Jim Werngren

Reevaluation of the Critical Concentration for Drug Susceptibility Testing of Mycobacterium tuberculosis against Pyrazinamide Using Wild-Type MIC Distributions and pncA Gene Sequencing

ABSTRACT 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 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) <or= 1.0 mg/L as the tentative epidemiological cut-off (ECOFF) for ofloxacin and ciprofloxacin, and S <or= 0.5 mg/L for levofloxacin and moxifloxacin, although it is possible that adding more MIC data could shift the ECOFFs for ofloxacin and levofloxacin one dilution upwards. The proposed ECOFFs may be more appropriate if used as clinical breakpoints on Middlebrook 7H10 agar than the current critical concentrations of S <or= 2.0 mg/L for ciprofloxacin, ofloxacin and levofloxacin, and S <or= 0.5 mg/L could be considered as a clinical breakpoint for moxifloxacin, provided other investigators can confirm our findings.

Wild-Type MIC Distributions for Aminoglycoside and Cyclic Polypeptide Antibiotics Used for Treatment of Mycobacterium tuberculosis Infections

ABSTRACT 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.

Proficiency of drug susceptibility testing of Mycobacterium tuberculosis against pyrazinamide: the Swedish experience

BACKGROUND Pyrazinamide (PZA) is a key drug in the treatment of tuberculosis (TB), including multidrug-resistant TB. Drug susceptibility testing (DST) of Mycobacterium tuberculosis against PZA is not included in the World Health Organizations yearly proficiency testing. There is an increasing need to establish quality control of PZA DST. OBJECTIVE To evaluate the performance of PZA DST and to introduce a quality assurance system for the test in Sweden. METHOD Panels with PZA-susceptible and -resistant isolates were used in three rounds of proficiency testing in all five Swedish clinical TB laboratories and our reference laboratory. All laboratories used the MGIT 960 system. Minimum inhibitory concentrations (MICs) were determined and the pncA gene was sequenced to further characterise the 52 panel strains. RESULTS Good agreement was seen between the phenotypic PZA DST and pncA sequence data, and MIC determination confirmed high levels of resistance. However, in contrast to other drugs, for which correct proficiency test results were observed, specificity problems occurred for PZA DST in some laboratories. CONCLUSIONS In Sweden, using panel testing, differences were seen in the proficiency of TB laboratories in correctly identifying PZA susceptibility. Improved results were noted in the third round; PZA has therefore been included in yearly proficiency testing.

Ebselen and analogs as inhibitors of Bacillus anthracis thioredoxin reductase and bactericidal antibacterials targeting Bacillus species, Staphylococcus aureus and Mycobacterium tuberculosis

BACKGROUND Bacillus anthracis is the causative agent of anthrax, a disease associated with a very high mortality rate in its invasive forms. METHODS We studied a number of ebselen analogs as inhibitors of B. anthracis thioredoxin reductase and their antibacterial activity on Bacillus subtilis, Staphylococcus aureus, Bacillus cereus and Mycobacterium tuberculosis. RESULTS The most potent compounds in the series gave IC(50) values down to 70 nM for the pure enzyme and minimal inhibitory concentrations (MICs) down to 0.4 μM (0.12 μg/ml) for B. subtilis, 1.5 μM (0.64 μg/ml) for S. aureus, 2 μM (0.86 μg/ml) for B. cereus and 10 μg/ml for M. tuberculosis. Minimal bactericidal concentrations (MBCs) were found at 1-1.5 times the MIC, indicating a general, class-dependent, bactericidal mode of action. The combined bacteriological and enzymological data were used to construct a preliminary structure-activity-relationship for the benzoisoselenazol class of compounds. When S. aureus and B. subtilis were exposed to ebselen, we were unable to isolate resistant mutants on both solid and in liquid medium suggesting a high resistance barrier. CONCLUSIONS These results suggest that ebselen and analogs thereof could be developed into a novel antibiotic class, useful for the treatment of infections caused by B. anthracis, S. aureus, M. tuberculosis and other clinically important bacteria. Furthermore, the high barrier against resistance development is encouraging for further drug development. GENERAL SIGNIFICANCE We have characterized the thioredoxin system from B. anthracis as a novel drug target and ebselen and analogs thereof as a potential new class of antibiotics targeting several important human pathogens.

Determination of MIC Breakpoints for Second-Line Drugs Associated with Clinical Outcomes in Multidrug-Resistant Tuberculosis Treatment in China

ABSTRACT Our study aims to identify the clinical breakpoints (CBPs) of second-line drugs (SLDs) above which standard therapy fails in order to improve multidrug-resistant tuberculosis (MDR-TB) treatment. MICs of SLDs were determined for M. tuberculosis isolates cultured from 207 MDR-TB patients in a prospective cohort study in China between January 2010 and December 2012. Classification and regression tree (CART) analysis was used to identify the CBPs predictive of treatment outcome. Of the 207 MDR-TB isolates included in the present study, the proportion of isolates above the critical concentration recommended by WHO ranged from 5.3% in pyrazinamide to 62.8% in amikacin. By selecting pyrazinamide as the primary node (CBP, 18.75 mg/liter), 72.1% of sputum culture conversions at month four could be predicted. As for treatment outcome, pyrazinamide (CBP, 37.5 mg/liter) was selected as the primary node to predict 89% of the treatment success, followed by ofloxacin (CBP, 3 mg/liter), improving the predictive capacity of the primary node by 10.6%. Adjusted by identified confounders, the CART-derived pyrazinamide CBP remained the strongest predictor in the model of treatment outcome. Our findings indicate that the critical breakpoints of some second-line drugs and PZA need to be reconsidered in order to better indicate MDR-TB treatment outcome.

Antimicrobial Susceptibility of Mycobacterium marinum Determined by E-test and Agar Dilution

Mycobacterium marinum is recognized as a cutaneous pathogen requiring antibiotic treatment. We compared the E-test with a reference agar dilution method for susceptibility testing of M. marinum to amikacin, ciprofloxacin, clarithromycin, doxycycline, rifampicin, trimethoprim-sulfamethoxazole and ethambutol. MICs obtained after 6 d showed agreement between the E-test and agar dilution within +/- 2 dilutions in 95% of all cases for amikacin, ciprofloxacin, doxycycline and rifampicin. Inhibitory concentrations of trimethoprim-sulfamethoxazole were difficult to define using the E-test because of gradually decreased growth in the presence of increasing concentrations. For clarithromycin, results were generally 1-3 dilution steps lower with the E-test and for ethambutol they were often > 3 dilution steps lower. These differences always appeared in the low MIC range and did not affect the categorization of the strains as susceptible to these 2 antimicrobial agents. All strains were interpreted as susceptible to all tested antibiotics, except for doxycycline, according to recommended breakpoints. Overall, our results suggest that the E-test can be considered an alternative for susceptibility testing of certain antibacterial agents against M. marinum.

In Vitro Activity of AZD5847 against Geographically Diverse Clinical Isolates of Mycobacterium tuberculosis

ABSTRACT The MIC of the novel antituberculosis (anti-TB) drug AZD5847 was determined against 146 clinical isolates from diverse geographical regions, including eastern Europe, North America, Africa, and Asia, using the automated Bactec Mycobacterial Growth Indicator Tube (MGIT) 960 system. These isolates originated from specimen sources such as sputum, bronchial alveolar lavage fluid, pleural fluid, abscess material, lung biopsies, and feces. The overall MIC90 was 1.0 mg/liter (range, 0.125 to 4 mg/liter). The MICs of AZD5847 for isolates of Mycobacterium tuberculosis were similar among drug-sensitive strains, multidrug-resistant (MDR) strains, and extensively drug resistant (XDR) strains. The good in vitro activity of AZD5847 against M. tuberculosis and the lack of cross-resistance make this agent a promising anti-TB drug candidate.

Protecting Pyrazinamide, a Priority for Improving Outcomes in Multidrug-Resistant Tuberculosis Treatment.

We read with great interest the mathematical model presented by Fofana et al. ([1][1]) on the role of pyrazinamide (PZA) in the emergence of multidrug-resistant tuberculosis (MDR TB), particularly as the results of their model mirror our concerns regarding the amplification of PZA resistance during

New insights into the mechanism of action of pyrazinamide, implications for susceptibility testing, and future regimens☆

Pyrazinamide (PZA) is included in the 2016 World Health Organization multidrug-resistant tuberculosis treatment guidelines and is a key component of most ongoing clinical trials investigating novel antibiotic combinations. PZA resistance is associated with worse tuberculosis treatment outcomes. Unfortunately, for such an important drug, phenotypic susceptibility testing is extremely challenging. The exacting bacterial growth conditions required to induce susceptibility to the drug reduce the accuracy of the susceptibility assay, even in experienced laboratories, and widespread testing is not performed. This situation is unacceptable for such a valuable and important drug. A more complete understanding of the mechanism of action of PZA would be expected to lead to improvements in this situation. Although the exact mechanism of action of PZA is not known yet, it is widely accepted that PZA is a prodrug requiring transformation to pyrazinoic acid, the active form, by the mycobacterial enzyme encoded by the pncA gene. Most clinical resistance indeed appears to be a result of a diverse range of mutations in this gene and sequencing of the pncA gene has been shown to have excellent predictive power for PZA resistance. The wider availably of pncA sequencing in combination with databases of the phenotypic implications of these mutations has helped make genetic testing for PZA resistance a practical proposition. For the past decades, it has been generally accepted that an extracellular low pH is required for PZA activity but work in our laboratory [1] and others [2] has recently challenged this assumption. Alternative bacterial stresses, apart from a reduced pH of the growth media (such as reduced temperature), can also induce a PZA-susceptible phenotype. The characterization of spontaneous in vitro-resistant pyrazinoic acid mutants selected under neutral pH conditions suggests a key role for the pantothenate/coenzyme A biosynthetic pathway. This has profound implications for the mechanism of action of PZA as well as potentially the bacterial population against which PZA is active in the host. These findings will be discussed as well as their implications for further research and the future of PZA susceptibility testing.