Innovative Methodology for Antimicrobial Susceptibility Determination in <i>Mycoplasma</i> Biofilms
<i>Mycoplasma</i> spp. are facultative pathogens that contribute to the pathogenesis of multiple bovine diseases, including the bovine respiratory disease complex, and have been shown to form biofilms. Biofilm formation is associated with increased antibiotic resistance in many organisms...
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MDPI AG
2024-12-01
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| author | B. Tegner Jacobson Jessica DeWit-Dibbert Eli T. Selong McKenna Quirk Michael Throolin Chris Corona Sobha Sonar LaShae Zanca Erika R. Schwarz Diane Bimczok |
| author_facet | B. Tegner Jacobson Jessica DeWit-Dibbert Eli T. Selong McKenna Quirk Michael Throolin Chris Corona Sobha Sonar LaShae Zanca Erika R. Schwarz Diane Bimczok |
| author_sort | B. Tegner Jacobson |
| collection | DOAJ |
| description | <i>Mycoplasma</i> spp. are facultative pathogens that contribute to the pathogenesis of multiple bovine diseases, including the bovine respiratory disease complex, and have been shown to form biofilms. Biofilm formation is associated with increased antibiotic resistance in many organisms, but accurate determination of antimicrobial susceptibility in biofilms is challenging. In <i>Mycoplasma</i> spp., antimicrobial susceptibility is routinely determined using metabolic pH-dependent color change. However, biofilm formation can lead to reduced metabolism, making interpretation of metabolic readouts difficult. Therefore, we developed and optimized a new flow cytometry-based method for antimicrobial susceptibility testing in biofilm-forming <i>Mycoplasma</i>, termed the live/dead antimicrobial susceptibility test (LD-AST). The LD-AST measures the proportion of live bacteria upon exposure to antibiotics, works robustly with both planktonic and biofilm cultures, and enables the determination of the minimum bactericidal concentration (MBC) for a given antibiotic. We used two strains of <i>Mycoplasma bovis</i> (Donetta PG45 and Madison) and two clinical <i>Mycoplasma bovoculi</i> isolates (MVDL1 and MVDL2) to determine the impact of biofilm growth on antimicrobial susceptibility for gentamicin, enrofloxacin, or tetracycline. All <i>Mycoplasma</i> strains were susceptible to all antibiotics when cultured as planktonic cells, with MBCs in the expected range. However, three out of four strains (Donetta PG45, MVDL1, and MVDL2) were completely resistant to all three antibiotics when newly adhered biofilms were analyzed, whereas <i>M. bovis</i> Madison gave variable results. For mature biofilms that were cultured for 4–5 days before antibiotic exposure, results also were variable, with some strains showing an increased resistance with certain antibiotics and a decreased resistance with others. Overall, these results are consistent with earlier reports that biofilms can exhibit increased antimicrobial resistance. |
| format | Article |
| id | doaj-art-2d37b9d73d464ecab9632eeda99d37ea |
| institution | DOAJ |
| issn | 2076-2607 |
| language | English |
| publishDate | 2024-12-01 |
| publisher | MDPI AG |
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| series | Microorganisms |
| spelling | doaj-art-2d37b9d73d464ecab9632eeda99d37ea2025-08-20T02:50:41ZengMDPI AGMicroorganisms2076-26072024-12-011212265010.3390/microorganisms12122650Innovative Methodology for Antimicrobial Susceptibility Determination in <i>Mycoplasma</i> BiofilmsB. Tegner Jacobson0Jessica DeWit-Dibbert1Eli T. Selong2McKenna Quirk3Michael Throolin4Chris Corona5Sobha Sonar6LaShae Zanca7Erika R. Schwarz8Diane Bimczok9Department of Microbiology and Cell Biology, Montana State University, Bozeman, MT 59718, USADepartment of Microbiology and Cell Biology, Montana State University, Bozeman, MT 59718, USADepartment of Chemistry and Biochemistry, Montana State University, Bozeman, MT 59718, USADepartment of Microbiology and Cell Biology, Montana State University, Bozeman, MT 59718, USADepartment of Mathematical Sciences, Montana State University, Bozeman, MT 59718, USADepartment of Mathematical Sciences, Montana State University, Bozeman, MT 59718, USADepartment of Microbiology and Cell Biology, Montana State University, Bozeman, MT 59718, USADepartment of Microbiology and Cell Biology, Montana State University, Bozeman, MT 59718, USAMontana Veterinary Diagnostic Laboratory, Montana Department of Livestock, Bozeman, MT 59718, USADepartment of Microbiology and Cell Biology, Montana State University, Bozeman, MT 59718, USA<i>Mycoplasma</i> spp. are facultative pathogens that contribute to the pathogenesis of multiple bovine diseases, including the bovine respiratory disease complex, and have been shown to form biofilms. Biofilm formation is associated with increased antibiotic resistance in many organisms, but accurate determination of antimicrobial susceptibility in biofilms is challenging. In <i>Mycoplasma</i> spp., antimicrobial susceptibility is routinely determined using metabolic pH-dependent color change. However, biofilm formation can lead to reduced metabolism, making interpretation of metabolic readouts difficult. Therefore, we developed and optimized a new flow cytometry-based method for antimicrobial susceptibility testing in biofilm-forming <i>Mycoplasma</i>, termed the live/dead antimicrobial susceptibility test (LD-AST). The LD-AST measures the proportion of live bacteria upon exposure to antibiotics, works robustly with both planktonic and biofilm cultures, and enables the determination of the minimum bactericidal concentration (MBC) for a given antibiotic. We used two strains of <i>Mycoplasma bovis</i> (Donetta PG45 and Madison) and two clinical <i>Mycoplasma bovoculi</i> isolates (MVDL1 and MVDL2) to determine the impact of biofilm growth on antimicrobial susceptibility for gentamicin, enrofloxacin, or tetracycline. All <i>Mycoplasma</i> strains were susceptible to all antibiotics when cultured as planktonic cells, with MBCs in the expected range. However, three out of four strains (Donetta PG45, MVDL1, and MVDL2) were completely resistant to all three antibiotics when newly adhered biofilms were analyzed, whereas <i>M. bovis</i> Madison gave variable results. For mature biofilms that were cultured for 4–5 days before antibiotic exposure, results also were variable, with some strains showing an increased resistance with certain antibiotics and a decreased resistance with others. Overall, these results are consistent with earlier reports that biofilms can exhibit increased antimicrobial resistance.https://www.mdpi.com/2076-2607/12/12/2650antimicrobial resistance<i>Mycoplasma bovis</i><i>Mycoplasma</i> sp.biofilm formationflow cytometrylive/dead staining |
| spellingShingle | B. Tegner Jacobson Jessica DeWit-Dibbert Eli T. Selong McKenna Quirk Michael Throolin Chris Corona Sobha Sonar LaShae Zanca Erika R. Schwarz Diane Bimczok Innovative Methodology for Antimicrobial Susceptibility Determination in <i>Mycoplasma</i> Biofilms Microorganisms antimicrobial resistance <i>Mycoplasma bovis</i> <i>Mycoplasma</i> sp. biofilm formation flow cytometry live/dead staining |
| title | Innovative Methodology for Antimicrobial Susceptibility Determination in <i>Mycoplasma</i> Biofilms |
| title_full | Innovative Methodology for Antimicrobial Susceptibility Determination in <i>Mycoplasma</i> Biofilms |
| title_fullStr | Innovative Methodology for Antimicrobial Susceptibility Determination in <i>Mycoplasma</i> Biofilms |
| title_full_unstemmed | Innovative Methodology for Antimicrobial Susceptibility Determination in <i>Mycoplasma</i> Biofilms |
| title_short | Innovative Methodology for Antimicrobial Susceptibility Determination in <i>Mycoplasma</i> Biofilms |
| title_sort | innovative methodology for antimicrobial susceptibility determination in i mycoplasma i biofilms |
| topic | antimicrobial resistance <i>Mycoplasma bovis</i> <i>Mycoplasma</i> sp. biofilm formation flow cytometry live/dead staining |
| url | https://www.mdpi.com/2076-2607/12/12/2650 |
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