm formation. In this study we characterized biofilm formation of five selected strains isolated from raw fermented dry sausages, upon different growth conditions. Four strains exhibited a biofilm positive phenotype with strain-dependent intensities. Biofilm formation of S. xylosus was influenced by the addition of glucose, sodium chloride and lactate to the growth medium, respectively. It was further dependent on strain-specific cell surface properties. Three strains exhibited hydrophobic and two hydrophilic cell surface properties. The biofilm positive hydrophilic strain TMW 2.1523 adhered significantly better to hydrophilic than to hydrophobic supports, whereas the differences in adherence to hydrophobic versus hydrophilic supports were not as distinct for the hydrophobic strains TMW 2.1023, TMW 2.1323, and TMW 2.1521. Comparative genomics enabled prediction of functional biofilm-related genes and link these to phenotypic variations. While a wide range of biofilm associated factors/genes previously described for S. aureus and S. epidermidis were absent in the genomes of the five strains analyzed, they all possess the gene encoding biofilm associated protein Bap. The only biofilm negative strain TMW 2.1602 showed a mutation in the bap sequence. This study demonstrates that Bap and surface hydrophobicity are important factors in S. xylosus biofilm formation with potential impact on the assertiveness of a starter strain against autochthonous staphylococci by competitive exclusion during raw sausage fermentation.

Biodiversity of Photobacterium spp. Isolated From Meats.

Photobacteria are common psychrophilic bacteria found in marine environments. Recently, several studies revealed high numbers of Photobacterium (P.) spp. on packaged fresh meat. Their occurrence appears relevant for the spoilage of meat, since species of the genus are already known as potent fish spoilage organisms. Here we report on distribution, biodiversity, and specific traits of P. carnosum (n = 31), P. phosphoreum (n = 24), and P. iliopiscarium (n = 3) strains from different foods. Biodiversity was assessed by genomic fingerprinting, diversity index analysis, growth dynamics, comparison of metabolic activities, and antibiotic resistance. We observed a ubiquitous occurrence of the species on all common meats independent of packaging conditions and producer, suggesting contamination during an established processing or packaging step. Regarding biodiversity, the three species differed clearly in their growth properties and metabolic characteristics, with P. phosphoreum growing the fastest and showing the strongest alkalization of the media. On strain level we also recorded variations in enzymatic reactions, acid production, and antibiotic resistances not restricted to specific meat types. This depicts high biodiversity on species and strain level on each contaminated meat sample. Our analysis showed that meat-borne strains of P. phosphoreum and P. iliopiscarium clearly differ from their type strains from a marine habitat. Additionally, we report for the first time isolation of P. carnosum strains from packaged fish, which in contrast showed comparable phenotypic properties to meat-borne strains. This hints at different initial origins of P. phosphoreum/P. iliopiscarium (marine background) and P. carnosum (no demonstrated marine background) contaminations on fish and meat, respectively.

Quantitative Oxygen Consumption and Respiratory Activity of Meat Spoiling Bacteria Upon High Oxygen Modified Atmosphere.

High oxygen modified atmosphere packaging is a commonly applied method to prolong the minimum shelf life of fresh (red) meats. Upon spoilage, changes of the initial oxygen concentration and microbiome composition can be observed. Thus, we classified the typical representative meat spoiling bacteria Brochothrix (B.) thermosphacta TMW2.2101 and the four lactic acid bacteria (LAB) Carnobacterium (C.) divergens TMW2.1577, C. maltaromaticum TMW2.1581, Leuconostoc (L.) gelidum subsp. gelidum TMW2.1618, and L. gelidum subsp. gasicomitatum TMW2.1619 along their oxygen consuming capacity, which can indicate the timeline of microbiome and sensorial changes. All bacteria were grown in a model system employing gas tight glass bottles containing meat simulation media and under modified atmosphere (70% O2 and 30% CO2). Oxygen concentrations of media and headspaces were monitored over time and the oxygen uptake rate (OUR) was calculated for all species. All bacteria were able to consume dissolved oxygen, with B. thermosphacta TMW2.2101 exhibiting a 31-times higher OUR per single cell in 60 h. Furthermore, all strains showed significant growth enhancement in the presence of heme indicating respiratory activity. Comparative genomic and physiological analyses predict the activity of a respiratory chain for all species upon high oxygen atmosphere. An additional cytochrome aa3 oxidase is suggested to be responsible for the increased OUR of B. thermosphacta TMW2.2101. Furthermore, B. thermosphacta TMW2.2101 revealed highest oxidative stress tolerance compared to the other bacteria, facilitating a higher respiratory activity. Coupling of respiration and fermentation via regeneration of NADH can be a competitive advantage for meat spoiling bacteria resulting in a higher cell count and possibly accelerated spoilage. The exhibited highest capacity for oxygen consumption of B. thermosphacta compared to LAB in vitro also suggests a higher contribution of this bacterium to the change in the atmosphere upon spoilage of MAP meats in situ.

Prediction of in situ metabolism of photobacteria in modified atmosphere packaged poultry meat using metatranscriptomic data.

Modified atmosphere packaging (MAP) is widely used in food industry to extend the microbiological shelf life of meat. Common CO2-containing gas atmospheres for poultry meat packaging are either nearly O2-free or high O2 MAPs. In this work, we compared spoilage microbiota of skinless chicken breast in CO2/O2 (30/70%) and CO2/N2 (30/70%) MAP, which are culturable with conventional methods and identified isolates by MALDI-TOF MS. These data were compared to metatranscriptome sequencing enabling a culture-independent overview on the composition of microbiota at species level. While typical MAP meat spoilers were confirmed in the transcriptomic approach, we also found high numbers of transcripts mapping to Photobacterium spp. sequences in these samples. As photobacteria were recently shown to occur in different MAP and vacuum packaged meats, we used the respective part of the metatranscriptomic data for prediction of Photobacterium spp. major metabolic routes in situ, upon growth in MAP poultry meat. It is predicted that they employ similar metabolism in both atmospheres: In the lack of carbohydrates upon meat spoilage, the pyruvate pool is filled via glycerol originating from lipolysis and amino acid conversions. From the pyruvate pool, gluconeogenesis is fed enabling cell wall biosynthesis and growth as well as catabolism to lactate and other metabolites, or anaplerosis towards the citric acid cycle. Production is predicted of several biogenic amines including tyramine and cadaverine, enabling generation of proton motive force. Taken together, photobacteria express metabolic pathways upon growth on meat, which should lead to compounds overlapping with those of known potent meat spoilers.