Alicia Alonso-Hernando

Exposure of Escherichia coli ATCC 12806 to Sublethal Concentrations of Food-Grade Biocides Influences Its Ability To Form Biofilm, Resistance to Antimicrobials, and Ultrastructure

ABSTRACT Escherichia coli ATCC 12806 was exposed to increasing subinhibitory concentrations of three biocides widely used in food industry facilities: trisodium phosphate (TSP), sodium nitrite (SNI), and sodium hypochlorite (SHY). The cultures exhibited an acquired tolerance to biocides (especially to SNI and SHY) after exposure to such compounds. E. coli produced biofilms (as observed by confocal laser scanning microscopy) on polystyrene microtiter plates. Previous adaptation to SNI or SHY enhanced the formation of biofilms (with an increase in biovolume and surface coverage) both in the absence and in the presence (MIC/2) of such compounds. TSP reduced the ability of E. coli to produce biofilms. The concentration of suspended cells in the culture broth in contact with the polystyrene surfaces did not influence the biofilm structure. The increase in cell surface hydrophobicity (assessed by a test of microbial adhesion to solvents) after contact with SNI or SHY appeared to be associated with a strong capacity to form biofilms. Cultures exposed to biocides displayed a stable reduced susceptibility to a range of antibiotics (mainly aminoglycosides, cephalosporins, and quinolones) compared with cultures that were not exposed. SNI caused the greatest increase in resistances (14 antibiotics [48.3% of the total tested]) compared with TSP (1 antibiotic [3.4%]) and SHY (3 antibiotics [10.3%]). Adaptation to SHY involved changes in cell morphology (as observed by scanning electron microscopy) and ultrastructure (as observed by transmission electron microscopy) which allowed this bacterium to persist in the presence of severe SHY challenges. The findings of the present study suggest that the use of biocides at subinhibitory concentrations could represent a public health risk.

Effects of exposure to poultry chemical decontaminants on the membrane fluidity of Listeria monocytogenes and Salmonella enterica strains

There is a lack of work comparing the influence of various poultry chemical decontaminants on the membrane fluidity of pathogenic bacteria. In order to assess the possible role of physical membrane changes on bacterial adaptation to decontaminants, fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene (DPH) was measured in Listeria monocytogenes and Salmonella enterica strains before and after growth in the presence of increasing sub-lethal concentrations of decontaminants (trisodium phosphate - TSP, acidified sodium chlorite - ASC, citric acid - CA and peroxyacids - PA). Higher (P<0.05) anisotropy values (lower membrane fluidity) were observed, both before and after exposure to decontaminants, in strains of L. monocytogenes (average 0.206+/-0.008) than in those of S. enterica (0.188+/-0.013). Cells exposed to sub-inhibitory concentrations of acid decontaminants (CA or PA) showed higher (P<0.05) anisotropy values and percentages of survival to acid stress than unexposed cells, suggesting that adaptation to these compounds is related to changes in membrane fluidity. Minimal changes in anisotropy values were observed after growth in presence of TSP or ASC. After treatment with strong concentrations of acid decontaminants (0.05 and 1% ASC, 5% and 10% CA, and 0.1 and 0.25% PA) the highest anisotropy values (highest membrane rigidity) were shown by cells previously grown with sub-inhibitory concentrations of CA or PA, suggesting that the synergistic effects of successive applications of various acids should be minimized. Because of the relationship between high membrane rigidity and high resistance to different stresses, the fact that it is essential to ensure adequate, inhibitory, CA or PA concentrations during poultry decontamination treatments is underlined.

Effect of the temperature of the dipping solution on the antimicrobial effectiveness of various chemical decontaminants against pathogenic and spoilage bacteria on poultry.

The influence of the temperature of the dipping solution on the antimicrobial effectiveness of several chemical poultry decontaminants was assessed. A total of 765 poultry legs were inoculated with gram-positive bacteria (Listeria monocytogenes, Staphylococcus aureus, Bacillus cereus, or Brochothrix thermosphacta) or gram-negative bacteria (Salmonella enterica serotype Enteritidis, Escherichia coli, Yersinia enterocolitica, or Pseudomonas fluorescens). Samples were dipped for 15 min in solutions (wt/vol) of trisodium phosphate (12%), acidified sodium chlorite (1,200 ppm), citric acid (2%), peroxyacids (220 ppm), chlorine dioxide (50 ppm), or tap water or were left untreated (control). The temperatures of the dipping solutions were 4, 20, or 50°C. Microbiological analyses and pH determinations were carried out after 0, 1, 3, and 5 days of storage at 4°C. In comparison with the control samples, all chemical solutions were effective for reducing microbial loads. The temperature of treatment affected the microbial reductions caused by all chemicals (P < 0.001). The lowest average bacterial reductions caused by trisodium phosphate, acidified sodium chlorite, citric acid, and peroxyacids were observed at 4°C, all sampling days and microbial groups being considered simultaneously. The highest and the lowest effectiveness for chlorine dioxide were observed at 4 and 50°C, respectively. These results may be of use to meat processors for selecting the best conditions for decontamination treatments and may help the European Regulatory Authorities make their decisions for authorization of poultry decontamination treatments.

Adaptation and cross-adaptation of Listeria monocytogenes and Salmonella enterica to poultry decontaminants

Information on the potential for acquired reduced susceptibility of bacteria to poultry decontaminants occurring is lacking. Minimal Inhibitory Concentrations (MICs) were established for assessing the initial susceptibility and the adaptative and cross-adaptative responses of four bacterial strains (Listeria monocytogenes serovar l/2a, L. monocytogenes serovar 4b, Salmonella enterica serotype Typhimurium, and S. enterica serotype Enteritidis) to four poultry decontaminants (trisodium phosphate, acidified sodium chlorite -ASC-, citric acid, and peroxyacetic acid). The initial susceptibility was observed to differ among species (all decontaminants) and between Salmonella strains (ASC). These inter- and intra-specific variations highlight (1) the need for strict monitoring of decontaminant concentrations to inactivate all target pathogens of concern, and (2) the importance of selecting adequate test strains in decontamination studies. MICs of ASC (0.17±0.02 to 0.21±0.02 mg/ml) were higher than the U.S. authorized concentration when applied as a pre-chiller or chiller solution (0.05 to 0.15 mg/ml). Progressively increasing decontaminant concentrations resulted in reduced susceptibility of strains. The highest increase in MIC was 1.88 to 2.71-fold (ASC). All decontaminants were shown to cause cross-adaptation of strains between both related and unrelated compounds, the highest increase in MIC being 1.82-fold (ASC). Our results suggest that the in-use concentrations of ASC could, in certain conditions, be ineffective against Listeria and Salmonella strains. The adaptative and cross-adaptative responses of strains tested to poultry decontaminants are of minor concern. However, the observations being presented here are based on in vitro studies, and further research into practical applications are needed in order to confirm these findings.

Comparative analysis of acid resistance in Listeria monocytogenes and Salmonella enterica strains before and after exposure to poultry decontaminants. Role of the glutamate decarboxylase (GAD) system

Data on the ability of chemical poultry decontaminants to induce an acid stress response in pathogenic bacteria are lacking. This study was undertaken in order to compare the survival rates in acid broths of Listeria monocytogenes and Salmonella enterica strains, both exposed to and not exposed to decontaminants. The contribution of the glutamate decarboxylase (GAD) acid resistance system to the survival of bacteria in acid media was also examined. Four strains (L. monocytogenes serovar 1/2, L. monocytogenes serovar 4b, S. enterica serotype Typhymurium and S. enterica serotype Enteritidis) were tested before (control) and after exposure to trisodium phosphate, acidified sodium chlorite, citric acid, chlorine dioxide and peroxyacids (strains were repeatedly passed through media containing increasing concentrations of a compound). Stationary-phase cells (10(8) cfu/ml) were inoculated into tryptic soy broth (TSB) acidified with citric acid (pH 2.7 and 5.0) with or without glutamate (10 mM) added, and incubated at 37 degrees C for 15 min. Survival percentages (calculated from viable colonies) varied from 2.47 +/- 0.67% to 91.93 +/- 5.83%. L. monocytogenes cells previously exposed to acid decontaminants (citric acid and peroxyacids) showed, when placed in acid TSB, a higher (P < 0.05) percentage of survival (average 38.80 +/- 30.52%) than control and pre-exposed to non-acidic decontaminants strains (22.82 +/- 23.80%). Similar (P > 0.05) survival percentages were observed in previously exposed to different decontaminants and control Salmonella strains. The GAD acid resistance system did not apparently play any role in the survival of L. monocytogenes or S. enterica at a low pH. This study demonstrates for the first time that prior exposure to acidic poultry decontaminants increases the percentage of survival of L. monocytogenes exposed to severe acid stress. These results have important implications for the meat industry when considering which decontaminant treatment to adopt.

Behaviour of co-inoculated pathogenic and spoilage bacteria on poultry following several decontamination treatments.

The potential of chemical decontaminants to cause harmful effects on human health is among the causes of the rejection of antimicrobial treatments for removing surface contamination from poultry carcasses in the European Union. This study was undertaken to determine whether decontaminants might give a competitive advantage to pathogenic bacteria on poultry and involve a potential risk to consumer. A total of 144 chicken legs were co-inoculated with similar concentrations of pathogenic bacteria (Listeria monocytogenes, Staphylococcus aureus, Salmonella enterica serotype Enteritidis or Escherichia coli) and spoilage bacteria (Brochothrix thermosphacta or Pseudomonas fluorescens). Samples were dipped for 15min in solutions (w/v) of trisodium phosphate (12%; TSP), acidified sodium chlorite (1200ppm; ASC), citric acid (2%; CA), peroxyacids (220ppm; PA) or chlorine dioxide (50ppm; CD), or were left untreated (control). Microbiological analyses were carried out on day 0 and every 24h until day 7 of storage (at 10±1°C). The modified Gompertz equation was used as the primary model to fit observed data. TSP, ASC and CA were effective in extending the lag phase (L, ranging from 1.47±1.34days to 4.06±1.16days) and in decreasing the concentration of bacteria during the stationary phase (D, ranging from 2.46±0.51 log(10) cfu/cm(2) to 8.64±0.53 log(10) cfu/cm(2)), relative to the control samples (L values ranging from 0.59±0.38days and 2.52±2.28days, and D values ranging from 6.32±0.89 log(10) cfu/cm(2) to 9.39±0.39 log(10) cfu/cm(2), respectively). Both on untreated and on most decontaminated samples the overgrowth of spoilage bacteria among the species tested was observed throughout storage, suggesting that spoilage would occur prior to any noteworthy increase in the levels of pathogenic microorganisms. However, L. monocytogenes counts similar to, or higher than, those for spoilage bacteria were observed on samples treated with TSP, ASC or CA, suggesting that these treatments might pose a danger to consumers. However, these results derive from laboratory-based experiments testing artificially-inoculated bacteria. Further investigations of the natural micro-biota of poultry carcasses would be appropriate in order to substantiate these findings.

Decontamination treatments for psychrotrophic microorganisms on chicken meat during storage at different temperatures.

The antimicrobial effectiveness of five chemical decontaminants (12 % trisodium phosphate [TSP], 1,200 ppm acidified sodium chlorite [ASC], 2 % citric acid [CA], 220 ppm of peroxyacids [PA], or 50 ppm of chlorine dioxide [CD]) against psychrotrophic populations on skinned chicken legs was assessed throughout 120 h of storage under various temperature abuse scenarios. Three different simulated cold chain disruptions were used: T1 (12 h at 1 ± 1 °C, 6 h at 15 ± 1 °C, and 102 h at 4 ± 1 °C), T2 (18 h at 1 ± 1 °C, 6 h at 15 ± 1 °C, and 96 h at 10 ± 1 °C), or T3 (18 h at 4 ± 1 °C, 6 h at 20 ± 1 °C, and 96 h at 7 ± 1 °C). Microbiological analyses were carried out at 0, 24, 72, and 120 h of storage. Substantial microbial reductions, with respect to control (untreated) samples, were obtained in legs treated with TSP, ASC, and CA, with average values ranging from 1.54 ± 1.52 to 2.02 ± 2.19 log CFU/cm(2). TSP was the most effective compound under mild abuse temperature conditions (T1), with mean reductions of 2.01 ± 1.67 log CFU/cm(2), whereas ASC, followed by CA, proved to be particularly useful under moderate abuse conditions (T3; average reductions of 2.99 ± 2.27 and 1.98 ± 1.65 log CFU/cm(2), respectively). Treatment with PA or CD resulted in minimal microbial reductions.