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Oxygen limitation induces acid tolerance and impacts simulated gastro-intestinal transit in Listeria monocytogenes J0161.

Sewell D, Allen SCh, Phillips CA - Gut Pathog (2015)

Bottom Line: Using a gastro-intestinal transit model it was found that anaerobic growth induced an acid tolerance response which enhanced resistance to pH 2.5 simulated gastric juice (SGJ) compared to aerobically grown cells (p < 0.05).These findings provide an initial insight into the effects of anaerobiosis on stress response and survival potential in L. monocytogenes.While it appears anaerobiosis may impact these, further work is required to confirm these findings are not strain specific.

View Article: PubMed Central - PubMed

Affiliation: School of Health, University of Northampton, Northampton, UK.

ABSTRACT

Unlabelled: ᅟ: Listeria monocytogenes is a food-borne pathogen and the causative agent of listeriosis, a severe infection to those with a pre-disposition. Infections often arise through consumption of contaminated foods, where high intrinsic resistance to food processing practises permit survival and growth. Several practises, including refrigeration, acidification and oxygen limitation are ineffective in controlling L. monocytogenes, therefore foods which do not undergo thermal processing, e.g. ready-to-eat products, are considered high risk. While the responses to several food processing practises have been investigated, there are few reports on the responses of L. monocytogenes to oxygen limitation. Therefore the aim of this study was to investigate the effects of oxygen limitation on stress response andsurvival capacity during simulated gastro-intestinal transit.

Findings: Anaerobiosis induced an acid tolerance response, causing cells to be more resistant to organic and inorganic acids than aerobically grown counterparts (p < 0.05). Using a gastro-intestinal transit model it was found that anaerobic growth induced an acid tolerance response which enhanced resistance to pH 2.5 simulated gastric juice (SGJ) compared to aerobically grown cells (p < 0.05). This response was most pronounced in exponential phase cells. However, exposure of stationary phase cells to pH 3.5 SGJ enhanced bile tolerance, suggesting a link between acid and bile tolerance.

Conclusions: The responses of L. monocytogenes to oxygen limitation are not extensively studied. These findings provide an initial insight into the effects of anaerobiosis on stress response and survival potential in L. monocytogenes. While it appears anaerobiosis may impact these, further work is required to confirm these findings are not strain specific.

No MeSH data available.


Related in: MedlinePlus

Simulated gastro-intestinal transit of exponential phase cells. Cells were grown to mid-exponential phase at 37°C under aerobic () or anaerobic () conditions before being subjected to ‘short’ (A, B) or ‘long’ (C, D) simulated gastric transit. For ‘short’ simulated gastric transit 4 ml of growing culture was added to 16 ml simulated gastric juice at pH 2.5 (A) and pH 1.7 (B), for final pH values of 2.5 and 3.5 respectively. Suspensions were incubated at 37°C for 60 minutes and then neutralised to ~ pH 7.2 and 0.1x volumes of 3% (w/v) bile salt solution added (giving a final bile salt concentration of 0.3% w/v, as seen in vivo). Suspensions were incubated for a further 60 minutes at 37°C. For ‘long’ simulated gastric transit 4 ml of growing culture was added to 16 ml simulated gastric juice at pH 2.5 (C) and pH 1.7 (D), for final pH values of 2.5 and 3.5 respectively. Suspensions were incubated at 37°C for 120 minutes and then neutralised to ~ pH 7.2 and 0.1x volumes of 3% (w/v) bile salt solution added (giving a final bile salt concentration of 0.3% w/v, as seen in vivo). Suspensions were incubated for a further 120 minutes at 37°C. Throughout testing, samples were taken at half hourly intervals, diluted in BPW and spiral plated on BHI agar. Plates were enumerated after 24–48 hours incubation at 37°C. PBS (), SGJ () and bile () controls were also included. Limit of detection = 3x102cfu ml−1. Arrows indicate pH neutralization and addition of bile salts. Data is representative of three independent experiments conducted in duplicate.
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Fig2: Simulated gastro-intestinal transit of exponential phase cells. Cells were grown to mid-exponential phase at 37°C under aerobic () or anaerobic () conditions before being subjected to ‘short’ (A, B) or ‘long’ (C, D) simulated gastric transit. For ‘short’ simulated gastric transit 4 ml of growing culture was added to 16 ml simulated gastric juice at pH 2.5 (A) and pH 1.7 (B), for final pH values of 2.5 and 3.5 respectively. Suspensions were incubated at 37°C for 60 minutes and then neutralised to ~ pH 7.2 and 0.1x volumes of 3% (w/v) bile salt solution added (giving a final bile salt concentration of 0.3% w/v, as seen in vivo). Suspensions were incubated for a further 60 minutes at 37°C. For ‘long’ simulated gastric transit 4 ml of growing culture was added to 16 ml simulated gastric juice at pH 2.5 (C) and pH 1.7 (D), for final pH values of 2.5 and 3.5 respectively. Suspensions were incubated at 37°C for 120 minutes and then neutralised to ~ pH 7.2 and 0.1x volumes of 3% (w/v) bile salt solution added (giving a final bile salt concentration of 0.3% w/v, as seen in vivo). Suspensions were incubated for a further 120 minutes at 37°C. Throughout testing, samples were taken at half hourly intervals, diluted in BPW and spiral plated on BHI agar. Plates were enumerated after 24–48 hours incubation at 37°C. PBS (), SGJ () and bile () controls were also included. Limit of detection = 3x102cfu ml−1. Arrows indicate pH neutralization and addition of bile salts. Data is representative of three independent experiments conducted in duplicate.

Mentions: When challenged by SGJ at pH 3.5 L. monocytogenes J0161 cells remained viable. There were no differences in viable counts between those subjected to SGJ and control cells (p > 0.05), therefore pre-conditioning under oxygen limiting conditions had no effect on resistance to SGJ at pH 3.5 (p > 0.05) (Figure 2A and C). However, there was evidence of anaerobiosis induced acid tolerance when cells were subjected to SGJ at pH 2.5. When grown under oxygen limiting conditions, L. monocytogenes cells remained viable for 60 minutes following SGJ exposure. During both short and long simulated gastric transit, the number of recoverable cells was significantly greater following anaerobic growth compared to those grown aerobically (p < 0.05) (Figure 2B and D). While this demonstrates the potential for anaerobiosis induced acid tolerance to permit successful passage of L. monocytogenes through the low pH of the stomach, it should be noted that exponential phase cells were unable to survive bile salt exposure, irrespective of growth conditions. Bile is a potent antimicrobial which has been previously reported to rapidly inactivate exponential phase cells at physiological concentrations [13], growth under oxygen limiting conditions did not induce a cross protection against such exposure.Figure 2


Oxygen limitation induces acid tolerance and impacts simulated gastro-intestinal transit in Listeria monocytogenes J0161.

Sewell D, Allen SCh, Phillips CA - Gut Pathog (2015)

Simulated gastro-intestinal transit of exponential phase cells. Cells were grown to mid-exponential phase at 37°C under aerobic () or anaerobic () conditions before being subjected to ‘short’ (A, B) or ‘long’ (C, D) simulated gastric transit. For ‘short’ simulated gastric transit 4 ml of growing culture was added to 16 ml simulated gastric juice at pH 2.5 (A) and pH 1.7 (B), for final pH values of 2.5 and 3.5 respectively. Suspensions were incubated at 37°C for 60 minutes and then neutralised to ~ pH 7.2 and 0.1x volumes of 3% (w/v) bile salt solution added (giving a final bile salt concentration of 0.3% w/v, as seen in vivo). Suspensions were incubated for a further 60 minutes at 37°C. For ‘long’ simulated gastric transit 4 ml of growing culture was added to 16 ml simulated gastric juice at pH 2.5 (C) and pH 1.7 (D), for final pH values of 2.5 and 3.5 respectively. Suspensions were incubated at 37°C for 120 minutes and then neutralised to ~ pH 7.2 and 0.1x volumes of 3% (w/v) bile salt solution added (giving a final bile salt concentration of 0.3% w/v, as seen in vivo). Suspensions were incubated for a further 120 minutes at 37°C. Throughout testing, samples were taken at half hourly intervals, diluted in BPW and spiral plated on BHI agar. Plates were enumerated after 24–48 hours incubation at 37°C. PBS (), SGJ () and bile () controls were also included. Limit of detection = 3x102cfu ml−1. Arrows indicate pH neutralization and addition of bile salts. Data is representative of three independent experiments conducted in duplicate.
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Related In: Results  -  Collection

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Fig2: Simulated gastro-intestinal transit of exponential phase cells. Cells were grown to mid-exponential phase at 37°C under aerobic () or anaerobic () conditions before being subjected to ‘short’ (A, B) or ‘long’ (C, D) simulated gastric transit. For ‘short’ simulated gastric transit 4 ml of growing culture was added to 16 ml simulated gastric juice at pH 2.5 (A) and pH 1.7 (B), for final pH values of 2.5 and 3.5 respectively. Suspensions were incubated at 37°C for 60 minutes and then neutralised to ~ pH 7.2 and 0.1x volumes of 3% (w/v) bile salt solution added (giving a final bile salt concentration of 0.3% w/v, as seen in vivo). Suspensions were incubated for a further 60 minutes at 37°C. For ‘long’ simulated gastric transit 4 ml of growing culture was added to 16 ml simulated gastric juice at pH 2.5 (C) and pH 1.7 (D), for final pH values of 2.5 and 3.5 respectively. Suspensions were incubated at 37°C for 120 minutes and then neutralised to ~ pH 7.2 and 0.1x volumes of 3% (w/v) bile salt solution added (giving a final bile salt concentration of 0.3% w/v, as seen in vivo). Suspensions were incubated for a further 120 minutes at 37°C. Throughout testing, samples were taken at half hourly intervals, diluted in BPW and spiral plated on BHI agar. Plates were enumerated after 24–48 hours incubation at 37°C. PBS (), SGJ () and bile () controls were also included. Limit of detection = 3x102cfu ml−1. Arrows indicate pH neutralization and addition of bile salts. Data is representative of three independent experiments conducted in duplicate.
Mentions: When challenged by SGJ at pH 3.5 L. monocytogenes J0161 cells remained viable. There were no differences in viable counts between those subjected to SGJ and control cells (p > 0.05), therefore pre-conditioning under oxygen limiting conditions had no effect on resistance to SGJ at pH 3.5 (p > 0.05) (Figure 2A and C). However, there was evidence of anaerobiosis induced acid tolerance when cells were subjected to SGJ at pH 2.5. When grown under oxygen limiting conditions, L. monocytogenes cells remained viable for 60 minutes following SGJ exposure. During both short and long simulated gastric transit, the number of recoverable cells was significantly greater following anaerobic growth compared to those grown aerobically (p < 0.05) (Figure 2B and D). While this demonstrates the potential for anaerobiosis induced acid tolerance to permit successful passage of L. monocytogenes through the low pH of the stomach, it should be noted that exponential phase cells were unable to survive bile salt exposure, irrespective of growth conditions. Bile is a potent antimicrobial which has been previously reported to rapidly inactivate exponential phase cells at physiological concentrations [13], growth under oxygen limiting conditions did not induce a cross protection against such exposure.Figure 2

Bottom Line: Using a gastro-intestinal transit model it was found that anaerobic growth induced an acid tolerance response which enhanced resistance to pH 2.5 simulated gastric juice (SGJ) compared to aerobically grown cells (p < 0.05).These findings provide an initial insight into the effects of anaerobiosis on stress response and survival potential in L. monocytogenes.While it appears anaerobiosis may impact these, further work is required to confirm these findings are not strain specific.

View Article: PubMed Central - PubMed

Affiliation: School of Health, University of Northampton, Northampton, UK.

ABSTRACT

Unlabelled: ᅟ: Listeria monocytogenes is a food-borne pathogen and the causative agent of listeriosis, a severe infection to those with a pre-disposition. Infections often arise through consumption of contaminated foods, where high intrinsic resistance to food processing practises permit survival and growth. Several practises, including refrigeration, acidification and oxygen limitation are ineffective in controlling L. monocytogenes, therefore foods which do not undergo thermal processing, e.g. ready-to-eat products, are considered high risk. While the responses to several food processing practises have been investigated, there are few reports on the responses of L. monocytogenes to oxygen limitation. Therefore the aim of this study was to investigate the effects of oxygen limitation on stress response andsurvival capacity during simulated gastro-intestinal transit.

Findings: Anaerobiosis induced an acid tolerance response, causing cells to be more resistant to organic and inorganic acids than aerobically grown counterparts (p < 0.05). Using a gastro-intestinal transit model it was found that anaerobic growth induced an acid tolerance response which enhanced resistance to pH 2.5 simulated gastric juice (SGJ) compared to aerobically grown cells (p < 0.05). This response was most pronounced in exponential phase cells. However, exposure of stationary phase cells to pH 3.5 SGJ enhanced bile tolerance, suggesting a link between acid and bile tolerance.

Conclusions: The responses of L. monocytogenes to oxygen limitation are not extensively studied. These findings provide an initial insight into the effects of anaerobiosis on stress response and survival potential in L. monocytogenes. While it appears anaerobiosis may impact these, further work is required to confirm these findings are not strain specific.

No MeSH data available.


Related in: MedlinePlus