Antimicrobial Tolerance in Salmonella: Contributions to Survival and Persistence in Processing Environments.

Salmonella remains a top bacterial pathogen implicated in several food-borne outbreaks, despite the use of antimicrobials and sanitizers during production and processing. While these chemicals have been effective, Salmonella has shown the ability to survive and persist in poultry processing environments. This can be credited to its microbial ability to adapt and develop/acquire tolerance and/or resistance to different antimicrobial agents including oxidizers, acids (organic and inorganic), phenols, and surfactants. Moreover, there are several factors in processing environments that can limit the efficacy of these antimicrobials, thus allowing survival and persistence. This mini-review examines the antimicrobial activity of common disinfectants/sanitizers used in poultry processing environments and the ability of Salmonella to respond with innate or acquired tolerance and survive exposure to persists in such environments. Instead of relying on a single antimicrobial agent, the right combination of different disinfectants needs to be developed to target multiple pathways within Salmonella.

Low-Level Tolerance to Antibiotic Trimethoprim in QAC-Adapted Subpopulations of Listeria monocytogenes.

Between January and July 2021, there were as many as 30 recalls in the U.S. due to potential Listeria monocytogenes contamination from a variety of food products including muffins, kimchi, chicken salad, ready-to-eat chicken, smoked fish, mushrooms, queso fresco cheese, ice cream, turkey sandwiches, squash, and other foods. A contaminated food chain can serve as a potential vehicle for transmitting antibiotic resistant bacteria since there is a slow emergence of multi-drug antibiotic resistance in L. monocytogenes. Biocides are essential for safe food processing, but they may also induce unintended selective pressure at sublethal doses for the expression of antibiotic resistance in L. monocytogenes. To better understand the sources of such slow emergence of antibiotic resistance through biocide residues present in the food environments, we are working on the role of sublethal doses of commonly used biocides in defined broth and water models for understanding L. monocytogenes adaptation. We recently published the development of low-level tolerance to fluoroquinolone antibiotic ciprofloxacin in quaternary ammonium compound (QAC) adapted subpopulations of L. monocytogenes (Microorganisms 9, 1052). Of the six different antibiotics tested to determine heterologous stress adaptation in eight strains of L. monocytogenes, trimethoprim was the second one that exhibited low-level tolerance development after continuous exposure (by three approaches) to sublethal concentrations of QAC against actively growing planktonic cells of L. monocytogenes. When adapted to daily cycles of fixed or gradually increasing sublethal concentrations of QAC, we observed three main findings in eight L. monocytogenes strains against trimethoprim: (a) 3 of the 8 strains exhibited significant increase in short-range minimum inhibitory concentration (MIC) of trimethoprim by 1.7 to 2.5 fold in QAC-adapted subpopulations compared to non-adapted cells (p < 0.05); (b) 2 of the 8 strains exhibited significant increase in growth rate in trimethoprim (optical density (OD) by 600 nm at 12 h) by 1.4 to 4.8 fold in QAC-adapted subpopulations compared to non-adapted cells (p < 0.05); and (c) 5 of the 8 strains yielded significantly higher survival by 1.3-to-3.1 log CFU/mL in trimethoprim in QAC-adapted subpopulations compared to the non-adapted control (p < 0.05). However, for 3/8 strains of L. monocytogenes, there was no increase in the survival of QAC-adapted subpopulations compared to non-adapted control in trimethoprim. These findings suggest the potential formation of low-level trimethoprim tolerant subpopulations in some L. monocytogenes strains where QAC may be used widely. These experimental models are useful in developing early detection methods for tracking the slow emergence of antibiotic tolerant strains through food chain. Also, these findings are useful in understanding the predisposing conditions leading to slow emergence of antibiotic resistant strains of L. monocytogenes in various food production and food processing environments.

Low-Level Tolerance to Fluoroquinolone Antibiotic Ciprofloxacin in QAC-Adapted Subpopulations of Listeria monocytogenes.

There was a development of low-level tolerance to fluoroquinolone antibiotic ciprofloxacin in Listeria monocytogenes after sublethal adaptation to quaternary ammonium compound (QAC). Using eight L. monocytogenes strains, we determined the changes in short-range MIC, growth rate, and survival for heterologous stress response to ciprofloxacin, after sublethal exposure to daily cycles of fixed or gradually increasing concentration of QAC. Three main findings were observed. (1) MIC increase-QAC-adapted subpopulations exhibited a significant increase in short-range MIC of ciprofloxacin, by 1.5 to 2.9 fold, as compared to non-adapted control for 4/8 strains (p < 0.05). (2) Growth rate increase-QAC-adapted subpopulations exhibited significant 2.1- to 6.8- fold increase in growth rate (OD600 at 10 h) in ciprofloxacin-containing broth, as compared to non-adapted control for 5/8 strains (p < 0.05). (3) Survival increase-QAC-adapted subpopulations of L. monocytogenes yielded significantly higher survival in ciprofloxacin-containing agar by 2.2 to 4.3 log CFU/mL for 4/8 strains, as compared to non-adapted control (p ˂ 0.05). However, for other 4/8 strains of L. monocytogenes, there was no increase in survival of QAC-adapted subpopulations, as compared to non-adapted control in ciprofloxacin. These findings suggest the potential formation of low-level ciprofloxacin-tolerant subpopulations in some L. monocytogenes strains when exposed to residual QAC concentrations (where QAC might be used widely) and such cells if not inactivated might create food safety risk.

Decreased biofilm formation by planktonic cells of Listeria monocytogenes in the presence of sodium hypochlorite.

The objective of this study was to determine if the adaptation at planktonic stage to subinhibitory concentrations (SIC) of sodium hypochlorite (NaOCl) could modulate the biofilm forming ability of five Listeria monocytogenes strains V7, Scott A, FSL-N1-227, FSL F6-154 and ATCC 19116 representing serotypes 1/2a, 4b and 4c. Biofilm formation by NaOCl nonadapted and adapted L. monocytogenes planktonic cells was measured in the presence or absence of SIC of NaOCl. The biofilm formation ability of NaOCl nonadapted and adapted L. monocyotgenes planktonic cells was reduced only in the presence of NaOCl (P < 0.05). Scanning electron microscopy revealed that the continuous exposure of NaOCl induced morphological changes in the L. monocytogenes biofilm structure and reduced its attachment to polystyrene surface. The qRT-PCR results also showed that the subinhibitory NaOCl reduced biofilm formation related gene expression such as motility and quorum sensing signals (P < 0.05). These findings indicate that subinhibitory NaOCl can reduce the ability of L. monocytogenes planktonic cells to form biofilms on polystyrene surface.

Rugose Morphotype in Salmonella Typhimurium and Salmonella Heidelberg Induced by Sequential Exposure to Subinhibitory Sodium Hypochlorite Aids in Biofilm Tolerance to Lethal Sodium Hypochlorite on Polystyrene and Stainless Steel Surfaces.

Salmonella biofilms act as a continuous source for cross-contamination in the food processing environments. In this study, a stable rugose morphotype of Salmonella was first induced by sequential exposure to subinhibitory concentrations (SICs) of sodium hypochlorite (NaOCl) (ranging from 50 to 300 ppm over 18-day period) in tryptic soy broth. Then, rugose and smooth morphotypes of Salmonella Typhimurium ATCC 14028 and Salmonella Heidelberg ATCC 8326 were characterized for biofilm forming abilities on polystyrene and stainless steel surfaces. Rugose morphotype of both ATCC 14028 and ATCC 8326 exhibited higher Exopolysaccharide (EPS) formation than smooth morphotype (p ≤ 0.05). Also, the SICs of NaOCl (200 or 300 ppm in broth model) increased the biofilm formation ability of rugose morphotype of ATCC 8326 (p ≤ 0.05) but decreased that of ATCC 14028. The 2-day-old Salmonella biofilms were treated with biocidal concentrations of 50, 100, or 200 ppm NaOCl (pH 6.15) in water for 5, 10, or 20 min at room temperature. The biofilm reduction in CFU/cm2 for the rugose was lower than the smooth morphotype on both surfaces (p ≤ 0.05) by lethal NaOCl in water. Scanning electron micrographs on both polystyrene and stainless steel surfaces demonstrated that the rugose morphotype produced a denser biofilm than the smooth morphotype. Transmission electron micrographs revealed the cell wall roughness in rugose morphotype, which may help in tolerance to NaOCl. The gene expression data indicate that the expression of biofilm regulator (csgD), curli (csgA, csgB, and csgC), and cellulose (bcsE) was significantly increased in rugose morphotype when induced by sequential exposure of NaOCl SICs. These findings reveal that the rugose morphotype of S. Typhimurium and S. Heidelberg produced significantly denser biofilm on food contact surfaces, which also increased with sequential exposure to SICs of NaOCl in the case of S. Heidelberg, and these biofilms were more tolerant to biocidal NaOCl concentrations commonly used in the food processing plants.

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.

Listeria monocytogenes Response to Sublethal Chlorine Induced Oxidative Stress on Homologous and Heterologous Stress Adaptation.

The objective of this study was to determine the effect of chlorine induced sublethal oxidative stress against homologous and heterologous stress adaptations in five Listeria monocytogenes (Lm) strains. Lm cells were exposed to gradually increasing sublethal concentrations of total chlorine/day: 250 ppm (day 1), 270 ppm (day 2), 290 ppm (day 3), 310 ppm (day 4), 330 ppm (day 5), 350 ppm (day 6), and 375 ppm (day 7) in tryptic soy broth (TSB). Changes in minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of Lm cells exposed to chlorine and control (non-adapted cells) were determined by the macro-dilution method. Chlorine-adapted Lm cells were also evaluated for changes in antibiotic resistance using the Kirby-Bauer disk diffusion and MIC double dilution assay as per the Clinical and Laboratory Standards Institute (CLSI, 2016) guidelines. In four Lm strains (Scott A, V7, FSL-N1-227 and FSL-F6-154) after adapted to sublethal chlorine, the MIC (600 ppm) and MBC (700 ppm) values of chlorine were slightly higher as compared to control (500 ppm MIC, and 600 ppm MBC). The Kirby-Bauer and MIC double dilution assays showed some significant changes in antibiotic susceptibility patterns for antibiotics such as streptomycin, gentamicin and ceftriaxone (p < 0.05). However, the changes in zones of inhibition and MIC values to all antibiotics tested for the chlorine-adapted and non-adapted (control) Lm cells were still within the susceptible range. Transmission electron microscopy studies showed that changes in cell wall and membrane integrity resulting, from the elongation of cells, may contribute to the possible routes of its increase in tolerance to chlorine and selective antibiotics. These findings indicate that the continuous exposure of Lm cells to chlorine may lead to significant changes in homologs and heterologous stress adaptation.

Salmonella enterica growth and biofilm formation in flesh and peel cantaloupe extracts on four food-contact surfaces.

Salmonella enterica is responsible for the highest number of foodborne disease outbreaks pertaining to cantaloupe industry. The objective of this study was to examine the growth and biofilm formation by outbreak strains of S. enterica ser. Poona (S. Poona), S. enterica ser. Stanley (S. Stanley) and S. enterica ser. Montevideo (S. Montevideo) on different food-contact processing surfaces in cantaloupe flesh and peel extracts at 22 °C and 10 °C. The generation time of all S. enterica strains tested was shorter in the high concentration (50 mg/ml) of cantaloupe extract and high temperature. In 50 mg/ml of cantaloupe flesh or peel extract, the populations of S. enterica were increased by 5 log CFU/ml in 24 h at 22 °C and 1 log CFU/ml in 72 h at 10 °C. In 2 mg/ml of cantaloupe flesh or peel extracts, the populations of S. enterica were increased by 3.5 log CFU/ml in 56 h at 22 °C, but there were no changes in 72 h at 10 °C. The biofilm production of S. enterica was greater at 50 mg/ml of cantaloupe extract and 22 °C, but no major differences (P ≥ 0.05) were found among the strains tested. In 50 mg/ml cantaloupe extract, S. enterica produced 5-6 log CFU/cm2 biofilm in 4-7 d at 22 °C and approximately 3.5-4 log CFU/cm2 in 7 d at 10 °C. In 2 mg/ml of cantaloupe extract, S. enterica produced 4-4.5 log CFU/cm2 biofilms in 4-7 d at 22 °C and 3 log CFU/cm2 in 7 d at 10 °C. Biofilm formation by S. Poona (01A4754) was lowest on buna-n rubber compared to stainless steel, polyethylene and polyurethane surfaces under the majority of conditions tested. Overall, these findings show that S. enterica strains can grow rapidly and form biofilms on different cantaloupe processing surfaces in the presence of low concentrations of cantaloupe flesh or peel extracts.

Scanning electron microscopy of Salmonella biofilms on various food-contact surfaces in catfish mucus.

The objective of this study was to determine the growth and survival of Salmonella enterica in the presence of high and low concentrations (375 µg/ml and 15 µg/ml) of catfish mucus extract at 10 °C and 22 °C for 63 days. The second objective of this study was to investigate the biofilm formation of Salmonella enterica serovar Blockley (7175) in catfish mucus extract for 48 h at 22 °C on four food-contact surfaces and to observe the biofilm populations using Scanning Electron Microscopy (SEM). The surface properties, surface roughness and surface energies were determined using contact angle measurement and atomic force microscopy. In 375 µg/ml of catfish mucus extract that was inoculated with 3 log CFU/ml, the growth of Salmonella counts were increased to a maximum of 6-7 log CFU/ml at 10 °C and 7-8 log CFU/ml at 22 °C in 7-14 d and decreased by 1-2 log CFU/ml from these peak levels at both 10 °C and 22 °C from 21 to 63 d. In 15 µg/ml of catfish mucus extract, Salmonella counts were in the range of 4-5 log CFU/ml at 10 °C and 5-6 log CFU/ml at 22 °C over 7-63 d of storage. By contrast, Salmonella counts were non-detectable in the absence of catfish mucus by 21-28 d of storage at 10 °C or 22 °C. The biofilm counts of S. Blockley (7175) on a stainless steel surface were 4 log CFU/cm2 and 5.5 log CFU/cm2 in 15 µg/ml and 375 µg/ml of catfish mucus extract respectively after 48 h incubation at 22 °C. SEM revelead that biofilm formation by S. Blockley (7175) was less in 15 µg/ml than 375 µg/ml of catfish mucus extract on stainless steel. In addition, SEM indicated that the visible biofilms were least on buna-N rubber as compared to stainless steel, polyethylene and polyurethane surfaces. Contact angle and atomic force microscopy confirmed that buna-N rubber was highly hydrophobic with low surface energy and low roughness when compared to other three surfaces. These findings indicate that Salmonella can utilize catfish mucus as a nutrient source to survive for longer periods and promote biofilm formation for its persistence on different food-contact surfaces.

Growth and Biofilm Formation by Listeria monocytogenes in Catfish Mucus Extract on Four Food Contact Surfaces at 22 and 10°C and Their Reduction by Commercial Disinfectants.

The objective of this study was to determine the effect of strain and temperature on growth and biofilm formation by Listeria monocytogenes in high and low concentrations of catfish mucus extract on various food contact surfaces at 10 and 22°C. The second objective of this study was to evaluate the efficacy of disinfectants at recommended concentrations and contact times for removing L. monocytogenes biofilm cells from a stainless steel surface covered with catfish mucus extract. Growth and biofilm formation of all L. monocytogenes strains increased with higher concentrations of catfish mucus extract at both 10 and 22°C. When 15 µg/mL catfish mucus extract was added to 3 log CFU/mL L. monocytogenes, the biofilm levels of L. monocytogenes on stainless steel reached 4 to 5 log CFU per coupon at 10°C and 5 to 6 log CFU per coupon at 22°C in 7 days. With 375 µg/mL catfish mucus extract, the biofilm levels of L. monocytogenes on stainless steel reached 5 to 6 log CFU per coupon at 10°C and 6 to 7.5 log CFU per coupon at 22°C in 7 days. No differences ( P > 0.05) were observed between L. monocytogenes strains tested for biofilm formation in catfish mucus extract on the stainless steel surface. The biofilm formation by L. monocytogenes catfish isolate HCC23 was lower on Buna-N rubber than on stainless steel, polyethylene, and polyurethane surfaces in the presence of catfish mucus extract ( P < 0.05). Contact angle analysis and atomic force microscopy confirmed that Buna-N rubber was highly hydrophobic, with lower surface energy and less roughness than the other three surfaces. The complete reduction of L. monocytogenes biofilm cells was achieved on the stainless steel coupons with a mixture of disinfectants, such as quaternary ammonium compounds with hydrogen peroxide or peracetic acid with hydrogen peroxide and octanoic acid at 25 or 50% of the recommended concentration, in 1 or 3 min compared with use of the quaternary ammonium compounds, chlorine, or acid disinfectants alone, which were ineffective for removing all the L. monocytogenes biofilm cells.

Biofilm formation by Salmonella spp. in catfish mucus extract under industrial conditions.

The objective of this study was to determine the effect of strain and temperature on the growth and biofilm formation of Salmonella spp. in high and low concentrations of catfish mucus extract on different food-contact surfaces at 22 °C and 10 °C. The second objective of this study was to evaluate the efficacy of disinfectants at recommended concentrations and contact times for removing Salmonella biofilms cells on a stainless steel surface containing catfish mucus extract. Growth and biofilm formation of all Salmonella strains increased with higher concentrations of catfish mucus extract at both 10 °C and 22 °C. In 15 µg/ml of catfish mucus extract inoculated with 3 log CFU/ml, the biofilm levels of Salmonella on stainless steel surface reached to 3.5 log CFU/cm2 at 10 °C or 5.5 log CFU/cm2 at 22 °C in 7 days. In 375 µg/ml of catfish mucus extract inoculated with 3 log CFU/ml, the biofilm levels of Salmonella on the stainless steel surface reached 4.5 log CFU/cm2 at 10 °C and 6.5 log CFU/cm2 at 22 °C in 7 days. No differences were observed between Salmonella strains tested for biofilm formation in catfish mucus extract on the stainless steel surface. The biofilm formation by Salmonella Blockley (7175) in catfish mucus extract was less (P < 0.05) on buna-N rubber when compared to stainless steel, polyethylene and polyurethane surfaces. Salmonella biofilm cells were not detectable on the stainless steel surface after treatment with a mixture of disinfectants but were still present when single compound disinfectants were used.

Induction and stability of oxidative stress adaptation in Listeria monocytogenes EGD (Bug600) and F1057 in sublethal concentrations of H2O2 and NaOH.

Food processing and food handling environments may contain residual levels of sanitizers or cleaners which may trigger oxidative stress adaptation in Listeria monocytogenes. The aim of this study was to determine the induction and stability of oxidative stress adaptation in L. monocytogenes EGD (Bug600) (serotype 1/2a) and F1057 (serotype 4b) at different concentrations and times of sublethal oxidative stress induced by H2O2 or sublethal alkali stress induced by NaOH at 37°C. Both L. monocytogenes Bug600 and F1057 strains showed significantly higher survival in lethal oxidative stress (1000ppm H2O2) after pre-exposure to 50ppm H2O2 for 30min compared to control cells (no pre-exposure to H2O2). When the cells were pre-exposed to sublethal alkali stress by NaOH, the oxidative stress adaptation was induced within 5min in L. monocytogenes. The survival of both L. monocytogenes strains was increased by 2 to 4.5 logs in lethal oxidative stress when the cells were pre-exposed to sublethal alkali stress at pH9 from 5 to 120min by NaOH compared to control cells (no pre-exposure to sublethal alkali pH). Two other alkali reagents tested (KOH and NH4OH) also induced oxidative stress adaptation in L. monocytogenes. For both L. monocytogenes strains, the oxidative stress adaptation induced by sublethal H2O2 was reversible in 30min and that induced by sublethal alkali stress was reversible within 60min at 37°C in the absence of such sublethal stress. These findings show that sublethal oxidative or alkali stress conditions can induce oxidative stress adaptation that may increase the risk of survival of L. monocytogenes cells in lethal oxidative stress.

Stability of sublethal acid stress adaptation and induced cross protection against lauric arginate in Listeria monocytogenes.

The stability of acid stress adaptation in Listeria monocytogenes and its induced cross protection effect against GRAS (generally recognized as safe) antimicrobial compounds has never been investigated before. In the present study, the acid stress adaptation in L. monocytogenes was initially induced in pH 5.0 tryptic soy broth supplemented with 0.6% yeast extract (TSB-YE) at 37 °C. Subsequently, the stability of acid stress adaptation, which was defined as the capacity to maintain its acquired acid adaptation after induction in the absence of sublethal acid stress, was determined at 37 °C, 22 °C or 4 °C in broth and in different food substrates. Then, the acid stress adaptation induced cross protection against lauric arginate (LAE) and its stability was investigated in TSB-YE, milk and carrot juice. Our findings show that the acid stress adaptation was stable at 4 °C up to 24h but was reversed at 37 °C or 22 °C within 2h. In the cross protection assay with LAE, the acid stress adapted cells had approximately 2 log CFU/ml greater survival than non-adapted cells in broth at 22 °C or in milk and carrot juice at 4 °C. The acid adaptation induced cross protection against LAE in L. monocytogenes was reversible within 1h at 4 °C in the absence of sublethal acid stress. Our findings suggest that the stability of acid adaptation in L. monocytogenes under cold conditions should be taken into account when the risk analysis is performed during food processing.

Low, medium, and high heat tolerant strains of Listeria monocytogenes and increased heat stress resistance after exposure to sublethal heat.

A group of 37 strains representing all 13 serotypes of Listeria monocytogenes with an initial cell density of 10(7) CFU/ml were analyzed for their heat tolerance at 60°C for 10 min. These L. monocytogenes strains were categorized into three heat tolerance groups: low (<2 log CFU/ml survival), medium (2 to 4 log CFU/ml survival), and high (4 to 6 log CFU/ml survival). Serotype 1/2a strains had relatively low heat tolerance; seven of the eight tested strains were classified as low heat tolerant. Of the two serotype 1/2b strains tested, one was very heat sensitive (not detectable) and the other was very heat resistant (5.4 log CFU/ml survival). Among the 16 serotype 4b strains, survival ranged from not detectable to 4 log CFU/ml. When one L. monocytogenes strain from each heat tolerance group was subjected to sublethal heat stress at 48°C for 30 or 60 min, the survival of heat-stressed cells at 60°C for 10 min increased by 5 log CFU/ml (D60°C-values nearly doubled) compared with the nonstressed control cells. Sublethal heat stress at 48°C for 60 or 90 min increased the lag phase of L. monocytogenes in tryptic soy broth supplemented with 0.6% yeast extract at room temperature by 3 to 5 h compared with nonstressed control cells. The heat stress adaptation in L. monocytogenes was reversed after 2 h at room temperature but was maintained for up to 24 h at 4°C. Our results indicate a high diversity in heat tolerance among strains of L. monocytogenes, and once acquired this heat stress adaptation persists after cooling, which should be taken into account while conducting risk analyses for this pathogen.

Influence of temperature on acid-stress adaptation in Listeria monocytogenes.

Our findings show that temperature plays a significant role in the induction of acid-stress adaptation in Listeria monocytogenes, and two distinct patterns were observed: (1) Presence of sublethal acid at 37°C or 22°C significantly induced acid-stress adaptation; and (2) Presence of sublethal acid at 4°C did not induce any acid-stress adaptation. Both patterns were confirmed by two experimental models: (1) L. monocytogenes cells were first grown at 37°C and then exposed to sublethal acid at 37°C, 22°C, and 4°C prior to lethal acid challenge; (2) Alternatively, L. monocytogenes cells were first grown at 4°C for 20 days before pre-exposure to sublethal acid and then challenged with lethal acid. Regardless of whether L. monocytogenes cells were simultaneously exposed with both cold stress and sublethal acid stress, or subjected to cold growth first before exposure to sublethal acid, no acid-stress adaptation was induced at 4°C. We also found that acid-stress adaptation in L. monocytogenes did not occur in acidic whey at 4°C. Bead beating treatment prior to mild acid pre-exposure at 4°C partially induced acid adaptation in L. monocytogenes. Our findings suggest that cold temperature can prevent the risk of acid-stress adaptation in L. monocytogenes.

Synergistic activity between lauric arginate and carvacrol in reducing Salmonella in ground turkey.

In the present study, low concentrations of carvacrol (0.025 to 0.2%) and lauric arginate (LAE; 25 to 200 ppm) were tested at 4, 22, and 45°C in a broth model, and higher concentrations of carvacrol (0.1 to 5%) and LAE (200 to 5,000 ppm) were tested individually and in combination at 4°C in 3 different ground turkey samples (with 15, 7, and 1% fat content) for their effectiveness against a 3-strain mixture of Salmonella. A low concentration of 25 ppm of LAE or 0.025% carvacrol had no effect on Salmonella in a broth model, but their mixture showed a synergistic action by reducing 6 log cfu/mL Salmonella counts to a nondetectable level within 30 min of exposure. The US Food and Drug Administration-recommended 200 ppm of LAE was not sufficient for Salmonella reductions in ground turkey when applied internally. High concentrations of 2,000 to 5,000 ppm of LAE or 1 to 2% carvacrol were needed to reduce Salmonella counts by 2 to 5 log cfu/g in ground turkey by internal application. No specific relationship existed between fat content and LAE or carvacrol concentrations for Salmonella reductions. For example, 2,000 ppm of LAE could reduce Salmonella counts by 4 log cfu/g in 1% fat-containing turkey samples but very similar ~1.5 log cfu/g reductions in both 7 and 15% fat-containing ground turkey samples. For the total microbial load, about 2,000 ppm of LAE or 2% of carvacrol treatments were needed to achieve 2 to 3 log (P ≤ 0.05) cfu/g reductions in different turkey samples. A mixture of 1% carvacrol and 2,000 ppm of LAE exhibited a synergistic action in ground turkey containing 7% fat by reducing the Salmonella counts by 4 log cfu/g, which was not found with individual antimicrobial treatments.

Inhibition and inactivation of Salmonella typhimurium biofilms from polystyrene and stainless steel surfaces by essential oils and phenolic constituent carvacrol.

Persistence of Salmonella biofilms within food processing environments is an important source of Salmonella contamination in the food chain. In this study, essential oils of thyme and oregano and their antimicrobial phenolic constituent carvacrol were evaluated for their ability to inhibit biofilm formation and inactivate preformed Salmonella biofilms. A crystal violet staining assay and CFU measurements were utilized to quantify biofilm cell mass, with evaluating factors such as strain variation, essential oil type, their concentrations, exposure time, as well as biofilm formation surface. Of the three Salmonella strains, Salmonella Typhimurium ATCC 23564 and Salmonella Typhimurium ATCC 19585 produced stronger biofilms than Salmonella Typhimurium ATCC 14028. Biofilm formation by different Salmonella strains was 1.5- to 2-fold higher at 22°C than at 30 or 37°C. The presence of nonbiocidal concentrations of thyme oil, oregano oil, and phenolic carvacrol at 0.006 to 0.012% suppressed Salmonella spp. biofilm formation 2- to 4-fold, but could not completely eliminate biofilm formation. There was high correlation in terms of biofilm inactivation, as determined by the crystal violet-stained optical density (at a 562-nm wavelength) readings and the viable CFU counts. Reduction of biofilm cell mass was dependent on antimicrobial concentration. A minimum concentration of 0.05 to 0.1% of these antimicrobial agents was needed to reduce a 7-log CFU biofilm mass to a nondetectable level on both polystyrene and stainless steel surfaces within 1 h of exposure time.

Reduction of Listeria monocytogenes biofilms on stainless steel and polystyrene surfaces by essential oils.

Plant-derived essential oils were tested for their ability to eliminate biofilms of Listeria monocytogenes on polystyrene and stainless steel surfaces. Various concentrations of essential oils were tested with different contact times on biofilms of various ages. Preliminarily screening of nine essential oils and related phenolic compounds in a disk diffusion assay revealed that thyme oil, oregano oil, and carvacrol had the highest antimicrobial activity. Further screening of these three compounds against 21 L. monocytogenes strains representing all 13 serotypes indicated some strain-specific variations in antimicrobial activity. For 1-day-old biofilms of mixed L. monocytogenes strains produced at 22°C on polystyrene microtiter plates, only 0.1% concentrations of thyme oil, oregano oil, and carvacrol were needed to eliminate 7 log CFU per well. On the stainless steel coupons, a 0.5% concentration of these compounds was adequate to completely eliminate 4-day-old biofilms at 7 log CFU per coupon. Our findings indicate that these compounds are potential candidates for elimination of L. monocytogenes biofilms on stainless steel and polystyrene surfaces.

Reduction of Listeria monocytogenes in raw catfish fillets by essential oils and phenolic constituent carvacrol.

The antimicrobial activity of various essential oils and carvacrol was determined on fresh raw catfish fillets against a 4-strain Listeria monocytogenes mixture representing serotypes 1/2b, 3b, 4b, and 4c that were predominantly isolated from catfish processing environments. Thyme oil, oregano oil and carvacrol exhibited concentration and time dependent responses in broth against L. monocytogenes; for example 0.5% concentrations resulted in 4 log CFU/mL reduction within 30 min whereas 0.1% concentrations required more than 24 h for the same level of reduction. Lemon, orange, and tangerine oils, at 0.5% showed listeriostatic effect in which 4 log CFU/mL of the initial L. monocytogenes load was unchanged at 4 °C in 10 d whereas 1% concentrations were listericidal in a time dependent manner. Apart from carvacrol, efficacy of tested essential oils in reducing L. monocytogenes and total microbial load from catfish fillet was very limited. Dipping treatment of catfish fillets in 2% carvacrol solution for 30 min at 4 °C reduced L. monocytogenes to an undetectable level from their initial load of 5 log CFU/g and reduced total microbial load from catfish fillets by approximately 5 log CFU/g. In sensory analysis trained panelist preferred control samples over 2% carvacrol treated samples implying potential limitation in applicability of carvacrol for fillet treatments.

Reduction of Listeria monocytogenes in queso fresco cheese by a combination of listericidal and listeriostatic GRAS antimicrobials.

Single and combined effects of three GRAS (generally recognized as safe) antimicrobials including, bacteriophage P100 (phage P100), lauric arginate (LAE), and potassium lactate-sodium diacetate mixture (PL-SD) were evaluated against Listeria monocytogenes cold growth in queso fresco cheese (QFC). The fate of phage P100 when exposed to LAE (200 ppm) or PL-SD (2.8% PL and 0.2% SD) was determined at 4°C and 30°C in a broth model. Phage P100 was found to be stable in the presence of these antimicrobial agents as plaque forming units (PFU) did not vary between control, LAE or PL-SD treatments. When 9 log CFU/ml of stationary phase cells of L. monocytogenes was exposed to these antimicrobials in tryptic soy broth, there was a 3 to 5 log CFU/ml reduction with phage P100 and a complete 9 log CFU/ml reduction with LAE but no measurable reduction with PL-SD after 24h at 4°C or 30°C. In QFC, the L. monocytogenes populations increased from the initial 3.5 log CFU/cm(2) to 7.7 log CFU/cm(2) in 28 days at 4°C. Treatment with 7.8 log PFU/cm(2) of phage P100 or 200 ppm of LAE showed strong listericidal effect initially by reducing L. monocytogenes counts by 2 to 3.5-4 log CFU/cm(2) while there was a subsequent regrowth of L. monocytogenes at 4°C. Treatment with PL-SD showed strong listeriostatic effect without decreasing L. monocytogenes counts but growth was prevented for 28 days at 4°C. Only the combined treatment of listericidal phage P100 or LAE with listeriostatic PL-SD reduced the initial L. monocytogenes counts by 2-4 log CFU/cm(2) and also kept the L. monocytogenes counts at that reduced level in QFC for 28 days at 4°C.