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Enhancing Antimicrobial Susceptibility Testing for Acinetobacter baumannii Using Physiologically Relevant Culture Media and Biofilm Formation Assays

PMCID: PMC12452809

PMID: 40981720

Abstract

Abstract Acinetobacter baumannii is a high‐risk pathogen associated with increased patient morbidity and mortality. Host‐pathogen interactions amplify its virulence, in part by promoting biofilm formation—a crucial factor in antimicrobial resistance and persistence. Given the bacterium's strong propensity for acquiring resistance, antimicrobial susceptibility testing (AST) is essential for guiding effective therapeutic interventions. However, discrepancies have been observed between in vitro AST results and therapeutic outcomes, with some antimicrobials being deemed to show in vivo efficacy despite appearing ineffective in vitro . This discordance may stem from traditional AST protocols, which rely on bacteriological media such as Mueller Hinton broth (MHB) optimized for bacterial growth but not for mimicking the host environment. Moreover, conventional AST does not account for virulence traits such as biofilm formation, which further contribute to treatment failure. Incorporating physiologically relevant culture media, such as Roswell Park Memorial Institute (RPMI) 1640 medium, alongside assessment of biofilm formation may improve the predictive value of AST. This work outlines two complementary protocols for improving AST interpretation in A. baumannii infections. Basic Protocol 1 compares minimum inhibitory concentration (MIC) values generated using MHB and RPMI. Basic Protocol 2 evaluates biofilm formation in MHB, tryptic soy broth (TSB; control), and RPMI, with and without antimicrobial exposure. Together, these approaches aim to inform alternative AST strategies that better reflect in vivo conditions and optimize therapeutic decision‐making. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1 : Comparing A. baumannii minimum inhibitory concentration (MIC) results in bacteriological (MHB) versus physiological (RPMI) media Basic Protocol 2 : Comparing A. baumannii isolate(s) biofilm formation following assays completed in bacteriological culture media (MHB and control TSB) and physiological medium (RPMI)

Full Text


Acinetobacter baumannii, a non‐fermenting Gram‐negative organism, is a significant cause of nosocomial infections, including ventilator‐associated pneumonia and central‐line‐associated bloodstream infections (Gidey et al., 2023; Peleg et al., 2008; Shadan et al., 2023). Because of its ability to render most therapeutics—including salvage therapies such as carbapenems and polymyxins—ineffective, the World Health Organization (WHO) has designated A. baumannii a global priority pathogen urgently requiring novel treatments (Abdul‐Mutakabbir et al., 2021; Asokan et al., 2019). Notably, host‐pathogen interactions contribute to its resistance (Eze et al., 2018; Pires & Parker, 2019; Sato et al., 2020). For example, during A. baumannii infection, host cells mount a robust inflammatory response upon recognizing pathogen‐associated molecular patterns via receptors such as TLR4 (activated by lipooligosaccharide [LOS] lipid A) and TLR9 (which detects internalized bacterial DNA). These pathways initiate signaling cascades that elevate proinflammatory cytokines and chemokines, which are essential for pathogen clearance (Chen, 2020; Li et al., 2019). In turn, A. baumannii employs sophisticated immune evasion strategies, including subverting the complement cascade through outer membrane proteins such as OmpA and producing a capsule that impairs opsonization and complement deposition (Shadan et al., 2023; Zhang et al., 2022).
A hallmark of A. baumannii pathogenicity—and a key contributor to its therapeutic recalcitrance—is biofilm formation (Gedefie et al., 2021; Harding et al., 2018). Biofilms are structured microbial communities encased in an extracellular polymeric substance (EPS) matrix, in which bacteria are shielded from antimicrobial agents and immune effectors. Within biofilms, bacteria adopt a metabolically dormant and phenotypically reversible state. Biofilms can form on both biotic and abiotic surfaces, creating highly variable microenvironments characterized by nutrient and pH gradients, which result in heterogeneous bacterial populations (Ambrosi et al., 2020; Maure et al., 2023; Roy et al., 2022). Like OmpA, biofilms modulate host‐pathogen interactions by dampening local immune responses; the matrix impedes effector cell penetration and inhibits complement activation, thereby interfering with opsonization and reducing phagocytosis. The interplay between biofilm formation and immune evasion contributes to the development of chronic infections and complicates treatment in nosocomial settings (Eze et al., 2018; García‐Patiño et al., 2017; Li et al., 2020; Magda et al., 2022; Roy et al., 2022; Schulze et al., 2021).
Treatment of A. baumannii infections typically relies on antimicrobial susceptibility testing (AST) to guide therapy (Pierce et al., 2023). Accurate AST is therefore paramount for enabling prompt and effective clinical decision‐making. Historically, AST has evolved through iterative improvements aimed at enhancing diagnostic precision. Early methodologies, including broth and tube dilution techniques, established the framework for quantifying the minimum inhibitory concentration (MIC) of antimicrobials (Wheat, 2001). The broth microdilution method (BMD)—an adaptation that miniaturizes tube dilution to assess multiple drug concentrations simultaneously—has since become the gold standard for AST due to its reproducibility and quantitative rigor (Khan et al., 2019).
However, although BMD provides essential MIC data, it is optimized for in vitro conditions and does not fully replicate the complexities of the host environment (Heithoff et al., 2023; Miller et al., 2023; Nizet, 2017). Standard AST relies on Mueller Hinton broth (MHB), a bacteriological medium optimized for bacterial growth rather than physiological relevance (Nizet, 2017; Nussbaumer‐Pröll & Zeitlinger, 2020). Studies have demonstrated that the choice of testing medium can significantly influence the observed antimicrobial susceptibility (Heithoff et al., 2023; Makris et al., 2018). In contrast, Roswell Park Memorial Institute 1640 medium (RPMI 1640, hereafter RPMI), formulated initially for propagating immortalized cell lines, more closely mimics host physiology and is increasingly used in pharmacological research. RPMI contains components such as bicarbonate, essential for maintaining physiological pH, and glutathione, a vital intracellular antioxidant that neutralizes reactive oxygen species (ROS) in vivo—both absent in MHB (Berti et al., 2020; Nizet, 2017). This compositional difference enhances the physiological relevance of AST, as demonstrated in studies showing potent activities of antibiotics that were evident in RPMI but absent in MHB, and corroborated by effective bacterial killing in human serum and murine infection models (Miller et al., 2023; Nizet, 2017; Rubio et al., 2024).
In this article, we describe two protocols to assess the impact of culture medium on A. baumannii AST results and biofilm formation. Basic Protocol 1 details how to compare MIC values obtained via the traditional gold‐standard BMD methods following exposure of A. baumannii isolates to active agents in bacteriological (MHB) versus physiological (RPMI) media. Basic Protocol 2 outlines how to assess biofilm formation under host‐mimicking conditions by comparing the biofilm‐forming capacity of A. baumannii isolates in RPMI, tryptic soy broth (TSB), and MHB, with and without antimicrobial exposure, using a crystal violet assay
Basic Protocol 1 describes how to perform MIC testing with A. baumannii using active antimicrobial agents and the gold‐standard BMD method for AST. The protocol also outlines how to interpret and present MIC results obtained from testing in bacteriologic culture medium (Mueller Hinton Broth, MHB) and physiological culture medium (RPMI 1640); the workflow is outlined in Figure 1. To perform MIC testing via BMD, clinical isolates should be recovered from cryogenic stocks, diluted in sterile water, adjusted to a turbidity of 1.5 McFarland units (∼1.5 × 108 CFU/ml), and diluted 1:10. The isolates are then exposed to serially diluted A. baumannii‐active antimicrobials in both MHB and RPMI and incubated overnight at 37°C. MIC values from each medium should be recorded and analyzed to determine how culture conditions influence AST results. Although this protocol was applied specifically to evaluate MIC differences in 15 multidrug‐resistant (MDR) A. baumannii isolates collected during an outbreak at Loma Linda University Medical Center (LLUMC), it is readily adaptable to other Gram‐negative pathogens and antimicrobial agents. In this study, colistin (COL), a commonly used last‐resort agent for A. baumannii, was used as the test antimicrobial. Therefore, COL will be referenced throughout to illustrate the interpretation and presentation of results.
Refer to CLSI guidelines (Lewis et al., 2025) for MIC breakpoints, quality control strains, etc.
Ensure that final volume in each well is 100 µl (based on CLSI guidelines; Lewis et al., 2025).
Compare MICs generated in the different media, as shown in Figure 2.
Basic Protocol 2 describes how to assess the biofilm‐forming capacities of A. baumannii isolates in bacteriological (MHB and TSB) and physiological (RPMI) culture media, both in the presence and absence of active antimicrobial agents (for workflow, see Fig. 3). The protocol also outlines how to interpret and present the resulting data. To begin, overnight cultures of A. baumannii should be transferred into different bacteriological media, such as TSB, MHB, and RPMI, with TSB serving as a control to account for potential confounding effects of overnight growth conditions. The cultures are tested with and without A. baumannii‐active antimicrobials. After overnight incubation, bacterial suspensions are plated into wells of 96‐well microtiter plates and incubated statically at 37°C for 24 hr. Wells are then washed with distilled water, stained with 0.1% crystal violet (CV), rinsed, dried, and destained with ethanol. Biofilm biomass is quantified by measuring absorbance. This protocol was initially developed to assess biofilm formation in 15 multidrug‐resistant A. baumannii isolates. However, it is readily adaptable for evaluating biofilm dynamics in other Gram‐negative organisms. In our experiments, colistin (COL) was used as the representative antimicrobial; thus, COL is referenced throughout to illustrate how to present and interpret results.
Identify classification based of breakpoint value (according to CLSI guidelines; Lewis et al., 2025).
A p < .05 denotes statistical significance, as shown in Figure 4.
A p < .05 denotes statistical significance, and results can be visualized using a heat map as shown in Figure 5.
The current gold standard for antimicrobial susceptibility testing (AST) includes determining minimal inhibitory concentrations (MICs) using the broth microdilution (BMD) method in bacteriological medium (MHB), according to CLSI guidelines (Lewis, 2025). All experiments described in Basic Protocol 1 should follow these guidelines, with the only deviation being the substitution of MHB with physiological media (RPMI) in parallel testing.
The MIC values obtained are used to determine sub‐inhibitory concentrations for subsequent biofilm assays in Basic Protocol 2, making it essential to perform MIC testing before biofilm assessment. A. baumannii MIC testing via BMD has an approximate turnaround time of 24 hr. To ensure a consistent inoculum, clinical isolates should be cultured from cryogenic stock, and suspensions should be adjusted within the doubling time of the organism. The doubling time (DT) can be calculated using the formula:



In our study, the MDR A. baumannii isolates had an average doubling time of ∼50 min; thus, suspensions were seeded within ∼40 min of preparation (Antunes et al., 2011). Antimicrobial stock solutions should be prepared fresh daily to ensure consistency in final concentrations. Variability in stock preparation can affect reproducibility and interpretation of results.
During biofilm assays (Basic Protocol 2), include negative controls during day 2 overnight inoculations for quality control and contamination monitoring. Negative controls may contain drug, while sterile/blank wells should contain only medium. These should be clearly distinguished during data analysis. Handle microtiter plates gently to avoid biofilm disruption and minimize assay variance. Overstaining with CV can increase biofilm fixation but lead to uneven quantification; similarly, ensure that the destaining step is fully complete before measuring absorbance to achieve homogenous readings. Lastly, avoid using the outer wells of the 96‐well microtiter plates in testing for biofilm formation, as they are prone to evaporation, which can affect results.
For troubleshooting suggestions, refer to Table 1.
To assess the impact of culture medium on A. baumannii MIC values, we followed the procedures described in Basic Protocol 1. We observed a significant difference in colistin susceptibility (as shown in Fig. 2). Conducting a paired t‐test is recommended to account for intra‐isolate variability and increase statistical power.
To assess the impact of culture medium on A. baumannii biofilm formation, we followed the procedures described in Basic Protocol 2. In our study, we observed a significant difference in biofilm formation across media. Conducting a two‐way ANOVA to evaluate the independent and interactive effects of culture medium and antimicrobial exposure on biofilm mass is recommended. Results can be visualized in GraphPad Prism using a violin plot (as shown in Fig. 4) or a heatmap (as shown in Fig. 5) to illustrate isolate‐specific responses.

Sections

"[{\"pmc\": \"PMC12452809\", \"pmid\": \"40981720\", \"reference_ids\": [\"cpz170207-bib-0010\", \"cpz170207-bib-0023\", \"cpz170207-bib-0030\", \"cpz170207-bib-0001\", \"cpz170207-bib-0004\", \"cpz170207-bib-0007\", \"cpz170207-bib-0025\", \"cpz170207-bib-0028\", \"cpz170207-bib-0006\", \"cpz170207-bib-0015\", \"cpz170207-bib-0030\", \"cpz170207-bib-0032\"], \"section\": \"INTRODUCTION\", \"text\": \"\\nAcinetobacter baumannii, a non\\u2010fermenting Gram\\u2010negative organism, is a significant cause of nosocomial infections, including ventilator\\u2010associated pneumonia and central\\u2010line\\u2010associated bloodstream infections (Gidey et\\u00a0al., 2023; Peleg et\\u00a0al., 2008; Shadan et\\u00a0al., 2023). Because of its ability to render most therapeutics\\u2014including salvage therapies such as carbapenems and polymyxins\\u2014ineffective, the World Health Organization (WHO) has designated A. baumannii a global priority pathogen urgently requiring novel treatments (Abdul\\u2010Mutakabbir et\\u00a0al., 2021; Asokan et\\u00a0al., 2019). Notably, host\\u2010pathogen interactions contribute to its resistance (Eze et\\u00a0al., 2018; Pires & Parker, 2019; Sato et\\u00a0al., 2020). For example, during A. baumannii infection, host cells mount a robust inflammatory response upon recognizing pathogen\\u2010associated molecular patterns via receptors such as TLR4 (activated by lipooligosaccharide [LOS] lipid A) and TLR9 (which detects internalized bacterial DNA). These pathways initiate signaling cascades that elevate proinflammatory cytokines and chemokines, which are essential for pathogen clearance (Chen, 2020; Li et\\u00a0al., 2019). In turn, A. baumannii employs sophisticated immune evasion strategies, including subverting the complement cascade through outer membrane proteins such as OmpA and producing a capsule that impairs opsonization and complement deposition (Shadan et\\u00a0al., 2023; Zhang et\\u00a0al., 2022).\"}, {\"pmc\": \"PMC12452809\", \"pmid\": \"40981720\", \"reference_ids\": [\"cpz170207-bib-0009\", \"cpz170207-bib-0011\", \"cpz170207-bib-0002\", \"cpz170207-bib-0019\", \"cpz170207-bib-0026\", \"cpz170207-bib-0007\", \"cpz170207-bib-0008\", \"cpz170207-bib-0016\", \"cpz170207-bib-0017\", \"cpz170207-bib-0026\", \"cpz170207-bib-0029\"], \"section\": \"INTRODUCTION\", \"text\": \"A hallmark of A. baumannii pathogenicity\\u2014and a key contributor to its therapeutic recalcitrance\\u2014is biofilm formation (Gedefie et\\u00a0al., 2021; Harding et\\u00a0al., 2018). Biofilms are structured microbial communities encased in an extracellular polymeric substance (EPS) matrix, in which bacteria are shielded from antimicrobial agents and immune effectors. Within biofilms, bacteria adopt a metabolically dormant and phenotypically reversible state. Biofilms can form on both biotic and abiotic surfaces, creating highly variable microenvironments characterized by nutrient and pH gradients, which result in heterogeneous bacterial populations (Ambrosi et\\u00a0al., 2020; Maure et\\u00a0al., 2023; Roy et\\u00a0al., 2022). Like OmpA, biofilms modulate host\\u2010pathogen interactions by dampening local immune responses; the matrix impedes effector cell penetration and inhibits complement activation, thereby interfering with opsonization and reducing phagocytosis. The interplay between biofilm formation and immune evasion contributes to the development of chronic infections and complicates treatment in nosocomial settings (Eze et\\u00a0al., 2018; Garc\\u00eda\\u2010Pati\\u00f1o et\\u00a0al., 2017; Li et\\u00a0al., 2020; Magda et\\u00a0al., 2022; Roy et\\u00a0al., 2022; Schulze et\\u00a0al., 2021).\"}, {\"pmc\": \"PMC12452809\", \"pmid\": \"40981720\", \"reference_ids\": [\"cpz170207-bib-0024\", \"cpz170207-bib-0031\", \"cpz170207-bib-0013\"], \"section\": \"INTRODUCTION\", \"text\": \"Treatment of A. baumannii infections typically relies on antimicrobial susceptibility testing (AST) to guide therapy (Pierce et\\u00a0al., 2023). Accurate AST is therefore paramount for enabling prompt and effective clinical decision\\u2010making. Historically, AST has evolved through iterative improvements aimed at enhancing diagnostic precision. Early methodologies, including broth and tube dilution techniques, established the framework for quantifying the minimum inhibitory concentration (MIC) of antimicrobials (Wheat, 2001). The broth microdilution method (BMD)\\u2014an adaptation that miniaturizes tube dilution to assess multiple drug concentrations simultaneously\\u2014has since become the gold standard for AST due to its reproducibility and quantitative rigor (Khan et\\u00a0al., 2019).\"}, {\"pmc\": \"PMC12452809\", \"pmid\": \"40981720\", \"reference_ids\": [\"cpz170207-bib-0012\", \"cpz170207-bib-0020\", \"cpz170207-bib-0021\", \"cpz170207-bib-0021\", \"cpz170207-bib-0022\", \"cpz170207-bib-0012\", \"cpz170207-bib-0018\", \"cpz170207-bib-0005\", \"cpz170207-bib-0021\", \"cpz170207-bib-0020\", \"cpz170207-bib-0021\", \"cpz170207-bib-0027\"], \"section\": \"INTRODUCTION\", \"text\": \"However, although BMD provides essential MIC data, it is optimized for in vitro conditions and does not fully replicate the complexities of the host environment (Heithoff et\\u00a0al., 2023; Miller et\\u00a0al., 2023; Nizet, 2017). Standard AST relies on Mueller Hinton broth (MHB), a bacteriological medium optimized for bacterial growth rather than physiological relevance (Nizet, 2017; Nussbaumer\\u2010Pr\\u00f6ll & Zeitlinger, 2020). Studies have demonstrated that the choice of testing medium can significantly influence the observed antimicrobial susceptibility (Heithoff et\\u00a0al., 2023; Makris et\\u00a0al., 2018). In contrast, Roswell Park Memorial Institute 1640 medium (RPMI 1640, hereafter RPMI), formulated initially for propagating immortalized cell lines, more closely mimics host physiology and is increasingly used in pharmacological research. RPMI contains components such as bicarbonate, essential for maintaining physiological pH, and glutathione, a vital intracellular antioxidant that neutralizes reactive oxygen species (ROS) in vivo\\u2014both absent in MHB (Berti et\\u00a0al., 2020; Nizet, 2017). This compositional difference enhances the physiological relevance of AST, as demonstrated in studies showing potent activities of antibiotics that were evident in RPMI but absent in MHB, and corroborated by effective bacterial killing in human serum and murine infection models (Miller et\\u00a0al., 2023; Nizet, 2017; Rubio et\\u00a0al., 2024).\"}, {\"pmc\": \"PMC12452809\", \"pmid\": \"40981720\", \"reference_ids\": [\"cpz170207-prot-0001\", \"cpz170207-prot-0002\"], \"section\": \"INTRODUCTION\", \"text\": \"In this article, we describe two protocols to assess the impact of culture medium on A. baumannii AST results and biofilm formation. Basic Protocol 1 details how to compare MIC values obtained via the traditional gold\\u2010standard BMD methods following exposure of A. baumannii isolates to active agents in bacteriological (MHB) versus physiological (RPMI) media. Basic Protocol 2 outlines how to assess biofilm formation under host\\u2010mimicking conditions by comparing the biofilm\\u2010forming capacity of A. baumannii isolates in RPMI, tryptic soy broth (TSB), and MHB, with and without antimicrobial exposure, using a crystal violet assay\"}, {\"pmc\": \"PMC12452809\", \"pmid\": \"40981720\", \"reference_ids\": [\"cpz170207-prot-0001\", \"cpz170207-fig-0001\"], \"section\": \"COMPARING A. baumannii MINIMUM INHIBITORY CONCENTRATION (MIC) RESULTS IN BACTERIOLOGICAL (MHB) VERSUS PHYSIOLOGICAL (RPMI) MEDIA\", \"text\": \"Basic Protocol 1 describes how to perform MIC testing with A. baumannii using active antimicrobial agents and the gold\\u2010standard BMD method for AST. The protocol also outlines how to interpret and present MIC results obtained from testing in bacteriologic culture medium (Mueller Hinton Broth, MHB) and physiological culture medium (RPMI 1640); the workflow is outlined in Figure 1. To perform MIC testing via BMD, clinical isolates should be recovered from cryogenic stocks, diluted in sterile water, adjusted to a turbidity of 1.5 McFarland units (\\u223c1.5 \\u00d7 108 CFU/ml), and diluted 1:10. The isolates are then exposed to serially diluted A. baumannii\\u2010active antimicrobials in both MHB and RPMI and incubated overnight at 37\\u00b0C. MIC values from each medium should be recorded and analyzed to determine how culture conditions influence AST results. Although this protocol was applied specifically to evaluate MIC differences in 15 multidrug\\u2010resistant (MDR) A. baumannii isolates collected during an outbreak at Loma Linda University Medical Center (LLUMC), it is readily adaptable to other Gram\\u2010negative pathogens and antimicrobial agents.\\u00a0In this study, colistin (COL), a commonly used last\\u2010resort agent for A. baumannii, was used as the test antimicrobial. Therefore, COL will be referenced throughout to illustrate the interpretation and presentation of results.\"}, {\"pmc\": \"PMC12452809\", \"pmid\": \"40981720\", \"reference_ids\": [\"cpz170207-bib-0014\"], \"section\": \"Day 2: Perform MIC testing using the BMD method in both RPMI and MHB media\", \"text\": \"Refer to CLSI guidelines (Lewis et\\u00a0al., 2025) for MIC breakpoints, quality control strains, etc.\"}, {\"pmc\": \"PMC12452809\", \"pmid\": \"40981720\", \"reference_ids\": [\"cpz170207-bib-0014\"], \"section\": \"\", \"text\": \"Ensure that final volume in each well is 100 \\u00b5l (based on CLSI guidelines; Lewis et\\u00a0al., 2025).\"}, {\"pmc\": \"PMC12452809\", \"pmid\": \"40981720\", \"reference_ids\": [\"cpz170207-fig-0002\"], \"section\": \"\", \"text\": \"Compare MICs generated in the different media, as shown in Figure 2.\"}, {\"pmc\": \"PMC12452809\", \"pmid\": \"40981720\", \"reference_ids\": [\"cpz170207-prot-0002\", \"cpz170207-fig-0003\"], \"section\": \"COMPARING A. baumannii ISOLATE BIOFILM FORMATION FOLLOWING ASSAYS COMPLETED IN BACTERIOLOGICAL CULTURE MEDIA (MHB AND CONTROL TSB AND PHYSIOLOGICAL MEDIUM (RPMI)\", \"text\": \"Basic Protocol 2 describes how to assess the biofilm\\u2010forming capacities of A. baumannii isolates in bacteriological (MHB and TSB) and physiological (RPMI) culture media, both in the presence and absence of active antimicrobial agents (for workflow, see Fig. 3). The protocol also outlines how to interpret and present the resulting data. To begin, overnight cultures of A. baumannii should be transferred into different bacteriological media, such as TSB, MHB, and RPMI, with TSB serving as a control to account for potential confounding effects of overnight growth conditions. The cultures are tested with and without A. baumannii\\u2010active antimicrobials. After overnight incubation, bacterial suspensions are plated into wells of 96\\u2010well microtiter plates and incubated statically at 37\\u00b0C for 24 hr. Wells are then washed with distilled water, stained with 0.1% crystal violet (CV), rinsed, dried, and destained with ethanol. Biofilm biomass is quantified by measuring absorbance. This protocol was initially developed to assess biofilm formation in 15 multidrug\\u2010resistant A. baumannii isolates. However, it is readily adaptable for evaluating biofilm dynamics in other Gram\\u2010negative organisms. In our experiments, colistin (COL) was used as the representative antimicrobial; thus, COL is referenced throughout to illustrate how to present and interpret results.\"}, {\"pmc\": \"PMC12452809\", \"pmid\": \"40981720\", \"reference_ids\": [\"cpz170207-bib-0014\"], \"section\": \"\", \"text\": \"Identify classification based of breakpoint value (according to CLSI guidelines; Lewis et\\u00a0al., 2025).\"}, {\"pmc\": \"PMC12452809\", \"pmid\": \"40981720\", \"reference_ids\": [\"cpz170207-fig-0004\"], \"section\": \"\", \"text\": \"A p <\\u00a0.05 denotes statistical significance, as shown in Figure 4.\"}, {\"pmc\": \"PMC12452809\", \"pmid\": \"40981720\", \"reference_ids\": [\"cpz170207-fig-0005\"], \"section\": \"\", \"text\": \"A p <\\u00a0.05 denotes statistical significance, and results can be visualized using a heat map as shown in Figure 5.\"}, {\"pmc\": \"PMC12452809\", \"pmid\": \"40981720\", \"reference_ids\": [\"cpz170207-bib-0014\", \"cpz170207-prot-0001\"], \"section\": \"Critical Parameters\", \"text\": \"The current gold standard for antimicrobial susceptibility testing (AST) includes determining minimal inhibitory concentrations (MICs) using the broth microdilution (BMD) method in bacteriological medium (MHB), according to CLSI guidelines (Lewis, 2025). All experiments described in Basic Protocol\\u00a01 should follow these guidelines, with the only deviation being the substitution of MHB with physiological media (RPMI) in parallel testing.\"}, {\"pmc\": \"PMC12452809\", \"pmid\": \"40981720\", \"reference_ids\": [\"cpz170207-prot-0002\"], \"section\": \"Critical Parameters\", \"text\": \"The MIC values obtained are used to determine sub\\u2010inhibitory concentrations for subsequent biofilm assays in Basic Protocol 2, making it essential to perform MIC testing before biofilm assessment. A. baumannii MIC testing via BMD has an approximate turnaround time of 24 hr. To ensure a consistent inoculum, clinical isolates should be cultured from cryogenic stock, and suspensions should be adjusted within the doubling time of the organism. The doubling time (DT) can be calculated using the formula:\\n\\n\\n\"}, {\"pmc\": \"PMC12452809\", \"pmid\": \"40981720\", \"reference_ids\": [\"cpz170207-bib-0003\"], \"section\": \"Critical Parameters\", \"text\": \"In our study, the MDR A. baumannii isolates had an average doubling time of \\u223c50 min; thus, suspensions were seeded within \\u223c40 min of preparation (Antunes et\\u00a0al., 2011). Antimicrobial stock solutions should be prepared fresh daily to ensure consistency in final concentrations. Variability in stock preparation can affect reproducibility and interpretation of results.\"}, {\"pmc\": \"PMC12452809\", \"pmid\": \"40981720\", \"reference_ids\": [\"cpz170207-prot-0002\"], \"section\": \"Critical Parameters\", \"text\": \"During biofilm assays (Basic Protocol\\u00a02), include negative controls during day 2 overnight inoculations for quality control and contamination monitoring. Negative controls may contain drug, while sterile/blank wells should contain only medium. These should be clearly distinguished during data analysis. Handle microtiter plates gently to avoid biofilm disruption and minimize assay variance. Overstaining with CV can increase biofilm fixation but lead to uneven quantification; similarly, ensure that the destaining step is fully complete before measuring absorbance to achieve homogenous readings. Lastly, avoid using the outer wells of the 96\\u2010well microtiter plates in testing for biofilm formation, as they are prone to evaporation, which can affect results.\"}, {\"pmc\": \"PMC12452809\", \"pmid\": \"40981720\", \"reference_ids\": [\"cpz170207-tbl-0001\"], \"section\": \"Troubleshooting\", \"text\": \"For troubleshooting suggestions, refer to Table 1.\"}, {\"pmc\": \"PMC12452809\", \"pmid\": \"40981720\", \"reference_ids\": [\"cpz170207-prot-0001\", \"cpz170207-fig-0002\"], \"section\": \"Understanding Results\", \"text\": \"To assess the impact of culture medium on A. baumannii MIC values, we followed the procedures described in Basic Protocol 1. We observed a significant difference in colistin susceptibility (as shown in Fig. 2). Conducting a paired t\\u2010test is recommended to account for intra\\u2010isolate variability and increase statistical power.\"}, {\"pmc\": \"PMC12452809\", \"pmid\": \"40981720\", \"reference_ids\": [\"cpz170207-prot-0002\", \"cpz170207-fig-0004\", \"cpz170207-fig-0005\"], \"section\": \"Understanding Results\", \"text\": \"To assess the impact of culture medium on A. baumannii biofilm formation, we followed the procedures described in Basic Protocol 2. In our study, we observed a significant difference in biofilm formation across media. Conducting a two\\u2010way ANOVA to evaluate the independent and interactive effects of culture medium and antimicrobial exposure on biofilm mass is recommended. Results can be visualized in GraphPad Prism using a violin plot (as shown in Fig. 4) or a heatmap (as shown in Fig. 5) to illustrate isolate\\u2010specific responses.\"}]"

Metadata

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