Inactivation of Escherichia coli O157:H7 on stainless steel upon exposure to Paenibacillus polymyxa biofilms

We investigated the potential use of biofilm formed by a competitive-exclusion (CE) microorganism to inactivate Escherichia coli O157:H7 on a stainless steel surface. Five microorganisms showing inhibitory activities against E. coli O157:H7 were isolated from vegetable seeds and sprouts. The microorganism with the greatest antimicrobial activity was identified as Paenibacillus polymyxa (strain T5). In tryptic soy broth (TSB), strain T5 reached a higher population at 25 °C than at 12 or 37 °C without losing inhibitory activity against E. coli O157:H7. When P. polymyxa (6 log CFU/mL) was co-cultured with E. coli O157:H7 (2, 3, 4, or 5 log CFU/mL) in TSB at 25 °C, the number of E. coli O157:H7 decreased significantly within 24 h. P. polymyxa formed a biofilm on stainless steel coupons (SSCs) in TSB at 25 °C within 24 h, and cells in biofilms, compared to attached cells without biofilm formation, showed significantly increased resistance to a dry environment (43% relative humidity [RH]). With the exception of an inoculum of 4 log CFU/coupon at 100% RH, upon exposure to biofilm formed by P. polymyxa on SSCs, populations of E. coli O157:H7 (2, 4, or 6 log CFU/coupon) were significantly reduced within 48 h. Most notably, when E. coli O157:H7 at 2 log CFU/coupon was applied to SSCs on which P. polymyxa biofilm had formed, it was inactivated within 1 h, regardless of RH. These results will be useful when developing strategies using biofilms produced by competitive exclusion microorganisms to inactivate foodborne pathogens in food processing environments.     

Survival of foodborne pathogens at different relative humidities and temperatures and the effect of sanitizers on apples with different surface conditions

We investigated the effects of factors such as relative humidity (RH) and temperature on pathogen survival on apples with different surface conditions. Apples with different surface conditions (unblemished, bruised, or cut) were inoculated with three pathogens (Escherichia coli O157:H7, Salmonella Typhimurium, and Staphylococcus aureus) and stored at different RH levels (RH 100, 85, or 68%) at 4 °C or 15 °C for 2 days. S. aureus survived most readily on apple surfaces; it had no significant reduction on any of the apple surfaces for any of the three RH levels after 2 days of storage. The reduction levels of E. coli O157:H7 and S. Typhimurium on unblemished and bruised apple surfaces were higher at RH of 85% and 68% than at RH of 100% at 15 °C; and reduction levels were approximately 3 log₁₀ CFU/apple at 4 °C in RH of 68%. No significant reduction in any of the three pathogens on cut apple surfaces was observed for any RH level. The effectiveness of chemical sanitizers (chlorine sanitizer and 2% lactic acid) in reducing pathogens (E. coli O157:H7, S. Typhimurium, and S. aureus) on apple surfaces (unblemished, bruised, or cut) was also evaluated. Treatment with chlorine sanitizer and 2% lactic acid for 5 min significantly reduced pathogen levels on unblemished and bruised apple surfaces but not on cut apple surfaces. In conclusion, the surface conditions of the apple significantly affected pathogen survival and the effectiveness of sanitizing methods.     

Biofilm-producing ability of Staphylococcus aureus isolates from Brazilian dairy farms

This study aimed to investigate the in silico biofilm production ability of Staphylococcus aureus strains isolated from milking parlor environments on dairy farms from São Paulo, Brazil. The Staph. aureus isolates were obtained from 849 samples collected on dairy farms, as follows: milk from individual cows with subclinical mastitis or history of the disease (n = 220); milk from bulk tank (n = 120); surfaces of milking machines and utensils (n = 389); and milk handlers (n = 120). Thirty-one Staph. aureus isolates were obtained and categorized as pulsotypes by pulsed-field gel electrophoresis and submitted to assays for biofilm formation on polystyrene, stainless steel, rubber, and silicone surfaces. Fourteen (45.2%) pulsotypes were considered producers of biofilm on the polystyrene microplate assay, whereas 13 (41.9%) and 12 (38.7%) pulsotypes were biofilm producers on stainless steel and rubber, respectively. None of the pulsotypes evaluated produced biofilms on silicone. Approximately 45% of Staph. aureus pulsotypes isolated from different sources on dairy farms showed the ability to produce biofilms in at least one assay, indicating possible persistence of this pathogen in the milking environment. The potential involvement of Staph. aureus in subclinical mastitis cases and its occurrence in milk for human consumption emphasize the need to improve hygiene practices to prevent biofilm formation on the farms studied.     

Microbial biofilm detection on food contact surfaces by macro-scale fluorescence imaging

Hyperspectral fluorescence imaging methods were utilized to evaluate the potential detection of pathogenic bacterial biofilm formations on five types of food contact surface materials: stainless steel, high-density polyethylene (HDPE), plastic laminate (Formica), and two variations of polished granite. The main objective of this study was to determine a minimal number of spectral fluorescence bands suitable for detecting microbial biofilms on surfaces commonly used to process and handle food. Spots of biofilm growth were produced on sample surfaces by spot-inoculations of pathogenic Escherichia coli O157:H7 and Salmonella followed by room temperature storage for 3 days. Subsequently, hyperspectral fluorescence images were acquired from 421 to 700 nm using ultraviolet-A excitation. Both E. coli O157:H7 and Salmonella biofilms emitted fluorescence predominantly in the blue to green wavelengths with emission maxima at approximately 480 nm. A single-band image at 559 nm was able to detect the biofilm spots on stainless steel. On HDPE and granite, algorithms using different two-band ratios provided better separation of the biofilm spots from background areas than any single-band images did. The biofilm spots on stainless steel, HDPE, and granite could be detected with overall detection rate of 95%. On Formica, too many false positives were present to accurately determine an effective biofilm detection rate. This may have been due to the lower cell population density that was observed for the biofilm spots grown on Formica (approximately 4.3–6.4 log cfu cm⁻²) as compared to the other surfaces. These findings can be incorporated into developing portable hand-held imaging devices for sanitation inspection of food processing surfaces.     

Biofilm formation on stainless steel as a function of time and temperature and control through sanitizers

Enterococcus spp. contamination was screened from a Minas Frescal cheese processing line. Biofilm formation of Enterococcus faecium and Enterococcus faecalis isolates was evaluated and the effect of sanitization procedures in the control of these biofilms was investigated. Enterococcus spp. were detected in raw milk, milk machine, door handle, floor, drain, thermometer, and Minas Frescal cheese. Biofilm formation on stainless steel was modelled as a function of time (0, 1.2, 4, 6.8, and 8 days) and temperature (7, 13, 27, 41, and 47 °C) using response surface methodology. The model showed that E. faecium biofilms were formed from 1 to 8 days at 12–47 °C, while E. faecalis biofilms were formed from 1 to 8 days at 10–43 °C. None of the sanitizers (sodium hypochlorite 100 mg L⁻¹, peracetic acid 300 mg L⁻¹, and chlorhexidine digluconate 400 mg L⁻¹) was able to completely eliminate the biofilms.     

In situ characterization and analysis of Salmonella biofilm formation under meat processing environments using a combined microscopic and spectroscopic approach

Salmonella biofilm on food-contact surfaces present on food processing facilities may serve as a source of cross-contamination. In our work, biofilm formation by multi-strains of meat-borne Salmonella incubated at 20 °C, as well as the composition and distribution of extracellular polymeric substances (EPS), were investigated in situ by combining confocal laser scanning microscopy (CLSM), scanning electron microscope (SEM), attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) and Raman spectroscopy. A standard laboratory culture medium (tryptic soy broth, TSB) was used and compared with an actual meat substrate (meat thawing-loss broth, MTLB). The results indicated that Salmonella grown in both media were able to form biofilms on stainless steel surfaces via building a three-dimensional structure with multilayers of cells. Although the number of biofilm cells grown in MTLB was less than that in TSB, the cell numbers in MTLB was adequate to form a steady and mature biofilm. Salmonella grown in MTLB showed “cloud-shaped” morphology in the mature biofilm, whereas when grown in TSB appeared “reticular-shaped”. The ATR-FTIR and Raman analysis revealed a completely different chemical composition between biofilms and the corresponding planktonic cells, and some important differences in biofilms grown in MTLB and in TSB. Importantly, our findings suggested that the progress towards a mature Salmonella biofilm on stainless steel surfaces may be associated with the production of the EPS matrix, mainly consisting of polysaccharides and proteins, which may serve as useful markers of biofilm formation. Our work indicated that a combination of these non-destructive techniques provided new insights into the formation of Salmonella biofilm matrix.     

Role of shear stress on biofilm formation of Candida krusei in a rotating disk system

In industrial conditions, biofilms formed on pipes, joints and heat exchangers are exposed to varying shear stress conditions caused by fluid flow. In this study we examined the effect of shear, created by the tangential liquid flow in a rotating disk system (RDS) on adhesion and biofilm formation of Candida krusei.C. krusei biofilms were formed on stainless steel (AISI 304 2B food grade) while being exposed to different shear stresses (from 0 to 91 N m⁻²) generated by two rotational speeds (350 and 800 rpm). The coupons were examined by fluorescein diacetate (FDA) at 24-h interval for 4 days.The morphology of the biofilms and the disposition of C. krusei cells in laminar and transitional flow were markedly different. The morphology of biofilm features in the transitional flow revealed the influence of hydrodynamic drag.The early stage of biofilm development resulted practically unaffected by shear stress. However, in a mature biofilm, shear stress determined the disposition of biofilm cells onto the surface. Microcolonies began to appear approximately at 48 h, at all tested shear stresses, and biofilm formation continued throughout the entire experimental period. Moreover, shape of biofilms was probably governed by the continuous applied shear stress. Finally, biofilms formed under higher shear stress differs significantly in their arrangement, as compared with those formed under lower shear conditions.     

Resistances to benzalkonium chloride of bacteria dried with food elements on stainless steel surface

To confirm the importance of washing food sediments from the surface of food-related environments, we examined resistances against benzalkonium chloride of pathogenic bacterial (Escherichia coli O26, Pseudomonas aeruginosa and Staphylococcus aureus) cells dried and adhered on stainless steel dishes with milk, beef gravy or tuna gravy. Suspensions (0.1 ml) of these bacteria (8-9 log cfu/ml) were put on a 5 cm ? stainless steel dish and dried at room temperature (20-24 °C) for 90 min in a bio-clean bench with ventilation. Though these bacteria suspended with distilled water decreased 30-40 fold during the drying period, milk and the gravies protected the bacteria. Without the food elements, the adhered E. coli and Stap. aureus were decreased from 6 to<2 log cfu/dish by 0.5 mg/ml benzalkonium chloride (BKC) for 10 min treatment. Although Ps. aeruginosa showed resistance to BKC, the adhered cells were inactivated by 2.0 mg/ml BKC. However, the bactericidal effect disappeared by the food elements, particularly with milk, even at 1.0 and/or 2.0 mg/ml BKC levels. The protective efficiency of milk on bacteria disappeared if washed with water.     

Modelling survival kinetics of Staphylococcus aureus and Escherichia coli O157:H7 on stainless steel surfaces soiled with different substrates under static conditions of temperature and relative humidity

The survival of Escherichia coli O157:H7 and Staphylococcus aureus on stainless steel surfaces with Saline Solution (SS), Tryptone Soy Broth (TSB) and meat purge was studied, and based on results, mathematical models describing survival of pathogens as a function of time were proposed. Results indicated that S. aureus was able to survive longer than E. coli O157:H7 in all substrates. The type of substrate had a greater impact on the survival of E. coli O157:H7. This microorganism only remained viable for 8 and 50 h (hours) on surfaces with SS and TSB, respectively while on meat purge, the microorganism could be recovered after 200 h. For S. aureus, SS and TSB led to similar survival times (250 h) whereas on meat purge, survival capacity increased to 800 h. Survival data for S. aureus could be well described by a log-linear model or a Weibull model depending on the type of substrate (R² > 0.85). E. coli O157:H7 displayed an evident tail zone which made the Weibull model more appropriate (R² > 0.94). These survival models may be used in quantitative risk assessment to produce more accurate risk estimates. Finally, the results highlight the importance of performing effective cleaning procedures to prevent cross contamination.     

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.     

Candida krusei development on turbulent flow regimes: Biofilm formation and efficiency of cleaning and disinfection program

In food processing lines or in complex equipment such as pumps or valves, microorganisms are exposed to varying hydrodynamic conditions caused by the flow of liquid food, and biofilms are thus grown under a wide distribution of local hydrodynamic strengths. Using an industrially relevant strain of Candida krusei, we demonstrated that biofilms formed on stainless steel for 4 days at Reynolds (Re) numbers ranging from 294,000 to 1.2 × 10⁶ proceeds through three distinct developmental phases. These growth phases transform adherent blastospores to well-defined cellular communities encased in an extracellular matrix and biofilm formation increases when increasing Reynolds number and time. In all growth phases, the morphology of C. krusei biofilm revealed the influence of hydrodynamic drag. Indeed, we study the effect of cleaning and sanitation procedure in the control of turbulent flow-generated biofilm. This procedure involves alkali (NaOH 0.5%) and sodium hypochlorite (500 ppm). In terms of total biofilm mass, removal decreases with increasing biofilm age. The largest reduction post-treatment (between 57% and 62%) was observed, to all Reynolds numbers, on 24 and 48 h-old biofilms. Removal was between 39% and 46% on 72 h-old biofilms and was close to 30% for all Reynolds numbers on 96 h-old biofilm.     

Attachment and biofilm formation by Escherichia coli O157:H7 at different temperatures, on various food-contact surfaces encountered in beef processing

Escherichia coli O157:H7 attached to beef-contact surfaces found in beef fabrication facilities may serve as a source of cross-contamination. This study evaluated E. coli O157:H7 attachment, survival and growth on food-contact surfaces under simulated beef processing conditions. Stainless steel and high-density polyethylene surfaces (2 × 5 cm) were individually suspended into each of three substrates inoculated (6 log CFU/ml or g) with E. coli O157:H7 (rifampicin-resistant, six-strain composite) and then incubated (168 h) statically at 4 or 15 °C. The three tested soiling substrates included sterile tryptic soy broth (TSB), unsterilized beef fat-lean tissue (1:1 [wt/wt]) homogenate (10% [wt/wt] with sterile distilled water) and unsterilized ground beef. Initial adherence/attachment of E. coli O157:H7 (0.9 to 2.9 log CFU/cm²) on stainless steel and high-density polyethylene was not affected by the type of food-contact surface but was greater (p < 0.05) through ground beef. Adherent and suspended E. coli O157:H7 counts increased during storage at 15 °C (168 h) by 2.2 to 5.4 log CFU/cm² and 1.0 to 2.8 log CFU/ml or g, respectively. At 4 °C (168 h), although pathogen levels decreased slightly in the substrates, numbers of adherent cells remained constant on coupons in ground beef (2.4 to 2.5 log CFU/cm²) and increased on coupons in TSB and fat-lean tissue homogenate by 0.9 to 1.0 and 1.7 to 2.0 log CFU/cm², respectively, suggesting further cell attachment. The results of this study indicate that E. coli O157:H7 attachment to beef-contact surfaces was influenced by the type of soiling substrate and temperature. Notably, attachment occurred not only at a temperature representative of beef fabrication areas during non-production hours (15 °C), but also during cold storage (4 °C) temperatures, thus, rendering the design of more effective sanitation programs necessary.     

Biofilm Formation of O157 and Non‐O157 Shiga Toxin‐Producing Escherichia coli and Multidrug‐Resistant and Susceptible Salmonella Typhimurium and Newport and Their Inactivation by Sanitizers

This study compared biofilm formation by 7 serogroups of pathogenic Escherichia coli and 2 or 3 phenotypes of Salmonella (susceptible, multidrug‐resistant [MDR], and/or multidrug resistant with ampC gene [MDR‐AmpC]). One‐week mature biofilms were also exposed to water, quaternary ammonium compound‐based (QAC), and acid‐based (AB) sanitizers. Seven groups (strain mixture) of above‐mentioned pathogens were separately spot‐inoculated onto stainless steel coupons surfaces for target inoculation of 2 log CFU/cm², then stored statically, partially submerged in 10% nonsterilized meat homogenate at 4, 15, and 25 °C. Biofilm cells were enumerated on days 0, 1, 4, and 7 following submersion in 30 mL for 1 min in water, QAC, and AB. Counts on inoculation day ranged from 1.6 ± 0.4 to 2.4 ± 0.6 log CFU/cm² and changed to 1.2 ± 0.8 to 1.9 ± 0.8 on day 7 at 4 °C with no appreciable difference among the 7 pathogen groups. After treatment with QAC and AB on day 7, counts were reduced (P < 0.05) to less than 0.7 ± 0.6 and 1.2 ± 0.5, respectively, with similar trends among pathogens. Biofilm formation at higher temperatures was more enhanced; E. coli O157:H7, as an example, increased (P < 0.05) from 1.4 ± 0.6 and 2.0 ± 0.3 on day 0 to 4.8 ± 0.6 and 6.5 ± 0.2 on day 7 at 15 and 25 °C, respectively. As compared to 4 °C, after sanitation, more survivors were observed for 15 and 25 °C treatments with no appreciable differences among pathogens. Overall, we observed similar patterns of growth and susceptibility to QAC and AB sanitizers of the 7 tested pathogen groups with enhanced biofilm formation capability and higher numbers of treatment survivors at higher temperatures.     

Evaluation of anti-Listeria meat borne Lactobacillus for biofilm formation on selected abiotic surfaces

The ability of meat borne anti-Listeria Lactobacillus to form biofilms under different in vitro conditions and on abiotic surfaces was investigated. Biofilm formation by the adhesion to polystyrene microtiter plates was determined, this being higher for Lactobacillus curvatus CRL1532 and CRL705 and Lactobacillus sakei CRL1862. The physicochemical properties of the cell surface were relatively hydrophilic and acidic in character; L. sakei CRL1862 exhibiting the strongest autoaggregation. The adhesion of lactobacilli to stainless steel (SS) and polytetrafluoroethylene (PTFE) supports at 10 °C was found to be maximal for L. sakei CRL1862 on SS after 6 days. When biofilm architecture was characterized by epifluorescence and SEM, L. sakei CRL1862 homogeneously covered the SS surface while cell clusters were observed on PTFE; the extracellular polymeric substance matrix adapted to the topography and hydrophilic/hydrophobic characteristics of each material. The feasibility of L. sakei CRL1862 to form biofilm on materials used in meat processing highlights its potential as a control strategy for Listeria monocytogenes biofilms.     

The probability of growth of Listeria monocytogenes in cooked salmon and tryptic soy broth as affected by salt,smoke compound,and storage temperature

The objectives of this study were to examine and model the probability of growth of Listeria monocytogenes in cooked salmon containing salt and smoke (phenol) compound and stored at various temperatures. A growth probability model was developed, and the model was compared to a model developed from tryptic soy broth (TSB) to assess the possibility of using TSB as a substitute for salmon. A 6-strain mixture of L. monocytogenes was inoculated into minced cooked salmon and TSB containing 0–10% NaCl and 0–34 ppm phenol to levels of 10^2–3 cfu/g, and the samples were vacuum-packed and stored at 0-–25 °C for up to 42 days. A total 32 treatments, each with 16 samples, selected by central composite designs were tested. A logistic regression was used to model the probability of growth of L. monocytogenes as a function of concentrations of salt and phenol, and storage temperature. Resulted models showed that the probabilities of growth of L. monocytogenes in both salmon and TSB decreased when the salt and/or phenol concentrations increased, and at lower storage temperatures. In general, the growth probabilities of L. monocytogenes were affected more profoundly by salt and storage temperature than by phenol. The growth probabilities of L. monocytogenes estimated by the TSB model were higher than those by the salmon model at the same salt/phenol concentrations and storage temperatures. The growth probabilities predicted by the salmon and TSB models were comparable at higher storage temperatures, indicating the potential use of TSB as a model system to substitute salmon in studying the growth behavior of L. monocytogenes may only be suitable when the temperatures of interest are in higher storage temperatures (e.g., >12 °C). The model for salmon demonstrated the effects of salt, phenol, and storage temperature and their interactions on the growth probabilities of L. monocytogenes, and may be used to determine the growth probability of L. monocytogenes in smoked seafood.     

Escherichia coli O157:H7 survival,biofilm formation and acid tolerance under simulated slaughter plant moist and dry conditions

Microorganisms persisting in slaughter plant environments may develop acid resistance and be translocated to other environmental surfaces or products. The objective of this study was to evaluate the potential of Escherichia coli O157:H7 to form biofilms and maintain acid resistance, under different culture habituation scenarios, on stainless steel coupons (2 × 5 × 0.08 cm), in the presence of beef carcass decontamination runoff fluids (washings). Coupons were stored in test tubes with unsterilized water washings (WW; pH 6.94) or lactic acid washings (LAW; pH 4.98), which were inoculated with E. coli O157:H7 (10³–10⁴ CFU/ml) and incubated at 15 (24 or 48 h) or 35 °C (7 or 24 h), simulating different habituation scenarios on sites of a slaughter plant, including sanitation and overnight drying, during consecutive operational shifts. Acid resistance (AR) of planktonic and detached E. coli O157:H7 cells was assessed in tryptic soy broth adjusted to pH 3.5 with lactic acid. The highest pre-drying attachment and AR of E. coli O157:H7 were observed after 24 h at 35 °C and 48 h at 15 °C. Drying reduced (P < 0.05) recovery of attached E. coli O157:H7 cells; however, exposure of dried coupons to uninoculated washings allowed recovery of attached E. coli O157:H7, which restored AR, especially under conditions that favored post-drying growth. Exposure of attached cells to 50 ppm PAA for 45 s before drying, as well as habituation in LAW, reduced the recovery and AR of E. coli O157:H7. Therefore, incomplete removal of biofilms may result in cells of increased AR, especially in sites within a slaughter plant, in which liquid meat wastes may remain for long periods of time.     

Postharvest quality of arazá fruit during low temperature storage

Arazá (Eugenia stipitata Mc Vaugh) fruits at breaker stage of maturity were stored at 7, 10, 12, or 20 °C and 85-90% RH for 2 weeks, with or without an additional simulated shelf-life period (3 days at 20 °C and 70% RH). Some half-yellow (turning) arazá fruit were also stored at 7 or 12 °C. Respiration rate, ethylene production, quality traits and physiological disorders and decay were monitored. Arazá fruit of both stages of maturity showed a climacteric pattern of ripening, with the maximum levels of respiration being reached after 5 days at 20 °C for breaker fruit, while half-yellow fruit ripened totally after one day. Weight loss was the most limiting quality trait for arazá fruit. Chilling injury symptoms included skin scald (only at 7 °C), uneven ripening (at 7 or 10 °C, including uneven softening during storage, particularly in breaker fruit), and slight acidification at 7 °C. Decay in the post-storage shelf-life periods (mainly Gloesporium sp.) was particularly high after storage at 7 °C in breaker fruit. The storage of breaker arazá fruit at 12 °C is recommended because this prevents chilling injury and flesh acidification, and allows normal fruit ripening during a post-storage shelf-life at 20 °C, as revealed by the lower organic acids (mainly malic) content and increased sugar (glucose, fructose and sucrose) content.     

Temperature and nutrient effects on Campylobacter jejuni attachment on multispecies biofilms on stainless steel

Campylobacterjejuni is a thermophilic microaerophilic pathogen that is commonly found in the intestinal tract of chickens. In this study, attachment of C. jejuni 1221gfp in biofilms on stainless steel was assessed at various temperatures and with reduced nutrients. Bacteria collected from a saline rinse of processed broiler chicken carcasses were used to form initial biofilms. The whole carcass rinse (WCR) biofilms were formed by incubation of the bacteria for 16 h at 13, 20, 37, and 42 degrees C on stainless steel coupons in tryptic soy broth (TSB). The resulting biofilms were stained with Hoechst 33258 stain and visualized by epifluorescence microscopy. WCR biofilms formed at 13 degrees C yielded the highest surface area coverage (47.6%), and the lowest coverage (2.1%) was attained at 42 degrees C. C. jejuni transformed to produce green fluorescent protein (gfp) was allowed to attach to the preexisting biofilms (from WCR incubated for 16 h) at each of the four temperatures, and attached cells were enumerated by visualization with an epifluorescence microscope. Attachment of C. jejuni 1221gfp did not significantly differ (P > 0.05) among the four temperatures. C. jejuni 1221gfp was cultured only from coupons with biofilms formed at 13 and 20 degrees C. For nutrient limitation experiments, WCR biofilms were allowed to grow in 10- and 50-fold diluted TSB at 20 and 37 degrees C for 48 h. The WCR biofilm surface area coverage (approximately 2%) was greater at 37 degrees C than at 20 degrees C for both TSB concentrations. C. jejuni 1221gfp was incubated with the WCR biofilm for 48 h at 20 and 37 degrees C, and attached cells were enumerated. Attachment was significantly higher (P < 0.05) only for the treatments with 1:10 TSB at 20 degrees C and 1:50 TSB at 37 degrees C. Under reduced-nutrient conditions, C. jejuni 1221gfp was cultured only from biofilms formed at 20 degrees C. Under the conditions tested, the attachment of C. jejuni 1221gfp on stainless steel and biofilms was affected by a combination of temperature and nutrient availability, but C. jejuni culturability was affected solely by temperature.     

Inactivation of Clostridium sporogenes spores on stainless-steel using heat and an organic acidic chemical agent

The efficacy of a food grade acidic chemical agent for the reduction of Clostridium sporogenes spores on a stainless steel surface was investigated. The chemical agent was a combination of selected fatty acids and lactate esters. Distilled deionized water and 35% hydrogen peroxide were used as negative and positive controls, respectively. Approximately 3 log cfu reductions in viable spore numbers were detected on the steel surfaces for all treatments at room temperature, except the controls. Reductions in the viable spore numbers significantly increased with increasing exposure times and concentrations of the acidic agent. Five log reduction of viable spore number was achieved after 10 min treatment with the 10% agent solution at 68 °C. No viable spores were observed on the 10% agent treated sample after a 60 min exposure time at 75 °C. This research showed that the acidic sanitizer tested in this study could be used to reduce the number of C. sporogenes spores on stainless steel surfaces.     

Fate of inoculated Escherichia coli O157:H7, cultured under different conditions, on fresh and decontaminated beef transitioned from vacuum to aerobic packaging

This study evaluated the behavior of Escherichia coli O157:H7 during aerobic storage, after storage in vacuum packages, on beef inoculated with cultures prepared (35 degrees C, 24 h) in tryptic soy broth without dextrose (TSB), nonacid hot water carcass decontamination runoff fluids (washings; pH 6.0; WASH), cells from biofilms formed on stainless steel coupons in WASH (WETB), or WETB dried (25 degrees C, 12 h) before harvesting of cells (DRYB). These inocula were applied to fresh beef pieces (40 cm2), which were then left untreated or treated by immersion in hot water (75 degrees C) followed by 2% lactic acid (55 degrees C; hot water/lactic acid [HW/LA]), for 30 s each. Inoculated samples were vacuum packaged and stored at 0 (30, 60, or 90 days), 4 (7 or 14 days), or 12 degrees C (4 or 8 days) and subsequently transferred to retail packages for aerobic storage at 7 degrees C for 5 days. Populations of E. coli O157:H117, regardless of inoculum type, remained generally unchanged (P > 0.05) after aerobic storage (7 degrees C, 5 days) of untreated or HW/LA-treated beef samples previously stored in vacuum packages at 0 or 4 degrees C. However, reductions in E. coli O157:H7 levels were generally obtained when vacuum packaged, untreated beef samples previously stored at 12 degrees C were transitioned to aerobic conditions. Additionally, despite similar (P > 0.05) levels of E. coli O157:H7 cells of TSB, WASH, WETB, and DRYB origin on vacuum-packaged, untreated samples after 8 days of storage at 12 degrees C, subsequent aerobic storage resulted in larger (P < 0.05) reductions of cells of WETB and DRYB origin than for cells of TSB and WASH origin. For HW/LA-treated beef previously stored at 12 degrees C in vacuum packages, populations of E. coli O157:H7 remained largely unchanged after aerobic storage in retail packages. Results thus indicated that aerobic storage of beef (7 degees C, 5 days) previously stored in vacuum packages at 0 or 4 degrees C did not lead to E. coli O157:H7 population changes, whereas transition from vacuum packages stored under mildly abusive temperature (12 degrees C) to aerobic storage may have caused injury and death to the pathogen.