Florence Brossier

Assessment of Clarithromycin Susceptibility in Strains Belonging to the Mycobacterium abscessus Group by erm(41) and rrl Sequencing

ABSTRACT Clarithromycin was the drug of choice for Mycobacterium abscessus infections until inducible resistance due to erm(41) was described. Because M. abscessus was split into M. abscessus sensu stricto, Mycobacterium massiliense, and Mycobacterium bolletii, we looked for erm(41) in the three species and determined their clarithromycin susceptibility levels. Ninety strains were included: 87 clinical strains from cystic fibrosis patients (61%) and others (39%), representing 43 M. abscessus, 30 M. massiliense, and 14 M. bolletii strains identified on a molecular basis, and 3 reference strains. Clarithromycin and azithromycin MICs were determined by broth microdilution and Etest with a 14-day incubation period. Mutations in rrl (23S rRNA gene) known to confer acquired clarithromycin resistance were also sought. erm(41) was detected in all strains but with two deletions in all M. massiliense strains. These strains were indeed susceptible to clarithromycin (MIC90 of 1 μg/ml) except for four strains with rrl mutations. M. abscessus strains harbored an intact erm(41) but had a T/C polymorphism at the 28th nucleotide: T28 strains (Trp10 codon) demonstrated inducible clarithromycin resistance (MIC90 of >16 μg/ml), while C28 strains (Arg10) were susceptible (MIC90 of 2 μg/ml) except for two strains with rrl mutations. M. bolletii strains had erm(41) sequences similar to the sequence of the T28 M. abscessus group, associated with inducible clarithromycin resistance (MIC90 of >16 μg/ml). erm(41) sequences appeared species specific within the M. abscessus group and were fully concordant with clarithromycin susceptibility when erm(41) sequencing was associated with detection of rrl mutations. Clarithromycin-resistant strains, including the six rrl mutants, were more often isolated in cystic fibrosis patients, but this was not significantly associated with a previous treatment.

Detection by GenoType MTBDRsl Test of Complex Mechanisms of Resistance to Second-Line Drugs and Ethambutol in Multidrug-Resistant Mycobacterium tuberculosis Complex Isolates

ABSTRACT The GenoType MTBDRsl test rapidly detects resistance to ethambutol, fluoroquinolones, and second-line aminoglycosides (amikacin and kanamycin) and cyclic peptide (capreomycin) in Mycobacterium tuberculosis. A set of 41 multidrug-resistant (MDR) M. tuberculosis strains, 8 extensively drug-resistant (XDR) M. tuberculosis strains, and 3 non-MDR M. tuberculosis strains were tested by the MTBDRsl test and by DNA sequencing of the resistance-determining regions in gyrA and gyrB (fluoroquinolones [FQ]), rpsL (streptomycin), rrs and tlyA (aminoglycosides and/or cyclic peptide), and embB (ethambutol). The sensitivity and specificity of the MTBDRsl test were as follows: 87% and 96%, respectively, for fluoroquinolones; 100% for both for amikacin; 77% and 100%, respectively, for kanamycin, 80% and 98%, respectively, for capreomycin; and 57% and 92%, respectively, for ethambutol. Analysis of the discrepant results indicated that three FQ-resistant strains (including one XDR strain) with mutations in gyrB were missed by the MTBDRsl test and that one FQ-susceptible strain, identified as resistant by the MTBDRsl test, had a double mutation (T80A-A90G) in GyrA that did not confer resistance to FQ. Five strains (including two XDR strains) without mutations in rrs were monoresistant to aminoglycosides or cyclic peptide and were missed by the MTBDRsl test. Finally, 12/28 ethambutol-resistant strains had no mutation at codon 306 in embB, while 2/24 ethambutol-susceptible strains had such a mutation. In conclusion, the MTBDRsl test efficiently detects the most common mutations involved in resistance to fluoroquinolones, aminoglycosides/cyclic peptide, and ethambutol and accurately assesses susceptibility to amikacin. However, due to mutations not included in the test (particularly in gyrB) or resistance mechanisms not yet characterized (particularly those related to ethambutol resistance and to monoresistance to aminoglycosides or cyclic peptide), the wild-type results yielded by the MTBDRsl test should be confirmed by drug susceptibility testing.

Performance of the genotype MTBDR line probe assay for detection of resistance to rifampin and isoniazid in strains of Mycobacterium tuberculosis with low- and high-level resistance.

ABSTRACT We assessed the performance of the Genotype MTBDR line probe assay that offers the simultaneous identification of Mycobacterium tuberculosis and its resistance to rifampin (RIF) and isoniazid (INH) by detecting the most commonly found mutations in the rpoB and katG genes. One hundred thirteen M. tuberculosis isolates were tested. The nucleotide sequences of the katG and inhA genes and the mabA-inhA promoter region were also determined. The MTBDR assay detected 100% and 67% (n = 64) of the strains resistant to RIF and INH, respectively. Among the latter, 62 strains carried a Ser315Thr mutation in katG, 59 of them displaying a high level of resistance to INH. Two strains with a low level of INH resistance had a Ser315Asn mutation. No mutation was found by the MTBDR assay for 31 INH-resistant strains (33%), of which 24 showed a low level of resistance. By DNA sequencing, we found among them various mutations in the KatG protein for 7 strains, a C→T mutation in position −15 of the mabA-inhA promoter in 17 strains, and a Ser94Ala mutation in InhA for 7 strains. In conclusion, the MTBDR assay, which fits easily in the workflow of a routine laboratory, enabled the detection of 100% of the RIF-resistant strains and 89% of the INH-resistant strains with a high level of resistance but only 17% of the strains characterized by a low level of INH resistance, indicating that the test can be used as a rapid method to detect in the same experiment the rifampin-resistant and the high-level isoniazid-resistant strains of M. tuberculosis.

Outbreak of Nontuberculous Mycobacterial Subcutaneous Infections Related to Multiple Mesotherapy Injections

ABSTRACT We describe an outbreak of severe subcutaneous infections due to nontuberculous mycobacteria following mesotherapy. Epidemiological studies and molecular comparisons of Mycobacterium chelonae strains from different patients and the environment suggested that contamination may be associated with inappropriate cleaning of the multiple-injection device with tap water.

Imipenem, Meropenem, or Doripenem To Treat Patients with Pseudomonas aeruginosa Ventilator-Associated Pneumonia

ABSTRACT Only limited data exist on Pseudomonas aeruginosa ventilator-associated pneumonia (VAP) treated with imipenem, meropenem, or doripenem. Therefore, we conducted a prospective observational study in 169 patients who developed Pseudomonas aeruginosa VAP. Imipenem, meropenem, and doripenem MICs for Pseudomonas aeruginosa isolates were determined using Etests and compared according to the carbapenem received. Among the 169 isolates responsible for the first VAP episode, doripenem MICs were lower (P < 0.0001) than those of imipenem and meropenem (MIC50s, 0.25, 2, and 0.38, respectively); 61%, 64%, and 70% were susceptible to imipenem, meropenem, and doripenem, respectively (P was not statistically significant). Factors independently associated with carbapenem resistance were previous carbapenem use (within 15 days) and mechanical ventilation duration before VAP onset. Fifty-six (33%) patients had at least one VAP recurrence, and 56 (33%) died. Factors independently associated with an unfavorable outcome (recurrence or death) were a high day 7 sequential organ failure assessment score and mechanical ventilation dependency on day 7. Physicians freely prescribed a carbapenem to 88 patients: imipenem for 32, meropenem for 24, and doripenem for 32. The remaining 81 patients were treated with various antibiotics. Imipenem-, meropenem-, and doripenem-treated patients had similar VAP recurrence rates (41%, 25%, and 22%, respectively; P = 0.15) and mortality rates (47%, 25%, and 22%, respectively; P = 0.07). Carbapenem resistance emerged similarly among patients treated with any carbapenem. No carbapenem was superior to another for preventing carbapenem resistance emergence.

Standardized interpretation of antibiotic susceptibility testing and resistance genotyping for Mycobacterium abscessus with regard to subspecies and erm41 sequevar

OBJECTIVES The objective of this study was to provide standardized antibiotic susceptibility testing (AST) for Mycobacterium abscessus with regard to subspecies. METHODS One hundred and sixty-five clinical isolates were tested for susceptibility to 15 antibiotics using a commercial microdilution method, at two reading times: (i) early reading time (ERT), when the growth control was first positive; and (ii) late reading time (LRT), of 14 days, for detecting inducible resistance. In addition, genes or mutations involved in resistance were studied [erm(41), rrl and rrs]. RESULTS Three patterns were observed for clarithromycin: (i) MIC >16 mg/L at ERT (median 5 days) for 15 isolates [10 subsp. abscessus erm(41) sequevar T28, 3 subsp. bolletii and 2 subsp. massiliense] among which 9 harboured an a2058g/c rrl mutation; (ii) MIC ≤16 mg/L at ERT, but >16 mg/L at LRT, for 106 isolates [84 abscessus erm(41) T28 and 22 bolletii] showing intrinsic inducible resistance; and (iii) MIC ≤4 mg/L at ERT and LRT for 44 isolates [18 abscessus erm(41) C28 and 26 massiliense]. Amikacin MIC was >64 mg/L for eight isolates [five abscessus erm(41) T28, two massiliense and one bolletii] among which seven harboured the a1408g rrs mutation, but ≤64 mg/L for the remaining isolates without mutation. For the other antibiotics, only one WT pattern was observed, with cefoxitin, tigecycline and linezolid showing MIC values compatible with susceptibility. CONCLUSIONS Standard AST can predict clarithromycin and amikacin resistance using interpretation rules with regard to subspecies. For other antibiotics, since only one pattern is observed, there is no need for systematic phenotypic or genotypic testing.

The in vitro mechanisms of isoniazid and ethionamide resistance poorly reflect those in vivo in Mycobacterium tuberculosis.

HAL is a multi-disciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. The in vitro mechanisms of isoniazid and ethionamide resistance poorly reflect those in vivo in Mycobacterium tuberculosis Florence Brossier, Emmanuelle Cambau, Emilie Tessier, Vincent Jarlier, Wladimir Sougakoff

Assessing Primary and Secondary Resistance to Clarithromycin and Amikacin in Infections Due to Mycobacterium avium Complex

Mycobacterium avium and Mycobacterium intracellulare are the most clinically important species of the M. avium complex (MAC). From 2009 to 2011, 277 M. avium and 229 M. intracellulare isolates considered responsible for infection based on the ATS/IDSA criteria ([1][1]) were studied ([Table 1][2]).

Could the DiversiLab® semi-automated repetitive-sequence-based PCR be an acceptable technique for typing isolates of Pseudomonas aeruginosa? An answer from our experience and a review of the literature

Recently the DiversiLab® (DL) system (bioMérieux) was developed as an automated platform that uses repetitive element polymerase chain reaction (rep-PCR) technology for standardized, reproducible DNA fingerprinting of bacteria. The purpose of this study was to evaluate the usefulness of DL rep-PCR for typing Pseudomonas aeruginosa isolates. The performance of DL rep-PCR was compared with that of pulsed-field gel electrophoresis (PFGE) in a prospective multicenter study of patients with ventilator-associated pneumonia due to P. aeruginosa, conducted in 3 intensive care units over a 31-month period. In total, 203 P. aeruginosa isolates from 66 patients, from whom at least 2 consecutive respiratory samples each were collected more than 48 h apart, were typed using DL rep-PCR. Forty isolates (corresponding to 20 patients) were also typed using PFGE of SpeI-digested DNA. The typeability was 100% with DL rep-PCR and 95% with PFGE. The discriminatory power was close for DL rep-PCR and for PFGE (Simpsons diversity indices of 0.901 and 0.947, respectively). Insufficient agreement between DL rep-PCR and PFGE typing results was observed for the 40 selected isolates (adjusted Rand coefficient of 0.419), mostly due to isolates of the same DL rep-PCR type but of different PFGE types (adjusted Wallace coefficients of 0.306 for DL rep-PCR with PFGE, and of 0.667 for PFGE with DL rep-PCR). Considered together with published data, DL rep-PCR results should be interpreted with caution for the investigation of outbreaks caused by P. aeruginosa and evaluated in conjunction with epidemiological data.

Concomitant multidrug-resistant pulmonary tuberculosis and susceptible tuberculous meningitis.

To the Editor: In 2012, a 34-year-old HIV-seronegative man was hospitalized after several months of cough, fever, night sweats, 10-kg weight loss, and, in the past month, severe headache. The patient was born in Romania and had lived in France for 2 years. He had a history of pulmonary tuberculosis (TB) for which treatment was started in Romania in 2006 and 2008, but he did not complete treatment. The treatment he received in Romania was unknown. At hospital admission, the patient had a fever of 39°C, stiff neck, and swollen cervical and axillary lymph nodes. A chest radiograph showed multiple cavities and nodular opacities in both superior lobes. Sputum auramine staining indicated that acid-fast bacilli was positive, which supported the diagnosis of pulmonary TB. Examination of cerebrospinal fluid (CSF) revealed hypoglycorrachia (0.95 mmol/L, concentration ratio CSF/blood: 0.2 [reference range 0.5–0.75]), hyperproteinorrachia (1.3 g/L [reference range 0.2–0.4 g/L]), erythrocyte count 2,000 μL (reference value <10 μL), and leukocyte count 150 μL (reference value <10 μL). Auramine staining showed no acid-fast bacilli in CSF. Standard antituberculous therapy with rifampin (RIF), isoniazid (INH), pyrazinamide, and ethambutol was started. Genomic amplification–based assay (Xpert MTB/RIF; Cepheid, Maurens-Scopont, France), performed on sputum, confirmed the presence of the Mycobacterium tuberculosis genome and detected resistance to RIF (Table). The line probe assay Genotype MTBDRplus (Hain Lifescience, Bandol, France) performed on sputum showed a positive signal for all wild-type sequences and for rpoB (S531L associated with RIF resistance) and katG (S315T associated with INH resistance) mutations, suggesting the presence of mixed susceptible and resistant M. tuberculosis. Second-line treatment was started: moxifloxacin, amikacin, ethionamide, para-aminosalicylic acid, cycloserine and linezolid. The presence of mixed M. tuberculosis organisms in lungs was confirmed with culture methods and by phenotypic drug susceptibility testing (DST) that showed 1% resistant mutant to RIF and INH (proportion method) (1). The isolate was considered resistant to RIF and INH and was thus categorized as multidrug resistant (MDR). DST also showed that the sputum isolate had an elevated proportion of ofloxacin-resistant mutants (2 mg/L, 0.02% resistant mutants). Although no mutation in gyrA or gyrB was detected on colonies grown in the absence of fluoroquinolone, a gyrB N538D mutation was identified on colonies grown on ofloxacin medium. On the basis of the DST results, the treatment was changed to ethambutol, pyrazinamide, amikacin, moxifloxacin, para-aminosalicylic acid, and linezolid. CSF culture was eventually positive for M. tuberculosis on Lowenstein-Jensen medium after 30 days. DST performed on the CSF isolate showed a drug-sensitive phenotype; thus, RIF was reintroduced in addition to the other antimicrobial drugs. After 3 months, although the patient had improved, he left the hospital against medical advice without providing follow-up contact information. Table Localization, drug-susceptibility results, and MIRU-VNTR genotypes of Mycobacterium tuberculosis strains recovered from a man with both pulmonary tuberculosis and tuberculous meningitis, France, 2012* Mycobacterial interspersed repetitive unit–variable number tandem repeat (MIRU-VNTR) analysis was conducted on culture of the sputum and CSF sample and on colonies grown on INH, RIF, and ofloxacin–containing medium, as described (2) (Table). This analysis showed different genotypes for the lung MDR and the CSF-susceptible isolates. None of the MIRU-VNTR patterns were linked to known lineages. The mutation rate of MIRU-VNTR has been evaluated at 2.7 × 10–3/loci/year (3). Rather than natural evolution of 1 strain, the 5-locus difference observed for the MIRU-VNTR genotypes is probably attributable to infection with 2 M. tuberculosis strains. Surprisingly, MIRU-VNTR patterns were identical for the gyrB mutated lung isolate and to the antimicrobial drug–susceptible isolate obtained from CSF. Also, the gyrB mutated lung isolate was susceptible to other antimicrobial drugs, whereas the lung MDR isolate did not harbor any gyrB mutation. Taken together, both genotype and DST heterogeneity were shown (Table). None of the drug-resistant clones were isolated in CSF. This heterogenous infection raises 2 hypotheses: the patient was infected with both the MDR and the non-MDR strains or with 2 non-MDR strains and acquired additional drug resistance in the lung during treatment. Clonal differences among M. tuberculosis culture isolates obtained from pulmonary and CSF samples indicate compartmentalization (4). Heterogeneity in M. tuberculosis isolates already has been reported either for genotypes or for drug susceptibility (5–7). However, combination of genotype and resistance heterogeneity is a diagnostic and therapeutic challenge (8). In our laboratory, the line probe assay performed directly on sputum detected the 1% INH- and RIF-resistant mycobacteria, although a previous report suggest that this assay could not detect <5% resistant mycobacteria (9). The antimicrobial drug–resistant clones were isolated from only lung. The higher bacillary population in lungs than in central nervous system may account for an increased selection of resistant mutants in lungs. This finding raises the question of whether DST obtained from lung isolates should be used for establishing TB treatment at extrapulmonary localizations, especially in patients with meningitis, for whom effective treatment is an emergency. Because first-line treatments are more effective, we wonder whether treatment for MDR TB patients who have secondarily acquired resistance and both lung and central nervous system TB should target MDR and drug-susceptible TB until the CSF strain is proven to also be MDR.