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.

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.

On the Mechanism of Hydrophilicity of Graphene.

It is generally accepted that the hydrophilic property of graphene can be affected by the underlying substrate. However, the role of intrinsic vs substrate contributions and the related mechanisms are vividly debated. Here, we show that the intrinsic hydrophilicity of graphene can be intimately connected to the position of its Fermi level, which affects the interaction between graphene and water molecules. The underlying substrate, or dopants, can tune hydrophilicity by modulating the Fermi level of graphene. By shifting the Fermi level of graphene away from its Dirac point, via either chemical or electrical voltage doping, we show enhanced hydrophilicity with experiments and first principle simulations. Increased vapor condensation on graphene, induced by a simple shifting of its Fermi level, exemplifies applications in the area of interfacial transport phenomena.