RESUMO
Probiotics are widely prescribed for prevention of antibiotics-associated dysbiosis and related adverse effects. However, probiotic impact on post-antibiotic reconstitution of the gut mucosal host-microbiome niche remains elusive. We invasively examined the effects of multi-strain probiotics or autologous fecal microbiome transplantation (aFMT) on post-antibiotic reconstitution of the murine and human mucosal microbiome niche. Contrary to homeostasis, antibiotic perturbation enhanced probiotics colonization in the human mucosa but only mildly improved colonization in mice. Compared to spontaneous post-antibiotic recovery, probiotics induced a markedly delayed and persistently incomplete indigenous stool/mucosal microbiome reconstitution and host transcriptome recovery toward homeostatic configuration, while aFMT induced a rapid and near-complete recovery within days of administration. In vitro, Lactobacillus-secreted soluble factors contributed to probiotics-induced microbiome inhibition. Collectively, potential post-antibiotic probiotic benefits may be offset by a compromised gut mucosal recovery, highlighting a need of developing aFMT or personalized probiotic approaches achieving mucosal protection without compromising microbiome recolonization in the antibiotics-perturbed host.
Assuntos
Antibacterianos/farmacologia , Microbioma Gastrointestinal/efeitos dos fármacos , Probióticos/administração & dosagem , Adolescente , Adulto , Idoso , Animais , Transplante de Microbiota Fecal , Fezes/microbiologia , Feminino , Humanos , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/microbiologia , Lactobacillus/efeitos dos fármacos , Lactobacillus/genética , Lactobacillus/isolamento & purificação , Lactococcus/genética , Lactococcus/isolamento & purificação , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , RNA Ribossômico 16S/análise , RNA Ribossômico 16S/genética , RNA Ribossômico 16S/metabolismo , Adulto JovemRESUMO
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.
Assuntos
Proteínas de Bactérias , Regulação Bacteriana da Expressão Gênica , Redes Reguladoras de Genes , Lactococcus lactis , Lactococcus lactis/genética , Lactococcus lactis/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Transformação Bacteriana/genética , Lactococcus/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Competência de Transformação por DNA/genéticaRESUMO
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.
Assuntos
Bacteriófagos , Lactococcus , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Bacteriófagos/genética , Cristalografia por Raios X , Lactococcus/virologia , Lactococcus/genética , Modelos Moleculares , Domínios Proteicos , Multimerização Proteica , DNA Polimerase Dirigida por RNA/metabolismo , DNA Polimerase Dirigida por RNA/química , DNA Polimerase Dirigida por RNA/genética , Relação Estrutura-AtividadeRESUMO
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.
Assuntos
Parede Celular , Lactococcus , Polissacarídeos Bacterianos , Ramnose , Proteínas de Bactérias/metabolismo , Parede Celular/química , Parede Celular/metabolismo , Glicosiltransferases/metabolismo , Lactococcus/classificação , Lactococcus/citologia , Lactococcus/metabolismo , Lactococcus/virologia , Lipídeos , Peptidoglicano/metabolismo , Polissacarídeos Bacterianos/metabolismo , Conformação Proteica , Ramnose/metabolismo , Especificidade por Substrato , Bacteriófagos/fisiologiaRESUMO
Dairy industries apply selected lactococcal strains and mixed cultures to produce diverse fermented products with distinctive flavor and texture properties. Innovation of the starter culture functionality in cheese applications embraces natural biodiversity of the Lactococcus species to identify novel strains with alternative flavor or texture forming capacities and/or increased processing robustness and phage resistance. Mobile genetic elements (MGE), like integrative conjugative elements (ICEs) play an important role in shaping the biodiversity of bacteria. Besides the genes involved in the conjugation of ICEs from donor to recipient strains, these elements also harbor cargo genes that encode a wide range of functions. The definition of such cargo genes can only be achieved by accurate identification of the ICE boundaries (delimiting). Here, we delimited 25 ICEs in lactococcal genome sequences with low contig numbers using insertion-sites flanking single-copy core-genome genes as markers for each of the distinct ICE-integrases we identified previously within the conserved ICE-core genes. For ICEs in strains for which genome information with large numbers of contigs is available, we exemplify that CRISPR-Cas9 driven ICE-curing, followed by resequencing, allows accurate delimitation and cargo definition of ICEs. Finally, we compare and contrast the cargo gene repertoire of the 26 delimited lactococcal ICEs, identifying high plasticity among the cargo of lactococccal ICEs and a range of encoded functions that is of apparent industrial interest, including restriction modification, abortive infection, and stress adaptation genes.
Assuntos
Genoma Bacteriano , Lactococcus/genética , Sequências Repetitivas Dispersas/genética , Sistemas CRISPR-Cas , Conjugação GenéticaRESUMO
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.
Assuntos
Doenças dos Peixes , Infecções por Bactérias Gram-Positivas , Lactococcus , Lactococcus/genética , Lactococcus/isolamento & purificação , Lactococcus/classificação , Animais , Doenças dos Peixes/microbiologia , Infecções por Bactérias Gram-Positivas/microbiologia , Infecções por Bactérias Gram-Positivas/veterinária , Peixes/microbiologia , Sequenciamento Completo do Genoma , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
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.
Assuntos
Lactococcus lactis , Timosina , Vacinas , Animais , Interferons/metabolismo , Lactococcus , Leucócitos Mononucleares , Adjuvantes Imunológicos/metabolismo , Proteínas Recombinantes/metabolismo , Timosina/metabolismo , Vacinas/metabolismo , Galinhas , Lactococcus lactis/metabolismoRESUMO
AIMS: This study aimed to prospect and isolate lactic acid bacteria (LAB) from an artisanal cheese production environment, to assess their safety, and to explore their bacteriocinogenic potential against Listeria monocytogenes. METHODS AND RESULTS: Samples were collected from surfaces of an artisanal-cheese production facility and after rep-PCR and 16S rRNA sequencing analysis, selected strains were identified as to be belonging to Lactococcus garvieae (1 strain) and Enterococcus faecium (14 isolates, grouped into three clusters) associated with different environments (worktables, cheese mold, ripening wooden shelves). All of them presented bacteriocinogenic potential against L. monocytogenes ATCC 7644 and were confirmed as safe (γ-hemolytic, not presenting antibiotic resistance, no mucus degradation properties, and no proteolytic or gelatinase enzyme activity). Additionally, cell growth, acidification and bacteriocins production kinetics, bacteriocin stability in relation to different temperatures, pH, and chemicals were evaluated. According to performed PCR analysis all studied strains generated positive evidence for the presence of entA and entP genes (for production of enterocins A and enterocins P, respectively). However, pediocin PA-1 associated gene was recorded only in DNA obtained from E. faecium ST02JL and Lc. garvieae ST04JL. CONCLUSIONS: It is worth considering the application of these safe LAB or their bacteriocins in situ as an alternative means of controlling L. monocytogenes in cheese production environments, either alone or in combination with other antimicrobials.
Assuntos
Bacteriocinas , Queijo , Enterococcus faecium , Microbiologia de Alimentos , Lactococcus , Listeria monocytogenes , Queijo/microbiologia , Enterococcus faecium/genética , Enterococcus faecium/isolamento & purificação , Enterococcus faecium/metabolismo , Lactococcus/genética , Lactococcus/isolamento & purificação , Bacteriocinas/farmacologia , Brasil , Listeria monocytogenes/genética , Listeria monocytogenes/efeitos dos fármacos , RNA Ribossômico 16S/genética , Antibacterianos/farmacologiaRESUMO
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.
Assuntos
Queijo , Lactobacillales , Lactobacillus delbrueckii , Lactococcus lactis , Lactococcus , Animais , Bovinos , Feminino , Lactobacillales/genética , Leite , Reação em Cadeia da Polimerase em Tempo Real , Lactobacillus delbrueckii/genética , Lactococcus lactis/genéticaRESUMO
BACKGROUND: Although microorganisms are the main cause of spoilage in prepared beef steaks, very few deep spoilage mechanisms have been reported so far. Aiming to unravel the mechanisms during 12 days of storage at 4 °C affecting the quality of prepared beef steak, the present study investigated the changes in microbial dynamic community using a combined high-throughput sequencing combined and bioinformatics. In addition, gas chromatography-mass spectrometry combined with multivariate statistical analysis was utilized to identify marker candidates for prepared steaks. Furthermore, cloud platform analysis was applied to determine prepared beef steak spoilage, including the relationship between microbiological and physicochemical indicators and volatile compounds. RESULTS: The results showed that the dominant groups of Pseudomonas, Brochothrix thermosphacta, Lactobacillus and Lactococcus caused the spoilage of prepared beef steak, which are strongly associated with significant changes in physicochemical properties and volatile organic compounds (furan-2-pentyl-, pentanal, 1-octanol, 1-nonanol and dimethyl sulfide). Metabolic pathways were proposed, among which lipid metabolism and amino acid metabolism were most abundant. CONCLUSION: The present study is helpful with respect to further understanding the relationship between spoilage microorganisms and the quality of prepared beef steak, and provides a reference for investigating the spoilage mechanism of dominant spoilage bacteria and how to extend the shelf life of meat products. © 2024 Society of Chemical Industry.
Assuntos
Bactérias , Biologia Computacional , Compostos Orgânicos Voláteis , Bovinos , Animais , Compostos Orgânicos Voláteis/química , Compostos Orgânicos Voláteis/metabolismo , Compostos Orgânicos Voláteis/análise , Bactérias/genética , Bactérias/classificação , Bactérias/isolamento & purificação , Bactérias/metabolismo , Cromatografia Gasosa-Espectrometria de Massas , Microbiologia de Alimentos , Armazenamento de Alimentos , Pseudomonas/crescimento & desenvolvimento , Pseudomonas/metabolismo , Lactobacillus/metabolismo , Refrigeração , Brochothrix/metabolismo , Brochothrix/crescimento & desenvolvimento , Brochothrix/isolamento & purificação , Lactococcus , Carne Vermelha/microbiologia , Carne Vermelha/análiseRESUMO
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.
Assuntos
Doenças dos Peixes , Infecções por Bactérias Gram-Positivas , Lactococcus , Filogenia , Infecções Estreptocócicas , Streptococcus agalactiae , Animais , Streptococcus agalactiae/isolamento & purificação , Streptococcus agalactiae/efeitos dos fármacos , Streptococcus agalactiae/genética , Doenças dos Peixes/microbiologia , Doenças dos Peixes/epidemiologia , Tailândia/epidemiologia , Lactococcus/isolamento & purificação , Lactococcus/classificação , Lactococcus/genética , Infecções Estreptocócicas/veterinária , Infecções Estreptocócicas/microbiologia , Infecções Estreptocócicas/epidemiologia , Infecções por Bactérias Gram-Positivas/veterinária , Infecções por Bactérias Gram-Positivas/microbiologia , Infecções por Bactérias Gram-Positivas/epidemiologia , Tilápia/microbiologia , Ciclídeos , RNA Ribossômico 16S/genéticaRESUMO
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.
Assuntos
Inflamassomos , Mastite , Humanos , Feminino , Animais , Bovinos , Camundongos , Inflamassomos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Piroptose , Linfócitos T/metabolismo , Lactococcus/metabolismo , Mastite/microbiologia , Mastite/veterinária , InflamaçãoRESUMO
Efficient genome editing methods are essential for biotechnology and fundamental research. Homologous recombination (HR) is the most versatile method of genome editing, but techniques that rely on host RecA-mediated pathways are inefficient and laborious. Phage-encoded single-stranded DNA annealing proteins (SSAPs) improve HR 1,000-fold above endogenous levels. However, they are not broadly functional. Using Escherichia coli, Lactococcus lactis, Mycobacterium smegmatis, Lactobacillus rhamnosus and Caulobacter crescentus, we investigated the limited portability of SSAPs. We find that these proteins specifically recognize the C-terminal tail of the host's single-stranded DNA-binding protein (SSB) and are portable between species only if compatibility with this host domain is maintained. Furthermore, we find that co-expressing SSAPs with SSBs can significantly improve genome editing efficiency, in some species enabling SSAP functionality even without host compatibility. Finally, we find that high-efficiency HR far surpasses the mutational capacity of commonly used random mutagenesis methods, generating exceptional phenotypes that are inaccessible through sequential nucleotide conversions.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Edição de Genes/métodos , Recombinação Homóloga/fisiologia , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Bacteriófagos/genética , Bacteriófagos/metabolismo , Caulobacter crescentus/metabolismo , DNA/química , DNA/genética , Reparo do DNA , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/química , Escherichia coli/metabolismo , Recombinação Homóloga/genética , Lactococcus/metabolismo , Mycobacterium smegmatis/metabolismo , Domínios Proteicos/genéticaRESUMO
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.
Assuntos
Cloretos , Lactococcus , Fenótipo , Meios de Cultura , Ácido gama-AminobutíricoRESUMO
A gram-positive, facultatively anaerobic, and coccoid or ovoid-shaped bacterium designated M2458T was isolated from the intestinal contents of APPswe/PSΔE9 mouse model of Alzheimer's disease. With the polyphasic approach, the taxonomic position of the novel isolate was confirmed. Strain M2458T grew well at 37 °C on YCFA agar. Strain M2458T belongs to the family Streptococcaceae and class Bacilli, and it is closed to Lactococcus formosensis NBRC 109475T (97.59% sequence similarity) according to its 16S rRNA gene sequence. In a comparison of two housekeeping genes, rpoA and rpoB, strain M2458T was found to be well separated from Lactococcus formosensis NBRC 109475T. On the basis of whole genome sequences, the DNA G+C content was 38.29 mol%. The phylogentic analysis of the whole genome showed that a different branch was clearly formed in the phylogenetic tree of strain M2458T compared to other strains in the genus Lactococcus. A total of eight genes in strain M2458T are involved in the 'neurodegenerative disease' pathway, which involves an annotated protein (glyceraldehyde 3-phosphate dehydrogenase) involved in Alzheimer's disease. In terms of average nucleotide identity and digital DNA-DNA hybridization, strain M2458T was identified as a novel species of the genus Lacococcus. The major fatty acids (> 10% of the total fatty acids) were C18:1ω9c (39.68%), C16:0 (13.26%) and C18:1ω7c (11.52%). The polar lipids consisted of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, and an unidentified phospholipid. As a result of its genotypic and phenotypic characteristics, strain M2458T was considered to be a new species within the genus Lactococcus; the name Lactococcus intestinalis sp. nov. has been proposed, with type strain M2458T (=JCM 35706 = CGMCC 1.60066).
Assuntos
Doença de Alzheimer , Animais , Camundongos , Filogenia , RNA Ribossômico 16S/genética , Ácido Láctico , Conteúdo Gastrointestinal , DNA Bacteriano/genética , Fosfolipídeos , Ácidos Graxos , Lactococcus , Análise de Sequência de DNA , Técnicas de Tipagem Bacteriana , Hibridização de Ácido NucleicoRESUMO
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.
Assuntos
Doenças dos Peixes , Animais , Sorogrupo , Doenças dos Peixes/microbiologia , Peixes , Lactococcus/genética , JapãoRESUMO
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.
Assuntos
Doenças dos Peixes , Animais , Japão , Sorogrupo , Plasmídeos/genética , Lactococcus/genética , Eritromicina , GenômicaRESUMO
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.
Assuntos
Doenças dos Peixes , Penaeidae , Animais , Penaeidae/microbiologia , Filogenia , Salinidade , Doenças dos Peixes/microbiologia , Bactérias , Lactococcus , ÁguaRESUMO
Lactococcosis, caused by the Gram-positive bacterium Lactococcus garvieae, is a major concern in rainbow trout (Oncorhynchus mykiss) farms, which are regularly affected by outbreaks especially during the summer/fall months. In these farms, unvaccinated healthy and symptomatic fish can coexist with vaccinated fish. In the present study, innate (leukogram, serum lysozyme activity, peroxidase activity, antiprotease activity, bactericidal activity, total IgM and total proteins), and specific immune parameters (serum antibodies to L. garvieae) were assessed in unvaccinated adult rainbow trout naturally exposed to the pathogen, with or without evidence of clinical signs, or subjected to vaccination. Blood was drawn from all three groups, and blood smears were prepared. Bacteria were found in the blood smears of 70% of the symptomatic fish but not in any of the asymptomatic fish. Symptomatic fish showed lower blood lymphocytes and higher thrombocytes than asymptomatic fish (p ≤ .05). Serum lysozyme and bactericidal activity did not vary substantially among groups; however, serum antiprotease and peroxidase activity were significantly lower in the unvaccinated symptomatic group than in the unvaccinated and vaccinated asymptomatic groups (p ≤ .05). Serum total proteins and total immunoglobulin (IgM) levels in vaccinated asymptomatic rainbow trout were significantly higher than in unvaccinated asymptomatic and symptomatic groups (p ≤ .05). Similarly, vaccinated asymptomatic fish produced more specific IgM against L. garvieae than unvaccinated asymptomatic and symptomatic fish (p ≤ .05). This preliminary study provides basic knowledge on the immunological relationship occurring between the rainbow trout and L. garvieae, potentially predicting health outcomes. The approach we proposed could facilitate infield diagnostics, and several non-specific immunological markers could serve as reliable indicators of the trout's innate ability to fight infection.
Assuntos
Doenças dos Peixes , Infecções por Bactérias Gram-Positivas , Oncorhynchus mykiss , Animais , Oncorhynchus mykiss/microbiologia , Infecções por Bactérias Gram-Positivas/prevenção & controle , Infecções por Bactérias Gram-Positivas/veterinária , Infecções por Bactérias Gram-Positivas/microbiologia , Muramidase , Doenças dos Peixes/microbiologia , Lactococcus , Anticorpos Antibacterianos , Imunoglobulina M , PeroxidasesRESUMO
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.