Sources and contamination routes of seafood with human pathogenic Vibrio spp.: A Farm-to-Fork approach.

Vibrio spp., known human foodborne pathogens, thrive in freshwater, estuaries, and marine settings, causing vibriosis upon ingestion. The rising global vibriosis cases due to climate change necessitate a deeper understanding of Vibrio epidemiology and human transmission. This review delves into Vibrio contamination in seafood, scrutinizing its sources and pathways. We comprehensively assess the contamination of human-pathogenic Vibrio in the seafood chain, covering raw materials to processed products. A "Farm-to-Fork" approach, aligned with the One Health concept, is essential for grasping the complex nature of Vibrio contamination. Vibrio's widespread presence in natural and farmed aquatic environments establishes them as potential entry points into the seafood chain. Environmental factors, including climate, human activities, and wildlife, influence contamination sources and routes, underscoring the need to understand the origin and transmission of pathogens in raw seafood. Once within the seafood chain, the formation of protective biofilms on various surfaces in production and processing poses significant food safety risks, necessitating proper cleaning and disinfection to prevent microbial residue. In addition, inadequate seafood handling, from inappropriate processing procedures to cross-contamination via pests or seafood handlers, significantly contributes to Vibrio food contamination, thus warranting attention to reduce risks. Information presented here support the imperative for proactive measures, robust research, and interdisciplinary collaboration in order to effectively mitigate the risks posed by human pathogenic Vibrio contamination, safeguarding public health and global food security. This review serves as a crucial resource for researchers, industrials, and policymakers, equipping them with the knowledge to develop biosecurity measures associated with Vibrio-contaminated seafood.

Lack of correlation between growth rate and sequence type among Cronobacter sakazakii.

Species identification and growth rates for a collection of Cronobacter strains from clinical and non-clinical sources have been previously reported. However, advancements in DNA sequencing-based identification methods now allow for more accurate identification. Here we report the sequence types (STs) for 24 strains of Cronobacter sakazakii and examine any possible correlation between sequence type and growth rate, which could influence risk through greater pathogen multiplication and reach of infectious doses during time between formula preparation and feeding. The most common clonal complexes (CCs) identified were C. sakazakii CC1 and CC4. CC1 strains belonged to ST1 (n = 8) and ST391 (n = 1), while CC4 included ST4 (n = 4), ST255 (n = 1) and ST295 (n = 1). Three strains were found to belong to CC100 and two were found to belong to ST64. The remaining STs identified were represented by single strains. CC4 strains have a slightly not significant tendency for faster growth rates at 25 °C; however, the small sample size suggests that more strains need to be analysed to determine if this is a true result. In conclusion, the growth rates of C. sakazakii strains do not appear to be strongly correlated to ST.

Benzalkonium chloride disinfectant residues stimulate biofilm formation and increase survival of Vibrio bacterial pathogens.

Vibrio spp. are opportunistic human and animal pathogens found ubiquitously in marine environments. Globally, there is a predicted rise in the prevalence of Vibrio spp. due to increasing ocean temperatures, which carries significant implications for public health and the seafood industry. Consequently, there is an urgent need for enhanced strategies to control Vibrio spp. and prevent contamination, particularly in aquaculture and seafood processing facilities. Presently, these industries employ various disinfectants, including benzalkonium chloride (BAC), as part of their management strategies. While higher concentrations of BAC may be effective against these pathogens, inadequate rinsing post-disinfection could result in residual concentrations of BAC in the surrounding environment. This study aimed to investigate the adaptation and survival of Vibrio spp. exposed to varying concentrations of BAC residues. Results revealed that Vibrio bacteria, when exposed, exhibited a phenotypic adaptation characterized by an increase in biofilm biomass. Importantly, this effect was found to be strain-specific rather than species-specific. Exposure to BAC residues induced physiological changes in Vibrio biofilms, leading to an increase in the number of injured and alive cells within the biofilm. The exact nature of the "injured" bacteria remains unclear, but it is postulated that BAC might heighten the risk of viable but non-culturable (VBNC) bacteria development. These VBNC bacteria pose a significant threat, especially since they cannot be detected using the standard culture-based methods commonly employed for microbiological risk assessment in aquaculture and seafood industries. The undetected presence of VBNC bacteria could result in recurrent contamination events and subsequent disease outbreaks. This study provides evidence regarding the role of c-di-GMP signaling pathways in Vibrio adaptation mechanisms and suggests that c-di-GMP mediated repression is a potential avenue for further research. The findings underscore that the misuse and overuse of BAC may increase the risk of biofilm development and bacterial survival within the seafood processing chain.

Bacteria-nanoparticle interactions in the context of nanofouling.

The attachment of microbial communities to surfaces is a well-known problem recognized to be involved in a variety of critical issues in the sectors of food processing, chronic wounds, infection from implants, clogging of membranes and corrosion of equipment. Considering the importance of the detrimental impact of biofouling, it has received much attention in the scientific community and from concerned stakeholders. With the development of nanotechnology and the nowadays widespread use of engineered nanoparticles (ENPs), concerns have been raised regarding their fate in terrestrial and aquatic environments. Safety aspects and public health issues are critical in the management of handling nanomaterials and their nanowastes. The interactions of various types of nanoparticles (NPs) with planktonic bacteria have also received attention due to their antimicrobial properties. However, their behavior in regard to biofilms is not well understood although, in the environment, most of the bacteria prefer living in sessile communities. The question appears relevant considering the need to build knowledge on the fate of nanoparticles and the fact that no one can exclude the risk of accumulation of nanoparticles in biofilms and on surfaces leading to a form of nanofouling involving both engineered nanoparticles (ENPs) and nanoplastics. The present analysis of recent research accounts allows in identifying that (1) research activities related to water remediation systems have been mostly oriented on the impact of NPs on pre-existing biofilms, (2) experimental designs are restricted to few scenarios of exposure, usually limited to relative short-time periods although nanofouling could favour the development of multi-resistant bacterial species through sub-lethal exposures over prolong periods of time (3) nanofouling in other systems in which biofilms develop remains to be addressed, and (4) new research directions are required for investigating the mechanisms involved and the subsequent impact of nanofouling on bacterial consortium responses encountered in a variety of environments such as those prevailing in food production/processing settings. Finally, this review aims at providing recent information and insights on nanoparticle-bacterial interactions in the context of biofilms in order to supply an updated outlook of research perspectives that could help establish the framework for production, use and fate of nanomaterials as well as future research directions.

Interactions between spoilage bacteria in tri-species biofilms developed under simulated meat processing conditions.

The formation of biofilms in the food industry is a major issue, as they are a frequent source of contamination of products, which can result in significant economic losses for processors through spoilage of foods or pose serious health concerns for consumers when foodborne pathogens are present. In this study, experiments were carried out using CDC Biofilm Reactors to produce biofilms on two test surfaces (polystyrene and stainless steel coupons) under a regimen for simulated meat processing conditions (SMPC). This entailed a 12 day regimen of daily cycles of filling the reactors with a meat slurry and letting stand for 16 h, followed by draining and refilling with water for an 8 h period in order to mimic a possible scenario of fluctuating periods of nutrient availability and starvation in a meat processing facility. Strains of Pseudomonas fluorescens, Lactobacillus plantarum and Leuconostoc pseudomesenteroides were used for mono and mixed cultures biofilms as they are relevant spoilage bacteria in the meat processing industry. In monoculture, the viable cell densities (CFU/cm2) of the two lactic acid bacteria species tested were higher for biofilms grown on polystyrene as compared to those obtained on stainless steel, whereas viable cell numbers in P. fluorescens monoculture were surface-independent. Synergistic interactions were demonstrated during growth of multi-species biofilms. Results from experiments where one of the 3 strains was inoculated 24 h before introduction of the other two strains showed increased levels of L. plantarum within biofilms grown on both test surfaces. The model developed here serves as a baseline to study the interactions between potential spoilage bacteria during biofilm development.

Dynamics of Biofilm Formation by Salmonella Typhimurium and Beef Processing Plant Bacteria in Mono- and Dual-Species Cultures.

This study aimed to determine the impact of bacteria from a beef plant conveyor belt on the biofilm formation of Salmonella in dual-species cultures. Beef plant isolates (50) including 18 Gram-negative aerobes (GNA), 8 Gram-positive aerobes (GPA), 5 lactic acid bacteria (LAB), 9 Enterobacteriaceae (EB), and 10 generic Escherichia coli (GEC) were included for developing biofilms in mono- and co-culture with S. Typhimurium at 15 °C for 6 days. Five selected cultures in planktonic form and in biofilms were tested for susceptibility to two commonly used sanitizers (i.e. E-San and Perox-E Plus). In mono-cultures, ≥ 80, 67, 61, 20, and 13% of GEC, EB, GNA, LAB, and GPA, respectively, developed measurable biofilms after 2 days, while all co-culture pairings with S. Typhimurium achieved some level of biofilm production. The predominant effect of EB and only effect of GEC strains on the biofilm formation of S. Typhimurium was antagonistic, while that of Gram-positive bacteria was synergistic, with the effect being more prominent on day 6. The effect was highly variable for the GNA isolates. Six aerobic isolates that formed moderate/strong biofilms by day 2 greatly boosted the co-culture biofilm formation. Seven Gram-negative bacteria were antagonistic against the biofilm formation of the co-cultures. Both sanitizers completely inactivated the selected planktonic cultures, but were largely ineffective against biofilms. In conclusion, all beef plant isolates assessed formed biofilms when paired with S. Typhimurium. Aerobic biofilm formers may create a more favorable condition for Salmonella biofilm formation, while some beef plant isolates have potential as a biocontrol strategy for Salmonella biofilms.

Impact of Listeria Inoculation and Aerated Steam Sanitization on Volatile Emissions of Whole Fresh Cantaloupes.

Rapid methods to detect bacterial pathogens on food and strategies to control them are needed to mitigate consumer risk. This study assessed volatile emissions from whole cantaloupe melons (Cucumis melo) as an indicator of Listeria contamination and in response to steam vapor decontamination. Cantaloupe were inoculated with Listeria innocua, a nonpathogenic surrogate for L. monocytogenes, then exposed to 85 °C steam for 240 s (4 min) followed by rapid chilling and storage for 0, 7, 10, or 14 days at 4, 7, or 10 °C. Volatile emissions from whole melons were collected on Carbopack B/Carboxen 1000 headspace collection tubes and analyzed by gas chromatography-mass spectroscopy following thermal desorption. Introduction of L. innocua to cantaloupe rind resulted in a reduction of aromatic compound emission. However, this response was not unique to Listeria contamination in that steam vapor treatment also reduced emission of these compounds. As well, steam vapor treatment diminished the number of viable Listeria and indigenous microflora while causing physiological injury to melon rind. Heat treatment had no significant effects on flesh firmness, color, titratable acidity, or soluble solids, but the production of typical aroma volatiles during postharvest ripening was inhibited. No unique volatile compounds were detected in Listeria contaminated melons. While changes in volatile emissions were associated with Listeria inoculation, they could not be differentiated from heat treatment effects. Results indicate that volatile emissions cannot be used as a diagnostic tool to identify Listeria contamination in whole cantaloupe melons. PRACTICAL APPLICATION: The detection of pathogen contamination on fresh produce is a continuing challenge. Using a nondestructive screening method, the presence of surrogate Listeria innocua on fresh whole cantaloupes was shown to alter the emissions of aromatic volatiles from whole cantaloupes. However, these altered emissions were not found to be unique to Listeria spp. and therefore cannot be used as a definitive indicator of Listeria contamination.

Aerated Steam Sanitization of Whole Fresh Cantaloupes Reduces and Controls Rind-Associated Listeria but Enhances Fruit Susceptibility to Secondary Colonization.

Recent bacterial illnesses and outbreaks associated with the consumption of fresh and fresh-cut fruit and vegetables emphasize the need to supply produce that is microbiologically safe while retaining its quality and nutrient value. We assessed the capacity of aerated steam to reduce initial levels and control the posttreatment proliferation of a 4-strain mixture of Listeria innocua, a surrogate for L. monocytogenes, and microflora native to the rind of whole cantaloupes. Studies were conducted at the pilot-scale level by passing deliberately contaminated melons through a prototype stainless-steel, continuous-feed heating device. Exposure for 240 s to aerated steam heated to 85 °C achieved a mean reduction in surface-inoculated L. innocua of 3.9 ± 0.6 log10 CFU/cm2 (n = 3) and decreased background microorganisms (yeast, moulds, and coliforms) to undetectable levels. No significant outgrowth of surviving L. innocua or yeast and moulds was observed on heat-treated melons during their storage at 4, 7, and 10 °C for 14 days. Treated fruit continued to respire. Although rind quality was altered, edible fleshy portions remained largely unaffected. Cantaloupe inoculated with L. innocua subsequent to its exposure to aerated steam provided a suitable environment for surrogate growth (mean 3.3 log10 increase in rind density over 10 days at 7 °C), whereas its proliferation was restricted on nonheated cantaloupe (mean 0.7 log10 increase). Steam sanitization provides an effective means for the control of pathogen and spoilage organisms, but the proliferation of surrogate organisms on heated cantaloupes raises concern regarding the impact of postprocessing contamination on consumer health risk. PRACTICAL APPLICATION: Water vapor (steam) at a high temperature can be used to sanitize the surface of fresh, whole cantaloupe melons in a continuous-feed manner. Both Listeria bacteria and spoilage organisms are markedly reduced from initial levels and survivor outgrowth severely restricted during subsequent refrigerated storage. This approach to microorganism control is likely most applicable in situations where rinds and flesh are to be separated immediately via further processing.

Variations in biofilm formation, desiccation resistance and Benzalkonium chloride susceptibility among Listeria monocytogenes strains isolated in Canada.

Listeria monocytogenes is a pathogenic foodborne microorganism noted for its ability to survive in the environment and food processing facilities. Survival may be related to the phenotype of individual strains including the ability to form biofilms and resist desiccation and/or sanitizer exposure. The objectives of this research were to compare 14 L. monocytogenes strains isolated from blood (3), food (6) and water (5) with respect to their benzalkonium chloride (BAC) sensitivity, desiccation resistance, and ability to form biofilm. Correlations were tested between those responses, and the presence of the SSI-1 (Stress Survival Islet) and LGI1/CC8 (Listeria Genomic Island 1 in a clonal complex 8 background) genetic markers. Genetic sequences from four strains representing different phenotypes were also probed for predicted amino acid differences in biofilm, desiccation, and membrane related genes. The water isolates were among the most desiccation susceptible strains, while strains exhibiting desiccation resistance harboured SSI-1 or both the SSI-1 and LGI1/CC8 markers. BAC resistance was greatest in planktonic LGI1/CC8 cells (relative to non-LGI1/CC8 cells), and higher BAC concentrations were also needed to inhibit the formation of biofilm by LGI1/CC8 strains during incubation for 48h and 6days compared to other strains. Formation of biofilm on stainless steel was not significantly (p>0.05) different among the strains. Analysis of genetic sequence data from desiccation and BAC sensitive (CP4 5-1, CP5 2-3, both from water), intermediate (Lm568, food) and desiccation and BAC resistant (08 5578, blood, human outbreak) strains led to the finding of amino acid differences in predicted functional protein domains in several biofilm, desiccation and peptidoglycan related genes (e.g., lmo0263, lmo0433, lmo0434, lmo0771, lmo0973, lmo1080, lmo1224, lmo1370, lmo1744, and lmo2558). Notably, the LGI1/CC8 strain 08-5578 had a frameshift mutation in lmo1370, a gene previously associated with desiccation resistance. In conclusion, the more desiccation and BAC resistant LGI1/CC8 isolates may pose a challenge for sanitation efforts.

Modeling the behavior of Listeria monocytogenes during enrichment in half Fraser broth; impact of pooling and the duration of enrichment on the detection of L. monocytogenes in food.

A stochastic model describing the growth of Listeria monocytogenes during enrichment in half Fraser was developed for the purpose of estimating the effects of modifications to the first enrichment step of the EN ISO 11290-1 detection method. Information pertaining to the variability of growth rates, physiological state of the cell, and the behavior of individual cells contaminating the food were obtained from previously published studies. We used this model to investigate the impact of pooling enrichment broths (wet pooling) on the performance of the standard method. For validation of the model, the numbers of L. monocytogenes occurring in 88 naturally contaminated foods following pre-enrichment were compared to model-simulated microbial counts. The model was then used to perform simulations representative of the natural contamination observed for smoked salmon in the European baseline survey of 2010-2011. The model-estimated L. monocytogenes levels following individual enrichment or following the pooling of five broths where only one would be contaminated were compared. The model indicated a 10% loss of method sensitivity resulting from wet pooling. The model also predicted a 5% decrease in the sensitivity of the method when the duration of the enrichment was reduced from 24 to 22 h.

Role of sigB and osmolytes in desiccation survival of Listeria monocytogenes in simulated food soils on the surface of food grade stainless steel.

This research aimed to determine whether the SigB (σ(B)) regulon and osmolytes impact the survival of the foodborne pathogen, Listeria monocytogenes, during desiccation in simulated food soils with varying salt and nutrient contents on food grade stainless steel (SS) surfaces. L. monocytogenes 568 (Lm568, serotype 1/2a), its isogenic sigB mutant (ΔsigB) and the back-complemented ΔsigB were desiccated in BHI, TSB with 1% glucose (TSB-glu), peptone physiological saline (PPS) and minimal media (MM) for 21 days at 43% relative humidity (RH) and 15 °C on SS. The effect of food related osmolytes (proline, betaine and carnitine) on desiccation survival was studied by (a) pre-culturing strains in MM with an osmolyte followed by desiccation in MM and (b) by desiccating strains in MM with an osmolyte. Desiccation survival of L. monocytogenes was positively correlated to the nutrient and osmolyte concentrations in the desiccation substrates. Initial Lm568 levels of 8 Log(CFU/cm(2)) decreased by 0.9 Log(CFU/cm(2)) in BHI and 1.1-2.9 Log(CFU/cm(2)) in TSB-glu, PPS and MM after 21 days. Comparatively, the initial survival of ΔsigB was reduced in PS and MM, while no differences were observed among the three strains in BHI and TSB-glu. Pre-culture in osmolyte containing MM enhanced (p < 0.05) desiccation survival of all strains. Desiccation in osmolyte-containing MM improved desiccation survival of all strains, albeit the protection was less than that observed after pre-culture with the osmolytes. Complementation of the ΔsigB mutant restored the wildtype phenotype. In conclusion, this work shows the protective effect of osmolytes in desiccation survival of L. monocytogenes, while the σ(B) regulon only improved the initial survival in nutrient and osmolyte poor environments.

Attachment of Listeria innocua to polystyrene: effects of ionic strength and conditioning films from culture media and milk proteins.

It is recognized that bacterial adhesion usually occurs on conditioning films made of organic macromolecules absorbed to abiotic surfaces. The objectives of this study were to determine the extent to which milk protein-coated polystyrene (PS) pegs interfere with biofilm formation and the synergistic effect of this conditioning and hypertonic growth media on the bacterial adhesion and biofilm formation of Listeria innocua, used as a nonpathogenic surrogate for Listeria monocytogenes. PS pegs were uncoated (bare PS) or individually coated with whey proteins isolate (WPI), ß-lactoglobulin, bovine serum albumin, or tryptic soy broth (TSB) and were incubated in bacterial suspensions in modified Welshimer's broth. After 4 h, the number of adherent cells was dependent on the coating, as follows: TSB (10(7) CFU/ml) > bare PS > ß-lactoglobulin > bovine serum albumin ∼ WPI (10(4) CFU/ml). The sessile cell counts increased up to 24 h, reaching > 10(7) CFU per peg for all surfaces (P > 0.1), except for WPI-coated PS; this indicates that the inhibitory effects of milk protein conditioning films are transient, slowing down the adhesion process. The 4-h bacterial adhesion on milk protein-coated PS in modified Welshimer's broth supplemented with salt (0 to 10% [wt/vol]) did not vary (P > 0.1), indicating that conditioning with milk proteins was the major determinant for inhibition of bacterial adhesion and that the synergetic effect of salt and milk proteins on adhesion was minimal. Moreover, the presence of 5 to 10% salt significantly inhibited 24-h biofilm formation on the TSB-coated and bare PS, with a decrease of >3 log at 10% (wt/vol) NaCl and almost completely depleted viable sessile bacteria on the milk protein-coated PS.

An allelopathy based model for the Listeria overgrowth phenomenon.

In a standard procedure of food safety testing, the presence of the pathogenic bacterium Listeria monocytogenes can be masked by non-pathogenic Listeria. This phenomenon of Listeria overgrowth is not well understood. We present a mathematical model for the growth of a mixed population of L. innocua and L. monocytogenes that includes competition for a common resource and allelopathic control of L. monocytogenes by L. innocua when this resource becomes limited, which has been suggested as one potential explanation for the overgrowth phenomenon. The model is tested quantitatively and qualitatively against experimental data in batch experiments. Our results indicate that the phenomenon of masked pathogens can depend on initial numbers of each population present, and on the intensity of the allelopathic effect. Prompted by the results for the batch setup, we also analyze the model in a hypothetical chemostat setup. Our results suggest that it might be possible to operate a continuous growth environment such that the pathogens outcompete the non-pathogenic species, even in cases where they would be overgrown in a batch environment.

Increased thermal and osmotic stress resistance in Listeria monocytogenes 568 grown in the presence of trehalose due to inactivation of the phosphotrehalase-encoding gene treA.

The food-borne pathogen Listeria monocytogenes is a problem for food processors and consumers alike, as the organism is resistant to harsh environmental conditions and inimical barriers implemented to prevent the survival and/or growth of harmful bacteria. One mechanism by which listeriae mediate survival is through the accumulation of compatible solutes, such as proline, betaine and carnitine. In other bacteria, including Escherichia coli, the synthesis and accumulation of another compatible solute, trehalose, are known to aid in the survival of stressed cells. The objective of this research was to investigate trehalose metabolism in L. monocytogenes, where the sugar is thought to be transferred across the cytoplasmic membrane via a specific phosphoenolpyruvate phosphotransferase system and phosphorylation to trehalose-6-phosphate (T6P). The latter is subsequently broken down into glucose and glucose-6-phosphate by α,α-(1,1) phosphotrehalase, the putative product of the treA gene. Here we report on an isogenic treA mutant of L. monocytogenes 568 (568:ΔTreA) which, relative to the wild-type strain, displays increased tolerances to multiple stressors, including heat, high osmolarity, and desiccation. This is the first study to examine the putative trehalose operon in L. monocytogenes, and we demonstrate that lmo1254 (treA) in L. monocytogenes 568 indeed encodes a phosphotrehalase required for the hydrolysis of T6P. Disruption of the treA gene results in the accumulation of T6P which is subsequently dephosphorylated to trehalose in the cytosol, thereby contributing to the stress hardiness observed in the treA mutant. This study highlights the importance of compatible solutes for microbial survival in adverse environments.

Growth of Listeria spp. in shredded cabbage is enhanced by a mild heat treatment.

Mild thermal processing can enhance the shelf life of cut fruits and vegetables by delaying the onset of spoilage and preserving the organoleptic properties of shredded cabbage. However, food safety issues related to this process have not been fully investigated. Therefore, the survival and growth of Listeria spp. on cabbage treated in this manner was examined. Experimentally, 24 strains of Listeria spp. (including L. monocytogenes) were inoculated onto cut and intact cabbage tissues and stored at 5 degrees C. All strains on intact tissues exhibited a moderate decline in numbers (up to 1.0 log CFU/cm(2)) over the 28-day storage period. Conversely, cut tissue supported growth of most strains during the first 7 to 14 days of incubation with maximum increases of 1.2 log CFU/cm(2). Subsequently, the survival or growth on heat-treated (50 degrees C for 3 min) and untreated shredded cabbage of four L. monocytogenes and four nonpathogenic Listeria spp. strains were compared during storage for 21 days at 5 degrees C. Growth on untreated shred for all strains was similar to the results observed on cut tissue with a maximum increase of approximately 1.0 log CFU/g. However, in the heat-treated cabbage shred all strains displayed a rapid increase in growth (up to 2.5 log CFU/g) during the first 7 days of incubation, which may be indicative of the destruction of an endogenous growth-inhibiting compound within the cabbage. In conclusion, this study shows that mild thermal treatments of cut cabbage may promote pathogen growth if other inimical barriers are not implemented downstream of the thermal treatment.

Genotypic and phenotypic characterisation of a collection of Cronobacter (Enterobacter sakazakii) isolates.

Enterobacter sakazakii has been identified as the causative agent of serious neonatal infections, associated with high mortality rate. In many cases, powdered infant formula (PIF) has been identified as the source of infection. Recently, E. sakazakii was proposed to be classified in a new genus, Cronobacter. Since knowledge on this pathogen is still incomplete, there is a need for molecular characterization schemes in order to help with epidemiological investigation and evaluate strain variability. The objectives of this study were to combine genotypic (pulsed-field gel electrophoresis [PFGE], 16S rRNA gene sequencing, and automated ribotyping) methods with traditional phenotypic biochemical methods to characterize a collection of Cronobacter isolates from various origins. In addition, the relative growth dynamics were compared by estimating the growth rates for each isolate in non-selective broth (BHI) at 25 degrees C and 37 degrees C. According to biochemical test profiles the majority of isolates were identified as Cronobacter sakazakii, which seemed to be the most common species distributed in the environment of PIF production plants. Furthermore, the PFGE technique displayed very high discriminatory power as 61 distinct pulsotypes were revealed among the 150 Cronobacter isolates. Combining information on sample origin and pulse type, 64 isolates were deemed as unique strains. Although genetic typing data for the strains clearly delineated them into clusters closely corresponding to biochemical speciation results, it was not without discrepancies as some strains did not group as predicted. Important for quantitative risk assessment is the fact that despite the high genetic heterogeneity observed for this collection, most Cronobacter strains displayed similar growth rates irrespective of species designation.

Insertional mutagenesis of Listeria monocytogenes 568 reveals genes that contribute to enhanced thermotolerance.

The objectives of this study were to identify molecular mechanisms of thermotolerance using transposon mutants of Listeria monocytogenes 568, serotype 1/2a, and to compare their thermal death kinetics at 52, 56 and 60 degrees C. Sixteen Tn917 transposon mutants with enhanced heat resistance were acquired from a library of 4300 mutants following a multi-step screening process. Genetic regions with Tn917 insertions encompassed a broad range of functionalities including; transport, metabolism, replication and repair, general stress, and structural properties. Modeling of the heat inactivation data using the Geeraerd et al. and Whiting (Fermi) models showed that the mutants' enhanced thermal resistance was manifested mostly through a significant (p

Strain and growth temperature influence Listeria spp. attachment to intact and cut cabbage.

Twenty four Listeria strains representing three different species and two serotypes of L. monocytogenes were investigated for their ability to attach to and colonize cabbage tissue. All strains exhibited a preference to attach to cut tissues compared to the intact leaf surfaces. Most strains attached to cut surfaces at levels 1.0 to 1.2 log CFU/cm(2) above numbers on intact tissue. Although all strains demonstrated the ability to colonize both intact and cut surfaces, some strains consistently exhibited higher levels of attachment. This attribute was independent of species or serotype. Scanning electron microscopy (SEM) revealed the presence of increased cell numbers on the cut edges with numerous cells located within folds and crevices. The distribution of cells found on the intact surfaces appeared to be randomly distributed with no apparent affinity for specialized surface structures such as stomata. SEM analysis also revealed the increased presence of large clusters of cells on leaf surfaces after 4 and 24 h. These cell aggregates appeared to be in the early stages of biofilm development. L. moncytogenes strain Scott A was used to examine the effect of prior growth temperature on attachment at 10 degrees C. Cells attached to intact cabbage surfaces within 5 min of exposure, with numbers reaching 4.3 log CFU/cm(2) for cells grown at 22 degrees C and 37 degrees C, and 3.8 log CFU/cm(2) for 10 degrees C cultures. The culture growth temperature was shown to significantly (P<0.05) affect the strength of attachment (S(R) values) during the first 4 h of exposure to intact surfaces, as cells cultivated at 37 degrees C were more easily removed from leaf surfaces than those cultivated at 10 degrees C or 22 degrees C. However, after 24 h binding was not significantly different between temperatures (P>0.05) where more than 80% of cells, regardless of cultivation temperature, remained attached to the leaf surfaces following successive washes. Irrespectively of prior growth temperature, increasing exposure time to the cabbage resulted in increased attached cell numbers as well as increased binding strength. The increase in development of cell clusters and early biofilm structures may explain the decreased efficiency over time in removal of cells from the cabbage surfaces. The information presented in this study may have important implications for produce handling practices and the implementation of wash regimes intended to remove microorganisms from edible plant surfaces.