Efficacy of egg cleaning compounds on eggshells contaminated with Salmonella enterica serovar Enteritidis

Salmonella Enteritidis infections of egg contents can be related to external contamination of the shell. In this study, the efficacy of three commercial cleaning and/or sanitizing compounds (sodium carbonate, sodium hypochlorite, and potassium hydroxide) was evaluated for bactericidal activity at pH values of 10, 11, and 12 against various concentrations (10(2), 10(4), or 10(6) CFU/ml) of Salmonella Enteritidis inoculated onto the eggshell surface. Efficacy of these chemical agents was also assessed against Salmonella Enteritidis in aqueous suspension. Our results indicated that none of the chemicals applied at the recommended manufacturer's concentrations (sodium carbonate, 36 ppm; other treatments, 200 ppm) could eliminate Salmonella Enteritidis from eggshells artificially contaminated with the highest bacterial concentrations (10(4) or 10(6) CFU/ml). Higher concentrations of each product, at least 5 to 20 times greater than recommended doses, were needed to destroy the bacteria on egg surfaces. However, at or slightly above the manufacturer's recommended use concentrations, all three formulations were effective against Salmonella Enteritidis in aqueous suspension (10(8) CFU/ml) or on eggshells contaminated with 10(2) CFU/ml. For both shell and suspension assays, inactivation of Salmonella Enteritidis occurred at lower concentrations at pH 12 than at pH 11 and 10. Contact time between chemicals and Salmonella apparently influenced bacterial inactivation. Extended contact times (2 to 10 min) reduced minimum chemical concentrations necessary to inactivate the bacteria. However, neither pH nor contact time influenced Salmonella Enteritidis inactivation when the initial bacterial numbers on eggshells were high.     

Inactivation of Salmonella enterica serovar Enteritidis on shell eggs by ozone and UV radiation

The presence of Salmonella enterica serovar Enteritidis in shell eggs has serious public health implications. Several treatments have been developed to control Salmonella on eggs with mixed results. Currently, there is a need for time-saving, economical, and effective egg sanitization treatments. In this study, shell eggs externally contaminated with Salmonella (8.0 x 10(5) to 4.0 x 10(6) CFU/g of eggshell) were treated with gaseous ozone (O3) at 0 to 15 lb/in2 gauge for 0 to 20 min. In other experiments, contaminated shell eggs were exposed to UV radiation at 100 to 2,500 microW/cm2 for 0 to 5 min. Treatment combination included exposing contaminated eggs to UV (1,500 to 2,500 microW/cm2) for 1 min, followed by ozone at 5 lb/in2 gauge for 1 min. Eggs that were (i) noncontaminated and untreated, (ii) contaminated and untreated, and (iii) contaminated and treated with air were used as controls. Results indicated that treating shell eggs with ozone or UV, separately or in combination, significantly (P < 0.05) reduced Salmonella on shell eggs. For example, contaminated eggs treated with ozone at 4 to 8 degrees C and 15 lb/in2 gauge for 10 min or with UV (1,500 to 2,500 microW/cm2) at 22 to 25 degrees C for 5 min produced 5.9- or 4.3-log microbial reductions or more, respectively, when compared with contaminated untreated controls. Combinations including UV followed by ozone treatment resulted in synergistic inactivation of Salmonella by 4.6 log units or more in about 2 min of total treatment time. Salmonella was effectively inactivated on shell eggs in a short time and at low temperature with the use of a combination of UV radiation and ozone.     

Effect of thermoultrasonication on Salmonella enterica serovar Enteritidis in distilled water and intact shell eggs

The combined effects of simultaneous application of ultrasonic waves and heat treatment (thermoultrasonication) on the survival of a strain of Salmonella enterica Enteritidis was studied in both distilled water and intentionally contaminated intact eggs immersed in water. Although minor differences were observed between parameters obtained for thermoultrasonic treatment of bacteria suspended in water and those attached to the shell egg, the thermoultrasonication effects were considered to be of the same level in the range of temperatures assayed (52 to 58 degrees C). This combined process presented a clearly higher killing effect than the heat treatment alone. It decreased the decimal reduction times (D-values) by 80 to 55%, respectively, in the range of temperatures for heat treatment when the organism was suspended in water, which means a 99.5% reduction (5D to >2D) of the original bacterial load versus a 90% reduction for the heat treatment alone. The practical implications of the phenomenon are discussed.     

Standardized laboratory-scale preparation of mayonnaise containing low levels of Salmonella enterica serovar Enteritidis

Salmonella enterica serovar Enteritidis PT4 and PT6 are associated with food poisoning outbreaks and are often found in food only in low concentrations. In this study a reproducible laboratory-scale procedure for preparation of mayonnaise is presented. The mayonnaise that simulates a naturally low-level contaminated product can be used for validation of new methods and is also suitable to study the behavior of low numbers of food pathogenic spoilage microorganisms in a food environment. During processing, liquid egg was artificially contaminated with low levels of S. enterica serovar Enteritidis that resulted in levels of 1 to 3 log10 CFU/g in the final mayonnaise. Cells of S. enterica serovar Enteritidis had increased stability in the mayonnaise when they were subjected to low pH in two stages, first to pH 5.8 and afterward to pH 4.5 before addition to the mayonnaise. The pH of the mayonnaise was between 4.2 to 4.5 and remained stable over the storage period. Low-level S. enterica serovar Enteritidis remained stable in artificially contaminated mayonnaise for 4 weeks at 4 degrees C.     

Protective action of Lactobacillus kefir carrying S-layer protein against Salmonella enterica serovar Enteritidis

Eight Lactobacillus kefir strains isolated from different kefir grains were tested for their ability to antagonize Salmonella enterica serovar Enteritidis (Salmonella enteritidis) interaction with epithelial cells. L. kefir surface properties such as autoaggregation and coaggregation with Salmonella and adhesion to Caco-2/TC-7 cells were evaluated. L. kefir strains showed significantly different adhesion capacities, six strains were able to autoaggregate and four strains coaggregated with Salmonella. Coincubation of Salmonella with coaggregating L. kefir strains significantly decreased its capacity to adhere to and to invade Caco-2/TC-7 cells. This was not observed with non coaggregating L. kefir strains. Spent culture supernatants of L. kefir contain significant amounts of S-layer proteins. Salmonella pretreated with spent culture supernatants (pH 4.5-4.7) from all tested L. kefir strains showed a significant decrease in association and invasion to Caco-2/TC-7 cells. Artificially acidified MRS containing lactic acid to a final concentration and pH equivalent to lactobacilli spent culture supernatants did not show any protective action. Pretreatment of this pathogen with spent culture supernatants reduced microvilli disorganization produced by Salmonella. In addition, Salmonella pretreated with S-layer proteins extracted from coaggregating and non coaggregating L. kefir strains were unable to invade Caco-2/TC-7 cells. After treatment, L. kefir S-layer protein was detected associated with Salmonella, suggesting a protective role of this protein on association and invasion.     

Eggshell penetration of various types of hens' eggs by Salmonella enterica serovar Enteritidis

Egg weight, shell thickness, number of pores, cuticle deposition, eggshell strength (dynamic stiffness and damping ratio), and the ability of Salmonella enterica serovar Enteritidis (SE) to penetrate the eggshell were determined. Penetration was assessed by filling the eggs with a selective medium that allowed viewing of Salmonella growth on the inside of the shell and membrane complex. After inoculation of each shell with on average 2.71 log CFU, the eggs were stored for up to 14 days at 20 degrees C and 60% relative humidity. Commercially available eggs were used. At 14 days of storage, only 6.0% of the eggs from free-range hens and 16.0% of the generic (i.e., eggs from hens in conventional battery cages that were given standard feed) white eggs were penetrated. The generic brown, organic, and omega-3-enriched eggs were penetrated at a frequency of 30 to 34%. In a second experiment it was shown that the layer strains of the hen (ISA-Brown Warren versus Bovans Goldline), which were kept in furnished cages, did not affect eggshell penetration by SE. For Bovans Goldline hens, the housing system (furnished cage versus aviary) did not affect penetration, while a trend was visible toward a higher fraction of penetrated eggshells when hens were fed corncob mix rather than standard feed. Eggshell penetration was observed more frequently in the absence of cuticle spots and for eggs having lower dynamic stiffness values. Shell contamination at the end of storage was highly correlated with SE penetration.     

Heat tolerance of Salmonella enterica serovars Agona, Enteritidis, and Typhimurium in peanut butter

Recent large foodborne outbreaks caused by Salmonella enterica serovars have been associated with consumption of foods with high fat content and reduced water activity, even though their ingredients usually undergo pasteurization. The present study was focused on the heat tolerance of Salmonella enterica serovars Agona, Enteritidis, and Typhimurium in peanut butter. The Salmonella serovars in the peanut butter were resistant to heat, and even at a temperature as high as 90 degrees C only 3.2-log reduction in CFU was observed. The obtained thermal inactivation curves were upwardly concave, indicating rapid death at the beginning (10 min) followed by lower death rates and an asymptotic tail. The curves fitted the nonlinear Weibull model with beta parameters < 1, indicating that the remaining cells have a lower probability of dying. beta at 70 degrees C (0.40 +/- 0.04) was significantly lower than beta at 80 degrees C (0.73 +/- 0.19) and 90 degrees C (0.69 +/- 0.17). Very little decrease in the viable population (less than 2-log decrease) was noted in cultures that were exposed to a second thermal treatment. Peanut butter is a highly concentrated colloidal suspension of lipid and water in a peanut meal phase. We hypothesized that differences in the local environments of the bacteria, with respect to fat content or water activity, explained the observed distribution and high portion of surviving cells (0.1%, independent of the initial cell number). These results demonstrate that thermal treatments are inadequate to consistently destroy Salmonella in highly contaminated peanut butter and that the pasteurization process cannot be improved significantly by longer treatment or higher temperatures.     

Survival, elongation, and elevated tolerance of Salmonella enterica serovar Enteritidis at reduced water activity

Growing microorganisms on dry surfaces, which results in exposure to low water activity (a(w)), may change their normal morphology and physiological activity. In this study, the morphological changes and cell viability of Salmonella enterica serovar Enteritidis challenged to low a(w) were analyzed. The results indicated that exposure to reduced a(w) induced filamentation of the cells. The amount of filamentous cells at a(w) 0.94 was up to 90% of the total number of cells. Surviving filamentous cells maintained their membrane integrity after exposure to low a(w) for 21 days. Furthermore, cells prechallenged to low a(w), obtained with an ionic humectant, demonstrated higher resistance to sodium hypochlorite than control cells. These resistant cells are able to survive disinfection more efficiently and can therefore cause contamination of foods coming in contact with surfaces. This points to the need for increased attention to cleaning of surfaces in household environments and disinfection procedures in processing plants.     

Effects of gamma irradiation on the viability and phenotypic characteristics of Salmonella Enteritidis inoculated into specific-pathogen-free eggs

The goal of this study was to determine the effects of various levels of gamma irradiation on the phenotypic characteristics of 20 strains of Salmonella Enteritidis inoculated separately into specific-pathogen-free shell eggs. Bacterial strains were inoculated into egg yolks and exposed to (60)Co radiation at doses of 0.49 to 5.0 kGy. The eggs were maintained at 25°C and analyzed for the presence of Salmonella on days 1, 2, 4, and 7, and the recovered Salmonella isolates were characterized biochemically. All strains were resistant to doses of 0.49, 0.54, 0.59, 0.8, and 1 kGy; colony counts were ≥10(5) CFU/ml of egg yolk except for one strain, which was detected at 96 h and at 7 days after irradiation at 1 kGy, with a population reduction of 2 log CFU/ml. For the other evaluated doses, 12 strains (60.0%) were resistant at 1.5 kGy and 7 strains (35.0%) were resistant at 3.0 kGy. Among all analyzed strains, 5.0 kGy was more effective for reducing and/or eliminating the inoculated bacteria; only two (10%) strains were resistant to this level of irradiation. Salmonella colony counts were significantly reduced (P < 0.01) with increasing doses from the day 1 to 7 of observation, when microbial growth peaked. Loss of mobility, lactose fermentation, citrate utilization, and hydrogen sulfide production occurred in some strains after irradiation independent of dose and postirradiation storage time. Increases in antibiotic susceptibility also occurred: seven strains became sensitive to β-lactams, two strains became sensitive to antifolates, and one strain each became sensitive to fluoroquinolone, phenicol, nitrofurans, tetracyclines, and aminoglycosides. The results indicate that up to 5.0 kGy of radiation applied to shell eggs inoculated with Salmonella Enteritidis at 4 log CFU per egg is not sufficient for complete elimination of this pathogen from this food matrix.     

Incubation of supplemented egg contents pools to support rapid detection of Salmonella enterica serovar Enteritidis

Detecting internal contamination of eggs with Salmonella enterica serovar Enteritidis (Salmonella Enteritidis) is an important aspect of efforts to identify infected laying flocks. When egg contents pools are tested for Salmonella Enteritidis, a preliminary incubation step is often employed to allow small initial populations of contaminants to multiply to more easily detectable numbers. Consistent detection of Salmonella Enteritidis in egg pools by direct plating requires the presence of at least 10(5) CFU/ml, whereas some very rapid methods can require as many as 10(7) CFU/ml. The present study determined the rates at which initial inocula of approximately 10 Salmonella Enteritidis cells multiplied in 10-egg pools, some of which were supplemented with concentrated nonselective enrichment broth or with a source of iron. At 37 degrees C, Salmonella Enteritidis concentrations in supplemented egg pools usually reached 10(5) CFU/ml within 12 h and 10(7) CFU/ml by 12 to 15 h of incubation. At 25 degrees C, Salmonella Enteritidis concentrations in supplemented egg pools typically attained 10(5) CFU/ml by 18 to 27 h and 10(7) CFU/ml by 27 to 36 h of incubation. At both temperatures, Salmonella Enteritidis multiplication was significantly slower in unsupplemented pools. Accordingly, the length of incubation time necessary for consistent detection of small numbers of Salmonella Enteritidis in egg contents pools depends on the incubation temperature used, on whether the egg pools are supplemented to increase the rate of bacterial multiplication, and on the sensitivity of subsequent tests applied to the incubated pools.     

Effects of carob bean gum on performance,nutrient digestibility and Salmonella enterica var. Enteritidis colonisation in chickens

Salmonella is presently one of the microorganisms of higher concern for food safety in poultry products. The present study examined the effect of feeding galactomannans from carob bean gum on nutrient digestibility and performance in chickens, and on the prevalence of Salmonella enterica var. Enteritidis in challenged animals. Four experiments were performed with either broiler or leghorn chickens, challenged with 10⁶ CFU (colony-forming units) of S. Enteritidis at 1 day of life, and feeding carob bean gum at different concentrations (25, 50 or 100 g/kg, depending on the experiment), alone or in combination with β-mannanase, cellulase or α-galactosidase at 8.3 U/g; or feeding D-mannose at 25 g/kg, or depolymerized carob bean gum or guar gum at 100 mg/kg. Trials lasted 3 or 4 weeks. Body weight and feed intake were determined and feed conversion ratio calculated (feed:gain). Faeces were collected during the last week on trial for evaluation of nutrient balance (energy, lipids and protein), using chromium oxide as inert marker. Viscosity of the ileal content was also determined at the end of the second experiment. Salmonella presence in caeca was determined two and 3 weeks after challenge. Performance and nutritive value of diets were impaired in birds fed carob bean gum, with higher effect at higher inclusion rates. D-mannose impaired performance variables only whereas depolymerized gums did not affect bird performance or nutritive value of the diets. Of the enzymes tested, only β-mannanase significantly decreased the viscosity of the intestinal contents of birds fed carob bean gum and partly counteracted the impairment in bird performance and the reduction in the nutritive value of the diets. The number of Salmonella-positive birds varied among experiments and was lower in the third week post-challenge compared to the second week post-challenge. However, the reduction in the number of Salmonella-positive birds was more constant and marked when carob bean gum was present in the diet. The inclusion of carob bean gum in the diet of chickens at the high concentrations used in the present experiment reduced the presence of Salmonella in challenged birds, but it also impaired performance and nutrient digestibility. These impairments were partially counteracted by the addition of β-mannanase to the diet. Carob bean gum might be used to reduce the incidence of Salmonella in chickens, while its negative effects on performance and nutrient digestibility could be counteracted by β-mannanase.     

Phenotypic Characterization and Genotypic Subtyping of Salmonella enterica Serovars Enteritidis and Gallinarum Isolated from Human and Poultry-Related Samples

This study aimed to assess the serotypes, antibiotic susceptibility, and molecular subtyping of Salmonella enterica isolated from food products and human patients with gastroenteritis. A total of 59 isolates were investigated, and the results revealed a predominance of S. Enteritidis with 57.63% (34/59) S. Gallinarum held second with 15.62% (5/32) of total food borne. While, isolates from humans showed 18.51% (5/27) of S. Typhimurium. High level of resistance to nalidixic acid was noted among food strains and 35.29% of human isolates were found to be multidrug resistant. Eight representative isolates were subtyping using three molecular approaches, ribotyping, Multilocus sequence typing (MLST) and Multiple-locus variable number tandem repeat analysis (MLVA). MLST showed three sequence types corresponding to two clonal complexes, (ST-78, CC-4) for S. Gallinarum, (ST-11, CC-4) for S. Enteritidis and (S-367, CC-37) for S. Cerro. While, MLVA generated six different profiles targeting nine loci for S. Enteritidis and S. Gallinarum.     

Studies on the effects of phosphine on Salmonella enterica serotype Enteritidis in culture medium and in black pepper (Piper nigrum)

The effect of phosphine on Salmonella enterica serotype Enteritidis inoculated in culture medium and in black pepper grains (Piper nigrum), as well as on the reduction of the microbial load of the dried and moisturized product, was verified. The postfumigation effect was verified in inoculated samples with 0.92 and 0.97 water activity (a(w)) exposed to 6 g/m(3) phosphine for 72 h, dried to 0.67 a(w), and stored for 24, 48, and 72 h. No decreases were observed in Salmonella Enteritidis populations in culture medium when fumigant concentrations up to 6 g/m(3) were applied for 48 h at 35°C. However, the colonies showed reductions in size and atypical coloration as the phosphine concentration increased. No reduction in Salmonella counts occurred on the inoculated dried samples after fumigation. On the other hand, when phosphine at concentrations of 6 g/m(3) was applied on moisturized black pepper for 72 h, decreases in Salmonella counts of around 80% were observed. The counts of total aerobic mesophilic bacterium populations of the dried and moisturized black pepper were not affected by the fumigant treatment. The results of the postfumigation studies indicated that Salmonella Enteritidis was absent in the fumigated grains after drying and storage for 72 h, indicating a promising application for this technique. It was concluded that for Salmonella Enteritidis control, phosphine fumigation could be applied to black pepper grains before drying and the producers should rigidly follow good agricultural practices, mainly during the drying process, in order to avoid product recontamination. Additional work is needed to confirm the findings with more Salmonella serotypes and strains.     

A pulsed field gel electrophoresis (PFGE) study that suggests a major world-wide clone of Salmonella enterica serovar Enteritidis

Since human infections by Salmonella enterica serovar Enteritidis (Salmonella Enteritidis) have been increasing world-wide over the past years and epidemiological studies have implicated the consumption of meat, poultry, eggs and egg products, elucidation of the predominant subtypes for this Salmonella spp. is important. In this study, 107 poultry and food isolates of Salmonella Enteritidis obtained from Germany were analyzed by pulsed field gel electrophoresis (PFGE), and the subtypes were compared with those of the 124 human isolates obtained in Taiwan. Results showed that for these 107 poultry and food isolates, when XbaI, SpeI and NotI were used for chromosomal DNA digestion followed by PFGE analysis, a total of 19, 20 and 19 PFGE patterns, respectively, were identified. Of them, 51 (47.7%), 52 (48.6%) and 42 (39.3%) strains belong to a single pattern of X3, S3 and N3, respectively, and 34 strains belong to a pattern combination of X3S3N3, which was the major subtype. When PFGE patterns of these 107 German isolates were compared with those of the 124 human isolates obtained in Taiwan, pattern combination of X3S3N3 was found as the most common pattern shared by isolates from both areas. PT4 is a major phage type for German and Taiwan isolates. Although most of the X3S3N3 strains are of this phage type, some strains of other PFGE patterns are also of this phage type. Since strains used in this study were unrelated, i.e., they were isolated from different origins in areas geographically far apart from each other, the PFGE study suggests a major world-wide clone of S. enterica serovar Enteritidis.     

Growth of Salmonella enterica serovar Enteritidis in albumen and yolk contents of eggs inoculated with this organism onto the vitelline membrane

By using an in vitro model simulating the potential opportunities for Salmonella enterica serovar Enteritidis (SE) to proliferate within eggs contaminated with this organism following oviposition, we investigated growth of SE in eggs. Seventy to 140 CFU of one of three SE strains originating either from egg contents, chicken meat, or a human infection were experimentally inoculated onto the vitelline membrane of eggs collected from specific-pathogen-free flocks of chickens and incubated at 25 degrees C. SE organisms were detected in 6 of 71 yolk contents of the eggs inoculated with any of the test strains attaining levels ranging from 2.0 x 10(2) to 4.2 x 10(8) CFU/ml by day 6. The organisms were also detected in the albumen from 38 of 55 eggs tested, growing to levels ranging from 1.0 x 10(2) to 4.3 x 10(8) CFU/ml by day 6 after inoculation. An additional three yolk contents and 15 albumen samples were culture positive for SE following enrichment. There was no correlation between the number of the organisms in the yolk contents and that in the albumen from each of the eggs. When 73 to 91 CFU of the egg strain were inoculated into samples of separated albumen obtained from eggs that were stored at 4 degrees C for 1 to 4 weeks or at 25 degrees C for 1 week, slight growth (3.0 x 10(2) to 7.4 x 10(3) CFU/ml) was found in only 3 of the 60 albumen samples by day 6 after inoculation, but the organisms were recovered from 52 samples following enrichment. The results suggest that the environment on or near the vitelline membrane can be conducive to SE proliferation over time.     

Inactivation of Salmonella Enteritidis, Salmonella Typhimurium, and Salmonella Senftenberg by ultrasonic waves under pressure

The resistance of Salmonella Enteritidis (ATCC 13076), Salmonella Typhimurium (ATCC 13311), and Salmonella Senftenberg 775W (ATCC 43845) to ultrasonic waves under pressure treatments, at sublethal (manosonication) and lethal temperatures (manothermosonication) in citrate-phosphate buffer and in liquid whole egg was investigated. The influence of treatment parameters on the inactivation rate of manosonication was also studied. Decimal reduction times (Dt) of Salmonella Enteritidis, Salmonella Typhimurium, and Salmonella Senftenberg 775W corresponding to a heat treatment at 60 degrees C in pH 7 buffer and in liquid whole egg were 0.068, 0.12, and 1.0 min for buffer, and 0.12, 0.20, and 5.5 min for liquid whole egg. Those corresponding to a manosonication treatment (117 microns, 200 kPa, 40 degrees C) in both media were 0.73, 0.78, and 0.84 min, and 0.76, 0.84, and 1.4 min, respectively. When the amplitude of ultrasonic waves was increased linearly, the inactivation rate of manosonication increased exponentially. The inactivation rate also increased when pressure was raised. However, the magnitude of this increase was progressively smaller at higher pressures. The magnitude of the influence of the amplitude of ultrasonic waves and static pressure on the inactivation rate of manosonication was the same in the three serotypes investigated. Whereas a heat treatment at 60 degrees C only attained a 1/2-log cycle reduction in the number of Salmonella Senftenberg 775W survivors, a manothermosonication treatment (117 microns and 200 kPa) at this temperature attained a 3-log cycle reduction.     

Purified beta-glucan as an abiotic feed additive up-regulates the innate immune response in immature chickens against Salmonella enterica serovar Enteritidis

Functionally, the innate immune system of immature chickens is inefficient during the first week posthatch. This immunological inefficiency enables pathogens such as Salmonella enterica serovar Enteritidis (SE) to invade and colonize the visceral organs of immature chickens. The objective of this study was to evaluate the effect of purified beta-glucan as an immunomodulator of the innate immune response. beta-glucan, as a feed additive, significantly provided protection against SE organ invasion in young chickens (P<0.05). The functional efficiency of heterophils isolated from neonatal chickens fed a beta-glucan ration was significantly (P<0.05) up-regulated when compared to heterophils isolated from chickens fed a control ration as determined with an array of functional assays. Phagocytosis, bactericidal killing, and oxidative burst were significantly increased in heterophils isolated from chickens fed the purified beta-glucan ration (P<0.05). To our knowledge, this is the first report of a purified beta-glucan feed additive significantly decreasing the incidence of SE organ invasion in immature chickens and up-regulating the functional abilities of heterophils isolated from immature chickens against an invading pathogen, SE.     

Biofilm formation by multidrug-resistant Salmonella enterica serotype typhimurium phage type DT104 and other pathogens

The biofilm-forming capability of Salmonella enterica serotypes Typhimurium and Heidelberg, Pseudomonas aeruginosa, Listeria monocytogenes, Escherichia coli O157:H7, Klebsiella pneumoniae, and Acinetobacter baumannii isolated from humans, animal farms, and retail meat products was evaluated by using a microplate assay. The tested bacterial species showed interstrain variation in their capabilities to form biofilms. Strong biofilm-forming strains of S. enterica serotypes, E. coli O157: H7, P. aeruginosa, K. pneumoniae, and A. baumannii were resistant to at least four of the tested antibiotics. To understand their potential in forming biofilms in food-processing environments, the strong biofilm formers grown in beef, turkey, and lettuce broths were further investigated on stainless steel and glass surfaces. Among the tested strains, Salmonella Typhimurium phage type DT104 (Salmonella Typhimurium DT104) isolated from retail beef formed the strongest biofilm on stainless steel and glass in beef and turkey broths. K. pneumoniae, L. monocytogenes, and P. aeruginosa were also able to form strong biofilms on the tested surface materials. Salmonella Typhimurium DT104 developed a biofilm on stainless steel in beef and turkey broths through (i) initial attachment to the surface, (ii) formation of microcolonies, and (iii) biofilm maturation. These findings indicated that Salmonella Typhimurium DT104 alongwith other bacterial pathogens could be a source of cross-contamination during handling and processing of food.     

Effect of temperature, pH, and water activity on biofilm formation by Salmonella enterica enteritidis PT4 on stainless steel surfaces as indicated by the bead vortexing method and conductance measurements

An assay was developed in an effort to elucidate the effect of important environmental parameters (temperature, pH, and water activity [aw]) on Salmonella Enteritidis biofilm formation on stainless steel surfaces. To achieve this, a modified microbiological technique used for biofilm studying (the bead vortexing method) and a rapid method based on conductivity measurements were used. The ability of the microorganism to generate biofilm on the stainless surfaces was studied at three temperatures (5, 20, and 37 degrees C), four pH values (4.5, 5.5, 6.5, and 7.4), and four aw values (0.5, 1.5, 5.5, and 10.5% NaCl). Results obtained by the bead vortexing method show that maximum numbers of adherent bacteria per square centimeter (106 CFU/cm2) were attained in 6 days at 20 degrees C. Biofilm formation after 7 days of incubation at 20 degrees C was found to be independent of the pH value. In addition, the high concentration of sodium chloride (10.5% NaCl, aw = 0.94) clearly inhibited the adherence of cells to the coupons. Conductance measurements were used as a supplementary tool to measure indirectly the attachment and biofilm formation of bacterial cells on stainless steel surfaces via their metabolic activity (i.e., changes in the conductance of the growth medium due to microbial growth or metabolism). Results obtained by conductance measurements corresponded well to those of the bead vortexing method. Furthermore, we were able to detect cells that remained attached on the metal surfaces even after vortexing via their metabolic activity. The results, except for demonstrating environmental-dependent Salmonella Enteritidis biofilm formation, indicated that traditional vortexing with beads did not remove completely biofilm cells from stainless steel; hence, conductance measurements seem to provide a more sensitive test capable to detect down to one single viable organism.