We sought to further investigate

We sought to further investigate the phenomenon of amoxicillin/clavulanate susceptibility using comparative genomics. A phylogenetic analysis indicated that a large proportion of MDR and XDR isolates in our dataset were part of a LAM4 clade, which was responsible for the famed Tugela Ferry XDR outbreak in KwaZulu-Natal in 2005 (Gandhi et al., 2006), and still accounts for a considerable burden of XDR (Chihota et al., 2012; Müller et al., 2013; Gandhi et al., 2014). Additionally, a large proportion of amoxicillin/clavulanate susceptible isolates were also members of this clade, raising the possibility that this drug combination may be of benefit for individuals harboring these strains. The pharmacodynamics of co-amoxiclav in the treatment of M. tuberculosis have not be formally studied. However for most bacteria the time above MIC (T>MIC) is considered the principal determinant of efficacy (Turnidge, 1998). The slow growth of M. tuberculosis means an extended T>MIC is likely to be critical for this organism. Despite this multiple and high dosing of co-amoxiclav could achieve drug levels expected to be active against M. tuberculosis strains with an MIC of ≤4μg/mL/2.5μg/mL amoxicillin/clavulanic purchase Dioscin (Chierakul et al., 2006). Next we identified putative genomic markers of amoxicillin/clavulanate susceptibility through application of a phylogenetically-aware linear mixed model (LMM), which is suitable for samples wherein there is a large phenotypic variability within a group of closely related isolates (Supplementary material). The LMM identified 12 candidate variant patterns, comprising 38 unique variants (Table 3, Table S5). It should be remembered that despite the use of a phylogenetically-aware LMM, association analyses cannot separate the effects of genomic variants that show linkage disequilibrium (i.e. those that represent the same pattern), a factor which must be considered when interpreting our results. Consequently, we do not infer causality, but rather suggest that the variants identified here represent genomic markers of amoxicillin/clavulanate susceptibility, which warrant further investigation. Although our analyses did identify a number of interesting variants, we did not identify any associations between amoxicillin/clavulanate susceptibility and variation in genes previously implicated for this phenotype e.g. mycobacterial l,d-transpeptidases, carboxypeptidases, penicillin-binding proteins (Dubée et al., 2012; Datta et al., 2006; Kumar et al., 2012; Flores et al., 2005; Bhakta and Basu, 2002; Danilchanka et al., 2008; Dinesh et al., 2013; Fukuda et al., 2013; McDonough et al., 2005). Furthermore, we found no evidence that altered β-lactamase genetics or activity accounts for observed differences in amoxicillin/clavulanate susceptibility. All 91 strains had a wild type blaC, the gene that encodes for the primary β-lactamase of M. tuberculosis. All strains in which β-lactamase activity was assessed—including strains with low MICs to amoxicillin/clavulanate—demonstrated the ability to hydrolyze nitrocefin, a β-lactam. Despite these findings, it is still conceivable that differences in BlaC expression may account for the observed range of β-lactam susceptibility, a hypothesis that will need to be tested in future experiments. For M. tuberculosis, first and second-line drug susceptibility conforms to a classic model of drug resistance: wild type M. tuberculosis is susceptible to first and second-line antitubercular drugs (e.g. isoniazid, rifampin, ofloxacin, kanamycin etc.), and exposure to these same agents selects for resistant organisms. In contrast, our documentation of M. tuberculosis susceptibility to amoxicillin/clavulanate does not fit this classical model. Drug susceptible reference strains of M. tuberculosis and clinical isolates in basal phylogenetic groups (Figs. 2 and 3) had innate resistance to amoxicillin/clavulanate, yet clinical isolates were observed to be susceptible, suggesting that they had acquired susceptibility to these agents. To distinguish this phenomenon from the classic model of drug resistance, we have named this concept “paradoxical hypersusceptibility,” which we define as phenotypic drug susceptibility observed in an organism in which its wild type state has innate resistance to that respective drug.