Desiccation of adhering and biofilm Listeria monocytogenes on stainless steel: Survival and transfer to salmon products.

The foodborne bacterial pathogen, Listeria monocytogenes, commonly contaminates foods during processing, where the microorganisms are potentially subjected to low relative humidity (RH) conditions for extended periods of time. The objective of this study was to examine survival during desiccation (43% RH and 15 °C) of biofilm L. monocytogenes N53-1 cells on stainless steel coupons and to assess subsequent transfer to salmon products. Formation of static biofilm (2 days at 100% RH and 15 °C) prior to desiccation for 23 days significantly (P<0.05) improved survival of cells desiccated in initial low salt concentrations (0.5%) compared to the survival for non-biofilm cells also desiccated in low salt, indicating the protective effect of the biofilm matrix. Osmoadaptation of cells in 5% NaCl before formation of the static biofilm significantly (P<0.05) increased long-term desiccation survival (49 days) irrespectively of the initial salt levels (0.5% and 5% NaCl). The efficiency of transfer (EOT) of desiccated biofilm cells was significantly (P<0.05) lower than EOTs for desiccated non-biofilm bacteria, however, as biofilm formation enhanced desiccation survival more bacteria were still transferred to smoked and fresh salmon. In conclusion, the current work shows the protective effect of biofilm formation, salt and osmoadaptation on the desiccation survival of L. monocytogenes, which in turn increases the potential for cross-contamination during food processing.

The survival of Listeria monocytogenes during long term desiccation is facilitated by sodium chloride and organic material.

One specific DNA-subtype, as determined by RAPD, of Listeria monocytogenes persisted in a fish slaughterhouse for years, even during months with no production where the plant was cleaned and kept dry. We hypothesised that tolerance to desiccation could be a factor in explaining the persistence of L. monocytogenes in food processing environments and the purpose of the present study was to determine ability of L. monocytogenes to survive desiccation on stainless steel under simulated food processing conditions. Viable counts of eight different L. monocytogenes strains exposed to different soils and relative humidities (RHs) during desiccation decreased significantly (p<0.05) during the first week but subsequently remained constant at a plateau for weeks or even months thereafter. Desiccation in physiological peptone saline (PPS) reduced survivors by 3-5 log units whereas bacterial cells suspended in bacteriological growth substrates (tryptone soy broth with 1% glucose, TSB-glu) or PPS with 5% NaCl only were reduced by 1-3 log units. At RHs of 2, 43 and 75%, surfaces were visibly dry after 1, 3 and 5days of incubation, respectively. The lowest RH resulted in the most significant loss of viability, however, 10(3)-10(4)CFU/cm(2) remained viable regardless of the desiccation treatment (i.e., presence of TSB-glu and/or salt). At 75% RH, the bacterial counts remained almost constant when desiccated in TSB-glu. When bacteria were grown and desiccated (15 degrees C, 43% RH) in salmon or smoked salmon juice, survivors decreased slowly resulting in low numbers (10(2)-10(3)CFU/cm(2)) from all eight strains remaining viable after 3months. Whilst conditions during desiccation had a pronounced influence on inactivation kinetics and the number of survivors, persistent L. monocytogenes were not more tolerant to desiccation than presumed non-persistent isolates. Our study shows that the ability to survive for months during desiccated conditions may be a factor explaining the ability of L. monocytogenes to persist in food processing environments.

Influence of processing steps in cold-smoked salmon production on survival and growth of persistent and presumed non-persistent Listeria monocytogenes.

Cold-smoked salmon is a ready-to-eat product in which Listeria monocytogenes sometimes can grow to high numbers. The bacterium can colonize the processing environment and it is believed to survive or even grow during the processing steps. The purpose of the present study was to determine if the steps in the processing of cold-smoked salmon affect survival and subsequent growth of a persistent strain of L. monocytogenes to a lesser degree than presumed non-persistent strains. We used a sequence of experiments increasing in complexity: (i) small salmon blocks salted, smoked or dried under model conditions, (ii) fillets of salmon cold-smoked in a pilot plant and finally, (iii) assessment of the bacterial levels before and after processing during commercial scale production. L. monocytogenes proliferated on salmon blocks that were brined or dipped in liquid smoke and left at 25 degrees C in a humidity chamber for 24 h. However, combining brining and liquid smoke with a drying (25 degrees C) step reduced the bacterium 10-100 fold over a 24 h period. Non-salted, brine injected or dry salted salmon fillets were surface inoculated with L. monocytogenes and cold-smoked in a pilot plant. L. monocytogenes was reduced from 10(3) to 10-10(2) CFU/cm(2) immediately after cold-smoking. The greatest reductions were observed in dry salted and brine injected fillets as compared to cold-smoking of non-salted fresh fillets. Levels of L. monocytogenes decreased further when the cold-smoked fish was vacuum-packed and stored at 5 degrees C. A similar decline was seen when inoculating brine injected fillets after cold-smoking. High phenol concentrations are a likely cause of this marked growth inhibition. In a commercial production facility, the total viable count of salmon fillets was reduced 10-1000 fold by salting, cold-smoking and process-freezing (a freezing step after smoking and before slicing). The prevalence of L. monocytogenes in the commercial production facility was too low to determine any quantitative effects, however, one of nine samples was positive before processing and none after. Taken together, the processing steps involved in cold-smoking of salmon are bactericidal and reduce, but do not eliminate L. monocytogenes. A persistent strain was no less sensitive to the processing steps than a clinical strain or strain EGD.

Sodium chloride enhances adherence and aggregation and strain variation influences invasiveness of Listeria monocytogenes strains.

Some subtypes of Listeria monocytogenes can persist in the food-processing industry, but the reasons for such persistence are not known. In the present study, 10 strains of L. monocytogenes representing known persistent randomly amplified polymorphic DNA (RAPD) types from fish processing plants were compared to eight strains of different RAPD type and origin (clinical, food, and animal). All 18 strains of L. monocytogenes had similar growth patterns at different temperatures (5 or 37 degrees C) or different salinities (0.5 or 5% NaCl), and all strains formed a thin layer of adhered cells on a plastic surface when cultured in tryptone soya broth (TSB) with a total of 1% glucose. Many ready-to-eat foods, such as cold-smoked fish, contain NaCl at concentrations of 2 to 5%, and NaCl is present in the processing environment. Adding NaCl to TSB changed the adhesion patterns of all strains, and all adhered better when NaCl was added. Also, the addition of NaCl caused a marked aggregation of 13 of the strains; however, 5 of the 18 strains did not aggregate in the presence of up to 5% NaCl. The aggregates stuck to the plastic surface, and this occurred in all but one of the persistent RAPD types. Four strains represented one particular RAPD type that has been isolated as a persistent RAPD type in several fish processing plants for up to 10 years. Because this RAPD type often can contaminate fish products, it is important to address its potential virulence. The 18 strains differed markedly in their ability to invade Caco-2 cells, and the four strains representing the universal persistent RAPD type were the least invasive (10(2) to 10(3) CFU/ml), whereas other strains invaded Caco-2 cells at levels of 10(4) to 10(5) CFU/ml. Five of the 18 strains belonged to the genetic lineage 1 and were the most invasive. Although the most commonly isolated persistent RAPD type was low invasive, it is important to understand why moderate salinity facilitates aggregation and biofilm formation, for this understanding can be beneficial in developing procedures to reduce processing plant contamination.

Description of Shewanella glacialipiscicola sp. nov. and Shewanella algidipiscicola sp. nov., isolated from marine fish of the Danish Baltic Sea, and proposal that Shewanella affinis is a later heterotypic synonym of Shewanella colwelliana.

Two novel species belonging to the genus Shewanella are described on the basis of a polyphasic taxonomic approach. A total of 40 strains of Gram-negative, psychrotolerant, H2S-producing bacteria were isolated from marine fish (cod and plaice) caught in the Baltic Sea off Denmark. Strains belonging to group 1 (seven strains) were a lactate-assimilating variant of Shewanella morhuae with a G+C content of 44 mol%. The strains of group 2 (33 strains) utilized lactate, N-acetylglucosamine and malate but did not produce DNase or ornithine decarboxylase. Their G+C content was 47 mol%. Phylogenetic analysis of the 16S rRNA gene sequence data placed the two novel species within the genus Shewanella. Group 1 showed greatest sequence similarity with S. morhuae ATCC BAA-1205T (99.9 %). However, gyrB gene sequence analysis and DNA-DNA hybridization differentiated these isolates from S. morhuae, with 95.6 % sequence similarity and less than 57 % DNA relatedness, respectively. Group 2 strains shared more than 99 % 16S rRNA gene sequence similarity with the type strains of Shewanella colwelliana and Shewanella affinis, but gyrB sequence similarity ( approximately 85 %) and the results of DNA hybridization ( approximately 28 %) indicated that the new isolates represented a novel species. Furthermore, when compared to each other, the type strains of S. colwelliana and S. affinis had almost identical gyrB sequences and significantly high DNA reassociation values (76-83 %), indicating that they belonged to the same species. Based on the conclusions of this study, we propose the novel species Shewanella glacialipiscicola sp. nov. (type strain T147T=LMG 23744T=NBRC 102030T) for group 1 strains and Shewanella algidipiscicola sp. nov. (type strain S13T=LMG 23746T=NBRC 102032T) for group 2 strains, and we propose that Shewanella affinis as a later heterotypic synonym of Shewanella colwelliana.

Prevalence and survival of Listeria monocytogenes in Danish aquatic and fish-processing environments.

Listeria monocytogenes contamination of ready-to-eat food products such as cold-smoked fish is often caused by pathogen subtypes persisting in food-processing environments. The purpose of the present study was to determine whether these L. monocytogenes subtypes can be found in the outside environment, i.e., outside food processing plants, and whether they survive better in the aquatic environment than do other strains. A total of 400 samples were collected from the outside environment, fish slaughterhouses, fish farms, and a smokehouse. L. monocytogenes was not detected in a freshwater stream, but prevalence increased with the degree of human activity: 2% in seawater fish farms, 10% in freshwater fish farms, 16% in fish slaughterhouses, and 68% in a fish smokehouse. The fish farms and slaughterhouses processed Danish rainbow trout, whereas the smokehouse was used for farm-raised Norwegian salmon. No variation with season was observed. Inside the processing plants, the pattern of randomly amplified polymorphic DNA (RAPD) types was homogeneous, but greater diversity existed among isolates from the outside environments. The RAPD type dominating the inside of the fish smokehouse was found only sporadically in outside environments. To examine survival in different environments, L. monocytogenes or Listeria innocua strains were inoculated into freshwater and saltwater microcosms. Pathogen counts decreased over time in Instant Ocean and remained constant in phosphate-buffered saline. In contrast, counts decreased rapidly in natural seawater and fresh water. The count reduction was much slower when the natural waters were autoclaved or filtered (0.2-microm pore size), indicating that the pathogen reduction in natural waters was attributable to a biological mechanism, e.g., protozoan grazing. A low prevalence of L. monocytogenes was found in the outside environment, and the bacteria did not survive well in natural environments. Therefore, L. monocytogenes in the outer environment associated with Danish fish processing is probably of minor importance to the environment inside a fish production plant.

Potassium lactate combined with sodium diacetate can inhibit growth of Listeria monocytogenes in vacuum-packed cold-smoked salmon and has no adverse sensory effects.

Growth of Listeria monocytogenes in ready-to-eat fish products such as cold-smoked salmon is an important food safety issue. The objective of this study was to evaluate the antilisterial activity of potassium lactate (PL) in combination with sodium acetate (SA) or sodium diacetate (SDA) in cold-smoked salmon and to determine whether these compounds could be incorporated easily into the formulations and technology currently used by processors. A commercial brine injector was used to inject salmon filets with either saturated saline brine or saturated saline brine supplemented with combinations of PL and SA (PURASAL Opti. Form PA 4) or PL and SDA (PURASAL Opti. Form PD 4). In the brine-injected cold-smoked salmon, 2.1% (water phase) PL and 0.12% (water phase) SDA delayed the growth of L. monocytogenes for up to 42 days of vacuum-packaged storage at 10 degrees C. Storage at 25 degrees C for 6 h resulted in only a 1-log CFU/g increase in L. monocytogenes. Treatments with lower concentrations of PL and SDA or similar concentrations of PL and SA resulted in an extended lag phase and slower growth of L. monocytogenes. It was not possible to incorporate more than 2% (water phase) PL while ensuring a minimum of 3% (water phase) NaCl in the finished product because PL decreased the solubility of NaCl. Sensory analyses revealed that the preservatives did not negatively affect flavor or odor. The combination of PL and SDA is therefore a viable technology for preventing L. monocytogenes growth on cold-smoked salmon.

One group of genetically similar Listeria monocytogenes strains frequently dominates and persists in several fish slaughter- and smokehouses.

Contamination of foods with the human pathogen Listeria monocytogenes may occur during processing, and the purpose of this study was to determine whether genetically similar strains colonize different processing plants or whether specific persistent strains are unique to each processing plant. We hypothesized that specific L. monocytogenes strains may be better adapted to specific environmental niches in the processing environment. L. monocytogenes contamination patterns were identified by the collection of 686 and 267 samples from the processing environments: raw fish and products of four fish smokehouses and four fish slaughterhouses, respectively. Samples were collected both during production and after cleaning and disinfection. Typically, these samplings were separated by 1 to 3 months. Sampling sites were targeted toward areas likely to harbor the bacterium. L. monocytogenes was isolated from 213 samples, and one strain from each positive sample was typed by RAPD (random amplified polymorphic DNA) analysis with four different primers. The 213 strains were divided into 37 RAPD types. One RAPD type was predominant; 86 of 213 strains belonged to this type. This type was found in three smokehouses and two slaughterhouses and was predominant in three of these plants. A subset of 35 strains was also analyzed by amplified fragment length polymorphism typing, which confirmed the genetic similarity of the groups. Moreover, strains of the dominant RAPD type were indistinguishable from strains isolated frequently from smoked fish products 10 years ago. One smokehouse was surveyed for a year and a half, and the dominant RAPD type persisted throughout the survey period and accounted for 94 of 118 isolates. Our study indicates that strains of L. monocytogenes that are genetically very closely related may be especially adapted to colonizing the processing equipment or especially resistant to cleaning and disinfection.

Shewanella hafniensis sp. nov. and Shewanella morhuae sp. nov., isolated from marine fish of the Baltic Sea.

Two novel species belonging to the genus Shewanella are described on the basis of their phenotypic characteristics, phylogenetic analyses of 16S rRNA and gyrB gene sequences and levels of DNA-DNA hybridization. A total of 47 strains belonging to two novel Gram-negative, psychrotolerant, H2S-producing bacterial species were isolated from marine fish (cod and flounder) caught from the Baltic Sea off Denmark. The phenotypic characteristics of strains belonging to group 1 (14 strains) indicated that these represented a non-sucrose-assimilating variant of Shewanella baltica with a DNA G+C content of 47.0 mol%. Strains of group 2 (33 isolates) did not utilize the carbon substrates assimilated by S. baltica except gluconate, N-acetylglucosamine and malate. Their DNA G+C content was 44.0 mol%. Phylogenetic analysis of the 16S rRNA gene sequence data placed the two novel species within the genus Shewanella. Group 1 strains showed greatest sequence similarity to Shewanella putrefaciens ATCC 8071T (99.0 %) and with S. baltica NCTC 10375(T) (98.3 %). However, gyrB gene sequence analysis showed these isolates to share only 90.0 % sequence similarity with S. putrefaciens ATCC 8071T and 93.9 % with S. baltica NCTC 10375T. Similarly, DNA-DNA hybridization experiments revealed DNA relatedness levels of 38 % between the group 1 isolates and S. putrefaciens ATCC 8071T and 43 % with S. baltica NCTC 10375T. The group 2 strains shared less than 97 % 16S rRNA gene sequence similarities with recognized Shewanella species. Comparisons between the two novel species indicated 16S rRNA gene sequence similarity of approximately 98 %, gyrB gene sequence similarity of approximately 89 % and DNA-DNA reassociation values of 20-34 %. Based on the evidence presented, two novel species, Shewanella hafniensis sp. nov. (type strain P010T = ATCC BAA-1207T = NBRC 100975T) and Shewanella morhuae sp. nov. (type strain U1417T = ATCC BAA-1205T = NBRC 100978T), are described.

Antilisterial activity of a Carnobacterium piscicola isolated from Brazilian smoked fish (surubim [Pseudoplatystoma sp.]) and its activity against a persistent strain of Listeria monocytogenes isolated from surubim.

Data on the prevalence and growth of Listeria monocytogenes in lightly preserved fish products from subtropical and tropical regions are very scarce. Our research describes L. monocytogenes that was detected in 5% of the packages of cold-smoked surubim, a native Brazilian freshwater fish that we analyzed, and shows that the strains isolated were of the same random amplified polymorphic DNA subtype as the strains that were isolated from the same factory 4 years earlier. A bacteriocinogenic strain of Carnobacterium piscicola (strain C2), isolated from vacuum-packed cold-smoked surubim, and two C. piscicola strains, isolated from vacuum-packed, cold-smoked salmon, were capable of limiting or completely inhibiting the growth of an L. monocytogenes (strain V2) isolated from surubim in fish peptone model systems incubated at 10 degrees C. Monocultures of L. monocytogenes reached 108 CFU/ml (g), whereas the growth of L. monocytogenes was completely inhibited by C. piscicola C2. The bacteriocinogenic C. piscicola A9b+ and its nonbacteriocinogenic mutant A9b- reduced maximum Listeria levels by 2 to 3 log units. Both bacteriocinogenic C. piscicola strains prevented listerial growth in cold-smoked fish juices (surubim and salmon). Although the carnobacteria grew poorly on cold-smoked surubim at 10 degrees C, the strains were able to reduce maximum Listeria counts by 1 to 3 log units in an artificially inoculated product (surubim). We conclude that Brazilian smoked fish products harbor L. monocytogenes and should be stabilized against the growth of the organism. C. piscicola C2 has the potential for use as a bioprotective culture in surubim and other lightly preserved fish, but further studies are required to optimize its effect.

Bias in the Listeria monocytogenes enrichment procedure: lineage 2 strains outcompete lineage 1 strains in University of Vermont selective enrichments.

Listeria monocytogenes can be isolated from a range of food products and may cause food-borne outbreaks or sporadic cases of listeriosis. L. monocytogenes is divided into three genetic lineages and 13 serotypes. Strains of three serotypes (1/2a, 1/2b, and 4b) are associated with most human cases of listeriosis. Of these, strains of serotypes 1/2b and 4b belong to lineage 1, whereas strains of serotype 1/2a and many other strains isolated from foods belong to lineage 2. L. monocytogenes is isolated from foods by selective enrichment procedures and from patients by nonselective methods. The aim of the present study was to investigate if the selective enrichment procedure results in a true representation of the subtypes of L. monocytogenes present in a sample. Eight L. monocytogenes strains (four lineage 1 strains and four lineage 2 strains) and one Listeria innocua strain grew with identical growth rates in the nonselective medium brain heart infusion (BHI), but differed in their growth rate in the selective medium University of Vermont medium I (UVM I). When coinoculated in UVM I, some strains completely outgrew other strains. This outcome was dependent on the lineage of L. monocytogenes rather than the individual growth rate of the strains. When inoculated at identical cell densities in UVM I, L. innocua outcompeted L. monocytogenes lineage 1 strains but not lineage 2 strains. In addition, lineage 2 L. monocytogenes strains outcompeted lineage 1 L. monocytogenes strains in all combinations tested, indicating a bias in strains selected by the enrichment procedures. Bias also occurred when coinoculating two lineage 2 or lineage 1 strains; however, it did not appear to correlate with origin (clinical versus food). Identical coinoculation experiments in BHI suggested that the selective compounds in UVM I and II influenced this bias. The results of the present study demonstrate that the selective procedures used for isolation of L. monocytogenes may not allow a true representation of the types present in foods. Our results could have a significant impact on epidemiological studies, as lineage 1 strains, which are often isolated from clinical cases of listeriosis, may be suppressed during enrichment by other L. monocytogenes lineages present in a food sample.

Comparison of sodium hypochlorite-based foam and peroxyacetic acid-based fog sanitizing procedures in a salmon smokehouse: survival of the general microflora and Listeria monocytogenes.

The effects of fog sanitization with peroxyacetic acid (hydrogen peroxide, peracetic acid, and acetic acid in combination) on general hygiene (aerobic plate count) and on Listeria monocytogenes were assessed in a slicing area at a salmon smokehouse and compared with the effects of foam sanitization with sodium hypochlorite (routinely performed at the smokehouse). Two hundred twenty-three environmental samples were collected with sponges and swabs after each of the sanitization procedures, and 68 samples were collected during production. The total culturable aerobic plate count was determined for each sample, and a total of 288 bacterial strains were randomly isolated and tentatively identified to genus level by physiological and biochemical tests. The microflora was dominated by Neisseriaceae, Enterobacteriaceae, and lactic acid bacteria during production. Foam sanitization caused a change in the composition of the flora, with Pseudomonas spp. and Alcaligenes spp. being the dominant gram-negative bacteria and Kurthia spp. and Bacillus spp. being the surviving gram-positive bacteria. Bacteria were very sensitive to fog sanitization, and yeasts accounted for almost half of the surviving flora. By a selective isolation method, strains of L. monocytogenes were isolated and subsequently characterized by random amplified polymorphic DNA (RAPD) typing. Following foam sanitization, 14 to 42% of the samples contained <10 CFU per site, whereas 29 to 78% of the samples collected after fog sanitization contained this level of bacteria. The prevalence of L. monocytogenes was unchanged, but L. monocytogenes was found only in poorly cleaned areas such as drains. The RAPD types for all positive samples were identical to the type that had persisted in the smokehouse since 1995, indicating the importance of drains as a niche.