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

We investigated the activity of meropenem-clavulanic acid (MEM-CLA) against 68 Mycobacterium tuberculosis isolates. We included predominantly multi- and extensively drug-resistant tuberculosis (MDR/XDR-TB) isolates, since the activity of MEM-CLA for resistant isolates has previously not been studied extensively. Using Middlebrook 7H10 medium, all but four isolates showed an MIC distribution of 0.125 to 2 mg/liter for MEM-CLA, below the non-species-related breakpoint for MEM of 2 mg/liter defined by EUCAST. MEM-CLA is a potential treatment option for MDR/XDR-TB.

Little difference between minimum inhibitory concentrations of Mycobacterium tuberculosis wild-type organisms determined with BACTEC MGIT 960 and Middlebrook 7H10.

The MIC wild-type (WT) distribution for Mycobacterium tuberculosis in BACTEC 960 MGIT is not defined, which may result in poor reproducibility for drug susceptibility testing (DST), as several DST methods with different breakpoints are in use. In a comparison between MGIT and Middlebrook 7H10 medium of seven first- and second-line drugs, including 133 MIC determinations of 15 WT isolates, we found an agreement of 91.7% within ± one MIC dilution step. The results confirm the agreement in MIC testing between 7H10 and MGIT and indicate that breakpoints could be harmonized in order to avoid misclassification.

Meropenem-clavulanate has high in vitro activity against multidrug-resistant Mycobacterium tuberculosis.

AIMS AND OBJECTIVES: With the relentless increase in multidrug- and extensively-drug resistant tuberculosis (MDR/XDR-TB), new treatment strategies are necessary. Favorable results have been reported by combining a ß-lactam antibiotic and a ß-lactamase inhibitor. The ß-lactamase encoded by the blaC gene of Mycobacterium tuberculosis (MTB) is the major mechanism of resistance to ß-lactam antibiotics (e.g., penicillin). Meropenem, a ß-lactam antibiotic of the carbapenem group, is a relatively weak substrate for the ß-lactamase of MTB. The ß-lactamase inhibitor clavulanate irreversibly inactivates the ß-lactamase encoded by the blaC gene, thus making the combination of meropenem and clavulanate an interesting treatment alternative for MTB. However, very few isolates of MTB have been tested for this drug combination and few clinical reports exist. Thus, the present study investigates the in vitro activity of meropenem-clavulanate for drug-resistant MTB isolates, including MDR/XDR-TB. METHODS: The minimum inhibitory concentration (MIC) distribution of meropenem-clavulanate was determined using Middlebrook 7H10, including MDR and XDR strains of MTB (n=68). Meropenem was prepared in a stock solution with a final concentration range of 0.002-512mg/L. Clavulanate was added at a fixed concentration of 64mg/L, to avoid a decline of the ß-lactamase to insufficient levels during the experiment. All isolates were evaluated after three weeks of growth. The pan-susceptible strain H37Rv was used as a control. RESULTS: There was a Gaussian MIC-distribution between 0.125 and 2mg/L of meropenem-clavulanate (expressed as the concentration of meropenem), but four isolates had very high MIC levels (16 and 32mg/L), which is likely to be out of reach in clinical doses (Fig. 1). The susceptibility of the isolates to meropenem-clavulanate was not correlated to the level of resistance to first- or second-line anti-tuberculous drugs. The MIC of the pan-susceptible control strain H37Rv was 1mg/L of meropenem, when combined with clavulanate. CONCLUSIONS: The present study shows that meropenem-clavulanate has low MICs against MTB in vitro, including MDR and XDR-TB isolates. Meropenem has good tissue penetration and low protein-binding, but requires an intravenous access and is relatively expensive. Meropenem-clavulanate may be a treatment option in selected cases of MDR/XDR-TB, although further clinical studies are warranted.

Intra- and extracellular activities of trimethoprim-sulfamethoxazole against susceptible and multidrug-resistant Mycobacterium tuberculosis.

We investigated the activity of trimethoprim-sulfamethoxazole (SXT) against Mycobacterium tuberculosis, the pathogen that causes tuberculosis (TB). The MIC distribution of SXT was 0.125/2.4 to 2/38 mg/liter for the 100 isolates tested, including multi- and extensively drug-resistant isolates (MDR/XDR-TB), whereas the intracellular MIC90 of sulfamethoxazole (SMX) for the pansusceptible strain H37Rv was 76 mg/liter. In an exploratory analysis using a ratio of the unbound area under the concentration-time curve from 0 to 24 h over MIC (fAUC0-24/MIC) using ≥ 25 as a potential target, the cumulative fraction response was ≥ 90% at doses of ≥ 2,400 mg of SMX. SXT is a potential treatment option for MDR/XDR-TB.

Rifampicin-resistant and rifabutin-susceptible Mycobacterium tuberculosis strains: a breakpoint artefact?

OBJECTIVES: It has long been assumed that some rifampicin-resistant Mycobacterium tuberculosis strains are susceptible to, and thus treatable with, rifabutin. However, clinical breakpoints for susceptibility testing of rifabutin as well as the evidence for a clinical effect of rifabutin in rifampicin-resistant strains remains poorly defined. The objective of this study was to re-evaluate the breakpoint for rifabutin in relation to its MIC wild-type distribution and the presence of mutations in rpoB. METHODS: The MIC in 7H10 Middlebrook medium was determined for clinical isolates of M. tuberculosis (n = 95), where a majority were multidrug resistant. Additionally, all strains were screened for rpoB mutations by sequencing and the GenoType MTBDRplus assay. RESULTS: Rifampicin resistance was confirmed by genotypical and/or phenotypical tests in 73 isolates (76.8%). Nineteen isolates, defined as rifampicin resistant and rifabutin susceptible according to the present breakpoint, exhibited significantly higher MICs of rifabutin (0.064-0.5 mg/L) than rifabutin-susceptible isolates without any detectable mutations in rpoB (P < 0.001). These 19 isolates were clearly resistant to rifampicin (MIC 2-256 mg/L) and all but one had mutations in rpoB, with 9 (47.4%) specifically in Asp516Val. CONCLUSIONS: Our results indicate that rifampicin-resistant but rifabutin-susceptible isolates according to the present breakpoints harbour rpoB mutations and have a rifabutin MIC significantly higher than strains without any detectable mutations in rpoB. So far there are no clinical, pharmacological or microbiological data to confirm that such isolates can be treated with rifabutin and we suggest a revision of the current breakpoints.

Reevaluation of the critical concentration for drug susceptibility testing of Mycobacterium tuberculosis against pyrazinamide using wild-type MIC distributions and pncA gene sequencing.

Pyrazinamide (PZA) is a potent first-line agent for the treatment of tuberculosis (TB) with activity also against a significant part of drug-resistant Mycobacterium tuberculosis strains. Since PZA is active only at acid pH, testing for susceptibility to PZA is difficult and insufficiently reproducible. The recommended critical concentration for PZA susceptibility (MIC, 100 mg/liter) used in the Bactec systems (460 and MGIT 960) has not been critically evaluated against wild-type MIC distributions in clinical isolates of Mycobacterium tuberculosis. Using the Bactec MGIT 960 system, we determined the PZA MICs for 46 clinical M. tuberculosis isolates and compared the results to pncA sequencing and previously obtained Bactec 460 data. For consecutive clinical isolates (n = 15), the epidemiological wild-type cutoff (ECOFF) for PZA was 64 mg/liter (MIC distribution range, ≤ 8 to 64 mg/liter), and no pncA gene mutations were detected. In strains resistant in both Bactec systems (n = 18), the PZA MICs ranged from 256 to ≥ 1,024 mg/liter. The discordances between pncA sequencing, susceptibility results in Bactec 460, and MIC determinations in Bactec MGIT 960 were mainly observed in strains with MICs close to or at the ECOFF. We conclude that in general, wild-type and resistant strains were clearly separated and correlated to pncA mutations, although some isolates with MICs close to the ECOFF cause reproducibility problems within and between methods. To solve this issue, we suggest that isolates with MICs of ≤ 64 mg/liter be classified susceptible, that an intermediary category be introduced at 128 mg/liter, and that strains with MICs of >128 mg/liter be classified resistant.

Wild-type distributions of seven oral second-line drugs against Mycobacterium tuberculosis.

OBJECTIVES: To determine wild-type minimum inhibitory concentration (MIC) distributions for Mycobacterium tuberculosis, as the background data for defining susceptibility breakpoints are limited. METHODS: We determined wild-type MIC distributions of M. tuberculosis using a 96-stick replicator in Middlebrook 7H10 (7H10) medium for ethionamide (ETH), prothionamide, thiacetazone, cycloserine, rifabutin (RFB), clofazimine and linezolid in consecutive susceptible clinical isolates (n = 78). RESULTS: Tentative epidemiological wild-type cut-offs (ECOFF) were determined for all investigated drugs where World Health Organization recommended critical concentrations for 7H10 are lacking, except for ETH. As the ECOFF was closely related to the non-wild-type strains for ETH and thiacetazone, the use of an intermediary (I) category in drug susceptibility testing could increase reproducibility. The cross-resistance between ETH and isoniazid was 21%. Applying 0.5 mg/l as a breakpoint for RFB classified two non-wild type and rpoB mutated isolates as susceptible for RFB and resistant against rifampicin. CONCLUSIONS: We propose that wild-type MIC distributions should be used as a tool to define clinical breakpoints against second-line drugs. This is increasingly important considering the rapid emergence of drug resistance.

Wild-type MIC distributions for aminoglycoside and cyclic polypeptide antibiotics used for treatment of Mycobacterium tuberculosis infections.

The aminoglycosides and cyclic polypeptides are essential drugs in the treatment of multidrug-resistant tuberculosis, underscoring the need for accurate and reproducible drug susceptibility testing (DST). The epidemiological cutoff value (ECOFF) separating wild-type susceptible strains from non-wild-type strains is an important but rarely used tool for indicating susceptibility breakpoints against Mycobacterium tuberculosis. In this study, we established wild-type MIC distributions on Middlebrook 7H10 medium for amikacin, kanamycin, streptomycin, capreomycin, and viomycin using 90 consecutive clinical isolates and 21 resistant strains. Overall, the MIC variation between and within runs did not exceed +/-1 MIC dilution step, and validation of MIC values in Bactec 960 MGIT demonstrated good agreement. Tentative ECOFFs defining the wild type were established for all investigated drugs, including amikacin and viomycin, which currently lack susceptibility breakpoints for 7H10. Five out of seven amikacin- and kanamycin-resistant isolates were classified as susceptible to capreomycin according to the current critical concentration (10 mg/liter) but were non-wild type according to the ECOFF (4 mg/liter), suggesting that the critical concentration may be too high. All amikacin- and kanamycin-resistant isolates were clearly below the ECOFF for viomycin, and two of them were below the ECOFF for streptomycin, indicating that these two drugs may be considered for treatment of amikacin-resistant strains. Pharmacodynamic indices (peak serum concentration [Cmax]/MIC) were more favorable for amikacin and viomycin compared to kanamycin and capreomycin. In conclusion, our data emphasize the importance of establishing wild-type MIC distributions for improving the quality of drug susceptibility testing against Mycobacterium tuberculosis.

Wild-type MIC distributions of four fluoroquinolones active against Mycobacterium tuberculosis in relation to current critical concentrations and available pharmacokinetic and pharmacodynamic data.

OBJECTIVES: To describe wild-type distributions of the MIC of fluoroquinolones for Mycobacterium tuberculosis in relation to current critical concentrations used for drug susceptibility testing and pharmacokinetic/pharmacodynamic (PK/PD) data. METHODS: A 96-stick replicator on Middlebrook 7H10 medium was used to define the MICs of ciprofloxacin, ofloxacin, moxifloxacin and levofloxacin for 90 consecutive clinical strains and 24 drug-resistant strains. The MICs were compared with routine BACTEC 460 susceptibility results and with MIC determinations in the BACTEC MGIT 960 system in a subset of strains using ofloxacin as a class representative. PK/PD data for each drug were reviewed in relation to the wild-type MIC distribution. RESULTS: The wild-type MICs of ciprofloxacin, ofloxacin, moxifloxacin and levofloxacin were distributed from 0.125 to 1, 0.25 to 1, 0.032 to 0.5 and 0.125 to 0.5 mg/L, respectively. The MIC data correlated well with the BACTEC 960 MGIT and BACTEC 460 results. PD indices were the most favourable for levofloxacin, followed by moxifloxacin, ofloxacin and ciprofloxacin. CONCLUSIONS: We propose S (susceptible)