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.     

Identification of Carnobacterium species by restriction fragment length polymorphism of the 16S-23S rRNA gene intergenic spacer region and species-specific PCR

The genus Carnobacterium is currently divided into the following eight species: Carnobacterium piscicola, C. divergens, C. gallinarum, C. mobile, C. funditum, C. alterfunditum, C. inhibens, and C. viridans. An identification tool for the rapid differentiation of these eight Carnobacterium species was developed, based on the 16S-23S ribosomal DNA (rDNA) intergenic spacer region (ISR). PCR-restriction fragment length polymorphism (PCR-RFLP) analysis of this 16S-23S rDNA ISR was performed in order to obtain restriction profiles for all of the species. Three PCR amplicons, which were designated small ISR (S-ISR), medium ISR (M-ISR), and large ISR (L-ISR), were obtained for all Carnobacterium species. The L-ISR sequence revealed the presence of two tRNA genes, tRNA(Ala) and tRNA(Ile), which were separated by a spacer region that varied from 24 to 38 bp long. This region was variable among the species, allowing the design of species-specific primers. These primers were tested and proved to be species specific. The identification method based on the 16S-23S rDNA ISR, using PCR-RFLP and specific primers, is very suitable for the rapid low-cost identification and discrimination of all of the Carnobacterium species from other phylogenetically related lactic acid bacteria.     

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.     

alpha-Chitinase activity among lactic acid bacteria

Chitin is a polysaccharide widely distributed in nature. Among 115 strains from 29 species of lactic acid bacteria only strains belonging to Carnobacterium divergens and Carnobacterium maltaromaticum hydrolyzed alpha-chitin. This activity was not affected by temperature (10 degrees C versus 30 degrees C) and in most cases not subject to glucose catabolite repression.     

The differentiation of Carnobacterium divergens using the random amplification of polymorphic DNA polymerase chain reaction technique

The potential of the randomly amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) technique to differentiate Carnobacterium divergens from other members of the genus Carnobacterium was examined. A numerical analysis of the genomic profiles obtained demonstrated that it was possible to differentiate the C. divergens strains from other Carnobacterium strains using this technique. The heterogeneity observed in the representatives of the species C. piscicola adds further weight to the suggestion in other taxonomic studies that subspecies of this species exist.     

Differentiation of Carnobacterium divergens and Carnobacterium piscicola by numerical analysis of total soluble cell protein patterns and DNA-DNA hybridizations

Fifteen strains of Carnobacterium divergens and Carnobacterium piscicola, isolated from different meat products, were differentiated from Carnobacterium gallinarum and Carnobacterium mobile by numerical analysis of total soluble cell protein patterns. This technique proved more reliable than conventional physiological and biochemical tests and grouped a genetically heterogeneous collection of species with low DNA homology values (less than 25%) into well-defined clusters.     

Identification of Carnobacterium spp. and Leuconostoc spp. in meat by genus-specific 16S rRNA probes

Oligonucleotide probes specific for Carnobacterium and Leuconostoc species were constructed from the variable regions of 16S rRNA obtained from the literature and sequence data bases. The probes were hybridized with crude nucleic acid extract from 32 type strains of lactic acid bacteria (LAB) commonly found on meat. Two of the probes hybridized only to the four Carnobacterium species whereas the other two hybridized only to five of the six Leuconostoc species tested. The probes were also hybridized with nucleic acids from unknown strains of LAB. The identification was consistent with the results of biochemical tests used to characterize the two genera.     

Random amplification of polymorphic DNA (RAPD) typing of carnobacteria isolated from hindgut chamber and large intestine of Atlantic cod (Gadus morhua l.)

Autochthonous and allochthonous bacteria were isolated from hindgut chamber and large intestine of fed and starved Atlantic cod (Gadus morhua L.). All bacterial strains isolated from hindgut chamber belong to carnobacteria. However, only 10.2% of the bacteria strains from the large intestine belong to carnobacteria. Random amplification of polymorphic DNA (RAPD) analysis using three selective primers, was performed to further identify the carnobacteria strains. Nine of these were isolated from hindgut chamber contents, ten associated with epithelial cells of the hindgut chamber, and six isolated from the large intestines of fed and starved fish. The 25 isolates segregated into eight clusters. The major cluster comprised nine strains isolated from the hindgut chamber of both fed and starved fish showing low similarity with the reference strains. The other strains isolated from the hindgut were located in clusters showing high similarity with Carnobacterium gallinarum or Carnobacterium piscicola. Strains isolated from large intestine appeared more divergent and were located in five different clusters. Autochthonous (indigenous) bacteria were clearly demonstrated in the hindgut chamber as transmission electron microscopy revealed rod-shaped bacteria between adjacent microvilli. Endocytosis of bacteria by epithelial cells was observed in the hindgut chamber.     

Psychrotrophic,lactic acid-producing bacteria from anoxic waters in Ace Lake,Antarctica; Carnobacterium funditum sp. nov. and Carnobacterium alterfunditum sp. nov.

Heterofermentative, lactic acid-producing, gram-positive, motile bacteria were isolated from the waters of Ace Lake, Antarctica. All strains produced virtually only l(+)lactic acid from d(+)glucose. d(–)ribose was fermented to lactic, acetic, and formic acids, and ethanol. Cell walls contained meso-diaminopimaleic acid. The strains did not grow at 30°C and were psychrotrophic. Whole cells contained 18:1cis 9 as a major component of their fatty acids. At 20°C, the strains grew better anaerobically than aerobically and all strains lacked catalase, oxidase and respiratory lipoquinones. DNA that coded for most of the 16S rRNA gene of one of the strains was amplified by the polymerase chain reaction and sequenced. The strain was phylogenetically most closely related to Carnobacterium mobile (K_nuc=0.0214). The isolates separated into two phenotypes. DNA/DNA homology studies determined on a representative from each phenotype showed low homology between the phenotypes (38±8%), and with Carnobacterium mobile (26±2%, 34±2%). Carnobacterium funditum sp. nov. produced acid from mannitol, trehalose, but not amygdalin. The G+C content of the DNA was 32–34%, and the Type strain is DSM 5970 (=ACAM 312). Carnobacterium alterfunditum sp. nov. produced acid weakly from amygdalin but not from mannitol or trehalose. The G+C content was 33–34%, and the Type strain is DSM 5972 (=ACAM 313).     

Heterologous production of bisucaberin using a biosynthetic gene cluster cloned from a deep sea metagenome

A siderophore biosynthetic gene cluster was cloned from a metagenomic library generated from deep sea sediment. The gene cluster was successfully expressed in Escherichia coli to produce bisucaberin, a siderophore originally reported from the marine bacterium Alteromonas haloplanktis. The cloned bisucaberin biosynthetic gene cluster was moderately similar to that of the known bisucaberin producer Vibrio salmonicida. However, the cloned gene cluster consists of four genes rather than three genes found in the V. salmonicida cluster. The low overall homology of the amino acid and nucleotide sequences with those of other species suggests that the cloned genes were derived from one of the unsequenced bacteria including uncultured species.     

Evidence on inhibition of Listeria monocytogenes by divercin V41 action

AIMS: The aim of this study was to investigate the role of divercin V41 in inhibition and prevention of Listeria monocytogenes. METHODS AND RESULTS: Carnobacterium divergens V41 deficient in bacteriocin production was isolated and characterized by enzyme-liked immunosorbent assay, multiplex polymerase chain reaction and bacteriocin diffusion test. Carnobacterium divergens V41 (divercin+) and Carnobacterium divergens V41C9 (divercin-) were grown in the presence of L. monocytogenes in smoked salmon model medium. Carnobacterium divergens V41, but not C. divergens V41C9, was able to inhibit growth of L. monocytogenes. The results indicate that inhibition of L. monocytogenes in the presence of C. divergens V41 is because of the production of divercin V41 and not to a nutritional advantage. CONCLUSIONS: Carnobacterium divergens V41 may be a promising agent in food safety. SIGNIFICANCE AND IMPACT OF THE STUDY: The study demonstrates a potential use of a bacteriocin producing lactic acid bacteria in the area food protection.     

Bacteriocin production by spray-dried lactic acid bacteria

AIMS: Cell survival and antagonistic activity against Listeria innocua, Listeria monocytogenes and Staphylococcus aureus were investigated after spray-drying three bacteriocin-producing strains of lactic acid bacteria: Carnobacterium divergens, Lactobacillus salivarius and Lactobacillus sakei. METHODS AND RESULTS: Bacterial cell concentrates were spray-dried and stored at 4 degrees C and 18 degrees C and 0.3% ERH (equilibrium relative humidity). Enumeration and antagonistic activity were evaluated before and after spray-drying and at regular intervals during storage. CONCLUSIONS: A higher survival rate was obtained when survival was performed at 4 degrees C. With the exception of Carnobacterium divergens which lost the inhibitory activity against Staph. aureus after drying, antagonistic production was not affected by the process nor by the storage. Of the three species studied, Lact. salivarius showed the highest resistance to the spray-drying and storage processes. SIGNIFICANCE AND IMPACT OF THE STUDY: Spray-drying is a potentially useful process for large scale production of dried powders containing viable organisms with antagonistic activity against pathogens.     

Explorative multivariate analyses of 16S rRNA gene data from microbial communities in modified-atmosphere-packed salmon and coalfish

Modified-atmosphere packaging (MAP) of foods in combination with low-temperature storage extends product shelf life by limiting microbial growth. We investigated the microbial biodiversity of MAP salmon and coalfish by using an explorative approach and analyzing both the total amounts of bacteria and the microbial group composition (both aerobic and anaerobic bacteria). Real-time PCR analyses revealed a surprisingly large difference in the microbial loads for the different fish samples. The microbial composition was determined by examining partial 16S rRNA gene sequences from 180 bacterial isolates, as well as by performing terminal restriction fragment length polymorphism analysis and cloning 92 sequences from PCR products of DNA directly retrieved from the fish matrix. Twenty different bacterial groups were identified. Partial least-squares (PLS) regression was used to relate the major groups of bacteria identified to the fish matrix and storage time. A strong association of coalfish with Photobacterium phosphoreum was observed. Brochothrix spp. and Carnobacterium spp., on the other hand, were associated with salmon. These bacteria dominated the fish matrixes after a storage period. Twelve Carnobacterium isolates were identified as either Carnobacterium piscicola (five isolates) or Carnobacterium divergens (seven isolates), while the eight Brochothrix isolates were identified as Brochothrix thermosphacta by full-length 16S rRNA gene sequencing. Principal-component analyses and PLS analysis of the growth characteristics (with 49 different substrates) showed that C. piscicola had distinct substrate requirements, while the requirements of B. thermosphacta and C. piscicola were quite divergent. In conclusion, our explorative multivariate approach gave a picture of the total microbial biodiversity in MAP fish that was more comprehensive than the picture that could be obtained previously. Such information is crucial in controlled food production when, for example, the hazard analysis of critical control points principle is used.     

The effect of diet on aerobic bacterial flora associated with intestine of Arctic charr (Salvelinus alpinus L.)

In order to extend the knowledge on the possible effect of diet on the gastrointestinal microbial community of fish, Arctic charr (Salvelinus alpinus L.) were fed diets containing high (23.7%) and low (6.4%) levels of carbohydrate. The number of viable aerobic and facultative aerobic bacteria associated with the digestive tract were not influenced by dietary regimen. A wide range of bacterial species was isolated, and the predominant bacterial species of both rearing groups were identified as Staphylococcus. There were, however, some differences in bacterial composition between the rearing groups, as well as inter-individual variations. For example, atypical Aeromonas salmonicida were isolated from the small and large intestine of two fish fed low dietary carbohydrate, while Aer. caviae-like isolates were found in the small intestine of four fish fed high carbohydrate. Non-motile Aeromonas spp. were found in the rearing group fed high dietary carbohydrate, but at low frequencies. Dietary manipulation seemed to influence the species composition of carnobacteria, Gram-positive rods, oxidase and catalase-negative and fermentative metabolism. Carnobacterium piscicola-like bacteria were only found in the small intestine, while C. mobile-like and Carnobacterium spp. were isolated from the large intestine of fish fed high carbohydrate. On the contrary, C. divergens-like isolates were found associated with the small and large intestine of fish fed low dietary carbohydrate.     

Biological properties of some nitrogen-fixing associative enterobacteria

Enterobacteria isolates from potato tubers were able to fix nitrogen, to protect plants against phytopathogens and to produce phytohormones thus increasing the plant yield. These isolates were previously phenotypically identified as Erwinia carotovora; however, they differed from typical E. carotovora in a number of biological characteristics and were found to be nonphytopathogenic (avirulent) due to the lack of pectate lyase activity. A data matrix, containing 31 strains and 105 biological characteristics was used for computer cluster analysis. The avirulent strains formed a separate cluster more closely related to Klebsiella spp. strains (with a 0.67 level of similarity) than to typical phytopathogenic bacteria of the E. carotovora group (with a 0.48 level of similarity). A phylogenetic analysis based on restriction polymorphisms of an amplified ribosomal DNA spacer region revealed that the avirulent strains studied here were different from all Erwinia, Klebsiella and other enterobacteria species strains. The AP PCR/hybridization technique showed cross homology of amplified DNA of these avirulent strains and a lack of such homology with the DNA from strains of other species. Numerical taxonomy data, rDNA analysis and AP PCR/hybridization assays confirmed that these avirulent bacteria may be regarded as an independent group of enterobacteria.     

Production of histamine and tyramine by lactic acid bacteria isolated from vacuum-packed sugar-salted fish

The incidence of histamine- or tyramine-producing lactic acid bacteria was examined in several products of vacuum-packed sugar-salted fish (salmon, halibut, mackerel). No histamine-producing isolates were observed, whereas the majority of tyramine-producing isolates were identified as Carnobacterium spp. These organisms were shown to be important members of the microbial flora during storage of vacuum-packed sugar-salted salmon at 5°C. The amount of tyramine produced was reduced by lowering the temperature from 9°C to 4°C for all of five strains of carnobacteria or lactobacilli. The majority of tyramine was produced during the exponential growth phase for Carnobacterium piscicola N 5 and Lactobacillus viridescens N 69. The ability of these bacteria to produce tyramine may be used as an index of microbial quality/acceptability of stored vacuum-packed sugar-salted fish.     

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.     

Differentiation of closely related Carnobacterium food isolates based on 16S-23S ribosomal DNA intergenic spacer region polymorphism

A novel strategy for identification of Carnobacterium food isolates based on restriction fragment length polymorphism (RFLP) of PCR-amplified 16S-23S ribosomal intergenic spacer regions (ISRs) was developed. PCR amplification from all Carnobacterium strains studied always yielded three ISR amplicons, which were designated the small ISR (S-ISR), the medium ISR (M-ISR), and the large ISR (L-ISR). The lengths of these ISRs varied from one species to another. Carnobacterium divergens NCDO 2763(T) and C. mobile DSM 4849(T) generated one major S-ISR band (ca. 400 bp) and minor M-ISR and L-ISR bands (ca. 500 and ca. 600 bp, respectively). The ISRs amplified from C. gallinarum NCFB 2766(T) and C. piscicola NCDO 2762(T) were larger (S-ISR, ca. 600 bp; M-ISR, ca. 700 bp; and L-ISR, ca. 800 bp). The L-ISR contained two tDNAs coding for tRNA(Ile) and tRNA(Ala) genes. The M-ISR included one tRNA(Ala) gene, and the S-ISR did not contain a tDNA gene. The RFLP scheme devised involves estimation of variable PCR product sizes together with HinfI, TaqI, and HindIII restriction analysis. Forty-two isolates yielded four unique band patterns that correctly resolved these isolates into four Carnobacterium species. This method is very suitable for rapid, low-cost identification of a wide variety of Carnobacterium species without sequencing.     

Simultaneous detection of Carnobacterium and Leuconostoc in meat products by multiplex PCR

AIMS: To develop a multiplex PCR approach for simultaneous detection of Leuconostoc and Carnobacterium and its validation in meat products. METHODS AND RESULTS: Two multiplex PCR assays were developed using newly designed 16S rDNA-directed primers adapted to the current taxonomic situation of genera Leuconostoc and Carnobacterium that allow: (i) simultaneous detection of both genera, and members of the nonmotile species of genus Carnobacterium and (ii) identification in a single assay of the nonmotile species C. divergens, C. maltaromicum and C. gallinarum. Sensitivity values of 10(3) and 10(4) CFU g(-1) were determined for multiplex PCR detection of Carnobacterium and Leuconostoc, respectively, following artificially inoculated meat trials. In addition, both multiplex PCR assays were validated in 14 naturally contaminated samples covering nine types of meat products. Results obtained by colony identification were confirmed by PCR detection. CONCLUSIONS: The methods described in this study provide a rapid and reliable tool for PCR detection of Carnobacterium and Leuconostoc, in meat products, and for colony identification. SIGNIFICANCE AND IMPACT OF THE STUDY: This multiplex PCR approach will help in the analysis of the spoilage microbiota of refrigerated vacuum-packaged meat product in order to determine the appropriate preservation method.