Meropenem - clavulanic acid has high in vitro activity against multidrug - 1 resistant Mycobacterium tuberculosis

18 We investigated the activity of meropenem-clavulanic acid (MEM-CLA) against 68 M. 19 tuberculosis isolates. We included predominantly multiand extensively drug-resistant 20 tuberculosis isolates (MDR/XDR-TB), since the activity of MEM-CLA for resistant isolates 21 has not been studied extensively previously. Using Middlebrook 7H10 medium, all but four 22 isolates showed a MIC distribution of 0.125-2 mg/L of MEM-CLA, below the non-species 23 related breakpoint for MEM of 2 mg/L defined by EUCAST. MEM-CLA is a potential 24 treatment option for MDR/XDR-TB. 25 on July 9, 2017 by gest httpaac.asm .rg/ D ow nladed fom Multidrugresistant and extensively drug-resistant tuberculosis (MDR/XDR-TB) is 26 unrelentingly increasing worldwide. As MDR/XDR-TB is notoriously difficult to treat, 27 already approved drugs, such as trimethoprim-sulfamethoxazole, are being investigated as 28 treatment options (1). The activity of penicillin against Mycobacterium tuberculosis was 29 investigated already in the 1940s (2), but β-lactams were deemed ineffective. However, it was 30 later shown that the β-lactamase BlaC causes the hydrolysis of β-lactam antibiotics (3-6). 31 This hydrolysis can be inhibited by the β-lactamase-inhibitor clavulanic acid (CLA), which 32 irreversibly inactivates BlaC (6, 7). Meropenem (MEM) is a β-lactam antibiotic of the 33 carbapenem group. Even though MEM is a relatively poor substrate for BlaC (8), MEM on 34 its own shows conflicting evidence regarding antituberculous activity (9-12). Hence, the 35 combination meropenem-clavulanic acid (MEM-CLA) is an interesting treatment alternative 36 for drug-resistant TB, but there is a lack of both in vitro and in vivo studies for this 37 combination. The aim of this study was to investigate the in vitro effect of MEM-CLA 38 against M. tuberculosis, predominantly MDR/XDR-TB isolates. 39 Using Middlebrook 7H10, 94 M. tuberculosis isolates were studied. The isolates consisted of 40 clinical isolates and isolates submitted to the Public Health Agency of Sweden for 41 proficiency drug susceptibility testing, with all isolates being globally sourced. A total of 68 42 isolates showed sufficient growth to be studied further and they were categorized into three 43 resistance groups and consisted of 36 MDR-TB, 13 XDR-TB and 19 with mixed resistance 44 patterns (non-MDR/XDR-TB). H37Rv (ATCC 27294) was used as control. Middlebrook 45 7H10 agar (BD AB, Stockholm, Sweden) enriched with OADC (10% oleic 46 acid/albumin/dextrose/catalase) and 5% glycerol was prepared in 14 cm Petri dishes, each 47 dish containing 60 ml of agar. A stock solution was prepared by diluting MEM with water, 48 and then applied in serial two-step dilutions, reaching a final antibiotic concentration range of 49 0.002-512 mg/L of MEM. CLA was added to all dishes at a concentration of 64 mg/L, in 50 on July 9, 2017 by gest httpaac.asm .rg/ D ow nladed fom order to ensure a sufficient concentration of the β-lactamase inhibitor throughout the whole 51 experiment. Due to the short half-life of CLA in solid media (1.4 days) (13), the 52 concentration of CLA after the first week of our experiment, was expected to be around 2 53 mg/L, similar to the concentrations of CLA seen in serum after a dose of 500 mg/125 mg of 54 amoxicillin-clavulanic acid (AMX-CLA) (14). 55 The MICs of the 68 M. tuberculosis isolates were determined by inoculating bacterial 56 suspensions onto the Middlebrook 7H10 medium using a 96-stick replicator, as previously 57 described (15). The mycobacterial cultures were incubated at 37oC and growth was evaluated 58 after three weeks. The MIC was defined as the lowest concentration with less growth than the 59 1:100 diluted control. Sterile water was used as a negative control (15). 60 The MIC distribution of MEM-CLA (expressed as the concentration of MEM) was 0.125-32 61 mg/L (Figure 1). All but four isolates had MICs of ≤ 2 mg/L of MEM-CLA, which is the 62 non-species related susceptibility breakpoint of MEM defined by EUCAST (16). The MIC90 63 and the MIC of the control strain H37Rv were both 1 mg/L. 64 Overall, we observed low MICs of MEM-CLA against M. tuberculosis in vitro, even for 65 highly drug-resistant isolates. The majority of the isolates followed a Gaussian wild-type 66 distribution, with MICs below or equal to the EUCAST non-species related susceptibility 67 breakpoint of MEM of 2 mg/L (16). Four isolates (two MDR-TB and two XDR-TB isolates) 68 had very high MIC levels (16 and 32 mg/L), a drug concentration unlikely to be achieved in 69 serum even with high-dose regimens (17). The four M. tuberculosis isolates with very high 70 MICs (16 and 32 mg/L) of MEM-CLA were already highly resistant to other 71 pharmacologically unrelated anti-TB drugs, which could be due to a generally lower 72 permeability, as previously suggested for highly resistant M. tuberculosis isolates (13). 73 on July 9, 2017 by gest httpaac.asm .rg/ D ow nladed fom β-lactam antibiotics have little intrinsic activity against M. tuberculosis (18-21), but are 74 effective in vitro when combined with a β-lactamase inhibitor (18, 20-23). The combination 75 of β-lactam antibiotics and β-lactamase inhibitors has shown anti-TB effect in humans (24, 76 25), rodents (26), within macrophages (10) as well as against non-replicating M. tuberculosis 77 in vitro (8). MEM has poor intrinsic activity (12), but is bactericidal in vitro when combined 78 with CLA (8, 27). The in vitro effect of MEM-CLA has been investigated previously for 79 H37Rv (10, 26) and 13 isolates of M. tuberculosis, all XDR-TB (8). All isolates were 80 susceptible to ≤1mg/L of MEM-CLA, thus in line with our tentative breakpoint of 2 mg/L. 81 However, the concentration of the β-lactamase inhibitor seems important. When Gonzalo et 82 al used 2.5 mg/L of CLA, they found a higher MIC of ≤3 mg/L of MEM-CLA for 28 mainly 83 drug-resistant M. tuberculosis isolates. On the other hand, a synergistic effect was seen when 84 AMX-CLA and MEM were combined, resulting in only 3/28 M. tuberculosis isolates with 85 higher MICs than 1.25 mg/L (11). CLA is presently only available in combination with AMX 86 (i.e. Augmentin®), necessitating the use of MEM in combination with AMX-CLA for drug87