Acute Cholecystitis Due to Lactococcus lactis and Single-Center Experience With Infections Due to Lactococcus spp.

Background: Lactococcus species are used to ferment milk to yogurt, cheese, and other products. The gram-positive coccus causes diseases in amphibia and fish and is a rare human pathogen. Patients and Methods: A 51-year-old male underwent laparoscopic cholecystectomy for acute and chronic calculous cholecystitis. Lactococcus lactis was isolated from pus from his gallbladder empyema. Results: Our institutional database was searched for other cases of Lactococcus spp. infections and four patients (2 males, 2 females; aged 51, 64, 78, and 80 years) were identified during a four-year period. The three other patients had positive blood cultures associated with pneumonia, toxic megacolon, and severe gastroenteritis. All isolates were monocultures with Lactococcus lactis (2), Lactococcus garvieae (1) and Lactococcus raffinolactis (1). Two patients died related to their sepsis. We report the second case of cholecystitis involving Lactococcus. Conclusions: Lactococcus is a very rare pathogen mainly causing blood stream infections but needs to be considered to cause serious surgical infections in humans.

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.

CLI: A new protocol for the isolation of Lactic Acid Bacteria from complex plant samples.

Lactic Acid Bacteria (LAB) are predominantly probiotic microorganisms and the most are Generally Recognized As Safe (GRAS). LAB inhabit in the human gut ecosystem and are largely found in fermented foods and silage. In the last decades, LAB have also has been found in plant microbiota as a new class of microbes with probiotic activity to plants. For this reason, today the scientific interest in the study and isolation of LAB for agronomic application has increased. However, isolation protocols from complex samples such as plant tissues are scarce and inefficient. In this study, we developed a new protocol (CLI, Complex samples LAB Isolation) which yields purified LAB from plants. The sensitivity of CLI protocol was sufficient to isolate representative microorganisms of LAB genera (i.e. Leuconostoc, Lactococcus and Enterococcus). CLI protocol consists on five steps: i) sample preparation and pre-incubation in 1% sterile peptone at 30 °C for 24-48 h; ii) Sample homogenization in vortex by 10 min; iii) sample serial dilution in quarter-strength Ringer solution, iv) incubation in MRS agar plates with 0.2% of sorbic acid, with 1% of CaCO3, O2 < 15%, at pH 5.8 and 37 °C for 48 h.; v) Selection of single colonies with LAB morphology and CaCO3-solubilization halo. Our scientific contribution is that CLI protocol could be used for several complex samples and represents a useful method for further studies involving native LAB.

Lactococcus garvieae-associated septicemia in a central bearded dragon.

Lactococcus garvieae is the causative agent of lactococcosis in fish and an emerging zoonotic pathogen with high levels of antimicrobial resistance. We report a case of L. garvieae-associated septicemia in a central bearded dragon (Pogona vitticeps) confirmed via whole-blood PCR and direct sequencing. Following a 30-d course of ceftazidime (20 mg/kg IM q72h), the animal's clinical condition had not resolved; leukopenia persisted, with heterophil toxic change. Coelomic ultrasound findings were consistent with preovulatory follicular stasis, folliculitis, and coelomitis. Following surgical ovariectomy and an additional 30-d course of ceftazidime, the animal's behavior and appetite returned to normal, the animal tested negative via whole-blood PCR assay, and the CBC was unremarkable. To our knowledge, L. garvieae with L. garvieae-associated clinical disease has not been reported previously in a bearded dragon. We conclude that L. garvieae should be considered as a possible etiologic agent in cases of septicemia in bearded dragons, with the potential for zoonotic transmission warranting further investigation.

Impact of Electrolyzed Water on the Microbial Spoilage Profile of Piedmontese Steak Tartare.

A low initial contamination level of the meat surface is the sine qua non to extend the subsequent shelf life of ground beef for as long as possible. Therefore, the short- and long-term effects of a pregrinding treatment with electrolyzed water (EW) on the microbiological and physicochemical features of Piedmontese steak tartare were here assessed on site, by following two production runs through storage under vacuum packaging conditions at 4°C. The immersion of muscle meat in EW solution at 100 ppm of free active chlorine for 90 s produced an initial surface decontamination with no side effects or compositional modifications, except for an external color change that was subsequently masked by the grinding step. However, the initially measured decontamination was no longer detectable in ground beef, perhaps due to a quick recovery by bacteria during the grinding step from the transient oxidative stress induced by the EW. We observed different RNA-based metataxonomic profiles and metabolomic biomarkers (volatile organic compounds [VOCs], free amino acids [FAA], and biogenic amines [BA]) between production runs. Interestingly, the potentially active microbiota of the meat from each production run, investigated through operational taxonomic unit (OTU)-, oligotyping-, and amplicon sequence variant (ASV)-based bioinformatic pipelines, differed as soon as the early stages of storage, whereas microbial counts and biomarker dynamics were significantly distinguishable only after the expiration date. Higher diversity, richness, and abundance of Streptococcus organisms were identified as the main indicators of the faster spoilage observed in one of the two production runs, while Lactococcus piscium development was the main marker of shelf life end in both production runs. IMPORTANCE Treatment with EW prior to grinding did not result in an effective intervention to prolong the shelf life of Piedmontese steak tartare. Our RNA-based approach clearly highlighted a microbiota that changed markedly between production runs but little during the first shelf life stages. Under these conditions, an early metataxonomic profiling might provide the best prediction of the microbiological fate of each batch of the product.

Isolation and Identification of Lactic Acid Bacteria from Cow Milk and Milk Products.

Lactic acid bacteria (LAB) have long been consumed by people in several fermented foods such as dairy products. A study was conducted on lactating dairy cows to isolate and characterize LAB from dairy products found in and around Bahir-Dar city, North Western Ethiopia. Milk and milk products were randomly collected from dairy farms, milk vending shops, individual households, and supermarkets for bacteriological investigations. A total of sixteen samples were taken from different sources and cultured on different selective media: de Man, Rogosa, and Sharpe (MRS) agar for Lactobacillus spp.; M17 agar for Lactococcus spp.; Rogasa SL agar for Streptococci spp.; and MRS supplemented with cysteine (0.5%) for Bifidobacteria spp. Different laboratory techniques were implemented for LAB isolation and identification. A total of 41 bacterial isolates were grouped under five different genera of LAB and Bifidobacteria spp. were identified based on the growth morphology on the selective media, growth at a different temperature, gas production from glucose, carbohydrate fermentation, and other biochemical tests. LAB genera such as Lactobacillus, Lactococcus, Leuconostoc, Pediococcus, Streptococcus, and Bifidobacterium spp. were isolated and identified from raw milk, cheese, and yogurt. Based on the current study, the majority of the LAB (24.38%) was isolated from cheese and yogurt. Among these, Lactobacillus, Lactococcus (21.94%), Streptococcus (19.51%), Leuconostoc (14.64%), Bifidobacteria (12.19%), and Pediococcus (7.31%) spp. were also identified from these products. Furthermore, based on the bacterial load count and different identification methodologies, our study revealed that Lactobacillus spp. were the dominant LAB isolated from milk and milk products. As a result, since there are few studies on the isolation and identification of lactic acid bacteria from dairy products in Ethiopia, more research studies are needed to complete the identification and characterization to species level and their possible role as probiotics.

Complete genome sequence and probiotic properties of Lactococcus petauri LZys1 isolated from healthy human gut.

Introduction. Lactococcus petauri LZys1 (L. petauri LZys1) is a type of lactic acid bacteria (LAB), which was initially isolated from healthy human gut.Hypothesis/Gap Statement. It was previously anticipated that L. petauri LZys1 has potential characteristics of probiotic properties. The genetic structure and the regulation functions of L. petauri LZys1 need to be better revealed.Aim. The aim of this study was to detect the probiotic properties L. petauri LZys1 and to reveal the genome information related to its genetic adaptation and probiotic profiles.Methodology. Multiple in vitro experiments were carried out to evaluate its lactic acid-producing ability, resistance to pathogenic bacterial strains, auto-aggregation and co-aggregation ability, and so on. Additionally, complete genome sequencing, gene annotation, and probiotic associated gene analysis were performed.Results. The complete genome of L. petauri LZys1 comprised of 1 985 765 bp, with a DNA G+C content of 38.07 %, containing 50 tRNA, seven rRNA, and four sRNA. A total of 1931 genes were classified into six functional categories by Kyoto Encyclopaedia of Genes and Genomes (KEGG) database. The neighbour-joining phylogeny tree based on the whole genome of L. petauri LZys1 and other probiotics demonstrated that L. petauri LZys1 has a significant similarity to Lactococcus garvieae. The functional genes were detected to expound the molecular mechanism and biochemical processes of its potential probiotic properties, such as atpB gene.Conclusion. All the results described in this study, together with relevant information previously reported, made L. prtauri LZys1 a very interesting potential strain to be considered as a prominent candidate for probiotic use.

Identification of Potential Probiotics Producing Bacteriocins Active against Listeria monocytogenes by a Combination of Screening Tools.

Listeria monocytogenes is an important food-borne pathogen and a serious concern to food industries. Bacteriocins are antimicrobial peptides produced naturally by a wide range of bacteria mostly belonging to the group of lactic acid bacteria (LAB), which also comprises many strains used as starter cultures or probiotic supplements. Consequently, multifunctional strains that produce bacteriocins are an attractive approach to combine a green-label approach for food preservation with an important probiotic trait. Here, a collection of bacterial isolates from raw cow's milk was typed by 16S rRNA gene sequencing and MALDI-Biotyping and supernatants were screened for the production of antimicrobial compounds. Screening was performed with live Listeria monocytogenes biosensors using a growth-dependent assay and pHluorin, a pH-dependent protein reporting membrane damage. Purification by cation exchange chromatography and further investigation of the active compounds in supernatants of two isolates belonging to the species Pediococcus acidilactici and Lactococcus garvieae suggest that their antimicrobial activity is related to heat-stable proteins/peptides that presumably belong to the class IIa bacteriocins. In conclusion, we present a pipeline of methods for high-throughput screening of strain libraries for potential starter cultures and probiotics producing antimicrobial compounds and their identification and analysis.

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.

Can sucrose-substitutes increase the antagonistic activity against foodborne pathogens, and improve the technological and functional properties of sheep milk kefir?

The effect of different types of sugar (sucrose, demerara, brown, fructose, coconut sugar, and honey) on sheep milk kefir was evaluated. Microbial counts (Lactobacillus, Lactococcus, Leuconostoc, yeast), antagonistic activity against foodborne pathogens, microstructure (scanning electron microscopy), and antiproliferative activity of cancer cells were evaluated. Furthermore, the antioxidant activity (DPPH), inhibitory activity of angiotensin-converting enzyme (ACE), α-amylase, and α-glucosidase, lactose content, lactic and acetic acids and ethanol, fatty acid profile and volatile organic compounds were determined. The addition of sugars increased the Lactobacillus population (up to 2.24 log CFU/mL), metabolites concentration, antagonistic activity against pathogens, antioxidant activity (11.1 to 24.1%), ACE inhibitory activity (27.5 to 37.6%), α-amylase inhibition (18 to 37.4%), and anti-proliferative activity. Furthermore, it improved the fatty acid profile and volatile compounds. The results suggest that the replacement of sucrose with different types of sugar constitute an interesting option to be used in sheep milk kefir formulations.

Assessing the efficacy of eDNA metabarcoding for measuring microbial biodiversity within forest ecosystems.

We investigated the nascent application and efficacy of sampling and sequencing environmental DNA (eDNA) in terrestrial environments using rainwater that filters through the forest canopy and understory vegetation (i.e., throughfall). We demonstrate the utility and potential of this method for measuring microbial communities and forest biodiversity. We collected pure rainwater (open sky) and throughfall, successfully extracted DNA, and generated over 5000 unique amplicon sequence variants. We found that several taxa including Mycoplasma sp., Spirosoma sp., Roseomonas sp., and Lactococcus sp. were present only in throughfall samples. Spiroplasma sp., Methylobacterium sp., Massilia sp., Pantoea sp., and Sphingomonas sp. were found in both types of samples, but more abundantly in throughfall than in rainwater. Throughfall samples contained Gammaproteobacteria that have been previously found to be plant-associated, and may contribute to important functional roles. We illustrate how this novel method can be used for measuring microbial biodiversity in forest ecosystems, foreshadowing the utility for quantifying both prokaryotic and eukaryotic lifeforms. Leveraging these methods will enhance our ability to detect extant species, describe new species, and improve our overall understanding of ecological community dynamics in forest ecosystems.

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.

Fermentation Enhanced Biotransformation of Compounds in the Kernel of Chrysophyllum albidum.

Chrysophyllum albidum Linn (African star apple) is a fruit with extensive nutritional and medicinal benefits. The fruit and kernel in the seed are both edible. Strains of lactic acid bacteria (LAB) were isolated from fermented seeds and assessed for probiotic characteristics. The extracts in both the unfermented and the fermented aqueous extracts from the kernels obtained from the seeds of C. albidum were subjected to analysis using the gas chromatography/mass spectrometry (GC-MS) method. This analysis identified the bioactive compounds present as possible substrate(s) for the associated organisms inducing the fermentation and the resultant biotransformed products formed. Three potential probiotic LAB strains identified as Lactococcus raffinolactis (ProbtA1), Lactococcus lactis (ProbtA2a), and Pediococcus pentosaceus (ProbtA2b) were isolated from the fermented C. albidum seeds. All strains were non hemolytic, which indicated their safety, Probt (A1, A2a, and A2b) grew in an acidic environment (pH 3.5) during the 48-h incubation time, and all three strains grew in 1% bile, and exhibited good hydrophobicity and auto-aggregation properties. Mucin binding proteins was not detected in any strain, and bile salt hydrolase was detected in all the strains. l-lactic acid (28.57%), norharman (5.07%), formyl 7E-hexadecenoate (1.73%), and indole (1.51%) were the four major constituents of the fermented kernel of the C. albidum, while 2,5-dimethylpyrazine (C1, 1.27%), 3,5-dihydroxy-6-methyl-2,3-dihydropyran-4-one (C2, 2.90%), indole (C3, 1.31%), norharman (C4, 3.01%), and methyl petroselinate (C5, 4.33%) were the five major constituents of the unfermented kernels. The isolated LAB are safe for consumption. The fermenting process metabolized C1, C2, and C5, which are possible starter cultures for the growth of probiotics. Fermentation is an essential tool for bioengineering molecules in foods into safe and health beneficial products.

Evaluation of lactic acid bacteria strains isolated from fructose-rich environments for their mannitol-production and milk-gelation abilities.

Mannitol is a sugar alcohol, or polyol, widely used in the food industry because of its low-calorie properties. Industrial production of mannitol is difficult and expensive. However, certain bacterial species are known to produce mannitol naturally, including certain lactic acid bacteria and fructophilic lactic acid bacteria (LAB). In this study, bacterial strains isolated from fructose-rich sources, including flowers, leaves, and honey, were identified by 16S rRNA sequence analysis as Leuconostoc, Fructobacillus, Lactococcus, and Lactobacillus species and 4 non-LAB species. DNA profiles generated by pulsed-field gel electrophoresis discriminated 32 strains of Leuconostoc mesenteroides and 6 Fructobacillus strains. Out of 41 LAB strains isolated, 32 were shown to harbor the mdh gene, which encodes the mannitol dehydrogenase enzyme, and several showed remarkable fructose tolerance even at 50% fructose concentrations, indicating their fructophilic nature. Several of the strains isolated, including Leuconostoc mesenteroides strains DPC 7232 and DPC 7261, Fructobacillus fructosus DPC 7237, and Fructobacillus fructosus DPC 7238, produced higher mannitol concentrations than did the positive control strain Limosilactobacillus reuteri DSM 20016 during an enzymatic screening assay. Mannitol concentrations were also examined via HPLC in 1% fructose de Man, Rogosa, and Sharpe medium (FMRS) or 1% fructose milk (FM). Among the strains, Fructobacillus fructosus DPC 7238 displayed high fructose utilization (9.27 g/L), high mannitol yield (0.99 g of mannitol/g of fructose), and greatest volumetric productivities (0.46 g/L per h) in FMRS. However, Leuconostoc mesenteroides DPC 7261 demonstrated the highest fructose utilization (8.99 g/L), mannitol yield (0.72 g of mannitol/g of fructose), and volumetric productivities (0.04 g/L per h) in FM. Storage modulus G' (>0.1 Pa) indicated a shorter gelation time for Limosilactobacillus reuteri DSM 20016 (8.73 h), followed by F. fructosus DPC 7238 (11.57 h) and L. mesenteroides DPC 7261 (14.52 h). Our results show that fructose-rich niches can be considered important sources of fructophilic LAB strains, with the potential to be used as starter cultures or adjunct cultures for the manufacture of mannitol-enriched fermented dairy products and beverages.

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).