Item No. 1 of 1

INVESTIGATOR: Nannapaneni, R.

PERFORMING INSTITUTION:
MISSISSIPPI STATE UNIV
MISSISSIPPI STATE, MISSISSIPPI 39762

EMERGENCE OF ANTIBIOTIC RESISTANCE IN FOODBORNE BACTERIAL PATHOGENS AFTER PROLONGED EXPOSURE TO SUBLETHAL STRESS ENVIRONMENTS

NON-TECHNICAL SUMMARY: The factors that accelerate the emergence and spread of antibiotic-resistant bacteria in humans, animals and in the environment are not clearly established. Recent evidence suggests that sublethal concentrations of antibiotics play an important role in the evolution of antibiotic resistance. However, all our current understanding about the genetics and evolution of antibiotic-resistance is based on the selections of rare mutants with strong phenotypes when exposed to lethal concentrations of drugs. But this may not be the case under natural environments in which foodborne pathogenic bacteria may be frequently exposed to concentration gradients of antibiotics. Also, foodborne bacterial pathogens may be exposed to sublethal concentrations of various acids, alkali, heat or oxidative stress that may put selection pressure prior to antibiotic exposure. Under sublethal environments, any resistance mutations if induced are rapidly enriched upon coming in contact with low antibiotic concentrations which may lead a step-wide development of antibiotic resistance. Keeping this hypothesis in view, we will measure the degree and stability of increase or decrease in resistance in diverse strains of two foodborne bacterial pathogens, Listeria monocytogenes and Salmonella strains, against selected antibiotics after their exposure to sublethal acid, alkali, and oxidative stress environments prior to exposure to lethal antibiotics. This study will lead to important evidence on the role sublethal acid, alkali, oxidative and heat stress that may be prevalent in food production and processing environments on the emergence of antibiotic cross-resistance risk in foodborne bacterial pathogens. We will detect hypermutable clones that exhibit antibiotic resistance.On September 18, 2014, an executive order has been signed by the President of the United States on combating antibiotic-resistant bacteria. A new task force has been setup to detect, prevent, and control illness and death related to antibiotic-resistant infections by implementing measures that reduce the emergence and spread of antibiotic-resistant bacteria. Our work will demonstrate the linkage between unintended mild stress exposure leading to antibiotic-resistant phenotypes of L. monocytogenes and Salmonella in food production and processing environments. Our work will determine the sublethal triggers for stress-hardened L. monocytogenes and Salmonella in high-risk serotypes that may lead to emergence of highly stable antibiotic resistance clones. This will lead to improvements in risk analysis and subsequent intervention technologies for various food production and processing systems for elimination of these emerging antibiotic resistant foodborne bacterial pathogens.

OBJECTIVES: Emergence of antibiotic resistance has become an increasing public health concern worldwide. All our current understanding about the genetics and evolution of antibiotic-resistance is based on the selections of rare mutants with strong phenotypes when exposed to lethal concentrations of drugs. But this may not be the case under natural environments in which foodborne pathogenic bacteria may be frequently exposed to concentration gradients of antibiotics or sublethal concentrations of various acids, alkali, heat or oxidative stress that may put selection pressure prior to antibiotic exposure. Under those sublethal environments, any resistance mutations if induced are rapidly enriched upon coming in contact with low antibiotic concentrations which may lead to a step-wide development of antibiotic resistance. Keeping this hypothesis in view, we will measure the degree and stability of increase or decrease in resistance in diverse strains of two foodborne bacterial pathogens, Gram-positive Listeria monocytogenes and Gram-negative Salmonella strains, against selected antibiotics after their adaption to sublethal acid, alkali, and oxidative stress environments prior to exposure to antibiotics with the following objectives:Objective 1. Determine the degree and stability of resistance to ampicillin, amoxicillin, and trimethoprim/sulfamethoxazole in genetically diverse L. monocytogenes serotypes isolated from fresh fruits and vegetables, dairy and poultry products after exposure to sublethal acid, alkali and oxidative stress environments.Objective 2. Determine the degree and stability of resistance to ceftriaxone, ciprofloxacin, and trimethoprim/sulfamethoxazole in genetically diverse Salmonella serotypes isolated from fresh fruits and vegetables, dairy and poultry products after exposure to sublethal acid, alkali and oxidative stress environments.

APPROACH: Objective (1). A maximum of 10 strains of L. monocytogenes will be investigated that represent a genetically diverse backgrounds based on their serotypes and antibiotic resistance profiles. A diverse L. monocytogenes strain bank containing isolates from 2011 cantaloupe outbreak (1/2a - 2011L-2625, 1/2b - 2011L-2624) or from celery (4b - F8027) or carrots (1/2b - F8385) or coleslaw (4b - G1091) have been obtained from the University of Georgia. L. monocytogenes isolated from dairy or poultry as well as other reference stains of L. monocytogenes Scott A (4b, human clinical, FDA) and L. monocytogenes Bug600 (1/2a - clinical, Institute Pasteur, France) are in our culture collection. A well-defined sublethal stress adaptation will be induced in 1 h at 37ºC by pre-exposing107 CFU/ml of L. monocytogenes cells to pH 5 with 1M lactic acid (acid adapted cells) or pH 9.0 with 4 N NaOH (alkali adapted cells) or pH 7.2 (non-adapted cells) or by 50 ppm H2O2 (oxidative stress adapted cells) in TSB-YE that will survive post exposure lethal acid (pH 3.5), alkali (pH 11.5), or oxidative stress (1000 ppm H2O2). For a longer sublethal stress exposure, cell suspensions of 107 CFU/ml will be exposed for 7 cycles of 24 h each by transferring into a fresh medium containing that sublethal stress at the end of each cycle. The control non-stressed L. monocytogenes cells will be prepared using the same generation cycles except that the growth will be at a normal pH of 7.0 in the absence of target stress. The sublethal stress inducing substances will be eliminated by centrifugation step prior to lethal antibiotic exposure. We will then screen acid, alkali, and oxidative stress resistant phenotypes of L. monocytogenes serotypes against a panel of 3 antibiotics on Mueller-Hinton agar as described by the Clinical Laboratory Standards Institute (CLSI). The three antibiotics against L. monocytogenes are ampicillin, amoxicillin, and trimethoprim/sulfamethoxazole. The antibiotics will be dissolved to prepare selected stock concentrations and serial two fold dilutions of antibiotic stock solution will be prepared in MHB in sterile 96-well microdilution plates for MIC determinations of stressed cells and non-stressed cells of L. monocytogenes. If the CLSI 2 fold broth microdilution MIC method prove to be insensitive, three other methods will be attempted in order to observe the dynamic picture of the bactericidal activity of antibiotics against L. monocytogenes after sublethal stress adaptation. These assays include: (a) Antibiotic concentration to kill (CTK) studies which will yield the survival counts of stress-adapted and non-adapted L. monocytogenesafter 2 h of lethal exposure in different lethal antibiotic concentrations (1xMBC, 2xMBC, 4xMBC, 8xMBC) by plating cell suspensions on agar; (b) Time to kill (TTK) studies which will yield the survival counts of stress-adapted and non-adapted L. monocytogenesafter 0 min, 30 min, 1 h and 2 h of lethal exposure at a single antibiotic concentration by plating cell suspensions on agar. If there is a significant increase in survival of L. monocytogenesafter sublethal stress adaptation, it will be distinguished by the TKC and CTK assays where control cells without prior exposure to sublethal stresses will be killed by a rapid bactericidal activity prior to plating; and (c) Also, MICs and MBCs determined by the agar dilution method may detect significant differences between stress adapted and non-adapted cells if such differences are confounded by the 2-fold broth microdilution method or by the Kirby Bauer agar disc diffusion assay. All isolates of L. monocytogeneswill be characterized for antibiotic resistance profiles by broth microdilution and agar dilution methods according to the approved CLSI guidelines prior to sublethal stress treatments.Objective (2). A maximum of 10 strains each of Salmonella will be investigated that represent a genetically diverse backgrounds based on their serotypes and antibiotic resistance profiles. A diverse collection of Salmonella serotypes isolated from raw poultry products representing, S. Typhimurium, S. Heidelberg, S. Enteritidis, S. Saintpaul S. Newport, S. Reading and S. Schwarzengrund exhibiting different antibiotic resistance profiles are in our culture collection. This objective will focus on three antibiotics against Salmonella which includes ceftriaxone, ciprofloxacin, and trimethoprim/sulfamethoxazole. All other methods for the determination of increase or decrease in resistance or susceptibility to these antibiotics by acid, alkali and oxidative stressed cells of Salmonella serotypes are same described as above in Objective 1.A two-way, two-factorial within a completely randomized design (ANOVA using SPSS), will be utilized to study the effect of non-adapted and sublethal stress adaptation on the survival of L. monocytogenes and Salmonella in multiple lethal antibiotic concentrations at one time point (CTK studies), or a single lethal antibiotic concentration at multiple time points (TTK studies), separately for shorter and longer stress adapted cells at 37°C. For each group of acid, alkali and oxidative stress adapted cells, the significant differences between strains in their survival in lethal antibiotic concentrations will be assessed for L. monocytogenes and Salmonella serotypes. An antibiotic concentration will be considered to have a significant reduction in bactericidal effect (killing value) when the decrease in cell viability between the stress adapted and the corresponding non-adapted samples is statistically significant (P < 0.05). All experiments will be conducted in three independent replicates and the mean values (MICs, MBCs or cell viability) for stress adapted and non-adapted will be calculated using the SPSS statistical software package and these means will be compared using a Tukey's Honestly Significant Difference (HSD) test. The results will be considered statistically significant with P-values reported at P < 0.05.

PROGRESS: 2016/01 TO 2019/12
Target Audience:Results of this work was disseminated to USDA, FDA and academic scientists and food industry clients through the peer-reviewed publications and by presentations at the annual meetings of the International Poultry Science Forum andInternationalAssociation for Food Protection. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Two graduate students have received an extensive training in foodborne bacterial pathogen sublethal stress adaptation and food safety by undertaking thesis projects focusing in this area. How have the results been disseminated to communities of interest?The results were dessiminated by peer-reviewed publications in the international journals and by oral and poster presentations of this work to USDA, FDA and academic scientists and food industry clients at the annual meetings of the International Association for Food Protection for three consecutive years. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

IMPACT: 2016/01 TO 2019/12
What was accomplished under these goals? Heterologous stress adaptation (changes in antibiotic susceptibility) after sublethal oxidative stress induced by chlorine in Listeria monocytogenes:L. monocytogenesstrains were screened for susceptibility to different classes of antibiotics before and after gradual exposure to the increasing concentrations of sodium hypochlorite. All five strains tested were found to have intrinsic resistance to nalidixic acid while 2 of the 5 strains tested (Scott A and N1-227) have intrinsic resistance to ceftriaxone (a third-generation cephalosporin). The average zone of inhibition was slightly decreased by 0.5 to 2.2 mm compared to controls for all 11 antibiotics tested. Interestingly, the disk diffusion assay showed that all five-strains studied in the present study develop significant tolerance for streptomycin. Significant antibiotic tolerance development was also observed for antibiotics such as ampicillin, amoxicillin, sulfamethoxazole and trimethoprim, ciprofloxacin, rifampicin, and ceftriaxone after sublethal chlorine exposure toL. monocytogenesby disk diffusion assay. Results showed that the effect of sublethal chlorine exposure onL. monocytogeneshas significantly increased its zone of inhibition for few of antibiotics however, it did not influence antibiotic susceptibility zone. Similarly, development of antibiotic tolerance was measured after sublethal chlorine exposure toL. monocytogeneswith double dilution MIC assay. The assay showed that significant antibiotic tolerance was developed for ampicillin (1 strain), tetracycline (2 strains), streptomycin (1 strain), and ceftriaxone (3 strains). A major change in MIC was observed for the reference strain ATCC 19116 which exhibited a 4-fold increase in MIC to ceftriaxone after sublethal chlorine adaptation. A minor but significant change in antibiotic susceptibility range for ceftriaxone was also observed in two intrinsic resistant strains, Scott A and N1-227, in chlorine oxidative stress adapted cells ofL. monocytogenes.The decrease in zone of inhibition and increase in MIC for antibiotics after sublethal chlorine exposure could be related with phenotypic antibiotic tolerance development inL. monocytogenes. Sublethal chlorine induced oxidative stress caused significant and consistent morphological changes inL. monocytogenesstrains Scott A, V7 and N1-227 which included: (1) changes in cell length, (2) changes in cell wall thickness, (3) changes in cell membrane shape; and (4) cytoplasmic changes. Sublethal chlorine adaptedL. monocytogenesexhibited a consistent elongation of cells, presence of multiple chromosomes and membrane bleb formations which may indicate transient inhibition of cell division. Along with the elongated cells, outer membrane bleb formation and wavy cell envelop structures were frequently observed in chlorine stress adapted cells ofL. monocytogeneswhich may be associated with the activation of a bacterial phenotypic response to sublethal oxidative stress. In conclusion, our results show that the exposure to sublethal concentrations of chlorine could potentially have impact on the cross-responses ofL. monocytogenesto commonly used antibiotics, enabling microorganisms to adapt to adverse environmental conditions and enhance antibiotic resistance. The changes in cell wall and membrane integrity resulting from the elongation of cells may contribute to possible routes ofL. monocytogenesresponse for minor but significant increase in tolerance to chlorine and selective antibiotics. Heterologous stress adaptation (changes in antibiotic susceptibility) after sublethal oxidative stress induced by quaternary ammonium compound (QAC)in Listeria monocytogenes: Antibiotic susceptibility of eight strains of L. monocytogenes exposed to sublethal oxidative stress induced by QAC was determined by the MIC dilution assay as per CLSI (2008) guidelines. No significant antibiotic tolerance development was observed against ampicillin and amoxicillin by MIC broth dilution assay for QAC-adapted cells of L. monocytogenes. However, there were changes in antibiotic susceptibility against ciprofloxacin, gentamicin, and trimethoprim after sublethal oxidative stress induced by QAC in L. monocytogenes strains as explained below: Heterologous stress response against gentamicin: Three major findings were observed: (1) No significant changes in short-range MIC of gentamicin was observed between QAC adapted and non-adapted cells; (2) However, growth rate of QAC adapted cells was significantly higher (P<0.05) for up to 16 h than non-adapted cells in 50% MIC of gentamicin by OD600nm ; and (3) Survival of QAC adapted cells was higher by approximately 1 log CFU in concentration-to-kill assay in lethal gentamicin in TSA compared to non-adapted cells for one of the four strains but these differences were not statistically significant. Heterologous stress response against ciprofloxacin: Four main findings were observed: (1) The MIC of ciprofloxacin against L. monocytogenes was increased by 1 to 4 µg/ml for QAC-adapted cells compared to non-adapted cells for 5 out of 8 strains. A two-fold increase in MIC of ciprofloxacin was observed for one strain of L.monocytogenes adapted to QAC; (2) The growth rate (OD600 nm) of QAC-adapted cells was significantly higher than non-adapted cells in 2 µg/ml of ciprofloxacin in TSBYE for 5 out of 8 strains; (3) The lag phase of L. monocytogenes was decreased by 5-6 h in 2 µg/ml of ciprofloxacin for QAC-adapted cells compared to non-adapted cells for 5 out of 8 strains; and (4) The survival counts of QAC-adapted cells was significantly higher by 2 to 4 log CFU/ml in TSAYE containing ciprofloxacin (2 µg/ml) as compared to non-adapted cells for 5 out of 8 strains of L.monocytogenes. Greater than two fold increase in survival for 3 out of 8 QAC-adapted strains of L.monocytogenes was observed. Heterologous stress response against trimethoprim:Four main findings were observed: (1) The MIC of trimethoprim againstL. monocytogeneswas increased by 0.2 to 0.5 µg/ml for QAC-adapted cells compared to non-adapted cells for 6 out of 8 strains. Following adaptation, a two-fold increase in MIC of trimethoprim was observed for two strains ofL.monocytogenesadapted to QAC; (2) The growth rate (OD600 nm) of QAC-adapted cells was significantly higher for up to 5-8 h than non-adapted cells in TSBYE containing trimethoprim (0.125 µg/ml) for 5 out of 8 strains; (3) The lag phase ofL. monocytogeneswas decreased in 0.125 µg/ml of trimethoprim for QAC-adapted cells compared to non-adapted cells for 6 out of 8 strains; and (4) Six QAC-adaptedL.monocytogenesstrains showed significantly increased survival by 1.5 to 2.5 logs CFU/ml against trimethoprim (0.125 µg/ml) in agar model as comparison to non-adapted cells. Heterologous stress adaptation (changes in antibiotic susceptibility) after sublethal oxidative stress induced by chlorine or QAC in Salmonella strains: Slight differences in antibiotic susceptibility patterns of chlorine-adapted (rugose and smooth) of Salmonella Typhimurium were observed as compared to non-adapted control cells. The rugose cells of S. Typhimurium exhibited a slight reduction (≤ 2mm) in susceptibility to streptomycin, nalidixic acid, ciprofloxacin, and ceftriaxone when compared to smooth cells. A slight increase in MIC was observed for rugose cells against all the antibiotics tested except ciprofloxacin compared to smooth cells. In another study, no significant antibiotic tolerance development was observed against ceftriaxone and ciprofloxacin by MIC broth dilution assay for QAC-adapted cells of Salmonella. However, there were changes in antibiotic susceptibility against ampicillin and trimethoprim after sublethal oxidative stress induced by QAC in 2of the 8 Salmonella strains tested.

PUBLICATIONS (not previously reported): 2016/01 TO 2019/12
1. Type: Conference Papers and Presentations Status: Submitted Year Published: 2020 Citation: Kode, D., Nannapaneni, R., Bansal, M., Cheng, W-H., Sharma, C.S., Kiess, A. 2020. Changes in susceptibility to trimethoprim in Listeria monocytogenes strains after exposure to gradually increasing sublethal concentrations of quaternary ammonium compound. Submitted for presentations at the Annual Meeting of the International Association for Food Protection, August 2-5, 2020 in Cleveland, OH.
2. Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Bansal, M., Nannapaneni, R., McDaniel, C., Sharma, C.S. 2017. Influence of chlorine induced sub-lethal oxidative stress on homologous stress adaptation and biofilm formation in Listeria monocytogenes. International Poultry Scientific Forum, January 30-31, 2017 at Georgia World Congress Center, Atlanta, Georgia.
3. Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Bansal, M., Sharma, C.S., Nannapaneni, R. 2017. Changes in zones of inhibition and minimum inhibitory concentrations of antibiotics in Listeria monocytogenes strains after exposure to chlorine induced sublethal oxidative stress. Annual Meetings of the International Association for Food Protection, July 9-12, 2017 in Tampa, Florida.
4. Type: Theses/Dissertations Status: Published Year Published: 2017 Citation: Bansal, Mohit. 2017. Listeria monocytogenes response to sublethal sodium hypochlorite induced oxidative stress on its biofilm forming ability and antibiotic resistance. MS Thesis, Mississippi State University (submitted November 2017).
5. Type: Journal Articles Status: Published Year Published: 2018 Citation: Bansal, M., Nannapaneni, R., Sharma, C.S., and Kiess, A. 2018. Listeria monocytogenes response to sublethal chlorine induced oxidative stress on homologous and heterologous stress adaptation. Frontiers in Microbiology 9:2050
6. Type: Journal Articles Status: Published Year Published: 2019 Citation: Bansal, M., Nannapaneni, R., Kode, D., Chang, S., Sharma, C.S., McDaniel, C., and Kiess, A. 2019. Rugose morphotype in Salmonella Typhimurium and S. Heidelberg induced by sequential exposure to subinhibitory NaOCl aids in biofilm tolerance to lethal NaOCl on polystyrene and stainless steel surfaces. Frontiers in Microbiology 10:2704.
7. Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Kode, D., Nannapaneni, R., Bansal, M., Cheng, W-H., Sharma, C.S., and Kiess, A. 2019. Homologous stress adaptation in four strains of Listeria monocytogenes to quaternary ammonium compounds after sublethal exposure, P1-148. Annual Meeting of the International Association for Food Protection, July 21-24, 2019 in Louisville, KY.
8. Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Kode, D., Nannapaneni, R., Bansal, M., Cheng, W-H., Sharma, C.S., and Kiess, A. 2019. Heterologous stress adaptation to gentamicin in four strains of Listeria monocytogenes after sublethal adaptive response to quaternary ammonium compound (QAC), P1-146, Annual Meeting of the International Association for Food Protection, July 21-24, 2019 in Louisville, KY.
9. Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Kode, D., Nannapaneni, R. 2019. Homologous stress adaptation in four strains of Listeria monocytogenes to quaternary ammonium compound (QAC) after sublethal exposure. Summer Student Science Symposium of the Mississippi Academy of Sciences, July 11, 2019 held at Mississippi State University.
10. Type: Conference Papers and Presentations Status: Submitted Year Published: 2020 Citation: Kode, D., Nannapaneni, R., Bansal, M., Cheng, W-H., Sharma, C.S., Kiess, A. 2020. Homologous stress adaptive response in eight strains of Listeria monocytogenes after gradual exposure to increasing sublethal concentration of quaternary ammonium compound. Submitted for presentations at the Annual Meeting of the International Association for Food Protection, August 2-5, 2020 in Cleveland, OH.
11. Type: Conference Papers and Presentations Status: Submitted Year Published: 2020 Citation: Kode, D., Nannapaneni, R., Bansal, M., Cheng, W-H., Sharma, C.S., Kiess, A. 2020. Changes in susceptibility to ciprofloxacin in Listeria monocytogenes strains after exposure to gradually increasing sublethal concentrations of quaternary ammonium compound. Submitted for presentations at the Annual Meeting of the International Association for Food Protection, August 2-5, 2020 in Cleveland, OH.

PROGRESS: 2017/01/01 TO 2017/12/31
Target Audience:Results of this work was disseminated to USDA, FDA and academic scientists and food industry clients at the 2017 annual meetings of the International Poultry Science Forum, International Association for Food Protection, and Poultry Science Association. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?One graduate student has received an extensive training in foodborne bacterial pathogen sublethal stress adaptation and food safety by undertaking the thesis project focusing in this area. How have the results been disseminated to communities of interest?Oral and poster presentations were submittedfor disseminating theresults of this workto USDA, FDA and academic scientists and food industry clients at theannual meetings of the International Poultry Science Forum, International Association for Food Protection, and Poultry Science Association. What do you plan to do during the next reporting period to accomplish the goals?One journal article of this work will bepublished in 2018 and will be disseminated to various food industry professionals and USDA, FDA and academic scientists working on foodborne bacterial sublethal stress adaptation and antibiotic resistance.

IMPACT: 2017/01/01 TO 2017/12/31
What was accomplished under these goals? The objective of this study was to determine the effect of sublethal oxidative stress induced by chlorine against homologous and heterologous stress adaptation in five Listeria monocytogenes strains. L. monocytogenes cells were exposed to gradually increasing sub-inhibitory concentrations of total chlorine/day starting from 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 L. monocytogenes cells exposed to sublethal chlorine at 250 ppm (1/2 MIC) for one day, 250 ppm to 330 ppm (1/2 to 2/3 MIC) over 5 days, and 250 ppm to 375 ppm (1/2 to 3/4 MIC) over 7 days, and control (non-adapted cells) were determined by the macro-dilution method. Chlorine-adapted L. monocytogenes cells were also evaluated for changes in antibiotic resistance using the Kirby-Bauer disk diffusion and MIC double dilution assay as per the Clinical & Laboratory Standards Institute (CLSI, 2008) guidelines. In four L. monocytogenes strains (Scott A, V7, FSL-N1-227 and FSL-F6-154) after adapted to sublethal chlorine, the chlorine MIC (600 ppm) and MBC (700 ppm) values were slightly higher as compared to control (500 ppm MIC, and 600 ppm MBC). All five L. monocytogenes strains were screened for susceptibility to different classes of antibiotics before and after gradual exposure to the increasing concentrations of sodium hypochlorite. All five strains tested were found to have intrinsic resistance to nalidixic acid while 2 of the 5 strains tested (Scott A and N1-227) have intrinsic resistance to ceftriaxone (a third-generation cephalosporin). The disk diffusion assay results showed that the sublethal oxidative stress induced by sublethal sodium hypochlorite influenced the zones of inhibition in L. monocytogenes. The average zone of inhibition was slightly decreased by 0.5 to 2.2 mm compared to controls for all 11 antibiotics tested. However, adapted cells were slightly more susceptible to rifampicin where the zone of inhibition was increased by 0.3-0.5 mm. The double dilution MIC assay revealed a minor change in average MIC values for ampicillin, tetracycline, amoxicillin and ciprofloxacin. However, the average MIC values were doubled for streptomycin and gentamicin against oxidative stress adapted L. monocytogenes strain V7 compared to control. A major change in MIC was observed for the reference strain ATCC 19116 which exhibited a significant increase in resistance to ceftriaxone. A slight change in antibiotic susceptibility range was found for two more strains, Scott A and N1-227, for ceftriaxone in chlorine oxidative stress adapted cells of L. monocytogenes. In summary, the Kirby-Bauer and MIC double dilution assays showed some changes in antibiotic susceptibility patterns for antibiotics such as streptomycin, gentamicin and ceftriaxone. 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 Lm response for its increase in slight tolerance to chlorine and selective antibiotics. These findings indicate that the continuous exposure of Lm cells to chlorine may induce slight changes in homologues and heterologous stress adaptation.

PUBLICATIONS: 2017/01/01 TO 2017/12/31
1. Type: Theses/Dissertations Status: Submitted Year Published: 2017 Citation: Bansal, Mohit. 2017. Listeria monocytogenes response to sublethal sodium hypochlorite induced oxidative stress on its biofilm forming ability and antibiotic resistance. MS Thesis, Mississippi State University (submitted November 2017).
2. Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Bansal, M., Nannapaneni, R., Sharma, C. S. (July 10, 2017)."Changes in zones of inhibition and minimum inhibitory concentration of antibiotics in Listeria monocytogenes strains after exposure to chlorine induced sublethal oxidative stress." 2017 Annual Meeting of the International Association for Food Protection, International Association for Food Protection, Tampa, FL.

PROGRESS: 2016/01/01 TO 2016/12/31
Target Audience:Results of this work will be disseminated to USDA, FDA and academic scientists and food industry clients at the 2017 annual meetings of the International Poultry Science Forum or International Association for Food Protection, and Institute of Food Technologists. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?One graduate student is currently receiving extensive training in foodborne bacterial pathogen sublethal stress adaptation and food safety by undertaking the thesis project focusing in this area. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?Two journal articles of this work will be published in 2017 and will be disseminated to various food industry professionals and USDA, FDA and academic scientists working on foodborne bacterial sublethal stress adaptation and antibiotic resistance.

IMPACT: 2016/01/01 TO 2016/12/31
What was accomplished under these goals? The prevalence of antibiotic resistance in Listeria monocytogenes was estimated at 1.27% among clinical isolates but some studies have recently reported an increased rate of resistance to one or several clinically relevant antibiotics in environmental isolates. The objective of this study was to determine the influence of sublethal chlorine stress on the changes in homologous and heterologous stress adaptation in L. monocytogenes. In the first part of this study, we have determined whether the sublethal exposure of chlorine (sodium hypochlorite) can influence the homologous stress adaptation and biofilm formation ability of two L. monocytogenes strains, FSL F6-154 (serotype 1/2a) and Scott A (serotype 4b). L monocytogenes cells were exposed to increasing (20 ppm/day) sub-inhibitory concentrations of total chlorine from 250 ppm to 375 ppm in tryptic soy broth (TSB). Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of L. monocytogenes cells exposed to chlorine and control (not-exposed to chlorine) were determined at 250 (1/2 MIC), 330 (2/3 MIC) and 375 (3/4 MIC) ppm of chlorine by macro-dilution method. The biofilm forming ability of chlorine treated and control cells were determined by crystal violet assay in 96-well microtiter at 37°C after 48 h. After exposure to all three sublethal chlorine treatments, there was an increase in MIC/MBC to 600-700 ppm of chlorine from the initial 500-600 ppm (non-exposed to chlorine) in both L. monocytogenes FSL F6-154 and ScottA strains indicating that L. monocytogenes planktonic cells can develop resistance to chlorine after exposure to sublethal chlorine concentrations. However, the chlorine induced oxidative stress significantly (p ? 0.05) reduced biofilm forming ability of both the strains on polystyrene surface as compared to control cells. Further, the anti-biofilm effect of chlorine was clearly observed at 330 (2/3 MIC) and 375 (3/4 MIC) ppm concentration for the oxidative stressed and non-stressed cells in the presence of chlorine for Scott A strain (p ? 0.05). Scanning electron microscopy revealed that the continuous exposure of chlorine induces morphological changes in bacterial morphology and biofilm structure of L. monocytogenes. The findings from the study indicate that continuous exposure of chlorine induces homologues stress adaptation and reduces biofilm forming ability of L. monocytogenes. In the second part of this study, we have determined whether the sublethal exposure to chlorine (sodium hypochlorite) can influence the heterologous stress adaptation or alter the antibiotic resistance in five L. monocytogenes strains including, FSL F6-154 (serotype 1/2a), FSL N1-227(serotype 4b), ATCC 19116 (serotype 4C), V7 (serotype ½ a) and Scott A (serotype 4b) by disc-diffusion assay as per CLSI method. L monocytogenes cells were exposed to increasing (20 ppm/day) sublethal concentrations of total chlorine from 250 ppm to 375 ppm in TSB at 37°C in 24-h cycles to obtain cells adapted to 250 (1/2 MIC), 330 (2/3 MIC) and 375 (3/4 MIC) of chlorine and control non-adapted cells. Ten antibiotics discs used for Kirby-Bauer disc-diffusion test were as follows: amoxicillin (20 ug) and clavulanic acid (10 μg), ampicillin (10 μg) ceftriaxone (30 μg), streptomycin (10 μg), nalidixic acid (30 μg), gentamicin (10 μg), sulphamethoxazole (1.25 μg) and trimethoprim (23.75 μg), ciprofloxacin (5 μg), rifampin (5 μg) and vancomycin (30 μg). Our current findings show that the disc-diffusion (zones of inhibition) test did not detect any changes in antibiotic resistance in all the five L. monocytogenes strains tested after any sublethal oxidative stress treatments induced by chlorine versus control non-stressed cells. In the third part of this study, we have determined if there were any changes in minimum inhibitory concentration (MIC) to four antibiotics in two L. monocytogenes strains (Scott A and V7) as per CLSI method after exposure to sublethal chlorine stress versus control not-stressed cells. L monocytogenes cells were exposed to increasing (20 ppm/day) sublethal concentrations of total chlorine from 250 ppm to 375 ppm in TSB at 37°C in 24-h cycles to obtain cells adapted to 250 (1/2 MIC), 330 (2/3 MIC) and 375 (3/4 MIC) of chlorine. Antibiotic stocks (10,000 X concentrations) were prepared in sterile distilled water and working stocks (4X concentrations) were prepared in Mueller Hinton broth. Our current results show that there were no changes in the minimum inhibitory concentration to gentamicin, streptomycin, tetracycline and ampicillin in two L. monocytogenes strain after sublethal oxidative stress treatments induced by chlorine versus control non-stressed cells. Further studies will determine if there are any differences in survival of L. monocytogenes cells in lethal concentrations of selected antibiotics by broth and agar-assay models after exposure to sublethal oxidative stress induced by chlorine compared to non-stressed cells.

PUBLICATIONS: 2016/01/01 TO 2016/12/31
Type: Conference Papers and Presentations Status: Submitted Year Published: 2017 Citation: Bansal M, Nannapaneni R, McDaniel C, and Sharma CS. 2017. Influence of chlorine induced sub-lethal oxidative stress on homologous stress adaptation and biofilm formation in Listeria monocytogenes. Submitted for presentation at the 2017 International Poultry Scientific Forum, January 31 to February 2, 2017, Atlanta, GA.