Growth inhibition of Listeria monocytogenes by a nonbacteriocinogenic Carnobacterium piscicola

AIMS: This study elucidates the mechanisms by which a nonbacteriocinogenic Carnobacterium piscicola inhibits growth of Listeria monocytogenes. METHODS AND RESULTS: Listeria monocytogenes was exposed to live cultures of a bacteriocin-negative variant of C. piscicola A9b in co-culture, in a diffusion chamber system, and to a cell-free supernatant. Suppression of maximum cell density (0-3.5 log units) of L. monocytogenes was proportional to initial levels of C. pisciola (10(3)-10(7) CFU ml(-1)). Cell-to-cell contact was not required to cause inhibition. The cell-free C. piscicola supernatant caused a decrease in L. monocytogenes maximum cell density, which was abolished by glucose addition but not by amino acid, vitamin or mineral addition. The fermentate also gave rise to a longer lag phase and a reduction in growth rate. These effects were independent of glucose and may have been caused by acetate production by C. piscicola. 2D gel-electrophoretic patterns of L. monocytogenes exposed to C. piscicola or to L. monocytogenes fermentate did not differ. Treatment with C. piscicola fermentate resulted in down-regulation (twofold) of genes involved in purine- or pyrimidine metabolism, and up-regulation (twofold) of genes from the regulon for vitamin B12 biosynthesis and propanediol and ethanolamine utilization. CONCLUSIONS: A nonbacteriocinogenic C. piscicola reduced growth of L. monocytogenes partly by glucose depletion. SIGNIFICANCE AND IMPACT OF THE STUDY: Understanding the mechanism of microbial interaction enhances prediction of growth in mixed communities as well as use of bioprotective principles for food preservation.     

Specific molecular detection of Carnobacterium piscicola SF668 in cold smoked salmon

AIMS: To establish a rapid and reliable multiplex PCR (mPCR)-based method allowing specific identification of Carnobacterium piscicola SF668 during storage of cold smoked salmon (CSS). METHODS AND RESULTS: CSS was inoculated with C. piscicola SF668 and stored at 4 degrees C. Samples were withdrawn at regular time intervals and analysed by counting the number of viable cells. About 25-100% of colonies grown on Elliker plates were subjected to mPCR amplification. The results show that strains presumably identified as C. piscicola SF668 were predominant over the test period. CONCLUSIONS: mPCR is a powerful tool to study competitiveness of C. piscicola SF668, which inhibits the growth of Listeria monocytogenes. SIGNIFICANCE AND IMPACT OF THE STUDY: The present study demonstrates the importance of molecular methods in studying competitiveness of strains with potential food applications.     

Simple method to identify bacteriocin induction peptides and to auto-induce bacteriocin production at low cell density

The production of some bacteriocins by lactic acid bacteria is regulated by induction peptides (IPs) that are secreted by a dedicated secretion system. The IP gene cbaX, for carnobacteriocin A production by Carnobacterium piscicola LV17A, and a presumptive IP gene (orf6), associated with the genetic locus for enterocin B production in Enterococcus faecium BFE 900, were fused to the signal peptide of the bacteriocin divergicin A from Carnobacterium divergens LV13 to access the general secretory pathway. The culture supernatants of C. piscicola UAL26 and Lactococcus lactis MG1363 containing either of these constructs were used to induce bacteriocin production by Bac(-) cultures of C. piscicola LV17A or E. faecium CTC492. The cbaX fusion product induced bacteriocin production by Bac(-) C. piscicola LV17A, but the orf6 fusion product did not induce bacteriocin production by E. faecium CTC492. This represents a relatively simple method of confirming the role of presumptive IPs. The transformation of C. piscicola LV17A with the CbaX gene under expression of the P32 promoter from L. lactis resulted in constitutive production of bacteriocin by either the dedicated transport apparatus or the general secretory pathway.     

Mobilization and expression of bacteriocin plasmids from Carnobacterium piscicola isolated from meat

Mobilization and expression of bacteriocin plasmids from Carnobacterium piscicola isolated from meat. The nonconjugative plasmids pCP40 and pCP49 associated with bacteriocin production in Carnobacterium piscicola LV17, a lactic acid bacterium isolated from meat, were mobilized by the wide host range conjugative plasmid pAMβ1 by two stage conjugation. At the first stage, pAMβ1 was conjugally transferred into C. piscicola LV17 containing the two plasmids associated with bacteriocin production and a cryptic plasmid. Mobilization of the two bacteriocin plasmids by pAMβ1 was done by the second stage conjugation between the pAMβ1-containing C. piscicola LV17 and chloramphenicol (Cm)-resistant Bac^- mutant of C. piscicola LV17. The transconjugants had either partial bacteriocin activity associated with acquisition of pCP40 or pCP49, or complete bacteriocin activity associated with acquisition of all three of the resident plasmids from C. piscicola LV17 or an 89 MDa cointegrated plasmid derived from pCP40 and pCP49. Further manipulation of the transconjugants and a mutant strain of C. piscicola LV17 resulted in separate strains with only pCP40 or pCP49 which produce different bacteriocins. The bacteriocin gene from pCP49 was cloned into pCaT, a chloramphenicol resistance-encoding vector, and electrotransformed into another bacteriocin-producing strain of C. piscicola , enhancing the antagonistic spectrum of the recipient strain.     

Heterologous expression of the lactacin F peptides by Carnobacterium piscicola LV17.

The lactacin F complex, composed of LafA and LafX peptides, is produced by Lactobacillus johnsonii VPI 11088 and is active against five other Lactobacillus species and Enterococcus faecalis. The genetic determinants encoding the lactacin F complex are organized in a 1-kb polycistronic operon which comprises three genes, lafA, lafX, and ORFZ (encoding the putative immunity protein). The lafA and lafX genes encode the bacteriocin precursors with N-terminal extensions characterized by a Gly-Gly-1*Xaa+1 cleavage site (*). The Gly-Gly motif is conserved in several other bacteriocins, including carnobacteriocins A, BM1, and B2. Carnobacterium piscicola LV17 produces carnobacteriocins which are active against Listeria monocytogenes and other lactic acid bacteria. In this study, the lactacin F operon was introduced into C. piscicola LV17. The transformants produced lactacin F concurrently with the carnobacteriocins. When the lafA and lafX genes were separated and cloned individually into LV17, production of either LafA or LafX by C. piscicola LV17 was detected by complementation with L. johnsonii clones producing LafX or LafA, respectively. Transformants of C. piscicola LV17 which produced lactacin F, LafA, or LafX, in combination with the carnobacteriocins, were assayed for an increased and expanded inhibitory spectrum. The recombinant organisms were only active against lactacin F- and carnobacteriocin-sensitive strains. A plasmidless derivative of LV17 which does not produce the carnobacteriocins failed to produce lactacin F, LafA, or LafX when transformed with the appropriate recombinant plasmids. The ability of C. piscicola LV17 to produce lactacin F demonstrates that the machinery for the carnobacteriocins is capable of processing and exporting bacteriocins from both systems.     

New species of Tereancistrum Kritsky,Thatcher & Kayton, 1980 (Monogenea: Dactylogyridae: Ancyrocephalinae) from the gills of Prochilodus lineatus (Osteichthyes: Prochilodontidae) from the upper Paraná River floodplain,Brazil

Two new species of Tereancistrum Kritsky, Thatcher & Kayton, 1980 are described from the gills of Prochilodus lineatus (Prochilodontidae) collected from the upper Paraná River floodplain, Brazil. T. toksonum n. sp. is characterised by a slim ventral bar with a sclerotised membrane along the anterior margin. Another characteristic is the dorsal anchor with an elongated deep root. T. curimba n. sp. is similar to T. ornatus Kritsky, Thatcher & Kayton, 1980 but possesses a sclerotised structure between the accessory sclerites of the ventral anchors. Moreover, the accessory anchor sclerites of this new species are longer than those described for T. ornatus.     

The contribution of bacteriocin to inhibition of Listeria monocytogenes by Carnobacterium piscicola strains in cold-smoked salmon systems

AIMS: To study the importance of bacteriocin production for the antilisterial effect of a bacteriocinogenic Carnobacterium piscicola strain A9b on growth of Listeria monocytogenes in broth and cold-smoked salmon systems. METHODS AND RESULTS: Acriflavin treatment of strain A9b resulted in loss of bacteriocin production and of immunity to carnobacteriocin B2. Two plasmids present in the wild-type were lost in the variant that was also more sensitive to bavaricin and leucocin A than the wild-type indicating cross-resistance to class IIa bacteriocins. The growth rate of the bac- mutant was higher than that of the wild-type at 5 and 37 degrees C but not at 25 or 30 degrees C. In salmon juice the maximum cell density of L. monocytogenes was suppressed 3 and 6 log by co-culture with C. piscicola A9b bac- and bac+, respectively, as compared with the control. Sterile filtered cultures of C. piscicola A9b bac- caused a limited suppression of the maximum cell density of L. monocytogenes similar to that observed when sterile buffer was added in equal amounts. Semi-purified carnobacteriocin B2 caused a 3.5 log decline in viable cell count after 6 day of incubation in cold-smoked salmon juice at 5 degrees C. High resistance level to carnobacteriocin B2 was observed for L. monocytogenes cells exposed to semi-purified and in situ produced carnobacteriocin B2. CONCLUSIONS: The presence of bacteriocin production in C. piscicola enhances its inhibition of L. monocytogenes. SIGNIFICANCE AND IMPACT OF THE STUDY: Due to the emergence of resistance, a bacteriocin negative lactic acid bacteria may be more suited for practical use as a bioprotective agent against L. monocytogenes in ready-to-eat foods.     

Efflux of ions and ATP depletion induced by pediocin PA-1 are concomitant with cell death in Listeria monocytogenes Scott A

Domination of Carnobacterium divergens LV13 by a bacteriocin-producing (bac⁺) organism Carnobacterium piscicola LV17 was dependent on the level of inoculum of the producer strain and its bacteriocin production. When C. piscicola LV17 was grown in APT broth from an initial inoculum of α-10⁴ cfu ml^-1, bacteriocin was not produced (bac^-) although maximum population was reached. The culture remained bac^- during subsequent inoculation at 10²-10⁷ cfu ml^-1 unless it was first grown on solid medium or if heat-treated supernatant fluids from a bac⁺ culture of C. piscicola LV17, LV17A or LV17B were added to the culture prior to the stationary phase of growth. Use of purified carnobacteriocins from C. piscicola LV17A and LV17B confirmed their role in regulation of the bac⁺ phenotype. The need for induction might account in part for differences in bacteriocin production by cultures in liquid and on solid growth media.     

Factors affecting production of an antilisterial bacteriocin by Carnobacterium piscicola strain A9b in laboratory media and model fish systems

AIMS: To investigate factors influencing bacteriocin production and bacteriocin stability of the bioprotective culture Carnobacterium piscicola strain A9b. METHODS AND RESULTS: Maximum activity was obtained in MRS7 broth (MRS adjusted to pH 7.2), with or without glucose. No bacteriocin was produced in APT broth when a low inoculum level (0.001%) was used. In contrast, inoculum level did not influence bacteriocin production in BHI and MRS7 without glucose. Bacteriocin production in APT was induced by the presence of an extracellular compound present in the sterile, filtered, cell-free supernatant fluid of a stationary-phase culture. Increasing concentrations of NaCl (2-7%) reduced bacteriocin production and maximum cell density of C. piscicola A9b when grown in cooked fish juice at 4 degrees C. CONCLUSION: Media composition, inoculum level and sodium chloride concentration affected production. SIGNIFICANCE AND IMPACT OF THE STUDY: The influence of NaCl on bacteriocin production may negate the inhibitory effect of C. piscicola A9b against Listeria monocytogenes in salty foods.     

Use of the polymerase chain reaction and oligonucleotide probes for the rapid detection and identification of Carnobacterium species from meat.

The polymerase chain reaction (PCR) was used selectively to amplify specific rDNA sequences of Carnobacterium divergens, C. mobile, C. piscicola and C. gallinarum in purified DNA extracts, crude cell lysates and food samples. The PCR products were visualized by agarose gel electrophoresis and identified, at species level, by hybridization reactions with three specific oligonucleotide probes for C. divergens, C. mobile and C. piscicola/C. gallinarum designed from 16S rRNA sequence data. The PCR was sufficiently sensitive to amplify DNA from a single bacterium to detectable levels after 30 cycles of amplification. Both radioactive (32P) and non-radioactive alkaline phosphatase labelled probes was able to detect the PCR products. Detection was highly specific and the probes did not hybridize with DNA samples from any other of the bacterial species tested. These methods enabled the rapid and specific detection and identification of carnobacteria from pure cultures and samples of meat.     

Purification and characterization of brochocin A and brochocin B(10-43), a functional fragment generated by heterologous expression in Carnobacterium piscicola

Brochothrix campestris ATCC 43754 produces a heat-stable, two-component, nonlantibiotic, class IIb bacteriocin, brochocin C (BrcC), that is active against a broad range of gram-positive bacteria, including spores of Clostridium botulinum. An improved purification method was developed for BrcC, in which n-butanol and chloroform extraction are used. Mass spectral characterization of the two components, brochocin A (BrcA) and brochocin B (BrcB), showed that both components are excreted into the medium by B. campestris as mature peptides consisting of 59 and 43 amino acids, respectively. Separate expression clones of BrcA and BrcB were constructed previously in Carnobacterium piscicola LV17C, but the products were not chemically characterized. Purification by the new protocol showed that BrcA is expressed as the mature 59-amino-acid peptide but that BrcB is produced by C. piscicola as a fragment, BrcB(10-43), which is cleaved at an internal Gly-Gly site. This fragment is not antimicrobial by itself, but in combination with BrcA it displays the full activity of the BrcC complex. Circular dichroism measurements revealed a high beta-sheet content in the secondary structure of both BrcA and BrcB(10-43), as well as in a 1:1 BrcA-BrcB(10-43) mixture. Separate expression clones of brcA and brcB were also constructed in Escherichia coli, but these clones only produced multiple fragments of the desired peptides with little or no activity.     

Long-term changes in growth and age at maturity of mackerel, Scomber scombrus L., from the North Sea

Mean length and mean weight at age of Atlantic mackerel in the North Sea increased significantly between 1960 and 1979, then, decreased markedly during the 1980s. In contrast, median age at maturity decreased (c. 1-1.5 years) from 1960. Median length at maturity remained stable until the 1980s, when it declined sharply. The significance of these changes is discussed in relation to variation in sea temperature, food availability and stock size. It is speculated that the changes during the 1980s resulted from immigration of western stock mackerel.     

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.     

Lactic acid bacteria associated with the digestive tract of Atlantic salmon (Salmo salar L.)

The present study reports the effect of excessive handling stress and starvation on the lactic acid bacteria associated with the digestive tract of Atlantic salmon (Salmo salar L.). A relatively low population level (approximately 2 x 103 bacteria per gram wet tissue) of viable adherent heterotrophic bacteria was associated with the digestive tract (foregut, midgut and hindgut). Of the 752 bacterial isolates isolated from diet, water and the digestive tract, 201 isolates belonged to the carnobacteria. Of these isolates, one from the diet, one from the rearing water and 80 from the gastrointestinal tract, were further identified on the basis of 16S rDNA sequence analysis. All these isolates were identified as being Carnobacterium piscicola-like. Daily repeated stress and starvation of the fish over 11 d had no influence on the total culturable bacterial numbers or population level of C. piscicola associated with the digestive tract. C. piscicola-like isolates colonizing the various intestinal regions (foregut, midgut and hindgut) were also screened for their ability to produce growth inhibitory compounds active against the fish pathogen Aeromonas salmonicida. Of the 199 C. piscicola isolates tested, 139 inhibited growth of the pathogen.     

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)     

Elimination of foreign material by epidermal malpighian cells during wound healing in fish skin

Cultured epidermal malpighian cells and experimental wounds of Atlantic salmon Salmo salar were challenged with a variety of particulate materials. Latex beads and the bacteria Carnobacterium piscicola, Pseudomonas fluorescens and Aeromonas salmonicida salmonicida were engulfed by cultured cells, whereas Staphylococcus intermedius were not. The cells engulfed bacteria that proliferated in culture medium devoid of antibiotics and melanin granules and other cellular debris. Cells at wound margins engulfed latex beads and C. piscicola, P. fluorescens and A. s. salmonicida , but not S. intermedius. Malpighian cells thus appear to be both phagocytic and discriminatory. The results support the hypothesis that malpighian cells remove foreign material from fish skin by sloughing after becoming laden with engulfed material.     

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)     

Nucleic acid relatedness studies on the genus Carnobacterium and related taxa

None of the species Carnobacterium piscicola, C. divergens, C. mobile or C. gallinarum showed significant DNA-DNA homology between themselves and other lactic acid bacteria [corrected]. Nevertheless, the Carnobacterium species were found to belong to the same ribosomal RNA homology cluster. The species in this cluster were distant from the other bacteria tested.     

Isolation and properties of a bacteriocin-producing Carnobacterium piscicola isolated from fish.

A facultative psychrotrophic lactic acid bacterium isolated from fresh fish was identified as Carnobacterium piscicola on the basis of carbohydrate utilization, G + C content and 16S RNA analysis. Its bacteriocin, designated carnocin UI49, is produced during the mid-exponential phase of growth at temperatures between 15 degrees C and 34 degrees C. Carnocin UI49 is active against a large number of closely-related lactic acid bacteria including carnobacteria, lactobacilli, pediococci and lactococci. Furthermore, the bacteriocin has a bactericidic mode of action which results in lysis of sensitive cells. Maximum bactericidal activity is observed at 34 degrees C with a decrease in activity down to 15 degrees C where it is completely abolished.     

Characterization of two new glycosyl hydrolases from the lactic acid bacterium Carnobacterium piscicola strain BA.

Three genes with homology to glycosyl hydrolases were detected on a DNA fragment cloned from a psychrophilic lactic acid bacterium isolate, Carnobacterium piscicola strain BA. A 2.2-kb region corresponding to an alpha-galactosidase gene, agaA, was followed by two genes in the same orientation, bgaB, encoding a 2-kb beta-galactosidase, and bgaC, encoding a structurally distinct 1.76-kb beta-galactosidase. This gene arrangement had not been observed in other lactic acid bacteria, including Lactococcus lactis, for which the genome sequence is known. To determine if these sequences encoded enzymes with alpha- and beta-galactosidase activities, we subcloned the genes and examined the enzyme properties. The alpha-galactosidase, AgaA, hydrolyzes para-nitrophenyl-alpha-D-galactopyranoside and has optimal activity at 32 to 37 degrees C. The beta-galactosidase, BgaC, has an optimal activity at 40 degrees C and a half-life of 15 min at 45 degrees C. The regulation of these enzymes was tested in C. piscicola strain BA and activity on both alpha- and beta-galactoside substrates decreased for cells grown with added glucose or lactose. Instead, an increase in activity on a phosphorylated beta-galactoside substrate was found for the cells supplemented with lactose, suggesting that a phospho-galactosidase functions during lactose utilization. Thus, the two beta-galactosidases may act synergistically with the alpha-galactosidase to degrade other polysaccharides available in the environment.