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1.  Lack of Antimicrobial Bactericidal Activity in Mycobacterium abscessus 
Antibiotic therapy of infections caused by the emerging pathogen Mycobacterium abscessus is challenging due to the organism's 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.
PMCID: PMC4068550  PMID: 24752273
2.  Identification and Evaluation of Improved 4′-O-(Alkyl) 4,5-Disubstituted 2-Deoxystreptamines as Next-Generation Aminoglycoside Antibiotics 
mBio  2014;5(5):e01827-14.
The emerging epidemic of drug resistance places the development of efficacious and safe antibiotics in the spotlight of current research. Here, we report the design of next-generation aminoglycosides. Discovery efforts were driven by rational synthesis focusing on 4′ alkylations of the aminoglycoside paromomycin, with the goal to alleviate the most severe and disabling side effect of aminoglycosides—irreversible hearing loss. Compounds were evaluated for target activity in in vitro ribosomal translation assays, antibacterial potency against selected pathogens, cytotoxicity against mammalian cells, and in vivo ototoxicity. The results of this study produced potent compounds with excellent selectivity at the ribosomal target, promising antibacterial activity, and little, if any, ototoxicity upon chronic administration. The favorable biocompatibility profile combined with the promising antibacterial activity emphasizes the potential of next-generation aminoglycosides in the treatment of infectious diseases without the risk of ototoxicity.
The ever-widening epidemic of multidrug-resistant infectious diseases and the paucity of novel antibacterial agents emerging from modern screening platforms mandate the reinvestigation of established drugs with an emphasis on improved biocompatibility and overcoming resistance mechanisms. Here, we describe the preparation and evaluation of derivatives of the established aminoglycoside antibiotic paromomycin that effectively remove its biggest deficiency, ototoxicity, and overcome certain bacterial resistance mechanisms.
PMCID: PMC4196235  PMID: 25271289
3.  Use of the Bruker MALDI Biotyper for Identification of Molds in the Clinical Mycology Laboratory 
Journal of Clinical Microbiology  2014;52(8):2797-2803.
Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) is increasingly used for the identification of bacteria and fungi in the diagnostic laboratory. We evaluated the mold database of Bruker Daltonik (Bremen, Germany), the Filamentous Fungi Library 1.0. First, we studied 83 phenotypically and molecularly well-characterized, nondermatophyte, nondematiaceous molds from a clinical strain collection. Using the manufacturer-recommended interpretation criteria, genus and species identification rates were 78.3% and 54.2%, respectively. Reducing the species cutoff from 2.0 to 1.7 significantly increased species identification to 71.1% without increasing misidentifications. In a subsequent prospective study, 200 consecutive clinical mold isolates were identified by the MALDI Biotyper and our conventional identification algorithm. Discrepancies were resolved by ribosomal DNA (rDNA) internal transcribed spacer region sequence analysis. For the MALDI Biotyper, genus and species identification rates were 83.5% and 79.0%, respectively, when using a species cutoff of 1.7. Not identified were 16.5% of the isolates. Concordant genus and species assignments of MALDI-TOF MS and the conventional identification algorithm were observed for 98.2% and 64.2% of the isolates, respectively. Four erroneous species assignments were observed using the MALDI Biotyper. The MALDI Biotyper seems highly reliable for the identification of molds when using the Filamentous Fungi Library 1.0 and a species cutoff of 1.7. However, expansion of the database is required to reduce the number of nonidentified isolates.
PMCID: PMC4136132  PMID: 24850347
4.  Validation of Antibiotic Susceptibility Testing Guidelines in a Routine Clinical Microbiology Laboratory Exemplifies General Key Challenges in Setting Clinical Breakpoints 
This study critically evaluated the new European Committee for Antimicrobial Susceptibility Testing (EUCAST) antibiotic susceptibility testing guidelines on the basis of a large set of disk diffusion diameters determined for clinical isolates. We report several paradigmatic problems that illustrate key issues in the selection of clinical susceptibility breakpoints, which are of general importance not only for EUCAST but for all guidelines systems, i.e., (i) the need for species-specific determinations of clinical breakpoints/epidemiological cutoffs (ECOFFs), (ii) problems arising from pooling data from various sources, and (iii) the importance of the antibiotic disk content for separating non-wild-type and wild-type populations.
PMCID: PMC4068562  PMID: 24777093
5.  Structure-Activity Relationships among the Kanamycin Aminoglycosides: Role of Ring I Hydroxyl and Amino Groups 
Antimicrobial Agents and Chemotherapy  2012;56(12):6104-6108.
The kanamycins form an important subgroup of the 4,6-disubstituted 2-deoxystreptamine aminoglycoside antibiotics, comprising kanamycin A, kanamycin B, tobramycin, and dibekacin. These compounds interfere with protein synthesis by targeting the ribosomal decoding A site, and they differ in the numbers and locations of amino and hydroxy groups of the glucopyranosyl moiety (ring I). We synthesized kanamycin analogues characterized by subtle variations of the 2′ and 6′ substituents of ring I. The functional activities of the kanamycins and the synthesized analogues were investigated (i) in cell-free translation assays on wild-type and mutant bacterial ribosomes to study drug-target interaction, (ii) in MIC assays to assess antibacterial activity, and (iii) in rabbit reticulocyte translation assays to determine activity on eukaryotic ribosomes. Position 2′ forms an intramolecular H bond with O5 of ring II, helping the relative orientations of the two rings with respect to each other. This bond becomes critical for drug activity when a 6′-OH substituent is present.
PMCID: PMC3497201  PMID: 22948879
6.  Evaluation of the Bruker MALDI Biotyper for Identification of Gram-Positive Rods: Development of a Diagnostic Algorithm for the Clinical Laboratory 
Journal of Clinical Microbiology  2014;52(4):1089-1097.
Reported matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) identification rates of Gram-positive rods (GPR) are low compared to identification rates of Gram-positive cocci. In this study, three sample preparation methods were compared for MALDI-TOF MS identification of 190 well-characterized GPR strains: direct transfer, direct transfer-formic acid preparation, and ethanol-formic acid extraction. Using the interpretation criteria recommended by the manufacturer, identification rates were significantly higher for direct transfer-formic acid preparation and ethanol-formic acid extraction than for direct transfer. Reducing the species cutoff from 2.0 to 1.7 significantly increased species identification rates. In a subsequent prospective study, 215 clinical GPR isolates were analyzed by MALDI-TOF MS, and the results were compared to those for identification using conventional methods, with discrepancies being resolved by 16S rRNA and rpoB gene analysis. Using the direct transfer-formic acid preparation and a species cutoff of 1.7, congruencies on the genus and species levels of 87.4% and 79.1%, respectively, were achieved. In addition, the rate of nonidentified isolates dropped from 12.1% to 5.6% when using an extended database, i.e., the Bruker database amended by reference spectra of the 190 GPR of the retrospective study. Our data demonstrate three ways to improve GPR identification by the Bruker MALDI Biotyper, (i) optimize sample preparation using formic acid, (ii) reduce cutoff scores for species identification, and (iii) expand the database. Based on our results, we suggest an identification algorithm for the clinical laboratory combining MALDI-TOF MS with nucleic acid sequencing.
PMCID: PMC3993486  PMID: 24452159
7.  The mitochondrion: A perpetrator of acquired hearing loss 
Hearing research  2013;303:12-19.
Age, drugs, and noise are major causes of acquired hearing loss. The involvement of reactive oxygen species (ROS) in hair cell death has long been discussed, but there is considerably less information available as to the mechanisms underlying ROS formation. Most cellular ROS arise in mitochondria and this review will evaluate evidence for mitochondrial pathology in general and dysfunction of the mitochondrial respiratory chain in particular in acquired hearing loss. We will discuss evidence that different pathways can lead to the generation of ROS and that oxidative stress might not necessarily be causal to all three pathologies. Finally, we will detail recent advances in exploiting knowledge of aminoglycoside-mitochondria interactions for the development of non-ototoxic antibacterials.
PMCID: PMC3681877  PMID: 23361190
acquired hearing loss; age; noise; aminoglycosides; reactive oxygen species; mitochondria; ribosomes
8.  Spectinamides: A New Class of Semisynthetic Anti-Tuberculosis Agents that Overcome Native Drug Efflux 
Nature medicine  2014;20(2):152-158.
Although the classical antibiotic spectinomycin is a potent bacterial protein synthesis inhibitor, poor antimycobacterial activity limits its clinical application for treating tuberculosis. Using structure-based design, a novel semisynthetic series of spectinomycin analogs was generated with selective ribosomal inhibition and excellent narrow-spectrum antitubercular activity. In multiple murine infection models, these spectinamides were well tolerated, significantly reduced lung mycobacterial burden and increased survival. In vitro studies demonstrated a lack of cross-resistance with existing tuberculosis therapeutics, activity against MDR/XDR-tuberculosis, and an excellent pharmacological profile. Key to their potent antitubercular properties was their structural modification to evade the Rv1258c efflux pump, which is upregulated in MDR strains and is implicated in macrophage induced drug tolerance. The antitubercular efficacy of spectinamides demonstrates that synthetic modifications to classical antibiotics can overcome the challenge of intrinsic efflux pump-mediated resistance and expands opportunities for target based tuberculosis drug discovery.
PMCID: PMC3972818  PMID: 24464186
9.  4′-O-substitutions determine selectivity of aminoglycoside antibiotics 
Nature communications  2014;5:3112.
Clinical use of 2-deoxystreptamine aminoglycoside antibiotics, which target the bacterial ribosome, is compromised by adverse effects related to limited drug selectivity. Here, we present a series of 4′,6′-O-acetal and 4′-O-ether modifications on glucopyranosyl ring I of aminoglycosides. Chemical modifications were guided by measuring interactions between the compounds synthesized and ribosomes harbouring single point mutations in the drug binding site, resulting in aminoglycosides that interact poorly with the drug-binding pocket of eukaryotic mitochondrial or cytosolic ribosomes. Yet, these compounds largely retain their inhibitory activity for bacterial ribosomes and show antibacterial activity. Our data indicate that 4′-O-substituted aminoglycosides possess increased selectivity towards bacterial ribosomes and little activity for any of the human drug-binding pockets.
PMCID: PMC3942853  PMID: 24473108
Aminoglycosides; Ribosome; Toxicity; Chemical Synthesis
10.  Evaluation of Cobas TaqMan MTB for Direct Detection of the Mycobacterium tuberculosis Complex in Comparison with Cobas Amplicor MTB 
Journal of Clinical Microbiology  2013;51(7):2112-2117.
The Roche Cobas Amplicor MTB assay, recently replaced by the Roche Cobas TaqMan MTB assay, was one of the first commercially available assays for detection of the Mycobacterium tuberculosis complex based on nucleic acid amplification. We reported previously on the limited specificity of the Cobas Amplicor MTB assay, in particular for positive samples with an optical density at 660 nm (OD660) of <2.0. Using a selected set of respiratory samples, which were scored as false positive by the Cobas Amplicor test, we demonstrate here that the specificity of the Cobas TaqMan assay is significantly improved. In addition, our study of a set of 133 clinical samples revealed that the Cobas TaqMan MTB assay showed significantly less PCR inhibition than the Cobas Amplicor test. An overall concordance of 98.2% was observed between the two assays. In a subsequent prospective study, we evaluated the performance of the Roche Cobas TaqMan MTB assay on 1,143 clinical specimens, including respiratory (n = 838) and nonrespiratory (n = 305) specimens. Using culture as the gold standard, we found a sensitivity of 88.4% and a specificity of 98.8% for the 838 respiratory specimens, compared to a sensitivity of 63.6% and a specificity of 94.6% for the 305 nonrespiratory specimens. We conclude that the Cobas TaqMan MTB assay is a significantly improved tool for the direct detection of M. tuberculosis DNA in clinical specimens.
PMCID: PMC3697670  PMID: 23616457
11.  Integrating the Xpert MTB/RIF Assay into a Diagnostic Workflow for Rapid Detection of Mycobacterium tuberculosis in a Low-Prevalence Area 
Journal of Clinical Microbiology  2013;51(7):2396-2399.
The Xpert MTB/RIF assay is a rapid and fully automated real-time PCR assay. The performance of the Xpert MTB/RIF assay as a primary screening test for urgent clinical specimens was evaluated during a 2-year period. The results showed that replacing smear microscopy with the Xpert MTB/RIF assay facilitates laboratory handling and improves the sensitivity and specificity of Mycobacterium tuberculosis detection.
PMCID: PMC3697699  PMID: 23616455
12.  Influence of Clinical Breakpoint Changes from CLSI 2009 to EUCAST 2011 Antimicrobial Susceptibility Testing Guidelines on Multidrug Resistance Rates of Gram-Negative Rods 
Journal of Clinical Microbiology  2013;51(7):2385-2387.
Multidrug resistance (MDR) rates of Gram-negative rods were analyzed comparing CLSI 2009 and EUCAST 2011 antibiotic susceptibility testing guidelines. After EUCAST 2011 was applied, the MDR rates increased for Klebsiella pneumoniae (2.2%), Enterobacter cloacae (1.1%), Pseudomonas aeruginosa (0.7%), and Escherichia coli (0.4%). A total of 24% of Enterobacteriaceae MDR isolates and 12% of P. aeruginosa MDR isolates were categorized as MDR due to breakpoint changes.
PMCID: PMC3697730  PMID: 23596246
13.  Identification of Gram-Positive Cocci by Use of Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry: Comparison of Different Preparation Methods and Implementation of a Practical Algorithm for Routine Diagnostics 
Journal of Clinical Microbiology  2013;51(6):1834-1840.
This study compared three sample preparation methods (direct transfer, the direct transfer-formic acid method with on-target formic acid treatment, and ethanol-formic acid extraction) for the identification of Gram-positive cocci with matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS). A total of 156 Gram-positive cocci representing the clinically most important genera, Aerococcus, Enterococcus, Staphylococcus, and Streptococcus, as well as more rare genera, such as Gemella and Granulicatella, were analyzed using a Bruker MALDI Biotyper. The rate of correct genus-level identifications was approximately 99% for all three sample preparation methods. The species identification rate was significantly higher for the direct transfer-formic acid method and ethanol-formic acid extraction (both 77.6%) than for direct transfer (64.1%). Using direct transfer-formic acid compared to direct transfer, the total time to result was increased by 22.6%, 16.4%, and 8.5% analyzing 12, 48, and 96 samples per run, respectively. In a subsequent prospective study, 1,619 clinical isolates of Gram-positive cocci were analyzed under routine conditions by MALDI-TOF MS, using the direct transfer-formic acid preparation, and by conventional biochemical methods. For 95.6% of the isolates, a congruence between conventional and MALDI-TOF MS identification was observed. Two major limitations were found using MALDI-TOF MS: the differentiation of members of the Streptococcus mitis group and the identification of Streptococcus dysgalactiae. The Bruker MALDI Biotyper system using the direct transfer-formic acid sample preparation method was shown to be a highly reliable tool for the identification of Gram-positive cocci. We here suggest a practical algorithm for the clinical laboratory combining MALDI-TOF MS with phenotypic and molecular methods.
PMCID: PMC3716085  PMID: 23554198
14.  Standardisation of disk diffusion results for antibiotic susceptibility testing using the sirscan automated zone reader 
BMC Microbiology  2013;13:225.
Standardisation of disk diffusion readings could improve reproducibility and accuracy of antibiotic susceptibility testing (AST). This study evaluated accuracy, reproducibility, and precision of automated inhibition zone reading using the “Sirscan automatic” zone reader (i2a, Perols Cedex, France).
In a first step we compared Sirscan results with manual calliper measurements for comparability and accuracy. Sirscan readings were checked and adjusted on-screen as recommended by the manufacturer. One hundred clinical bacterial isolates representing a broad spectrum of organisms routinely isolated in a clinical laboratory were tested, and zone diameter values and interpretation according to EUCAST guidelines were compared. In a second step we analysed, whether fully automated zone reading can decrease standard deviation of diameter measurements and, thus, improve reproducibility and precision of the disk diffusion method. Standard deviations of manual measurements, on-screen adjusted Sirscan measurements, and fully automated Sirscan readings were compared for 19 repeat independent measurements of inhibition zones of S. aureus ATCC 29213, E. coli ATCC 25922, and P. aeruginosa ATCC 27853 (EUCAST quality control strains).
On-screen adjusted Sirscan and calliper measurements displayed high comparability. No significant differences were detected comparing the results of both reading methods. Standard deviations of inhibition zone diameters were significantly lower for fully automated Sirscan measurements compared with both adjusted Sirscan readings and the manual method, resulting in better reproducibility and precision of the automated readings.
Our results indicate that fully automated zone reading can further improve standardisation of AST by decreasing standard deviation and, thus, improve precision of inhibition zone diameter results.
PMCID: PMC3852248  PMID: 24099061
Inhibition zone diameter; Kirby-Bauer; Automation
15.  The Rationale for Using Rifabutin in the Treatment of MDR and XDR Tuberculosis Outbreaks 
PLoS ONE  2013;8(3):e59414.
Genetically related Mycobacterium tuberculosis strains with alterations at codon 516 in the rpoB gene were observed amongst a substantial number of patients with drug resistant tuberculosis in the Eastern Cape Province (ECP) of South Africa. Mutations at codon 516 are usually associated with lower level rifampicin (RIF) resistance, while susceptibility to rifabutin (RFB) remains intact. This study was conducted to assess the rationale for using RFB as a substitution for RIF in the treatment of MDR and XDR tuberculosis outbreaks. Minimum inhibitory concentrations (MICs) of 34 drug resistant clinical isolates of M tuberculosis were determined by MGIT 960 and correlated with rpoB mutations. RFB MICs ranged from 0.125 to 0.25 µg/ml in the 34 test isolates thereby confirming phenotypic susceptibility as per critical concentration (CC) of 0.5 µg/ml. The corresponding RIF MICs ranged between 5 and 15 µg/ml, which is well above the CC of 1.0 µg/ml. Molecular-based drug susceptibility testing provides important pharmacogenetic insight by demonstrating a direct correlation between defined rpoB mutation and the level of RFB susceptibility. We suggest that isolates with marginally reduced susceptibility as compared to the epidemiological cut-off for wild-type strains (0.064 µg/ml), but lower than the current CC (≤0.5 µg/ml), are categorised as intermediate. Two breakpoints (0.064 µg/ml and 0.5 µg/ml) are recommended to distinguish between susceptible, intermediate and RFB resistant strains. This concept may assist clinicians and policy makers to make objective therapeutic decisions, especially in situations where therapeutic options are limited. The use of RFB in the ECP may improve therapeutic success and consequently minimise the risk of ongoing transmission of drug resistant M. tuberculosis strains.
PMCID: PMC3602005  PMID: 23527189
16.  HIV Infection Disrupts the Sympatric Host–Pathogen Relationship in Human Tuberculosis 
PLoS Genetics  2013;9(3):e1003318.
The phylogeographic population structure of Mycobacterium tuberculosis suggests local adaptation to sympatric human populations. We hypothesized that HIV infection, which induces immunodeficiency, will alter the sympatric relationship between M. tuberculosis and its human host. To test this hypothesis, we performed a nine-year nation-wide molecular-epidemiological study of HIV–infected and HIV–negative patients with tuberculosis (TB) between 2000 and 2008 in Switzerland. We analyzed 518 TB patients of whom 112 (21.6%) were HIV–infected and 233 (45.0%) were born in Europe. We found that among European-born TB patients, recent transmission was more likely to occur in sympatric compared to allopatric host–pathogen combinations (adjusted odds ratio [OR] 7.5, 95% confidence interval [95% CI] 1.21–infinity, p = 0.03). HIV infection was significantly associated with TB caused by an allopatric (as opposed to sympatric) M. tuberculosis lineage (OR 7.0, 95% CI 2.5–19.1, p<0.0001). This association remained when adjusting for frequent travelling, contact with foreigners, age, sex, and country of birth (adjusted OR 5.6, 95% CI 1.5–20.8, p = 0.01). Moreover, it became stronger with greater immunosuppression as defined by CD4 T-cell depletion and was not the result of increased social mixing in HIV–infected patients. Our observation was replicated in a second independent panel of 440 M. tuberculosis strains collected during a population-based study in the Canton of Bern between 1991 and 2011. In summary, these findings support a model for TB in which the stable relationship between the human host and its locally adapted M. tuberculosis is disrupted by HIV infection.
Author Summary
Human tuberculosis (TB) caused by Mycobacterium tuberculosis kills 1.5 million people each year. M. tuberculosis has been affecting humans for millennia, suggesting that different strain lineages may be adapted to specific human populations. The combination of a particular strain lineage and its corresponding patient population can be classified as sympatric (e.g. Euro-American lineage in Europeans) or allopatric (e.g. East-Asian lineage in Europeans). We hypothesized that infection with the human immunodeficiency virus (HIV), which impairs the human immune system, will interfere with this host–pathogen relationship. We performed a nation-wide molecular-epidemiological study of HIV–infected and HIV–negative TB patients between 2000 and 2008 in Switzerland. We found that HIV infection was associated with the less adapted allopatric lineages among patients born in Europe, and this was not explained by social or other patient factors such as increased social mixing in HIV–infected individuals. Strikingly, the association between HIV infection and less adapted M. tuberculosis lineages was stronger in patients with more pronounced immunodeficiency. Our observation was replicated in a second independent panel of M. tuberculosis strains collected during a population-based study in the Canton of Bern. In summary, our study provides evidence that the sympatric host–pathogen relationship in TB is disrupted by HIV infection.
PMCID: PMC3591267  PMID: 23505379
17.  Important Role for Mycobacterium tuberculosis UvrD1 in Pathogenesis and Persistence apart from Its Function in Nucleotide Excision Repair 
Journal of Bacteriology  2012;194(11):2916-2923.
Mycobacterium tuberculosis survives and replicates in macrophages, where it is exposed to reactive oxygen and nitrogen species that damage DNA. In this study, we investigated the roles of UvrA and UvrD1, thought to be parts of the nucleotide excision repair pathway of M. tuberculosis. Strains in which uvrD1 was inactivated either alone or in conjunction with uvrA were constructed. Inactivation of uvrD1 resulted in a small colony phenotype, although growth in liquid culture was not significantly affected. The sensitivity of the mutant strains to UV irradiation and to mitomycin C highlighted the importance of the targeted genes for nucleotide excision repair. The mutant strains all exhibited heightened susceptibility to representatives of reactive oxygen intermediates (ROI) and reactive nitrogen intermediates (RNI). The uvrD1 and the uvrA uvrD1 mutants showed decreased intracellular multiplication following infection of macrophages. Most importantly, the uvrA uvrD1 mutant was markedly attenuated following infection of mice by either the aerosol or the intravenous route.
PMCID: PMC3370636  PMID: 22467787
18.  Effect of Mutation and Genetic Background on Drug Resistance in Mycobacterium tuberculosis 
Bacterial factors may contribute to the global emergence and spread of drug-resistant tuberculosis (TB). Only a few studies have reported on the interactions between different bacterial factors. We studied drug-resistant Mycobacterium tuberculosis isolates from a nationwide study conducted from 2000 to 2008 in Switzerland. We determined quantitative drug resistance levels of first-line drugs by using Bactec MGIT-960 and drug resistance genotypes by sequencing the hot-spot regions of the relevant genes. We determined recent transmission by molecular methods and collected clinical data. Overall, we analyzed 158 isolates that were resistant to isoniazid, rifampin, or ethambutol, 48 (30.4%) of which were multidrug resistant. Among 154 isoniazid-resistant strains, katG mutations were associated with high-level and inhA promoter mutations with low-level drug resistance. Only katG(S315T) (65.6% of all isoniazid-resistant strains) and inhA promoter −15C/T (22.7%) were found in molecular clusters. M. tuberculosis lineage 2 (includes Beijing genotype) was associated with any drug resistance (adjusted odds ratio [OR], 3.0; 95% confidence interval [CI], 1.7 to 5.6; P < 0.0001). Lineage 1 was associated with inhA promoter −15C/T mutations (OR, 6.4; 95% CI, 2.0 to 20.7; P = 0.002). We found that the genetic strain background influences the level of isoniazid resistance conveyed by particular mutations (interaction tests of drug resistance mutations across all lineages; P < 0.0001). In conclusion, M. tuberculosis drug resistance mutations were associated with various levels of drug resistance and transmission, and M. tuberculosis lineages were associated with particular drug resistance-conferring mutations and phenotypic drug resistance. Our study also supports a role for epistatic interactions between different drug resistance mutations and strain genetic backgrounds in M. tuberculosis drug resistance.
PMCID: PMC3370767  PMID: 22470121
19.  Mycobacterium tuberculosis Transmission in a Country with Low Tuberculosis Incidence: Role of Immigration and HIV Infection 
Journal of Clinical Microbiology  2012;50(2):388-395.
Immigrants from high-burden countries and HIV-coinfected individuals are risk groups for tuberculosis (TB) in countries with low TB incidence. Therefore, we studied their role in transmission of Mycobacterium tuberculosis in Switzerland. We included all TB patients from the Swiss HIV Cohort and a sample of patients from the national TB registry. We identified molecular clusters by spoligotyping and mycobacterial interspersed repetitive-unit–variable-number tandem-repeat (MIRU-VNTR) analysis and used weighted logistic regression adjusted for age and sex to identify risk factors for clustering, taking sampling proportions into account. In total, we analyzed 520 TB cases diagnosed between 2000 and 2008; 401 were foreign born, and 113 were HIV coinfected. The Euro-American M. tuberculosis lineage dominated throughout the study period (378 strains; 72.7%), with no evidence for another lineage, such as the Beijing genotype, emerging. We identified 35 molecular clusters with 90 patients, indicating recent transmission; 31 clusters involved foreign-born patients, and 15 involved HIV-infected patients. Birth origin was not associated with clustering (adjusted odds ratio [aOR], 1.58; 95% confidence interval [CI], 0.73 to 3.43; P = 0.25, comparing Swiss-born with foreign-born patients), but clustering was reduced in HIV-infected patients (aOR, 0.49; 95% CI, 0.26 to 0.93; P = 0.030). Cavitary disease, male sex, and younger age were all associated with molecular clustering. In conclusion, most TB patients in Switzerland were foreign born, but transmission of M. tuberculosis was not more common among immigrants and was reduced in HIV-infected patients followed up in the national HIV cohort study. Continued access to health services and clinical follow-up will be essential to control TB in this population.
PMCID: PMC3264153  PMID: 22116153
20.  Phenylethyl Butyrate Enhances the Potency of Second-Line Drugs against Clinical Isolates of Mycobacterium tuberculosis 
Ethionamide (ETH) is a second-line drug for the treatment of tuberculosis. As a prodrug, ETH has to be activated by EthA. ethA is controlled by its repressor EthR. 2-Phenylethyl-butyrate (2-PEB) inhibits EthR binding, enhances expression of EthA, and thereby enhances the growth-inhibitory effects of ethionamide, isoxyl, and thiacetazone in Mycobacterium tuberculosis strains with resistance to ETH due to inhA promoter mutations but not ethA mutations.
PMCID: PMC3264283  PMID: 22106218
21.  Molecular Basis for the Selectivity of Antituberculosis Compounds Capreomycin and Viomycin▿† 
Antimicrobial Agents and Chemotherapy  2011;55(10):4712-4717.
Capreomycin and the structurally similar compound viomycin are cyclic peptide antibiotics which are particularly active against Mycobacterium tuberculosis, including multidrug resistant strains. Both antibiotics bind across the ribosomal interface involving 23S rRNA helix 69 (H69) and 16S rRNA helix 44 (h44). The binding site of tuberactinomycins in h44 partially overlaps with that of aminoglycosides, and they share with these drugs the side effect of irreversible hearing loss. Here we studied the drug target interaction on ribosomes modified by site-directed mutagenesis. We identified rRNA residues in h44 as the main determinants of phylogenetic selectivity, predict compensatory evolution to impact future resistance development, and propose mechanisms involved in tuberactinomycin ototoxicity, which may enable the development of improved, less-toxic derivatives.
PMCID: PMC3187005  PMID: 21768509
22.  Tuberculosis in HIV-Negative and HIV-Infected Patients in a Low-Incidence Country: Clinical Characteristics and Treatment Outcomes 
PLoS ONE  2012;7(3):e34186.
In Switzerland and other developed countries, the number of tuberculosis (TB) cases has been decreasing for decades, but HIV-infected patients and migrants remain risk groups. The aim of this study was to compare characteristics of TB in HIV-negative and HIV-infected patients diagnosed in Switzerland, and between coinfected patients enrolled and not enrolled in the national Swiss HIV Cohort Study (SHCS).
Methods and Findings
All patients diagnosed with culture-confirmed TB in the SHCS and a random sample of culture-confirmed cases reported to the national TB registry 2000–2008 were included. Outcomes were assessed in HIV-infected patients and considered successful in case of cure or treatment completion. Ninety-three SHCS patients and 288 patients selected randomly from 4221 registered patients were analyzed. The registry sample included 10 (3.5%) coinfected patients not enrolled in the SHCS: the estimated number of HIV-infected patients not enrolled in the SHCS but reported to the registry 2000–2008 was 146 (95% CI 122–173). Coinfected patients were more likely to be from sub-Saharan Africa (51.5% versus 15.8%, P<0.0001) and to present disseminated disease (23.9% vs. 3.4%, P<0.0001) than HIV-negative patients. Coinfected patients not enrolled in the SHCS were asylum seekers or migrant workers, with lower CD4 cell counts at TB diagnosis (median CD4 count 79 cells/µL compared to 149 cells/µL among SHCS patients, P = 0.07). There were 6 patients (60.0%) with successful outcomes compared to 82 (88.2%) patients in the SHCS (P = 0.023).
The clinical presentation of coinfected patients differed from HIV-negative TB patients. The number of HIV-infected patients diagnosed with TB outside the SHCS is similar to the number diagnosed within the cohort but outcomes are poorer in patients not followed up in the national cohort. Special efforts are required to address the needs of this vulnerable population.
PMCID: PMC3316631  PMID: 22479556
23.  Phylogenetic Sequence Variations in Bacterial rRNA Affect Species-Specific Susceptibility to Drugs Targeting Protein Synthesis▿‡ 
Antibiotics targeting the bacterial ribosome typically bind to highly conserved rRNA regions with only minor phylogenetic sequence variations. It is unclear whether these sequence variations affect antibiotic susceptibility or resistance development. To address this question, we have investigated the drug binding pockets of aminoglycosides and macrolides/ketolides. The binding site of aminoglycosides is located within helix 44 of the 16S rRNA (A site); macrolides/ketolides bind to domain V of the 23S rRNA (peptidyltransferase center). We have used mutagenesis of rRNA sequences in Mycobacterium smegmatis ribosomes to reconstruct the different bacterial drug binding sites and to study the effects of rRNA sequence variations on drug activity. Our results provide a rationale for differences in species-specific drug susceptibility patterns and species-specific resistance phenotypes associated with mutational alterations in the drug binding pocket.
PMCID: PMC3165350  PMID: 21730122
24.  UvrD2 Is Essential in Mycobacterium tuberculosis, but Its Helicase Activity Is Not Required ▿  
Journal of Bacteriology  2011;193(17):4487-4494.
UvrD is an SF1 family helicase involved in DNA repair that is widely conserved in bacteria. Mycobacterium tuberculosishas two annotated UvrD homologues; here we investigate the role of UvrD2. The uvrD2gene at its native locus could be knocked out only in the presence of a second copy of the gene, demonstrating that uvrD2is essential. Analysis of the putative protein domain structure of UvrD2 shows a distinctive domain architecture, with an extended C terminus containing an HRDC domain normally found in SF2 family helicases and a linking domain carrying a tetracysteine motif. Truncated constructs lacking the C-terminal domains of UvrD2 were able to compensate for the loss of the chromosomal copy, showing that these C-terminal domains are not essential. Although UvrD2 is a functional helicase, a mutant form of the protein lacking helicase activity was able to permit deletion of uvrD2at its native locus. However, a mutant protein unable to hydrolyze ATP or translocate along DNA was not able to compensate for lack of the wild-type protein. Therefore, we concluded that the essential role played by UvrD2 is unlikely to involve its DNA unwinding activity and is more likely to involve DNA translocation and, possibly, protein displacement.
PMCID: PMC3165537  PMID: 21725019
25.  Practical Approach for Reliable Detection of AmpC Beta-Lactamase-Producing Enterobacteriaceae ▿  
Journal of Clinical Microbiology  2011;49(8):2798-2803.
In this prospective study all Enterobacteriaceae isolates (n = 2,129) recovered in the clinical microbiology laboratory during October 2009 to April 2010 were analyzed for AmpC production. Clinical and Laboratory Standards Institute (CLSI) cefoxitin and cefotetan susceptibility breakpoints and CLSI critical ESBL diameters were used to screen for potential AmpC producers. In total, 305 isolates (211 potential AmpC producers and 94 AmpC screen-negative isolates as a control group) were further analyzed by multiplex PCR for the detection of plasmid-encoded ampC beta-lactamase genes and by ampC promoter sequence analysis (considered as the gold standard). Cefoxitin and cefotetan were assessed as primary screening markers. The sensitivities of cefoxitin and cefotetan for the detection of AmpC production were 97.4 and 52.6%, respectively, and the specificities were 78.7 and 99.3%, respectively. As a phenotypic confirmation test, the Etest AmpC and the cefoxitin-cloxacillin double-disk synergy method (CC-DDS) were compared. The sensitivities for the Etest AmpC and the CC-DDS method were 77.4 and 97.2%, respectively, and the specificity was 100% for both methods. The results of the Etest AmpC were inconclusive for 10 isolates. With the CC-DDS method 2 inconclusive results were observed. Based on this study, we propose a comprehensive diagnostic flow chart for the detection of AmpC production consisting of a simple phenotypic screening and a single phenotypic confirmation test with inconclusive results being resolved by molecular analysis. For the proposed flow chart using (i) cefoxitin as a screening marker for AmpC production, (ii) the CC-DDS method as phenotypic confirmation, and (iii) molecular methods in case of inconclusive results, the sensitivity and specificity for AmpC detection would have been 97.4 and 100%, respectively, with respect to the studied isolates. The phenotypic methods used in the AmpC algorithm are simple to perform and easy to implement in the diagnostic laboratory.
PMCID: PMC3147735  PMID: 21632895

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