Antimicrobial susceptibility testing to evaluate minimum inhibitory concentration values of clinically relevant antibiotics

Summary Antimicrobial susceptibility testing is used to determine the minimum inhibitory concentration (MIC), the standard measurement of antibiotic activity. Here, we present a protocol for evaluating MIC values of clinically relevant antibiotics against bacterial isolates cultured in standard bacteriologic medium and in mammalian cell culture medium. We describe steps for pathogen identification, culturing bacteria, preparing MIC plates, MIC assay incubation, and determining MIC. This protocol can potentially optimize the use of existing antibiotics while enhancing efforts to discover new ones. For complete details on the use and execution of this protocol, please refer to Heithoff et al.1


SUMMARY
Antimicrobial susceptibility testing is used to determine the minimum inhibitory concentration (MIC), the standard measurement of antibiotic activity. Here, we present a protocol for evaluating MIC values of clinically relevant antibiotics against bacterial isolates cultured in standard bacteriologic medium and in mammalian cell culture medium. We describe steps for pathogen identification, culturing bacteria, preparing MIC plates, MIC assay incubation, and determining MIC. This protocol can potentially optimize the use of existing antibiotics while enhancing efforts to discover new ones. For complete details on the use and execution of this protocol, please refer to Heithoff et al. 1

BEFORE YOU BEGIN
Antimicrobial resistance (AMR) to existing medications is one of the biggest challenges facing public healthcare. 2 Antimicrobial susceptibility testing (AST) is used to determine the minimum inhibitory concentration (MIC), the standard measurement of antibiotic activity. MICs define the clinical breakpoint, the concentration of antibiotic used to indicate whether an infection with a particular bacterial isolate is likely to be treatable in a patient. Clinical breakpoints are used by clinical microbiological laboratories to define patient isolates as susceptible (S), intermediate (I), or resistant (R) to a panel of antibiotics. Thus, the MIC assay is the gold standard for guiding physician treatment practices.
This protocol evaluates MIC values of clinically relevant antibiotics against bacterial isolates cultured in standard bacteriologic medium (cation-adjusted Mueller-Hinton broth [CAMHB]) and in mammalian cell culture medium (Dulbecco's modified Eagle's medium [DMEM]). 1,3,4 Before commencing AST, it is essential to prepare the required reagents (media, buffers, antibiotics) ( Table 1); identify the pathogen to be tested; select antibiotic concentration test ranges; and determine pathogen growth conditions to obtain adequate densities for reliable MIC determination. iii. Select antibiotic and view predicted susceptibility profile for each pathogen (e.g., Staphylococcus aureus susceptibility to ciprofloxacin) ( Figure 1). iv. Select a continuous range of ten 2-fold dilutions that encompass the clinical breakpoints used to categorize bacterial isolates as susceptible (S) or resistant (R) (if available) using the CLSI 6 and EUCAST 7 databases; e.g., https://www.eucast.org/clinical_breakpoints (Figures 1 and 2). i. Serially dilute bacterial culture 1:10; repeat 5-7 times; plate 100 mL of last 3 dilutions on bacteriological media (step-by-step method details, Step 1). ii. Count colonies after 18 h incubation. iii. Calculate colony forming units (cfu/mL) according to the dilution factor (avg. of 3 replicates).
Note: Alternatively, OD 600 can be used to estimate cfu/mL; however, cfu/mL equivalents can vary between and within bacterial species.  Environmental sensitization to physiologic conditions during bacterial culture and AST can have up to a 1000-fold effect on antibiotic susceptibility. 15 Consequentially, physiologic conditions should be implemented for any standardized AST protocol for widespread clinical utility. Detailed below is an AST protocol whereby both bacterial culture and MIC assays are performed in standard CAMHB and in DMEM cell culture medium. i. Pipette 50 mL of antibiotic from wells in column 1 into column 2. ii. Pipette up and down 3 times; repeat serial dilutions from wells in columns 3 through 10. iii. Discard 50 mL from wells in column 10.
Note: Cfu/mL for each pathogen/media was already determined by direct colony count (see before you begin, Step 4).
i. Transfer 100 mL to microfuge tube to verify inoculum cfu/mL in Step 6.
ii. Decant remaining 7 mL to sterile Petri dish (to facilitate pipetting). b. Add 50 mL of 2 3 bacterial inoculum to all wells except column 12 (media only). c. Add 50 mL of additional media to wells in column 12 (media only).

Alternate AST protocol for human sera or urine
Timing: same as CAMHB/DMEM protocol AST in human sera and urine presents a formidable challenge as these host fluids can be inhibitory to bacterial culture. Some pathogens form bacterial cell-to-cell aggregates in sera and/or do not grow to adequate bacterial cell densities in sera or urine for reliable MIC determination. Detailed below is an AST protocol developed for pooled human donor sera or urine ( Figure 5).
Step 1, step-by-step method details. 10. Culture bacterium in undiluted pooled human donor sera or urine.
Step 3, step-by-step method details.
Step 7, step-by-step method details. 16. Interpret MIC value with respect to clinical breakpoints. a.
Step 8, step-by-step method details.

EXPECTED OUTCOMES
Clinical implementation of testing in cell culture medium may identify existing antibiotics for the potential treatment of AMR infections that are rejected by standard testing based on standard

OPEN ACCESS
bacteriologic medium; and antibiotics that are ineffective despite indicated use by standard testing. Testing in DMEM revealed that b-lactam antibiotics were effective for the treatment of S. aureus in murine models of sepsis despite being rejected by testing in CAMHB (R to S, Table 2). Reciprocally, testing in DMEM revealed that colistin was ineffective for the treatment of A. baumannii, K. pneumoniae, or P. aeruginosa despite indicated use by testing in CAMHB (S to I/R). These data suggest that an AST experimental pipeline based on cell culture medium may improve the means by which antibiotics are tested, developed and prescribed. The protocol enables growth support for most bacterial isolates observed in clinical practice, and can be readily adapted to existing protocols and instrumentation. These features make the methodological transition to cell culture medium simple, scalable and affordable. Additionally, the experimental AST protocol based on human sera or urine has potential application for the translational development of precision personalized medicine that optimizes the identification and prescription of appropriate antibiotics for individual patients. Taken together, the experimental AST protocols described herein provide a platform for the discovery and development of new compounds as more accurate testing streamlines the identification of lead candidates early in the discovery process, potentially leading to significant time, cost and life savings. 1

LIMITATIONS
The AST experimental pipeline has the following limitations. First, MIC assays performed in vitro do not recapitulate all interactions between antibiotics and the host/bacterial pathogen, which can have a marked impact on drug potency. Second, results from the AST experimental pipeline cannot be generalized for MIC determinations within a species until a large number of clinical isolates are tested to ensure sufficient clinical representation. Third, clinical outcomes derived from systemic infection may not apply to localized infections (respiratory, skin, UTI) and thus, testing in physiologic media more representative of the corresponding site of infection might increase diagnostic accuracy. Last, the safety and efficacy of antibiotics identified by the experimental pipeline in animals must be confirmed in human studies before they can be generalized for patient treatment.

TROUBLESHOOTING Problem 1
Insufficient bacterial growth during cell culture and/or MIC assay (Step 2, 7, step-by-step method details).

Potential solution
Media supplementation with rich media (LB, CAMHB or TSB) at 30% v/v. Increase supplemented above 30% v/v.

Potential solution
MIC > highest drug concentration tested (all test wells are turbid); retest with higher drug concentration range.
MIC < lowest drug concentration tested (none of the test wells are turbid); retest with lower drug concentration range.

Problem 3
Inconsistent bacterial growth in sera during cell culture and/or MIC assay (Step 10, 15, alternate AST protocol for human sera or urine).

Potential solution
Increase vortex time to disrupt bacterial cell-to-cell aggregates.
Minimize standing time before cell dilution series and bacterial plating. MICs and susceptibility designations were determined by broth microdilution in CAMHB and DMEM. [16][17][18] Virulence assays: discordant MICs derived from AST in CAMHB and DMEM were tested for diagnostic accuracy in murine sepsis models (n = 10). 10