46c0a792fda43ef3ef6825c48e35eabb 322590.pdf 8bfa6c7389c4a902735f9a460500aaa0492547e9 322590.pdf 0addb7d0882874042d2403a067ed7bb96692e2cfcc7af94e11d55c028c6b2f27 322590.pdf Title: Design and Validation of a Simplified Method to Determine Minimum Bactericidal Concentration in Nontuberculous Mycobacteria Subject: Background/Objectives: Nontuberculous mycobacteria (NTM) infections are rising, particularly those by Mycobacterium avium complex (MAC) and Mycobacterium abscessus complex (MAB). Treating NTM infections is challenging due to their poor response to antibiotics. This study aimed to optimize the treatment of NTM infection by selecting antibiotics with bactericidal activity for combination therapy. To do this, we used the minimum bactericidal concentration (MBC) determination approach to define bactericidal or bacteriostatic activity. We developed three main objectives: validate a new method to determine MBC based on a reincubation method, determine MBC values of 229 NTM clinical isolates using the reincubation method, and evaluate antibiotic stability in preincubated microtiter plates. Methods: First, we assessed the stability of the antibiotics included in SLOWMYCOI Sensititre™ microtiter plates. Five strains of MAC were studied comparing the minimum inhibitory concentrations (MICs) of those preincubated for seven days vs. non-incubated plates. Then, we evaluated the percentage of reproducibility of MBC values using two methods, reincubation and subculturing (standard or traditional method) in 30 MAC isolates. Finally, we validated the reincubation method and prospectively determined the MBC values of the 229 NTM clinical strains. Results: Antibiotic stability: The MIC was equivalent after 7 and 14 days for all the antibiotics, except rifampicin, for which the MIC increased by 2- to 3-fold after preincubation. Reincubation method: The percentage of reproducibility of the MBC values between the two methods was 95.2% (range 76.6% to 100%). Prospective validation: MBC/MIC ratios revealed differential bactericidal activity for most antibiotics according to the different species, being bactericidal in M. avium and Mycobacterium xenopi, and predominantly bacteriostatic in MAB. Conclusions: Preincubation of Sensititre™ microtiter plates did not alter the MIC values of the antibiotics included except for rifampicin, suggesting a loss of activity. MBC determination can be easily performed by the Reincubation method presented. MBC values provide useful additional information regarding MIC values since the MBC/MIC ratio reveals whether antibiotics have bactericidal or bacteriostatic activity according to the species, which is pivotal for selecting the most adequate antibiotic combination to ensure efficient treatment management. Keywords: nontuberculous mycobacteria (NTM); reincubation method; subculturing method; SLOMYCOI Sensititre; antibiotics; minimum bactericidal concentration (MBC); bacteriostatic/bactericidal activity Author: Sara Batista, Mariana Fernández-Pittol, Lorena San Nicolás, Diego Martínez, Sofía Narváez, Mateu Espasa, Elena Garcia Losilla, Marc Rubio, Montserrat Garrigo, Griselda Tudó and Julian González-Martin Creator: LaTeX with hyperref Producer: pdfTeX-1.40.25 CreationDate: Mon Apr 7 07:42:22 2025 CEST ModDate: Mon Apr 7 08:52:23 2025 CEST Custom Metadata: no Metadata Stream: no Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 12 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 1358992 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- PLOLAK+VnURWPalladioL Type 1 Custom yes yes yes 10 0 BCXLPW+URWPalladioL-Roma Type 1 Custom yes yes yes 16 0 WBOJEO+URWPalladioL-Bold Type 1 Custom yes yes yes 22 0 WELHUF+URWPalladioL-Ital Type 1 Custom yes yes yes 27 0 FHBIAZ+EURB7 Type 1 Builtin yes yes yes 56 0 VRITEV+CMSY10 Type 1 Builtin yes yes yes 61 0 BVBEYZ+URWPalladioL-BoldItal Type 1 Custom yes yes yes 66 0 CAMXTK+EURM10 Type 1 Builtin yes yes yes 75 0 BCDEEE+PalatinoLinotype-Bold TrueType WinAnsi yes yes no 84 0 BCDFEE+PalatinoLinotype-Roman TrueType WinAnsi yes yes no 88 0 BCDGEE+PalatinoLinotype-Italic TrueType WinAnsi yes yes no 92 0 BCDHEE+PalatinoLinotype-Italic CID TrueType Identity-H yes yes yes 96 0 BCDIEE+PalatinoLinotype-Bold CID TrueType Identity-H yes yes yes 102 0 BCDJEE+PalatinoLinotype-Roman CID TrueType Identity-H yes yes yes 108 0 BCDEEE+PalatinoLinotype-Bold TrueType WinAnsi yes yes no 124 0 BCDFEE+PalatinoLinotype-Roman TrueType WinAnsi yes yes no 128 0 BCDGEE+PalatinoLinotype-Italic TrueType WinAnsi yes yes no 132 0 BCDHEE+PalatinoLinotype-Italic CID TrueType Identity-H yes yes yes 136 0 BCDIEE+PalatinoLinotype-Bold CID TrueType Identity-H yes yes yes 142 0 BCDJEE+PalatinoLinotype-Roman CID TrueType Identity-H yes yes yes 148 0 BCDEEE+PalatinoLinotype-Bold TrueType WinAnsi yes yes no 161 0 BCDFEE+PalatinoLinotype-Roman TrueType WinAnsi yes yes no 165 0 BCDGEE+PalatinoLinotype-Italic TrueType WinAnsi yes yes no 169 0 BCDHEE+PalatinoLinotype-Italic CID TrueType Identity-H yes yes yes 173 0 BCDIEE+PalatinoLinotype-Bold CID TrueType Identity-H yes yes yes 179 0 BCDJEE+PalatinoLinotype-Roman CID TrueType Identity-H yes yes yes 185 0 Jhove (Rel. 1.28.0, 2023-05-18) Date: 2025-12-03 04:05:42 CET RepresentationInformation: 322590.pdf ReportingModule: PDF-hul, Rel. 1.12.4 (2023-03-16) LastModified: 2025-12-02 10:14:46 CET Size: 1358992 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 342 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: section.1 Item: Title: Results Destination: section.2 Children: Item: Title: Antibiotic Stability Destination: subsection.2.1 Item: Title: Pilot Assay Destination: subsection.2.2 Item: Title: Implementation of the Reincubation Method Destination: subsection.2.3 Item: Title: Discussion Destination: section.3 Item: Title: Materials and Methods Destination: section.4 Children: Item: Title: Antibiotic Stability Destination: subsection.4.1 Children: Item: Title: Isolate Selection Destination: subsubsection.4.1.1 Item: Title: MIC Determination Destination: subsubsection.4.1.2 Item: Title: The New Reincubation Method to Evaluate MBC Determination: Pilot Assay Destination: subsection.4.2 Children: Item: Title: Isolate Selection Destination: subsubsection.4.2.1 Item: Title: MIC Determination Destination: subsubsection.4.2.2 Item: Title: MBC Determination by the Standard Method Destination: subsubsection.4.2.3 Item: Title: MBC Determination by the Reincubation Method Destination: subsubsection.4.2.4 Item: Title: Implementation of the Reincubation Method Destination: subsection.4.3 Children: Item: Title: Isolate Recruitment Destination: subsubsection.4.3.1 Item: Title: MIC Determination Destination: subsubsection.4.3.2 Item: Title: MBC Determination by the Reincubation Method Destination: subsubsection.4.3.3 Item: Title: Conclusions Destination: section.5 Item: Title: References Destination: appendix.A. Info: Title: Design and Validation of a Simplified Method to Determine Minimum Bactericidal Concentration in Nontuberculous Mycobacteria Author: Sara Batista, Mariana Fernández-Pittol, Lorena San Nicolás, Diego Martínez, Sofía Narváez, Mateu Espasa, Elena Garcia Losilla, Marc Rubio, Montserrat Garrigo, Griselda Tudó and Julian González-Martin Subject: Background/Objectives: Nontuberculous mycobacteria (NTM) infections are rising, particularly those by Mycobacterium avium complex (MAC) and Mycobacterium abscessus complex (MAB). Treating NTM infections is challenging due to their poor response to antibiotics. This study aimed to optimize the treatment of NTM infection by selecting antibiotics with bactericidal activity for combination therapy. To do this, we used the minimum bactericidal concentration (MBC) determination approach to define bactericidal or bacteriostatic activity. We developed three main objectives: validate a new method to determine MBC based on a reincubation method, determine MBC values of 229 NTM clinical isolates using the reincubation method, and evaluate antibiotic stability in preincubated microtiter plates. Methods: First, we assessed the stability of the antibiotics included in SLOWMYCOI Sensititre™ microtiter plates. Five strains of MAC were studied comparing the minimum inhibitory concentrations (MICs) of those preincubated for seven days vs. non-incubated plates. Then, we evaluated the percentage of reproducibility of MBC values using two methods, reincubation and subculturing (standard or traditional method) in 30 MAC isolates. Finally, we validated the reincubation method and prospectively determined the MBC values of the 229 NTM clinical strains. Results: Antibiotic stability: The MIC was equivalent after 7 and 14 days for all the antibiotics, except rifampicin, for which the MIC increased by 2- to 3-fold after preincubation. Reincubation method: The percentage of reproducibility of the MBC values between the two methods was 95.2% (range 76.6% to 100%). Prospective validation: MBC/MIC ratios revealed differential bactericidal activity for most antibiotics according to the different species, being bactericidal in M. avium and Mycobacterium xenopi, and predominantly bacteriostatic in MAB. Conclusions: Preincubation of Sensititre™ microtiter plates did not alter the MIC values of the antibiotics included except for rifampicin, suggesting a loss of activity. MBC determination can be easily performed by the Reincubation method presented. MBC values provide useful additional information regarding MIC values since the MBC/MIC ratio reveals whether antibiotics have bactericidal or bacteriostatic activity according to the species, which is pivotal for selecting the most adequate antibiotic combination to ensure efficient treatment management. 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