1432. Exploring Cell Wall Targets to Overcome Mycobacterium tuberculosis (Mtb): Ceftriaxone (CRO) Inhibits Ldt(Mt2), a Major Peptidoglycan (PG) Synthase

BACKGROUND: Drug-resistant tuberculosis (DR TB) is a deadly, difficult-to-treat infection, and new treatment strategies are needed. Despite the wide success of β-lactams (BLs), DR TB guidelines only include meropenem (MEM) and imipenem (IPM), given with clavulanate (CLA). BlaC, the Mtb β-lactamase, hydrolyzes CRO less efficiently than other cephems and β-lactamase inhibitors improve the in vitro susceptibility of Mtb to CRO. Surprisingly, CRO has not been evaluated in DR TB clinical studies. Moreover, the mechanisms by which CRO disrupts Mtb PG synthesis are not well characterized. CRO inhibits Ldt(Mt1­), but activity against Ldt(Mt2), an important Mtb PG synthase, is unknown. To explore this knowledge gap, we examined CRO inhibition of Ldt(Mt2). In addition, we investigated if combining CRO with MEM or IPM would lower minimum inhibitory concentrations (MICs) more than each agent alone. METHODS: A panel of Mtb isolates was selected for susceptibility testing with a broth microdilution method. Timed electrospray ionization-mass spectrometry (ESI-MS) and inhibition kinetic assays were performed. RESULTS: CRO MICs ranged 0.25 to 16 µg/mL and lowered ≤ 0.06 to 2 µg/mL with CLA (Table 1). Fractional inhibitory concentration indices for CRO + MEM or IPM was < 0.5 for six isolates, suggesting synergy. ESI-MS captured CRO-Ldt(Mt2­) acyl-complexes at timepoints 5 to 120 min, and a 158 Da fragment loss was observed; MEM and IPM were unchanged (Table 2, Figure 1). When Ldt(Mt2) was co-incubated with MEM and CRO together, only MEM complexes were captured. Interestingly, K(i)(app)­ with CRO (0.07 ± 0.01 µM) was comparable to that with MEM (0.09 ± 0.01 µM). [Figure: see text] [Figure: see text] [Figure: see text] CONCLUSION: CRO was effective in lowering MICs with MEM, IPM, and CLA for our Mtb isolates. Based upon ESI-MS, we found that CRO forms a stable complex with Ldt(Mt2) and the R2 side chain is eliminated, while kinetic observations support inhibition of Ldt(Mt2) by CRO (Figure 2). Previous work found MEM and IPM also inhibit multiple other PG synthases (e.g., PonA1, Ldt(Mt1), Ldt(Mt3)). We hypothesize that CRO + MEM/IPM inhibits the growth of Mtb by the combined inactivation of multiple cell wall enzymes. Our observations support the further exploration of the notion of “target redundancy” as an approach to treat multidrug-resistant mycobacteria with BLs. [Figure: see text] DISCLOSURES: Krisztina Papp-Wallace, Ph.D, Merck: Grant/Research Support|Venatorx: Grant/Research Support|Wockhardt: Advisor/Consultant Robert A. Bonomo, MD, NIH VA: Grant/Research Support|VenatoRx Merck Wockhardt Cystic Fibrosis Foundation: Grant/Research Support.