Florian P. Maurer

Acquisition of clarithromycin resistance mutations in the 23S rRNA gene of Mycobacterium abscessus in the presence of inducible erm(41)

OBJECTIVES Antibiotic therapy of pulmonary Mycobacterium abscessus infection is based on a combination treatment including clarithromycin. Recent data demonstrated that M. abscessus may carry a chromosomal, inducible erm gene coding for the ribosomal methylase Erm(41). The purpose of this study was to investigate whether in patients with chronic M. abscessus infection undergoing clarithromycin therapy, M. abscessus acquires clarithromycin resistance mutations in the rrl gene in addition to the presence of an inducible Erm(41) methylase. METHODS We determined clarithromycin MICs, erm(41) and rrl sequences for 29 clinical M. abscessus subsp. abscessus isolates of five different patients. The isolates were obtained between 2007 and 2011 covering a longitudinal observation period of 2-4 years for the individual patients. RESULTS In three out of five patients with an initial rrl wild-type isolate, follow-up isolates demonstrated acquisition of resistance mutations in the rrl gene in addition to the presence of an inducible Erm methylase. CONCLUSIONS Our results show that in M. abscessus, clarithromycin resistance mutations in the 23S rRNA peptidyltransferase region provide an additional selective advantage independent of a functional erm(41) gene.

Erm(41)-dependent inducible resistance to azithromycin and clarithromycin in clinical isolates of Mycobacterium abscessus

OBJECTIVES The ribosomal methylase Erm(41) confers inducible resistance to macrolides in Mycobacterium abscessus. The aim of this work was to systematically study and compare drug susceptibility to clarithromycin and azithromycin in M. abscessus and Mycobacterium chelonae clinical isolates with a particular focus on inducible drug resistance. METHODS Clinical isolates of M. abscessus subsp. abscessus (n = 21), M. abscessus subsp. bolletii (n = 16), M. abscessus subsp. massiliense (n = 10) and M. chelonae (n = 22) were characterized regarding their erm(41) and rrl genotypes and subjected to drug susceptibility testing (DST) for clarithromycin and azithromycin. Microdilution DST was performed in cation-adjusted Mueller-Hinton broth (pH 7.4) with readings at days 3, 7 and 12 and with pre-incubation at subinhibitory macrolide concentrations for erm(41) induction. In addition, the influence of variations in pH and growth medium on DST results was examined. RESULTS MICs of azithromycin were consistently higher than those of clarithromycin. In strains with an inducible erm(41) gene, high median MICs of ≥256 mg/L on day 12 were observed for both clarithromycin and azithromycin. Inducible resistance was at least as pronounced for azithromycin as for clarithromycin. CONCLUSIONS Our findings do not support the suggestion of a preferential use of azithromycin over clarithromycin in order to limit inducible macrolide resistance. Both compounds provoked a comparable resistance phenotype in M. abscessus. Caution is needed when using either azithromycin or clarithromycin for treatment of M. abscessus infections.

Lack of antimicrobial bactericidal activity in Mycobacterium abscessus

ABSTRACT Antibiotic therapy of infections caused by the emerging pathogen Mycobacterium abscessus is challenging due to the organisms natural resistance toward most clinically available antimicrobials. We investigated the bactericidal activity of antibiotics commonly administered in M. abscessus infections in order to better understand the poor therapeutic outcome. Time-kill curves were generated for clinical M. abscessus isolates, Mycobacterium smegmatis, and Escherichia coli by using antibiotics commonly categorized as bactericidal (amikacin and moxifloxacin) or bacteriostatic (tigecycline and linezolid). In addition, the impact of aminoglycoside-modifying enzymes on the mode of action of substrate and nonsubstrate aminoglycosides was studied by using M. smegmatis as a model organism. While amikacin and moxifloxacin were bactericidal against E. coli, none of the tested compounds showed bactericidal activity against M. abscessus. Further mechanistic investigations of the mode of action of aminoglycosides in M. smegmatis revealed that the bactericidal activity of tobramycin and gentamicin was restored by disruption of the chromosomal aac(2′) gene in the mycobacterial genome. The lack of bactericidal antibiotics in currently recommended treatment regimens provides a reasonable explanation for the poor therapeutic outcome in M. abscessus infection. Our findings suggest that chromosomally encoded drug-modifying enzymes play an important role in the lack of aminoglycoside bactericidal activity against rapidly growing mycobacteria.

Evaluation of Carbapenemase Screening and Confirmation Tests with Enterobacteriaceae and Development of a Practical Diagnostic Algorithm

ABSTRACT Reliable identification of carbapenemase-producing members of the family Enterobacteriaceae is necessary to limit their spread. This study aimed to develop a diagnostic flow chart using phenotypic screening and confirmation tests that is suitable for implementation in different types of clinical laboratories. A total of 334 clinical Enterobacteriaceae isolates genetically characterized with respect to carbapenemase, extended-spectrum β-lactamase (ESBL), and AmpC genes were analyzed. A total of 142/334 isolates (42.2%) were suspected of carbapenemase production, i.e., intermediate or resistant to ertapenem (ETP) and/or meropenem (MEM) and/or imipenem (IPM) according to EUCAST clinical breakpoints (CBPs). A group of 193/334 isolates (57.8%) showing susceptibility to ETP, MEM, and IPM was considered the negative-control group in this study. CLSI and EUCAST carbapenem CBPs and the new EUCAST MEM screening cutoff were evaluated as screening parameters. ETP, MEM, and IPM with or without aminophenylboronic acid (APBA) or EDTA combined-disk tests (CDTs) and the Carba NP-II test were evaluated as confirmation assays. EUCAST temocillin cutoffs were evaluated for OXA-48 detection. The EUCAST MEM screening cutoff (<25 mm) showed a sensitivity of 100%. The ETP APBA CDT on Mueller-Hinton agar containing cloxacillin (MH-CLX) displayed 100% sensitivity and specificity for class A carbapenemase confirmation. ETP and MEM EDTA CDTs showed 100% sensitivity and specificity for class B carbapenemases. Temocillin zone diameters/MIC testing on MH-CLX was highly specific for OXA-48 producers. The overall sensitivity, specificity, positive predictive value, and negative predictive value of the Carba NP-II test were 78.9, 100, 100, and 98.7%, respectively. Combining the EUCAST MEM carbapenemase screening cutoff (<25 mm), ETP (or MEM), APBA, and EDTA CDTs, and temocillin disk diffusion on MH-CLX promises excellent performance for carbapenemase detection.

Detection of Candidatus Neoehrlichia mikurensis, Borrelia burgdorferi sensu lato genospecies and Anaplasma phagocytophilum in a tick population from Austria

Candidatus Neoehrlichia mikurensis DNA was discovered in Ixodes ricinus ticks in 1999 and is referred to as an emerging human pathogen since its first detection in patients with febrile illness reported in 2010. In recent years, Ca. Neoehrlichia mikurensis has been detected in ticks from several European, Asian, and African countries. However, no epidemiological data exist for Austria, which is a highly endemic region for tick-transmitted diseases. To assess the geographic spread and prevalence of Ca. Neoehrlichia mikurensis sympatric with other tick-transmitted pathogens, we analysed 518 I. ricinus ticks collected in 2002 and 2003 in Graz, Austria. The prevalence of Ca. Neoehrlichia mikurensis was 4.2%, that of Borrelia burgdorferi sensu lato 25.7%, and that of Anaplasma phagocytophilum 1%. Coinfections with Ca. Neoehrlichia mikurensis and B. burgdorferi sensu lato were found in 2.3% of all ticks. Thus, the results show a relatively high prevalence of Ca. Neoehrlichia mikurensis in Austrian ticks suggesting a high probability for the occurrence of undiagnosed human infections in Austria.

Advances in rapid identification and susceptibility testing of bacteria in the clinical microbiology laboratory: implications for patient care and antimicrobial stewardship programs

Early availability of information on bacterial pathogens and their antimicrobial susceptibility is of key importance for the management of infectious diseases patients. Currently, using traditional approaches, it usually takes at least 48 hours for identification and susceptibility testing of bacterial pathogens. Therefore, the slowness of diagnostic procedures drives prolongation of empiric, potentially inappropriate, antibacterial therapies. Over the last couple of years, the improvement of available techniques (e.g. for susceptibility testing, DNA amplification assays), and introduction of novel technologies (e.g. MALDI-TOF) has fundamentally changed approaches towards pathogen identification and characterization. Importantly, these techniques offer increased diagnostic resolution while at the same time shorten the time-to-result, and are thus of obvious importance for antimicrobial stewardship. In this review, we will discuss recent advances in medical microbiology with special emphasis on the impact of novel techniques on antimicrobial stewardship programs.

Emergence of ceftazidime/avibactam non-susceptibility in an MDR Klebsiella pneumoniae isolate.

Background: Avibactam is a novel broad‐range &bgr;‐lactamase inhibitor active against Ambler class A (including ESBL and KPC) and some Ambler class C and D (e.g. OXA‐48) enzymes. We here report on the emergence of ceftazidime/avibactam resistance in clinical, multiresistant, OXA‐48 and CTX‐M‐14‐producing Klebsiella pneumoniae isolate DT12 during ceftazidime/avibactam treatment. Methods and results: Comparative whole‐genome sequence analysis identified two SNPs in the CTX‐M‐14‐encoding gene leading to two amino acid changes (P170S and T264I). Compared with WT CTX‐M‐14, expression of the CTX‐M‐14&Dgr;170&Dgr;264 isoform in Escherichia coli led to a >64‐ and 16‐fold increase in ceftazidime and ceftazidime/avibactam MICs, respectively, functionally linking the observed SNPs and elevated MICs. The mutated CTX‐M‐14 isoform exhibited augmented ceftazidime hydrolytic activity, which was a reasonable cause for impaired susceptibility to avibactam inhibition. The P170S exchange in CTX‐M‐14 was found in association with elevated ceftazidime/avibactam MICs for independent K. pneumoniae isolates, but was not sufficient for full resistance. Apparently, additional CTX‐M‐independent mechanisms contribute to ceftazidime/avibactam resistance in K. pneumoniae DT12. Conclusions: This study on the molecular basis of ceftazidime/avibactam resistance in clinical K. pneumoniae emerging in vivo underscores the need for continuous monitoring of ceftazidime/avibactam susceptibility during therapy. Despite sustained inhibition of OXA‐48, rapid development of CTX‐M‐14 isoforms exhibiting augmented ceftazidime hydrolytic activity may limit the usefulness of ceftazidime/avibactam monotherapies in infections caused by isolates carrying blaCTX‐M‐14 and blaOXA‐48.

Survey of macrolide-resistant Mycoplasma pneumoniae in children with community-acquired pneumonia in Switzerland.

Mycoplasma pneumoniae is a leading cause of communityacquired pneumonia (CAP) in children and macrolides are recommended for this entity [1]. Extensive macrolide use led to the rapid, worldwide emergence of macrolide-resistant M. pneumoniae (MRMP) [2] with rates of over 90% in Asia (China, 2012 [3]) and up to 26% in Europe (Italy, 2010 [4]). The first two cases of MRMP in Switzerland were reported in adults in 2012 [5]. We aimed to assess the presence of MRMP in children with CAP.

Relative contribution of biological variation and technical variables to zone diameter variations of disc diffusion susceptibility testing

OBJECTIVES Disc diffusion is still largely based on manual procedures. Technical variations originate from inoculum preparation, variations in materials, individual operator plate streaking and reading accuracy. Resulting measurement imprecision contributes to categorization errors. Biological variation resembles the natural fluctuation of a measured parameter such as antibiotic susceptibility around a mean value. It is deemed to originate from factors such as genetic background or metabolic state. This study analysed the relative contribution of different technical and biological factors to total disc diffusion variation. METHODS For calculation of relative error factor contribution to disc diffusion variability, five experiments were designed keeping different combinations of error factors constant. A mathematical model was developed to analyse the individual error factor contribution to disc diffusion variation for each of the tested drug-species combinations. RESULTS The contribution of biological variation to total diameter variance ranged from 10.4% to 98.8% for different drug-species combinations. Highest biological variation was found for Enterococcus faecalis WT and vancomycin (98.8%) and for penicillinase-producing Staphylococcus aureus and penicillin G (96.0%). Average imprecision of automated zone reading revealed that 1.4%-5.3% of total imprecision was due to technical variation, while materials, i.e. antibiotic discs and agar plates, contributed between 2.6% and 3.9%. Inoculum preparation and manual plate streaking contributed 6.8%-24.8% and 6.6%-24.3%, respectively, to total imprecision. CONCLUSIONS This study illustrates the relative contributions of technical factors that account for a significant part of total variance in disc diffusion. The highest relative contribution originated from the operator, i.e. manual inoculum preparation and plate streaking. Further standardization of inoculum preparation and plate streaking by automation could potentially increase the precision of disc diffusion and improve the correlation of susceptibility reports with clinical outcome.

Integrating Forecast Probabilities in Antibiograms: a Way To Guide Antimicrobial Prescriptions More Reliably?

ABSTRACT Antimicrobial susceptibility testing (AST) assigns pathogens to “susceptible” or “resistant” clinical categories based on clinical breakpoints (CBPs) derived from MICs or inhibition zone diameters and indicates the likelihood for therapeutic success. AST reports do not provide quantitative measures for the reliability of such categorization. Thus, it is currently impossible for clinicians to estimate the technical forecast uncertainty of an AST result regarding clinical categorization. AST error rates depend on the localization of pathogen populations in relation to CBPs. Bacterial species are, however, not homogeneous, and subpopulations behave differently with respect to AST results. We addressed how AST reporting errors differ between isolates with and without acquired drug resistance determinants. Using as an example the beta-lactams and their most important resistance mechanisms, we analyzed different pathogen populations for their individual reporting error probabilities. Categorization error rates were significantly higher for bacterial populations harboring resistance mechanisms than for the wild-type population. Reporting errors for amoxicillin-clavulanic acid and piperacillin-tazobactam in Escherichia coli infection cases were almost exclusively due to the presence of broad-spectrum- and extended-spectrum-beta-lactamase (ESBL)-producing microorganisms (79% and 20% of all errors, respectively). Clinicians should be aware of the significantly increased risk of erroneous AST reports for isolates producing beta-lactamases, particularly ESBL and AmpC. Including probability indicators for interpretation would improve AST reports.