Virulence variations between clonal complexes of Melisococcus plutonius and the possible causes.

Melissococcus plutonius is a pathogenic bacterium that affects honeybee brood triggering colony collapse in severe cases. The bacterium causes a European foulbrood (EFB) disease in the honeybee populations, impacting beekeeping and agricultural industries. The pathogenesis, epidemiology, and variants of M. plutonius have been studied, but the virulence factors involved in larval infection are still unknown. Recently, an in-silico study suggested putative genes that might play a role in the pathogenesis of EFB. However, studies are required to determine their function as virulence factors. In addition, the few studies of clonal complexes (CCs), virulence factors, and variation in the honeybee larvae mortality have interfered with the development of more efficient control methods. The research, development, and differences in virulence between genetic variants (CCs) of M. plutonius and potential virulence factors implicated in honeybee larval mortality are discussed in this review.

Metatranscriptome-based investigation of flavor-producing core microbiota in different fermentation stages of dajiang, a traditional fermented soybean paste of Northeast China.

Dajiang, or naturally fermented soybean paste, has a unique flavor that is influenced by the resident microflora. However, the association between flavor and the core microbiota is unclear. Recent advances in RNA sequencing have identified genes that are actively expressed in complex microbial communities. To this end, we analyzed the time-dependent changes in the microbiota and the metabolite profiles of Dajiang using metatranscriptome sequencing, HS-SPME-GC-MS and amino acid analysis identified 10 volatile compounds that contribute to the development of soybean paste flavor. Further analysis of the correlation between the active microorganisms and the physicochemical characteristics and flavor substances in soybean paste indicated that Lactobacillus and Tetragenococcus were the core genera affecting chromaticity and flavor. These microorganisms produce enzymes that catalyze a series of metabolic pathways that generate flavor substances. Our findings provide new insights into the role of the microbiota in the development of flavor in fermented foods.

Genomic Insight into the Salt Tolerance of Enterococcus faecium, Enterococcus faecalis and Tetragenococcus halophilus.

To shed light on the genetic basis of salt tolerance in Enterococcus faecium, Enterococcus faecalis, and Tetragenococcus halophilus, we performed comparative genome analysis of 10 E. faecalis, 11 E. faecium, and three T. halophilus strains. Factors involved in salt tolerance that could be used to distinguish the species were identified. Overall, T. halophilus contained a greater number of potassium transport and osmoprotectant synthesis genes compared with the other two species. In particular, our findings suggested that T. halophilus may be the only one among the three species capable of synthesizing glycine betaine from choline, cardiolipin from glycerol and proline from citrate. These molecules are well-known osmoprotectants; thus, we propose that these genes confer the salt-tolerance of T. halophilus.

Effect of co-culture with Tetragenococcus halophilus on the physiological characterization and transcription profiling of Zygosaccharomyces rouxii.

Zygosaccharomyces rouxii and Tetragenococcus halophilus are widely existed and play vital roles during the manufacture of fermented foods such as soy sauce. The aim of this study was to elucidate the effect of T. halophilus CGMCC 3792 on the physiological characterizations and transcription profiling of Z. rouxii CGMCC 3791. Salt tolerance analysis revealed that co-culture with T. halophilus enhanced the salt tolerance of Z. rouxii during salt stress. Analysis of the volatile compounds revealed that co-culture reduced the level of 1-butanol, improved the level of octanoic acid which all were produced by T. halophilus and reduced the level of phenylethyl alcohol produced by Z. rouxii. The presence of Z. rouxii decreased the contents of 3,4-dimethylbenzaldehyde and acetic acid produced by T. halophilus. In addition, co-culture improved the content of benzyl alcohol significantly. Analysis of membrane fatty acid showed that co-culture improved the content of palmitic (C16:0) and stearic (C18:0) acids in cells of Z. rouxii, and reduced the contents of myristic (C14:0), palmitoleic acid (C16:1) and oleic acid (C18:1). In order to further explore the interactions between the two strains, RNA-seq technology was used to investigate the effect of co-culture with T. halophilus on the transcription profiling of Z. rouxii. By comparing cells incubated in co-culture group with cells incubated in single-culture group, a total of 967 genes were considered as differentially expressed genes (DEGs). Among the DEGs, 72 genes were up-regulated, while 895 genes were down-regulated. These DEGs took party in various activities in cells of Z. rouxii, and the result showed co-culture with T. halophilus had a positive effect on proteolysis, the attachment of a cell to another cell, extracellular protein accumulation, energy metabolism, and a negative effect on oxidative phosphorylation, small molecular substances metabolism, DNA replication and repair, and transcription in cells of Z. rouxii. Results presented in this study may contribute to further understand the interactions between Zygosaccharomyces rouxii and Tetragenococcus halophilus.

Tetragenococcus halophilus MJ4 as a starter culture for repressing biogenic amine (cadaverine) formation during saeu-jeot (salted shrimp) fermentation.

Biogenic amines (BAs) are frequently present in traditionally fermented salted foods. In this study, a Tetragenococcus halophilus strain (MJ4) with no BA-producing ability was isolated from a fish (anchovy) sauce. Strain MJ4 did not produce BAs from supplied precursors and no BA-producing genes were identified in its genome. Bacterial community analysis showed that in non-inoculated saeu-jeot (shrimp sauce) fermentation, Tetragenococcus predominated after 82 days, while in strain MJ4-inoculated saeu-jeot, Tetragenococcus predominated during the entire fermentation. Strain MJ4 repressed the growth of T. muriaticus, a known BA producer, during fermentation, but metabolite analysis demonstrated that metabolite profiles, including amino acids, were similar regardless of MJ4 inoculation. The metabolite analysis also showed that strain MJ4 clearly repressed the formation of cadaverine during fermentation. This study suggests that the use of strain MJ4 as a starter culture in salted fish fermentation may be a good strategy for the reduction of BA formation.

Prevalence, typing and phylogenetic analysis of Melissococcus plutonius strains from bee colonies of the State of Chihuahua, Mexico.

European foulbrood (EFB) caused by Melissococcus plutonius is an important bee brood disease but, in Mexico, information about this bacterium is limited. We evaluated the prevalence of typical and atypical strains in beehives of seven apicultural regions of the state of Chihuahua, Mexico. We performed MLST and phylogenetic analysis to characterize the isolates. Prevalence was highest 59%, in the region of Chihuahua, and lowest, 14%, in the regions of Cuauhtémoc and Nuevo Casas Grandes. Typical and atypical strains were identified in hives from all regions; however, in the regions of Parral, Cuauhtémoc and Aldama, the atypical strains were only detected in combination with typical strains. We obtained 81 isolates of M. plutonius and identified seven sequence types, of which three were new types. Additionally, we observed a relation between sequence type and the region where the strain was isolated. Phylogenetic analysis and multilocus sequence typing using goeBURST analysis showed that 97.5% of the isolates correspond to the Clonal Complex (CC) 12 and 2.5% to the CC3. Our work is the first molecular characterization of M. plutonius in Mexico and contributes to global information about the epidemiology of this pathogen.

The molecular mechanism and post-transcriptional regulation characteristic of Tetragenococcus halophilus acclimation to osmotic stress revealed by quantitative proteomics.

Tetragenococcus halophilus is a moderate halophilic bacterium which was widely used in fermentation processes, growing in a broad range of salinity conditions, and can survive a saturated 26.47% w/w NaCl concentration. However, the mechanism of this outstanding ability to acclimate to extracellular osmotic stress still remains unknown. The current study firstly conducted a quantitative proteomic analysis to identify alterations of the cellular proteome under both hypo-osmotic and hyper-osmotic stress conditions. A total of 1405 proteins were identified and differentially accumulated proteins were analyzed, further functional annotations were performed using Gene Ontology and Kyoto Encyclopedia of Genes and Genomes. The results revealed that both hypo- and hyper-osmotic stresses have prominent impacts on the synthesis of proteins involving in multiple cellular functions. Further analyses of the differentially accumulated proteins suggested that the adaptation strategies T. halophilus applies to deal with hypo- and hyper-osmotic stress conditions may be distinct. Comparison of the differentially accumulated proteins in both transcriptomic and proteomic study indicated the existence of post-transcriptional modification during salinity adaptation of T. halophilus. The current study generated a proteomic atlas of differentially accumulated proteins under both hypo- and hyper-osmotic stress conditions, provided an overview of the molecular mechanism of osmotic acclimation of T. halophilus. SIGNIFICANCE: The current study aimed to reveal how the moderately halophilic Tetragenococcus halophilus adapt to extracellular salinity stress, which is the first proteomic study analyzing the differences in proteome of Tetragenococcus halophilus between hypo- and hyper-osmotic stress to our knowledge. By analyzing the differences in the accumulating levels of the proteome via isobaric labeling-based quantitative proteomic study, we identified proteins with significantly different accumulation levels which may play important roles in the adaptation process to extracellular salinity stress. Examining the cellular functions of these proteins according to Gene Ontology and Kyoto Encyclopedia of Genes and Genomes, a draft view of how the bacterium act to acclimate to osmotic stress has been drawn. Further analysis revealing the differences between the transcriptome and proteome suggested that some proteins may undergo post-transcriptional regulation during acclimation process, which still remains unstudied and needs further investigations. The results of the current study can help researchers to gain insights and further reveal the halophilic mechanism of halophiles.

Influence of yeast and lactic acid bacterium on the constituent profile of soy sauce during fermentation.

Soy sauce is a Japanese traditional seasoning composed of various constituents that are produced by various microbes during a long-term fermentation process. Due to the complexity of the process, the investigation of the constituent profile during fermentation is difficult. Metabolomics, the comprehensive study of low molecular weight compounds in biological samples, is thought to be a promising strategy for deep understanding of the constituent contribution to food flavor characteristics. Therefore, metabolomics is suitable for the analysis of soy sauce fermentation. Unfortunately, only few and unrefined studies of soy sauce fermentation using metabolomics approach have been reported. Therefore, we investigated changes in low molecular weight hydrophilic and volatile compounds of soy sauce using gas chromatography/mass spectrometry (GC/MS)-based non-targeted metabolic profiling. The data were analyzed by statistical analysis to evaluate influences of yeast and lactic acid bacterium on the constituent profile. Consequently, our results suggested a novel finding that lactic acid bacterium affected the production of several constituents such as cyclotene, furfural, furfuryl alcohol and methional in the soy sauce fermentation process.

Bacterial communities associated with invasive populations of Bactrocera dorsalis (Diptera: Tephritidae) in China.

The oriental fruit fly Bactrocera dorsalis (Hendel) is a destructive insect pest of a wide range of fruits and vegetables. This pest is an invasive species and is currently distributed in some provinces of China. To recover the symbiotic bacteria of B. dorsalis from different invasion regions in China, we researched the bacterial diversity of this fruit fly among one laboratory colony (Guangdong, China) and 15 wild populations (14 sites in China and one site in Thailand) using DNA-based approaches. The construction of 16S rRNA gene libraries allowed the identification of 24 operational taxonomic units of associated bacteria at the 3% distance level, and these were affiliated with 3 phyla, 5 families, and 13 genera. The higher bacterial diversity was recovered in wild populations compared with the laboratory colony and in samples from early term invasion regions compared with samples from late term invasion regions. Moreover, Klebsiella pneumoniae and Providencia sp. were two of the most frequently recovered bacteria, present in flies collected from three different regions in China where B. dorsalis is invasive. This study for the first time provides a systemic investigation of the symbiotic bacteria of B. dorsalis from different invasion regions in China.

Infection of Melissococcus plutonius clonal complex 12 strain in European honeybee larvae is essentially confined to the digestive tract.

Melissococcus plutonius is an important pathogen that causes European foulbrood (EFB) in honeybee larvae. Recently, we discovered a group of M. plutonius strains that are phenotypically and genetically distinct from other strains. These strains belong to clonal complex (CC) 12, as determined by multilocus sequence typing analysis, and show atypical cultural and biochemical characteristics in vitro compared with strains of other CCs tested. Although EFB is considered to be a purely intestinal infection according to early studies, it is unknown whether the recently found CC12 strains cause EFB by the same pathomechanism. In this study, to obtain a better understanding of EFB, we infected European honeybee (Apis mellifera) larvae per os with a well-characterized CC12 strain, DAT561, and analyzed the larvae histopathologically. Ingested DAT561 was mainly localized in the midgut lumen surrounded by the peritrophic matrix (PM) in the larvae. In badly affected larvae, the PM and midgut epithelial cells degenerated, and some bacterial cells were detected outside of the midgut. However, they did not proliferate in the deep tissues actively. By immunohistochemical analysis, the PM was stained with anti-M. plutonius serum in most of the DAT561-infected larvae. In some larvae, luminal surfaces of the PM were more strongly stained than the inside. These results suggest that infection of CC12 strain in honeybee larvae is essentially confined to the intestine. Moreover, our results imply the presence of M. plutonius-derived substances diffusing into the larval tissues in the course of infection.

Immunohistochemical evaluation of experimental Vagococcus salmoninarum infection in rainbow trout (Oncorhynchus mykiss, Walbaum 1792).

The aim of this study was to investigate the pathogenesis and histopathological and immunohistochemical findings in rainbow trout (Oncorhynchus mykiss) following experimental vagococcosis. For this purpose, 60 rainbow trout were used. The experimental study used the pathogen Vagococcus salmoninarum. The fish were intraperitoneally (IP) administered with an inoculate containing 0.1 mL of the bacteria, resulting in a dose of 1.2 × 10(9) cfu mL(-1) per fish. For histopathological observations, tissue samples were taken from fish that died during the experiment and fish that survived until the end of the trial (60th day). All the tissue samples were immunohistochemically stained by the avidin-biotin-peroxidase complex and immunofluorescence methods using polyclonal antibody to detect V. salmoninarum antigens. In immunoperoxidase staining, positive reactions to bacterial antigens were most commonly seen in the kidney, heart and liver. In the immunofluorescence analysis, the distribution of antigens in the tissue and organs was similar to that observed with the immunoperoxidase staining. The results reveal an important correlation between histochemical and immunohistochemical staining in demonstrating the distribution of V. salmoninarum antigens in the affected tissues.

Comparative decay of Catellicoccus marimmalium and enterococci in beach sand and seawater.

Most studies characterize microbial source tracking (MST) target performance using sensitivity and specificity metrics. However, it is important to also consider the temporal stability of MST targets in relation to regulated microbial pollutants. Differences among bacterial target stabilities may lead to erroneous conclusions about sources of contamination. The present study evaluates the relative stability of MST targets and fecal indicator organisms using the gull/pigeon-associated Catellicoccus marimammalium (CAT) marker and enterococci (ENT). The decay rates of CAT and ENT measured by culture (cENT) and QPCR (tENT) were compared in sand and seawater laboratory microcosms under environmentally relevant conditions (subject to tidal wetting versus no wetting in sand, and sunlit versus dark conditions in seawater). Bacterial targets were more persistent in beach sand than in seawater with decay rates on the order of 0.01-0.1 per day and 1 to 10 per day, respectively. Targets were more persistent in unwetted compared to wetted sand, and dark compared to sunlit seawater. During the first 8 days of the sand experiment, the decay rate k of CAT was greater than that of cENT. The decay rates of CAT, tENT, and cENT were similar in sand after day 8 and in dark seawater. In sunlit seawater, the decay rates were different between targets with kcENT > kCAT > ktENT. The decay rates presented here are useful for fate and transport models and also inform the use of MST marker concentrations to infer ENT sources in the environment.

Inhibitory effect of gut bacteria from the Japanese honey bee, Apis cerana japonica, against Melissococcus plutonius, the causal agent of European foulbrood disease.

European foulbrood is a contagious bacterial disease of honey bee larvae. Studies have shown that the intestinal bacteria of insects, including honey bees, act as probiotic organisms. Microbial flora from the gut of the Japanese honey bee, Apis cerana japonica F. (Hymenoptera: Apidae), were characterized and evaluated for their potential to inhibit the growth of Melissococcus plutonius corrig. (ex White) Bailey and Collins (Lactobacillales: Enterococcaceae), the causative agent of European foulbrood. Analysis of 16S rRNA gene sequences from 17 bacterial strains isolated by using a culture-dependent method revealed that most isolates belonged to Bacillus, Staphylococcus, and Pantoea. The isolates were screened against the pathogenic bacterium M. plutonius by using an in vitro growth inhibition assay, and one isolate (Acja3) belonging to the genus Bacillus exhibited inhibitory activity against M. plutonius. In addition, in vivo feeding assays revealed that isolate Acja3 decreased the mortality of honey bee larvae infected with M plutonius, suggesting that this bacterial strain could potentially be used as a probiotic agent against European foulbrood.

Enterococci in the environment.

Enterococci are common, commensal members of gut communities in mammals and birds, yet they are also opportunistic pathogens that cause millions of human and animal infections annually. Because they are shed in human and animal feces, are readily culturable, and predict human health risks from exposure to polluted recreational waters, they are used as surrogates for waterborne pathogens and as fecal indicator bacteria (FIB) in research and in water quality testing throughout the world. Evidence from several decades of research demonstrates, however, that enterococci may be present in high densities in the absence of obvious fecal sources and that environmental reservoirs of these FIB are important sources and sinks, with the potential to impact water quality. This review focuses on the distribution and microbial ecology of enterococci in environmental (secondary) habitats, including the effect of environmental stressors; an outline of their known and apparent sources, sinks, and fluxes; and an overview of the use of enterococci as FIB. Finally, the significance of emerging methodologies, such as microbial source tracking (MST) and empirical predictive models, as tools in water quality monitoring is addressed. The mounting evidence for widespread extraenteric sources and reservoirs of enterococci demonstrates the versatility of the genus Enterococcus and argues for the necessity of a better understanding of their ecology in natural environments, as well as their roles as opportunistic pathogens and indicators of human pathogens.

Characterization of Tetragenococcus strains from sugar thick juice reveals a novel species, Tetragenococcus osmophilus sp. nov., and divides Tetragenococcus halophilus into two subspecies, T. halophilus subsp. halophilus subsp. nov. and T. halophilus subsp. flandriensis subsp. nov.

Most bacteria recovered so far from sugar thick juice during storage represent strains of the species Tetragenococcus halophilus. Recently, several Gram-positive, non-motile, non-spore-forming cocci with other physiological and genetic traits were isolated from sugar thick juice samples from different origins. In this study, representative isolates were investigated using a polyphasic taxonomic approach. The 16S rRNA gene sequence similarity between these isolates and their closest relative, Tetragenococcus muriaticus, was 97.4%. The level of DNA-DNA relatedness between isolate T1(T), representing the newly found Tetragenococcus isolates, and T. muriaticus was 57%. Isolate T1(T) had a DNA G+C content of 36.7 mol%. Phylogenetic data and genomic and phenotypic features demonstrated that the isolates represent a novel species, for which the name Tetragenococcus osmophilus sp. nov. is proposed with T1(T) as the type strain (=LMG 26041(T) =DSM 23765(T)). Additionally, T. halophilus isolates from high-salt and high-sugar environments showed clear differences in several physiological and genetic characteristics like RAPD fingerprints and 16S rRNA gene sequences. DNA-DNA hybridizations, however, showed 79 to 80% relatedness between osmophilic and halophilic T. halophilus isolates, demonstrating that the different strains belong to the same species. Based on the phenotypic and genotypic differences observed, as well as the different origins of the strains and the industrial relevance of thick juice degradation, two subspecies of T. halophilus are described in this manuscript: T. halophilus subsp. halophilus subsp. nov. for the strains isolated from salt media and T. halophilus subsp. flandriensis subsp. nov. for the strains isolated from sugar-rich environments, which were first isolated in Flanders, Belgium. The type strains for the subspecies are IAM 1676(T) (=LMG 11490(T) =DSM 20339(T)) and T5(T) (=LMG 26042(T) =DSM 23766(T)), respectively.