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1.  Classification Algorithm for Subspecies Identification within the Mycobacterium abscessus Species, Based on Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry 
Journal of Clinical Microbiology  2014;52(9):3362-3369.
Mycobacterium abscessus, as a species, has been increasingly implicated in respiratory infections, notably in cystic fibrosis patients. The species comprises 3 subspecies, which can be difficult to identify. Since they differ in antibiotic susceptibility and clinical relevance, developing a routine diagnostic tool discriminating Mycobacterium abscessus at the subspecies level is a real challenge. Forty-three Mycobacterium abscessus species isolates, previously identified by multilocus sequence typing, were analyzed by matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS). A subspecies identification algorithm, based on five discriminating peaks, was drawn up and validated by blind identification of a further 49 strains, 94% of which (n = 46) were correctly identified. Two M. abscessus subsp. massiliense strains were misidentified as M. abscessus subsp. abscessus, and for 1 other strain identification failed. Inter- and intralaboratory reproducibility tests were conclusive. This study presents, for the first time, a classification algorithm for MALDI-TOF MS identification of the 3 M. abscessus subspecies. MALDI-TOF MS proved effective in discriminating within the M. abscessus species and might be easily integrated into the workflow of microbiology labs.
PMCID: PMC4313163  PMID: 25009048
2.  Clonal Relationship and Differentiation among Mycobacterium abscessus Isolates as Determined Using the Semiautomated Repetitive Extragenic Palindromic Sequence PCR-Based DiversiLab System 
Journal of Clinical Microbiology  2014;52(6):1969-1977.
Mycobacterium abscessus is a rapidly growing mycobacterium that causes respiratory tract infections in predisposed patients, such as those with cystic fibrosis and nosocomial skin and soft tissue infections. In order to investigate the clonal relationships between the strains causing epidemic episodes, we evaluated the discriminatory power of the semiautomated DiversiLab (DL) repetitive extragenic palindromic sequence PCR (REP-PCR) test for M. abscessus genotyping. Since M. abscessus was shown to be composed of subspecies (M. abscessus subsp. massiliense, M. abscessus subsp. bolletii, and M. abscessus subsp. abscessus), we also evaluated the ability of this technique to differentiate subspecies. The technique was applied to two collections of clinical isolates, (i) 83 M. abscessus original isolates (43 M. abscessus subsp. abscessus, 12 M. abscessus subsp. bolletii, and 28 M. abscessus subsp. massiliense) from infected patients and (ii) 35 repeated isolates obtained over 1 year from four cystic fibrosis patients. The DL REP-PCR test was standardized for DNA extraction, DNA amplification, and electrophoresis pattern comparisons. Among the isolates from distinct patients, 53/83 (62%) isolates showed a specific pattern, and 30 were distributed in 11 clusters and 6 patterns, with 2 to 4 isolates per pattern. The clusters and patterns did not fully correlate with multilocus sequence typing (MLST) analysis results. This revealed a high genomic diversity between patients, with a discriminatory power of 98% (Simpson's diversity index). However, since some isolates shared identical patterns, this raises the question of whether it is due to transmission between patients or a common reservoir. Multiple isolates from the same patient showed identical patterns, except for one patient infected by two strains. Between the M. abscessus subspecies, the indexes were <70%, indicating that the DL REP-PCR test is not an accurate tool for identifying organisms to the subspecies level. REP-PCR appears to be a rapid genotyping method that is useful for investigating epidemics of M. abscessus infections.
PMCID: PMC4042767  PMID: 24671795
3.  Xpert GBS Assay for Rapid Detection of Group B Streptococcus in Gastric Fluid Samples from Newborns 
Journal of Clinical Microbiology  2014;52(2):657-659.
The Xpert GBS real-time PCR assay was applied to gastric fluid samples from 143 newborns, and it detected group B streptococcus (GBS) within 1 h for 16 (11.2%) cases, while microscopic examination detected only 2 cases. The sensitivity and specificity of the Xpert GBS were 80% and 100%, respectively, with regard to 20 cases of GBS colonization or infection. Concordance of Xpert GBS results versus culture was 92.3%. This test detects in a timely manner newborns at risk for invasive GBS disease.
PMCID: PMC3911324  PMID: 24478506
4.  Mobile Insertion Cassette Elements Found in Small Non-Transmissible Plasmids in Proteeae May Explain qnrD Mobilization 
PLoS ONE  2014;9(2):e87801.
qnrD is a plasmid mediated quinolone resistance gene from unknown origin, recently described in Enterobacteriaceae. It encodes a pentapeptide repeat protein 36–60% different from the other Qnr (A, B, C, S and VC). Since most qnrD-positive strains were described as strains belonging to Proteus or Providencia genera, we hypothesized that qnrD originated in Proteeae before disseminating to other enterobacterial species. We screened 317 strains of Proteeae for qnrD and its genetic support by PCR. For all the seven qnrD-positive strains (4 Proteus mirabilis, 1 Proteus vulgaris and 2 Providencia rettgeri) the gene was carried onto a small non-transmissible plasmid, contrarily to other qnr genes that are usually carried onto large multi-resistant plasmids. Nucleotide sequences of the qnrD-bearing plasmids were 96% identical. Plasmids contained 3 ORFs apart from qnrD and belonged to an undescribed incompatibility group. Only one plasmid, in P. vulgaris, was slightly different with a 1,568-bp insertion between qnrD and its promoter, leading to absence of quinolone resistance. We sought for similar plasmids in 15 reference strains of Proteeae, but which were tested negative for qnrD, and found a 48% identical plasmid (pVERM) in Providencia vermicola. In order to explain how qnrD could have been inserted into such native plasmid, we sought for gene mobilization structures. qnrD was found to be located within a mobile insertion cassette (mic) element which sequences are similar to one mic also found in pVERM. Our conclusions are that (i) the small non-transmissible qnrD-plasmids described here may result from the recombination between an as-yet-unknown progenitor of qnrD and pVERM, (ii) these plasmids are maintained in Proteeae being a qnrD reservoir (iii) the mic element may explain qnrD mobilization from non-transmissible plasmids to mobilizable or conjugative plasmids from other Enterobacteriaceae, (iv) they can recombined with larger multiresistant plasmids conjugated in Proteeae.
PMCID: PMC3913671  PMID: 24504382
5.  atpE gene as a new useful specific molecular target to quantify Mycobacterium in environmental samples 
BMC Microbiology  2013;13:277.
The environment is the likely source of many pathogenic mycobacterial species but detection of mycobacteria by bacteriological tools is generally difficult and time-consuming. Consequently, several molecular targets based on the sequences of housekeeping genes, non-functional RNA and structural ribosomal RNAs have been proposed for the detection and identification of mycobacteria in clinical or environmental samples. While certain of these targets were proposed as specific for this genus, most are prone to false positive results in complex environmental samples that include related, but distinct, bacterial genera. Nowadays the increased number of sequenced genomes and the availability of software for genomic comparison provide tools to develop novel, mycobacteria-specific targets, and the associated molecular probes and primers. Consequently, we conducted an in silico search for proteins exclusive to Mycobacterium spp. genomes in order to design sensitive and specific molecular targets.
Among the 3989 predicted proteins from M. tuberculosis H37Rv, only 11 proteins showed 80% to 100% of similarity with Mycobacterium spp. genomes, and less than 50% of similarity with genomes of closely related Corynebacterium, Nocardia and Rhodococcus genera. Based on DNA sequence alignments, we designed primer pairs and a probe that specifically detect the atpE gene of mycobacteria, as verified by quantitative real-time PCR on a collection of mycobacteria and non-mycobacterial species. The real-time PCR method we developed was successfully used to detect mycobacteria in tap water and lake samples.
The results indicate that this real-time PCR method targeting the atpE gene can serve for highly specific detection and precise quantification of Mycobacterium spp. in environmental samples.
PMCID: PMC4219376  PMID: 24299240
Mycobacteria; atpE gene; Environmental samples
6.  Genetic Diversity and Population Structure of Mycobacterium marinum: New Insights into Host and Environmental Specificities 
Journal of Clinical Microbiology  2012;50(11):3627-3634.
Mycobacterium marinum causes a systemic tuberculosis-like disease in fish and skin infections in humans that can spread to deeper structures, resulting in tenosynovitis, arthritis, and osteomyelitis. However, little information is available concerning (i) the intraspecific genetic diversity of M. marinum isolated from humans and animals; (ii) M. marinum genotype circulation in the different ecosystems, and (iii) the link between M. marinum genetic diversity and hosts (humans and fish). Here, we conducted a genetic study on 89 M. marinum isolates from humans (n = 68) and fish (n = 21) by using mycobacterial interspersed repetitive units-variable number of tandem repeats (MIRU-VNTR) typing. The results show that the M. marinum population is genetically structured not only according to the host but also according to the ecosystem as well as to tissue tropism in humans. This suggests the existence of different genetic pools in the function of the biological and ecological compartments. Moreover, the presence of only certain M. marinum genotypes in humans suggests a different zoonotic potential of the M. marinum genotypes. Considering that the infection is linked to aquarium activity, a significant genetic difference was also detected when the human tissue tropism of M. marinum was taken into consideration, with a higher genetic polymorphism in strains isolated from patients with cutaneous forms than from individuals with deeper-structure infection. It appears that only few genotypes can produce deeper infections in humans, suggesting that the immune system might play a filtering role.
PMCID: PMC3486196  PMID: 22952269
7.  A systematic review of gyrase mutations associated with fluoroquinolone-resistant Mycobacterium tuberculosis and a proposed gyrase numbering system 
Fluoroquinolone resistance in Mycobacterium tuberculosis has become increasingly important. A review of mutations in DNA gyrase, the fluoroquinolone target, is needed to improve the molecular detection of resistance. We performed a systematic review of studies reporting mutations in DNA gyrase genes in clinical M. tuberculosis isolates. From 42 studies that met inclusion criteria, 1220 fluoroquinolone-resistant M. tuberculosis isolates underwent sequencing of the quinolone resistance-determining region (QRDR) of gyrA; 780 (64%) had mutations. The QRDR of gyrB was sequenced in 534 resistant isolates; 17 (3%) had mutations. Mutations at gyrA codons 90, 91 or 94 were present in 654/1220 (54%) resistant isolates. Four different GyrB numbering systems were reported, resulting in mutation location discrepancies. We propose a consensus numbering system. Most fluoroquinolone-resistant M. tuberculosis isolates had mutations in DNA gyrase, but a substantial proportion did not. The proposed consensus numbering system can improve molecular detection of resistance and identification of novel mutations.
PMCID: PMC3299416  PMID: 22279180
gyrA; gyrB; QRDRs; M. tuberculosis
8.  Detection of Antibiotic Resistance in Leprosy Using GenoType LepraeDR, a Novel Ready-To-Use Molecular Test 
Although leprosy is efficiently treated by multidrug therapy, resistance to first-line (dapsone, rifampin) and to second-line drugs (fluoroquinolones) was described worldwide. Since Mycobacterium leprae is not growing in vitro, phenotypic susceptibility testing requires a one year experiment in the mouse model and this is rarely performed. Genetics on antibiotic resistance provide the basis for molecular tests able to detect for antibiotic resistance in leprosy.
Methodology/Principal Findings
A reverse hybridization DNA strip test was developed as the GenoType LepraeDR test. It includes DNA probes for the wild-type sequence of regions of rpoB, gyrA and folP genes and probes for the prevalent mutations involved in acquired resistance to rifampin, fluoroquinolones and dapsone, respectively. The performances of the GenoType LepraeDR test were evaluated by comparing its results on 120 M. leprae strains, previously studied for resistance by the reference drug in vivo susceptibility method in the mouse footpad and for mutations in the gene regions described above by PCR-sequencing. The results of the test were 100% concordant with those of PCR sequencing and the mouse footpad test for the resistant strains: 16 strains resistant to rifampin, 22 to dapsone and 4 to ofloxacin with mutations (numbering system of the M. leprae genome) in rpoB (10 S456L, 1 S456F, 1 S456M + L458V, 1 H451Y, 1 G432S + H451D, 1 T433I + D441Y and 1 Q438V), in folP1 (8 P55L, 3 P55R, 7 T53I, 3 T53A, 1 T53V) and gyrA (4 A91V), respectively. Concordance was 98.3% for the susceptible strains, two strains showing a mutation at the codon 447 that in fact was not conferring resistance as shown by the in vivo method.
The GenoType LepraeDR test is a commercially available test that accurately detects for antibiotic resistance in leprosy cases. The test is easy to perform and could be implemented in endemic countries.
Author Summary
Although leprosy is a curable disease using a combination of antibiotics for one year, the transmission is still active with 230,000 new cases in 2010. Drug resistance has been described and may prevent eradication of the disease. The infectious agent causing leprosy, Mycobacterium leprae, is not growing in vitro and antibiotic susceptibility testing is possible only in the mouse footpad model that requires a one year experiment. Consequently this testing is rarely done and antibiotic resistance rates in leprosy are unknown. This is the reason why we endeavored to set a new diagnosis test that detects for antibiotic resistance in M. leprae. The test is based on the method of a DNA strip test with a multiplex PCR followed by reverse hybridization. It was developed as an easy-to-use test and it will be available in endemic countries, where these kinds of strip tests are already used for detection of drug resistance in tuberculosis. The results of the new test, Genotype LepraeDR, performed on 120 M. leprae strains were concordant with those of the standard PCR sequencing and mouse footpad susceptibility testing.
PMCID: PMC3409109  PMID: 22860144
9.  Description of a 2,683-Base-Pair Plasmid Containing qnrD in Two Providencia rettgeri Isolates 
qnr genes are plasmid-mediated quinolone resistance genes mainly harbored on large conjugative multiresistant plasmids. The qnrD gene was recently observed in Salmonella enterica on a small nonconjugative plasmid (p2007057). We describe two strains of Providencia rettgeri harboring qnrD on nonconjugative plasmids. The plasmids were 99% identical, with 2,683 bp and four open reading frames, including qnrD, but exhibited only 53% identity with the plasmid found in S. enterica.
PMCID: PMC3256040  PMID: 21986831
10.  Plasmidic qnrA3 Enhances Escherichia coli Fitness in Absence of Antibiotic Exposure 
PLoS ONE  2011;6(9):e24552.
The widespread presence of plasmid-mediated quinolone resistance determinants, particularly qnr genes, has become a current issue. By protecting DNA-gyrase from quinolones, Qnr proteins confer a low level quinolone resistance that is not sufficient to explain their emergence. Since Qnr proteins were hypothesized to act as DNA-binding protein regulators, qnr genes could have emerged by providing a selective advantage other than antibiotic resistance. We investigated host fitness of Escherichia coli isogenic strains after acquisition of the qnrA3 gene, inserted either alone onto a small plasmid (pBR322), or harbored on a large conjugative native plasmid, pHe96(qnrA3) found in a clinical isolate. The isogenic strains were derived from the susceptible E. coli CFT073, a virulent B2 group strain known to infect bladder and kidneys in a mouse model of pyelonephritis. In vitro experiments included growth analysis by automatic spectrophotometry and flow cytometry, and competitions with CFU enumeration. In vivo experiments included infection with each strain and pairwise competitions in absence of antimicrobial exposure. As controls for our experiments we used mutations known to reduce fitness (rpsL K42N mutation) or to enhance fitness (tetA deletion in pBR322). E. coli CFT073 transformed with pBRAM(PBR322-qnrA3) had significantly higher maximal OD than E. coli CFT073 transformed with pBR322 or pBR322ΔtetA, and in vivo competitions were more often won by the qnrA3 carrying strain (24 victories vs. 9 loss among 42 competitions, p = 0.001). In contrast, when pHe96(qnrA3) was introduced by conjugation in E. coli CFT073, it exerted a fitness cost shown by an impaired growth observed in vitro and in vivo and a majority of lost competitions (33/35, p<0.0001). In conclusion, qnrA3 acquisition enhanced bacterial fitness, which may explain qnr emergence and suggests a regulation role of qnr. However, fitness was reduced when qnrA3 was inserted onto multidrug-resistant plasmids and this can slow down its dissemination without antibiotic exposure.
PMCID: PMC3168526  PMID: 21915350
11.  Assessment of Clarithromycin Susceptibility in Strains Belonging to the Mycobacterium abscessus Group by erm(41) and rrl Sequencing▿  
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.
PMCID: PMC3028756  PMID: 21135185
12.  Multilocus Sequence Analysis and rpoB Sequencing of Mycobacterium abscessus (Sensu Lato) Strains▿ 
Journal of Clinical Microbiology  2011;49(2):491-499.
Mycobacterium abscessus, Mycobacterium bolletii, and Mycobacterium massiliense (Mycobacterium abscessus sensu lato) are closely related species that currently are identified by the sequencing of the rpoB gene. However, recent studies show that rpoB sequencing alone is insufficient to discriminate between these species, and some authors have questioned their current taxonomic classification. We studied here a large collection of M. abscessus (sensu lato) strains by partial rpoB sequencing (752 bp) and multilocus sequence analysis (MLSA). The final MLSA scheme developed was based on the partial sequences of eight housekeeping genes: argH, cya, glpK, gnd, murC, pgm, pta, and purH. The strains studied included the three type strains (M. abscessus CIP 104536T, M. massiliense CIP 108297T, and M. bolletii CIP 108541T) and 120 isolates recovered between 1997 and 2007 in France, Germany, Switzerland, and Brazil. The rpoB phylogenetic tree confirmed the existence of three main clusters, each comprising the type strain of one species. However, divergence values between the M. massiliense and M. bolletii clusters all were below 3% and between the M. abscessus and M. massiliense clusters were from 2.66 to 3.59%. The tree produced using the concatenated MLSA gene sequences (4,071 bp) also showed three main clusters, each comprising the type strain of one species. The M. abscessus cluster had a bootstrap value of 100% and was mostly compact. Bootstrap values for the M. massiliense and M. bolletii branches were much lower (71 and 61%, respectively), with the M. massiliense cluster having a fuzzy aspect. Mean (range) divergence values were 2.17% (1.13 to 2.58%) between the M. abscessus and M. massiliense clusters, 2.37% (1.5 to 2.85%) between the M. abscessus and M. bolletii clusters, and 2.28% (0.86 to 2.68%) between the M. massiliense and M. bolletii clusters. Adding the rpoB sequence to the MLSA-concatenated sequence (total sequence, 4,823 bp) had little effect on the clustering of strains. We found 10/120 (8.3%) isolates for which the concatenated MLSA gene sequence and rpoB sequence were discordant (e.g., M. massiliense MLSA sequence and M. abscessus rpoB sequence), suggesting the intergroup lateral transfers of rpoB. In conclusion, our study strongly supports the recent proposal that M. abscessus, M. massiliense, and M. bolletii should constitute a single species. Our findings also indicate that there has been a horizontal transfer of rpoB sequences between these subgroups, precluding the use of rpoB sequencing alone for the accurate identification of the two proposed M. abscessus subspecies.
PMCID: PMC3043527  PMID: 21106786
13.  Development of a Real-Time qPCR Method for Detection and Enumeration of Mycobacterium spp. in Surface Water ▿ †  
Applied and Environmental Microbiology  2010;76(21):7348-7351.
A real-time quantitative PCR method was developed for the detection and enumeration of Mycobacterium spp. from environmental samples and was compared to two other methods already described. The results showed that our method, targeting 16S rRNA, was more specific than the two previously published real-time quantitative PCR methods targeting another 16S rRNA locus and the hsp65 gene (100% versus 44% and 91%, respectively).
PMCID: PMC2976254  PMID: 20851986
14.  Comparison of Culture Methods for Isolation of Nontuberculous Mycobacteria from Surface Waters▿ †  
Applied and Environmental Microbiology  2010;76(11):3514-3520.
The environment is the likely source of most nontuberculous mycobacteria (NTM) involved in human infections, especially pulmonary, skin, and soft tissue infections. In order to measure the prevalence of NTM in different aquatic ecosystems, we tried to standardize the culture methods used for surface water testing since many procedures have been described previously. Cultivation of mycobacteria requires long-term incubation in rich media and inactivation of rapidly growing microorganisms whose growth impedes observation of mycobacterial colonies. Consequently, the two criteria used for evaluation of the methods examined were (i) the rate of inhibition of nontarget microorganisms and (ii) the efficiency of recovery of mycobacteria. We compared the competitive growth of Mycobacterium chelonae and M. avium with nontarget microorganisms on rich Middlebrook 7H11-mycobactin medium after treatment by several chemical decontamination methods that included acids, bases, detergent, or cetylpyridinium chloride (CPC) with and without an antibiotic cocktail, either PANTA (40 U/ml polymyxin, 4 μg/ml amphotericin B, 16 μg/ml nalidixic acid, 4 μg/ml trimethoprim, and 4 μg/ml azlocillin) or PANTAV (PANTA plus 10 μg/ml vancomycin). Our results showed that treatment for 30 min with CPC (final concentration, 0.05%) of water concentrated by centrifugation, followed by culture on a rich medium supplemented with PANTA, significantly decreased the growth of nontarget microorganisms (the concentrations were 6.2 ± 0.4 log10 CFU/liter on Middlebrook 7H11j medium and 4.2 ± 0.2 log10 CFU/liter on Middlebrook 7H11j medium containing PANTA [P < 0.001]), while the effect of this procedure on NTM was not as great (the concentrations of M. chelonae on the two media were 7.0 ± 0.0 log10 CFU/liter and 6.9 ± 0.0 log10 CFU/liter, respectively, and the concentrations of M. avium were 9.1 ± 0.0 log10 CFU/liter and 8.9 ± 0.0 log10 CFU/liter, respectively). We propose that this standardized culture procedure could be used for detection of NTM in aquatic samples.
PMCID: PMC2876437  PMID: 20363776
15.  Evaluation of a New Test, GenoType HelicoDR, for Molecular Detection of Antibiotic Resistance in Helicobacter pylori▿  
Journal of Clinical Microbiology  2009;47(11):3600-3607.
The eradication rate of Helicobacter pylori by standard therapy is decreasing due to antibiotic resistance, mainly to clarithromycin. Our aim was to provide a new molecular test to guide the treatment of new and relapsed cases. We first studied 126 H. pylori strains for phenotypic (MIC) and genotypic resistance to clarithromycin (rrl mutation) and levofloxacin (gyrA mutation) and then developed a DNA strip genotyping test on the basis of the correlation results and literature data. Clinical strains (n = 92) and gastric biopsy specimens containing H. pylori (n = 105) were tested blindly with the new molecular test GenoType HelicoDR. The presence of mutations or the absence of hybridization with wild-type sequences was predictive, in rrl for clarithromycin resistance in 91 cases (mostly the A2147G mutation) and in gyrA for levofloxacin resistance in 58 cases (mutations at codon 87 or 91). Genotyping revealed a mix of genotypes in 33% of the cases, reflecting a coinfection or selection for resistant mutants. The sensitivity and specificity of detecting resistance were 94% and 99% for clarithromycin and 87% and 98.5% for levofloxacin, respectively. The concordance scores were 0.96 for clarithromycin and 0.94 for levofloxacin. With global resistance rates of 46% for clarithromycin and 25% for levofloxacin, which were observed for consecutive positive biopsy specimens from 2007 and 2008, the positive and negative predictive values for detecting resistance were 99% and 94% for clarithromycin and 96% and 96% for fluoroquinolone. GenoType HelicoDR is efficient at detecting mutations predictive of antibiotic resistance in H. pylori when applied to strains or directly to gastric biopsy specimens.
PMCID: PMC2772597  PMID: 19759218
16.  Impact of Low-Level Resistance to Fluoroquinolones Due to qnrA1 and qnrS1 Genes or a gyrA Mutation on Ciprofloxacin Bactericidal Activity in a Murine Model of Escherichia coli Urinary Tract Infection▿  
Antimicrobial Agents and Chemotherapy  2009;53(10):4292-4297.
We investigated the impact of low-level resistance to fluoroquinolones on the bactericidal activity of ciprofloxacin in a murine model of urinary tract infection. The susceptible Escherichia coli strain CFT073 (ciprofloxacin MIC [CIP MIC] of 0.008 μg/ml) was compared to its transconjugants harboring qnrA1 or qnrS1 and to an S83L gyrA mutant. The three derivatives showed similar low-level resistance to fluoroquinolones (CIP MICs, 0.25 to 0.5 μg/ml). Bactericidal activity measured in vitro after 1, 3, and 6 h of exposure to 0.5 μg/ml of ciprofloxacin was significantly lower for the derivative strains (P < 0.01). In the murine model of urinary tract infection (at least 45 mice inoculated per strain), mice were treated with a ciprofloxacin regimen of 2.5 mg/kg, given subcutaneously twice daily for 2 days. In mice infected with the susceptible strain, ciprofloxacin significantly decreased viable bacterial counts (log10 CFU/g of tissue) in the bladder (4.2 ± 0.5 versus 5.5 ± 1.3; P = 0.001) and in the kidney (3.6 ± 0.8 versus 5.0 ± 1.1; P = 0.003) compared with those of untreated mice. In contrast, no significant decrease in viable bacterial counts was observed with any of the three derivative strains. The area under the concentration-time curve from 0 to 24 h/MIC and the maximum concentration of drug in serum/MIC ratios measured in plasma were indeed equal to 827 and 147, respectively, for the parental strain, and only 12.4 to 24.8 and 2.2 to 4.4, respectively, for the derivative strains. In conclusion, low-level resistance to fluoroquinolones conferred by a qnr gene is associated with decreased bactericidal activity of ciprofloxacin, similar to that obtained with a gyrA mutation.
PMCID: PMC2764221  PMID: 19635960
17.  Outbreak of Nontuberculous Mycobacterial Subcutaneous Infections Related to Multiple Mesotherapy Injections▿  
Journal of Clinical Microbiology  2009;47(6):1961-1964.
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.
PMCID: PMC2691096  PMID: 19386853
18.  The Pentapeptide Repeat Proteins MfpAMt and QnrB4 Exhibit Opposite Effects on DNA Gyrase Catalytic Reactions and on the Ternary Gyrase-DNA-Quinolone Complex▿ †  
Journal of Bacteriology  2008;191(5):1587-1594.
MfpAMt and QnrB4 are two newly characterized pentapeptide repeat proteins (PRPs) that interact with DNA gyrase. The mfpAMt gene is chromosome borne in Mycobacterium tuberculosis, while qnrB4 is plasmid borne in enterobacteria. We expressed and purified the two PRPs and compared their effects on DNA gyrase, taking into account host specificity, i.e., the effect of MfpAMt on M. tuberculosis gyrase and the effect of QnrB4 on Escherichia coli gyrase. Whereas QnrB4 inhibited E. coli gyrase activity only at concentrations higher than 30 μM, MfpAMt inhibited all catalytic reactions of the M. tuberculosis gyrase described for this enzyme (supercoiling, cleavage, relaxation, and decatenation) with a 50% inhibitory concentration of 2 μM. We showed that the D87 residue in GyrA has a major role in the MfpAMt-gyrase interaction, as D87H and D87G substitutions abolished MfpAMt inhibition of M. tuberculosis gyrase catalytic reactions, while A83S modification did not. Since MfpAMt and QnrB4 have been involved in resistance to fluoroquinolones, we measured the inhibition of the quinolone effect in the presence of each PRP. QnrB4 reversed quinolone inhibition of E. coli gyrase at 0.1 μM as described for other Qnr proteins, but MfpAMt did not modify M. tuberculosis gyrase inhibition by fluoroquinolones. Crossover experiments showed that MfpAMt also inhibited E. coli gyrase function, while QnrB4 did not reverse quinolone inhibition of M. tuberculosis gyrase. In conclusion, our in vitro experiments showed that MfpAMt and QnrB4 exhibit opposite effects on DNA gyrase and that these effects are protein and species specific.
PMCID: PMC2648189  PMID: 19060136
19.  Mutagenesis in the α3α4 GyrA Helix and in the Toprim Domain of GyrB Refines the Contribution of Mycobacterium tuberculosis DNA Gyrase to Intrinsic Resistance to Quinolones▿ †  
The replacement of M74 in GyrA, A83 in GyrA, and R447 in GyrB of Mycobacterium tuberculosis gyrase by their Escherichia coli homologs resulted in active enzymes as quinolone susceptible as the E. coli gyrase. This demonstrates that the primary structure of gyrase determines intrinsic quinolone resistance and was supported by a three-dimensional model of N-terminal GyrA.
PMCID: PMC2493125  PMID: 18426901
20.  A Plasmid-Borne Shewanella algae Gene, qnrA3, and Its Possible Transfer In Vivo between Kluyvera ascorbata and Klebsiella pneumoniae▿  
Journal of Bacteriology  2008;190(15):5217-5223.
The plasmid-borne quinolone resistance gene qnrA1 is prevalent in multidrug-resistant Enterobacteriaceae. A chromosomally encoded homologue in Shewanella algae, qnrA3, has been described. We isolated two qnrA3-positive strains, one of Klebsiella pneumoniae (He96) and one of Kluyvera ascorbata (Kas96), from the feces of an immunocompromised outpatient. The qnrA3 allele was identical to that of S. algae except for 5 nucleotides and differed from qnrA1 by 29 nucleotides affecting three amino acids. The analysis of the qnrA3 genetic environment showed that qnrA3 was inserted downstream from an ISCR1 element at a recombination crossover site described for other resistance genes, including qnrA1, and immediately upstream from IS26, a situation not described before. IS26 preceded an incomplete class 1 integron which contained, among other genes, aac(6′)-Ib-cr, another transferable quinolone resistance gene, and the β-lactamase gene blaOXA-1/30. The 10-kb fragment encompassing qnrA3 was compared to previously described qnrA1-containing plasmids and multidrug-resistant plasmids; it shares identical sequences with pC15a, pHSH2, pQR1, pQKp311H, and pSAL-1 but with rearrangements, deletions, and mutations. Conjugal transfer of qnrA3 was highly efficient (10−2) from K. pneumoniae He96 or K. ascorbata Kas96 to Escherichia coli J53 but less so (10−5) from either donor to a clinical strain of Enterobacter cloacae. This first description of a plasmid-borne copy and of the in vitro transfer of qnrA3 is taken to illustrate its likely in vivo transfer from S. algae to the Enterobacteriaceae.
PMCID: PMC2493261  PMID: 18515416
21.  Are All the DNA Gyrase Mutations Found in Mycobacterium leprae Clinical Strains Involved in Resistance to Fluoroquinolones?▿ † 
Mycobacterium leprae DNA gyrases carrying various mutations, previously described in clinical strains, were investigated for quinolone susceptibility by inhibition of supercoiling and DNA cleavage promotion. We demonstrated that the gyrA mutations leading to G89C or A91V confer fluoroquinolone resistance whereas the gyrB mutation leading to D205N does not.
PMCID: PMC2224767  PMID: 18070975
22.  Expression and Purification of an Active Form of the Mycobacterium leprae DNA Gyrase and Its Inhibition by Quinolones▿  
Mycobacterium leprae, the causative agent of leprosy, is noncultivable in vitro; therefore, evaluation of antibiotic activity against M. leprae relies mainly upon the mouse footpad system, which requires at least 12 months before the results become available. We have developed an in vitro assay for studying the activities of quinolones against the DNA gyrase of M. leprae. We overexpressed in Escherichia coli the M. leprae GyrA and GyrB subunits separately as His-tagged proteins by using a pET plasmid carrying the gyrA and gyrB genes. The soluble 97.5-kDa GyrA and 74.5-kDa GyrB subunits were purified by nickel chelate chromatography and were reconstituted as an enzyme with DNA supercoiling activity. Based on the drug concentrations that inhibited DNA supercoiling by 50% or that induced DNA cleavage by 25%, the 13 quinolones tested clustered into three groups. Analysis of the quinolone structure-activity relationship demonstrates that the most active quinolones against M. leprae DNA gyrase share the following structural features: a substituted carbon at position 8, a cyclopropyl substituent at N-1, a fluorine at C-6, and a substituent ring at C-7. We conclude that the assays based on DNA supercoiling inhibition and drug-induced DNA cleavage on purified M. leprae DNA gyrase are rapid, efficient, and safe methods for the screening of quinolone derivatives with potential in vivo activities against M. leprae.
PMCID: PMC1855561  PMID: 17325221
23.  Functional Analysis of DNA Gyrase Mutant Enzymes Carrying Mutations at Position 88 in the A Subunit Found in Clinical Strains of Mycobacterium tuberculosis Resistant to Fluoroquinolones▿  
Antimicrobial Agents and Chemotherapy  2006;50(12):4170-4173.
We investigated the enzymatic efficiency and inhibition by quinolones of Mycobacterium tuberculosis DNA gyrases carrying the previously described GyrA G88C mutation and the novel GyrA G88A mutation harbored by two multidrug-resistant clinical strains and reproduced by site-directed mutagenesis. Fluoroquinolone MICs and 50% inhibitory concentrations for both mutants were 2- to 43-fold higher than for the wild type, demonstrating that these mutations confer fluoroquinolone resistance in M. tuberculosis.
PMCID: PMC1694005  PMID: 17015625
24.  Genetic Basis for Natural and Acquired Resistance to the Diarylquinoline R207910 in Mycobacteria 
The atpE gene encoding the subunit c of the ATP synthase of Mycobacterium tuberculosis, the target of the new diarylquinoline drug R207910, has been sequenced from in vitro mutants resistant to the drug. The previously reported mutation A63P and a new mutation, I66M, were found. The genetic diversity of atpE in 13 mycobacterial species was also investigated, revealing that the region involved in resistance to R207910 is conserved, except in Mycobacterium xenopi in which the highly conserved residue Ala63 is replaced by Met, a modification that may be associated with the natural resistance of M. xenopi to R207910.
PMCID: PMC1538646  PMID: 16870785
25.  Novel Gyrase Mutations in Quinolone-Resistant and -Hypersusceptible Clinical Isolates of Mycobacterium tuberculosis: Functional Analysis of Mutant Enzymes 
Mutations in the DNA gyrase GyrA2GyrB2 complex are associated with resistance to quinolones in Mycobacterium tuberculosis. As fluoroquinolones are being used increasingly in the treatment of tuberculosis, we characterized several multidrug-resistant clinical isolates of M. tuberculosis carrying mutations in the genes encoding the GyrA or GyrB subunits associated with quinolone resistance or hypersusceptibility. In addition to the reported putative quinolone resistance mutations in GyrA, i.e., A90V, D94G, and D94H, we found that the GyrB N510D mutation was also associated with ofloxacin resistance. Surprisingly, several isolates bearing a novel combination of gyrA T80A and A90G changes were hypersusceptible to ofloxacin. M. tuberculosis GyrA and GyrB subunits (wild type [WT] and mutants) were overexpressed in Escherichia coli, purified to homogeneity, and used to reconstitute highly active gyrase complexes. Mutant proteins were produced similarly from engineered gyrA and gyrB alleles by mutagenesis. MICs, enzyme inhibition, and drug-induced DNA cleavage were determined for moxifloxacin, gatifloxacin, ofloxacin, levofloxacin, and enoxacin. Mutant gyrase complexes bearing GyrA A90V, D94G, and D94H and GyrB N510D were resistant to quinolone inhibition (MICs and 50% inhibitory concentrations [IC50s] at least 3.5-fold higher than the concentrations for the WT), and all, except the GyrB mutant, were less efficiently trapped as a quinolone cleavage complex. In marked contrast, gyrase complexes bearing GyrA T80A or A90G were hypersusceptible to the action of many quinolones, an effect that was reinforced for complexes bearing both mutations (MICs and IC50s up to 14-fold lower than the values for the WT). This is the first detailed enzymatic analysis of hypersusceptibility and resistance in M. tuberculosis.
PMCID: PMC1346799  PMID: 16377674

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