Birte Fonnesbech Vogel

Elucidation of Listeria monocytogenes Contamination Routes in Cold-Smoked Salmon Processing Plants Detected by DNA-Based Typing Methods

ABSTRACT The contamination routes of Listeria monocytogenes in cold-smoked salmon processing plants were investigated by analyzing 3,585 samples from products (produced in 1995, 1996, 1998, and 1999) and processing environments (samples obtained in 1998 and 1999) of two Danish smokehouses. The level of product contamination in plant I varied from 31 to 85%, and no L. monocytogenes was found on raw fish (30 fish were sampled). In plant II, the levels of both raw fish and product contamination varied from 0 to 25% (16 of 185 raw fish samples and 59 of 1,000 product samples were positive for L. monocytogenes). A total of 429 strains of L. monocytogenes were subsequently compared by random amplified polymorphic DNA (RAPD) profiling, and 55 different RAPD types were found. The RAPD types detected on the products were identical to types found on the processing equipment and in the processing environment, suggesting that contamination of the final product (cold-smoked salmon) in both plants (but primarily in plant I) was due to contamination during processing rather than to contamination from raw fish. However, the possibility that raw fish was an important source of contamination of the processing equipment and environment could not be excluded. Contamination of the product occurred in specific areas (the brining and slicing areas). In plant I, the same RAPD type (RAPD type 12) was found over a 4-year period, indicating that an established in-house flora persisted and was not eliminated by routine hygienic procedures. In plant II, where the prevalence of L. monocytogenes was much lower, no RAPD type persisted over long periods of time, and several differentL. monocytogenes RAPD types were isolated. This indicates that persistent strains may be avoided by rigorous cleaning and sanitation; however, due to the ubiquitous nature of the organism, sporadic contamination occurred. A subset of strains was also typed by using pulsed-field gel electrophoresis and amplified fragment length polymorphism profiling, and these methods confirmed the type division obtained by RAPD profiling.

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

ABSTRACT 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.

Identification of Shewanella baltica as the Most Important H2S-Producing Species during Iced Storage of Danish Marine Fish

ABSTRACT Shewanella putrefaciens has been considered the main spoilage bacteria of low-temperature stored marine seafood. However, psychrotropic Shewanella have been reclassified during recent years, and the purpose of the present study was to determine whether any of the new Shewanella species are important in fish spoilage. More than 500 H2S-producing strains were isolated from iced stored marine fish (cod, plaice, and flounder) caught in the Baltic Sea during winter or summer time. All strains were identified as Shewanella species by phenotypic tests. Different Shewanella species were present on newly caught fish. During the warm summer months the mesophilic human pathogenic S. algae dominated the H2S-producing bacterial population. After iced storage, a shift in the Shewanella species was found, and most of the H2S-producing strains were identified as S. baltica. The 16S rRNA gene sequence analysis confirmed the identification of these two major groups. Several isolates could only be identified to the genus Shewanella level and were separated into two subgroups with low (44%) and high (47%) G+C mol%. The low G+C% group was isolated during winter months, whereas the high G+C% group was isolated on fish caught during summer and only during the first few days of iced storage. Phenotypically, these strains were different from the type strains of S. putrefaciens, S. oneidensis, S. colwelliana, and S. affinis, but the high G+C% group clustered close to S. colwelliana by 16S rRNA gene sequence comparison. The low G+C% group may constitute a new species. S. baltica, and the low G+C% group of Shewanella spp. strains grew well in cod juice at 0°C, but three high G+C Shewanella spp. were unable to grow at 0°C. In conclusion, the spoilage reactions of iced Danish marine fish remain unchanged (i.e., trimethylamine-N-oxide reduction and H2S production); however, the main H2S-producing organism was identified as S. baltica.

Control options for Listeria monocytogenes in seafoods.

At least three outbreaks of listeriosis associated with seafood have been reported. Listeria monocytogenes is widely distributed in the general environment including fresh water, coastal water and live fish from these areas. Contamination or recontamination of seafood may also take place during processing and low levels (< 100 cfu/g) of L. monocytogenes are frequently found on seafood including ready-to-eat (RTE) products. Apart from heat treatment, which is very effective, there are few options for eliminating L. monocytogenes from foods and equipment. It is essential therefore, that growth of L. monocytogenes in the final product be inhibited. The preventive measures include the formulation of a cleaning and sanitising program specifically designed at reducing the presence of L. monocytogenes in the factory environment, the safe elimination of L. monocytogenes from heat treated products and prevention of growth in RTE products within the normal shelf life and conditions stated on the label. If any sampling is required, the sampling plans suggested by the International Commission on Microbiological Specifications for Foods [Int. J. Food Microbiol., 22 (1994) 89-96] are useful.

Diversity of Listeria monocytogenes isolates from cold-smoked salmon produced in different smokehouses as assessed by Random Amplified Polymorphic DNA analyses

One hundred and forty-eight Listeria monocytogenes isolates originating from vacuum packed cold-smoked salmon produced in 10 different Danish smokehouses were compared by Random Amplified Polymorphic DNA (RAPD) profiling. A total of 16 different reproducible RAPD profiles were obtained using a standardised RAPD analysis by four primers separately. The grouping of the 148 strains was exactly the same for the four primers used. For a sub-set of 20 strains typed by Pulsed Field Gel Electrophoresis (PFGE), only one strain was allocated into a different group as compared to the grouping by RAPD typing. Different RAPD types dominated in products from different smokehouses. Some identical RAPD types were isolated in several smokehouses. In each of four smokehouses, one particular RAPD type could be repeatedly isolated from products. Each smokehouse/product carried its own specific RAPD type and this may indicate a possible persistence of closely related strains of L. monocytogenes in smokehouses.

Bias in the Listeria monocytogenes Enrichment Procedure: Lineage 2 Strains Outcompete Lineage 1 Strains in University of Vermont Selective Enrichments

ABSTRACT 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.

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.

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.

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.

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.