Unveiling the regulatory network controlling natural transformation in lactococci.

Lactococcus lactis is a lactic acid bacterium of major importance for food fermentation and biotechnological applications. The ability to manipulate its genome quickly and easily through competence for DNA transformation would accelerate its general use as a platform for a variety of applications. Natural transformation in this species requires the activation of the master regulator ComX. However, the growth conditions that lead to spontaneous transformation, as well as the regulators that control ComX production, are unknown. Here, we identified the carbon source, nitrogen supply, and pH as key factors controlling competence development in this species. Notably, we showed that these conditions are sensed by three global regulators (i.e., CcpA, CodY, and CovR), which repress comX transcription directly. Furthermore, our systematic inactivation of known signaling systems suggests that classical pheromone-sensing regulators are not involved. Finally, we revealed that the ComX-degrading MecA-ClpCP machinery plays a predominant role based on the identification of a single amino-acid substitution in the adaptor protein MecA of a highly transformable strain. Contrasting with closely-related streptococci, the master competence regulator in L. lactis is regulated both proximally by general sensors and distantly by the Clp degradation machinery. This study not only highlights the diversity of regulatory networks for competence control in Gram-positive bacteria, but it also paves the way for the use of natural transformation as a tool to manipulate this biotechnologically important bacterium.

Transcriptome responses of Lactococcus paracarnosus to different gas compositions and co-culture with Brochothrix thermosphacta.

Lactococcus (Lc.) paracarnosus and the phylogenetically closely related Lc. carnosus species are common members of the microbiota in meat stored under modified atmosphere and at low temperature. The effect of these strains on meat spoilage is controversially discussed. While some strains are known to cause spoilage, others are being studied for their potential to suppress the growth of spoilage and pathogenic bacteria. In this study, Lc. paracarnosus DSM 111017T was selected based on a previous study for its ability to suppress the growth of meat spoilers, including Brochothrix thermosphacta. The mechanism by which this bioprotective strain inhibits competing bacteria and how it contributes to spoilage are not yet known. To answer these two questions, we investigated the effect of four different headspace gas mixtures (simulated air (21 % O2/79 % N2); HiOx-MAP (70 % O2/30 % CO2); nonOx-MAP (70 % N2/ 30 % CO2); simulated vacuum (100 % N2) and the presence of Brochothrix (B.) thermosphacta TMW 2.2101 on the growth and transcriptional response of Lc. paracarnosus DSM 111017T when cultured on a meat simulation agar surface at 4 °C. Analysis of genes specifically upregulated by the gas mixtures used revealed metabolic pathways that may lead to different levels of spoilage metabolites production. We propose that under elevated oxygen levels, Lc. paracarnosus preferentially converts pyruvate from glucose and glycerol to uncharged acetoin/diacetyl instead of lactate to counteract acid stress. Due to the potential production of a buttery off-flavour, the strain may not be suitable as a protective culture in meat packaged under high­oxygen conditions. 70 % N2/ 30 % CO2, simulated vacuum- and the presence of Lc. paracarnosus inhibited the growth of B. thermosphacta TMW 2.2101. However, B. thermosphacta did not affect gene regulation of metabolic pathways in Lc. paracarnosus, and genes previously predicted to be involved in B. thermosphacta growth suppression were not regulated at the transcriptional level. In conclusion, the study indicates that the gas mixture used in packaging significantly affects the metabolism and spoilage potential of Lc. paracarnosus and its ability to inhibit B. thermosphacta growth.

Behavioral transcriptomic effects of triploidy and probiotic therapy (Bifidobacterium, Lactobacillus, and Lactococcus mixture) on juvenile Chinook salmon (Oncorhynchus tshawytscha).

Aquaculturists use polyploid fish to maximize production albeit with some unintended consequences including compromised behaviors and physiological function. Given benefits of probiotic therapies (e.g., improved immune response, growth, and metabolism), we explored probiotic supplementation (mixture of Bifidobacterium, Lactobacillus, and Lactococcus), to overcome drawbacks. We first examined fish gut bacterial community composition using 16S metabarcoding (via principal coordinate analyses and PERMANOVA) and determined probiotics significantly impacted gut bacteria composition (p = 0.001). Secondly, we examined how a genomic disruptor (triploidy) and diet supplements (probiotics) impact gene transcription and behavioral profiles of hatchery-reared Chinook salmon (Oncorhynchus tshawytscha). Juveniles from four treatment groups (diploid-regular feed, diploid-probiotic feed, triploid-regular feed, and triploid-probiotic feed; n = 360) underwent behavioral assays to test activity, exploration, neophobia, predator evasion, aggression/sociality, behavioral sensitivity, and flexibility. In these fish, transcriptional profiles for genes associated with neural functions (neurogenesis/synaptic plasticity) and biomarkers for stress response and development (growth/appetite) were (i) examined across treatments and (ii) used to describe behavioral phenotypes via principal component analyses and general linear mixed models. Triploids exhibited a more active behavioral profile (p = 0.002), and those on a regular diet had greater Neuropeptide Y transcription (p = 0.02). A growth gene (early growth response protein 1, p = 0.02) and long-term neural development genes (neurogenic differentiation factor, p = 0.003 and synaptysomal-associated protein 25-a, p = 0.005) impacted activity and reactionary profiles, respectively. Overall, our probiotic treatment did not compensate for triploidy. Our research highlights novel applications of behavioral transcriptomics for identifying candidate genes and dynamic, mechanistic associations with complex behavioral repertoires.

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.

Characterization of Lactococcus garvieae and Streptococcus agalactiae in cultured red tilapia Oreochromis sp. in Thailand.

OBJECTIVE: Acute mortality with clinical symptoms of streptococcal-like infections was observed in red tilapia Oreochromis sp. cultured in floating cages in Prachin Buri Province, Thailand, during May 2023. Herein, we identified an emerging pathogen, Lactococcus garvieae, as the etiological agent. METHODS: After bacterial isolation from the brain and kidney of diseased fish, identification was performed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and the VITEK 2 system. Sequencing of the 16S ribosomal RNA (rRNA) gene and phylogenetic analysis were applied to confirm bacterial species. Antimicrobial susceptibility testing was conducted. Histopathological findings in the brain, kidney, spleen, liver, and heart were evaluated. RESULT: From 20 fish samples, L. garvieae (n = 18 isolates) and Streptococcus agalactiae (n = 2 isolates) were identified. A phylogenetic tree of the 16S rRNA gene revealed that Thai isolates of either L. garvieae or S. agalactiae clustered with reference piscine isolates from intercontinental locations. Our isolates showed resistance against quinolones while being susceptible to other antimicrobials. Histopathological changes demonstrated severe septicemic conditions, with more invasive lesions-especially in the heart and liver-being apparent in L. garvieae-infected fish compared to S. agalactiae-infected fish. CONCLUSION: This study represents the first reported outbreak of L. garvieae with a concurrent S. agalactiae infection in farmed red tilapia in Thailand.

Whole genome analysis, thermal and UV-tolerance of Lactococcus phage BIM BV-114 isolated from cheese brine.

Lactococcus phages that belong to the genus Ceduovirus are among the three most frequently isolated phage groups infecting Lactococcus lactis starter strains in dairy plants. In this study, we characterized virulent Lactococcus phage BIM BV-114 isolated from industrial cheese brine in Belarus and identified as Ceduovirus. The bacteriophage demonstrated a relatively short lytic cycle (latent period of 23 ± 5 min, lysis time of 90 ± 5 min), high thermal stability (inactivation after 7 min at 95 °C in skimmed milk) and tolerance to UV radiation (inactivation time - 15 min), indicating adaptation for better persistence in dairy facilities. The genome of the phage BIM BV-114 (21 499 bp; 37 putative open reading frames) has a similar organization to that of other Ceduovirus phages. RLf1_00140 and RLf_00050 gene products, found in the early genes region, may be involved in the sensitivity of phage to the lactococcal abortive infection mechanisms AbiV and AbiQ, respectively. Furthermore, nucleotide deletion, observed in the middle region of the gene encoding putative tape measure protein (RLf1_00300), is possibly responsible for increased thermal tolerance of phage BIM BV-114. Together, these findings will contribute to a better knowledge of virulent Lactococcus phages and the development of effective methods of their control for dairy technologies.

Structure-functional characterization of Lactococcus AbiA phage defense system.

Bacterial reverse transcriptases (RTs) are a large and diverse enzyme family. AbiA, AbiK and Abi-P2 are abortive infection system (Abi) RTs that mediate defense against bacteriophages. What sets Abi RTs apart from other RT enzymes is their ability to synthesize long DNA products of random sequences in a template- and primer-independent manner. Structures of AbiK and Abi-P2 representatives have recently been determined, but there are no structural data available for AbiA. Here, we report the crystal structure of Lactococcus AbiA polymerase in complex with a single-stranded polymerization product. AbiA comprises three domains: an RT-like domain, a helical domain that is typical for Abi polymerases, and a higher eukaryotes and prokaryotes nucleotide-binding (HEPN) domain that is common for many antiviral proteins. AbiA forms a dimer that distinguishes it from AbiK and Abi-P2, which form trimers/hexamers. We show the DNA polymerase activity of AbiA in an in vitro assay and demonstrate that it requires the presence of the HEPN domain which is enzymatically inactive. We validate our biochemical and structural results in vivo through bacteriophage infection assays. Finally, our in vivo results suggest that AbiA-mediated phage defense may not rely on AbiA-mediated cell death.

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.

Identification of ISVlu1-derived translocatable units containing optrA and/or fexA genes generated by homologous or illegitimate recombination in Lactococcus garvieae of porcine origin.

The optrA gene encodes an ABC-F protein which confers cross-resistance to oxazolidinones and phenicols. Insertion sequence ISVlu1, a novel ISL3-family member, was recently reported to be involved in the transmission of optrA in Vagococcus lutrae. However, the role of ISVlu1 in mobilizing resistance genes has not yet fully explored. In this study, two complete and three truncated copies of ISVlu1 were found on plasmid pBN62-optrA from Lactococcus garvieae. Analysis of the genetic context showed that both optrA and the phenicols resistance gene fexA were flanked by the complete or truncated ISVlu1 copies. Moreover, three different-sized ISVlu1-based translocatable units (TUs) carrying optrA and/or fexA, were detected from pBN62-optrA. Sequence analysis revealed that the TU-optrA was generated by homologous recombination while TU-fexA and TU-optrA+fexA were the products of illegitimate recombinations. Importantly, conjugation assays confirmed that pBN62-optrA was able to successfully transfer into the recipient Enterococcus faecalis JH2-2. To our knowledge, this is the first report about an optrA-carrying plasmid in L. garvieae which could horizontally transfer into other species. More importantly, the ISVlu1-flanked genetic structures containing optrA and/or fexA were also observed in bacteria of different species, which underlines that ISVlu1 is highly active and plays a vital role in the transfer of some important resistance genes, such as optrA and fexA.

Attenuation of Immunogenicity in MOG-Induced Oligodendrocytes by the Probiotic Bacterium Lactococcus Sp. PO3.

Background and Objectives: Milk is healthy and includes several vital nutrients and microbiomes. Probiotics in milk and their derivatives modulate the immune system, fight inflammation, and protect against numerous diseases. The present study aimed to isolate novel bacterial species with probiotic potential for neuroinflammation. Materials and Methods: Six milk samples were collected from lactating dairy cows. Bacterial isolates were obtained using standard methods and were evaluated based on probiotic characteristics such as the catalase test, hemolysis, acid/bile tolerance, cell adhesion, and hydrophobicity, as well as in vitro screening. Results: Nine morphologically diverse bacterial isolates were found in six different types of cow's milk. Among the isolates, PO3 displayed probiotic characteristics. PO3 was a Gram-positive rod cell that grew in an acidic (pH-2) salty medium containing bile salt and salinity (8% NaCl). PO3 also exhibited substantial hydrophobicity and cell adhesion. The sequencing comparison of the 16S rRNA genes revealed that PO3 was Lactococcus raffinolactis with a similarity score of 99.3%. Furthermore, PO3 was assessed for its neuroanti-inflammatory activity on human oligodendrocyte (HOG) cell lines using four different neuroimmune markers: signal transducer and activator of transcription (STAT-3), myelin basic protein (MBP), glial fibrillary acidic protein (GFAP), and GLAC in HOG cell lines induced by MOG. Unlike the rest of the evaluated neuroimmune markers, STAT-3 levels were elevated in the MOG-treated HOG cell lines compared to the untreated ones. The expression level of STAT-3 was attenuated in both PO3-MOG-treated and only PO3-treated cell lines. On the contrary, in PO3-treated cell lines, MBP, GFAP, and GLAC were significantly expressed at higher levels when compared with the MOG-treated cell lines. Conclusions: The findings reported in this article are to be used as a foundation for further in vivo research in order to pave the way for the possible use of probiotics in the treatment of neuroinflammatory diseases, including multiple sclerosis.

Detection and virulence of Lactococcus garvieae and L. petauri from four lakes in southern California.

OBJECTIVE: The first objective of the study aimed to detect the presence of Lactococcus petauri, L. garvieae, and L. formosensis in fish (n = 359) and environmental (n = 161) samples from four lakes near an affected fish farm in California during an outbreak in 2020. The second objective was to compare the virulence of the Lactococcus spp. in Rainbow Trout Oncorhynchus mykiss and Largemouth Bass Micropterus salmoides. METHODS: Standard bacterial culture methods were used to isolate Lactococcus spp. from brain and posterior kidney of sampled fish from the four lakes. Quantitative PCR (qPCR) was utilized to detect Lactococcus spp. DNA in fish tissues and environmental samples from the four lakes. Laboratory controlled challenges were conducted by injecting fish intracoelomically with representative isolates of L. petauri (n = 17), L. garvieae (n = 2), or L. formosensis (n = 4), and monitored for 14 days postchallenge (dpc). RESULT: Lactococcus garvieae was isolated from the brains of two Largemouth Bass in one of the lakes. Lactococcus spp. were detected in 14 fish (8 Bluegills Lepomis macrochirus and 6 Largemouth Bass) from 3 out of the 4 lakes using a qPCR assay. Of the collected environmental samples, all 4 lakes tested positive for Lactococcus spp. in the soil samples, while 2 of the 4 lakes tested positive in the water samples through qPCR. Challenged Largemouth Bass did not show any signs of infection postinjection throughout the challenge period. Rainbow Trout infected with L. petauri showed clinical signs within 3 dpc and presented a significantly higher cumulative mortality (62.4%; p < 0.0001) at 14 dpc when compared to L. garvieae (0%) and L. formosensis (7.5%) treatments. CONCLUSION: The study suggests that qPCR can be used for environmental DNA monitoring of Lactococcus spp. and demonstrates virulence diversity between the etiological agents of piscine lactococcosis.

Resistance and genomic characterization of a plasmid pkh2101 harbouring erm(B) isolated from emerging fish pathogen Lactococcus garvieae serotype II in Japan.

The emergence of antibiotic-resistant pathogenic strains of Lactococcus garvieae serotype II isolated from fish in Japan has become a growing concern in recent years. The data on drug susceptibility and its associated resistance mechanism are limited. Therefore, the present study was conducted to determine the minimum inhibitory concentrations (MICs) of chemotherapeutic agents against 98 pathogenic strains of emerging Lactococcus garvieae serotype II isolated from fish from six different prefectures in Japan from 2018 to 2021. The tested strains were resistant to erythromycin, lincomycin and tiamulin. PCR amplification revealed the presence of erm(B) in all erythromycin-resistant strains, while a conjugation experiment confirmed that these strains carried erm(B) that could be transferred to recipient Enterococcus faecalis OG1RF with frequencies from 10-4 to 10-6 per donor cells. Nucleotide sequencing of the representative isolated plasmid pkh2101 from an erythromycin-resistant strain showed that it was a 26,850 bp molecule with an average GC content of 33.49%, comprising 31 CDSs, 13 of which remained without any functional annotation. Comparative genomic analysis suggested that pkh2101 shared the highest similarity (97.57% identity) with the plasmid pAMbeta1, which was previously isolated clinically from Enterococcus faecalis DS-5. This study provides potential evidence that the plasmid harbouring erm(B) could be a source of antibiotic resistance transmission in emerging L. garvieae infection in aquaculture.

Comparative genome analyses of three serotypes of Lactococcus bacteria isolated from diseased cultured striped jack (Pseudocaranx dentex).

Lactococcosis, caused by members of the genus Lactococcus, represents a devastating disease inducing mass mortalities and economic losses in many fish species worldwide. The present work aimed to compare the whole genome sequences of three different serotypes of Lactococcus garvieae isolated from diseased cultured striped jack (Pseudocaranx dentex) in Ehime prefecture, Japan. The three serotypes showed different virulence in the challenge test using Japanese amberjack (Seriola quinqueradiata). The genome sequencing revealed that two of the strains (serotype I and serotype III) were identified as L. garvieae, while the third strain (serotype II) was identified as L. formosensis. The chromosome sizes of the three serotypes ranged from 1.9 to 2.0 Mb; the GC content ranges were 38.2 to 38.9%; and the numbers of predicted protein-coding sequences (CDSs) were from 1922 to 1959. Only the serotype II harbours two plasmids, sizes of around 14 kb and 9 kb. The detected virulence factors varied among the different serotypes with some shared factors like adherence, anti-phagocytosis, secretion system, toxin (haemolysin), serum resistance, antimicrobial resistance and others. The genomes also contained factors responsible for resistance to toxic compounds. The genome of the serotype III tended to encode more prophage regions than the other serotypes.

Crystal structure and biochemical analysis of acetylesterase (LgEstI) from Lactococcus garvieae.

Esterase, a member of the serine hydrolase family, catalyzes the cleavage and formation of ester bonds with high regio- and stereospecificity, making them attractive biocatalysts for the synthesis of optically pure molecules. In this study, we performed an in-depth biochemical and structural characterization of a novel microbial acetylesterase, LgEstI, from the bacterial fish pathogen Lactococcus garvieae. The dimeric LgEstI displayed substrate preference for the short acyl chain of p-nitrophenyl esters and exhibited increased activity with F207A mutation. Comparative analysis with other esterases indicated that LgEstI has a narrow and shallow active site that may exhibit substrate specificity to short acyl chains. Unlike other esterases, LgEstI contains bulky residues such as Trp89, Phe194, and Trp217, which block the acyl chain channel. Furthermore, immobilized LgEstI retained approximately 90% of its initial activity, indicating its potential in industrial applications. This study expands our understanding of LgEstI and proposes novel ideas for improving its catalytic efficiency and substrate specificity for various applications.

Isolation of Lactococcus garvieae NEP21 from raw cow (Bos indicus) milk in Nepal.

Lactococcus garvieae is an emerging zoonotic pathogen impacting both humans and animals. Infection of this bacterium is known to cause mastitis in cattle, and endocarditis, osteomyelitis, liver abscess, and gastrointestinal problems are reported in immunocompromised and elderly people that regularly consume or handle raw meat, milk, dairy products, and seafood. This study aimed at investigating and detecting lactic acid bacteria in raw cow (Bos indicus) milk samples from a smallholder farm in Nepal. Based on the plate culture, biochemical tests, and molecular sequencing of 16 s ribosomal RNA coding nuclear DNA region followed by phenotypic and genotypic analyses, L. garvieae NEP21 was detected and identified for the first time in Nepal in raw cow milk samples. This finding suggests the prevalence of L. garvieae NEP21 in raw cow milk and recommends further research and surveillance for understanding the extent of its presence in Nepal and globally for informed management of its infection in cattle and humans.

NGS analysis revealed that Lactococcus garvieae Lg-Per was Lactococcus petauri in Türkiye.

Lactococcus garvieae Lg-per was originally isolated from rainbow trout cultured in cages located on the Turkish coast of the Black Sea in 2011. A whole genome sequence of Lg-per was performed in the present study. The complete genome of Lg-per mapped to the reference genomes of L. garvieae (GCF_000269925.1) and Lactococcus petauri (GCF_014830225.1) had a total of 1,694,407 and 1,945,297 base pairs, respectively. Lg-per had 1955 protein-coding genes and 4 rRNA, 46 tRNA and 1 tmRNA operons. The orthoANI value was 98.30% between Lg-per and L. petauri (GCF_014830225.1) and 93.1% between Lg-per and L. garvieae (GCF_000269925.1). A phylogenetic tree generated from the whole genome sequences (WGS) of several Lactococcus species found that L. petauri (GCA 002154895) was closely related to the Lg-per strain with 98% similarity. Although L. garvieae Lg-per was confirmed as L. garvieae based on phenotypical, biochemical and 16S rRNA sequence, WGS of the Lg-per strain revealed that Lg-per was L. petauri. Using a 16S rRNA-based PCR detection approach, Lg-per was misdiagnosed as L. garvieae since its 16S rRNA gene was 99.9% similar to that of L. garvieae strains. Consequently, the 16S rRNA-based PCR detection approach may not be adequate for the identification of the Lactococcus genus. This is the first study to document the presence of L. petauri in Türkiye. L. garvieae isolates should be analysed using WGS since the same issue might occur in other countries.

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.

Expression, purification, and characterization of glutamate decarboxylase from human gut-originated Lactococcus garvieae MJF010.

Human gut-originated lactic acid bacteria were cultivated, and high γ-aminobutyric acid (GABA)-producing Lactococcus garvieae MJF010 was identified. To date, despite the importance of GABA, no studies have investigated GABA-producing Lactococcus species, except for Lc. lactis. A recombinant glutamate decarboxylase of the strain MJF010 (rLgGad) was successfully expressed in Escherichia coli BL21(DE3) with a size of 53.9 kDa. rLgGad could produce GABA, which was verified using the silylation-derivative fragment ions of GABA. The purified rLgGad showed the highest GABA-producing activity at 35 °C and pH 5. rLgGad showed a melting temperature of 43.84 °C. At 30 °C, more than 80% of the activity was maintained even after 7 h; however, it rapidly decreased at 50 °C. The kinetic parameters, Km, Vmax, and kcat, of rLgGad were 2.94 mM, 0.023 mM/min, and 12.3 min- 1, respectively. The metal reagents of CaCl2, MgCl2, and ZnCl2 significantly had positive effects on rLgGad activity. However, most coenzymes including pyridoxal 5'-phosphate showed no significant effects on enzyme activity. In conclusion, this is the first report of Gad from Lc. garvieae species and provides important enzymatic information related to GABA biosynthesis in the Lactococcus genus.

Genotyping and phenotyping of Lactococcus garvieae isolates from fish by pulse-field gel electrophoresis (PFGE) and electron microscopy indicate geographical and capsular variations.

Lactococcus garvieae is the etiological agent of Lactococcosis, an evolving disease affecting many fish species and causing significant economic losses worldwide. Assessing pathogen relatedness and bacterial population structure is critical for determining the epidemiology of L. garvieae infections and in establishing effective pathogen management methods. The previously published morphological and genetic studies point to a clonal population structure, as seen in other fish bacteria. In the present study, the pulsed-field gel electrophoresis (PFGE) method was utilized to define a population of 41 Taiwanese isolates from outbreaks with comparisons to four well-characterized non-Taiwanese isolates previously published. Two restriction enzymes (ApaI and SmaI) were utilized individually for PFGE analysis (cut-off value = 90.0%), revealing genetic heterogeneity across L. garvieae isolates, with ApaI and SmaI yielding 12 and seven distinct PFGE band patterns, respectively. The phylogenic analysis using internal transcribed spacer region clustered all L. garvieae isolates in the same clad. Furthermore, the electron microscopic results confirmed the absence of capsular gene cluster (CGC) in previously characterized Taiwanese vaccine strain (S3) from grey mullet. Overall, our findings emphasize the importance of analysing the morphological and genetic diversity in L. garvieae being correlated for proper taxonomic classification in vaccine strain selection and epidemiological studies.

Development of a quantitative polymerase chain reaction assay for detection of the aetiological agents of piscine lactococcosis.

Piscine lactococcosis is an emergent bacterial disease that is associated with high economic losses in many farmed and wild aquatic species worldwide. Early and accurate detection of the causative agent of piscine lactococcosis is essential for management of the disease in fish farms. In this study, a TaqMan quantitative polymerase chain reaction (qPCR) targeting the 16S-23S rRNA internal transcribed spacer region was developed and validated. Validation of the qPCR was performed with DNA of previously typed L. petauri and L. garvieae recovered from different aquatic hosts from distinct geographical locations, closely related bacterial species and common pathogens in trout aquaculture. Further diagnostic sensitivity and specificity was investigated by screening of fish, water and faecal samples. The developed qPCR assay showed high specificity, sensitivity and accuracy in detection of L. petauri and L. garvieae with lack of signals from non-target pathogens, and in screening of rainbow trout (Oncorhynchus mykiss) posterior kidney and environmental samples. The detection limit of the qPCR was four amplicon copies. Moreover, the sensitivity of the qPCR assay was not affected by presence of non-target DNA from either fish or environmental samples. The robustness, specificity and sensitivity of the developed qPCR will facilitate fast and accurate diagnosis of piscine lactococcosis to establish appropriate control measures in fish farms and aquaria.