A Combined Phenotypic-Genotypic Predictive Algorithm for In Vitro Detection of Bicarbonate: β-Lactam Sensitization among Methicillin-Resistant Staphylococcus aureus (MRSA).

Selvi C Ersoy,Robin Patel,Warren E Rose,Youngju Pak,Richard A Proctor,Ewan M Harrison,Arnold S Bayer,Henry F Chambers

doi:10.3390/antibiotics10091089

Abstract

Antimicrobial susceptibility testing (AST) is routinely used to establish predictive antibiotic resistance metrics to guide the treatment of bacterial pathogens. Recently, a novel phenotype termed “bicarbonate (NaHCO3)-responsiveness” was identified in a relatively high frequency of clinical MRSA strains, wherein isolates demonstrate in vitro “susceptibility” to standard β-lactams (oxacillin [OXA]; cefazolin [CFZ]) in the presence of NaHCO3, and in vivo susceptibility to these β-lactams in experimental endocarditis models. We investigated whether a targeted phenotypic-genotypic screening of MRSA could rule in or rule out NaHCO3 susceptibility upfront. We studied 30 well-characterized clinical MRSA bloodstream isolates, including 15 MIC-susceptible to CFZ and OXA in NaHCO3-supplemented Mueller–Hinton Broth (MHB); and 15 MIC-resistant to both β-lactams in this media. Using a two-tiered strategy, isolates were first screened by standard disk diffusion for susceptibility to a combination of amoxicillin-clavulanate [AMC]. Isolates then underwent genomic sequence typing: MLST (clonal complex [CC]); agr; SCCmec; and mecA promoter and coding region. The combination of AMC disk susceptibility testing plus mecA and spa genotyping was able to predict MRSA strains that were more or less likely to be NaHCO3-responsive in vitro, with a high degree of sensitivity and specificity. Validation of this screening algorithm was performed in six strains from the overall cohort using an ex vivo model of endocarditis. This ex vivo model recapitulated the in vitro predictions of NaHCO3-responsiveness vs. nonresponsiveness above in five of the six strains.

Highlights

The susceptible 2 genotype has a T at the mecA promoter -7 ribosomal-binding site (RBS), whereas the resistant 2 genotype has a G at this site. This promoter single nucleotide polymorphism (SNP) is associated with decreased mecA transcription in susceptible 2 MRSA as compared to resistant 2 isolates [29]. Both Chi-squared and Fisher’s exact analysis revealed that the susceptible 2 genotype were significantly associated with a NaHCO3 -responsive phenotype (Table S2, p < 0.01)
We investigated the correlation between NaHCO3 -responsiveness and susceptibility to amoxicillin + clavulanate (AMC) by standard disk diffusion assay
Current clinical guidelines discourage the use of β-lactams in lactams in MRSA infections because of their uniform “in vitro resistance” to such agents

Summary

Introduction

Antibiotic resistance is a major threat to healthcare initiatives worldwide [1,2,3]. Despite the availability of extensive in vitro testing methods and international consensus committee interpretive guidelines to aid in treatment practices, antibiotic resistance has not abated [3,4,5,6]. There has been a lack of innovation in the development of in vitro methods that mimic the host microenvironment and milieu during active infection [7,8,9]. Guideline committees, such as the Clinical and Laboratory Standards Institute (CLSI)

Methods

Results

Conclusion