RESUMO
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
Co-infection of Lactococcus garvieae and Aeromonas hydrophila, has been confirmed from diseased Nile Tilapia (Oreochromis niloticus), Chithralada strain cultured in a freshwater rearing pond of Alappuzha district of Kerala, India. The aetiological agents behind the disease outbreak were bacteriologically proven and confirmed by 16SrRNA sequencing and phylogenetic analysis. PCR detection of the virulent genes, showed existence of adhesin and hemolysin in L. garvieae and aerolysin in A. hydrophila strain obtained. To fulfil Koch's postulates, challenge experiments were conducted and median lethal dose (LD50) of L. garvieae and A. hydrophila was calculated as 1 × 105.91 CFU per mL and 1 × 105.2 CFU per mL respectively. Histopathologically, eyes, spleen, and kidney were the predominantly infected organs by L. garvieae and A. hydrophila. Out of the 13 antibiotics tested to check antibiotic susceptibility, L. garvieae showed resistance to almost 7 antibiotics tested, with a resistance to Ciprofloxacin while A. hydrophila was found resistant to Streptomycin and Erythromycin. Understanding the complex interaction between Gram-positive and Gram-negative bacteria in the disease process and pathogenesis in fish host will contribute to efficient treatment strategies. As a preliminary investigation into this complex interaction, the present study is aimed at phenotypic and genotypic characterization, pathogenicity evaluation, and antibiotic susceptibility of the co-infecting pathogens in a diseased sample of freshwater-farmed Nile tilapia.
Assuntos
Aeromonas hydrophila , Antibacterianos , Ciclídeos , Coinfecção , Doenças dos Peixes , Infecções por Bactérias Gram-Negativas , Lactococcus , Filogenia , Animais , Aeromonas hydrophila/genética , Aeromonas hydrophila/isolamento & purificação , Aeromonas hydrophila/patogenicidade , Aeromonas hydrophila/classificação , Aeromonas hydrophila/efeitos dos fármacos , Ciclídeos/microbiologia , Índia , Doenças dos Peixes/microbiologia , Lactococcus/genética , Lactococcus/isolamento & purificação , Lactococcus/classificação , Lactococcus/patogenicidade , Infecções por Bactérias Gram-Negativas/microbiologia , Infecções por Bactérias Gram-Negativas/veterinária , Coinfecção/microbiologia , Coinfecção/veterinária , Antibacterianos/farmacologia , Testes de Sensibilidade Microbiana , Infecções por Bactérias Gram-Positivas/microbiologia , Infecções por Bactérias Gram-Positivas/veterinária , RNA Ribossômico 16S/genética , Aquicultura , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/metabolismoRESUMO
The present study aimed to investigate the virulence, antibiotic resistance and biogenic amine production in bacteriocinogenic lactococci and enterococci isolated from goat milk in order to evaluate their safety. Twenty-nine bacteriocinogenic lactic acid bacteria (LAB: 11 Lactococcus spp., and 18 Enterococcus spp.) isolated from raw goat milk were selected and subjected to PCR to identify gelE, cylA, hyl, asa1, esp, efaA, ace, vanA, vanB, hdc1, hdc2, tdc and odc genes. The expression of virulence factors (gelatinase, hemolysis, lipase, DNAse, tyramine, histamine, putrescine) in different incubation temperatures was assessed by phenotypic methods, as well as the resistance to vancomycin, gentamicin, chloramphenicol, ampicillin and rifampicin (using Etest®). The tested isolates presented distinct combinations of virulence related genes, but not necessarily the expression of such factors. The relevance of identifying virulence-related genes in bacteriocinogenic LAB was highlighted, demanding for care in their usage as starter cultures or biopreservatives due to the possibility of horizontal gene transfer to other bacteria in food systems.
Assuntos
Aminas Biogênicas/análise , Resistência Microbiana a Medicamentos/genética , Enterococcus , Lactococcus , Leite/microbiologia , Fatores de Virulência/genética , Animais , Enterococcus/química , Enterococcus/genética , Enterococcus/isolamento & purificação , Enterococcus/patogenicidade , Perfilação da Expressão Gênica , Cabras , Lactococcus/química , Lactococcus/genética , Lactococcus/isolamento & purificação , Lactococcus/patogenicidade , Reação em Cadeia da PolimeraseRESUMO
Citrate is present in many natural substrates, such as milk, vegetables and fruits, and its metabolism by lactic acid bacteria (LAB) plays an important role in food fermentation. The industrial importance of LAB stems mainly from their ability to convert carbohydrates into lactic acid and, in some species, like Lactococcus lactis and Leuconostoc mesenteroides, to produce C4 flavor compounds (diacetyl, acetoin) through citrate metabolism. Three types of genetic organization and gene locations, involving citrate metabolism, have been found in LAB. Citrate uptake is mediated by a citrate permease, which leads to a membrane potential upon electrogenic exchange of divalent citrate and monovalent lactate. The internal citrate is cleaved into acetate and oxaloacetate by a citrate lyase, and oxaloacetate is decarboxylated into pyruvate by an oxaloacetate decarboxylase, yielding a pH gradient through the consumption of scalar protons.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Transporte/genética , Ácido Láctico/metabolismo , Lactococcus/enzimologia , Complexos Multienzimáticos/genética , Transportadores de Ânions Orgânicos/genética , Oxo-Ácido-Liases/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Proteínas de Transporte/metabolismo , DNA Bacteriano/genética , Diacetil/metabolismo , Regulação Enzimológica da Expressão Gênica/genética , Lactococcus/genética , Dados de Sequência Molecular , Complexos Multienzimáticos/metabolismo , Fases de Leitura Aberta , Transportadores de Ânions Orgânicos/metabolismo , Oxo-Ácido-Liases/metabolismo , SimportadoresRESUMO
Many genetically modified Lactococcus strains have been constructed in research laboratories around the world. Most of these have originated from laboratory strains and therefore there are several barriers to using them in an industrial setting. Laboratory strains are often plasmid-free and consequently Lac- and Prt-, rendering them unable to grow in milk. Many of the commonly used techniques have been optimised for laboratory strains and their application to industrial strains may require a great deal of effort. Often genetically modified organisms produced in the laboratory do not fit the published definition of 'food-grade' (Johansen, 1999, Encyclopedia of Food Microbiology, Academic Press, London, pp. 917-921) and a great deal of effort is required to eliminate undesirable DNA sequences. As a consequence, it is often necessary to recreate the strains in industrial backgrounds before the innovations described in the scientific literature can be applied to the real-world dairy industry.
Assuntos
Laticínios , Indústria Alimentícia , Lactococcus/genética , Biotecnologia , Organismos Geneticamente Modificados , Plasmídeos , Pesquisa , Especificidade da EspécieRESUMO
Many genetically modified Lactococcus strains have been constructed in research laboratories around the world. Most of these have originated from laboratory strains and therefore there are several barriers to using them in an industrial setting. Laboratory strains are often plasmid-free and consequently Lac- and Prt-, rendering them unable to grow in milk. Many of the commonly used techniques have been optimised for laboratory strains and their application to industrial strains may require a great deal of effort. Often genetically modified organisms produced in the laboratory do not fit the published definition of 'food-grade' (Johansen, 1999, Encyclopedia of Food Microbiology, Academic Press, London, pp. 917-921) and a great deal of effort is required to eliminate undesirable DNA sequences. As a consequence, it is often necessary to recreate the strains in industrial backgrounds before the innovations described in the scientific literature can be applied to the real-world dairy industry
Assuntos
Laticínios , Indústria Alimentícia , Lactococcus/genética , Biotecnologia , Especificidade da Espécie , Organismos Geneticamente Modificados , Plasmídeos , PesquisaRESUMO
The phenotypic and genetic analysis results for 84 isolates of Lactococcus garvieae (including 62 strains from trout with lactococcosis from four different countries, 7 strains from cows and water buffalos with subclinical mastitis, 3 from water, and 10 from human clinical samples) are presented. There was great phenotypic heterogeneity (13 different biotypes) based on the acidification of saccharose, tagatose, mannitol, and cyclodextrin and the presence of the enzymes pyroglutamic acid arylamidase and N-acetyl-beta-glucosaminidase. L. garvieae also exhibited high genetic diversity by pulsed-field gel electrophoresis (PFGE), with 19 different pulsotypes among the isolates of L. garvieae studied. Only epidemiologically related strains, like the Spanish and Italian fish isolates and the cow and water buffalo isolates, displayed a close genetic relationship by PFGE, while the strains isolated from sporadic clinical cases, like the human isolates, were genetically unrelated. Overall, a general correlation between phenotypic and genetic data was observed. Epidemiological analysis of biotype and PFGE results indicated that the trout lactococcosis outbreaks in Spain and Portugal and those in France and Italy were produced by genetically unrelated clones. In Spain, two different clones were detected; the outbreaks diagnosed from 1995 onward were produced by a clone (biotype 2, pulsotype A1) which, although genetically related, was different from the one that was responsible for the outbreaks studied between 1991 and 1994 (biotype 1, pulsotype B). The Portuguese isolate had a biochemical profile identical to that of the Spanish strain isolated from 1995 onward and is also genetically closely related to this strain (pulsotype A2). There was a close relationship between the two pulsotypes (E and F) found in the Italian isolates. The French isolate (biotype 3, pulsotype D) was not genetically related to any other L. garvieae fish isolate. These results suggest the existence of diverse infection sources for the different lactococcosis outbreaks.
Assuntos
Surtos de Doenças , Infecções por Bactérias Gram-Positivas/epidemiologia , Lactococcus/genética , Lactococcus/isolamento & purificação , Filogenia , Animais , Brasil , Búfalos/microbiologia , Bovinos , França/epidemiologia , Genótipo , Geografia , Infecções por Bactérias Gram-Positivas/microbiologia , Humanos , Itália/epidemiologia , Japão , Fenótipo , Portugal/epidemiologia , Espanha/epidemiologia , Truta/microbiologia , Estados Unidos , Microbiologia da ÁguaAssuntos
Enterococcus/classificação , Animais , Proteínas de Bactérias/metabolismo , Técnicas de Tipagem Bacteriana , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , Enterococcus/genética , Enterococcus/metabolismo , Humanos , Lactococcus/classificação , Lactococcus/genética , Lactococcus/metabolismo , Fenótipo , Especificidade da EspécieAssuntos
Búfalos/microbiologia , Enterococcus/genética , Enterococcus/isolamento & purificação , Lactococcus/genética , Lactococcus/isolamento & purificação , Mastite/veterinária , Animais , Brasil/epidemiologia , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , Eletroforese em Gel de Campo Pulsado , Feminino , Humanos , Mastite/epidemiologia , Mastite/microbiologia , Epidemiologia Molecular , Filogenia , Especificidade da EspécieRESUMO
During a survey of bacterial agents that cause subclinical mastitis in water buffalos, we isolated several strains of gram-positive cocci that appeared to be enterococci except that they grew very slowly at 45 degrees C and grew slowly in broth containing 6.5% NaCl. On the basis of the results of conventional physiologic tests, these strains were identified as Enterococcus durans. However, none of the strains reacted with the AccuProbe Enterococcus genetic probe. The whole-cell protein profiles of these organisms were compared with the profiles of Enterococcus and Lactococcus reference strains. apart from minor quantitative differences, the mastitis isolates had indistinguishable protein profiles that were similar to the profiles of the Lactococcus garvieae and Enterococcus seriolicida type strains. The results of DNA relatedness studies performed by using the hydroxyapatite method at 55 and 70 degrees C indicated that all of the mastitis isolates were related to the type strain of L. garvieae at the species level, despite the fact that they exhibited several uncommon phenotypic characteristics (growth at 45 degrees C, growth in broth containing 6.5% NaCl, and failure to produce acid from mannitol and sucrose). The high levels of DNA relatedness between strains of L. garvieae is a senior synonym of E. seriolicida, L. garvieae should be retained as the species name and strain ATCC 43921 should remain the type strain of this species.