Correcting a Fundamental Flaw in the Paradigm for Antimicrobial Susceptibility Testing

Abstract

The emergence and prevalence of antibiotic-resistant bacteria are an increasing cause of death worldwide, resulting in a global 'call to action' to avoid receding into an era lacking effective antibiotics. Despite the urgency, the healthcare industry still relies on a single in vitro bioassay to determine antibiotic efficacy. This assay fails to incorporate environmental factors normally present during host-pathogen interactions in vivo that significantly impact antibiotic efficacy. Here we report that standard antimicrobial susceptibility testing (AST) failed to detect antibiotics that are in fact effective in vivo; and frequently identified antibiotics that were instead ineffective as further confirmed in mouse models of infection and sepsis. Notably, AST performed in media mimicking host environments succeeded in identifying specific antibiotics that were effective in bacterial clearance and host survival, even though these same antibiotics failed in results using standard test media. Similarly, our revised media further identified antibiotics that were ineffective in vivo despite passing the AST standard for clinical use. Supplementation of AST medium with sodium bicarbonate, an abundant in vivo molecule that stimulates global changes in bacterial structure and gene expression, was found to be an important factor improving the predictive value of AST in the assignment of appropriate therapy. These findings have the potential to improve the means by which antibiotics are developed, tested, and prescribed.

Keywords: Antibiotic resistance; Antimicrobial susceptibility testing (AST); Antimicrobial therapy; MIC testing; Multidrug-resistant pathogens.

Graphical abstract

Fig. 1

Comparison of pathogen-antibiotic combinations that exhibited altered MICs derived from host-mimicking media relative to standard MHB medium. A panel of antibiotics was screened for altered MICs against (a) Staphylococcus spp., (b) S. pneumoniae, and (c) Gram-negative bacteria when tested in Dulbecco's Modified Eagle Medium (DMEM), Lacks medium, modified Lacks medium (MLM), low-phosphate, low-magnesium medium (LPM pH 5.5) relative to standard MHB medium, according to CLSI guidelines (Clinical and Laboratory Standards Institute, 2012a, Wiegand et al., 2008). Values depict the fold-change in MICs when derived in host-mimicking media relative to standard MHB medium (test/standard condition). Increased susceptibility depicted in blue; increased resistance depicted in red. MIC values were obtained from at least 6 independent determinations.

Fig. 2

Comparison summary of MICs derived from host-mimicking media versus standard MHB medium. (a) Colored regions depict the fraction of pathogen-antibiotic combinations tested that exhibited a fold-change in MICs (increased susceptibility or resistance) when derived in host-mimicking media (DMEM, MLM, LPM pH 5.5) relative to standard MHB medium (test/standard condition); ≤ 2-fold (green), 4-fold (yellow), ≥ 8-fold (red). (b) Depicted are percentages of pathogen-antibiotic combinations that resulted in altered MICs that crossed clinical breakpoint designations, used to define isolates as susceptible (“S”), intermediate (“I”), or resistant (“R”), that can impact clinical decision making on appropriate antibiotic therapy (Clinical and Laboratory Standards Institute, 2012a, European Committee on Antibiotic Susceptibility Testing, 2014).

Fig. 3

Antibiotic susceptibility testing in host-mimicking media improves the predictive value of AST in the assignment of appropriate antibiotic therapy in murine models of sepsis. Pathogen-antibiotic combinations that exhibited altered MICs in host-mimicking media relative to standard MHB medium and whose MICs crossed clinical breakpoint designations were evaluated in murine sepsis models of (a) S. aureus (MRSA [USA300]; MSSA Wound [MT3307]; MRSA Blood [MT3302]); (b) S. pneumoniae (SPN Daw25); and (c) S. Typhimurium (ST 14028) and K. pneumoniae (KPN ATCC13883). (d) MSSA (MT3307) clearance from blood circulation was examined following treatment with antibiotics predicted as highly effective via testing in standard MHB medium (co-trimoxazole, open boxes) or tissue culture medium (DMEM) (azithromycin, closed boxes), respectively. Untreated mice (open circles); expired mice (gray region); Colony Forming Units (CFU); Limit of Detection (LOD) = 100 CFU/ml (Patterson et al., 2013). Ten mice were evaluated per cohort. ***P < 0.001, **P < 0.01, or *P < 0.05.

Fig. 4

Supplementation of standard MHB medium with physiological levels of NaHCO₃ improves the predictive value of AST in the assignment of appropriate antibiotics for therapeutic intervention. (a) S. aureus exhibiting at least an 8-fold change in MIC in tissue culture medium (DMEM) vs. MHB medium were subjected to susceptibility tests in the presence and absence of physiological levels of NaHCO₃. Values represent MIC fold-change when derived in MHB medium in the presence/absence of NaHCO₃ (test/standard condition; left of slash); and in DMEM medium in the absence/presence of NaHCO₃ (test/standard condition; right of slash). Increased susceptibility is depicted in blue; increased resistance is depicted in red. Stippled boxes represent those that exhibited < 8-fold altered susceptibility between MHB and DMEM media. To control for pH and buffer considerations, strains were grown in MHB pH 7.2; MHB adjusted to pH 7.2 w/100 mM Tris; and DMEM liquid pH 7.4 (containing 44 mM NaHCO₃); all other media conditions were adjusted to pH 7.4 with 100 mM Tris including: MHB w/NaHCO₃; and NaHCO₃-free powdered DMEM w/wo NaHCO₃ (Supplementary Table 2a). (b) Dose response analysis of MRSA (USA300; MT3302) and MSSA (MT3307) antibiotic susceptibility following exposure to increasing concentrations of NaHCO₃ in standard MHB medium. AZM (azithromycin); ERY (erythromycin); CFX (ceftriaxone). (c) Susceptibility of S. pneumoniae and Salmonella spp. in the presence/absence of physiological levels of NaHCO₃ in MHB and/or DMEM media. For S. pneumoniae, values represent fold-change between MICs derived in MHB medium in the presence/absence of NaHCO₃ (test/standard condition). For Salmonella spp. values represent fold-change between MICs derived in MHB medium in the presence/absence of NaHCO₃ (test/standard condition; left of slash); and DMEM in the absence/presence of NaHCO₃ (test/standard condition; right of slash). No change (NC), Resistant (R). MICs were a consensus of at least 6 independent isolates.