Characterization of Leuconostoc gasicomitatum sp. nov., associated with spoiled raw tomato-marinated broiler meat strips packaged under modified-atmosphere conditions

Lactic acid bacteria (LAB) associated with gaseous spoilage of modified-atmosphere-packaged, raw, tomato-marinated broiler meat strips were identified on the basis of a restriction fragment length polymorphism (RFLP) (ribotyping) database containing DNAs coding for 16S and 23S rRNAs (rDNAs). A mixed LAB population dominated by a Leuconostoc species resembling Leuconostoc gelidum caused the spoilage of the product. Lactobacillus sakei, Lactobacillus curvatus, and a gram-positive rod phenotypically similar to heterofermentative Lactobacillus species were the other main organisms detected. An increase in pH together with the extreme bulging of packages suggested a rare LAB spoilage type called "protein swell." This spoilage is characterized by excessive production of gas due to amino acid decarboxylation, and the rise in pH is attributed to the subsequent deamination of amino acids. Protein swell has not previously been associated with any kind of meat product. A polyphasic approach, including classical phenotyping, whole-cell protein electrophoresis, 16 and 23S rDNA RFLP, 16S rDNA sequence analysis, and DNA-DNA reassociation analysis, was used for the identification of the dominant Leuconostoc species. In addition to the RFLP analysis, phenotyping, whole-cell protein analysis, and 16S rDNA sequence homology indicated that L. gelidum was most similar to the spoilage-associated species. The two spoilage strains studied possessed 98.8 and 99.0% 16S rDNA sequence homology with the L. gelidum type strain. DNA-DNA reassociation, however, clearly distinguished the two species. The same strains showed only 22 and 34% hybridization with the L. gelidum type strain. These results warrant a separate species status, and we propose the name Leuconostoc gasicomitatum sp. nov. for this spoilage-associated Leuconostoc species.     

A genus-specific PCR method for differentiation between Leuconostoc and Weissella and its application in identification of heterofermentative lactic acid bacteria from coffee fermentation

A genus-specific PCR analysis method was developed for a rapid and reliable differentiation between the two heterofermentative lactic acid bacteria genera Leuconostoc and Weissella. Primer sets specific for target regions of the 16S rRNA genes were designed and the specificity of the PCR was evaluated using the type strains of 13 species of Leuconostoc and 11 species of Weissella. In addition, the newly developed genus-specific PCR analysis was applied to characterize 72 lactic acid bacteria (LAB) strains isolated from coffee fermentation and which were presumptively classified as Leuconostoc or Weissella species. Additionally, a total of 34 LAB isolates from various other fermented foods were included. The investigations of these strains were conducted to test the effectiveness of correct characterization of field isolates using the genus-specific PCR approach. The correct assignment to one of these two genera by the application of the genus-specific primers was confirmed by further identifying the strains using repetitive extragenic palindromic-PCR and 16S rRNA gene sequencing.     

Identification of Carnobacterium, Lactobacillus, Leuconostoc and Pediococcus by rDNA-based techniques

Ribosomal DNA-based techniques including the analysis of profiles generated by ISR amplification, ISR restriction and ARDRA have been evaluated as molecular tools for identifying Carnobacterium, Lactobacillus, Leuconostoc and Pediococcus. They have been applied for the molecular characterization of 91 strains with the following identities: eight Carnobacterium including the eight type species of the genus; 61 Lactobacillus including 40 type strains out of 45 species, 13 Leuconostoc, out of them 11 are type strains and three are subspecies of Lc. mesenteroides; and nine strains representing the six species of genus Pediococcus. The genetic relationship displayed between these species by rrn-based profiles is sustained by their phylogenetic relationships and can therefore be considered useful for taxonomic purposes. Profiles obtained by ISR amplification allowed identification at genus level of Carnobacterium and Leuconostoc, and even at species level in genus Carnobacterium. Genera Lactobacillus and Pediococcus could not be distinguished from each other by applying this technique. The Lactobacillus species analysed here (45) were differentiated using ARDRA-DdeI and ISR-DdeI profiles, sequentially, and Pediococcus species by ISR-DdeI profiles. It was necessary to combine profiles generated by restriction of ISR-DdeI, ARDRA-DdeI and ARDRA-HaeIII in order to complete the identification of Leuconostoc species.     

The use of multiplex PCR reactions to characterize populations of lactic acid bacteria associated with meat spoilage

A rapid, systematic and reliable approach for identifying lactic acid bacteria associated with meat was developed, allowing for detection of Carnobacterium spp., Lactobacillus curvatus, Lact. sakei and Leuconostoc spp. Polymerase chain reaction primers specific for Carnobacterium and Leuconostoc were created from 16S rRNA oligonucleotide probes and used in combination with species-specific primers for the 16S/23S rRNA spacer region of Lact. curvatus and Lact. sakei in multiplex PCR reactions. The method was used successfully to characterize lactic acid bacteria isolated from a vacuum-packaged pork loin stored at 2 degrees C. Seventy isolates were selected for identification and 52 were determined to be Lact. sakei, while the remaining 18 isolates were identified as Leuconostoc spp.     

Role of broiler carcasses and processing plant air in contamination of modified-atmosphere-packaged broiler products with psychrotrophic lactic acid bacteria

Some psychrotrophic lactic acid bacteria (LAB) are specific meat spoilage organisms in modified-atmosphere-packaged (MAP), cold-stored meat products. To determine if incoming broilers or the production plant environment is a source of spoilage LAB, a total of 86, 122, and 447 LAB isolates from broiler carcasses, production plant air, and MAP broiler products, respectively, were characterized using a library of HindIII restriction fragment length polymorphism (RFLP) patterns of the 16 and 23S rRNA genes as operational taxonomic units in numerical analyses. Six hundred thirteen LAB isolates from the total of 655 clustered in 29 groups considered to be species specific. Sixty-four percent of product isolates clustered either with Carnobacterium divergens or with Carnobacterium maltaromaticum type strains. The third major product-associated cluster (17% of isolates) was formed by unknown LAB. Representative strains from these three clusters were analyzed for the phylogeny of their 16S rRNA genes. This analysis verified that the two largest RFLP clusters consisted of carnobacteria and showed that the unknown LAB group consisted of Lactococcus spp. No product-associated LAB were detected in broiler carcasses sampled at the beginning of slaughter, whereas carnobacteria and lactococci, along with some other specific meat spoilage LAB, were recovered from processing plant air at many sites. This study reveals that incoming broiler chickens are not major sources of psychrotrophic spoilage LAB, whereas the detection of these organisms from the air of the processing environment highlights the role of processing facilities as sources of LAB contamination.     

Role of Broiler Carcasses and Processing Plant Air in Contamination of Modified-Atmosphere-Packaged Broiler Products with Psychrotrophic Lactic Acid Bacteria

Some psychrotrophic lactic acid bacteria (LAB) are specific meat spoilage organisms in modified-atmosphere-packaged (MAP), cold-stored meat products. To determine if incoming broilers or the production plant environment is a source of spoilage LAB, a total of 86, 122, and 447 LAB isolates from broiler carcasses, production plant air, and MAP broiler products, respectively, were characterized using a library of HindIII restriction fragment length polymorphism (RFLP) patterns of the 16 and 23S rRNA genes as operational taxonomic units in numerical analyses. Six hundred thirteen LAB isolates from the total of 655 clustered in 29 groups considered to be species specific. Sixty-four percent of product isolates clustered either with Carnobacterium divergens or with Carnobacterium maltaromaticum type strains. The third major product-associated cluster (17% of isolates) was formed by unknown LAB. Representative strains from these three clusters were analyzed for the phylogeny of their 16S rRNA genes. This analysis verified that the two largest RFLP clusters consisted of carnobacteria and showed that the unknown LAB group consisted of Lactococcus spp. No product-associated LAB were detected in broiler carcasses sampled at the beginning of slaughter, whereas carnobacteria and lactococci, along with some other specific meat spoilage LAB, were recovered from processing plant air at many sites. This study reveals that incoming broiler chickens are not major sources of psychrotrophic spoilage LAB, whereas the detection of these organisms from the air of the processing environment highlights the role of processing facilities as sources of LAB contamination.     

Isolation and identification of cultivable lactic acid bacteria in traditional yak milk products of Gansu Province in China

Various traditional fermented yak milk and raw milk foods could be considered as an abundant resource for obtaining novel lactic acid bacteria (LAB) with unique properties. Eighty-eight samples of yak milk products were collected from Gansu Province in China. Three hundred and nineteen strains of LAB isolated from these samples were identified by phenotypic methods, 16S rRNA gene sequence analysis and PCR-Denaturing Gradient Gel Electrophoresis (PCR-DGGE) technology. Among the isolates, one hundred and sixty-four isolates (51.41% of the total) were classified under Lactobacilli, and one hundred and fifty-five (48.59%) belonged to cocci. All the isolates were classified to six genera (Lactobacillus, Lactococcus, Leuconostoc, Streptococcus, Enterococcus and Weissella) and twenty-one species. Lactobacillus helveticus (87 strains), Leuconostoc mesenteroides subsp. mesenteroides (49 strains), Streptococcus thermophilus (39 strains), Lactobacillus casei (31 strains) and Lactococcus lactis subsp. lactis (19 strains) were considered as the predominant populations in the yak milk products. The results showed that there were abundant genus and species LAB existing in yak milk products in Gansu Province in China. The obtained LAB pure cultures may be a valuable source for further starter selection.     

Lactic acid bacteria associated with vacuum-packed cooked meat product spoilage: population analysis by rDNA-based methods

AIM: To determine the lactic acid bacteria (LAB) implicated in bloating spoilage of vacuum-packed and refrigerated meat products. METHODS AND RESULTS: A total of 18 samples corresponding to four types of meat products, with and without spoilage symptoms, were studied. In all, 387 colonies growing on de Man, Rogosa and Sharpe, yeast glucose lactose peptone and trypticase soy yeast extract plates were identified by internal spacer region (ISR), ISR-restriction fragment length polymorphism and rapid amplified ribosomal DNA restriction analysis profiles as Lactobacillus (37%), Leuconostoc (43%), Carnobacterium (11%), Enterococcus (4%) and Lactococcus (2%). Leuconostoc mesenteroides dominated the microbial population of spoiled products and was always present at the moment bloating occurred. Lactobacillus sakei, Lactobacillus plantarum and Lactobacillus curvatus were found in decreasing order of abundance. The analysis of two meat products, 'morcilla' and 'fiambre de magro adobado' obtained from production lines revealed a common succession pattern in LAB populations in both products and showed that Leuc. mesenteroides became the main species during storage, despite being below the detection level of culture methods after packing. CONCLUSIONS: Our results pointed to Leuc. mesenteroides as the main species responsible for bloating spoilage in vacuum-packed meat products. SIGNIFICANCE AND IMPACT OF THE STUDY: Prevention of bloating spoilage in vacuum-packed cooked meat products requires the sensitive detection of Leuc. mesenteroides (i.e. by PCR).     

Characterization of lactic acid bacteria isolated from seafood

Lactic acid bacteria were isolated from various samples of seafood: fresh pollock, brine shrimp, gravad fish, vacuum-packed seafood (surimi, smoked tuna, salted cod), and fish stored under 100% CO₂ at 5°C (smoked tuna, fresh and salted cod, salmon). Eighty-six independent isolates were obtained and were grouped according to cell morphology, presence or absence of diaminopimelic acid in the cell wall, and lactate configuration. Fifty-four isolates were identified as belonging to the genus Lactococcus and most of them exhibited DNA homologies with L. lactis subsp. lactis. Four strains were identified as Lactobacillus plantarum , eight strains as genus Leuconostoc and 16 belonged to the genus Carnobacterium. One facultative heterofermentative Lactobacillus and three other isolates were not identified. Of the strains 47% showed similar patterns of carbohydrate fermentations especially among strains belonging to the genera Lactococcus and Carnobacterium. Most of the strains (64%) grew at 5°C, in salted media and in fish extract medium without added sugar. Carnobacterium piscicola and Carn. divergens were the only reference strains able to grow in the same conditions as well as psychrotroph strains isolated from seafood. A numerical analysis could not be used because of the divergent properties of isolates of the same genus and strong similarities between different genera.     

Identification and characterization of lactic acid bacteria isolated from traditional pickles in Sichuan, China

The pickle, a traditional fermented vegetable product, is popular in Sichuan Province of China. The objective of this study was to investigate the diversity of dominant lactic acid bacteria (LAB) in pickles by analyzing 36 samples collected from 6 different regions in Sichuan Province. The LAB counts in these samples varied from 3.90 to 8.40 log cfu ml^-1. In total, 185 presumptive LAB with Gram-positive and catalase-negative properties were obtained from these samples using MRS agar, and those strains were identified at the species level by physiological tests, 16S rRNA gene sequencing and multiplex PCR assay. The results revealed that all isolates were accurately identified as Enterococcus thailandicus (2 strains), Lactobacillus alimentarius (16 strains), L. brevis (24 strains), L. paracasei (9 strains), L. plantarum (81 strains), L. pentosus (38 strains), L. sakei (8 strains), L. spicheri (1 strain), Leuconostoc lactis (1 strain) and Pediococcus ethanolidurans (5 strains). The predominant LAB in Sichuan pickle was L. plantarum, which were isolated from most samples. The results also demonstrated that different regions in Sichuan Province have complex compositions of LAB species, and such a rich resource of LAB strains provides raw data for further studies involving probiotic strain selection.     

Identification and characterization of Lactic Acid Bacteria isolated from Tibetan Qula cheese

Fourteen strains of Lactic Acid Bacteria (LAB) isolated from Qula, a Tibetan traditional yak cheese, were divided into four groups (A-D) according to morphological and biochemical characteristics. On the basis of the 16S rRNA gene sequence analysis, group A and group B strains were placed in the cluster making up the genus Leuconostoc, which together with Leuconostoc mesenteroides and Leuconostoc pseudomesenteroides, formed a distinct cluster. The group C strain was clearly identified as Enterococcus faecium by forming a very well defined cluster with this species. The group D strains were placed in the lactobacilli cluster with Lactobacillus plantarum and Lactobacillus pentosus being the closely related species. On the basis of DNA-DNA hybridization, strains in the groups A, B, C and D were identified as Leuconostoc mesenteroides subsp. dextranicum, Leuconostoc pseudomesenteroides, Enterococcus faecium and Lactobacillus plantarum, respectively. Leuconostoc mesenteroides subsp. dextranicum was the dominate member of the population.     

Specific enumeration of lactic acid bacteria in fermenting grape must and wine by colony hybridization with non-isotopic DNA probes

A. LONVAUD-FUNEL, A. JOYEUX AND O. LEDOUX. 1991. Total DNA extracted from lactic acid bacteria commonly found in musts and wines was randomly labelled with digoxigenin. It was assayed for the detection of several species by dot-blot hybridization. The method proved to be specific as there was no cross-hybridization between most of the species belonging to the genera Leuconostoc, Pediococcus and Lactobacillus , homofermentative and heterofermentative ( Lact. plantarum, Lact. casei, Leuc. mesenteroides, Leuc. oenos, Ped. damnosus, Ped. pentosaceus ). However, it failed for some Lact. brevis strains which strongly hybridized with Lact. hilgardii.
Colony hybridization was performed directly on plates soon after enumeration. Eight probes of the most common species were used; it was possible to follow the evolution of each species during the vinification of two red wines. According to the phase of alcoholic fermentation, then malolactic fermentation, the predominance or regression of bacilli and cocci could be established.     

Production of phenyllactic acid by lactic acid bacteria: an approach to the selection of strains contributing to food quality and preservation

The ability of lactic acid bacteria (LAB) to produce phenyllactic (PLA) and 4-hydroxy-phenyllactic (OH-PLA) acids, metabolites involved in food quality and preservation, has been evaluated by HPLC analysis in 29 LAB strains belonging to 12 species widely used in the production of fermented foods. Metabolite production was demonstrated for all strains of the species Lactobacillus plantarum, Lactobacillus alimentarius, Lactobacillus rhamnosus, Lactobacillus sanfranciscensis, Lactobacillus hilgardii, Leuconostoc citreum, and for some strains of Lactobacillus brevis, Lactobacillus acidophilus and Leuconostoc mesenteroides subsp. mesenteroides. Strains were distinguished by analysis of variance in three groups including 15 strains that produced both metabolites (0.16-0.46 mM PLA and 0.07-0.29 mM OH-PLA), five strains accumulating in culture only PLA (0.17-0.57 mM) and nine non-producer strains (< or = 0.10 mM PLA and < or = 0.02 mM OH-PLA). Improvement of phenyllactic acid production was obtained in a selected L. plantarum strain by increasing the concentration of phenylalanine in culture and using low amounts of tyrosine.     

Detection of Lactobacillus, Pediococcus, Leuconostoc, and Weissella species in human feces by using group-specific PCR primers and denaturing gradient gel electrophoresis

Denaturing gradient gel electrophoresis (DGGE) of DNA fragments generated by PCR with 16S ribosomal DNA-targeted group-specific primers was used to detect lactic acid bacteria (LAB) of the genera Lactobacillus, Pediococcus, Leuconostoc, and Weissella in human feces. Analysis of fecal samples of four subjects revealed individual profiles of DNA fragments originating not only from species that have been described as intestinal inhabitants but also from characteristically food-associated bacteria such as Lactobacillus sakei, Lactobacillus curvatus, Leuconostoc mesenteroides, and Pediococcus pentosaceus. Comparison of PCR-DGGE results with those of bacteriological culture showed that the food-associated species could not be cultured from the fecal samples by plating on Rogosa agar. On the other hand, all of the LAB species cultured from feces were detected in the DGGE profile. We also detected changes in the types of LAB present in human feces during consumption of a milk product containing the probiotic strain Lactobacillus rhamnosus DR20. The analysis of fecal samples from two subjects taken before, during, and after administration of the probiotic revealed that L. rhamnosus was detectable by PCR-DGGE during the test period in the feces of both subjects, whereas it was detectable by culture in only one of the subjects.     

Bacteriophage ecology in commercial sauerkraut fermentations

Knowledge of bacteriophage ecology in vegetable fermentations is essential for developing phage control strategies for consistent and high quality of fermented vegetable products. The ecology of phages infecting lactic acid bacteria (LAB) in commercial sauerkraut fermentations was investigated. Brine samples were taken from four commercial sauerkraut fermentation tanks over a 60- or 100-day period in 2000 and 2001. A total of 171 phage isolates, including at least 26 distinct phages, were obtained. In addition, 28 distinct host strains were isolated and identified as LAB by restriction analysis of the intergenic transcribed spacer region and 16S rRNA sequence analysis. These host strains included Leuconostoc, Weissella, and Lactobacillus species. It was found that there were two phage-host systems in the fermentations corresponding to the population shift from heterofermentative to homofermentative LAB between 3 and 7 days after the start of the fermentations. The data suggested that phages may play an important role in the microbial ecology and succession of LAB species in vegetable fermentations. Eight phage isolates, which were independently obtained two or more times, were further characterized. They belonged to the family Myoviridae or Siphoviridae and showed distinct host ranges and DNA fingerprints. Two of the phage isolates were found to be capable of infecting two Lactobacillus species. The results from this study demonstrated for the first time the complex phage ecology present in commercial sauerkraut fermentations, providing new insights into the bioprocess of vegetable fermentations.     

Identification of lactic acid bacteria from spoilage associations of cooked and brined shrimps stored under modified atmosphere between 0 degrees C and 25 degrees C

AIMS: To evaluate spoilage and identify lactic acid bacteria (LAB) from spoilage associations of cooked and brined shrimps stored under modified atmosphere packaging (MAP) at 0, 5, 8, 15 and 25 degrees C. METHODS AND RESULTS: Bacterial isolates (102) from spoilage associations of cooked and brined MAP shrimps were characterized by phenotypic tests and identified as lactic acid bacteria (78 isolates), other Gram-positive bacteria (13 isolates) and Gram-negative bacteria (11 isolates). A selection of 48 LAB isolates were further characterized and identified by phenotypic tests and SDS-PAGE electrophoresis of whole cell proteins. Selected clusters of LAB isolates were analysed by plasmid profiling, pulsed field gel electrophoresis and 16S rRNA gene sequencing. Enterococcus faecalis was identified in spoilage associations at 15 degrees C and 25 degrees C, and its metabolic activity corresponded to chemical changes in spoiled products. Carnobacterium divergens, a non-motile Carnobacterium sp. nov. and Lactobacillus curvatus were the LAB species observed in spoilage associations of products stored at 0 degrees C, 5 degrees C and 8 degrees C. CONCLUSIONS: Enterococcus spp. and Carnobacterium spp. were the dominant parts of spoilage associations of cooked and brined MAP shrimps stored at high and low temperatures, respectively. SIGNIFICANCE AND IMPACT OF THE STUDY: The SDS-PAGE technique and simple biochemical keys allowed the majority of LAB isolates from spoilage associations of cooked and brined MAP shrimps to be identified at the species level.     

Biochemical and serological characterization of Carnobacterium spp. isolated from farmed and natural populations of striped bass and catfish.

A comparative analysis of the phenotypic and serological properties of Carnobacterium strains associated with mortalities of cultured striped bass and channel catfish and the properties of isolates from wild brown bullhead catfish in the Chesapeake Bay area in Maryland was conducted. All of the strains were gram-positive, facultatively anaerobic, nonmotile, non-spore-forming rods occurring singly or in short chains. They did not produce cytochrome oxidase or catalase, did not reduce nitrate, failed to produce H2S, were unable to grow on acetate medium, and did not produce gas from glucose or gluconate. The temperature and salinity ranges for most of the strains were 10 to 37 degrees C and 0 to 6% NaCl, respectively. The strains all fermented mannitol and inulin and were arginine dihydrolase positive; these are typical characteristics of Carnobacterium piscicola. The carbohydrate fermentation pattern exhibited by all of the isolates with the API-50 CHL system was also very similar to that shown by C. piscicola. Acid was produced from ribose, glucose, fructose, mannose, mannitol, N-acetylglucosamine, amygdaline, arbutin, esculin, salicin, cellobiose, maltose, sucrose, trehalose, and gentiobiose. The Carnobacterium strains did not show proteolytic, lipolytic, amylolytic, or hemolytic activity. Eighteen drugs were tested; all strains proved to be resistant to chloramphenicol, gentamicin, kanamycin, streptomycin, trimethoprim, quinolones, and nitrofurans. The analysis of membrane proteins supported the phenotypic similarities, two main patterns were established, one shared by the striped bass isolates and the reference strain of C. piscicola and another shared by most of the catfish strains. However, the agglutination assays demonstrated that only one Carnobacterium strain from striped bass was serologically related to C. piscicola ATCC 35586.(ABSTRACT TRUNCATED AT 250 WORDS)     

Common occurrence of plasmid DNA and vancomycin resistance in Leuconostoc spp.

Resistance to vancomycin permitted detection, in a culture of Streptococcus cremoris 290PC, of a contaminant gram-positive coccus. Morphological and physiological characteristics indicated that this bacterium was a strain of Leuconostoc sp., designated PO184. This strain contained four plasmid species, which were distinct from those harbored by S. cremoris 290PC. Antibiotic disk susceptibility tests indicated that Leuconostoc sp. strain PO184 was also resistant to sulfathiazole and trimethoprim and susceptible to 17 other antimicrobials. The MIC of vancomycin for this strain was greater than 2,000 micrograms/ml, and resistance did not depend on drug inactivation. Leuconostoc sp. strain PO184 produced a substance which was inhibitory to S. cremoris U134, but not to S. lactis ATCC 11454. Five other leuconostocs produced substances with antibacterial activity. Of 18 strains of Leuconostoc sp., 14 were resistant to at least 500 micrograms of vancomycin per ml, including four L. oenos strains which harbored no plasmid DNA in the 1- to 76-megadalton range. Twelve Leuconostoc sp. strains contained at least one plasmid species in this mass range. These findings are discussed from the physiological, taxonomical, and ecological standpoints and with regard to their potential applications.     

Carnobacterium divergens and Carnobacterium maltaromaticum as spoilers or protective cultures in meat and seafood: phenotypic and genotypic characterization

Carnobacterium, a genus of lactic acid bacteria, frequently dominate the microflora of chilled vacuum- or modified atmosphere-packed meat and seafood. In this study Carnobacterium isolates were characterized by phenotypic and molecular methods in order to investigate the association of species and intra-species groups with distinct kinds of meat and seafood. Of 120 test strains, 50 originated from meat (beef and pork products, including 44 strains isolated during this study and 6 strains obtained from culture collections) and 52 from seafoods (cod, halibut, salmon, shrimps and roe products). In addition, 9 reference strains of Carnobacterium spp from other sources than meat and fish and 9 reference strains of lactic acid bacteria belonging to other genera than Carnobacterium were included. Numerical taxonomy relying on classical biochemical reactions, carbohydrate fermentation and inhibition tests (temperature, salt, pH, chemical preservatives, antibiotics, bacteriocins), SDS-PAGE electrophoresis of whole cell proteins, plasmid profiling, intergenic spacer region (ISR) analysis and examination of amplified-fragment length polymorphism (AFLP) were employed to characterize the strains. The numerical taxonomic approach divided the carnobacteria strains into 24 groups that shared less than 89% similarity. These groups were identified as Carnobacterium divergens with one major cluster (40 strains) and 7 branches of one to four strains, Carnobacterium maltaromaticum (previous C. piscicola) with one major cluster (37 strains) and 9 branches of one to four strains and Carnobacterium mobile (three branches consisting in total of 4 strains). Branches consisting of references strains of the remaining Carnobacterium spp. were separated from clusters and branches of C. divergens, C. maltaromaticum and C. mobile. Isolates from the main clusters of C. divergens and C. maltaromaticum were found both in fresh and lightly preserved meat and seafood products. High phenotypic intra-species variability was observed for C. divergens and C. maltaromaticum but despite this heterogeneity in phenotypic traits a reliable identification to species levels was obtained by SDS-PAGE electrophoresis of whole cell proteins and by ISR based on 16S-23S rDNA intergenic spacer region polymorphism. With AFLP, two distinct clusters were observed for C. divergens but only one for C. maltaromaticum. The two C. divergens clusters were not identical to any of the clusters observed by numerical taxonomy. A limited number of C. divergens and C. maltaromaticum isolates possessed a biopreservative potential due to their production of bacteriocins with a wide inhibition spectrum. This study serves as a base-line for further investigations on the potential role of species of Carnobacterium in foods where they predominate the spoilage microflora.     

Biochemical and serological characterization of Carnobacterium spp. isolated from farmed and natural populations of striped bass and catfish.

A comparative analysis of the phenotypic and serological properties of Carnobacterium strains associated with mortalities of cultured striped bass and channel catfish and the properties of isolates from wild brown bullhead catfish in the Chesapeake Bay area in Maryland was conducted. All of the strains were gram-positive, facultatively anaerobic, nonmotile, non-spore-forming rods occurring singly or in short chains. They did not produce cytochrome oxidase or catalase, did not reduce nitrate, failed to produce H2S, were unable to grow on acetate medium, and did not produce gas from glucose or gluconate. The temperature and salinity ranges for most of the strains were 10 to 37 degrees C and 0 to 6% NaCl, respectively. The strains all fermented mannitol and inulin and were arginine dihydrolase positive; these are typical characteristics of Carnobacterium piscicola. The carbohydrate fermentation pattern exhibited by all of the isolates with the API-50 CHL system was also very similar to that shown by C. piscicola. Acid was produced from ribose, glucose, fructose, mannose, mannitol, N-acetylglucosamine, amygdaline, arbutin, esculin, salicin, cellobiose, maltose, sucrose, trehalose, and gentiobiose. The Carnobacterium strains did not show proteolytic, lipolytic, amylolytic, or hemolytic activity. Eighteen drugs were tested; all strains proved to be resistant to chloramphenicol, gentamicin, kanamycin, streptomycin, trimethoprim, quinolones, and nitrofurans. The analysis of membrane proteins supported the phenotypic similarities, two main patterns were established, one shared by the striped bass isolates and the reference strain of C. piscicola and another shared by most of the catfish strains. However, the agglutination assays demonstrated that only one Carnobacterium strain from striped bass was serologically related to C. piscicola ATCC 35586.(ABSTRACT TRUNCATED AT 250 WORDS)