Growth Rates of Vibrio parahaemolyticus Sequence Type 36 Strains in Live Oysters and in Culture Medium.

The pathogenic marine bacterium Vibrio parahaemolyticus can cause seafood-related gastroenteritis via the consumption of raw or undercooked seafood. Infections originating from relatively cool waters in the northeast United States are typically rare, but recently, this region has shown an increase in infections attributed to the ecological introduction of pathogenic sequence type 36 (ST36) strains, which are endemic to the cool waters of the Pacific Northwest. A 2005 risk assessment performed by the Food and Drug Administration (FDA) modeled the postharvest growth of V. parahaemolyticus in oysters as a function of air temperature and the length of time the oysters remained unrefrigerated. This model, while useful, has raised questions about strain growth differences in oyster tissue and whether invasive pathogenic strains exhibit different growth rates than nonclinical strains, particularly at lower temperatures. To investigate this question, live eastern oysters were injected with ST36 clinical strains and non-ST36 nonclinical strains, and growth rates were measured using the most probable number (MPN) enumeration. The presence of V. parahaemolyticus was confirmed using PCR by targeting the thermolabile hemolysin gene (tlh), thermostable direct hemolysin (tdh), tdh-related hemolysin (trh), and a pathogenesis-related protein (prp). The growth rates of the ST36 strains were compared to the FDA model and several other data sets of V. parahaemolyticus growth in naturally inoculated oysters harvested from the Chesapeake Bay. Our data indicate that the growth rates from most studies fall within the mean of the FDA model, but with slightly higher growth at lower temperatures for ST36 strains injected into live oysters. These data suggest that further investigations of ST36 growth capability in oysters at temperatures previously thought unsuitably low for Vibrio growth are warranted. IMPORTANCE Vibrio parahaemolyticus is the leading cause of seafood-related gastroenteritis in the United States, with an estimated 45,000 cases per year. Most individuals who suffer from vibriosis consume raw or undercooked seafood, including oysters. While gastroenteritis vibriosis is usually self-limiting and treatable, V. parahaemolyticus infections are a stressor on the growing aquaculture industry. Much effort has been placed on modeling the growth of Vibrio cells in oysters in order to aid oyster growers in designing harvesting best practices and ultimately, to protect the consumer. However, ecological invasions of nonnative bacterial strains make modeling their growth complicated, as these strains are not accounted for in current models. The National Shellfish Sanitation Program (NSSP) considers 10°C (50°F) a temperature too low to enable Vibrio growth, where 15°C is considered a cutoff temperature for optimal Vibrio growth, with temperatures approaching 20°C supporting higher growth rates. However, invasive strains may be native to cooler waters. This research aimed to understand strain growth in live oysters by measuring growth rates when oysters containing ST36 strains, which may be endemic to the U.S. Pacific Northwest, were exposed to multiple temperatures postharvest. Our results will be used to aid future model development and harvesting best practices for the aquaculture industry.

Vibrio parahaemolyticus risk assessment in the Pacific Northwest: it's not what's in the water.

The Gram-negative bacterium Vibrio parahaemolyticus (Vp) is a major cause of illness associated with the consumption of raw or undercooked seafood, primarily oysters. This species is a natural member of the bacterial community in brackish waters and is bioaccumulated by oysters through filter feeding. Only a subset of strains is thought to be pathogenic. Currently known virulence markers include the gene for the thermostable direct hemolysin (tdh). In this work we analyzed water and oysters for total Vp and strains encoding tdh from 26 oyster-growing areas of the Puget Sound and Pacific coast of Washington state in 2007 and 2008. In addition, possible plankton-associated Vp were assessed from net tow samples. The density of both total and tdh+ Vp in the water column were considerably higher in 2008 than 2007. However, the concentrations of both total and tdh+ Vp in the oyster tissue was similar for both years. A high proportion of Vp strains in the water column was found to be tdh+ in both 2007 and 2008; however, tdh+ strains were detected at much lower levels in oysters. The data show that analysis of Vp density in the oysters is a better risk assessment tool than density in the overlying water column.

Comparative Genomic Analysis of Vibrio diabolicus and Six Taxonomic Synonyms: A First Look at the Distribution and Diversity of the Expanded Species.

Vibrio is a diverse genus of Gammaproteobacteria autochthonous to marine environments worldwide. Vibrio diabolicus and V. antiquarius were originally isolated from deep-sea hydrothermal fields in the East Pacific Rise. These species are closely related to members of the Harveyi clade (e.g., V. alginolyticus and V. parahaemolyticus) that are commonly isolated from coastal systems. This study reports the discovery and draft genome sequence of a novel isolate (Vibrio sp. 939) cultured from Pacific oysters (Crassostrea gigas). Questions surrounding the identity of Vibrio sp. 939 motivated a genome-scale taxonomic analysis of the Harveyi clade. A 49-genome phylogeny based on 1,109 conserved coding sequences and a comparison of average nucleotide identity (ANI) values revealed a clear case of synonymy between Vibrio sp. 939, V. diabolicus Art-Gut C1 and CNCM I-1629, V. antiquarius EX25 and four V. alginolyticus strains (E0666, FF273, TS13, and V2). This discovery expands the V. diabolicus species and makes available six additional genomes for comparative genomic analyses. The distribution of the expanded species is thought to be global given the range of isolation sources (horse mackerel, seawater, sediment, dentex, oyster, artemia and polycheate) and origins (China, India, Greece, United States, East Pacific Rise, and Chile). A subsequent comparative genomic analysis of this new eight-genome subclade revealed a high degree of individual genome plasticity and a large repertoire of genes related to virulence and defense. These findings represent a significant revision to the understanding of V. diabolicus and V. antiquarius as both have long been regarded as distinct species. This first look at the expanded V. diabolicus subclade suggests that the distribution and diversity of this species mirrors that of other Harveyi clade species, which are notable for their ubiquity and diversity.

Environmental influences on the seasonal distribution of Vibrio parahaemolyticus in the Pacific Northwest of the USA.

Populations of Vibrio parahaemolyticus in the environment can be influenced by numerous factors. We assessed the correlation of total (tl+) and potentially virulent (tdh+) V. parahaemolyticus in water with three harmful algal bloom (HAB) genera (Pseudo-nitzschia, Alexandrium and Dinophysis), the abundance of diatoms and dinoflagellates, chlorophyll-a and temperature, salinity and macronutrients at five sites in Washington State from 2008-2009. The variability in V. parahaemolyticus density was explained predominantly by strong seasonal trends where maximum densities occurred in June, 2 months prior to the highest seasonal water temperature. In spite of large geographic differences in temperature, salinity and nutrients, there was little evidence of corresponding differences in V. parahaemolyticus density. In addition, there was no evident relationship between V. parahaemolyticus and indices of HAB genera, perhaps due to a lack of significant HAB events during the sampling period. The only nutrient significantly associated with V. parahaemolyticus density after accounting for the seasonal trend was silicate. This negative relationship may be caused by a shift in cell wall structure for some diatom species to a chitinous substrate preferred by V. parahaemolyticus. Results from our study differ from those in other regions corroborating previous findings that environmental factors that trigger vibrio and HAB events may differ depending on geographic locations. Therefore caution should be used when applying results from one region to another.

Zebrafish as a model for Vibrio parahaemolyticus virulence.

Vibrio parahaemolyticus is a Gram-negative, naturally occurring marine bacterium. Subpopulations of strains belonging to this species cause an acute self-limiting gastroenteritis in humans and, less commonly, wound infections. In vivo models to differentiate avirulent and virulent strains and evaluate the pathogenic potential of strains of this species have been largely focused on the presence of known virulence factors such as the thermostable direct haemolysin (TDH), the TDH-related haemolysin (TRH) or the contributions of the type 3 secretion systems. However, virulence is likely to be multifactorial, and additional, yet to be identified factors probably contribute to virulence in this bacterium. In this study, we investigated an adult zebrafish model to assess the overall virulence of V. parahaemolyticus strains. The model could detect differences in the virulence potential of strains when animals were challenged intraperitoneally, based on survival time. Differences in survival were noted irrespective of the source of isolation of the strain (environmental or clinical) and regardless of the presence or absence of the known virulence factors TDH and TRH, suggesting the influence of additional virulence factors. The model was also effective in comparing differences in virulence between the wild-type V. parahaemolyticus strain RIMD2210633 and isogenic pilin mutants ΔpilA and ΔmshA, a double mutant ΔpilA : ΔmshA, as well as a putative chitin-binding protein mutant, ΔgbpA.

Population structure of clinical and environmental Vibrio parahaemolyticus from the Pacific Northwest coast of the United States.

Vibrio parahaemolyticus is a common marine bacterium and a leading cause of seafood-borne bacterial gastroenteritis worldwide. Although this bacterium has been the subject of much research, the population structure of cold-water populations remains largely undescribed. We present a broad phylogenetic analysis of clinical and environmental V. parahaemolyticus originating largely from the Pacific Northwest coast of the United States. Repetitive extragenic palindromic PCR (REP-PCR) separated 167 isolates into 39 groups and subsequent multilocus sequence typing (MLST) separated a subset of 77 isolates into 24 sequence types. The Pacific Northwest population exhibited a semi-clonal structure attributed to an environmental clade (ST3, N = 17 isolates) clonally related to the pandemic O3:K6 complex and a clinical clade (ST36, N = 20 isolates) genetically related to a regionally endemic O4:K12 complex. Further, the identification of at least five additional clinical sequence types (i.e., ST43, 50, 65, 135 and 417) demonstrates that V. parahaemolyticus gastroenteritis in the Pacific Northwest is polyphyletic in nature. Recombination was evident as a significant source of genetic diversity and in particular, the recA and dtdS alleles showed strong support for frequent recombination. Although pandemic-related illnesses were not documented during the study, the environmental occurrence of the pandemic clone may present a significant threat to human health and warrants continued monitoring. It is evident that V. parahaemolyticus population structure in the Pacific Northwest is semi-clonal and it would appear that multiple sequence types are contributing to the burden of disease in this region.

Vibrio parahaemolyticus type IV pili mediate interactions with diatom-derived chitin and point to an unexplored mechanism of environmental persistence.

Vibrio parahaemolyticus is a naturally occurring bacterium common in coastal waters where it concentrates in shellfish through filter feeding. The bacterium is a human pathogen and the leading cause of seafood-borne gastroenteritis. Presently there is little information regarding mechanisms of environmental persistence of V.parahaemolyticus or an accurate early warning system for outbreak prediction. Vibrios have been shown to adhere to several substrates in the environment, including chitin, one of the most abundant polymers in the ocean. Diatoms are abundant in estuarine waters and some species produce chitin as a component of the silica cell wall or as extracellular fibrils. We examined the role of specific surface structures on the bacterium, the type IV pilins PilA and MshA, in adherence to diatom-derived chitin. Biofilm formation and adherence of V.parahaemolyticus to chitin is mediated by the ability of the bacterium to express functional type IV pili. The amount of adherence to diatom-derived chitin is controlled by increased chitin production that occurs in later stages of diatom growth. The data presented here suggest late-stage diatom blooms may harbour high concentrations of V.parahaemolyticus and could serve as the foundation for a more accurate early warning system for outbreaks of this human pathogen.

Genetic diversity of clinical and environmental Vibrio parahaemolyticus strains from the Pacific Northwest.

Since 1997, cases of Vibrio parahaemolyticus-related gastroenteritis from the consumption of raw oysters harvested in Washington State have been higher than historical levels. These cases have shown little or no correlation with concentrations of potentially pathogenic V. parahaemolyticus (positive for the thermostable direct hemolysin gene, tdh) in oysters, although significant concentrations of tdh(+) V. parahaemolyticus strains were isolated from shellfish-growing areas in the Pacific Northwest (PNW). We compared clinical and environmental strains isolated from the PNW to those from other geographic regions within the United States and Asia for the presence of virulence-associated genes, including the thermostable direct hemolysin (tdh), the thermostable-related hemolysin (trh), urease (ureR), the pandemic group specific markers orf8 and toxRS, and genes encoding both type 3 secretion systems (T3SS1 and T3SS2). The majority of clinical strains from the PNW were positive for tdh, trh, and ureR genes, while a significant proportion of environmental isolates were tdh(+) but trh negative. Hierarchical clustering grouped the majority of these clinical isolates into a cluster distinct from that including the pandemic strain RIMD2210633, clinical isolates from other geographical regions, and tdh(+), trh-negative environmental isolates from the PNW. We detected T3SS2-related genes (T3SS2ß) in environmental strains that were tdh and trh negative. The presence of significant concentrations of tdh(+), trh-negative environmental strains in the PNW that have not been responsible for illness and T3SS2ß in tdh- and trh-negative strains emphasizes the diversity in this species and the need to identify additional virulence markers for this bacterium to improve risk assessment tools for the detection of this pathogen.

Ecology of Vibrio parahaemolyticus and Vibrio vulnificus in the coastal and estuarine waters of Louisiana, Maryland, Mississippi, and Washington (United States).

Vibrio parahaemolyticus and Vibrio vulnificus, which are native to estuaries globally, are agents of seafood-borne or wound infections, both potentially fatal. Like all vibrios autochthonous to coastal regions, their abundance varies with changes in environmental parameters. Sea surface temperature (SST), sea surface height (SSH), and chlorophyll have been shown to be predictors of zooplankton and thus factors linked to vibrio populations. The contribution of salinity, conductivity, turbidity, and dissolved organic carbon to the incidence and distribution of Vibrio spp. has also been reported. Here, a multicoastal, 21-month study was conducted to determine relationships between environmental parameters and V. parahaemolyticus and V. vulnificus populations in water, oysters, and sediment in three coastal areas of the United States. Because ecologically unique sites were included in the study, it was possible to analyze individual parameters over wide ranges. Molecular methods were used to detect genes for thermolabile hemolysin (tlh), thermostable direct hemolysin (tdh), and tdh-related hemolysin (trh) as indicators of V. parahaemolyticus and the hemolysin gene vvhA for V. vulnificus. SST and suspended particulate matter were found to be strong predictors of total and potentially pathogenic V. parahaemolyticus and V. vulnificus. Other predictors included chlorophyll a, salinity, and dissolved organic carbon. For the ecologically unique sites included in the study, SST was confirmed as an effective predictor of annual variation in vibrio abundance, with other parameters explaining a portion of the variation not attributable to SST.

Comparative evolutionary analysis of the major structural subunit of Vibrio vulnificus type IV pili.

Type IV pili contribute to virulence in Vibrio vulnificus, the bacterium responsible for the majority of fatal seafood-related infections. Here, we performed within- and between-species evolutionary analysis of the gene that encodes the major structural subunit of the pilus, pilA, by comparing it with pilD and gyrB, the genes encoding the type IV prepilin peptidase and beta subunit of DNA gyrase, respectively. Although the diversity in pilD and gyrB is similar to each other and likely to have accumulated after speciation of V. vulnificus, pilA is several times more diverse at both nonsynonymous and synonymous levels. Also, in contrast to pilD and gyrB, there are virtually unrestricted and highly localized horizontal movements of pilA alleles between the major phylogenetic groups of V. vulnificus. The frequent movement of pilA involves homologous recombination of the entire gene with no evidence for intragenic recombination between the alleles. We propose that pilA allelic diversity and horizontal movement is maintained in the population by both diversifying and frequency-dependent selection most likely to escape shellfish innate immunity defense or lytic phages. Other possibilities leading to such selection dynamics of V. vulnificus pilA could involve adaptation to diverse host populations or within-host compartments, or natural DNA uptake and transformation. We show that the history of nucleotide diversification in pilA predates V. vulnificus speciation and this diversification started at or before the time of the last common ancestor for V. vulnificus, Vibrio parahaemolyticus, and Vibrio cholerae. At the same time, it appears that within the various pilA groups of V. vulnificus, there is no positive selection for structural mutations and consequently no evidence for source-sink selection. In contrast, pilD has accumulated a number of apparently adaptive mutations in the regions encoding the membrane-spanning portions of the prepilin peptidase, possibly affecting fimbrial expression and/or function, and is being subjected to source-sink selection dynamics.

Role of type IV pilins in persistence of Vibrio vulnificus in Crassostrea virginica oysters.

Vibrio vulnificus is part of the natural estuarine microflora and accumulates in shellfish through filter feeding. It is responsible for the majority of seafood-associated fatalities in the United States mainly through consumption of raw oysters. Previously we have shown that a V. vulnificus mutant unable to express PilD, the type IV prepilin peptidase, does not express pili on the surface of the bacterium and is defective in adherence to human epithelial cells (R. N. Paranjpye, J. C. Lara, J. C. Pepe, C. M. Pepe, and M. S. Strom, Infect. Immun. 66:5659-5668, 1998). A mutant unable to express one of the type IV pilins, PilA, is also defective in adherence to epithelial cells as well as biofilm formation on abiotic surfaces (R. N. Paranjpye and M. S. Strom, Infect. Immun. 73:1411-1422, 2005). In this study we report that the loss of PilD or PilA significantly reduces the ability of V. vulnificus to persist in Crassostrea virginica over a 66-h interval, strongly suggesting that pili expressed by this bacterium play a role in colonization or persistence in oysters.

A Vibrio vulnificus type IV pilin contributes to biofilm formation, adherence to epithelial cells, and virulence.

Vibrio vulnificus expresses a multitude of cell-associated and secreted factors that potentially contribute to pathogenicity, although the specific roles of most of these factors have been difficult to define. Previously we have shown that a mutation in pilD (originally designated vvpD), which encodes a type IV prepilin peptidase/N-methyltransferase, abolishes expression of surface pili, suggesting that they belong to the type IV class. In addition, a pilD mutant exhibits reduced adherence to HEp-2 cells, a block in secretion of several exoenzymes that follow the type II secretion pathway, and decreased virulence. In this study, we have cloned and characterized a V. vulnificus type IV pilin (PilA) that shares extensive homology to group A type IV pilins expressed by many pathogens, including Vibrio cholerae (PilA), Pseudomonas aeruginosa (PilA), and Aeromonas hydrophila (TapA). The V. vulnificus pilA gene is part of an operon and is clustered with three other pilus biogenesis genes, pilBCD. Inactivation of pilA reduces the ability of V. vulnificus to form biofilms and significantly decreases adherence to HEp-2 cells and virulence in iron dextran-treated mice. Southern blot analysis demonstrates the widespread presence of both pilA and pilD in clinical as well as environmental strains of V. vulnificus.

Inactivation of Listeria monocytogenes on Hot-smoked Salmon by the Interaction of Heat and Smoke or Liquid Smoke †.

L. monocytogenes was inoculated onto the surface of brined salmon steaks and heat processed in a commercial smokehouse to simulate a hot process for preparing smoked fish. The minimum temperature required for inactivation of L. monocytogenes was 153°F (67.2°C) when generated smoke was applied throughout the entire process. When generated smoke was added only during the last half of the process, L. monocytogenes was recovered from steaks heated to temperatures as high as 176°F (80.0°C). When smoke was not applied during the process, L. monocytogenes survived on steaks heated to internal temperatures between 131°F and 181°F (55.0 to 82.8°C) but was not isolated from steaks heated above 181°F (82.8°C). When liquid smoke CharSol C-l0 was applied as a full-strength (100%) dip before processing, L. monocytogenes was inactivated in samples processed at temperatures as low as 138°F (58.9°C). When liquid smoke l0-Poly or CharSol C-l0 was applied at a concentration of 50%, the lethal temperature was increased to the range of 145 to 150°F (62.8 to 65.6°C). With further dilution of C-l0 to 25% and 10% the inactivation temperatures increased to 156°F (68.9°C) and 163°F (72.8°C). A full-strength dip of CharOil, the oil-soluble fraction of CharSol C-l0, was less effective, and L. monocytogenes survived in salmon steaks processed to an internal temperature of 166°F (74.4°C), the highest temperature tested with this liquid smoke. This study provides evidence that heat alone is not reliable for inactivation of L. monocytogenes during the hot-smoking process. The proper stage and duration of smoke application or proper composition and concentration of liquid smoke in combination with heat are critical for inactivation of the organism.

Incidence and Sources of Listeria monocytogenes in Cold-Smoked Fishery Products and Processing Plants.

Cold-smoked salmon processing plants were surveyed to determine the occurrence and sources of L. monocytogenes contamination. Sanitation and cleanup procedures adequately eliminated L. monocytogenes from the processing line and equipment, but recontamination occurred soon after resumption of processing. The primary source of contamination proved to be the surface areas of frozen or fresh raw fish coming into the plant. Listeria species were not isolated from the flesh except when they were introduced during the filleting operation, by the injection of recirculated brine, or in localized areas where there were pre-existing bruises. Penetration of L. monocytogenes into intact flesh via the vascular system did not occur when fresh fish were immobilized with carbon dioxide and bled, nor when frozen, headed, and eviscerated fish were thawed for 20 h in water inoculated with 44 L. monocytogenes organisms per ml. Populations of L. monocytogenes inoculated onto the surface of brined salmon portions changed very little during a cold-smoke process at 72 to 87°F (22.2 to 30.6°C) for 20 h, with or without applied smoke; but when the processing temperature was lowered to 63 to 70°F (17.2 to 21.1°C), populations decreased 10- to 25-fold when smoke was applied. L. monocytogenes injected into the interior of these portions increased 2- to 6-fold at 63 to 70°F (17.2 to 21.1°C) and 100-fold at 72 to 87°F (22.2 to 30.6°C), regardless of the presence of smoke. L. monocytogenes was enumerated in 48 contaminated finished products collected from six different processing plants. L. monocytogenes populations ranged from 0.3 to 34.3 cells per g, with a mean of 6.2 per g and a median of 3.2 per g.

Inhibition of Listeria monocytogenes in Cold-process (Smoked) Salmon by Sodium Nitrite and Packaging Method.

The behavior of Listeria monocytogenes in relation to sodium nitrite (NaNO2) in combination with sodium chloride (NaCl) was evaluated in cold-process (smoked) salmon during storage at 5 or 10°C in either oxygen-permeable film or vacuum-sealed impermeable film. Salmon slices containing either 3 or 5% water-phase NaCl, with or without 190-200 ppm of NaNO2, were inoculated with 10 or 327 CFU/g (150 or 4.9 × 103 CFU/15-g sample) of strain Scott A. The inhibitory contribution of NaNO2 was relative to inoculum size, storage time and temperature, packaging method, and concentration of NaCl. There was less growth of L. monocytogenes in vacuum-packaged samples as compared to those packaged in oxygen-permeable film. The most inhibition was achieved in vacuum-packaged products stored at 5°C, where NaNO2 in combination with 5% water-phase NaCl prevented any increase in a 10 CFU/g-inoculum during 34 d storage. At 10°C, inhibition was initially enhanced by NaNO2, but by 32 d L. monocytogenes populations had increased from a 10 CFU/g-inoculum to the range of 106 CFU/g in vacuum-packaged products and 108 CFU/g in permeable-film packaged products, regardless of NaNO2 or NaCl concentration. Growth of naturally occurring aerobic microorganisms was also inhibited by NaNO2 but to a lesser degree than L. monocytogenes .

Selective and Differential Medium for Isolation of Listeria monocytogenes from Foods.

Hemolytic ceftazidime lithium chloride agar (HCLA) has been developed as a selective and differential plating medium for the isolation of Listeria monocytogenes from fishery products. Selectivity is based upon lithium chloride, colistin methane sulfonate, and ceftazidime. When horse blood was incorporated in the agar overlay, L. monocytogenes colonies were easily distinguished by their characteristic blue-gray color surrounded by narrow zones of ß-hemolysis. Listeria innocua , a species commonly present in foods, does not produce hemolysis on the medium. Therefore, one or more colonies of L. monocytogenes were easily distinguished from large populations of L. innocua . When used in combination with Food and Drug Administration and U.S. Department of Agriculture enrichment methodology, HCLA was effective in inhibiting competitive organisms, differentiating colonies of L. monocytogenes by their ß-hemolysis, and shortening the incubation time at 35°C for presumptive identification to 17-24 h.

Parameters for Control of Listeria monocytogenes in Smoked Fishery Products: Sodium Chloride and Packaging Method.

The behavior of Listeria monocytogenes (10 Scott A cells per g) in cold-process (smoked) salmon containing 3, 5, or 6% water-phase NaCl was evaluated during 30 to 40 d storage at 5 or 10°C in either oxygen-permeable film or vacuum-sealed impermeable film. At 10°C, L. monocytogenes grew to 106 to 108 CFU/g by the second week, with no differences attributed to NaCl concentration except for an initial lag in the 6% NaCl samples. Vacuum packaging suppressed growth of L. monocytogenes by 10- to 100-fold in samples with 3 or 5% NaCl. Inhibition related to NaCl concentration was most apparent at 5°C and L. monocytogenes populations were held below 102 CFU/g by 6% NaCl. Growth of a 327 CFU/g inoculum was about 10-fold greater than a 10 CFU/g inoculum at 10°C and 100-fold greater at 5°C. Growth of two strains isolated from naturally contaminated, commercially prepared, cold-smoked fish did not differ from Scott A. The use of sugar in the product did not influence growth of L. monocytogenes . Maximum populations of aerobic microorganisms reached at 5 and 10°C were similar, although the rate of growth was somewhat delayed at 5°C, and some inhibition was shown by 5 and 6% NaCl and by vacuum packaging.

Comparison of Selective Direct Plating Media for Enumeration and Recovery of L. monocytogenes from Cold-process (smoked) Fish.

Three selective media were evaluated for direct plating recovery and enumeration of Listeria monocytogenes in the presence of high levels of a variety of microorganisms occurring on cold-process (smoked) salmon products. Sliced salmon was brined to contain either no added salt, 3, or 5% water-phase NaCl, or 3 or 5% NaCl plus 140 ppm NaNO2. The slices were packaged in oxygen-permeable film or sealed under vacuum in oxygen-impermeable film, and stored at 10°C or 5°C until total microbial loads reached 106 to 109 CFU/g. Oxford formulation of Listeria selective agar and Lee's modification of Listeria selective agar achieved quantitative recovery of 102 cells per ml of L. monocytogenes strain Scott A in the presence of diluted slurries of these fish containing 104 to 108 CFU/ml of background organisms. A modification of lithium chloride-phenylethanol-moxalactam agar containing an iron-esculin indicator system sometimes failed because of interfering growth by the background microflora.