Lactococcus Garvieae Strains Research Articles

Bacteriocinogenic anti-listerial properties and safety assessment of Enterococcus faecium and Lactococcus garvieae strains isolated from Brazilian artisanal cheesemaking environment.

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. 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. 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.

Isolation, identification, and fermentation characteristics of endogenous lactic acid bacteria derived from edible mushrooms

A total of 83 acid-producing strains were isolated from fresh Lentinus edodes, Pleurotus eryngii, Flammulina velutipes, Agaricus bisporus, Pleurotus ostreatus, Pleurotus djamor, Pleurotus abalones, and Pleurotus citrinopileatus by CaCO3-MRS plate medium. These 83 strains were divided into 9 species, including 52 strains of Lactococcus lactis, 13 strains of Pediococcus pentosaceus, 8 strains of Enterococcus faecium, 3 strains of Lactococcus garvieae, 2 strains of Enterococcus casseliflavus, 2 strains of Lactobacillus plantarum, 1 strain of Enterococcus lactis, 1 strain of Pediococcus acidilactici, and 1 strain of Lactobacillus pentosus based on the catalase test, Gram staining, and 16S rDNA molecular identification. Among these P. acidilactici, P. pentosus, and L. plantarum could be used in food. P. acidilactici had a strong acid-producing capacity and fast growth rate, thus showing preferable fermentation characteristics in MRS broth and edible mushroom medium than P. pentosus and L. plantarum. In the medium of L. edodes, P. eryngii, and F. velutipes, the best sensory state can be reached within 12-24 h, among them P. eryngii had the best fermentation effect, which was characterized by uniform and bright color, moderate acidity, good flavor, and tight tissue state. This study investigated the types and fermentation characteristics of lactic acid bacteria (LAB) derived from edible mushrooms, enriched the resources of LAB suitable for the fermentation, and provided a theoretical reference for the application of LAB fermentation technology of edible mushrooms.

Prevalence and fingerprinting of lactic acid bacteria community during 180 days of ripening in traditional Turkish goatskin bag Tulum cheeses produced in the mountainous region of Karaman using culture-dependent and -independent methods

The lactic acid bacteria (LAB) prevalence and dynamics of goatskin Tulum cheese produced in mountainous region of Karaman province of Turkey throughout 180 days of ripening were investigated using culture-dependent and -independent (PCR-DGGE) methods. This study used the V3-region of the 16S rRNA gene amplification and sequencing to characterise the LAB community of this cheese. Microbial counts were variable throughout the ripening. In the culture-dependent method, Enterococcus faecium shared dominance with Enterococcus hirae species at the end of the ripening, followed by Lactobacillus plantarum species. On the other hand, Streptococcus strains were predominant at the initial stage of the ripening, while E. faecium strains were identified, as well as Enterococcus faecalis and Lactococcus garvieae strains, using molecular techniques at the end of ripening. It was inferred that using these two methods together gave more information about the biodiversity and dynamics of LAB in artisanal goatskin Tulum cheeses during ripening.

Lactococcus garvieae Isolates from Rainbow Trout (Oncorhynchus mykiss, W.) Compared by PLG and SA1B10 PCR Primer Pairs

The aim of this study was to identify and compare Lactococcus garvieae strains using the PLG primer pair and SA1B10 primer pair. Also, the antibiotic resistance of the strains was investigated in the study. For this aim, commercial trout farms at Kemer, Korkuteli, and Manavgat, Antalya Province, Turkey were visited every month from June to September 2018. Thirty sick fish were sampled in the study. Lethargy, anorexia, darkening of skin color, unilateral or bilateral exophthalmos, opacification and hemorrhages in the eyes of the sick fish, and abdominal dropsy were observed. At necropsy, hemorrhages in the internal organs, splenomegaly, and darkening of the spleen and ascites were present. Seventy-five strains from sick fish samples verified on the basis of the biochemical characterization and PCR studies. The strains showed homogeneity in terms of phenotypic characteristics and all strains were identified as Lactococcus garvieae. The PCR technique was applied to 50 of 75 strains using PLG primer pair. While 47 of 50 strains gave positive results, amplification was not observed in 3 strains. When the PCR technique using SA1B10 primer pair was applied to a total of 25 strains, 3 of which were negative, 22 of which gave positive amplification, and 25 strains gave positive results. Resistance was determined by the disk diffusion method. All seventy-five strains were resistant to ampicillin and all strains were susceptible to bacitracin and tetracycline. It was found that the resistance and susceptibility of the strains showed variation to other antibiotics used in the study.Keywords: Lactococcus garvieae; Rainbow Trout; PCR; Antibiotic

Screening of Lactococcal Adhesion Genes and Two Pneumococcal Genes as Genetic Determinants of Virulence in Lactococcus garvieae Strains

Nine lactococcal adhesin and two pneumococcal virulence genes were characterized in twenty Lactococcus garvieae strains obtained from rainbow trout, and also one human isolate by PCR. Findings showed that all strains carried adhPsaA (522 bp), LPxTG-1 (947 bp), adhCI (490 bp), and purB (864 bp) while some of the strains had adhPav (1048 bp), LPxTG-2 (767 bp), LPxTG-3 (231 bp), adhCII (732 bp), adh (398 bp) and SP_0121 (966 bp). High nucleotide homologies (85-99%) of adhCI, adhCII, adh, adhPav, adhPsaA, LPxTG-1, LPxTG-3 involved in bacterial adhesion were determined between L. garvieae strains and reference Lg2. Genes containing polymorphisms among strains were not considered to be directly involved in bacterial pathogenesis. The amplification of LPxTG-3 only in fish isolates showed that it might be responsible for coding the host-specific virulence factor. However, undetermined amplicons demonstrated that LPxTG-4 could not be used as a host-specific gene marker. Pneumococcal purB and SP_0121 have been experimentally detected in L. garvieae genome for the first time. Consequently, both purB and SP_0121 can be used as a virulence marker for L. garvieae. Findings provided valuable knowledge about L. garvieae pathogenesis and, will contribute to identify the appropriate genomic targets to develop new therapeutics against the lactococcosis.

Determination of serotypic differences of Lactococcus garvieae isolates obtained from rainbow trout farms

This study aimed to determine the serotypic differences between Lactococcus garvieae strains isolated from rainbow trout obtained from different fish farms in Turkey. For this purpose, some phenotypic properties of isolates were determined and then ELISA test was performed to determine serotypic differences. It was determined that all 22 L. garvieae isolates used in the study gave a cream colored, bright, rounded smooth S type colony in Triptic Soy Agar (TSA) and all strains were nonmobile in the native examination. Morphologically, all isolates were found to be Gram positive, nonmobile, α-hemolytic, to have a growth of 0-6.5% NaCl salinity, 21, 37 and 45℃ temperature, and to be oxidase and catalase negative. After examining the biochemical made with API Rapid ID 32 Strep test, it was observed that two L. garvieae isolates were different from the other isolates in terms of sucrose use and one L. garvieae isolate was different from other isolates in terms of maltose profile. According to the results of ELISA test used to determine the serotypical differences of the isolates, two L. garvieae isolates were serotypically different from the other isolates and L. garvieae isolates used in the study formed two different serotypic groups.

Dialogue between Staphylococcus aureus SA15 and Lactococcus garvieae strains experiencing oxidative stress

BackgroundStaphylococcus aureus is an important foodborne pathogen. Lactococcus garvieae is a lactic acid bacterium found in dairy products; some of its strains are able to inhibit S. aureus growth by producing H2O2. Three strains of L. garvieae from different origins were tested for their ability to inhibit S. aureus SA15 growth. Two conditions were tested, one in which H2O2 was produced (high aeration) and another one in which it was not detected (low aeration). Several S. aureus genes related to stress, H2O2-response and virulence were examined in order to compare their level of expression depending on the inoculated L. garvieae strain. Simultaneous L. garvieae H2O2 metabolism gene expression was followed.ResultsThe results showed that under high aeration condition, L. garvieae strains producing H2O2 (N201 and CL-1183) inhibited S. aureus SA15 growth and impaired its ability to deal with hydrogen peroxide by repressing H2O2-degrading genes. L. garvieae strains induced overexpression of S. aureus stress-response genes while cell division genes and virulence genes were repressed. A catalase treatment partially or completely restored the SA15 growth. In addition, the H2O2 non-producing L. garvieae strain (Lg2) did not cause any growth inhibition. The SA15 stress-response genes were down-regulated and cell division genes expression was not affected. Under low aeration condition, while none of the strains tested exhibited H2O2-production, the 3 L. garvieae strains inhibited S. aureus SA15 growth, but to a lesser extent than under high aeration condition.ConclusionTaken together, these results suggest a L. garvieae strain-specific anti-staphylococcal mechanism and an H2O2 involvement in at least two of the tested L. garvieae strains.

Identification and characterization of Lactococcus starter strains in milk-based traditional fermented products in the region of Iran

The aim of the present research was identification and investigation of technological attributes of Lactococcus starter strains from traditional dairy products collected from the countryside of Boroujerd in Iran. 33 samples were cultured on selective media M17 and typical colonies surveyed for morphological properties. Totally, 37 strains were isolated based on the diversity in cell morphology and identified using API galleries and carbohydrate fermentation includes 17 strains of Lactococcus lactis (45.96%), 12 strains of Lactococcus garvieae (32.43%) and 8 strains of Lactococcusplantarum (21.62%). Strains were appraised for hydrolysis of L-arginine, casein and starch. Furthermore, strains were evaluated for the ability to grow at temperature 10 °C, 45 °C and presence of 4% and 6.5% NaCl, antibiotic sensitivity, acidification ability, proteolytic and lipolytic activities. Generally, 3 strains of Lc.garvieae (GYLC1, BWLC1, DCLC1) and 7 strains of Lc. lactis (GCLC4, GWLC2, GWLC3, SWLC1, SWLC3, BCLC5, DYLC1) exposed the highest levels of technological properties in order to use as starter cultures.

Control of Shigatoxin-producing Escherichia coli in cheese by dairy bacterial strains

Bio-preservation could be a valuable way to control Shigatoxin-producing Escherichia coli (STEC) in cheese. To this end, 41 strains were screened for their inhibitory potential on model cheese curd and on pasteurized and raw milk uncooked pressed cheeses. Strains of Lactococcus lactis, Lactococcus garvieae, Leuconostoc pseudomesenteroides, Leuconostoc citreum, Lactobacillus sp, Carnobacterium mobile, Enterococcus faecalis, Enterococcus faecium, Macrococcus caseolyticus and Hafnia alvei reduced STEC O26:H11 counts by 1.4–2.5 log cfu g−1 and to a lesser extent STEC O157:H7 counts in pasteurized milk cheeses. Some strains can act in synergy to inhibit STEC in raw milk uncooked pressed cheeses. Inhibitory associations had no adverse effect on the sensory characteristics of these cheeses. The association of H. alvei, Lactobacillus plantarum and Lc. lactis was the most inhibitory: after inoculation of this consortium into milk, STEC O26:H11 and O157:H7, inoculated at 2 log cfu ml−1, were reduced by up to 3 log cfu g−1 in ripened cheese. Inhibition in cheese cannot be predicted from H2O2 production in BHI medium, decreased pH or milk reduction. It is not clear what role the rapid decrease in pH during the first 6 h may play in the inhibition. Further studies will be needed to determine the nature of the inhibition.

Antimicrobial susceptibility of the strains of Lactococcus garvieae isolated from cultured fish of the genus Seriola (yellowtail, amberjack and kingfish) in Japan from 2004 to 2009

Antimicrobial susceptibility of the strains of Lactococcus garvieae isolated from cultured fish of the genus Seriola (yellowtail, amberjack and kingfish) in Japan from 2004 to 2009

Detection of Tetracycline Resistance Genes in Lactococcus garvieae Strains Isolated from Rainbow Trout

Detection of Tetracycline Resistance Genes in Lactococcus garvieae Strains Isolated from Rainbow Trout

Inhibition of fish pathogens by the microbiota from rainbow trout (Oncorhynchus mykiss, Walbaum) and rearing environment

This work reports the isolation and taxonomic identification of the cultivable total microbiota (TM) and Lactic Acid Bacteria (LAB) from rainbow trout (Oncorhynchus mykiss, Walbaum) and rearing environment from selected stages of the life-cycle, and the evaluation of the LAB antimicrobial activity against the main fish pathogens. TM and LAB isolates were randomly selected and identified by 16S rRNA and/or superoxide dismutase gene sequencing. Although a great diversity in the TM was observed, Enterobacteriaceae and Aeromonadaceae were clearly prevalent, while the genus Lactococcus was the predominant LAB. From a total of 1620 randomly selected LAB, 1159 isolates (71.5%) showed antimicrobial activity. From these, 248 isolates (21.4%) selected for their activity against, at least, four fish pathogens, were taxonomically identified, being Lactococcus lactis the most common species (164 isolates, 66.1%). Interestingly, 88 isolates (35.5%), including 55 L. lactis isolates, exerted activity against four strains of the rainbow trout pathogen Lactococcus garvieae. Our results demonstrate that rainbow trout and rearing environment are potential sources for the isolation of LAB, mainly lactococci, active against L. garvieae and other fish pathogens. Moreover, this is the first study describing the cultivable TM and LAB from rainbow trout intestine and rearing environment along the fish life-cycle. The host-derived LAB active against fish pathogens comprise potential candidates as probiotics in rainbow trout farming as an alternative or complementary strategy to antibiotics and vaccines for disease prevention.

Evolution of proteolysis in Urfa cheese made from ewe's milk by wild type starter culture systems

The evolution of proteolysis in Urfa cheese made from ewe's milk by using various combinations of wild-type strains of Lactococcus lactis (strain 1B4), Lactococcus garvieae (strain IMAU 50157), Enterococcus faecalis (strain KLDSO.0341), Enterococcus faecium (strain ATCC 19434) and Enterococcus durans (strain IMAU 60200) was monitored for 180 days. The strains used in the study were isolated from traditional raw ewe's milk Urfa cheese and characterized by phenotypic and genotypic studies previously. Three different combinations of the strains were employed in the manufacture of cheeses from pasteurized milk. These are: cheese A (strains 1B4+ATCC 19434+IMAU 50157), cheese B (strains 1B4+IMAU 60200+ATCC 1934) and cheese C (strains ATCC 19434+1B4+IMAU 50157+IMAU 60200+KLDSO.0341). The control cheese (cheese D) was produced from raw ewe's milk without starter culture. Proteolysis in the cheeses was evaluated using urea-PAGE of pH 4.6-insoluble and RP-HPLC of both 70% ethanol-insoluble (EtOH-i) and ethanol-soluble (EtOH-s) nitrogen fractions. The variations in water soluble nitrogen (WSN) and trichloroacetic acid-soluble nitrogen (TCA-SN) were also monitored during ripening as well as gross compositions of the cheeses. Results indicated that the degradation of casein fractions were faster in the cheeses A, B and C than the control. The peptide profiles of 70% EtOH-i and EtOH-s nitrogen of the cheeses A, B and C were close to each other but far more extensive than the control cheese. The proteolytic capacities of the strains employed in the cheeses A, B and C were superior to that of raw milk flora. To conclude, any of the combinations of the wild-type strains could be used in the production of Urfa cheese from pasteurized ewe's milk, as long as the safe use of enterococcal strains is assured.

A comparative study of detection methods for Lactococcus garvieae in experimentally infected rainbow trout (Oncorhynchus mykiss, W.)

This study was carried out to detect Lactococcus garvieae from experimentally infected rainbow trout (Oncorhynchus mykiss, W.) using bacteriological, serological, and histopathological techniques. Rainbow trout were experimentally infected with two Lactococcus garvieae strains (L1 and L2) by intraperitonal injection. These strains caused bilateral exophthalmia with periocular hemorrhage, corneal opacification, darkening of the skin, strong internal congestion in the visceral organs, and enlargement of the spleen in the infected rainbow trout. The two L. garvieae strains were re-isolated from experimentally infected fish groups and identified with standard biochemical methods and API 20 STREP system. The presence of L. garvieae in the tissue of experimentally infected fish was confirmed by Indirect Fluorescent Antibody Technique (IFAT). The humoral antibody response of the infected fish was confirmed by slide agglutination, IFAT, and Enzyme Linked Immunosorbent Assay (ELISA). Histopathologically, liquefactive necrosis in the liver, spleen, and kidney, epithelial cells hyperplasia, hemorrhages and telangiectasia of the gill filaments, and sub-retinal hemorrhages in the eyes were observed.

Selected lactic acid bacteria as a hurdle to the microbial spoilage of cheese: Application on a traditional raw ewes' milk cheese

To evaluate the efficacy of lactic acid bacteria (LAB) to improve the hygienic safety of a traditional raw milk cheese, the raw ewes' milk protected denomination of origin (PDO) Pecorino Siciliano cheese was used as a model system. Different Pecorino Siciliano curds and cheeses were used as sources of autochthonous LAB subsequently used as starter and non-starter LAB. These were screened for their acidification capacity and autolysis. Starter LAB showing the best performance were genotypically differentiated and identified: two strains of Lactococcus lactis subsp. lactis were selected. From the non-starter LAB, Enterococcus faecalis, Lactococcus garvieae and Streptococcus macedonicus strains were selected. The five cultures were used in individual or dual inocula to produce experimental cheeses in a dairy factory for which production was characterised by high numbers of undesirable bacteria. At 5-month of ripening, the experimental cheeses produced with LAB were characterised by undetectable levels of enterobacteria and pseudomonads and the typical sensory attributes.

Genome Sequences of Two Lactococcus garvieae Strains Isolated from Meat

Lactococcus garvieae is an important fish pathogen and an emerging opportunistic human pathogen, as well as a component of natural microbiota in dairy and meat products. We present the first report of genome sequences of L. garvieae I113 and Tac2 strains isolated from a meat source.

Screening and characterization of LAB-produced bacteriocin-like substances from the intestine of grey mullet (Mugil cephalus L.) as potential biocontrol agents in aquaculture

To isolate and characterize bacteriocin-like inhibitory substance (BLIS)-producing lactic acid bacteria from the intestine of grey mullet. Inhibitory activity against at least one or more indicator strains was observed in one Enterococcus thailandicus, one Enterococcus faecium and two Lactococcus garvieae strains. Enterococcus faecium B3-8 and Ent. thailandicus B3-22 showed the greatest inhibitory activities against Listeria monocytogenes ATCC 19111 and were therefore further characterized. The results suggested that the inhibitory substances from the two strains showed similar characteristics with respect to sensitivity to heat and proteolytic enzymes. BLIS from Ent. thailandicus B3-22 was characterized by a broader inhibitory spectrum than that from Ent. faecium B3-8. SDS-PAGE revealed that the molecular size of partially purified BLISs from Ent. faecium B3-8 and Ent. thailandicus B3-22 was c. 5 and 3 kDa, respectively. The molecular mass of purified bacteriocin from Ent. thailandicus B3-22 was further determined to be 6319 Da by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The results indicated that BLIS from Ent. thailandicus B3-22 can effectively inhibit the growth of all tested L. garvieae strains. The findings obtained in this study suggest the potential use of Ent. thailandicus B3-22 as a biocontrol agent against pathogenic L. garvieae in the aquaculture. This is the first report describing the characteristics of BLIS from Ent. thailandicus that showed potential for use as a biocontrol agent in the aquaculture.

Genetic investigation within Lactococcus garvieae revealed two genomic lineages

The diversity of a collection of 49 Lactococcus garvieae strains, including isolates of dairy, fish, meat, vegetable and cereal origin, was explored using a molecular polyphasic approach comprising PCR-ribotyping, REP and RAPD-PCR analyses and a multilocus restriction typing (MLRT) carried out on six partial genes (atpA, tuf, dltA, als, gapC, and galP). This approach allowed high-resolution cluster analysis in which two major groups were distinguishable: one group included dairy isolates, the other group meat isolates. Unexpectedly, of the 12 strains coming from fish, four grouped with dairy isolates, whereas the others with meat isolates. Likewise, strains isolated from vegetables allocated between the two main groups. These findings revealed high variability within the species at both gene and genome levels. The observed genetic heterogeneity among L.garvieae strains was not entirely coherent with the ecological niche of origin of the strains, but rather supports the idea of an early separation of L.garvieae population into two independent genomic lineages.

Anti-Listeria activity of lactic acid bacteria isolated from golka, a regional cheese produced in Poland

Eight hundred lactic acid bacteria (LAB) strains isolated from golka, one of the oldest and most popular regional cheese produced in Poland, were screened for anti-Listeria activity.Nearly one third of isolates showed antagonistic activity against Listeria monocytogenes. Seven strains produced other substances active against L. monocytogenes. These substances were detected in neutralized cell-free supernatant fluids and were classified as bacteriocins. 16S rDNA identification of the selected bacteriocin-producing bacteria was performed. By using primers specific to a fragment of class IIa bacteriocin-coding genes, developed by the authors, presence of the bacteriocin structural gene was confirmed for all Lactococcus garvieae strains, Leuconostoc mesenteroides 453Lab and Lactobacillus plantarum Lab572. Presence of the class IIa bacteriocin-coding genes within genomes of non-bacteriocin-producing strains Lactococcus lactis subsp. lactis was also detected. Bacteriocins with the highest activity against Listeria, i.e. bacteriocins of L. garvieae Lab428, Lc. mesenteroides Lab25 and Lb. plantarum Lab572 strains, were partially characterized. The bacteriocins did not adhere to surface of the producer cells and exhibit a bactericidal mode of action. Bacteriocin of L. garvieae 428Lab was produced at much higher levels (12.800 AU/ml), as compared to the other bacteriocins (3.200 AU/ml).Our results indicate that golka cheese is a rich source of LAB active against L. monocytogenes. The bacteriocinogenic isolates from golka described in this work may potentially find application as starter or protective cultures and could constitute a superb genetic pool for production of new bacteriocins.

Application of suppressive subtractive hybridization to the identification of genetic differences between two Lactococcus garvieae strains showing distinct differences in virulence for rainbow trout and mouse

Lactococcus garvieae is the causative microbial agent of lactococcosis, an important and damaging fish disease in aquaculture. This bacterium has also been isolated from vegetables, milk, cheese, meat and sausages, from cow and buffalo as a mastitis agent, and even from humans, as an opportunistic infectious agent. In this work pathogenicity experiments were performed in rainbow trout and mouse models with strains isolated from human (L. garvieae HF) and rainbow trout (L. garvieae UNIUDO74; henceforth referred to as 074). The mean LD(50) value in rainbow trout obtained for strain 074 was 2.1 × 10(2) ± 84 per fish. High doses of the bacteria caused specific signs of disease as well as histological alterations in mice. In contrast, strain HF did not prove to be pathogenic either for rainbow trout or for mice. Based on these virulence differences, two suppressive subtractive hybridizations were carried out to identify unique genetic sequences present in L. garvieae HF (SSHI) and L. garvieae 074 (SSHII). Differential dot-blot screening of the subtracted libraries allowed the identification of 26 and 13 putative ORFs specific for L. garvieae HF and L. garvieae 074, respectively. Additionally, a PCR-based screening of 12 of the 26 HF-specific putative ORFs and the 13 074-specific ones was conducted to identify their presence/absence in 25 L. garvieae strains isolated from different origins and geographical areas. This study demonstrates the existence of genetic heterogeneity within L. garvieae isolates and provides a more complete picture of the genetic background of this bacterium.