Paradoxical Hypersusceptibility of Drug-resistant Mycobacteriumtuberculosis to β-lactam Antibiotics

Abstract

Mycobacterium tuberculosis (M. tuberculosis) is considered innately resistant to β-lactam antibiotics. However, there is evidence that susceptibility to β-lactam antibiotics in combination with β-lactamase inhibitors is variable among clinical isolates, and these may present therapeutic options for drug-resistant cases. Here we report our investigation of susceptibility to β-lactam/β-lactamase inhibitor combinations among clinical isolates of M. tuberculosis, and the use of comparative genomics to understand the observed heterogeneity in susceptibility. Eighty-nine South African clinical isolates of varying first and second-line drug susceptibility patterns and two reference strains of M. tuberculosis underwent minimum inhibitory concentration (MIC) determination to two β-lactams: amoxicillin and meropenem, both alone and in combination with clavulanate, a β-lactamase inhibitor. 41/91 (45%) of tested isolates were found to be hypersusceptible to amoxicillin/clavulanate relative to reference strains, including 14/24 (58%) of multiple drug-resistant (MDR) and 22/38 (58%) of extensively drug-resistant (XDR) isolates. Genome-wide polymorphisms identified using whole-genome sequencing were used in a phylogenetically-aware linear mixed model to identify polymorphisms associated with amoxicillin/clavulanate susceptibility. Susceptibility to amoxicillin/clavulanate was over-represented among isolates within a specific clade (LAM4), in particular among XDR strains. Twelve sets of polymorphisms were identified as putative markers of amoxicillin/clavulanate susceptibility, five of which were confined solely to LAM4. Within the LAM4 clade, 'paradoxical hypersusceptibility' to amoxicillin/clavulanate has evolved in parallel to first and second-line drug resistance. Given the high prevalence of LAM4 among XDR TB in South Africa, our data support an expanded role for β-lactam/β-lactamase inhibitor combinations for treatment of drug-resistant M. tuberculosis.

Keywords: Antimicrobial chemotherapy; Beta-lactam antibiotics; Extensively drug resistant (XDR); Multi-drug resistant (MDR); Recombination; pks12; tuberculosis.

Fig. 1A–1D

Clinical isolates of M. tuberculosis exhibit a range of susceptibility to β-lactam/β-lactamase inhibitors. 91 isolates of M. tuberculosis underwent MIC determination to A) amoxicillin, B) amoxicillin/clavulanate, C) meropenem, and D) meropenem/clavulanate. MICs are reported by β-lactam concentration, and clavulanate concentration was held constant at 2.5 μg/mL.

Fig. 2

Amoxicillin/clavulanate hypersusceptible isolates were largely clustered within a monophyletic XDR group within the LAM4 clade. A Bayesian phylogeny of 91 M. tuberculosis isolates, rooted with the M. canettii outgroup, is shown. Bootstrap support values > 80% are marked by an asterisk. Globally recognised M. tuberculosis lineages are indicated. Amoxicillin/clavulanate MICs are represented in a horizontal bar graph to the right of the tree. MICs are reported by amoxicillin concentration, and clavulanate concentration was held constant at 2.5 μg/mL. As indicated in the figure key, the MIC bar graph is color-coded according to the first and second-line drug susceptibility patterns of the corresponding strain. The shaded box indicates LAM4 strains; a monophyletic cluster of spoligotype LAM4 strains with MDR and XDR level resistance is observed within lineage 4. While a significant number of isolates with hypersusceptibility to amoxicillin/clavulanate were concentrated within the LAM4 XDR clade, there are other notable examples of hypersusceptible strains that pertain to other global lineages and spoligotypes.

Fig. 3

Tracing the emergence of amoxicillin/clavulanate susceptibility within the LAM4 clade. A. Bayesian phylogeny of 91 M. tuberculosis isolates as represented in Fig. 2. The LAM4 clade is marked by the blue box and is magnified in B. Nodes with bootstrap support values > 80% are marked by an asterisk. Nodes A-D define subclade divisions for which the corresponding pattern of genomic variants listed in Table 3 is conserved among all members of the subclade. Amoxicillin/clavulanate MICs (amoxicillin μg/mL, clavulanate concentration fixed at 2.5 μg/mL) are represented in a horizontal bar graph to the right of the tree. The bar graph is color-coded according to first and second-line drug susceptibility patterns as in Fig. 2. Historic LAM4 strains are identified by name: KZN605 (XDR), KZN1435 (MDR) and KZN4207 (first/second-line drug susceptible).