A Novel Propidium Monoazide-Based PCR Assay Can Measure Viable Uropathogenic E. coli In Vitro and In Vivo

BackgroundPolymerase chain reaction (PCR) is an important means by which to study the urine microbiome and is emerging as possible alternative to urine cultures to identify pathogens that cause urinary tract infection (UTI). However, PCR is limited by its inability to differentiate DNA originating f...

Full description

Bibliographic Details
Main Authors: Albert S. Lee, Olivia K. Lamanna, Kenji Ishida, Elaise Hill, Andrew Nguyen, Michael H. Hsieh
Format: Article
Language:English
Published: Frontiers Media S.A. 2022-02-01
Series:Frontiers in Cellular and Infection Microbiology
Subjects:
Online Access:https://www.frontiersin.org/articles/10.3389/fcimb.2022.794323/full
_version_ 1819281519607283712
author Albert S. Lee
Olivia K. Lamanna
Kenji Ishida
Elaise Hill
Elaise Hill
Andrew Nguyen
Michael H. Hsieh
Michael H. Hsieh
Michael H. Hsieh
author_facet Albert S. Lee
Olivia K. Lamanna
Kenji Ishida
Elaise Hill
Elaise Hill
Andrew Nguyen
Michael H. Hsieh
Michael H. Hsieh
Michael H. Hsieh
author_sort Albert S. Lee
collection DOAJ
description BackgroundPolymerase chain reaction (PCR) is an important means by which to study the urine microbiome and is emerging as possible alternative to urine cultures to identify pathogens that cause urinary tract infection (UTI). However, PCR is limited by its inability to differentiate DNA originating from viable, metabolically active versus non-viable, inactive bacteria. This drawback has led to concerns that urobiome studies and PCR-based diagnosis of UTI are confounded by the presence of relic DNA from non-viable bacteria in urine. Propidium monoazide (PMA) dye can penetrate cells with compromised cell membranes and covalently bind to DNA, rendering it inaccessible to amplification by PCR. Although PMA has been shown to differentiate between non-viable and viable bacteria in various settings, its effectiveness in urine has not been previously studied. We sought to investigate the ability of PMA to differentiate between viable and non-viable bacteria in urine.MethodsVarying amounts of viable or non-viable uropathogenic E. coli (UTI89) or buffer control were titrated with mouse urine. The samples were centrifuged to collect urine sediment or not centrifuged. Urine samples were incubated with PMA and DNA cross-linked using blue LED light. DNA was isolated and uidA gene-specific PCR was performed. For in vivo studies, mice were inoculated with UTI89, followed by ciprofloxacin treatment or no treatment. After the completion of ciprofloxacin treatment, an aliquot of urine was plated on non-selective LB agar and another aliquot was treated with PMA and subjected to uidA-specific PCR.ResultsPMA’s efficiency in excluding DNA signal from non-viable bacteria was significantly higher in bacterial samples in phosphate-buffered saline (PBS, dCT=13.69) versus bacterial samples in unspun urine (dCT=1.58). This discrepancy was diminished by spinning down urine-based bacterial samples to collect sediment and resuspending it in PBS prior to PMA treatment. In 3 of 5 replicate groups of UTI89-infected mice, no bacteria grew in culture; however, there was PCR amplification of E. coli after PMA treatment in 2 of those 3 groups.ConclusionWe have successfully developed PMA-based PCR methods for amplifying DNA from live bacteria in urine. Our results suggest that non-PMA bound DNA from live bacteria can be present in urine, even after antibiotic treatment. This indicates that viable but non-culturable E. coli can be present following treatment of UTI, and may explain why some patients have persistent symptoms but negative urine cultures following UTI treatment.
first_indexed 2024-12-24T01:00:59Z
format Article
id doaj.art-67796091d68149599e37ca10ca142a8d
institution Directory Open Access Journal
issn 2235-2988
language English
last_indexed 2024-12-24T01:00:59Z
publishDate 2022-02-01
publisher Frontiers Media S.A.
record_format Article
series Frontiers in Cellular and Infection Microbiology
spelling doaj.art-67796091d68149599e37ca10ca142a8d2022-12-21T17:23:22ZengFrontiers Media S.A.Frontiers in Cellular and Infection Microbiology2235-29882022-02-011210.3389/fcimb.2022.794323794323A Novel Propidium Monoazide-Based PCR Assay Can Measure Viable Uropathogenic E. coli In Vitro and In VivoAlbert S. Lee0Olivia K. Lamanna1Kenji Ishida2Elaise Hill3Elaise Hill4Andrew Nguyen5Michael H. Hsieh6Michael H. Hsieh7Michael H. Hsieh8Division of Pediatric Urology, Children’s National Hospital, Washington, DC, United StatesSheikh Zayed Institute, Children’s National Hospital, Washington, DC, United StatesSheikh Zayed Institute, Children’s National Hospital, Washington, DC, United StatesSchool of Medicine and Health Sciences, The George Washington University, Washington, DC, United StatesCenter for Cancer and Immunology Research, Children’s National Hospital, Washington, DC, United StatesSchool of Medicine and Health Sciences, The George Washington University, Washington, DC, United StatesDivision of Pediatric Urology, Children’s National Hospital, Washington, DC, United StatesSheikh Zayed Institute, Children’s National Hospital, Washington, DC, United StatesSchool of Medicine and Health Sciences, The George Washington University, Washington, DC, United StatesBackgroundPolymerase chain reaction (PCR) is an important means by which to study the urine microbiome and is emerging as possible alternative to urine cultures to identify pathogens that cause urinary tract infection (UTI). However, PCR is limited by its inability to differentiate DNA originating from viable, metabolically active versus non-viable, inactive bacteria. This drawback has led to concerns that urobiome studies and PCR-based diagnosis of UTI are confounded by the presence of relic DNA from non-viable bacteria in urine. Propidium monoazide (PMA) dye can penetrate cells with compromised cell membranes and covalently bind to DNA, rendering it inaccessible to amplification by PCR. Although PMA has been shown to differentiate between non-viable and viable bacteria in various settings, its effectiveness in urine has not been previously studied. We sought to investigate the ability of PMA to differentiate between viable and non-viable bacteria in urine.MethodsVarying amounts of viable or non-viable uropathogenic E. coli (UTI89) or buffer control were titrated with mouse urine. The samples were centrifuged to collect urine sediment or not centrifuged. Urine samples were incubated with PMA and DNA cross-linked using blue LED light. DNA was isolated and uidA gene-specific PCR was performed. For in vivo studies, mice were inoculated with UTI89, followed by ciprofloxacin treatment or no treatment. After the completion of ciprofloxacin treatment, an aliquot of urine was plated on non-selective LB agar and another aliquot was treated with PMA and subjected to uidA-specific PCR.ResultsPMA’s efficiency in excluding DNA signal from non-viable bacteria was significantly higher in bacterial samples in phosphate-buffered saline (PBS, dCT=13.69) versus bacterial samples in unspun urine (dCT=1.58). This discrepancy was diminished by spinning down urine-based bacterial samples to collect sediment and resuspending it in PBS prior to PMA treatment. In 3 of 5 replicate groups of UTI89-infected mice, no bacteria grew in culture; however, there was PCR amplification of E. coli after PMA treatment in 2 of those 3 groups.ConclusionWe have successfully developed PMA-based PCR methods for amplifying DNA from live bacteria in urine. Our results suggest that non-PMA bound DNA from live bacteria can be present in urine, even after antibiotic treatment. This indicates that viable but non-culturable E. coli can be present following treatment of UTI, and may explain why some patients have persistent symptoms but negative urine cultures following UTI treatment.https://www.frontiersin.org/articles/10.3389/fcimb.2022.794323/fullpropidium monoazideviabilityurinemicrobiomenon-culturable bacteriaurobiome
spellingShingle Albert S. Lee
Olivia K. Lamanna
Kenji Ishida
Elaise Hill
Elaise Hill
Andrew Nguyen
Michael H. Hsieh
Michael H. Hsieh
Michael H. Hsieh
A Novel Propidium Monoazide-Based PCR Assay Can Measure Viable Uropathogenic E. coli In Vitro and In Vivo
Frontiers in Cellular and Infection Microbiology
propidium monoazide
viability
urine
microbiome
non-culturable bacteria
urobiome
title A Novel Propidium Monoazide-Based PCR Assay Can Measure Viable Uropathogenic E. coli In Vitro and In Vivo
title_full A Novel Propidium Monoazide-Based PCR Assay Can Measure Viable Uropathogenic E. coli In Vitro and In Vivo
title_fullStr A Novel Propidium Monoazide-Based PCR Assay Can Measure Viable Uropathogenic E. coli In Vitro and In Vivo
title_full_unstemmed A Novel Propidium Monoazide-Based PCR Assay Can Measure Viable Uropathogenic E. coli In Vitro and In Vivo
title_short A Novel Propidium Monoazide-Based PCR Assay Can Measure Viable Uropathogenic E. coli In Vitro and In Vivo
title_sort novel propidium monoazide based pcr assay can measure viable uropathogenic e coli in vitro and in vivo
topic propidium monoazide
viability
urine
microbiome
non-culturable bacteria
urobiome
url https://www.frontiersin.org/articles/10.3389/fcimb.2022.794323/full
work_keys_str_mv AT albertslee anovelpropidiummonoazidebasedpcrassaycanmeasureviableuropathogenicecoliinvitroandinvivo
AT oliviaklamanna anovelpropidiummonoazidebasedpcrassaycanmeasureviableuropathogenicecoliinvitroandinvivo
AT kenjiishida anovelpropidiummonoazidebasedpcrassaycanmeasureviableuropathogenicecoliinvitroandinvivo
AT elaisehill anovelpropidiummonoazidebasedpcrassaycanmeasureviableuropathogenicecoliinvitroandinvivo
AT elaisehill anovelpropidiummonoazidebasedpcrassaycanmeasureviableuropathogenicecoliinvitroandinvivo
AT andrewnguyen anovelpropidiummonoazidebasedpcrassaycanmeasureviableuropathogenicecoliinvitroandinvivo
AT michaelhhsieh anovelpropidiummonoazidebasedpcrassaycanmeasureviableuropathogenicecoliinvitroandinvivo
AT michaelhhsieh anovelpropidiummonoazidebasedpcrassaycanmeasureviableuropathogenicecoliinvitroandinvivo
AT michaelhhsieh anovelpropidiummonoazidebasedpcrassaycanmeasureviableuropathogenicecoliinvitroandinvivo
AT albertslee novelpropidiummonoazidebasedpcrassaycanmeasureviableuropathogenicecoliinvitroandinvivo
AT oliviaklamanna novelpropidiummonoazidebasedpcrassaycanmeasureviableuropathogenicecoliinvitroandinvivo
AT kenjiishida novelpropidiummonoazidebasedpcrassaycanmeasureviableuropathogenicecoliinvitroandinvivo
AT elaisehill novelpropidiummonoazidebasedpcrassaycanmeasureviableuropathogenicecoliinvitroandinvivo
AT elaisehill novelpropidiummonoazidebasedpcrassaycanmeasureviableuropathogenicecoliinvitroandinvivo
AT andrewnguyen novelpropidiummonoazidebasedpcrassaycanmeasureviableuropathogenicecoliinvitroandinvivo
AT michaelhhsieh novelpropidiummonoazidebasedpcrassaycanmeasureviableuropathogenicecoliinvitroandinvivo
AT michaelhhsieh novelpropidiummonoazidebasedpcrassaycanmeasureviableuropathogenicecoliinvitroandinvivo
AT michaelhhsieh novelpropidiummonoazidebasedpcrassaycanmeasureviableuropathogenicecoliinvitroandinvivo