Redefining piscine lactococcosis.

Piscine lactococcosis is a significant threat to cultured and wild fish populations worldwide. The disease typically presents as a per-acute to acute hemorrhagic septicemia causing high morbidity and mortality, recalcitrant to antimicrobial treatment or management interventions. Historically, the disease was attributed to the gram-positive pathogen Lactococcus garvieae. However, recent work has revealed three distinct lactococcosis-causing bacteria (LCB)-L. garvieae, L. petauri, and L. formosensis-which are phenotypically and genetically similar, leading to widespread misidentification. An update on our understanding of lactococcosis and improved methods for identification are urgently needed. To this end, we used representative isolates from each of the three LCB species to compare currently available and recently developed molecular and phenotypic typing assays, including whole-genome sequencing (WGS), end-point and quantitative PCR (qPCR) assays, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), API 20 Strep and Biolog systems, fatty acid methyl ester analysis (FAME), and Sensititre antimicrobial profiling. Apart from WGS, sequencing of the gyrB gene was the only method capable of consistent and accurate identification to the species and strain level. A qPCR assay based on a putative glycosyltransferase gene was also able to distinguish L. petauri from L. garvieae/formosensis. Biochemical tests and MALDI-TOF MS showed some species-specific patterns in sugar and fatty acid metabolism or protein profiles but should be complemented by additional analyses. The LCB demonstrated overlap in host and geographic range, but there were relevant differences in host specificity, regional prevalence, and antimicrobial susceptibility impacting disease treatment and prevention. IMPORTANCE: Lactococcosis affects a broad range of host species, including fish from cold, temperate, and warm freshwater or marine environments, as well as several terrestrial animals, including humans. As such, lactococcosis is a disease of concern for animal and ecosystem health. The disease is endemic in European and Asian aquaculture but is rapidly encroaching on ecologically and economically important fish populations across the Americas. Piscine lactococcosis is difficult to manage, with issues of vaccine escape, ineffective antimicrobial treatment, and the development of carrier fish or biofilms leading to recurrent outbreaks. Our understanding of the disease is also widely outdated. The accepted etiologic agent of lactococcosis is Lactococcus garvieae. However, historical misidentification has masked contributions from two additional species, L. petauri and L. formosensis, which are indistinguishable from L. garvieae by common diagnostic methods. This work is the first comprehensive characterization of all three agents and provides direct recommendations for species-specific diagnosis and management.

Genomic investigation of Lactococcus formosensis, Lactococcus garvieae, and Lactococcus petauri reveals differences in species distribution by human and animal sources.

Lactococcus garvieae is a fish pathogen that can cause diseases in humans and cows. Two genetically related species, Lactococcus formosensis and Lactococcus petauri, may be misidentified as L. garvieae. It is unclear if these species differ in host specificity and virulence genes. This study analyzed the genomes of 120 L. petauri, 53 L. formosensis, and 39 L. garvieae isolates from various sources. The genetic diversity and virulence gene content of these isolates were compared. The results showed that 77 isolates previously reported as L. garvieae were actually L. formosensis or L. petauri. The distribution of the three species varied across different collection sources, with L. petauri being predominant in human infections, human fecal sources, and rainbow trout, while L. formosensis was more common in bovine isolates. The genetic diversity of isolates within each species was high and similar. Using a genomic clustering method, L. petauri, L. formosensis, and L. garvieae were divided into 45, 22, and 13 clusters, respectively. Most rainbow trout and human isolates of L. petauri belonged to different clusters, while L. formosensis isolates from bovine and human sources were also segregated into separate clusters. In L. garvieae, most human isolates were grouped into three clusters that also included isolates from food or other sources. Non-metric multidimensional scaling ordination revealed the differential association of 15 virulence genes, including 14 adherence genes and a bile salt hydrolase gene, with bacterial species and certain collection sources. In conclusion, this work provides evidence of host specificity among the three species. IMPORTANCE: Lactococcus formosensis and Lactococcus petauri are two newly discovered bacteria, which are closely related to Lactococcus garvieae, a pathogen that affects farmed rainbow trout, as well as causes cow mastitis and human infections. It is unclear whether the three bacteria differ in their host preference and the presence of genes that contribute to the development of disease. This study shows that L. formosensis and L. petauri were commonly misidentified as L. garvieae. The three bacteria showed different distribution patterns across various sources. L. petauri was predominantly found in human infections and rainbow trout, while L. formosensis was more commonly detected in cow mastitis. Fifteen genes displayed a differential distribution among the three bacteria from certain sources, indicating a genetic basis for the observed host preference. This work indicates the importance of differentiating the three bacteria in diagnostic laboratories for surveillance and outbreak investigation purposes.

Molecular mechanisms underlying the structural diversity of rhamnose-rich cell wall polysaccharides in lactococci.

In Gram-positive bacteria, cell wall polysaccharides (CWPS) play critical roles in bacterial cell wall homeostasis and bacterial interactions with their immediate surroundings. In lactococci, CWPS consist of two components: a conserved rhamnan embedded in the peptidoglycan layer and a surface-exposed polysaccharide pellicle (PSP), which are linked together to form a large rhamnose-rich CWPS (Rha-CWPS). PSP, whose structure varies from strain to strain, is a receptor for many bacteriophages infecting lactococci. Here, we examined the first two steps of PSP biosynthesis, using in vitro enzymatic tests with lipid acceptor substrates combined with LC-MS analysis, AlfaFold2 modeling of protein 3D-structure, complementation experiments, and phage assays. We show that the PSP repeat unit is assembled on an undecaprenyl-monophosphate (C55P) lipid intermediate. Synthesis is initiated by the WpsA/WpsB complex with GlcNAc-P-C55 synthase activity and the PSP precursor GlcNAc-P-C55 is then elongated by specific glycosyltransferases that vary among lactococcal strains, resulting in PSPs with diverse structures. Also, we engineered the PSP biosynthesis pathway in lactococci to obtain a chimeric PSP structure, confirming the predicted glycosyltransferase specificities. This enabled us to highlight the importance of a single sugar residue of the PSP repeat unit in phage recognition. In conclusion, our results support a novel pathway for PSP biosynthesis on a lipid-monophosphate intermediate as an extracellular modification of rhamnan, unveiling an assembly machinery for complex Rha-CWPS with structural diversity in lactococci.

Evidence of pediatric sepsis caused by a drug resistant Lactococcus garvieae contaminated platelet concentrate.

Owing to an increasing number of infections in adults, Lactococcus (L.) garvieae has gained recognition as an emerging human pathogen, causing bacteraemia and septicaemia. In September 2020, four paediatric onco-hematologic patients received a platelet concentrate from the same adult donor at Bambino Gesù Children's Hospital IRCCS, Rome. Three of four patients experienced L. garvieae sepsis one day after transfusion. The L. garvieae pediatric isolates and the donor's platelet concentrates were retrospectively collected for whole-genome sequencing and shot-gun metagenomics, respectively (Illumina HiSeq). By de novo assembly of the L. garvieae genomes, we found that all three pediatric isolates shared a 99.9% identity and were characterized by 440 common SNPs. Plasmid pUC11C (conferring virulence properties) and the temperate prophage Plg-Tb25 were detected in all three strains. Core SNP genome-based maximum likelihood and Bayesian trees confirmed their phylogenetic common origin and revealed their relationship with L. garvieae strains affecting cows and humans (bootstrap values >100 and posterior probabilities = 1.00). Bacterial reads obtained by the donor's platelet concentrate have been profiled with MetaPhlAn2 (v.2.7.5); among these, 29.9% belonged to Firmicutes, and 5.16% to Streptococcaceae (>97% identity with L. garvieae), confirming the presence of L. garvieae in the platelet concentrate transfusion. These data showed three episodes of sepsis for the first time due to a transfusion-associated transmission of L. garvieae in three pediatric hospitalized hematology patients. This highlights the importance to implement the screening of platelet components with new human-defined pathogens for ensuring the safety of blood supply, and more broadly, for the surveillance of emerging pathogens.

Proposal of Lactococcus garvieae subsp. bovis Varsha and Nampoothiri 2016 as a later heterotypic synonym of Lactococcus formosensis Chen et al. 2014 and Lactococcus formosensis subsp. bovis comb. nov.

Currently, Lactococcus garvieae contains two subspecies: L. garvieae subsp. bovis and L. garvieae subsp. garvieae. In a study by Varsha and Nampoothiri, high pheS (99.7 %) and rpoA (99.6 %) sequence similarities indicated that L. garvieae subsp. bovis and Lactococcus formosensis probably have a close taxonomic relationship; low pheS (92.2 %) and rpoA (97.8 %) sequence similarities and relatively low DNA-DNA hybridization value (75.8 %) indicated that L. garvieae subsp. bovis and L. garvieae subsp. garvieae probably represent two different species. In the present study, the taxonomic relationships between L. garvieae subsp. bovis, L. garvieae subsp. garvieae and L. formosensis were re-examined based on sequence analyses of 16S rRNA, pheS, recA, rpoA and rpoB genes, average nucleotide identity (ANI), digital DNA-DNA hybridization (dDDH) values, average amino acid identity (AAI), fatty acid methyl ester analysis and phenotypic characterization. L. garvieae subsp. bovis LMG 30663T exhibited 97.3 % ANI, 78.3 % dDDH and 96.4 % AAI values to L. formosensis NBRC 109475T, higher than the threshold for species demarcation (95-96, 70 and 95-96 %, respectively), indicating that L. garvieae subsp. bovis LMG 30663T and L. formosensis NBRC 109475T belong to the same species. L. garvieae subsp. bovis LMG 30663T had 91.2 % ANI, 43.3 % dDDH and 92.9-93.0% AAI values with the type strain of L. garvieae subsp. garvieae, indicating that they represent two different species. Because L. formosensis has been proposed and validated before L. garvieae subsp. bovis, L. garvieae subsp. bovis is transferred to L. formosensis as L. formosensis subsp. bovis comb. nov. The type strain of L. formosensis subsp. bovis is BSN307T (=DSM 100577T=MCC 2824T=KCTC 21083T=LMG 30663T). The type strain of L. formosensis subsp. formosensis is 516T (=NBRC 109475T=BCRC 80576T).

Genomic relatedness of a canine Lactococcus garvieae to human, animal and environmental isolates.

Lactococcus (L.) garvieae is a zoonotic fish pathogen that can also cause bacteraemia and endocarditis in humans and has been isolated from healthy or diseased domestic animals. Nevertheless L. garvieae is more an opportunistic, than a primary pathogen since most affected humans have predisposing conditions and comorbidities. L. garvieae is also present in other animal species, most frequently cattle, but also sheep, goats, water buffaloes, and pigs, and much more rarely dogs, cats, horses, camel, turtle, snake and crocodile. The purpose of this study was to genomically (i) confirm the identification by MALDI-TOF MS® of a L. garvieae from the nasal discharge of a dog with chronic respiratory disorders and (ii) compare this canine isolate with human and animal L. garvieae isolates. According to the BLAST analysis after Whole Genome Sequencing, this canine isolate was more than 99% identical to 3 L. garvieae and belonged to a new Multi-Locus Sequence Type (ST45). MLST and whole genomes-based phylogenetic analysis were performed on the canine isolate and the 40 genomes available in Genbank. The canine L. garvieae was most closely related to an Australian camel and an Indian fish L. garvieae and more distantly to human L. garvieae. Twenty-five of the 29 putative virulence-associated genes searched for were detected, but not the 16 capsule-encoding genes. The heterogeneity of the L. garvieae species is reflected by the diversity of the MLSTypes and virulotypes identified and by the phylogenetic analysis.

Evaluation of seed associated endophytic bacteria from tolerant chilli cv. Firingi Jolokia for their biocontrol potential against bacterial wilt disease.

In this study, the seed endosphere of a bacterial wilt tolerant chilli cv. Firingi Jolokia was explored in order to find effective agents for bacterial wilt disease biocontrol. A total of 32 endophytic bacteria were isolated from freshly collected seeds and six isolates were selected based on R. solanacearum inhibition assay. These isolates were identified as Bacillus subtilis (KJ-2), Bacillus velezensis (KJ-4), Leuconostoc mesenteroides (KP-1), Lactococcus lactis (LB-3), Bacillus amyloliquefaciens (WK-2), and Bacillus subtilis (WK-3) by 16S rRNA gene sequencing. In the in planta R. solanacearum inhibition assay carried out by seedling root bacterization method, Bacillus subtilis (KJ-2) exhibited highest biocontrol efficacy of 86.6 % on 7th day post R. solanacearum inoculation and a minimum biocontrol efficacy of 52.9 % was noted for Leuconostoc mesenteroides (KP-1). GC-HRMS analysis detected several known antimicrobial compounds in the extract of the culture supernatant of Bacillus subtilis (KJ-2); which may contribute to inhibition of R. solanacearum. In the growth promotion assay conducted using these isolates, only two of them namely Bacillus subtilis (KJ-2) and Bacillus amyloliquefaciens (WK-2) showed growth promotion in true leafed tomato plants. All the selected seed endophytic isolates were able to control bacterial wilt of tomato at the seedling stage and Bacillus subtilis (KJ-2) was found to be most effective in controlling the disease. The results of the present study highlighted that seed endosphere of bacterial wilt tolerant cultivar is a rich source of R. solanacearum antagonizing bacterial isolates.

Comparative analysis of the pulmonary microbiome in healthy and diseased pigs.

The lungs possess an effective antimicrobial system and a strong ability to eliminate microorganisms in healthy organisms, and were once considered sterile. With the development of culture-independent sequencing technology, the richness and diversity of porcine lung microbiota have been gaining attention. In order to study the relationship between lung microbiota and porcine respiratory disease complex (PRDC), the lung microbiota in healthy and diseased swine bronchoalveolar lavage fluids were analyzed and compared using the Illumina MiSeq sequencing platform. The predominant microbial communities of healthy and diseased swine were similar at the phylum level, mainly composed of Proteobacteria, Firmicutes, Tenericutes, and Bacteroidetes. However, the bacterial taxonomic communities of healthy and diseased swine differed at the genus level. The higher relative abundances of Lactococcus, Enterococcus, Staphylococcus, and Lactobacillus genera in healthy swine might provide more benefits for lung health, while the enhanced richness of Streptococcus, Haemophilus, Pasteurella, and Bordetella genera in diseased swine might be closely related to pathogen invasion and the occurrence of respiratory disease. In conclusion, the observed differences in the richness and diversity of lung microbiota can provide novel insights into their relationship with PRDC. Analyses of swine lung microbiota communities might produce an effective strategy for the control and prevention of respiratory tract infections.

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).

Lactococcus insecticola sp. nov. and Lactococcus hodotermopsidis sp. nov., isolated from the gut of the wood-feeding lower termite Hodotermopsis sjostedti.

Two strains of lactic acid bacteria, designated Hs20B0-1T and Hs30E4-3T, were isolated from the gut of the damp-wood termite Hodotermopsis sjostedti. These strains were characterized genetically and phenotypically. Strain Hs20B0-1T was related to Lactococcus piscium DSM 6634T showing 96.3 and 84.2 % sequence similarity in 16S rRNA gene and rpoB gene sequences, respectively. Strain Hs30E4-3T was related to Lactococcus plantarum DSM 20686T showing 94.8 and 82.2 % sequence similarity in 16S rRNA gene and rpoB gene sequences, respectively. The 16S rRNA gene sequence similarity between strains Hs20B0-1T and Hs30E4-3T was 95.7 %. Furthermore, genomic comparisons using pairwise average nucleotide identity (ANI) and digital DNA-DNA hybridization (DDH) analyses between strain Hs20B0-1T and L. piscium DSM 6634T resulted in values of 73.5 and 20.1 %, respectively. Strain Hs30E4-3T had 72.8 % ANI similarity and 21.3 % DDH similarity to L. plantarum DSM 20686T. Strains Hs20B0-1T and Hs30E4-3T had 75.4 % ANI similarity and 21.1 % DDH similarity to each other. The cell-wall peptidoglycan types of strains Hs20B0-1T and Hs30E4-3T were A4α, Lys-Asp and A3α, Lys-Thr-Ala, respectively. The two strains, Hs20B0-1T and Hs30E4-3T, are distinguishable from each other and other established Lactococcus species phylogenetically and phenotypically. In conclusion, two novel species of the genus Lactococcus are proposed, namely Lactococcus insecticola Hs20B0-1T (=JCM 33485T=DSM 110147T) and Lactococcus hodotermopsidis Hs30E4-3T (=JCM 33486T=DSM 110148T), respectively.

Different growth kinetics in blood components and genetic analysis of Lactococcus garvieae isolated from platelet concentrates.

BACKGROUND: In 2014, we experienced the first isolation of Lactococcus garvieae from a platelet concentrate (PC). Thereafter, L. garvieae contamination of PCs occurred in two more cases in Japan. It is rare that bacterial contamination with uncommon strains like this species occurs frequently within a short period. Therefore, we performed a detailed analysis of the characteristics of these strains. STUDY DESIGN AND METHODS: Three bacterial strains were identified by biochemical testing and molecular analysis. Genomic diversity was characterized by multilocus sequence typing (MLST). To observe growth kinetics in blood components, PCs were inoculated with the three different strains. RESULTS: All three strains were identified as L. garvieae by molecular analysis. Each strain belonged to a different phylogenetic group according to MLST analysis. In the spiking trial, the three strains demonstrated differences in their final concentrations and changes in appearance of PCs. CONCLUSION: In this study, all three L. garvieae strains were correctly identified by molecular analysis. Since the three strains were collected in different regions of Japan and belonged to different phylogenetic groups according to MLST analysis, it is suggested that L. garvieae have a wide distribution with diversity in Japan. In PCs, the three L. garvieae strains showed clear differences in growth kinetics and changes in appearance of PCs. These differences may have been the primary determinant of whether PC contamination was detected before transfusion. Moreover, L. garvieae represents an emerging foodborne bacterium that can cause transfusion-transmitted bacteremia. Understanding our cases may help prevent bacterial contamination of blood products.

Characterization of a potential probiotic bacterium Lactococcus raffinolactis WiKim0068 isolated from fermented vegetable using genomic and in vitro analyses.

BACKGROUND: Lactococcus members belonging to lactic acid bacteria are widely used as starter bacteria in the production of fermented dairy products. From kimchi, a Korean food made of fermented vegetables, Lactococcus raffinolactis WiKim0068 was isolated and its genome was analyzed. RESULTS: The complete genome of the strain WiKim0068 consists of one chromosome and two plasmids that comprises 2,292,235 bp, with a G + C content of 39.7 mol%. Analysis of orthoANI values among Lactococcus genome sequences showed that the strain WiKim0068 has > 67% sequence similarity to other species and subspecies. In addition, it displayed no antibiotic resistance and can metabolize nicotinate and nicotinamide (vitamin B3). CONCLUSION: These results augments our understanding of the genus Lactococcus and suggest that this new strain has potential industrial applications.

Lactococcus protaetiae sp. nov. and Xylanimonas protaetiae sp. nov., isolated from gut of larvae of Protaetia brevitarsis seulensis.

Two bacterial strains, designated 2DFWM-2T and FW10M-9T, were isolated from gut of larva of Protaetia brevitarsis seulensis grown at the National Institute of Agricultural Sciences, Wanju-gun, South Korea. 16S rRNA and rpoB gene sequences showed that strain 2DFWM-2T formed a separate branch with Lactococcus allomyrinae 1JSPR-7T in the genus Lactococcus, adjacent to a group of Lactococcus lactis subspecies. ANI and dDDH values between 2DFWM-2T and Lactococcus allomyrinae 1JSPR-7T were 93.30% and 53.20%, respectively. Based on the 16S rRNA gene sequence analysis, strain FW10M-9T was classified into the genus Xylanimonas revealing 96.9-98.5% sequence similarities with the Xylanimonas species. The ANI values of strain FW10M-9T with the closely species Xylanimonas pachnodae NBRC 107786T, Xylanimonas allomyrinae 2JSPR-7T, Isoptericola variabilis JCM 11754T and Xylanimonas cellulosilytica DSM 15894T was 81.5%, 81.2%, 81.0% and 84.1%, respectively, and the dDDH values estimated by GGDC was 24.3%, 24.3%, 29.3% and 28.1%, respectively. On the basis of the phenotypic and genotypic data, it is proposed that strain 2DFWM-2T represents a novel species of the genus Lactococcus, for which the name Lactococcus protaetiae sp. nov. is proposed, and the type strain is 2DFWM-2T (= KACC 19320T = NBRC 113069T). And, strain FW10M-9T represents a novel species of the genus Xylanimonas, for which the name Xylanimonas protaetiae sp. nov. is proposed, and the type strain is FW10M-9T (= KACC 19331T = NBRC 113053T).

The effects of a multispecies synbiotic on microbiome-related side effects of long-term proton pump inhibitor use: A pilot study.

Side effects of proton pump inhibitors (PPI) can be linked to the changes in the intestinal microbiome that occur during therapy, especially in long-term users. Therefore, the microbiome might also be a key player in the reduction of PPI side effects. We tested the effects of a three-month intervention with a multispecies synbiotic on intestinal inflammation, gut barrier function, microbiome composition, routine laboratory parameters and quality of life in patients with long-term PPI therapy. Thirty-six patients received a daily dose of a multispecies synbiotic for three months and were clinically observed without intervention for another three months. After intervention 17% of patients reached normal calprotectin levels; the overall reduction did not reach statistical significance (-18.8 ng/mg; 95%CI: -50.5; 12.9, p = 0.2). Elevated zonulin levels could be significantly reduced (-46.3 ng/mg; 95%CI: -71.4; -21.2; p < 0.001). The abundance of Stomatobaculum in the microbiome was reduced and Bacillus increased during the intervention. Furthermore, albumin, alkaline phosphatase and thrombocyte count were significantly increased and aspartate transaminase was significantly decreased during intervention. Gastrointestinal quality of life showed significant improvements. In conclusion, microbiome-related side effects of long-term PPI use can be substantially reduced by synbiotic intervention. Further studies are warranted to optimize dosage and duration of the intervention.

First Report of Isolation and Genome Sequence of L. petauri Strain from a Rainbow Trout Lactococcosis Outbreak.

Lactococcosis is a disease encountered in a wide variety of fish species causing mortalities and having great economic impact on farmed fish. In this study, we report for the first time the isolation of a strain of the recently described novel species Lactococcus petauri, from rainbow trout suffering from lactococcosis. The aim of this study was to determine the complete genome sequence of L. petauri strain LG_SAV_20 and to characterize its antimicrobial resistance and virulence. The genome of L. petauri LG_SAV_20 consists of 2,078,949 base pair (bp) with a GC content of 38.05%, 1950 predicted coding sequence (CDS), and 60 RNAs (51 tRNAs, 3 ncRNAs, and 6 rRNAs). Phylogenetic analysis revealed that L. petauri LG_SAV_20 shares most of its genome with L. garvieae strains isolated from rainbow trout. Detection of genes associated with antimicrobial resistance indicated that the isolate possesses the multidrug transporter mdt(A) gene, while using comparative analysis we identified several genes that might be related to bacterial pathogenesis. This genomic information provides new insights into the role of this novel species as an etiological agent of lactococcosis.

Lactococcus kimchii sp. nov., a new lactic acid bacterium isolated from kimchi.

A coccus strain designated S-13T was isolated from commercial baechu-kimchi in Korea. Comparison of the 16S rRNA gene sequence indicated that strain S-13T had the highest similarity to Lactococcus taiwanensis 0905C15T (97.9 %), Lactococcus lactis subsp. tructae L105T (97.6 %), Lactococcus lactis subsp. cremoris NCDO 607T (97.5 %), Lactococcus lactis subsp. hordniae NBRC 100931T (97.2 %), and Lactococcus lactis subsp. lactis JCM 5805T (97.2 %). The detailed phylogenetic analyses based on the 16S rRNA, rpoB and recA genes indicated that S-13T was separated from the other species and subspecies in the genus Lactococcus. The DNA-DNA relatedness between S-13T and closely related type strains, such as L. taiwanensis 0905C15T, L. lactis subsp. tructae L105T, L. lactis subsp. cremoris NCDO 607T, L. lactis subsp. hordniae NBRC 100931T, and L. lactis subsp. lactis JCM 5805T was 25.6, 20.4, 25.1, 20.2 and 21.7 %, respectively. The major fatty acids were C16 : 0, cyclo-C19 : 0ω8c and C 14 : 0. The DNA G+C content of S-13T was 39.4 mol%. From the results of the phenotypic characteristics and chemotaxonomic analysis, it was concluded that strain S-13T represents a novel species in the genus Lactococcus for which the name Lactococcus kimchii sp. nov. (=KCTC 21096T=NBRC 113348T) is proposed.

Lactococcus allomyrinae sp. nov., isolated from gut of larvae of Allomyrina dichotoma.

Strain 1JSPR-7T, a facultatively anaerobic bacterium isolated from the gut of larvae of Allomyrina dichotoma raised in Wanju-gun, Republic of Korea, was characterized using a polyphasic approach. Comparative analysis of 16S rRNA gene and rpoB gene sequences showed that strain 1JSPR-7T fell within the genus Lactococcus, forming a compact cluster with the type strain of four subspecies of Lactococcus lactis and Lactococcus taiwanensis. The 16S rRNA gene sequence of strain 1JSPR-7T revealed the highest homology with L. lactissubsp. lactis JCM 5805T (97.3 %) and L. lactissubsp. hordniae NBRC 100931T (97.1 %), and the rpoB gene sequence showed the highest similarity to L. lactissubsp. cremoris DSM 20069T (91.4 %) and L. lactissubsp. tructae L105T (91.4 %). The average nucleotide identity and digital DNA-DNA hybridization values indicated that strain 1JSPR-7T was a novel species of the genus Lacococcus. The major fatty acids (>10 % of the total fatty acids) were summed feature 7 (unknown 18.846, C19 : 1ω6c and/or C19 : 0cyclo ω10c), C16 : 0 and C14 : 0, and the predominant menaquinone was MK-8 with MK-7 as a minor one. The polar lipids consisted of diphosphatidylglycerol, phosphatidylglycerol, an unidentified phospholipid and three unidentified glycolipids with diphosphatidylglycerol as the major one. The cell-wall peptidoglycan was of the A4α type with an interpeptide bridge comprising l-Lys-d-Asp. The DNA G+C content based on the whole genome sequences was 37.4 mol%. Based on the data obtained, strain 1JSPR-7T represents a novel species of the genus Lactococcus, for which the name Lactococcusallomyrinae sp. nov. is proposed. The type strain is 1JSPR-7T (=KACC 19319T=NBRC 113068T).

Meta-Analysis Reveals Reproducible Gut Microbiome Alterations in Response to a High-Fat Diet.

Multiple research groups have shown that diet impacts the gut microbiome; however, variability in experimental design and quantitative assessment have made it challenging to assess the degree to which similar diets have reproducible effects across studies. Through an unbiased subject-level meta-analysis framework, we re-analyzed 27 dietary studies including 1,101 samples from rodents and humans. We demonstrate that a high-fat diet (HFD) reproducibly changes gut microbial community structure. Finer taxonomic analysis revealed that the most reproducible signals of a HFD are Lactococcus species, which we experimentally demonstrate to be common dietary contaminants. Additionally, a machine-learning approach defined a signature that predicts the dietary intake of mice and demonstrated that phylogenetic and gene-centric transformations of this model can be translated to humans. Together, these results demonstrate the utility of microbiome meta-analyses in identifying robust and reproducible features for mechanistic studies in preclinical models.

Lactococcus termiticola sp. nov., isolated from the gut of the wood-feeding higher termite Nasutitermes takasagoensis.

A strain of lactic acid bacteria, designated NtB2T, isolated from the gut of the wood-feeding higher termite Nasutitermes takasagoensis, was characterized genetically and phenotypically. Strain NtB2T was related to Lactococcus lacti subsp. tructae JCM 31125T isolated from brown trout, showing 93.2 and 81.0 % similarity in 16S rRNA gene and rpoB gene sequences, respectively. Furthermore, genomic comparisons using pairwise average nucleotide identity analysis and the Genome-to-Genome Distance Calculator between strain NtB2T and L. lacti subsp. tructae JCM 31125T gave values of 81.0 and 23.2 %, respectively. Major cellular fatty acids produced by strain NtB2T were C18 : 1ω9c and C16 : 0. The cell-wall peptidoglycan type of strain NtB2T was A3α, Lys-Gly-Ser-Ala2. Based on the data presented, the isolate represents a novel species of the genus Lactococcus, for which the name Lactococcus termiticola sp. nov. is proposed. The type strain is NtB2T (=JCM 32569T=DSM 107259T).

Genetic and phenotypic features defining industrial relevant Lactococcus lactis, L. cremoris and L. lactis biovar. diacetylactis strains.

Lactococcus lactis strains constitute one of the most important starter cultures for cheese production. In this study, a genome-wide analysis was performed including 68 available genomes of L. lactis group strains showing the existence of two species (L. lactis and L. cremoris) and two biovars (L. lactis biovar. diacetylactis and L. cremoris biovar. lactis). The proposed classification scheme revealed coherency among phenotypic (through in silico and in vivo bacterial function profiling), phylogenomic (through maximum likelihood trees) and genomic (using overall genome sequence-based parameters) approaches. Strain biodiversity for the industrial biovar. diacetylactis was also analyzed, finding they are formed by at least three variants with the CC1 clonal complex as the only one distributed worldwide. These findings and methodologies will help improve the selection of L. lactis group strains for industrial use as well as facilitate the interpretation of previous or future research studies on this diverse group of bacteria.