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.     

High-resolution amplified fragment length polymorphism typing of Lactococcus lactis strains enables identification of genetic markers for subspecies-related phenotypes

A high-resolution amplified fragment length polymorphism (AFLP) methodology was developed to achieve the delineation of closely related Lactococcus lactis strains. The differentiation depth of 24 enzyme-primer-nucleotide combinations was experimentally evaluated to maximize the number of polymorphisms. The resolution depth was confirmed by performing diversity analysis on 82 L. lactis strains, including both closely and distantly related strains with dairy and nondairy origins. Strains clustered into two main genomic lineages of L. lactis subsp. lactis and L. lactis subsp. cremoris type-strain-like genotypes and a third novel genomic lineage rooted from the L. lactis subsp. lactis genomic lineage. Cluster differentiation was highly correlated with small-subunit rRNA homology and multilocus sequence analysis (MLSA) studies. Additionally, the selected enzyme-primer combination generated L. lactis subsp. cremoris phenotype-specific fragments irrespective of the genotype. These phenotype-specific markers allowed the differentiation of L. lactis subsp. lactis phenotype from L. lactis subsp. cremoris phenotype strains within the same L. lactis subsp. cremoris type-strain-like genomic lineage, illustrating the potential of AFLP for the generation of phenotype-linked genetic markers.     

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.     

Biodiversity of lactic acid bacteria in Moroccan soft white cheese (Jben)

The bacterial diversity occurring in traditional Moroccan soft white cheese, produced in eight different regions in Morocco, was studied. A total of 164 lactic acid bacteria were isolated, purified and identified by whole-cell protein fingerprinting and rep-PCR genomic fingerprinting. The majority of the strains belonged to the genera Lactobacillus, Lactococcus, Leuconostoc and Enterococcus. Sixteen species were identified: Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus paracasei, Lactobacillus brevis, Lactobacillus buchneri, Lactococcus lactis, Lactococcus garvieae, Lactococcus raffinolactis, Leuconostoc pseudomesenteroides, Leuconostoc mesenteroides, Leuconostoc citreum, Eterococcus durans, Enterococcus faecalis, Enterococcus faecium, Enterococcus saccharominimus and Streptococcus sp.     

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.     

动物微生态制剂猪肠源乳球菌的分离与鉴定

试验的菌种是从宁夏平罗县边远农村基本自然生长的健康肉猪的小肠、大肠和盲肠中分离获得的,共分离出64株菌株。通过对这64株菌株的菌落形态观察和革兰氏染色镜检。筛选出9株进行了乳球菌属的生理生化鉴定,初步确定这9株属于乳球菌属(Lactococcus)。再通过糖醇类发酵产酸鉴定,确定2株为乳酸乳球菌乳酸亚种(L.lactissubsplactis),3株为植物乳球菌(L.plantarum),4株为棉籽糖乳球菌(L.raffi-nolactis)。各项鉴定结果均符合乳球菌属和相应种鉴定标准。     

A facile PCR method for detecting replication mode of lactococcial plasmids

A rapid and accurate method was developed for detecting replication type of plasmids of Lactococci. Two specific PCR primer sets were designed for rep gene of Lactococci, one (RC1-RCr1) is derived from the RC-type plasmid, pWV01 and the other (J1-Jr1) is derived from the theta-type plasmid, pVS40. The two primer sets could be used to differentiate the RC-type (pWV01) and the theta-type (pWV02, 03, 04, and 05) replication in Lactococcus lactis subsp. cremoris Wg2 and CCRC10791.     

Microsatellite retrieval in lettuce (Lactuca sativa L.).

By using enriched genomic libraries, microsatellite-containing sequences were isolated from lettuce (Lactuca sativa) with high efficiency. With this approach, a sizeable fraction (up to 55%) of the clones contained a microsatellite. In about half of these clones, primers could be designed for PCR amplification of the microsatellite. This yielded 28 primer sets amplifying unambiguously scorable products, of which 26 showed polymorphisms in a test set of six lettuce varieties. Practically all microsatellite-amplifying primer sets yielded products in lettuce's nearest relative, L. serriola, but only half of the primer sets yielded products in the more distant species L. saligna and L. virosa. An average polymorphism information content (PIC) value of 0.55 and an average number of 3.5 alleles per locus were in the normal range for a self-fertilizing species like lettuce. In addition, the incidental cloning of a microsatellite-containing repeat family, apparently specific for Lactuca, is reported and the implications for the efficient retrieval of single-locus microsatellite sequences are discussed. The microsatellite loci isolated will be useful for distinguishing lettuce cultivars and for screening diversity of genetic resources.     

Use of PCR-based methods for rapid differentiation of Lactobacillus delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis.

Two PCR-based methods, specific PCR and randomly amplified polymorphic DNA PCR (RAPD-PCR), were used for rapid and reliable differentiation of Lactobacillus delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis. PCR with a single combination of primers which targeted the proline iminopeptidase (pepIP) gene of L. delbrueckii subsp. bulgaricus allowed amplification of genomic fragments specific for the two subspecies when either DNA from a single colony or cells extracted from dairy products were used. A numerical analysis of the RAPD-PCR patterns obtained with primer M13 gave results that were consistent with the results of specific PCR for all strains except L. delbrueckii subsp. delbrueckii LMG 6412(T), which clustered with L. delbrueckii subsp. lactis strains. In addition, RAPD-PCR performed with primer 1254 provided highly polymorphic profiles and thus was superior for distinguishing individual L. delbrueckii strains.     

SdrI of Staphylococcus saprophyticus is a multifunctional protein: localization of the fibronectin-binding site

The transferability of a large plasmid that harbors a tetracycline resistance gene tet (S), to fish and human pathogens was assessed using electrotransformation and conjugation. The plasmid, originally isolated from fish intestinal Lactococcus lactis ssp. lactis KYA-7, has potent antagonistic activity against the selected recipients ( Lactococcus garvieae and Listeria monocytogenes ), preventing conjugation. Therefore the tetracycline resistance determinant was transferred via electroporation to L . garvieae . A transformant clone was used as the donor in conjugation experiments with three different L. monocytogenes strains. To our knowledge, this is the first study showing the transfer of an antibiotic resistance plasmid from fish-associated lactic bacteria to L. monocytogenes , even if the donor L. garvieae was not the original host of the tetracycline resistance but experimentally created by electroporation. These results demonstrate that the antibiotic resistance genes in the fish intestinal bacteria have the potential to spread both to fish and human pathogens, posing a risk to aquaculture and consumer safety.     

Classification and characterization of lactic acid bacteria isolated from the intestines of common carp and freshwater prawns

Fifty-four isolates of lactic acid bacteria were obtained from the intestines of the common carp (Cyprinus carpio) and freshwater prawns (Macrobrachium rosenbergii) in Nakorn-Pathom Province, Thailand. All isolates were Gram-positive and catalase-negative cocci that did not produce gas from glucose and formed dl or L(+) lactic acid only. Most isolates were able to grow in broth at pH 9.6, in 6.5% NaCl (w/v) and 40% (w/v) bile. These isolates were divided into six groups (A-F) by sugar fermentation patterns. Strains in the groups A, B, C, and D showed intergroup DNA homology values of above 73.8%, indicating that these groups were composed of a single species. Following phylogenetic analysis, strains E 1, E 7, and E 26 from groups A, E, and F were placed in the clusters of the genera Lactococcus, Pediococcus, and Enterococcus, respectively. The type strains of Lactococcus garvieae, Pediococcus acidilactici, and Enterococcus faecium were the most closely related species with E 1, E 7, and E 26 in the phylogenetic tree, respectively. The DNA-DNA hybridization results indicated that strains in groups A (including groups B, C, and D), E, and F could be identified as belonging to the species Lactococcus garvieae, Pediococcus acidilactici, and Enterococcus faecium, respectively. Lactococcus garvieae was the dominant member of the population, accounting for 90.7% of the isolates.     

Lactococcus lactis subspecies lactis also causes white muscle disease in farmed giant freshwater prawns Macrobrachium rosenbergii

From May to August 2001 in Taiwan, 27 farms for the giant freshwater prawn Macrobrachium rosenbergii experienced white tail disease outbreaks in animals approximately 3 to 5 mo old, with total lengths from 6 to 8 cm. Examination of the infected prawns revealed not only previously reported Lactococcus garvieae (16 farms) but also the novel L. lactis subsp. lactis (10 farms). One farm had shrimp infected with both bacteria. In the farms with L. lactis infections, the cumulative mortality was approximately 25 to 60%. Gross signs of disease were opaque and whitish muscles, while histopathology included marked edema and necrotic lesions, with inflammation in the muscles and hepatopancreas. Bacteria isolated using brain/heart infusion medium or tryptic soy agar were Gram-positive and ovoid. Eleven isolates from different farms were identified as L. lactis subsp. lactis using API 20 Strep and Rapid ID32 Strep tests and using PCR assays specific for the L. lactis subsp. lactis 16S rDNA gene (650 bp amplicon) and for the 16S to 23S rDNA interspacer region (380 bp amplicon). In addition, sequencing of the full 16S rDNA genes of 2 of the isolates (MR17 and MR26; GenBank Accession Numbers AF493058 and AF493057, respectively) revealed 99.9% identity between the isolates and 98.7% identity to several complete 16S rRNA sequences of L. lactis subsp. lactis at GenBank. Experimental infections with our isolates gave gross signs and histopathological changes similar to those seen in naturally infected prawns. The mean lethal dose of 4 isolates and the reference strain L. lactis subsp. lactis BCRC 10791 ranged from 4.2 x 10(6) to 2.5 x 10(7) colony-forming units prawn(-1), indicating virulence similar to that previously reported for L. garvieae. This is the first report confirming L. lactis subsp. lactis as a pathogen in juvenile and adult prawns from aquaculture.     

Diversity of lactic acid bacteria associated with fish and the fish farm environment, established by amplified rRNA gene restriction analysis

Lactic acid bacteria have become a major source of concern for aquaculture in recent decades. In addition to true pathogenic species of worldwide significance, such as Streptococcus iniae and Lactococcus garvieae, several species have been reported to produce occasional fish mortalities in limited geographic areas, and many unidentifiable or ill-defined isolates are regularly isolated from fish or fish products. To clarify the nature and prevalence of different fish-associated bacteria belonging to the lactic acid bacterium group, a collection of 57 isolates of different origins was studied and compared with a set of 22 type strains, using amplified rRNA gene restriction analysis (ARDRA). Twelve distinct clusters were delineated on the basis of ARDRA profiles and were confirmed by sequencing of sodA and 16S rRNA genes. These clusters included the following: Lactococcus raffinolactis, L. garvieae, Lactococcus l., S. iniae, S. dysgalactiae, S. parauberis, S. agalactiae, Carnobacterium spp., the Enterococcus "faecium" group, a heterogeneous Enterococcus-like cluster comprising indiscernible representatives of Vagococcus fluvialis or the recently recognized V. carniphilus, V. salmoninarum, and Aerococcus spp. Interestingly, the L. lactis and L. raffinolactis clusters appeared to include many commensals of fish, so opportunistic infections caused by these species cannot be disregarded. The significance for fish populations and fish food processing of three or four genetic clusters of uncertain or complex definition, namely, Aerococcus and Enterococcus clusters, should be established more accurately.     

Genome sequences of Lactococcus garvieae TB25, isolated from Italian cheese, and Lactococcus garvieae LG9, isolated from Italian rainbow trout

Lactococcus garvieae is a fish pathogen and an emerging zoonotic opportunistic pathogen as well as a component of natural microbiota in dairy products. Here, we present the first report of a genome sequence of L. garvieae TB25, isolated from a dairy source, and that of L. garvieae LG9, isolated from rainbow trout.     

Characterization of <Emphasis Type="Italic">Lactococcus lactis</Emphasis> subsp. <Emphasis Type="Italic">lactis</Emphasis> isolated from surface waters

A group of nine presumptive enterococci was isolated on enterococcal selective media Slanetz-Bartley agar and/or kanamycin-esculin-azide agar during a screening of Enterococcus spp. in surface waters. All strains formed a homogeneous cluster separated from all enterococcal species using rep-PCR fingerprinting with the (GTG)(5) primer but they matched fingerprints revealed by Lactococcus lactis subsp. lactis representatives. Further identification using extensive biotyping and automated ribotyping with EcoRI (RiboPrinter(R) microbial characterization system) confirmed all strains as L. lactis subsp. lactis in full correspondence with the (GTG)(5)-PCR. We demonstrated that L. lactis subsp. lactis strains occur in different surface waters and can be confused with enterococci due to their positive growth on selective enterococcal media as well as positive results in tests commonly used for identification of the genus Enterococcus (esculin hydrolysis, acetoin and pyrrolidonyl arylamidase production, growth at 10 degrees C and in 6.5% NaCl). The (GTG)(5)-PCR fingerprinting was revealed as a reliable and fast method for the identification of L. lactis subsp lactis while automated ribotyping with EcoRI proved to be a good tool for intrasubspecies typing purposes.     

A PCR fingerprinting technique to distinguish isolates of Lactococcus lactis

A fingerprinting technique similar to repetitive extragenic palindromic PCR was developed to identify strains of Lactococcus lactis. The method distinguishes closely related strains and discriminates among some with identical ldh sequences. The fingerprinting primer LL-Rep1 complements a moderately repeated sequence found in low G+C Gram-positive bacteria and may therefore prove useful for discriminating among strains of other low G+C Gram-positive species.     

Detection and characterization of a bacteriocin, garviecin L1-5, produced by Lactococcus garvieae isolated from raw cow's milk

AIMS: The identification of a bacteriocin-producing lactococcal strain isolated from raw cow's milk is reported, along with production conditions, physical and chemical properties, and mode of action of the bacteriocin. METHODS and RESULTS: On the basis of resistance to clindamycin, species-specific PCR and amplification of the 16S-23S rDNA spacer region, the strain was identified as Lactococcus garvieae. Its bacteriocin, designated garviecin L1-5, was bactericidal against closely related species and strains of species from different genera, including Listeria monocytogenes and Clostridium spp. Garviecin L1-5 was shown to be proteinaceous by protease inactivation and was unaffected by heat treatments, also at low pH values. When amplifying known lactococcal bacteriocin genes using DNA from strain L1-5 as template, no amplification products were observed on the agarose gel. The molecular weight of garviecin L1-5 was about 2.5 kDa. As far as is known, no bacteriocins have been detected from Lactococcus garvieae. CONCLUSION: The general properties of garviecin L1-5 are characteristic of the low-molecular-weight bactericidal peptide group. SIGNIFICANCE AND IMPACT OF THE STUDY: The survey of micro-organisms for novel antimicrobial substances provided valuable information on their physiology, ecology and practical application.