Drug susceptibility patterns of Mycobacterium abscessus and Mycobacterium massiliense isolated from respiratory specimens

In this study, we aimed to retrospectively investigate and compare the drug susceptibility patterns of two major Mycobacterium abscessus complex (MABC) species; M. abscessus and M. massiliense. A total of 546?MABC respiratory isolates (277?M. abscessus and 269?M. massiliense) from 2011 to 2016 were analyzed in this study. We estimated minimum inhibitory concentrations (MICs) using the broth microdilution method, and we calculated MIC₅₀ and MIC₉₀ values from the MIC distribution. Both M. abscessus and M. massiliense were highly susceptible to amikacin and linezolid. For M. abscessus, the proportions of inducible and acquired resistance to clarithromycin were 68.6% and 12.3%, respectively. Only 15.2% of M. abscessus remained susceptible at day 14. On the other hand, none of the M. massiliense showed inducible resistance and 6.3% showed acquired resistance to clarithromycin. A total of 92.6% of the M. massiliense remained susceptible at day 14. The resistance rate of M. abscessus to moxifloxacin (90.3%) was significantly higher than that of M. massiliense (83.3%; p?=?0.016). These susceptibility differences may explain the divergent treatment outcomes between patients with pulmonary disease caused by these two species.     

Standardization of multilocus sequence typing scheme for Mycobacterium abscessus and Mycobacterium massiliense

This study aims to develop a multilocus sequence typing (MLST) scheme for Mycobacterium abscessus complex for the typing of stains within each species. A total of 89 clinical isolates of M. abscessus complex from 71 patients of 2 tertiary care hospitals in South Korea were included. Forty-two isolates were identified as M. abscessus, and 29, as Mycobacterium massiliense through sequencing of 8 housekeeping genes and rpoB. The MLST scheme identified 26 different sequence types(STs) and 13 different clonal complexes (CCs) in M. abscessus and 12 different STs and 6 different CCs in M. massiliense. The MLST data showed high concordance with the XbaI-macrorestriction patterns of pulsed-field gel electrophoresis in the duplicated isolates. Our MLST schemes could identify different strains of M. abscessus and M. massiliense, and the schemes also showed a reliable reproducibility. Therefore, our MLST schemes may be useful in studying the epidemiology of M. abscessus and M. massiliense infections.     

Rapid identification of strains belonging to the Mycobacterium abscessus group through erm(41) gene pyrosequencing

Mycobacterium abscessus and Mycobacterium massiliense lung infections have different clarithromycin susceptibilities, making proper identification important; however, standard multi-gene sequencing in clinical laboratories is laborious and time consuming. We developed a pyrosequencing-based method for rapid identification of strains belonging to the M. abscessus group by targeting erm(41). We examined 55 isolates from new pulmonary M. abscessus infections and identified 28 M. abscessus, 25 M. massiliense, and 2 Mycobacterium bolletii isolates. Multi-gene sequencing of 16S rRNA, hsp65, rpoB, and the 16S–23S ITS region was concordant with the results of erm(41) pyrosequencing; thus, the M. abscessus group can be identified by single-nucleotide polymorphisms in erm(41). The method also enables rapid identification of polymorphic, inducible clarithromycin-resistant sequevars (T28 or C28). Pyrosequencing of erm(41) is a rapid, reliable, high-throughput alternative method for identifying and characterizing M. abscessus species. Further testing of a diverse collection of isolates is necessary to demonstrate the discriminatory power of erm(41) sequencing to differentiating species with this highly divergent group.     

Carbapenems and Rifampin Exhibit Synergy against Mycobacterium tuberculosis and Mycobacterium abscessus

An effective regimen for treatment of tuberculosis (TB) is comprised of multiple drugs that inhibit a range of essential cellular activities in Mycobacterium tuberculosis. The effectiveness of a regimen is further enhanced if constituent drugs act with synergy. Here, we report that faropenem (a penem) or biapenem, doripenem, or meropenem (carbapenems), which belong to the β-lactam class of antibiotics, and rifampin, one of the drugs that forms the backbone of TB treatment, act with synergy when combined. One of the reasons (carba)penems are seldom used for treatment of TB is the high dosage levels required, often at the therapeutic limits. The synergistic combination of rifampin and these (carba)penems indicates that (carba)penems can be administered at dosages that are therapeutically relevant. The combination of faropenem and rifampin also limits the frequency of resistant mutants, as we were unable to obtain spontaneous mutants in the presence of these two drugs. The combinations of rifampin and (carba)penems were effective not only against drug-sensitive Mycobacterium tuberculosis but also against drug-resistant clinical isolates that are otherwise resistant to rifampin. A combination of doripenem or biapenem and rifampin also exhibited synergistic activity against Mycobacterium abscessus. Although the MICs of these three drugs alone against M. abscessus are too high to be of clinical relevance, their concentrations in combinations are therapeutically relevant; therefore, they warrant further evaluation for clinical utility to treat Mycobacterium abscessus infection, especially in cystic fibrosis patients.     

A case of mediastinal abscess and infected aortic aneurysm caused by dissemination of Mycobacterium abscessus subsp. massiliense pulmonary disease

An 81-year-old man was admitted to our hospital because of fever and malaise that had persisted for 3 months. The patient had undergone two aortic valve replacements, 10 and 5 years previously, because of aortic valve regurgitation and infectious endocarditis. He also had had asymptomatic Mycobacterium abscessus complex (MABC) pulmonary disease for the two previous years. Contrast-enhanced computed tomography showed a mediastinal abscess and an ascending aortic aneurysm. Mycobacterium abscessus subsp. massiliense was cultured from his blood, suggesting the aortic aneurysm was secondary to infection of an implanted device. After enlargement over only a few days, a leakage of contrast medium to the mediastinal abscess was found on computed tomography. The patient was diagnosed with rupture of an infectious aortic aneurysm, and emergency aortic replacement and drainage of the mediastinal abscess were successful. The patient was treated with several antibiotics, including meropenem, amikacin, and clarithromycin, and his general condition improved. Cultures from both the mediastinal abscess and a pericardial patch that was placed at the time of surgery 5 years previously revealed MABC. In our case, the infected aortic aneurysm most likely resulted from MABC pulmonary disease rather than from previous intraoperative contamination. This route of infection is rare. Physicians should be aware of the possibility of dissemination and subsequent infection of implants related to MABC pulmonary disease.     

In vitro activity of tedizolid against the Mycobacterium abscessus complex

Infections due to Mycobacterium abscessus carry a poor prognosis since this rapidly growing mycobacterium is intrinsically resistant to most antibiotics. Here, we evaluate the in vitro activity of the new oxazolidinone tedizolid against a collection of 44 M. abscessus clinical isolates. The MIC₅₀s and MIC₉₀s of tedizolid (2 and 8 μg/mL, respectively) were 2- to 16-fold lower than those of linezolid. There was no difference between the 3M. abscessus subspecies. Time-kill assays did not show any bactericidal activity at 4- and 8-fold the MIC. Combination of tedizolid with clarithromycin was synergistic against 1 out of 6 isolates, while indifferent interactions were observed for tedizolid combined with tigecycline, ciprofloxacin, and amikacin.     

Mycobacterium abscessus and massiliense lung infection during macrolide treatment for bronchiolitis obliterans after allogeneic hematopoietic stem cell transplantation

In patients undergoing allogeneic hematopoietic stem cell transplantation (allo-SCT), post-transplant lung infection is critical for their prognosis. Mycobacterium abscessus complex is not fully recognized as a nontuberculous mycobacteria (NTM) pathogen of post-SCT lung infection. Here, we present three post-allogeneic SCT patients who developed pulmonary infection caused by M. abscessus complex including M. abscessus and M. massiliense. In all three cases, macrolide antibiotics had been administered for bronchiolitis obliterans syndrome (BOS) before the confirmation of their infection, and macrolide resistance was noted in the M. abscessus isolates, one of which resulted in an unfavorable treatment outcome. It is important to consider M. abscessus lung infection as well as other NTM in patients receiving allo-SCT, particularly those receiving macrolide therapy for BOS.     

In Vitro Synergy between Clofazimine and Amikacin in Treatment of Nontuberculous Mycobacterial Disease

Disease caused by nontuberculous mycobacteria (NTM) is increasing in frequency. The outcome of treatment for NTM lung disease is poor, particularly lung disease caused by Mycobacterium simiae and M. abscessus. Exploring synergy between active available drugs is a sensible way forward given the lack of new active drugs. We tested for synergy between amikacin and clofazimine, using standardized methods, in 564 consecutive clinical isolates identified as 21 species of rapidly growing mycobacteria, 16 clinical M. avium complex isolates, and 10 M. simiae isolates. Clofazimine and amikacin are each active in vitro against NTM; 97% (n = 548) of the rapid growers revealed MICs of clofazimine of ≤1 μg/ml, and 93% (n = 524) proved susceptible to amikacin. The combination showed significant synergistic activity in 56 of 68 (82%) eligible M. abscessus isolates, 4 of 5 M. chelonae isolates, and 1 M. fortuitum and 1 M. cosmeticum isolate, with 4- to 8-fold decreases in MICs to both drugs. Significant synergy could also be demonstrated against all M. avium complex and M. simiae isolates, with fractional inhibitory concentrations of <0.5. Clofazimine and amikacin show significant synergistic activity against both rapidly and slowly growing nontuberculous mycobacteria. The safety and tolerability of adding clofazimine to amikacin-containing regimens should be tested in clinical trials, and the results of susceptibility tests for these two compounds and their combination merit clinical validation. Synergy between clofazimine and other antibiotics with intracellular targets should be explored.     

In vitro susceptibility patterns for rapidly growing nontuberculous mycobacteria in the United States

Antimicrobial susceptibility testing for rapidly growing mycobacteria (RGM) is uncommon or only performed in large reference laboratories. Here we developed a cumulative antibiogram for 14 RGM using the largest sample size to date (N = 3860). All RGM showed 82% to 100% susceptibility to amikacin. Mycobacterium abscessus showed low percentages of susceptibility to most antimicrobials; of antimicrobials without interpretations, the minimum inhibitory concentration-90 for clofazimine was low (≤0.5mg/L). All three subspecies had ≤2.6% rrl resistance mutations, however intact erm(41) was detected in 70% to100% of M. abscessus abscessus and bolletii. Mycobacterium chelonae had a similar susceptibility pattern to M. abscessus subsp. massiliense and Mycobacterium immunogenum except that it was susceptible to tobramycin (87%). Mycobacterium fortuitum complex and similar organisms showed higher frequency of susceptibility to fluoroquinolones, beta-lactams, linezolid, and trimethoprim/sulfamethoxazole. Although relatively small published RGM antibiograms showed substantial variance, a comprehensive antibiogram can help influence treatment and monitoring patterns of resistance.     

结核分枝杆菌临床分离株药敏结果与耐药程度的关联分析

目的 探讨结核分枝杆菌临床分离株对12种抗结核药物的MIC值及其药敏检测结果 与耐药程度的关联规律,从而为临床制定治疗方案提供借鉴依据.方法 采用液体培养基联合MTT技术进行抗结核药物的MIC检测.对上海市肺科医院2009年1-6月间的163株结核分枝杆菌临床分离株进行RFP、INH、SM、EMB、OFLX、LVFX、MOX、AMK、CPM、PTA、CLA和PAIN的MIC检测和Bactec MGIT快速培养仪药敏检测,并进行MIC与耐药程度的关联性分析.结果 67%(42/62)的SM耐药株MIC≥16 μg/ml,63%(51/81)的INH耐药株MIC≥8 μg/ml,77%(50/65)的RFP耐药株MIC≥8 μg/ml,20%(12/60)的EMB耐药株MIC≥4 μg/ml;43%(25/58)的OFLX耐药株MIC≥8 μg/ml;41%(15/37)的AMK耐药菌株MIC≥16 μg/ml,41%(12/29)的CPM耐药菌株MIC≥4 μg/ml.OFLX耐药株的3个氟喹诺酮类药物的MIC(OFLX、LVFX和MOX的MIC分别为2～128、1～32和0.0625～1 μg/ml)差异有统计学意义(F=16.874,P<0.01);SM、INH、RFP、EMB、OFLX、AMK和CPM在任6种及7种药物同时耐药株中的MIC值(分别为0.5～128、2～64、0.25～128、1～32、1～64、0.5～128和1～128μg/ml)明显高于任1种及2种药物耐药菌株的MIC值(MIC值分别为0.25～128、0.0625～64、0.25～32、0.25～2、0.125～2、0.5～4和1～4 μg/ml,F值分别为20.066、40.499、47.197、70.373、91.432、41.840和21.547,P均<0.05);SM、INH、RFP、EMB在4种药物同时耐药菌株中MIC值(分别为1～128、2～64、0.25～128和1～32μg/ml)显著高于任1种及2种药物耐药株(MIC值分别为0.25～128、0.0625～64、0.25～64和0.25～2 μg/ml,F值分别为26.242、23.563、31.541和64.469,P均<0.05);RFP在MDR酶株的MIC值(2～64 μg/ml)显著高于非MDR的耐药菌株的MIC值(0.25 μg/ml,F=5.613,P<0.05).结论 本研究揭示了常用的12种抗结核药物的耐药程度与常规药敏检测结果 之间的关联规律,为临床医生根据常规药敏结果 制定更加有效的抗结核治疗方案提供重要的借鉴.     

Analysis of synergy between beta-lactams and anti-methicillin-resistant Staphylococcus aureus agents from the standpoint of strain characteristics and binding action

In light of the increasing number of clinical cases resistant to traditional monotherapies and the lack of novel antimicrobial agents, combination therapy is an appealing solution for the treatment of methicillin-resistant Staphylococcus aureus (MRSA) infections. Here, we evaluated the efficacy of anti-MRSA agents, such as vancomycin (VAN), daptomycin (DAP), and linezolid (LZD), in conjunction with 13 beta-lactams and non-beta-lactams. We assessed the in vitro activities of the various combinations against 40 MRSA strains based on the maximum synergistic effect (MSE), an index calculated from the MIC change with a combination agent. Nearly all the anti-MRSA agents, which were combined with beta-lactams as well as VAN and DAP, showed a synergistic effect with arbekacin. VAN also exhibited varying degrees of synergy depending on the type of beta-lactam, whereas DAP and LZD showed similar synergy with different beta-lactams. These effects were confirmed by antibiotic kill curves, except for the apparent interaction between LZD and beta-lactams. The MSE results were analyzed according to strain characteristics including susceptibility to combination agents, staphylococcal cassette chromosome mec type, and presence of the blaZ gene; however, no obvious correlations were observed. In a fluorescence binding assay, the fluorescence intensity of boron-dipyrromethene (BODIPY)-VAN decreased, whereas that of BODIPY-DAP increased in combination with a beta-lactam agent. These findings suggest that beta-lactam combinations are promising treatment options for MRSA infections and that the type of beta-lactam combined with VAN is important for the synergistic effect.     

Treatment outcomes of macrolide-susceptible Mycobacterium abscessus lung disease

Mycobacterium abscessus lung disease is difficult to treat due to inducible resistance to macrolides. However, 15%–20% of isolates are macrolide susceptible. In 14 patients with macrolide-susceptible M. abscessus lung disease, all isolates had nonfunctional erm(41) gene, and sputum culture conversion rate was achieved in 93% (13/14) following antibiotic therapy.     

Antipseudomonal Agents Exhibit Differential Pharmacodynamic Interactions with Human Polymorphonuclear Leukocytes against Established Biofilms of Pseudomonas aeruginosa

Pseudomonas aeruginosa is the most common pathogen infecting the lower respiratory tract of cystic fibrosis (CF) patients, where it forms tracheobronchial biofilms. Pseudomonas biofilms are refractory to antibacterials and to phagocytic cells with innate immunity, leading to refractory infection. Little is known about the interaction between antipseudomonal agents and phagocytic cells in eradication of P. aeruginosa biofilms. Herein, we investigated the capacity of three antipseudomonal agents, amikacin (AMK), ceftazidime (CAZ), and ciprofloxacin (CIP), to interact with human polymorphonuclear leukocytes (PMNs) against biofilms and planktonic cells of P. aeruginosa isolates recovered from sputa of CF patients. Three of the isolates were resistant and three were susceptible to each of these antibiotics. The concentrations studied (2, 8, and 32 mg/liter) were subinhibitory for biofilms of resistant isolates, whereas for biofilms of susceptible isolates, they ranged between sub-MIC and 2 × MIC values. The activity of each antibiotic alone or in combination with human PMNs against 48-h mature biofilms or planktonic cells was determined by XTT [2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide] assay. All combinations of AMK with PMNs resulted in synergistic or additive effects against planktonic cells and biofilms of P. aeruginosa isolates compared to each component alone. More than 75% of CAZ combinations exhibited additive interactions against biofilms of P. aeruginosa isolates, whereas CIP had mostly antagonistic interaction or no interaction with PMNs against biofilms of P. aeruginosa. Our findings demonstrate a greater positive interaction between AMK with PMNs than that observed for CAZ and especially CIP against isolates of P. aeruginosa from the respiratory tract of CF patients.     

Antimicrobial susceptibility testing of Mycobacteroides (Mycobacterium) abscessus complex,Mycolicibacterium (Mycobacterium) fortuitum,and Mycobacteroides (Mycobacterium) chelonae

The drug susceptibility of rapidly growing mycobacteria (RGM) varies among isolates. Treatment strategies similarly differ depending on the isolate, and for some, no clear strategy has been identified. This complicates clinical management of RGM. Following Clinical and Laboratory Standards Institute standard M24-A2, we assessed the susceptibility of 140 RGM isolates to 14 different antimicrobial drugs by measuring their minimal inhibitory concentrations (MICs). We also investigated the correlation of clarithromycin (CAM) MICs with the erm(41) and rrl gene mutations in the Mycobacteroides (Mycobacterium) abscessus complex, the rrl mutation in Mycobacteroides (Mycobacterium) chelonae, and the erm(39) mutation in Mycolicibacterium (Mycobacterium) fortuitum to determine the contribution of these mutations to CAM susceptibility. The five species and subspecies examined included 48 M. abscessus subsp. abscessus isolates (34.3%), 35 (25.0%) being M. abscessus subsp. massiliense, and two (1.4%) being M. abscessus subsp. bolletii. The M. abscessus complex accounted for 85 isolates (60.7%) in total, whereas 43 isolates (30.7%) were M. fortuitum, and 12 (8.6%) were M. chelonae. Our results demonstrated species-specific susceptibility to antimicrobials. In most cases, susceptibility to CAM could be predicted based on genetic pattern, but since one isolate did not fit that pattern, MIC values needed to be measured. Some isolates also exhibited rates of resistance to other drugs that differed from those previously reported in other locations, indicating that accurate identification of the bacterial isolate and use of the correct method for determining MIC are both important for the diagnosis of RGM.     

In vitro and in vivo activities of rifampin, streptomycin, amikacin, moxifloxacin, R207910, linezolid, and PA-824 against Mycobacterium ulcerans

Seven antimicrobials were tested in vitro against 29 clinical isolates of Mycobacterium ulcerans. R207910 demonstrated the lowest MIC(50) and MIC(90), followed by moxifloxacin (MXF), streptomycin (STR), rifampin (RIF), amikacin (AMK), linezolid (LZD), and PA-824. All but PA-824 demonstrated an MIC(90) significantly less than the clinically achievable peak serum level. Administered as monotherapy to mice, RIF, STR, AMK, MXF, R207910, and LZD demonstrated some degree of bactericidal activity, whereas PA-824 failed to prevent mortality and to reduce the mean number of CFU in the footpads. Because 4 or 8 weeks of treatment by the combinations RIF-MXF, RIF-R207910, and RIF-LZD displayed bactericidal effects similar to those of RIF-STR and RIF-AMK, these three combinations might be considered as orally administered combined regimens for treatment of Buruli ulcer. Taking into account the cost, potential toxicity, and availability, the combination RIF-MXF appears more feasible for application in the field; additional experiments with mice are warranted to define further its activity against M. ulcerans. In addition, a pilot clinical trial is proposed to test the efficacy of RIF-MXF for treatment of Buruli ulcer.     

Mycobacterium tuberculosis Mutations Associated with Reduced Susceptibility to Linezolid

Linezolid (LZD) has become increasingly important for the treatment of multidrug-resistant tuberculosis (MDR-TB), but its mechanisms of resistance are not well characterized. We isolated 32 mutants of Mycobacterium tuberculosis with reduced susceptibility to LZD, which was accounted for by rrl and rplC mutations in almost equal proportions, causing lower and higher MICs, respectively. Our findings provide useful information for the rapid detection of LZD resistance for improved treatment of MDR-TB.     

Clofazimine Prevents the Regrowth of Mycobacterium abscessus and Mycobacterium avium Type Strains Exposed to Amikacin and Clarithromycin

Multidrug therapy is a standard practice when treating infections by nontuberculous mycobacteria (NTM), but few treatment options exist. We conducted this study to define the drug-drug interaction between clofazimine and both amikacin and clarithromycin and its contribution to NTM treatment. Mycobacterium abscessus and Mycobacterium avium type strains were used. Time-kill assays for clofazimine alone and combined with amikacin or clarithromycin were performed at concentrations of 0.25× to 2× MIC. Pharmacodynamic interactions were assessed by response surface model of Bliss independence (RSBI) and isobolographic analysis of Loewe additivity (ISLA), calculating the percentage of statistically significant Bliss interactions and interaction indices (I), respectively. Monte Carlo simulations with predicted human lung concentrations were used to calculate target attainment rates for combination and monotherapy regimens. Clofazimine alone was bacteriostatic for both NTM. Clofazimine-amikacin was synergistic against M. abscessus (I = 0.41; 95% confidence interval [CI], 0.29 to 0.55) and M. avium (I = 0.027; 95% CI, 0.007 to 0.048). Based on RSBI analysis, synergistic interactions of 28.4 to 29.0% and 23.2 to 56.7% were observed at 1× to 2× MIC and 0.25× to 2× MIC for M. abscessus and M. avium, respectively. Clofazimine-clarithromycin was also synergistic against M. abscessus (I = 0.53; 95% CI, 0.35 to 0.72) and M. avium (I = 0.16; 95% CI, 0.04 to 0.35), RSBI analysis showed 23.5% and 23.3 to 53.3% at 2× MIC and 0.25× to 0.5× MIC for M. abscessus and M. avium, respectively. Clofazimine prevented the regrowth observed with amikacin or clarithromycin alone. Target attainment rates of combination regimens were >60% higher than those of monotherapy regimens for M. abscessus and M. avium. The combination of clofazimine with amikacin or clarithromycin was synergistic in vitro. This suggests a potential role for clofazimine in treatment regimens that warrants further evaluation.     

Impact of β-Lactamase Inhibition on the Activity of Ceftaroline against Mycobacterium tuberculosis and Mycobacterium abscessus

The production of β-lactamases Bla_Mab and BlaC contributes to β-lactam resistance in Mycobacterium abscessus and Mycobacterium tuberculosis, respectively. Ceftaroline was efficiently hydrolyzed by these enzymes. Inhibition of M. tuberculosis BlaC by clavulanate decreased the ceftaroline MIC from ≥256 to 16 to 64 μg/ml, but these values are clinically irrelevant. In contrast, the ceftaroline-avibactam combination should be evaluated against M. abscessus since it inhibited growth at lower and potentially achievable drug concentrations.     

Mycobacterium tuberculosis rrs A1401G mutation correlates with high-level resistance to kanamycin,amikacin, and capreomycin in clinical isolates from mainland China

Mutations correlating phenotypic resistance level with the injectable second-line anti-tuberculosis drugs (SLDs) including kanamycin (KAN), amikacin (AMK), and capreomycin (CAP) remain elusive. A collection of 114 Mycobacterium tuberculosis clinical isolates from mainland China was analyzed. The minimum inhibitory concentration (MIC) of each strain was determined and the sequences of rrs, tlyA, promoter of eis as well as 5′ untranslated region (UTR) of whiB7 were amplified and sequenced. No mutation in tlyA, promoter of eis and 5′ UTR of whiB7, was found to be associated with resistance among these samples. Sequencing data of 1400 rrs region demonstrated the A1401G mutation in rrs was prevalent, which presented in 84% of the KAN resistant isolates while only in about 50% of the AMK or CAP resistant isolates. Furthermore, most of the resistant isolates with A1401G mutation showed high-level resistance to these injectable SLDs. In conclusion, our results suggest the rrs A1401G mutation was related to high-level resistance to KAN, AMK, and CAP in M. tuberculosis isolates from mainland China.