Lactococcus carnosus sp. nov. and Lactococcus paracarnosus sp. nov., two novel species isolated from modified-atmosphere packaged beef steaks.

As part of a study investigating the spoilage microbiome of modified-atmosphere packaged beef from Germany, four novel strains of lactic acid bacteria were isolated and subsequently taxonomically characterized by a polyphasic approach, which revealed that they could not be assigned to known species. The isolates were Gram-staining-positive, coccoid, facultatively anaerobic, non-motile, catalase-negative and oxidase-negative. Morphological, physiological and phylogenetic analysis revealed a distinct lineage within the genus Lactococcus, with Lactococcus piscium and Lactococcus plantarum as closest relatives. Results indicated that they represented two different novel species with two strains, (TMW 2.1612T/TMW 2.1613 and TMW 2.1615T/TMW 2.1614), respectively. The two strains of both novel species shared identical 16S rRNA gene sequences but a MLSA allowed their intraspecies differentiation. The 16S rRNA gene sequences of TMW 2.1612T and TMW 2.1615T had a similarity of 99.85 % to each other and a similarity of 99.85 and 99.78 % the most closely related type strain of Lactococcus piscium, respectively. However, the ANIb value between the respective type strains TMW 2.1612T and TMW 2.1615T, and the type strain of Lactococcus piscium was only 94.3 and 92.0 %, respectively, and 92.9 % between TMW 2.1612T and TMW 2.1615T. The in silico DDH estimate value between the respective type strain TMW 2.1612T and TMW 2.1615T and the most closely related type strain of Lactococcus piscium was only 59.9 and 48.9 %, respectively, and 51.1 % between TMW 2.1612T and TMW 2.1615T. Peptidoglycan type of strain TMW 2.1612T is Lys-Thr-Ala and major fatty acids are summed feature 8 and C16 : 0. Peptidoglycan type of strain TMW 2.1615T is Lys-Ala and major fatty acids are C16 : 0, C19 : 0cyclo ω8c and summed feature 8. On the basis of polyphasic evidence, the meat isolates represent two novel species of the genus Lactococcus, for which the names Lactococcus carnosus sp. nov and Lactococcus paracarnosus sp. nov are proposed. The designated respective type strains are TMW 2.1612T (=DSM 111016T =CECT 30115T) and TWM 2.1615T (=DSM 111017T =CECT 30116T).

Lactococcus allomyrinae sp. nov., isolated from gut of larvae of Allomyrina dichotoma.

Strain 1JSPR-7T, a facultatively anaerobic bacterium isolated from the gut of larvae of Allomyrina dichotoma raised in Wanju-gun, Republic of Korea, was characterized using a polyphasic approach. Comparative analysis of 16S rRNA gene and rpoB gene sequences showed that strain 1JSPR-7T fell within the genus Lactococcus, forming a compact cluster with the type strain of four subspecies of Lactococcus lactis and Lactococcus taiwanensis. The 16S rRNA gene sequence of strain 1JSPR-7T revealed the highest homology with L. lactissubsp. lactis JCM 5805T (97.3 %) and L. lactissubsp. hordniae NBRC 100931T (97.1 %), and the rpoB gene sequence showed the highest similarity to L. lactissubsp. cremoris DSM 20069T (91.4 %) and L. lactissubsp. tructae L105T (91.4 %). The average nucleotide identity and digital DNA-DNA hybridization values indicated that strain 1JSPR-7T was a novel species of the genus Lacococcus. The major fatty acids (>10 % of the total fatty acids) were summed feature 7 (unknown 18.846, C19 : 1ω6c and/or C19 : 0cyclo ω10c), C16 : 0 and C14 : 0, and the predominant menaquinone was MK-8 with MK-7 as a minor one. The polar lipids consisted of diphosphatidylglycerol, phosphatidylglycerol, an unidentified phospholipid and three unidentified glycolipids with diphosphatidylglycerol as the major one. The cell-wall peptidoglycan was of the A4α type with an interpeptide bridge comprising l-Lys-d-Asp. The DNA G+C content based on the whole genome sequences was 37.4 mol%. Based on the data obtained, strain 1JSPR-7T represents a novel species of the genus Lactococcus, for which the name Lactococcusallomyrinae sp. nov. is proposed. The type strain is 1JSPR-7T (=KACC 19319T=NBRC 113068T).

Endocarditis por Lactococcus garvieae en un paciente sometido a hemodialisis crónica. Primer caso reportado en Chile y revisión de la literatura / Lactococcus garvieae endocarditis in a patient undergoing chronic hemodialysis. First case report in Chile and review of the literature

Resumen Los casos reportados de infección por Lactococcus garvieae son escasos y sólo uno asociado a hemodiálisis. Comunicamos el caso de endocarditis infecciosa de curso fatal por L. garvieae en un paciente con una enfermedad renal crónica sometido a hemodiálisis y portador de diverticulosis colónica no complicada. Se realiza una revisión de los casos publicados y se discuten los actuales desafíos diagnósticos y terapéuticos de este patógeno, capaz de producir infecciones graves y potencialmente fatales en pacientes susceptibles. Este sería el segundo caso de infección asociada a hemodiálisis y el primero reportado en Chile.

Reports of Lactococcus garvieae infections in humans are scarce, and only one of them in a patient under-going hemodialysis. We report the first case of Lactococcus garvieae infection in Chile, presenting as an infective endocarditis, ultimately fatal, in a patient with uncomplicated colonic diverticulosis and end stage renal failure undergoing chronic hemodialysis. We review the published cases and discuss the diagnostic and therapeutic challenges associated with this new, increasingly diagnosed pathogen, capable of producing serious infections in susceptible patients.

Lactococcus hircilactis sp. nov. and Lactococcus laudensis sp. nov., isolated from milk.

Two strains of lactic acid bacteria, designated 117(T) and 4195(T), were isolated from goat milk in Valtellina, Italy and from cow milk in Valle Trompia, Italy, respectively, and characterized taxonomically by a polyphasic approach. The strains were Gram-stain-positive, coccoid, non-spore-forming and catalase-negative bacteria. Morphological, physiological and phylogenetic data indicated that these isolates belonged to the genus Lactococcus. Strain 117(T) was closely related to Lactococcus fujiensis, Lactococcus lactis subsp. lactis, L. lactis subsp. cremoris, L. lactis subsp. hordniae, L. lactis subsp. tructae and Lactococcus taiwanensis, showing 93-94% and 82-89% 16S rRNA and rpoB gene sequence similarities, respectively. Strain 4195(T) was closely related to Lactococcus chungangensis, Lactococcus raffinolactis, Lactococcus plantarum and Lactococcus piscium, showing 92-98% and 86-99% 16S rRNA and rpoB gene sequence similarities, respectively. Based on this evidence and the data obtained in the present study, the milk isolates represent two novel species of the genus Lactococcus, for which the names Lactococcushircilactis sp. nov., and Lactococcuslaudensis sp. nov. are proposed. The respective type strains are 117(T) ( = LMG 28352(T) = DSM 28960(T)) and 4195(T )( = LMG 28353(T) = DSM 28961(T)).

Transcriptomic analysis of Lactococcus chungangensis sp. nov. and its potential in cheese making.

Lactococcus lactis has a played a prominent role in food industry from traditional milk fermentations to industrial scale processes. Extensive studies on the biochemical, physiological, and genetic aspects of L. lactis are evident from published literature. Recently, another novel species, Lactococcus chungangensis, was isolated from activated sludge as the sixth member of the genus to be discovered. To date, no study has been conducted to explore the functional aspects of L. chungangensis to identify features similar to those in L. lactis that are relevant to the dairy industry. Hence, the present study was undertaken to identify functional genes relevant to dairy application through comparative transcriptomic analysis of L. chungangensis with L. lactis. In expression microarray data, 415 genes were upregulated and 1,500 were downregulated of the total 1,915 probes analyzed. Interesting findings from this study were the identification of functional genes such as aminohydrolase and S-adenosylmethionine in L. chungangensis, which are useful in flavor production in cheese making. Probing these genes by PCR and analyzing the sequence confirmed the presence of these genes. Phenotypic analysis of these genes was also investigated by growing the strain in different concentrations of skim milk media to confirm the ability of L. chungangensis to degrade casein in milk, which is the major precursor for flavor enhancing compounds. Other adaptive and stress-response genes such as cold shock and heat shock proteins were also revealed. All experimental investigations at the functional level suggest that L. chungangensis possesses some interesting genes which are of commercial significance, especially in cheese production.

Psychrotrophic members of Leuconostoc gasicomitatum, Leuconostoc gelidum and Lactococcus piscium dominate at the end of shelf-life in packaged and chilled-stored food products in Belgium.

Previously, a considerable underestimation (+0.5-3.2 log CFU/g) on the contamination levels of psychrotrophic lactic acid bacteria (LAB) was observed for 33 retail, packaged food products stored at chilling temperature when the mesophilic enumeration technique was implemented as reference shelf-life parameter. In the present study, the microbial diversity of the dominant psychrotrophic LAB recovered after incubation of plates at 22 °C for 5 days was determined using a polyphasic taxonomic approach. A total of 212 LAB isolates were identified using a combination of rep-PCR fingerprinting, amplified fragment length polymorphism (AFLP) analysis and pheS gene sequencing. Leuconostoc gasicomitatum, Leuconostoc gelidum, Leuconostoc spp., Lactococcus piscium and Lactobacillus algidus proved to be the most competent and predominant species that may go undetected by the widely applied mesophilic enumeration protocols (ISO 4833:2003 and ISO 15214:1998). This study has assessed the interspecific variation among potential spoilage LAB, and highlights the significance of implementing a reference shelf-life parameter based on the enumeration of the total psychrotrophic bacterial load for industrial microbiological routine analyses.

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.

Endocardite por lactococcus garvieae: primeiro relato de caso da América Latina / Lactococcus garvieae endocarditis: first case report in Latin America / Endocarditis por lactococcus garvieae: primer relato de caso de América Latina

Lactococcus garvieae, patógeno zoonótico emergente, é responsável por mastite em ruminantes e septicemia em peixes. Embora seja considerado oportunista e raramente causar infecções em humanos, sua incidência deve estar subestimada devido à dificuldade do diagnóstico. Há pouquíssimos relatos de osteomielite, abscesso hepático e peritonite, e apenas nove casos descritos na literatura mundial de endocardite. Relatamos o primeiro caso de endocardite por Lactococcus garvieae da América Latina em paciente portadora de prótese valvar metálica, com quadro de febre diária, calafrios, nodos de Osler e seis hemoculturas positivas para Lactococcus garvieae, que preenchiam os critérios de Duke para o diagnóstico de "endocardite infecciosa definitiva".

Lactococcus garvieae, an emerging zoonotic pathogen, is responsible for mastitis in rodents and sepsis in fish. Although deemed opportunistic and hardly ever causing infections in humans, its incidence is probably underestimated due to the difficulty in diagnosis. There are very few reports of osteomyelitis, liver abscess, and peritonitis, and only nine cases of endocarditis described in worldwide literature. We describe the first case of Lactococcus garvieae endocarditis in Latin America, in a female patient with metallic prosthetic heart valve who presented with daily fever, chills, Osler nodes and six positive blood cultures for Lactococcus garvieae, which met Duke's criteria for the diagnosis of "definitive infective endocarditis".

Lactococcus garvieae, patógeno zoonótico emergente, es responsable por mastitis en rumiantes y septicemia en peces. Aunque sea considerado oportunista y raramente cause infecciones en humanos, su incidencia debe estar subestimada debido a la dificultad del diagnóstico. Hay poquísimos relatos de osteomielitis, absceso hepático y peritonitis, y apenas nueve casos descriptos en la literatura mundial de endocarditis. Relatamos el primer caso de endocarditis por Lactococcus garvieae de América Latina en paciente portadora de prótesis valvar metálica, con cuadro de fiebre diaria, escalofríos, nódulos de Osler y seis hemocultivos positivos para Lactococcus garvieae, que llenaban los criterios de Duke para el diagnóstico de "endocarditis infecciosa definitiva".

Lactococcus strains treated with heat and hen-egg-white lysozyme induce abundant interleukin-12 production by J774.1 macrophages and murine spleen cells.

The IL-12-inducing ability of lactic acid bacteria could be a critical index of immunomodulatory activity, especially in promoting T-helper-1 responses and in suppressing T-helper-2-mediated allergic responses. We aimed to develop a simple method for enhancing the IL-12-inducing ability of bacteria. We examined the in vitro effects of strains of lysozyme-modified Lactococcus (ML-LYS), prepared by heat treatment of the Lactococcus strain in the presence of lysozyme, on the ability of mouse macrophage-like J774.1 cells and spleen cells to produce IL-12. An IL-12-inducing ability greater than that of heat-killed bacteria was shown by 41 of 46 ML-LYS strains in J774.1 cells and by all 46 ML-LYS strains in mouse spleen cells. In contrast, bacteria modified by α-lactalbumin, ß-lactoglobulin, or ovalbumin did not enhance IL-12 production in J774.1 cells. Microscopically, ML-LYS showed stronger resistance to lysozyme and macrophage digestion than did heat-killed bacteria or the other modified bacteria. Addition of chitotriose, a lysozyme inhibitor, enhanced IL-12 production by J774.1 cells stimulated with heat-killed bacteria. Therefore, enhancement of resistance to lysozyme may be a key factor in the strong IL-12-inducing ability of ML-LYS. These findings have important implications for the design of dairy products that have an immunomodulatory effect using the modified bacteria.

Polyphasic study of microbial communities of two Spanish farmhouse goats' milk cheeses from Sierra de Aracena.

The microbial communities present in 2 different types of farmhouse goats' milk cheese from the Aracena mountains (southwest Spain), Quesailla Arochena (hard cheese) and Torta Arochena (soft cheese), have been studied using both culture-dependent and culture-independent techniques. All bacterial isolates were clustered by using randomly amplified polymorphic DNA (RAPD) and identified by 16S rRNA gene sequencing, species-specific PCR and multiplex PCR. Thus a total of 26 different species were identified, the majority belonging to the lactic-acid bacteria (LAB), mainly represented by Lactococcus lactis and Lactobacillus species such as Lactobacillus plantarum and Lactobacillus paracasei, together with a significant proportion of enterococci. Amongst the non-lactic-acid bacteria (NLAB), which represented 37% of the isolates in Torta Arochena, enterobacteria were the most important, Hafnia alvei and Serratia liquefaciens being the predominant species in Quesailla Arochena and Torta Arochena respectively. Moreover, RAPD analysis of the isolates revealed that most of the genotypes were specific to one of the cheeses, although a few genotypes common to both cheeses were found. The culture-independent study carried out by temporal-temperature-gradient gel electrophoresis (TTGE) with 2 target genes, rRNA 16S and rpoB, revealed less species diversity but L. lactis and Lb. plantarum were also predominant. Nevertheless, TTGE carried out using RNAr 16S also detected some organisms that had not been isolated by the culture-dependent method, such as Leuconostoc lactis and Mycoplasma agalactie in Quesailla Arochena. Although TTGE of the rpoB gene revealed less species diversity, it did lead to the detection of previously non-isolated species, such as Ln. lactis in Quesailla Arochena. Apart from this, the fingerprinting of Lactobacillus populations by length-heterogeneity PCR showed the predominance of the Lb. plantarum group, followed by Lactobacillus curvatus and, in smaller quantities, Lb. paracasei in Torta Arochena. From our results we may conclude that both types of methods complement each other and offer a more complete vision of the microbial diversity of these ecosystems.

Lactococcus chungangensis sp. nov., a lactic acid bacterium isolated from activated sludge foam.

The taxonomic position of a Gram-positive coccus, designated strain CAU 28T, isolated from activated sludge foam was determined by using a polyphasic approach. Based on its cellular morphology and the results of biochemical tests, strain CAU 28T was identified tentatively as a member of the genus Lactococcus. Comparative 16S rRNA gene sequence analysis showed that levels of similarity between strain CAU 28T and the type strains of recognized Lactococcus species ranged from 90.4 to 97.2 %. DNA-DNA hybridization studies showed that strain CAU 28T displayed less than 26.1 % relatedness to the type strains of recognized Lactococcus species. The rep-PCR fingerprints revealed that strain CAU 28T was well separated from reference Lactococcus species. The combined genotypic and phenotypic data indicate that strain CAU 28T represents a novel species of the genus Lactococcus, for which the name Lactococcus chungangensis sp. nov. is proposed. The type strain is CAU 28T (=KCTC 13185T =CCUG 55099T).

Polyphasic approach to bacterial dynamics during the ripening of Spanish farmhouse cheese, using culture-dependent and -independent methods.

We studied the dynamics of the microbial population during ripening of Cueva de la Magahá cheese using a combination of classical and molecular techniques. Samples taken during ripening of this Spanish goat's milk cheese in which Lactococcus lactis and Streptococcus thermophilus were used as starter cultures were analyzed. All bacterial isolates were clustered by using randomly amplified polymorphic DNA (RAPD) and identified by 16S rRNA gene sequencing, species-specific PCR, and multiplex PCR. Our results indicate that the majority of the 225 strains isolated and enumerated on solid media during the ripening period were nonstarter lactic acid bacteria, and Lactobacillus paracasei was the most abundant species. Other Lactobacillus species, such as Lactobacillus plantarum and Lactobacillus parabuchneri, were also detected at the beginning and end of ripening, respectively. Non-lactic-acid bacteria, mainly Kocuria and Staphylococcus strains, were also detected at the end of the ripening period. Microbial community dynamics determined by temporal temperature gradient gel electrophoresis provided a more precise estimate of the distribution of bacteria and enabled us to detect Lactobacillus curvatus and the starter bacteria S. thermophilus and L. lactis, which were not isolated. Surprisingly, the bacterium most frequently found using culture-dependent analysis, L. paracasei, was scarcely detected by this molecular approach. Finally, we studied the composition of the lactobacilli and their evolution by using length heterogeneity PCR.

Polyphasic characterization of the lactic acid bacteria in kefir.

The lactic acid bacteria of kefir were isolated and characterized using phenotypical, biochemical, and genotypical methods. Polyphasic analyses of results permitted the identification of the microflora to the strain level. The genus Lactobacillus was represented by the species Lb. kefir and Lb. kefiranofaciens. Both subspecies of Lactococcus lactis (lactis and cremoris) were isolated. Leuconostoc mesenteroides subsp. cremoris was also found. The kefir studied contained few species of lactic acid bacteria but showed a high number of different strains. We found that the polyphasic analysis approach increases the confidence in strain determination. It helped confirm strain groupings and it showed that it could have an impact on the phylogeny of the strains.