Inducible Macrolide Resistance Research Articles

Macrolones target bacterial ribosomes and DNA gyrase and can evade resistance mechanisms.

Growing resistance toward ribosome-targeting macrolide antibiotics has limited their clinical utility and urged the search for superior compounds. Macrolones are synthetic macrolide derivatives with a quinolone side chain, structurally similar to DNA topoisomerase-targeting fluoroquinolones. While macrolones show enhanced activity, their modes of action have remained unknown. Here, we present the first structures of ribosome-bound macrolones, showing that the macrolide part occupies the macrolide-binding site in the ribosomal exit tunnel, whereas the quinolone moiety establishes new interactions with the tunnel. Macrolones efficiently inhibit both the ribosome and DNA topoisomerase in vitro. However, in the cell, they target either the ribosome or DNA gyrase or concurrently both of them. In contrast to macrolide or fluoroquinolone antibiotics alone, dual-targeting macrolones are less prone to select resistant bacteria carrying target-site mutations or to activate inducible macrolide resistance genes. Furthermore, because some macrolones engage Erm-modified ribosomes, they retain activity even against strains with constitutive erm resistance genes.

Prevalence and antimicrobial susceptibility pattern of Mycobacterium abscessus complex isolates in Chongqing, Southwest China

ObjectivesTo investigate the prevalence of Mycobacterium abscessus complex (MABC), drug resistance characteristics, and the relationship between clarithromycin (CLA) susceptibility and MABC genotype in Chongqing, China. MethodsA total of 434 NTM patient isolates were collected between October 2018 and October 2019. Isolates confirmed to be non-tuberculous mycobacteria (NTM) were tested for minimal inhibitory concentrations of antimicrobial agents. In addition, rrl and erm(41) gene sequences were used to analyze the acquired macrolide resistance and inducible macrolide resistance. ResultsOverall, 17 different NTM species were detected, of which M. abscessus (22.6 %, 91/403) was most prevalent. Amikacin, CLA, azithromycin and cefoxitin exhibited potent activities against MABC organisms, but no significant differences were observed in drug resistance rates between M. abscessus and M. massiliense (P > 0.05). On day 3 of culture, the acquired resistance rate against CLA was 7.4 % (9/121). Of 41 MABC isolates with inducible CLA resistant, 95.1 % (39/41) isolates belonged to the erm(41) T28 sequevar, while the remaining 4.9 % (2/41) possessed the M. massiliense genotype. All erm(41) C28 sequevar isolates were sensitive to CLA on day3 and day 14 of culture. Meanwhile, of the 5 erm(41) T28 isolates with acquired resistance, all possessed rrl 2058/2059 mutations, including 3 isolates with A2058C mutation and 2 isolates with A2059G mutation. While 2 of the 4 M. massiliense isolates with acquired resistance possessed the A2059G mutation, and one isolate possessed the A2058G mutation. ConclusionErm(41) and rrl gene could serve as useful markers for predicting macrolide susceptibility of MABC complex isolates.

Erratum for Brown-Elliott et al., "Emergence of Inducible Macrolide Resistance in Mycobacterium chelonae Due to Broad-Host-Range Plasmid and Chromosomal Variants of the Novel 23S rRNA Methylase Gene, erm(55)".

Erratum for Brown-Elliott et al., "Emergence of Inducible Macrolide Resistance in Mycobacterium chelonae Due to Broad-Host-Range Plasmid and Chromosomal Variants of the Novel 23S rRNA Methylase Gene, erm(55)".

Treatment of Mycobacterium abscessus complex pulmonary disease

Background: In South Korea, bacteria in the <i>Mycobacterium abscessus</i> complex (MABC), a group of rapidly growing mycobacteria, are second to those in the <i>Mycobacterium avium</i> complex as a cause of non-tuberculous mycobacterial pulmonary disease. The MABC includes several subspecies, including <i>M. abscessus</i> subsp. <i>abscessus</i> (<i>M. abscessus</i>) and <i>M. abscessus</i> subsp. <i>massiliense</i> (<i>M. massiliense</i>), the former of which is difficult to treat owing to its antibiotic resistance.Current Concepts: <i>M. abscessus</i> encodes a functional erythromycin ribosomal methylase gene, <i>erm</i>(41), that causes inducible macrolide resistance. Contrastingly, <i>M. massiliense</i> lacks a functional <i>erm</i>(41) gene owing to a partial deletion. Accordingly, culture conversion rates using currently recommended macrolide-based antibiotic treatments are considerably higher among patients with <i>M. massiliense</i> infection than in those infected with <i>M. abscessus</i>. Phase therapy (intensive and continuous) is recommended for MABC pulmonary disease and, depending on the subspecies and antimicrobial susceptibility test results, should include an initial treatment of ≥3 to 4 injectable and oral antibiotics, followed by inhaled or intravenous amikacin and ≥1 to 2 oral antibiotics. Recommended injectable antibiotics include amikacin, imipenem or cefoxitin, and tigecycline, and oral antibiotics include macrolides (azithromycin or clarithromycin), clofazimine, linezolid, and rifabutin. For some patients, surgery should be considered as an adjunctive treatment option.Discussion and Conclusion: Given that <i>M. abscessus</i> expresses the inducible resistance gene <i>erm</i>(41) associated with macrolide resistance, the identification of MABC subspecies is important for disease management. However, despite the combined application of several injectable/oral antibiotics, the treatment outcomes for <i>M. abscessus</i> pulmonary disease remain unsatisfactory.

Emergence of Inducible Macrolide Resistance in Mycobacterium chelonae Due to Broad-Host-Range Plasmid and Chromosomal Variants of the Novel 23S rRNA Methylase Gene, erm(55).

Macrolides are a mainstay of therapy for infections due to nontuberculous mycobacteria (NTM). Among rapidly growing mycobacteria (RGM), inducible macrolide resistance is associated with four chromosomal 23S rRNA methylase (erm) genes. Beginning in 2018, we detected high-level inducible clarithromycin resistance (MICs of ≥16μg/mL) in clinical isolates of Mycobacterium chelonae, an RGM species not previously known to contain erm genes. Using whole-genome sequencing, we identified a novel plasmid-mediated erm gene. This gene, designated erm(55)P, exhibits <65% amino acid identity to previously described RGM erm genes. Two additional chromosomal erm(55) alleles, with sequence identities of 81% to 86% to erm(55)P, were also identified and designated erm(55)C and erm(55)T. The erm(55)T is part of a transposon. The erm(55)P allele variant is located on a putative 137-kb conjugative plasmid, pMchErm55. Evaluation of 133 consecutive isolates from 2020 to 2022 revealed 5 (3.8%) with erm(55). The erm(55)P gene was also identified in public data sets of two emerging pathogenic pigmented RGM species: Mycobacterium iranicum and Mycobacterium obuense, dating back to 2008. In both species, the gene appeared to be present on plasmids homologous to pMchErm55. Plasmid-mediated macrolide resistance, not described previously for any NTM species, appears to have spread to multiple RGM species. This has important implications for antimicrobial susceptibility guidelines and treatment of RGM infections. Further spread could present serious consequences for treatment of other macrolide-susceptible RGM. Additional studies are needed to determine the transmissibility of pMchErm55 and the distribution of erm(55) among other RGM species.

Inducible Mega-Mediated Macrolide Resistance Confers Heteroresistance in Streptococcus pneumoniae.

In Streptococcus pneumoniae (Spn), the 5.4 to 5.5 kb Macrolide Genetic Assembly (Mega) encodes an efflux pump (Mef[E]) and a ribosomal protection protein (Mel) conferring antibiotic resistance to commonly used macrolides in clinical isolates. We found the macrolide-inducible Mega operon provides heteroresistance (more than 8-fold range in MICs) to 14- and 15-membered ring macrolides. Heteroresistance is commonly missed during traditional clinical resistance screens but is highly concerning as resistant subpopulations can persist despite treatment. Spn strains containing the Mega element were screened via Etesting and population analysis profiling (PAP). All Mega-containing Spn strains screened displayed heteroresistance by PAP. The heteroresistance phenotype was linked to the mRNA expression of the mef(E)/mel operon of the Mega element. Macrolide induction uniformly increased Mega operon mRNA expression across the population, and heteroresistance was eliminated. A deletion of the 5' regulatory region of the Mega operon results in a mutant deficient in induction as well as in heteroresistance. The mef(E)L leader peptide sequence of the 5' regulatory region was required for induction and heteroresistance. Treatment with a noninducing 16-membered ring macrolide antibiotic did not induce the mef(E)/mel operon or eliminate the heteroresistance phenotype. Thus, inducibility of the Mega element by 14- and 15-membered macrolides and heteroresistance are linked in Spn. The stochastic variation in mef(E)/mel expression in a Spn population containing Mega provides the basis for heteroresistance.

Towards clinical breakpoints for non-tuberculous mycobacteria – Determination of epidemiological cut off values for the Mycobacterium avium complex and Mycobacterium abscessus using broth microdilution

ObjectiveFor non-tuberculous mycobacteria (NTM), minimum inhibitory concentration (MIC) distributions of wild-type isolates have not been systematically evaluated despite their importance for establishing antimicrobial susceptibility testing (AST) breakpoints. MethodsWe gathered MIC distributions for drugs used against the Mycobacterium avium complex (MAC) and Mycobacterium abscessus (MAB) obtained by commercial broth microdilution (SLOMYCOI and RAPMYCOI) from 12 laboratories. Epidemiological cut-off values (ECOFFs) and tentative ECOFFs (TECOFFs) were determined by EUCAST methodology including quality control (QC) strains. ResultsThe clarithromycin ECOFF was 16 mg/L for M. avium (n = 1271) whereas TECOFFs were 8 mg/L for M. intracellulare (n = 415) and 1 mg/L for MAB (n = 1014) confirmed by analysing MAB subspecies without inducible macrolide resistance (n = 235). For amikacin, the ECOFFs were 64 mg/L for MAC and MAB. For moxifloxacin, the WT spanned >8 mg/L for both MAC and MAB. For linezolid, the ECOFF and TECOFF were 64 mg/L for M. avium and M. intracellulare, respectively. Current CLSI breakpoints for amikacin (16 mg/L), moxifloxacin (1 mg/L) and linezolid (8 mg/L) divided the corresponding WT distributions. For QC M. avium and M. peregrinum, ≥95% of MIC values were well within recommended QC ranges. ConclusionAs a first step towards clinical breakpoints for NTM, (T)ECOFFs were defined for several antimicrobials against MAC and MAB. Broad wild-type MIC distributions indicate a need for further method refinement which is now under development within the EUCAST subcommittee for anti-mycobacterial drug susceptibility testing. In addition, we showed that several CLSI NTM breakpoints are not consistent in relation to the (T)ECOFFs.

1440. Local Antibiogram for Mycobacterium abscessus Shows Variability from Previously Published Susceptibility Data

Abstract Background Mycobacterium abscessus complex (MabsC) causes a wide variety of human disease and is frequently resistant to multiple agents. Empiric therapy is typically based on previously published susceptibility data which suggests amikacin (AMK), cefoxitin (FOX), clarithromycin (CLR), and imipenem (IPM) should be utilized. However, there is considerable variability in susceptibility patterns according to locale. We generated an antibiogram based on local MabsC isolates and compared it to previously reported susceptibility data to determine if local variation exists and assist in empiric therapy choice. Methods Non-duplicate local isolates of MabsC from 2011-2021 with in-vitro susceptibility data (via broth microdilution per CLSI guidance) available were included in the generation of a local antibiogram. This included standard 14 day incubation for inducible macrolide resistance. A review of the literature via PubMed search was undertaken to generate comparator susceptibility patterns with a focus on published reports with greater than 20 isolates. Results Sixty one unique local isolates of MabsC were identified, 44 of which were from lung, eight skin/soft tissue, five bone/joint, two blood, one urine, and one ascitic fluid. Susceptibilities are shown in Table 1. Twenty one manuscripts described susceptibility patterns with a range of isolates from 20 to 404 with a median of 70. These revealed susceptibility ranges for AMK range from 45-100%, FOX 0-72%, CLR 19-96%, and IPM 0%-71% as seen in Table 2. Susceptibilities of Mycobacterium abscessus isolates Results of antimicrobial susceptibility testing for 61 isolates of MabsC with percent resistant, intermediate, and susceptible shown, along with break points used per CLSI Comparison of Drug Susceptibility Rates from Previous Studies Conclusion We found high local resistance rates to cefoxitin and imipenem in contrast to empiric therapy recommendations and greatest susceptibility to amikacin. Published data shows a wide variation of susceptibility patterns to the recommended therapies based on location. It would be clinically advantageous for institutions to generate an antibiogram based on their local resistance data to assist empiric therapy choices in MabsC infections. Our data suggests that such local antibiograms could lead to substantial changes from the recommended empiric therapy at the institutional level for these infections which are historically challenging to treat. Disclosures Trevor C. Van Schooneveld, MD, bioMerieux: Advisor/Consultant|bioMerieux: Grant/Research Support|Insmed: Grant/Research Support|Merck: Grant/Research Support|Thermo-Fischer: Advisor/Consultant.

Impact on Macrolide Resistance of Genetic Diversity of Mycobacterium abscessus Species

Our previous study identified that the Mycobacterium abscessus subsp. abscessus T28 sequevar does not fully represent inducible macrolide resistance. Thus, we initiated a correlation study between genotypes and phenotypes. In total, 75 isolates from patients with skin and soft tissue infections were enrolled in the study. These strains were tested against 11 antimycobacterial agents using Sensitire RAPMYCO plates and the CLSI-recommended broth microdilution method. In order to analyze erm(41) and partial hsp65, rpoB, secA1, and rrl genes, bacterial genomic DNA was extracted from bacteria. The MEGA X software was used for phylogenetic analyses. The most active agents against most M. abscessus species were amikacin and tigecycline. Clarithromycin was effective toward M. abscessus subsp. massiliense and nearly all M. abscessus subsp. abscessus C28 sequevars. Two varieties of M. abscessus subsp. abscessus T28 sequevars did not represent inducible macrolide resistance. Most M. abscessus species showed intermediate susceptibility to cefoxitin and imipenem. Six additional agents were less effective against M. abscessus species. Following phylogenetic analyses, two outliers of M. abscessus subsp. abscessus T28 sequevars seem to represent no inducible macrolide resistance. In addition, we discovered genetic mosaicism of hsp65, rpoB, and secA1 in M. abscessus species was common. T28 sequevars of M. abscessus subsp. abscessus do not fully represent inducible macrolide resistance. The outlier of erm(41) phylogeny of the M. abscessus subsp. abscessus T28 sequevar is possibly due to macrolide susceptibility. Evaluation of the antimicrobial susceptibility of M. abscessus species is a reliable tool for assisting physicians in selecting the most effective antimycobacterial agent(s). IMPORTANCE Macrolides are the mainstays of the antimycobacterial regimens against Mycobacterium abscessus species (formerly Mycobacterium abscessus complex). erm(41) confers inducible macrolide resistance for M. abscessus subsp. bolletii strains, and the majority of M. abscessus subsp. abscessus T28 sequevars. Furthermore, the acquired macrolide resistance of M. abscessus species is due to a point mutation in rrl. However, not all M. abscessus subsp. abscessus T28 sequevars have inducible macrolide resistance. Exploration of the mechanism of macrolide resistance requires an understanding of genetic diversity. The genetic mosaicism of the erm(41), rpoB, hsp65, and secA1 genes within three subspecies of M. abscessus species is not uncommon. The T28 sequevar of erm(41) confers inducible macrolide resistance to the genetic mosaic strain. The development of new anti-M. abscessus species infection overcoming inducible macrolide resistance and/or acquired macrolide resistance is a crucial issue.

High Rates of Antimicrobial Resistance in Rapidly Growing Mycobacterial Infections in Taiwan.

Rapidly growing mycobacteria (RGM) has gained increasing clinical importance, and treatment is challenging due to diverse drug resistance. The minimum inhibitory concentrations (MIC) of 13 antimicrobial agents using modified broth microdilution and E-test were determined for 32 clinical isolates of RGM, including Mycobacterium abscessus (22 isolates) and Mycobacterium fortuitum (10 isolates). Our results showed high rates of resistance to available antimicrobial agents. Amikacin remained highly susceptible (87.5%). Clarithromycin was active against the isolates of M. abscessus (95.5%), and M. fortuitum (50%), but 36.4% and 20% had inducible macrolide resistance, respectively. Rates of susceptibility to tigecycline were 68.2–70%, and linezolid 45.5–50%, respectively. The quinolones (ciprofloxacin and moxifloxacin) showed better in vitro activity against M. fortuitum isolates (50% susceptibility) than the M. abscessus isolates (31.8% susceptibility). The susceptibilities to other conventional anti-mycobacterial agents were poor. The MICs of E-test were higher than broth microdilution and may result in reports of false resistance. In conclusion, the implementation of the modified broth microdilution plates into the routine clinical laboratory workflow to provide antimicrobial susceptibility early, allows for the timely selection of appropriate treatment of RGM infections to improve outcome.

New RAPMYCOI SensititreTM Antimicrobial Susceptibility Test for Atypical Rapidly Growing Mycobacteria (RGM).

Rapidly growing mycobacteria (RGM) cause an increasing international concern, mainly due to their natural resistance to many antibiotics. The aim of this study was to conduct species identification and determine the antimicrobial susceptibility profiles of RGM isolated in Poland. Antimicrobial susceptibility was tested using broth microdilution and the RAPMYCOI panel. A total of 60 strains were analysed, including the following species: M. fortuitum complex (30), M. abscessus subsp. abscessus (16), M. abscessus subsp. massiliense (7), M. chelonae (5), and M. mucogenicum (2). For 12 M. abscessus subsp. abscessus strains, the presence of the erm 41T28 genotype associated with inducible macrolide resistance and a functional erm gene was confirmed. A MUT2 mutation in the rrl gene (constitutive resistance) was identified for two strains from the subtype M. abscessus subsp. massiliense. Among the 15 tested antibiotics, amikacin and linezolid had the strongest antimycobacterial activity. Most of the tested strains were resistant to doxycycline and trimethoprim/sulfamethoxazole. Tigecycline MICs were low for all tested strains. Findings from our study highlight the importance of correct identification of clinical isolates and antimicrobial susceptibility testing.

In vitro activity of fidaxomicin against nontuberculosis mycobacteria.

Introduction. Nontuberculous mycobacteria (NTM) infections are increasing worldwide and are relatively resistant to many of the first- and second-line drugs to treat tuberculosis. Macrolide antibiotics, such as clarithromycin and azithromycin, are the key drugs for treating NTM infections. Fidaxomicin is a macrolide antibiotic that is widely used in treating Clostridium difficle (C.difficile) infections, and has high in vitro activity against Mycobacterium tuberculosis especially multidrug-resistant tuberculosis (MDR-TB) and has no cross-resistance with rifampicin.Hypothesis. Fidaxomicin may have in vitro activity against NTM strains.Aim. To find that whether the macrolide antibiotic fidaxomicin has in vitro activity against NTM strains.Methodology. Fidaxomicin used in this study was firstly tested on C. difficile reference strains and has shown to be effective and workable. And then 28 rapidly growing mycobacteria (RGM), 12 slowly growing mycobacteria (SGM) reference strains and 103 NTM clinical isolates were tested by the microplate-based AlamarBlue assay (MABA) method to determine the MICs. Fidaxomicin, rifampicin and clarithromycin were tested against M. abcessus complex subspecies 14 M. abscessus and 5 M. massiliense strains for inducible resistance determination.Results. In total, 21 out of 28 RGM and 9 of 12 SGM reference strains have the MICs of fidaxomicin at or below 1 µg ml-1. Fidaxomicin also showed low MIC values for some clinical isolates including M. abscessus complex, M. avium complex, M. fortuitum, M. kansasii and M. parascrofulaceum. Fidaxomicin also has no inducible macrolide resistance in M. abscessus complex in comparison with clarithromycin.Conclusion. Fidaxomicin has high in vitro activity against most of the NTM reference strains and some prevalent NTM clinical isolates. This promising finding warrants further investigation on the actions of fidaxomicn in vivo and as a potential antibiotic for NTM treatment.

Highly resistant Mycobacterium abscessus subsp. abscessus infection in a Swiss cat

AbstractA 2‐year‐old European shorthair cat was presented with a nonhealing skin lesion on the right abdominal wall. Repeated surgical excision and antimicrobial treatment with cefovecin, clindamycin, and trimethoprim‐sulfamethoxazole did not lead to clinical improvement. Histopathology of the skin lesion showed pyogranulomatous dermatitis and panniculitis. Definitive diagnosis was made by mycobacterial culture and rpoB sequencing, revealing Mycobacterium abscessus subsp. abscessus. To the authors’ knowledge, this is the first report of Mycobacterium abscessus subsp. abscessus infection in a cat in Switzerland and the second case in Europe. The isolated strain carried a functional erm(41) gene that confers inducible macrolide resistance. Further phenotypic resistances to doxycycline, minocycline, and imipenem were detected, resulting in little chance of successful antimicrobial treatment. Mycobacterium abscessus subsp. abscessus is an emerging pathogen in human medicine and poses a non‐negligible zoonotic risk for the owners of the cat.

PCR amplification of the erm(41) gene can be used to predict the sensitivity of Mycobacterium abscessus complex strains to clarithromycin.

A worldwide increase in the Mycobacterium abscessus (M. abscessus) complex has been observed. Therefore, the aim of the present study was to investigate the diversity of the rrl and erm(41) genes, both of which are associated with macrolide sensitivity in the M. abscessus complex. The current study also examined the efficacy of mass spectrometry as an alternative to molecular testing to classify subspecies of the M. abscessus complex. A total of 14 strains of the M. abscessus complex were obtained, and based on conventional analyses using housekeeping genes, 57% were determined to be M. abscessus subsp. abscessus, 43% were M. abscessus subsp. massiliense, and none were identified as M. abscessus subsp. bolletii. However, depending on the strain, it was not always possible to distinguish between the subspecies by mass spectrometry. Consequently, PCR products for the rrl and erm(41) genes were directly sequenced. Overall, 7.1% of the strains were identified to have a rrl mutation, and 92.9% carried a T at position 28 of erm(41). Results presented here suggest that the principal cause of treatment failure for M. abscessus complex infections is inducible macrolide resistance encoded by the erm(41) gene. From a strictly pragmatic standpoint, the phenotypic function of a putative erm(41) gene is the most important piece of information required by clinicians in order to prescribe an effective treatment. Although PCR amplification of erm(41) is not sufficient to differentiate between the M. abscessus complex subspecies, PCR can be easily and efficiently used to predict the sensitivity of members of the M. abscessus complex to clarithromycin.

Antimicrobial susceptibility patterns and MICs among non-photochromogenic rapidly growing Mycobacteroides and Mycolicibacterium species.

Introduction. Non-photochromogenic rapidly growing mycobacteria (NPRGM) that branch distinctly from Mycobacteroides (Myco) and Mycolicibacterium (Mycolici) are increasingly observed clinically and present a complicated treatment challenge; thus, appropriate in vitro susceptibility testing is required.Methodology. We evaluated the MICs of nine antimicrobials used in the treatment of infections of 25 NPRGM type strains. The relation of inducible macrolide resistance with functional erythromycin ribosomal methylase (erm) genes was also investigated.Results. The initial clarithromycin MIC reading at 3 days showed resistance in four of the Mycolici strains. In contrast, the presence of erm genes among Mycolici species differed from previous findings. Both Myco and Mycolici species were highly susceptible to amikacin and linezolid. Myco species were resistant to fluoroquinolones, while Mycolici species were susceptible. Meropenem showed low activity against Myco species, but high activity against Mycolici species.Conclusion. NPRGM clade-specific susceptibility patterns suggest an urgent need to establish distinct breakpoints for Myco and Mycolici species.

Species Distribution and Macrolide Susceptibility of Mycobacterium fortuitum Complex Clinical Isolates.

The understanding of species distribution and inducible macrolide resistance in the Mycobacterium fortuitum complex (MFC) is limited. Of 90 mostly respiratory MFC clinical isolates, half were M. fortuitum, followed by M. peregrinum, M. porcinum, M. septicum, and M. conceptionense Most M. fortuitum, M. porcinum, and M. septicum isolates were inducibly resistant to clarithromycin, whereas two-thirds of the M. peregrinum isolates were clarithromycin susceptible. Clarithromycin-resistant M. fortuitum isolates exhibited common mutations of erm(39), potentially involved in clarithromycin resistance.

Erythromycin-resistant Streptococcus pneumoniae: phenotypes, genotypes, transposons and pneumococcal vaccine coverage rates

To assess the antibiotic resistance, transposon profiles, serotype distribution and vaccine coverage rates in 110 erythromycin-resistant S. pneumoniae clinical isolates. Erythromycin, clindamycin, tetracycline, chloramphenicol and kanamycin susceptibilities were assessed using the E-test/disc diffusion method. Inducible macrolide resistance was tested using the erythromycin-clindamycin double disc diffusion test. Serogrouping and serotyping were performed using latex particle agglutination and the Quellung reaction, respectively. Drug resistance genes and transposon-specific genes were investigated by PCR. Of the isolates, 93 % were resistant to clindamycin; 81 % were resistant to tetracycline; 76 % were multi-drug-resistant, having resistance to both clindamycin and tetracycline; and 12 % had extended-drug resistance, being resistant to clindamycin, tetracycline, chloramphenicol and kanamycin. The majority of isolates (88.2 %) exhibited the cMLSB phenotype. The association between the cMLSB phenotype and tetracycline resistance was related to transposons Tn2010 (38.2 %), Tn6002 (21.8 %) and Tn3872 (18.2 %). M and iMLSB phenotypes were observed in 7 and 5 % of the isolates, respectively. The most frequent serotype was 19 F (40 %). Among the erythromycin-resistant pneumococci, vaccine coverage rates for the 13-valent pneumococcal conjugate vaccine (PCV-13) and the 23-valent pneumococcal polysaccharide vaccine (PPSV-23) were 76.4 and 79.1 %, respectively, compared to 82.2 and 85.1 % transposon-carrying isolates. Multi-drug resistance among erythromycin-resistant S. pneumoniae isolates mainly occurs due to the horizontal spread of the Tn916 family of transposons. The majority of the transposon-carrying isolates are covered by 13- and 23-valent pneumococcal vaccines. Since serotype distribution and transposons in S. pneumoniae isolates may change over time, close monitoring is essential.

Mycobacterium bolletii Lung Disease in Cystic Fibrosis

The cystic fibrosis (CF) pathogen, Mycobacterium abscessus complex, covers three subspecies: M.abscessus, M.massiliense, and M.bolletii. There are no clinical outcome data concerning M.bolletii. Our aim was to characterize M.bolletii lung infections in patients with CF. We included patients with M.bolletii lung infection recorded between 1994 and 2012 in France. Data were collected from the CF registry, medical records, and questionnaires submitted to the CF primary physician. Strains were typed by multilocus sequence typing analysis. Fifteen cases were identified in nine CF centers. Nine patients (60%) presented with nontuberculous mycobacterial pulmonary disease. Follow-up of 13 patients showed a trend to more rapid decline in FEV1 in the first year of colonization (-9.4%; SD 19.3) in comparison with noninfected control subjects (-2.3%; SD 12.1) (P= .16). Twelve patients were treated, and 11 received oral macrolides. Treatment-induced eradication occurred in five patients (41.7%). Four patients died (26.7%), including one patient with fatal nontuberculous mycobacterial pulmonary disease. Inducible macrolide resistance was demonstrated in all strains. Patients always harbored unique strains. Our study reports the largest study cohort of CF patients infected with M.bolletii. M. bolletii infection affects both children and young adults, is most often symptomatic, and may be fatal. Macrolide-based therapies have poor effectiveness. There is no evidence of patient-to-patient transmission.

RETROSPECTIVE ANALYSIS OF SLOVENIAN MYCOBACTERIUM AVIUM COMPLEX AND MYCOBACTERIUM ABSCESSUS COMPLEX ISOLATES AND MOLECULAR RESISTANCE PROFILE

Abstract. Mycobacteria belonging to Mycobacterium (M.) avium complex (MAC) and M. abscessus complex (MABSC) are the most frequent causes of mycobacteriosis in the world. In the last few years MAC and MABSC taxonomy was rapidly changing due to new molecular methods conveying the possibility to differentiate between species. New techniques are able to identify M. chimaera that was previously recognized as M. intracellulare and also differentiate subspecies of MABSC. Due to their natural habitat, non-tuberculous mycobacteria (NTM) are constantly exposed to various concentrations of antimicrobial drugs and other chemicals and consequently they had developed different mechanisms of resistance. Macrolides and aminoglycosides are frequently used drugs to treat MAC and MABSC infections. The aim of our nation-wide survey was to obtain information about MABSC subspecies prevalence in Slovenia and to assess the percentage of misidentifications of M. chimaera isolates as M. intracellulare in the past. Moreover, the purpose of our study was to reveal, which of the two species M. intracellulare or M. chimaera is clinically more relevant in Slovenia. Further, the aim of the study was to detect mutations in erm(41), rrl and rrs genes, which are known to convey macrolide resistance (erm(41) and rrl) and aminoglycoside resistance (rrs). One hundred and thirty-two Slovenian mycobacterial isolates obtained from the National Mycobacterial Collection that belong to MAC and MABSC were analysed. GenoType NTM-DR was used to differentiate M. intracellulare from M. chimaera and subspecies of MABSC. Our results showed that 48% of previously identified M. intracellulare isolates were actually M. chimaera isolates and that M. abscessus subsp. abscessus was the most frequent subspecies of MABSC. Most of the MABSC isolates carried the inducible macrolide resistance genes (erm(41) and rrl), however none of the isolates of MAC and MABSC had mutations in rrs genes for aminoglycoside resistance.

Predominance of clarithromycin-susceptible Mycobacterium massiliense subspecies: Characterization of the Mycobacterium abscessus complex at a tertiary acute care hospital.

To characterize members of the Mycobacterium abscessus complex, with an emphasis on the correlation between species identification and clarithromycin associated genetic polymorphisms that contribute to inducible and constitutive macrolide resistance. PCR and sequencing analysis was used to elucidate the subspecies, erm(41) genotypes and the presence of rrl mutations. M. abscessus subsp. massiliense was the dominant subspecies (70.2 %), followed by M. abscessus subsp. abscessus (23.8 %) and M. abscessus subsp. bolletii (5.9 %). The majority of M. abscessus and M. bolletii isolates possessed T28 erm(41) sequevar and were inducibly resistant to clarithromycin. All M. massiliense carried the truncated erm(41) and were largely clarithromycin-susceptible (98.3 %). Constitutive resistance involving rrl mutations was rare and seen in only 2 isolates (2.2 %). Subspecies identification was insufficient to predict clarithromycin susceptibility and required the genetic resistance to be determined via sequencing. In our context, rrl mutations were uncommon and may not be an essential test.