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.     

Biodiversity among Lactobacillus helveticus strains isolated from different natural whey starter cultures as revealed by classification trees

Lactobacillus helveticus is a homofermentative thermophilic lactic acid bacterium used extensively for manufacturing Swiss type and aged Italian cheese. In this study, the phenotypic and genotypic diversity of strains isolated from different natural dairy starter cultures used for Grana Padano, Parmigiano Reggiano, and Provolone cheeses was investigated by a classification tree technique. A data set was used that consists of 119 L. helveticus strains, each of which was studied for its physiological characters, as well as surface protein profiles and hybridization with a species-specific DNA probe. The methodology employed in this work allowed the strains to be grouped into terminal nodes without difficult and subjective interpretation. In particular, good discrimination was obtained between L. helveticus strains isolated, respectively, from Grana Padano and from Provolone natural whey starter cultures. The method used in this work allowed identification of the main characteristics that permit discrimination of biotypes. In order to understand what kind of genes could code for phenotypes of technological relevance, evidence that specific DNA sequences are present only in particular biotypes may be of great interest.     

Comparison of Lactococcus garvieae strains isolated in northern Italy from dairy products and fishes through molecular typing

Aims:  To investigate the genetic relatedness between Lactococcus garvieae strains isolated from fish and dairy samples collected in northern Italy, using random-amplified polymorphic DNA (RAPD)-polymerase chain reaction (PCR), Sau -PCR and amplified fragment length polymorphism (AFLP).
Methods and Results:  Eighty-one isolates from bovine and caprine dairy products ( n  = 53) and from diseased rainbow trouts and other fishes ( n  = 28) were examined. All methods showed a typeability of 100%, repeatability ranging from 84·4% to 97·5% and discriminatory powers from 0·798 to 0·986. Dairy and fish strains revealed a low genetic relatedness as they are often grouped into distinct clusters. RAPD analysis discriminated 52 genotypes when primer M13 was used, whereas with primer P5 only 27 genotypes were identified. When Sau -PCR was performed, 13 genotypes were detected while AFLP analysis allowed the differentiation of 32 genotypes.
Conclusion:  L. garvieae strains isolated from dairy samples are generally not related to those collected from fish lactococcosis outbreaks.
Significance and Impact of the Study:  L. garvieae strains exhibit a genetic diversity related to the specific animal host they colonize. RAPD M13 fingerprinting proved to be a molecular tool for comparing isolates, whereas Sau -PCR and AFLP analyses were useful techniques to investigate the distribution of L. garvieae populations in the environment.     

Characterization of Lactococcus garvieae isolated from radish and broccoli sprouts that exhibited a KG+ phenotype, lack of virulence and absence of a capsule

AIMS: To identify Lactococcus garvieae isolates from radish and broccoli sprouts and compare them with virulent and less virulent mutant strains obtained from yellowtails with regard to KG phenotype, presence of a capsule and virulence towards yellowtails and mice. METHODS AND RESULTS: Comparative 16S rRNA gene sequence analysis of six isolates obtained from radish and broccoli sprouts indicated that they were L. garvieae (similarity >99%). They were compared with KG9502, Lg2 and ATCC49156 strains obtained from yellowtails. A less virulent mutant strain Lg2-S was obtained by Lg2 subculture. Biochemical characterization of the six strains resembled that of KG9502, Lg2, ATCC49156 and Lg2-S, except for saccharose and tagatose acidification and the presence of hippuricase. These six strains were nonpathogenic towards yellowtails and mice, nonsusceptible to bacteriophages and demonstrated heterogeneity on pulsed-field gel electrophoresis analysis. Using transmission electron microscopy, a capsule was observed in KG9502 and Lg2 but not in ATCC49156 and Lg2-S. CONCLUSIONS: We isolated L. garvieae strains that lacked pathogenicity towards yellowtails and mice from radish and broccoli sprouts; these were noncapsulated and exhibited KG(+) phenotype. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first documentation of L. garvieae isolated from terrestrial plants. These isolates exhibited genetic diversity; however, they were noncapsulated and nonpathogenic towards yellowtails and mice.     

Characterization of closely related lactococcal starter strains which show differing patterns of bacteriophage sensitivity

AIMS: To characterize a group of closely related Lactococcus lactis subsp. lactis casein starter strains used commercially, which differ in their sensitivity to bacteriophages isolated from the same industrial environment. METHODS AND RESULTS: Nine strains of L. lactis, six of which had been used as starter cultures for lactic casein manufacture, were shown to be closely related by pulsed-field gel electrophoresis and total DNA profiles. Nineteen phages which propagated on one or more of these starter strains were isolated from industrial casein whey samples. The phages were all small isometric-headed and could be divided into five groups on the basis of host range on the nine strains. Most of the phages did not give a PCR product with primers designed to detect the two most common lactococcal small isometric phage species (936 and P335). The hosts could be divided into six groups depending on their phage sensitivity. Plasmids encoding genes for the cell envelope associated PI-type proteinase, lactose metabolism and specificity subunits of a type I restriction/modification system were identified. CONCLUSIONS: This work demonstrates how isolates of the same starter strain may come to be regarded as separate cultures because of their different origins, and how these closely related strains may differ in some of their industrially relevant characteristics. SIGNIFICANCE AND IMPACT OF THE STUDY: This situation may be very common among lactococci used as dairy starter cultures, and implies that the dairy industry worldwide depends on a small number of different strains.     

Drug resistance and pulsed-field gel electrophoresis patterns of Lactococcus garvieae isolates from cultured Seriola (yellowtail, amberjack and kingfish) in Japan

AIMS: To investigate the existing antimicrobial susceptibility and genetic characteristics of Lactococcus garvieae isolates from cultured Seriola in Japan. METHODS AND RESULTS: Minimum inhibitory concentrations (MICs) of 14 antimicrobial agents for 170 isolates were determined using the agar dilution method. Seventy-five isolates (44.1%) were simultaneously resistant to erythromycin (EM) (MIC>or=2 microg ml-1), lincomycin (LCM) (MIC>or=128 microg ml-1) and oxytetracycline (OTC) (MIC>or=4 microg ml-1). Resistance to EM was grouped as intermediate- and high-level resistant by MIC values. All resistant isolates possessed ermB and tet(S) genes. The number of different bands between pulsed-field gel electrophoresis patterns of 25 isolates and two ATCC strains (isolated in 1974), determined using two enzymes (ApaI and SmaI), did not exceed 3. CONCLUSIONS: The present resistance pattern observed with ermB and tet(S) is similar to that observed in previous reports. Moreover, the genetic characteristics of L. garvieae isolates from a wide area in Japan in 2002 and ATCC strains were closely related. SIGNIFICANCE AND IMPACT OF THE STUDY: This study suggests that EM-, LCM- and OTC-resistant isolates have been present for 15 years and that L. garvieae strains with same origin have spread among Seriola spp. in Japan since 1974.     

Resistance to serum killing may contribute to differences in the abilities of capsulate and non-capsulated isolates of lactococcus garvieae to cause disease in rainbow trout (Oncorhynchus mykiss L.)

Three capsulated and two non-capsulated isolates of Lactococcus garvieae were investigated in terms of their wall proteins, virulence and interactions with rainbow trout immunoglobulin (Ig). All isolates were similar in integral membrane protein profile, and all were able to bind non-immune rainbow trout Ig, although different proteins appeared to be involved in Ig binding. However, whilst capsulated isolates were highly virulent, non-capsulated isolates were avirulent. This appeared to correlate with susceptibility of the non-capsulated isolates to rainbow trout normal serum. In contrast, the capsulated isolates were resistant to both normal and immune serum killing. In spite of this, passive immunisation of rainbow trout with specific anti-serum to L. garvieae was able to protect against challenge by capsulated isolates of L. garvieae. This suggests the antibody may have some other role in protection against disease caused by this important Gram-positive bacterial fish pathogen.     

Molecular characterization, technological properties and safety aspects of enterococci from 'Hussuwa', an African fermented sorghum product

AIMS: To identify enterococci from Hussuwa, a Sudanese fermented sorghum product, and determine their technological properties and safety for possible inclusion in a starter culture preparation. METHODS AND RESULTS: Twenty-two Enterococcus isolates from Hussuwa were identified as Enterococcus faecium by using phenotypic and genotypic tests such as 16S rDNA gene sequencing, RAPD-PCR and restriction fragment length polymorphism of the 16S/23S intergenic spacer region fingerprinting. Genotyping revealed that strains were not clonally related and exhibited a considerable degree of genomic diversity. Some strains possessed useful technological properties such as production of bacteriocins and H2O2 or utilization of raffinose and stachyose. None produced alpha-amylase or tannase. A safety investigation revealed that all strains were susceptible to the antibiotics ampicillin, gentamicin, chloramphenicol, tetracycline and streptomycin, but some were resistant to ciprofloxacin, erythromycin, penicillin and vancomycin. Production of biogenic amines or presence of genes encoding virulence determinants occurred in some strains. CONCLUSIONS: Enterococcus faecium strains are associated with fermentation of Sudanese Hussuwa. Some strains exhibited useful technological properties such as production of antimicrobial agents and fermentation of indigestible sugars, which may aid in stabilizing and improving the digestibility of the product respectively. SIGNIFICANCE AND IMPACT OF THE STUDY: Enterococci were shown to play a role in the fermentation of African foods. While beneficial properties of these bacteria are indicated, their presence in this food may also imply a hygienic risk as a result of antimicrobial resistances or presence of virulence determinants.     

Virulence factors in food,clinical and reference Enterococci: A common trait in the genus?

The occurrence of several virulence traits (cytolysin, adhesins and hydrolytic enzymes) was investigated in a collection of 164 enterococci, including food and clinical isolates (from human and veterinary origin), as well as type and reference strains from 20 enterococcal species. Up to fifteen different cyl genotypes were found, as well as silent cyl genes. The occurrence of the cyl operon and haemolytic potential seems to be widespread in the genus. A significant association of this virulent trait with clinical isolates was found (p < 0.05). High levels of incidence were also observed for genes encoding surface adhesins (esp, efaA(fs), efaA(fm)), agg and gelE, irrespectively of species allocation and origin of strains. Although gelE behaves as silent in the majority of the strains, gelatinase activity predominates in clinical isolates, whereas lipase and DNase were mainly detected in food isolates pointing to their minor role as virulence determinants. No hyaluronidase activity was detected for all strains. Numerical hierarchic data analysis grouped the strains in three main clusters, two of them including a total of 50 strains with low number of virulence determinants (from 2 to 7) and the other with 114 strains with a high virulence potential (up to 12 determinants). No statistical association was found between virulence clusters and species allocation (p > 0.10), strongly suggesting that virulence determinants are a common trait in the genus Enterococcus. Clinical strains seem to be significantly associated with high virulence potential, whereas food, commensal and environmental strains harbour fewer virulence determinants (p < 0.01). A high level of relative diversity in virulence patterns was observed (Shannon's index varies from 0.95 to 1.0 among clusters), reinforcing the strain-specific nature of the association of virulence factors. Although a low risk seems to be associated with the use of enterococci in long-established artisanal cheeses, screening of virulence traits and their cross-synergies must be performed, particularly for commercial starters, probiotic strains and products to be used by high risk population groups.     

Genotypic and technological characterization of Lactococcus lactis isolates involved in processing of artisanal Manchego cheese

Aims:  Genotypic and technological characterization of wild lactococci isolated from artisanal Manchego cheese during the ripening process for selection of suitable starter cultures.
Methods and Results:  A total of 114 isolates of lactococci were typed using randomly amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR). Sixteen distinct RAPD-PCR patterns, at a similarity level of 73%, were obtained. On the basis of species-specific PCR reaction, the isolates were assigned to the species Lactococcus lactis subsp. lactis and Lactococcus lactis subsp. cremoris with L. lactis subsp. lactis being predominant at both dairies. Twenty-six isolates were technologically characterized to select those with the best properties. Most of them showed good technological properties although some could produce tyramine.
Conclusions:  The presence of coincident genotypes at both dairies has been demonstrated, which would suggest that they are well adapted to the Manchego cheese environment. Interesting differences were found in the technological characterization and the potential role of autochthonous lactococci strains as starter culture has been displayed.
Significance and Impact of the Study:  The great economic importance of Manchego cheese encouraged a deeper knowledge of its microbiota, to select strains with the best properties to use as starter cultures in industrial Manchego cheeses, preserving the autochthonous characteristics.     

Integrated polymerase chain reaction-based procedures for the detection and identification of species and subspecies of the Gram-positive bacterial genus Lactococcus

AIMS: Five species of the Gram-positive bacterial genus Lactococcus (Lactococcus lactis, L. garvieae, L. plantarum, L. piscium and L. raffinolactis) are currently recognized. The aim of this work was to develop a simple approach for the identification of these species, as well as to differentiate the industrially important dairy subspecies L. lactis subsp. lactis and L. lactis subsp. cremoris. METHODS AND RESULTS: Methods were devised based on specific polymerase chain reaction (PCR) amplifications that exploit differences in the sequences of the 16S ribosomal RNA genes of each species, followed by restriction enzyme cleavage of the PCR products. The techniques developed were used to characterize industrial cheese starter strains of L. lactis and the results were compared with biochemical phenotype and DNA sequence data. CONCLUSIONS: The PCR primers designed can be used simultaneously, providing a simple scheme for screening unknown isolates. Strains of L. lactis show heterogeneity in the 16S ribosomal RNA gene sequence. SIGNIFICANCE AND IMPACT OF THE STUDY: This work provides an integrated set of methods for differentiation and identification of lactococcal species associated with agricultural, veterinary, medical and processed food industries.     

Spread and variability of the integrase gene in Lactobacillus delbrueckii ssp. lactis strains and phages isolated from whey starter cultures

AIMS: To determine the presence, diffusion and variability of the integrase (int) gene in Lactobacillus delbrueckii ssp. lactis isolated from natural whey starters used for the production of Italian hard cheeses. METHODS AND RESULTS: A PCR-based protocol aimed to amplify an internal fragment of the int gene was optimized taking into account phage genome sequences available from public databases. Thirty-seven of the 39 strains tested showed the presence of the putative int gene. Southern blot hybridization experiments confirmed data obtained by PCR. The presence of the putative int gene was observed also in 20 of 23 Lact. delbrueckii ssp. lactis lytic phages isolated from the same starter cultures used to isolate strains. Phylogenetic analysis of partial int gene revealed a high similarity both within and between strain- and phage-derived sequences. Sixty per cent of the int-positive strains resulted inducible with mitomycin C, and two of them released active phage particles. CONCLUSIONS: Our preliminary findings seem to suggest that an important number of Lact. delbrueckii ssp. lactis strains associated with the whey starters are lysogenic. SIGNIFICANCE AND IMPACT OF THE STUDY: Further contribution to obtain a clearer picture of the complex relationship between thermophilic lactic acid bacteria phage and host in whey starters for Italian, hard-cooked cheeses.     

Screening of benzalkonium chloride resistance in Listeria monocytogenes strains isolated during cold smoked fish production

AIMS: To determine the susceptibility to disinfectants and cross-resistance to antibiotics in Listeria monocytogenes strains isolated from fish products and the fish-processing environment. METHODS AND RESULTS: Minimal inhibitory concentration assessment, using the agar dilution method, showed 108 of 255 L. monocytogenes isolates with low susceptibility to benzalkonium chloride (BC), commonly used in food industries. Most of them are from raw products of farmed fish during processing, while the remaining resistant isolates were mainly from the environment and finished products irrespective of the fish species. Two BC-resistant isolates were resistant to ethidium bromide (EB). The conservation of resistance after plasmid curing suggested that the resistance genes are not plasmid associated. EB accumulation assays demonstrated that the two BC(R) EB(R) isolates used an efflux pump to expel these substrates whereas a different mechanism was probably used by the majority of the strains with BC(R) EB(S) pattern. No cross-resistance was found with antibiotics. CONCLUSIONS: This study highlights the difference in susceptibilities to BC for L. monocytogenes strains isolated from fish-processing plants and in resistance mechanisms to BC developed by these bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: The presence of BC resistant L. monocytogenes strains could contribute to their adaptation and so explained their survival and persistence in the fish-processing environment.     

Population structure and safety aspects of Enterococcus strains isolated from artisanal dry fermented sausages produced in Argentina

Enterococci population from Argentinean artisanal dry fermented sausage was identified and their safety aspects were evaluated. Species-specific PCR was used to distinguish between Enterococcus faecium (56%) and Enterococcus faecalis (17%). Other isolates (27%) were identified as Enterococcus durans , Enterococcus casseliflavus and Enterococcus mundtii by using 16S RNA gene sequence. RAPD analyses showed different biotypes for Ent. faecium and Ent. faecalis species. Low incidence of antibiotic resistance and high virulence traits in Ent. casseliflavus and Ent. faecalis were found; the majority of the Ent. faecium strains were shown to be free of virulence factors. The absence of virulence/resistance traits and the anti-Listeria activity of Ent. faecium isolates may be exploited to enhance natural preservation thereby guaranteeing organoleptic/safety characteristics of artisanal fermented sausages.     

Complete genome sequence and comparative analysis of the fish pathogen Lactococcus garvieae

Lactococcus garvieae causes fatal haemorrhagic septicaemia in fish such as yellowtail. The comparative analysis of genomes of a virulent strain Lg2 and a non-virulent strain ATCC 49156 of L. garvieae revealed that the two strains shared a high degree of sequence identity, but Lg2 had a 16.5-kb capsule gene cluster that is absent in ATCC 49156. The capsule gene cluster was composed of 15 genes, of which eight genes are highly conserved with those in exopolysaccharide biosynthesis gene cluster often found in Lactococcus lactis strains. Sequence analysis of the capsule gene cluster in the less virulent strain L. garvieae Lg2-S, Lg2-derived strain, showed that two conserved genes were disrupted by a single base pair deletion, respectively. These results strongly suggest that the capsule is crucial for virulence of Lg2. The capsule gene cluster of Lg2 may be a genomic island from several features such as the presence of insertion sequences flanked on both ends, different GC content from the chromosomal average, integration into the locus syntenic to other lactococcal genome sequences, and distribution in human gut microbiomes. The analysis also predicted other potential virulence factors such as haemolysin. The present study provides new insights into understanding of the virulence mechanisms of L. garvieae in fish.     

Effectiveness of Chemometric Techniques in Discrimination of Lactobacillus helveticus Biotypes from Natural Dairy Starter Cultures on the Basis of Phenotypic Characteristics

Lactobacillus helveticus is the dominant organism in natural starter cultures used for the production of typical Italian cheeses. In this study, 74 L. helveticus strains, isolated from grana and provolone cheese natural whey starters, were distinguished with respect to their origin by using both cell wall protein profiles and chemometric evaluation of some phenotypic parameters, such as the ability to acidify cultures and the presence of nonspecific proteolytic and peptidase activities. Cell wall protein patterns allowed L. helveticus strains to be distinguished with respect to their source of isolation. Among the different phenotypes studied, no single specific parameter permitted the two groups of strains to be separated. A good discrimination between the two groups of L. helveticus species was obtained by multivariate statistical techniques, which permitted the extraction of all of the discriminating information retained in the phenotypic activities. Associations between strain phenotype expression and dairy environmental ecosystem source are discussed.     

Biodiversity in Lactobacillus helveticus strains present in natural whey starter used for Parmigiano Reggiano cheese

AIMS: Lactobacillus helveticus is the dominant microflora of the natural whey starters used for Parmigiano Reggiano cheese making. The aim of this work was to study the biodiversity of different strains of Lact. helveticus present in six cultures and to compare them with strains of the same species previously isolated from natural whey cultures used for Grana Padano and Provolone cheeses. METHODS AND RESULTS: Twenty different biotypes of Lact. helveticus strains were identified combining the results deriving from SDS-PAGE of cell surface proteins and PCR fingerprinting using M13 as a primer. The biotypes were present in varying amounts in the six natural whey starters and the biodiversity was demonstrated not only within the whey cultures, but also between the whey cultures. CONCLUSIONS: Lact. helveticus strains isolated from Parmigiano Reggiano whey cultures analysed by PCR M13, SDS-PAGE and RFLP were distinguishable from Lact. helveticus strains of different dairy origin, namely Grana Padano and Provolone natural whey starters. SIGNIFICANCE AND IMPACT OF THE STUDY: The presence of different Lact. helveticus biotypes seems to be related to the specific ecosystem of cheese making and may be considered as one of the elements contributing to the typicality of Parmigiano Reggiano cheese.     

Quantification of Enterococcus italicus in traditional Italian cheeses by fluorescence whole-cell hybridization

The objective of this work was to investigate the spread of Enterococcus italicus in cheese. For this purpose, a fluorescence whole-cell hybridization protocol (FWCH) with a 16S rRNA probe was optimized to evaluate the presence and abundance of this organism in artisanal Italian cheeses. The FWCH method avoided the quantification problems using classical plate count techniques related to the well-known difficulties to cultivate E. italicus in selective enterococci media. After probe and FWCH optimization, 10 commercially available Italian semi-hard cheeses made with raw ewe or cow milk without starter addition were analyzed. All of them were subjected to FWCH experiments and six of them gave positive results with the probe, i.e. the E. italicus content was >4 log cells/g according to the detection limit of FWCH. Counts showed that E. italicus was present at levels ranging from 5.91+/-0.17 to 7.34+/-0.14 log cells/g; such levels were similar to, or even higher than, the total enterococci counted from the corresponding cheeses using kanamycin aesculin azide agar. The overall reliability of the FWCH method was tested by species-specific PCR. The positive amplification of the expected 323 bp fragment from both a cheese matrix and cell bulks of cheese samples containing high loads of this organism (as determined by FWCH counts) and the successful isolation of E. italicus strains from the above cheeses provided definitive proof of both probe specificity and the presence of this organism in cheeses. Although there is very little available quantitative data on the incidence of E. italicus in cheese, or its role in product quality, this study showed a wide diffusion of this organism in artisanal cheeses, where secondary non-starter lactic acid bacterial microflora, which enterococci belong to, may become dominant during ripening.