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.

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.

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.