A Fast and Easy ATP-Based Approach Enables MIC Testing for Non-resuscitating VBNC Pathogens
Many bacteria enter the viable but non-culturable (VBNC) state to maximize resources and increase their tolerance to harmful conditions to cope with environmental stress, which has been described for a plethora of important human and foodborne pathogens. VBNC pathogens can potentially present a seri...
Main Authors: | , , , , , |
---|---|
Format: | Article |
Language: | English |
Published: |
Frontiers Media S.A.
2019-06-01
|
Series: | Frontiers in Microbiology |
Subjects: | |
Online Access: | https://www.frontiersin.org/article/10.3389/fmicb.2019.01365/full |
id |
doaj-fd2aa80e9da04179bcfabdc039322cbd |
---|---|
record_format |
Article |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Christian Robben Anna Kristina Witte Dagmar Schoder Dagmar Schoder Beatrix Stessl Peter Rossmanith Peter Rossmanith Patrick Mester |
spellingShingle |
Christian Robben Anna Kristina Witte Dagmar Schoder Dagmar Schoder Beatrix Stessl Peter Rossmanith Peter Rossmanith Patrick Mester A Fast and Easy ATP-Based Approach Enables MIC Testing for Non-resuscitating VBNC Pathogens Frontiers in Microbiology viable but non-culturable (VBNC) antibiotic resistance food safety antimicrobial tolerance minimum inhibitory concentration metabolic activity |
author_facet |
Christian Robben Anna Kristina Witte Dagmar Schoder Dagmar Schoder Beatrix Stessl Peter Rossmanith Peter Rossmanith Patrick Mester |
author_sort |
Christian Robben |
title |
A Fast and Easy ATP-Based Approach Enables MIC Testing for Non-resuscitating VBNC Pathogens |
title_short |
A Fast and Easy ATP-Based Approach Enables MIC Testing for Non-resuscitating VBNC Pathogens |
title_full |
A Fast and Easy ATP-Based Approach Enables MIC Testing for Non-resuscitating VBNC Pathogens |
title_fullStr |
A Fast and Easy ATP-Based Approach Enables MIC Testing for Non-resuscitating VBNC Pathogens |
title_full_unstemmed |
A Fast and Easy ATP-Based Approach Enables MIC Testing for Non-resuscitating VBNC Pathogens |
title_sort |
fast and easy atp-based approach enables mic testing for non-resuscitating vbnc pathogens |
publisher |
Frontiers Media S.A. |
series |
Frontiers in Microbiology |
issn |
1664-302X |
publishDate |
2019-06-01 |
description |
Many bacteria enter the viable but non-culturable (VBNC) state to maximize resources and increase their tolerance to harmful conditions to cope with environmental stress, which has been described for a plethora of important human and foodborne pathogens. VBNC pathogens can potentially present a serious risk to human health as they are invisible to routine microbiological culture-based methods. Of high importance is the increased tolerance to antibiotics or disinfectant measures while in the VBNC state. The greatest remaining challenge for such investigations is the lack of an appropriate, cost-effective multi-species screening method due to experimental constraints. In this study, we investigated if de novo ATP production of cells in the VBNC state is a suitable indicator for overall cell viability that can be utilized to determine the minimum ATP inhibitory concentration (MAIC) of antibiotics and other antimicrobials. To validate this approach, heat-stress time-kill experiments were performed with both culturable and VBNC cells. We developed a comprehensive experimental setup and demonstrated the applicability of this VBNC–MIC assay for testing the tolerance of 12 strains of 4 important bacterial species (Escherichia coli, Bacillus cereus, Pseudomonas aeruginosa, and Listeria monocytogenes) in the VBNC state to eight important antimicrobials including four different antibiotics. We confirmed that bacteria in the VBNC state were resistant to all tested antibiotics (ampicillin, imipenem, ciprofloxacin, and gentamicin) and additionally insensitive to disinfectants (benzalkonium chloride and trioctylmethylammonium chloride) and preservatives (bronopol and sodium azide). These data emphasize the need for further research regarding the characteristics of bacterial pathogens in the VBNC state and present the advantages and high-throughput capabilities of ATP determinations to investigate tolerance of VBNC pathogens to antimicrobials. The presented method should be helpful in order to identify appropriate countermeasures, treatments, or disinfectants when confronted with bacterial pathogens in the VBNC state. |
topic |
viable but non-culturable (VBNC) antibiotic resistance food safety antimicrobial tolerance minimum inhibitory concentration metabolic activity |
url |
https://www.frontiersin.org/article/10.3389/fmicb.2019.01365/full |
work_keys_str_mv |
AT christianrobben afastandeasyatpbasedapproachenablesmictestingfornonresuscitatingvbncpathogens AT annakristinawitte afastandeasyatpbasedapproachenablesmictestingfornonresuscitatingvbncpathogens AT dagmarschoder afastandeasyatpbasedapproachenablesmictestingfornonresuscitatingvbncpathogens AT dagmarschoder afastandeasyatpbasedapproachenablesmictestingfornonresuscitatingvbncpathogens AT beatrixstessl afastandeasyatpbasedapproachenablesmictestingfornonresuscitatingvbncpathogens AT peterrossmanith afastandeasyatpbasedapproachenablesmictestingfornonresuscitatingvbncpathogens AT peterrossmanith afastandeasyatpbasedapproachenablesmictestingfornonresuscitatingvbncpathogens AT patrickmester afastandeasyatpbasedapproachenablesmictestingfornonresuscitatingvbncpathogens AT christianrobben fastandeasyatpbasedapproachenablesmictestingfornonresuscitatingvbncpathogens AT annakristinawitte fastandeasyatpbasedapproachenablesmictestingfornonresuscitatingvbncpathogens AT dagmarschoder fastandeasyatpbasedapproachenablesmictestingfornonresuscitatingvbncpathogens AT dagmarschoder fastandeasyatpbasedapproachenablesmictestingfornonresuscitatingvbncpathogens AT beatrixstessl fastandeasyatpbasedapproachenablesmictestingfornonresuscitatingvbncpathogens AT peterrossmanith fastandeasyatpbasedapproachenablesmictestingfornonresuscitatingvbncpathogens AT peterrossmanith fastandeasyatpbasedapproachenablesmictestingfornonresuscitatingvbncpathogens AT patrickmester fastandeasyatpbasedapproachenablesmictestingfornonresuscitatingvbncpathogens |
_version_ |
1725320771043065856 |
spelling |
doaj-fd2aa80e9da04179bcfabdc039322cbd2020-11-25T00:32:07ZengFrontiers Media S.A.Frontiers in Microbiology1664-302X2019-06-011010.3389/fmicb.2019.01365462137A Fast and Easy ATP-Based Approach Enables MIC Testing for Non-resuscitating VBNC PathogensChristian Robben0Anna Kristina Witte1Dagmar Schoder2Dagmar Schoder3Beatrix Stessl4Peter Rossmanith5Peter Rossmanith6Patrick Mester7Christian Doppler Laboratory for Monitoring of Microbial Contaminants, Department of Farm Animal and Public Health in Veterinary Medicine, Institute for Milk Hygiene, Milk Technology and Food Science, University of Veterinary Medicine, Vienna, AustriaChristian Doppler Laboratory for Monitoring of Microbial Contaminants, Department of Farm Animal and Public Health in Veterinary Medicine, Institute for Milk Hygiene, Milk Technology and Food Science, University of Veterinary Medicine, Vienna, AustriaChristian Doppler Laboratory for Monitoring of Microbial Contaminants, Department of Farm Animal and Public Health in Veterinary Medicine, Institute for Milk Hygiene, Milk Technology and Food Science, University of Veterinary Medicine, Vienna, AustriaDepartment of Farm Animal and Public Health in Veterinary Medicine, Institute of Milk Hygiene, Milk Technology and Food Science, University of Veterinary Medicine, Vienna, AustriaDepartment of Farm Animal and Public Health in Veterinary Medicine, Institute of Milk Hygiene, Milk Technology and Food Science, University of Veterinary Medicine, Vienna, AustriaChristian Doppler Laboratory for Monitoring of Microbial Contaminants, Department of Farm Animal and Public Health in Veterinary Medicine, Institute for Milk Hygiene, Milk Technology and Food Science, University of Veterinary Medicine, Vienna, AustriaDepartment of Farm Animal and Public Health in Veterinary Medicine, Institute of Milk Hygiene, Milk Technology and Food Science, University of Veterinary Medicine, Vienna, AustriaChristian Doppler Laboratory for Monitoring of Microbial Contaminants, Department of Farm Animal and Public Health in Veterinary Medicine, Institute for Milk Hygiene, Milk Technology and Food Science, University of Veterinary Medicine, Vienna, AustriaMany bacteria enter the viable but non-culturable (VBNC) state to maximize resources and increase their tolerance to harmful conditions to cope with environmental stress, which has been described for a plethora of important human and foodborne pathogens. VBNC pathogens can potentially present a serious risk to human health as they are invisible to routine microbiological culture-based methods. Of high importance is the increased tolerance to antibiotics or disinfectant measures while in the VBNC state. The greatest remaining challenge for such investigations is the lack of an appropriate, cost-effective multi-species screening method due to experimental constraints. In this study, we investigated if de novo ATP production of cells in the VBNC state is a suitable indicator for overall cell viability that can be utilized to determine the minimum ATP inhibitory concentration (MAIC) of antibiotics and other antimicrobials. To validate this approach, heat-stress time-kill experiments were performed with both culturable and VBNC cells. We developed a comprehensive experimental setup and demonstrated the applicability of this VBNC–MIC assay for testing the tolerance of 12 strains of 4 important bacterial species (Escherichia coli, Bacillus cereus, Pseudomonas aeruginosa, and Listeria monocytogenes) in the VBNC state to eight important antimicrobials including four different antibiotics. We confirmed that bacteria in the VBNC state were resistant to all tested antibiotics (ampicillin, imipenem, ciprofloxacin, and gentamicin) and additionally insensitive to disinfectants (benzalkonium chloride and trioctylmethylammonium chloride) and preservatives (bronopol and sodium azide). These data emphasize the need for further research regarding the characteristics of bacterial pathogens in the VBNC state and present the advantages and high-throughput capabilities of ATP determinations to investigate tolerance of VBNC pathogens to antimicrobials. The presented method should be helpful in order to identify appropriate countermeasures, treatments, or disinfectants when confronted with bacterial pathogens in the VBNC state.https://www.frontiersin.org/article/10.3389/fmicb.2019.01365/fullviable but non-culturable (VBNC)antibiotic resistancefood safetyantimicrobial toleranceminimum inhibitory concentrationmetabolic activity |