Assessment of the antifungal efficacy of whey fermentate alone or in combination with citrus extract to control Aspergillus flavus mold in semi-moist pet food for dogs.

Semi-moist pet foods contain moisture levels ranging from 15 to 40%, making them ideal for mold growth and mycotoxin production. To control spoilage, synthetic mold inhibitors such as potassium sorbate have been used, but consumers prefer "natural" preservatives. Whey fermentate (WPF) is an efficient antifungal, but it requires large doses. Therefore, the objective of this study was to determine the antifungal effect of WPF alone or in combination with citrus extract oil (CEX) against Aspergillus flavus in semi-moist pet food. Nutritionally complete semi-moist pet foods were produced with WPF alone [0.25, 0.5, and 1.0% (w/w)] and in combination with CEX; 0.25% WPF+ 0.015% CEX, 0.25% WFP+ 0.15% CEX, 0.5% WPF+ 0.015% CEX, and 0.5% WFP+ 0.15% CEX (w/w). The negative control (NC) contained no antifungal additive and the positive control (PC) had potassium sorbate (0.1% w/w). The semi-moist pet food was thermally formed and was cut into 3 cm × 3 cm square pieces. Individual food pieces were inoculated with 0.1 mL of Aspergillus flavus (ATCC 204304) to achieve a final concentration of ~5.0 log CFU/piece. The inoculated pieces were individually incubated at 25°C. Fungal analysis was performed on day 3, 6, 9, 12, 15, 18, 21, 24, and 28 by surface plating on Potato Dextrose Agar (PDA) followed by incubation at 25°C for 72 h. The total log reductions were calculated by subtracting the initial inoculum from the final log counts on day 28. Higher log reductions of Aspergillus flavus (p < 0.05) were observed when WPF at 0.25 and 0.5% was combined with 0.15% CEX compared to when 0.015% CEX was used individually. All treatments were different from the NC (p < 0.05). Citrus extract at 0.15% potentiated the antifungal effect of WPF at 0.5% to give a similar log reduction (p > 0.05) to WPF at 1.0% in the food. In conclusion, CEX potentiated the antifungal efficacy and reduced the dose of WPF required to control Aspergillus flavus in semi-moist dog food.

Effects of liquid smoke preparations on shelf life and growth of wild type mold and Aspergillus flavus in a model semi moist pet food.

Liquid smoke is a naturally derived flavor component and preservative with known antimicrobial properties. To our knowledge, there is a paucity of information on antifungal potential of liquid smoke against toxigenic fungi like Aspergillus flavus that produce mycotoxins in human and pet foods. Semi-moist pet food with high moisture content (20-30%) is susceptible to mold contamination and requires intervention. The objectives of this study were to determine the effects of liquid smoke preparations on the growth of wild-type mold and A. flavus in semi-moist pet food. Semi-moist pet food was formulated with eight different liquid smoke preparations (S1-S8) containing varying amounts of organic acids, phenol and carbonyl compounds (ranging from low to high) at 0% (untreated), 0.5, 1, 2, and 4% (w/w). A positive control consisted of 0.2% potassium sorbate known to inhibit mold growth. Shelf life was estimated by storing the samples at 28°C and 65-70% RH over 30 days and recording the number of days until the appearance of visible wild-type mold. In another experiment, samples were spot inoculated with A. flavus (∼10,000 CFU/mL), incubated at 25°C, and analyzed for fungal growth at sampling intervals of 2 days over a 35-day period. Liquid smoke at 0.5, 1, 2, and 4% extended the shelf life of samples on an average by a total of 11.6, 12.5, 17.2, and 24.1 days when compared to the untreated samples (7.7 days). The smoke preparations Cloud S-C100 (S3) and Code-10 (S6) (high carbonyl, medium/low phenol) were the most effective (P < 0.05) in prolonging the number of days to visible mold growth (26-28 days). In the challenge study with A. flavus, Cloud S-C100 (S3), Cloud S-AC15 (S8) (high to medium carbonyl, low phenol), and Code 10 (S6) (base smoke) reduced (P < 0.05) mold counts by 1.0, 1.7, and 2.5 logs when compared to the untreated samples at 1, 2, and 4%, respectively. Addition of smoke at 0.5% did not reduce mold counts. The carbonyl preparations of liquid smoke were the most effective at enhancing shelf life of semi-moist pet food, and at inhibiting A. flavus growth.

Application of Acidulants to Control Salmonella spp. in Rendered Animal Fats and Oils with Different Levels of Unsaturation.

Salmonella-contaminated pet foods could potentially become a source of human salmonellosis. This study evaluated the survival of Salmonella without and with the addition of acidulants in different fat types (chicken fat (CF), canola oil (CO), Menhaden fish oil (FO), lard (La), and tallow (Ta)) commonly used to coat dry pet food kibbles. The minimum inhibitory concentration (MIC) of individual acidulants and the combination were determined using the broth microdilution method. Autoclave-sterilized rendered fats were treated with pre-determined concentrations of antimicrobial acidulants (0.5% sodium bisulfate (SBS), 0.5% phosphoric acid (PA), 0.25% lactic acid (LA), etc.) and incubated overnight at 45 °C. The treated fats were inoculated with approximately eight logs of a Salmonella cocktail. Microbiological analyses were conducted separately for the fat-phase and water-phase at predetermined time intervals (0, 2, 6, 12, and 24 h) by plating them onto TSA plates. After incubating at 37 °C for 24 h, the plate count results were expressed as log CFU/mL. The MIC of SBS was 0.3125%, and of PA and LA were both 0.1953% against cocktail Salmonella serotypes. We observed a possible synergistic effect when SBS and organic acid were combined. All the acidulant tested at targeted concentrations individually as well as in combination with organic acids were highly effective against Salmonella spp. (non-detectable within 2 h) across different fat types. A potent anti-bactericidal effect leading to non-detectable Salmonella immediately (<1 h) at 45 °C was observed in the aqueous phase of the fish oil system, even without the addition of acidulants. These findings are significant for the dry pet food industries, where potential post-processing contamination of Salmonella could be controlled by treating fats and oils with acidulants.

Use of Organic Acid Mixtures Containing 2-Hydroxy-4-(Methylthio) Butanoic Acid (HMTBa) to Mitigate Salmonella enterica, Shiga Toxin-Producing Escherichia coli (STEC) and Aspergillus flavus in Pet Food Kibbles.

Post-processing operations of extruded pet food kibbles involve coating the product with fats and flavorings. These processes increase the risk for cross-contamination with food-borne pathogens such as Salmonella and Shiga toxin-producing Escherichia coli (STEC), and mycotoxin-producing molds such as Aspergillus spp. after the thermal kill step. In this study, the antimicrobial effects of two types of organic acid mixtures containing 2-hydroxy-4-(methylthio) butanoic acid (HMTBa), Activate DA™ and Activate US WD-MAX™, against Salmonella enterica, STEC and Aspergillus flavus when used as a coating on pet food kibbles were evaluated. Using canola oil and dry dog digest as fat and flavor coatings, the efficacy of Activate DA (HMTBa + fumaric acid + benzoic acid) at 0%, 1% and 2%, and Activate US WD-MAX (HMTBa + lactic acid + phosphoric acid) at 0%, 0.5% and 1% was tested on kibbles inoculated with a cocktail of S. enterica serovars (Enteritidis, Heidelberg and Typhimurium) or Shiga toxin-producing E. coli (STEC) serovars (O121, and O26) at 37 °C for 0, 12, 24, 48, 72 h, 30 and 60 days. Similarly, their efficacy was tested against A. flavus at 25 °C for 0, 3, 7, 14, 21, 28 and 35 days. Activate DA at 2% and Activate US WD-MAX at 1% reduced Salmonella counts by ~3 logs after 12 h and 4-4.6 logs after 24 h. Similarly, STEC counts were reduced by ~2 logs and 3 logs after 12 h and 24 h, respectively. Levels of A. flavus did not vary up to 7 days, and afterwards started to decline by >2 logs in 14 days, and up to 3.8-log reduction in 28 days for Activate DA and Activate US WD-MAX at 2% and 1%, respectively. The results suggest that the use of these organic acid mixtures containing HMTBa during kibble coating may mitigate post-processing enteric pathogen and mold contamination in pet food kibbles, with Activate US WD-MAX being effective at a lower concentration (0.5-1%) compared to Activate DA.

Mitigation of Salmonella on Food Contact Surfaces by Using Organic Acid Mixtures Containing 2-Hydroxy-4-(methylthio) Butanoic Acid (HMTBa).

Contaminated surfaces can transmit pathogens to food in industrial and domestic food-handling environments. Exposure to pathogens on food contact surfaces may take place via the cross-contamination of pathogens during postprocessing activities. Formaldehyde-based commercial sanitizers in recent years are less commonly being used within food manufacturing facilities due to consumer perception and labeling concerns. There is interest in investigating clean-label, food-safe components for use on food contact surfaces to mitigate contamination from pathogenic bacteria, including Salmonella. In this study, the antimicrobial effects of two types of organic acid mixtures containing 2-hydroxy-4-(methylthio) butanoic acid (HMTBa), Activate DA™ and Activate US WD-MAX™, against Salmonella when applied onto various food contact surfaces were evaluated. The efficacy of Activate DA (HMTBa + fumaric acid + benzoic acid) at 1% and 2% and Activate US WD-MAX (HMTBa + lactic acid + phosphoric acid) at 0.5% and 1% against Salmonella enterica (serovars Enteritidis, Heidelberg, and Typhimurium) were evaluated on six different material surfaces: plastic (bucket elevator and tote bag), rubber (bucket elevator belt and automobile tire), stainless steel, and concrete. There was a significant difference in the Salmonella log reduction on the material surfaces due to the organic acid treatments when compared to the untreated surfaces. The type of material surface also had an effect on the log reductions obtained. Stainless steel and plastic (tote) had the highest Salmonella log reductions (3-3.5 logs), while plastic (bucket elevator) and rubber (tire) had the lowest log reductions (1-1.7 logs) after treatment with Activate US WD-MAX. For Activate DA, the lowest log reductions (~1.6 logs) were observed for plastic (bucket elevator) and rubber (tire), and the highest reductions were observed for plastic (tote), stainless steel, and concrete (2.8-3.2 logs). Overall, the results suggested that Activate DA at 2% and Activate US WD-MAX at 1% are potentially effective at reducing Salmonella counts on food contact surfaces by 1.6-3.5 logs.

Method validation for the recovery of the porcine respiratory and reproductive syndrome virus, a potential SARS-CoV-2 surrogate, from stainless steel.

Virus survival on fomites may represent a vehicle for transmission to humans. This study was conducted to optimize and validate a recovery method for the porcine respiratory and reproductive syndrome virus (PRRSV), a potential SARS-CoV-2 surrogate, from stainless steel. Coupons (1.5 × 1.5 cm) inoculated with ca. 7 logs TCID50 of PRRSV were dried for 15 min at room temperature, followed by incubation at 4°C and 35% relative humidity. After 1 h and 24 h, the coupons were processed by four different methods: vortex in DMEM media, vortex in DMEM media with beads, vortex in elution buffer, and shake in elution buffer. The rinsates were processed for titration using the TCID50 method in the MARC-145 cell line. All four methods were equally effective to recover the virus from the soiled SS surfaces (> 79% recovery). The amount of infectious virus recovered after 24 h was similar (P > 0.05) to that recovered after 1 h, indicating that the virus was stable at 4°C for up to 24 h. Using an elution buffer followed by shaking was the least labor-intensive and most economical method. Therefore, this method will be used for future experiments on PRRSV survival and transfer from food-contact surfaces.

Effects of antimicrobial addition on lipid oxidation of rendered chicken fat.

This study evaluated the effects of antimicrobial acidulant addition on lipid oxidation of rendered chicken fat. Chicken fat was untreated (control) or treated with either sodium bisulfate (SBS) or lactic acid (LA) at 0.5% w/w and incubated for 6 wk at 40 °C. Peroxide value (PV), p-anisidine value (AV), and free fatty acid (FFA) levels were measured at days 0 (D0), 1(D1), 3 (D3), 5 (D5), and 7 (D7), and weeks 2 (W2), 3 (W3), 4 (W4), 5 (W5), and 6 (W6). The FFA level of untreated-control fat was ~7% and remained consistent throughout the incubation until W6 (~8.5%; P < 0.05). The FFA values in SBS-treated fat were constant (range 7.25%-8.30%) throughout the incubation, whereas the FFA in LA-treated fat peaked at W5 (9.3%; P < 0.05). For the control fat, PVs were between 0.56 and 0.67 meq/100 g until W1 then declined. For the SBS-treated fat, the PVs remained low and similar to the control with the exception of a slight increase on W4 to 0.38 meqv/100 g (P < 0.05). In the LA-treated fat, the PV was greater than (P < 0.05) the control from W1 and increased to a peak on W5 (2.52 meq/100 g). The AV of control fat averaged 2.12 at D0 and increased through W2. In control and LA-treated fat, the AV values declined slightly thereafter, whereas SBS-treated fat increased (P < 0.05) to 10.28 on W5. This study indicates that when included at antimicrobial effective levels, LA may reduce the shelf-life of chicken fat, but SBS had a minimal effect over 6 wk of storage.

Temperature-Dependent Antimicrobial Activity of Menhaden Fish Oil In Vitro and on Pet Food Kibbles against Salmonella.

ABSTRACT: Fish oil inclusion into a dry pet food provides a source of long-chain omega-3 fatty acids. Polyunsaturated fatty acids in fish oil have antibacterial activity against various foodborne pathogens, such as Salmonella and pathogenic Escherichia coli. The purpose of this study was to determine the effect of temperature applied to dry pet food kibbles on the antimicrobial activity of menhaden fish oil against Salmonella spp. Sterile menhaden oil was inoculated with ∼8 log of a Salmonella cocktail (∼3% moisture; Salmonella Enteritidis, Salmonella Heidelberg, and Salmonella Typhimurium) and incubated at 25, 37, and 45°C. Microbiological evaluation of the water phase was done after 2 h on tryptic soy agar. Sterile kibbles were coated with fish oil (7.0%, w/w). Canola oil coating was kept as a control. One hour after coating, the kibbles were inoculated with ∼9 log of Salmonella and incubated at the respective temperature. The microbiological evaluation was conducted at 0, 2, 6, 12, and 24 h. The oil phase of the fish oil system was negative for Salmonella after 2 h of incubation and confirmed by enrichment and PCR. From the water phase, 8.1 and 7.3 log were recovered at 25 and 37°C, respectively, and no Salmonella was detected at 45°C. On the kibble, menhaden oil had higher antimicrobial (P ≤ 0.05) activity after 12 h at 25°C and throughout the experiment at 37°C. At 45°C, the fish oil had a superior antimicrobial activity against the Salmonella cocktail after 2 h. When the fish oil alone was compared at different temperatures, a higher antimicrobial activity was observed at 37 and 45°C across all time points. The results indicate antimicrobial activity of menhaden oil increases with temperature. This is an important finding to the pet food industry: a higher fat holding temperature (∼45°C) and the application process may help mitigate Salmonella on extruded kibbles.

Significance of Sodium Bisulfate (SBS) Tempering in Reducing the Escherichia coli O121 and O26 Load of Wheat and Its Effects on Wheat Flour Quality.

The occurrence of recalls involving pathogenic Escherichia coli-contaminated wheat flours show the need for incorporating antimicrobial interventions in wheat milling. The objectives of this study were to assess the efficacy of sodium bisulfate (SBS) tempering in reducing E. coli O121 (ATCC 2219) and O26 (ATCC 2196) wheat load and to evaluate the impact of effective (≥3.0 log reductions) SBS treatments on wheat flour quality. Wheat grains were inoculated with E. coli (~6 log CFU/g) and tempered (17% moisture, 24 h) using the following SBS concentrations (%wheat basis): 0, 0.5, 0.75, 1.0, 1.25, and 1.5% SBS. Reductions in E. coli O121 and O26 wheat load at different time intervals (0.5, 2, 6, 12, 18, and 24 h) during tempering were evaluated. The addition of SBS during tempering resulted in E. coli (O121 and O26) log reductions of 2.0 (0.5% SBS) to >4.0 logs (1.5% SBS) (p ≤ 0.05). SBS tempering (1.25 and 1.5% SBS) produced acidic wheat flours (pH = 4.51-4.60) but had comparable wheat flour properties in terms of composition, dough, and bread-making properties relative to the control (0% SBS). SBS tempering reduced the E. coli O121 and O26 load of wheat after tempering with minimal effects on wheat flour quality.

Survival of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and Herpes Simplex Virus 1 (HSV-1) on Foods Stored at Refrigerated Temperature.

Outbreaks of coronavirus infectious disease 2019 (COVID-19) in meat processing plants and media reports of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection on foods have raised concerns of a public health risk from contaminated foods. We used herpes simplex virus 1, a non-Biosafety Level 3 (non-BSL3) enveloped virus, as a surrogate to develop and validate methods before assessing the survival of infectious SARS-CoV-2 on foods. Several food types, including chicken, seafood, and produce, were held at 4 °C and assessed for infectious virus survival (herpes simplex virus 1 (HSV-1) and SARS-CoV-2) at 0 h, 1 h, and 24 h post-inoculation (hpi) by plaque assay. At all three time points, recovery of SARS-CoV-2 was similar from chicken, salmon, shrimp, and spinach, ranging from 3.4 to 4.3 log PFU/mL. However, initial (0 h) virus recovery from apples and mushrooms was significantly lower than that from poultry and seafood, and infectious virus decreased over time, with recovery from mushrooms becoming undetectable by 24 hpi. Comparing infectious virus titers with viral genome copies confirmed that PCR-based tests only indicate presence of viral nucleic acid, which does not necessarily correlate with the quantity of infectious virus. The survival and high recovery of SARS-CoV-2 on certain foods highlight the importance of safe food handling practices in mitigating any public health concerns related to potentially contaminated foods.

Sustainability and Pet Food: Is There a Role for Veterinarians?

Sustainability has become a watchword for a wide array of resource-intensive goods and services. This is promulgated by an increasing global population and concerns that natural resources and a hospitable climate will not be preserved for future generations. Life-cycle analysis is a tool that provides a framework to determine the magnitude products contribute to carbon emissions and depletion of natural resources. In this review, published research has been summarized to provide an overview of the impacts that pet food production and pet ownership have on the environment and the prospective role of veterinary practitioners in advocating for sustainability.

A Comparison of Salmonella Survival and Detection Using an Enrichment Technique in Dry- and Wet-Inoculated Rendered Chicken Fat Treated with Sodium Bisulfate.

ABSTRACT: The differences in the recovery of Salmonella from rendered chicken fat treated with sodium bisulfate (SBS) when inoculated with a dry versus wet inoculum were evaluated. Food-grade rendered chicken fat was inoculated with a dry inoculum and a wet inoculum containing a cocktail of Salmonella serovars (Enteritidis, Heidelberg, and Typhimurium). In addition, the effect of an antimicrobial treatment (SBS) against Salmonella in both the aqueous phase and fat phase of the chicken fat was evaluated. The untreated control samples in the aqueous phase had a consistent level of Salmonella (∼7 log) when both the dry and wet inocula were used. In the SBS-treated aqueous phase, Salmonella pathogens were not detectable after 6 h when the wet inoculum was used; when the dry inoculum was used, Salmonella pathogens were not detectable at 24 h. Salmonella pathogens were detected for up to 6 h in the SBS-treated fat phase when the dry inoculum was used compared with 2 h with the wet inoculum. The 24-h fat samples that failed to show growth on Trypticase soy agar were enriched for Salmonella isolation, followed by confirmation by PCR using primers for the invA gene. SBS-treated and control samples from the dry-inoculated rendered chicken fat and the inoculated control from the wet-inoculated rendered chicken fat tested positive for Salmonella. However, the SBS-treated sample from the wet-inoculated fat was negative for Salmonella. The use of dry SBS powder against dry Salmonella inoculum in the fat matrix caused only ∼2.8-log reduction after 24 h compared with ∼2.2-log reduction in the positive control. However, the recovery of Salmonella from untreated control fat was lower and was not different (P > 0.05) from that recovered from the SBS-treated fat. The results suggest that viable but nonculturable states of Salmonella may develop in rendered chicken fat or that injured cells may be present, which indicates that testing should include an enrichment and appropriate molecular confirmation instead of agar plating alone.

Use of Medium Chain Fatty Acids To Mitigate Salmonella Typhimurium (ATCC 14028) on Dry Pet Food Kibbles.

ABSTRACT: This study evaluated the antimicrobial effects of medium chain fatty acids (MCFAs) against Salmonella Typhimurium (ATCC 14028) when used on dry dog food kibbles. The MIC of three MCFAs, caproic (C6), caprylic (C8), and capric (C10), was determined using the broth micro- and macrodilution assay technique. Using canola oil as a fat coating, the efficacy of each MCFA was then tested on dry dog food kibbles at 37°C for up to 5 h. The MIC was found to be 0.3125, 0.3125, and 0.625% for C6, C8, and C10, respectively. When the MCFAs were tested on fat-coated dry kibbles, all three MCFAs reduced (P ≤ 0.05) Salmonella levels by >4.5 log after 5 h when the Salmonella recovery from a no-treatment control was ∼6.4 log. At each evaluation time point, the three treatments were effective in reducing (P ≤ 0.05) Salmonella loads. No countable colonies of Salmonella were detected at 4 h when the combination of C6+C8 was used on the kibbles (P ≤ 0.05), whereas with the C6+C10 combination, the Salmonella colonies were not detectable between 2 and 4 h after treatments (P ≤ 0.05). Different combinations of C8 and C10 caused Salmonella to drop to a nondetectable limit (1 CFU/g) between 1 and 5 h after treatment (P ≤ 0.05). This study suggests that the use of MCFAs during kibble coating may mitigate postprocessing Salmonella recontamination on dry dog food kibbles.

Assessing the Efficacy of Sodium Bisulfate and Organic Acid Treatments for Control of Salmonella Typhimurium in Rendered Chicken Fat Applied to Pet Foods.

This study was conducted to evaluate the effects of sodium bisulfate (SBS), lactic acid (LA), phosphoric acid (PA), and combinations of organic acids with SBS on Salmonella in rendered chicken fat and in water. The MICs of the antimicrobials individually and in combination were determined. Efficacies of the antimicrobials against Salmonella were tested in both media. The MICs of SBS, LA, and PA were 0.5, 0.5, and 0.25%, respectively. At the given concentrations in the water phase, 0.5% SBS was more effective (P < 0.05; 2.7-log reduction) than LA and PA at 0 h. SBS and LA were more effective (P < 0.05) than PA with >4-log reductions at 2 h and complete kill at 6 h. After 24 h, each of the chemicals completely eliminated the Salmonella. However, because of low recovery in the fat phase, Salmonella was not detected after 12 h and all three chemicals effectively reduced (P < 0.05) Salmonella at 6 h compared with the control. When combinations were used in the water phase, SBS plus butyric acid decreased (P < 0.05) Salmonella by >5.5 log CFU/mL after 12 h. The SBS+LA combinations were effective (P < 0.05) after 2 h. The combinations of SBS+PA resulted in ∼3.5-log reductions in Salmonella (P < 0.05) after 6 h. In the fat phase, except for the SBS+PA combination, Salmonella reduction was not different from that for the positive control. When SBS was combined with organic acids, Salmonella inhibition was achieved at a lower SBS concentration, indicating a possibly synergistic effect of these chemicals. These results suggest that inclusion of SBS or LA at 0.5% individually or a combination of SBS with organic acids could reduce Salmonella in rendered chicken fat contaminated by residual water encountered during storage and transport.

Effect of Chlorine-Induced Sublethal Oxidative Stress on the Biofilm-Forming Ability of Salmonella at Different Temperatures, Nutrient Conditions, and Substrates.

The present study was conducted to evaluate the effect of chlorine-induced oxidative stress on biofilm formation by various Salmonella strains on polystyrene and stainless steel (SS) surfaces at three temperatures (30, 25 [room temperature], and 4°C) in tryptic soy broth (TSB) and 1/10 TSB. Fifteen Salmonella strains (six serotypes) were exposed to a sublethal chlorine concentration (150 ppm of total chlorine) in TSB for 2 h at the predetermined temperatures. The biofilm-forming ability of the Salmonella strains was determined in 96-well polystyrene microtiter plates by using a crystal violet staining method and on SS coupons in 24-well tissue culture plates. All tested strains of Salmonella produced biofilms on both surfaces tested at room temperature and at 30°C. Of the 15 strains tested, none (chlorine stressed and nonstressed) formed biofilm at 4°C. At 30°C, Salmonella Heidelberg (ID 72), Salmonella Newport (ID 107), and Salmonella Typhimurium (ATCC 14028) formed more biofilm than did their respective nonstressed controls on polystyrene ( P ≤ 0.05). At room temperature, only stressed Salmonella Reading (ID 115) in 1/10 TSB had significantly more biofilm formation than did the nonstressed control cells ( P ≤ 0.05). Salmonella strains formed more biofilm in nutrient-deficient medium (1/10 TSB) than in full-strength TSB. At 25°C, chlorine-stressed Salmonella Heidelberg (ATCC 8326) and Salmonella Enteritidis (ATCC 4931) formed stronger biofilms on SS coupons ( P ≤ 0.05) than did the nonstressed cells. These findings suggest that certain strains of Salmonella can produce significantly stronger biofilms on plastic and SS upon exposure to sublethal chlorine.