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.

New Genetic Determinants for qPCR Identification and the Enumeration of Selected Lactic Acid Bacteria in Raw-Milk Cheese.

Lactic acid bacteria (LAB) play an important role in the ripening of cheeses and contribute to the development of the desired profile of aroma and flavor compounds. Therefore, it is very important to monitor the dynamics of bacterial proliferation in order to obtain an accurate and reliable number of their cells at each stage of cheese ripening. This work aimed to identify and conduct a quantitative assessment of the selected species of autochthonous lactic acid bacteria from raw cow's milk cheese by the development of primers and probe pairs based on the uniqueness of the genetic determinants with which the target microorganisms can be identified. For that purpose, we applied real-time quantitative PCR (qPCR) protocols to quantify Lactobacillus delbrueckii subsp. bulgaricus, Streptococcus thermophilus, and Lactococcus lactis subsp. cremoris cells in cheese directly after production and over three-month and six-month ripening periods. While L. lactis subsp. cremoris shows good acidification ability and the ability to produce antimicrobial compounds, L. delbrueckii subsp. bulgaricus has good proteolytic ability and produces exo-polysaccharides, and S. thermophilus takes part in the formation of the diacetyl flavor compound by metabolizing citrate to develop aroma, they all play an important role in the cheese ripening. The proposed qPCR protocols are very sensitive and reliable methods for a precise enumeration of L. delbrueckii subsp. bulgaricus, S. thermophilus, and L. lactis subsp. cremoris in cheese samples.

A high fiber diet or supplementation with Lactococcus lactis subspecies cremoris to pregnant mice confers protection against intestinal injury in adult offspring.

The diet during pregnancy, or antenatal diet, influences the offspring's intestinal health. We previously showed that antenatal butyrate supplementation reduces injury in adult murine offspring with dextran sulfate sodium (DSS)-induced colitis. Potential modulators of butyrate levels in the intestine include a high fiber diet or dietary supplementation with probiotics. To test this, we supplemented the diet of pregnant mice with high fiber, or with the probiotic bacteria Lactococcus lactis subspecies cremoris or Lactobacillus rhamnosus GG. We then induced chronic colitis with DSS in their adult offspring. We demonstrate that a high fiber antenatal diet, or supplementation with Lactococcus lactis subspecies cremoris during pregnancy diminished the injury from DSS-induced colitis in offspring. These data are evidence that antenatal dietary interventions impact offspring gut health and define the antenatal diet as a therapeutic modality to enhance offspring intestinal health.

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.

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 exerts a critical role in inducing inflammation in dairy mastitis by triggering NLRP3 inflammasome-mediated pyroptosis in MAC-T cells.

Lactococcus garvieae (L. garvieae) is a pathogenic bacterium that is Gram-positive and catalase-negative (GPCN), and it is capable of growing in a wide range of environmental conditions. This bacterium is associated with significant mortality and losses in fisheries, and there are concerns regarding its potential as a zoonotic pathogen, given its presence in cattle and dairy products. While we have identified and characterized virulent strains of L. garvieae through phenotyping and molecular typing studies, their impact on mammary tissue remains unknown. This study aims to investigate the pathogenicity of strong and weak virulent strains of L. garvieae using in vivo mouse models. We aim to establish MAC-T cell model to examine potential injury caused by the strong virulent strain LG41 through the TLR2/NLRP3/NF-kB pathway. Furthermore, we assess the involvement of NLRP3 inflammasome-mediated pyroptosis in dairy mastitis by silencing NLRP3. The outcomes of this study will yield crucial theoretical insights into the potential mechanisms involved in mastitis in cows caused by the L. garvieae-induced inflammatory response in MAC-T cells.

Characterization of a Panel of Constitutive Promoters from Lactococcus cremoris for Fine-Tuning Gene Expression.

Lactococcus cremoris (homotypic synonym: Lactococcus lactis) is receiving increasing attention as a prominent vehicle for the delivery of live vaccines. This can hardly be achieved without developing tools for the genetic manipulation of L. cremoris, and the paucity of studies on L. cremoris endogenous promoters has attracted our attention. Here, we report the discovery and characterization of 29 candidate promoters identified from L. cremoris subsp. cremoris NZ9000 by RNA sequencing analysis. Furthermore, 18 possible constitutive promoters were obtained by RT-qPCR screening from these 29 candidate promoters. Then, these 18 promoters were cloned and characterized by a reporter gene, gusA, encoding ß-glucuronidase. Eventually, eight endogenous constitutive promoters of L. cremoris were obtained, which can be applied to genetic manipulation of lactic acid bacteria.

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.

Ribosome profiling reveals downregulation of UMP biosynthesis as the major early response to phage infection.

Bacteria have evolved diverse defense mechanisms to counter bacteriophage attacks. Genetic programs activated upon infection characterize phage-host molecular interactions and ultimately determine the outcome of the infection. In this study, we applied ribosome profiling to monitor protein synthesis during the early stages of sk1 bacteriophage infection in Lactococcus cremoris. Our analysis revealed major changes in gene expression within 5 minutes of sk1 infection. Notably, we observed a specific and severe downregulation of several pyr operons which encode enzymes required for uridine monophosphate biosynthesis. Consistent with previous findings, this is likely an attempt of the host to starve the phage of nucleotides it requires for propagation. We also observed a gene expression response that we expect to benefit the phage. This included the upregulation of 40 ribosome proteins that likely increased the host's translational capacity, concurrent with a downregulation of genes that promote translational fidelity (lepA and raiA). In addition to the characterization of host-phage gene expression responses, the obtained ribosome profiling data enabled us to identify two putative recoding events as well as dozens of loci currently annotated as pseudogenes that are actively translated. Furthermore, our study elucidated alterations in the dynamics of the translation process, as indicated by time-dependent changes in the metagene profile, suggesting global shifts in translation rates upon infection. Additionally, we observed consistent modifications in the ribosome profiles of individual genes, which were apparent as early as 2 minutes post-infection. The study emphasizes our ability to capture rapid alterations of gene expression during phage infection through ribosome profiling. IMPORTANCE: The ribosome profiling technology has provided invaluable insights for understanding cellular translation and eukaryotic viral infections. However, its potential for investigating host-phage interactions remains largely untapped. Here, we applied ribosome profiling to Lactococcus cremoris cultures infected with sk1, a major infectious agent in dairy fermentation processes. This revealed a profound downregulation of genes involved in pyrimidine nucleotide synthesis at an early stage of phage infection, suggesting an anti-phage program aimed at restricting nucleotide availability and, consequently, phage propagation. This is consistent with recent findings and contributes to our growing appreciation for the role of nucleotide limitation as an anti-viral strategy. In addition to capturing rapid alterations in gene expression levels, we identified translation occurring outside annotated regions, as well as signatures of non-standard translation mechanisms. The gene profiles revealed specific changes in ribosomal densities upon infection, reflecting alterations in the dynamics of the translation process.

Resistance role of Lactobacillus sp. and Lactococcus sp. to copper ions in healthy children's intestinal microorganisms.

Heavy metal exposure is closely associated with gut microbe function and tolerance. However, intestinal microbe responses in children to different copper ion (Cu2+) concentrations have not yet been clarified. Here, in vitro cultivation systems were established for fecal microbe control and Cu2+-treated groups in healthy children. 16S rDNA high-throughput sequencing, meta-transcriptomics and metabolomics were used here to identify toxicity resistance mechanisms at microbiome levels. The results showed that Lactobacillus sp. and Lactococcus sp. exerted protective effects against Cu2+ toxicity, but these effects were limited by Cu2+ concentration. When the Cu2+ concentration was ≥ 4 mg/L, the abundance of Lactobacillus sp. and Lactococcus sp. significantly decreased, and the pathways of antioxidant activity and detoxification processes were enriched at 2 mg/L Cu2+, and beneficial metabolites accumulated. However, at high concentrations of Cu2+ (≥4 mg/L), the abundance of potential pathogen increased, and was accompanied by a downregulation of genes in metabolism and detoxification pathways, which meant that the balance of gut microbiota was disrupted and toxicity resistance decreased. From these observations, we identified some probiotics that are tolerant to heavy metal Cu2+, and warn that only when the concentration limit of Cu2+ in food is 2 mg/L, then a balanced gut microbiota can be guaranteed in children, thereby providing protection for their health.

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.

Construction of recombinant Lactococcus expressing thymosin and interferon fusion protein and its application as an immune adjuvant.

BACKGROUND: In recent years, biosafety and green food safety standards have increased the demand for immune enhancers and adjuvants. In the present study, recombinant food-grade Lactococcus lactis (r-L. lactis-Tα1-IFN) expressing thymosin Tα1 and chicken interferon fusion protein was constructed. RESULTS: The in vitro interactions with macrophages revealed a mixture of recombinant r-L. lactis-Tα1-IFN could significantly activate both macrophage J774-Dual™ NF-κB and interferon regulator (IRF) signaling pathways. In vitro interactions with chicken peripheral blood mononuclear cells (PBMCs) demonstrated that a mixture of recombinant r-L. lactis-Tα1-IFN significantly enhanced the expression levels of interferon (IFN)-γ, interleukin (IL)-10, CD80, and CD86 proteins in chicken PBMCs. Animal experiments displayed that injecting a lysis mixture of recombinant r-L. lactis-Tα1-IFN could significantly activate the proliferation of T cells and antigen-presenting cells in chicken PBMCs. Moreover, 16S analysis of intestinal microbiota demonstrated that injection of the lysis mixture of recombinant r-L. lactis-Tα1-IFN could significantly improve the structure and composition of chicken intestinal microbiota, with a significant increase in probiotic genera, such as Lactobacillus spp. Results of animal experiments using the lysis mixture of recombinant r-L. lactis-Tα1-IFN as an immune adjuvant for inactivated chicken Newcastle disease vaccine showed that the serum antibody titers of the experimental group were significantly higher than those of the vaccine control group, and the expression levels of cytokines IFN-γ and IL-2 were significantly higher than those of the vaccine control group. CONCLUSION: These results indicate that food-safe recombinant r-L. lactis-Tα1-IFN has potential as a vaccine immune booster and immune adjuvant. This study lays the foundation for the development of natural green novel animal immune booster or immune adjuvant.

Characteristics of Lactococcus petauri GB97 lysate isolated from porcine feces and its in vitro and in vivo effects on inflammation, intestinal barrier function, and gut microbiota composition in mice.

IMPORTANCE: Weaning is a crucial step in piglet management to improve pork production. During the weaning phase, disruption of epithelial barrier function and intestinal inflammation can lead to decreased absorption of nutrients and diarrhea. Therefore, maintaining a healthy intestine, epithelial barrier function, and gut microbiota composition in this crucial phase is strategic for optimal weaning in pigs. We isolated a lysate of Lactococcus petauri GB97 (LPL97) from healthy porcine feces and evaluated its anti-inflammatory activities, barrier integrity, and gut microbial changes in LPS-induced murine macrophages and DSS-induced colitis mice. We found that LPL97 regulated the immune response by downregulating the TLR4/NF-κB/MAPK signaling pathway both in vitro and in vivo. Furthermore, LPL97 alleviated the disruption of intestinal epithelial integrity and gut microbiota dysbiosis in colitis mice. This study indicates that LPL97 has the potential to be developed as an alternative feed additive to antibiotics for the swine industry.

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.

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.

Natural diversity of lactococci in γ-aminobutyric acid (GABA) production and genetic and phenotypic determinants.

BACKGROUND: γ-aminobutyric acid (GABA) is a bioactive compound produced by lactic acid bacteria (LAB). The diversity of GABA production in the Lactococcus genus is poorly understood. Genotypic and phenotypic approaches were therefore combined in this study to shed light on this diversity. A comparative genomic study was performed on the GAD-system genes (gadR, gadC and gadB) involved in GABA production in 36 lactococci including L. lactis and L. cremoris species. In addition, 132 Lactococcus strains were screened for GABA production in culture medium supplemented with 34 mM L-glutamic acid with or without NaCl (0.3 M). RESULTS: Comparative analysis of the nucleotide sequence alignments revealed the same genetic organization of the GAD system in all strains except one, which has an insertion sequence element (IS981) into the PgadCB promoter. This analysis also highlighted several deletions including a 3-bp deletion specific to the cremoris species located in the PgadR promoter, and a second 39-bp deletion specific to L. cremoris strains with a cremoris phenotype. Phenotypic analysis revealed that GABA production varied widely, but it was higher in L. lactis species than in L. cremoris, with an exceptional GABA production of up to 14 and 24 mM in two L. lactis strains. Moreover, adding chloride increased GABA production in some L. cremoris and L. lactis strains by a factor of up to 16 and GAD activity correlated well with GABA production. CONCLUSIONS: This genomic analysis unambiguously characterized the cremoris phenotype of L. cremoris species and modified GadB and GadR proteins explain why the corresponding strains do not produce GABA. Finally, we found that glutamate decarboxylase activity revealing GadB protein amount, varied widely between the strains and correlated well with GABA production both with and without chloride. As this protein level is associated to gene expression, the regulation of GAD gene expression was identified as a major contributor to this diversity.

First identification and histopathological analysis of Lactococcus garvieae infection in whiteleg shrimp, Penaeus vannamei cultured in low salinity water.

The isolation and characterization of bacterial species Lactococcus garvieae, previously unreported in whiteleg shrimp, Penaeus vannamei, has now been identified in the species. The pathogen was recovered from an affected shrimp farm in southern Taiwan. Bacterial characterization first identified the isolate as Gram-positive cocci, and biochemical profiles demonstrated that the causative agent of mortality was 97% L. garvieae. The bacterial cell DNA resulted in amplification of 1522 bp with 99.6% confirmation by PCR analysis. The phylogenetic tree revealed 100% evolutionary similarity among previously isolated strains. Experimental infection further confirmed higher susceptibility of whiteleg shrimp to L. garvieae in waters of lower salinity, particularly 5 ppt, than in higher salinity. Histopathological analysis showed severely damaged hepatopancreas with necrotized, elongated, collapsed tubules, dislodged membranes and granuloma formation in infected shrimp. Transmission electron microscopy observation indicated a hyaluronic acid capsular layer surrounding bacterial cell which is a virulence factor of L. garvieae and likely responsible for immunosuppression and higher mortality of shrimp cultured in lower salinity. Collectively, these findings report the first isolation of L. garvieae from whiteleg shrimp and shed new light on the disease that threatens the highly valuable species and accentuates the need for finding a solution.

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.