Escherichia coli is the species most frequently associated with clinical infections by extended-spectrum-β-lactamase (ESBL)-producing isolates, with the CTX-M ESBL enzymes being predominant and found in genetically diverse E. coli isolates. The main objective of this study was to compare, on the basis of a case-control design, the phylogenetic diversity of 152 CTX-M-producing and 152 non-ESBL-producing clinical E. coli isolates. Multilocus sequence typing revealed that even though CTX-M enzymes were largely disseminated across the diversity of E. coli isolates, phylogenetic group B2 showed a particularly heterogeneous situation. First, clone ST131 of group B2 was strongly associated with CTX-M production (55 [79%] of 70 isolates), with CTX-M-15 being predominant. Second, the remaining members of group B2 were significantly less frequently associated with CTX-M production (9 [12%] of 75) than E. coli phylogenetic groups A, B1, and D (88 [55%] of 159). CTX-M-producing ST131 E. coli isolates were significantly more frequent in patients hospitalized in geriatric wards or long-term care facilities. Besides, the non-ESBL ST131 isolates significantly more frequently showed resistance to penicillins than the non-ESBL, non-ST131 isolates did. In conclusion, the present study emphasizes the particular antimicrobial resistance and epidemiologic characteristics of clone ST131 within group B2, which could result from the higher antibiotic exposure of this clone, as it is the predominant clone of group B2 carried in the human gut.

TMC207 is a new antituberculous drug belonging to the diarylquinoline class which very efficiently inhibits the ATP synthase of mycobacteria such as Mycobacterium tuberculosis, one of the most important pathogens in the world. In order to map the amino acid residues involved in the binding of the drug, we have selected in vitro TMC207-resistant mutants from M. tuberculosis and diverse atypical mycobacteria. Six distinct mutations, Asp28→Gly, Asp28→Ala, Leu59→Val, Glu61→Asp, Ala63→Pro, and Ile66→Met, have been identified in the subunit c forming a C ring in the ATP synthase. They were studied by evaluating the levels of resistance that they confer in the selected clones and by using an isogenic complementation system in Mycobacterium smegmatis. The rates of increase of TMC207 MIC values (8- to 133-fold) were interpreted by constructing by homology modeling a structure of the mycobacterial C ring which was used for docking simulations with TMC207. Our results suggest that the residues found to be mutated in the resistant clones, together with a tyrosine specifically conserved at position 64 in mycobacteria, define a cleft located between two adjacent c subunits in the C ring. This cleft, which encompasses the proton-binding site (Glu61), is well fitted to bind TMC207 at the level of the bromoquinoline moiety, with the drug being anchored by several ionic, hydrogen, and halogen bonds with residues Glu61, Tyr64, and Asp28, respectively. These data shed light on the molecular interactions allowing TMC207 to bind specifically and efficiently at the level of the proton-binding site of the mycobacterial C ring.

Fluoroquinolone (FQ) resistance is emerging in Mycobacterium tuberculosis. The main mechanism of FQ resistance is amino acid substitution within the quinolone resistance-determining region (QRDR) of the GyrA subunit of DNA gyrase, the sole FQ target in M. tuberculosis. However, substitutions in GyrB whose implication in FQ resistance is unknown are increasingly being reported. The present study clarified the role of four GyrB substitutions identified in M. tuberculosis clinical strains, two located in the QRDR (D500A and N538T) and two outside the QRDR (T539P and E540V), in FQ resistance. We measured FQ MICs and also DNA gyrase inhibition by FQs in order to unequivocally clarify the role of these mutations in FQ resistance. Wild-type GyrA, wild-type GyrB, and mutant GyrB subunits produced from engineered gyrB alleles by mutagenesis were overexpressed in Escherichia coli, purified to homogeneity, and used to reconstitute highly active gyrase complexes. MICs and DNA gyrase inhibition were determined for moxifloxacin, gatifloxacin, ofloxacin, levofloxacin, and enoxacin. All these substitutions are clearly implicated in FQ resistance, underlining the presence of a hot spot region housing most of the GyrB substitutions implicated in FQ resistance (residues NTE, 538 to 540). These findings help us to refine the definition of GyrB QRDR, which is extended to positions 500 to 540.

Combination chemotherapy with rifampin and streptomycin (RIF-STR) for 8 weeks is currently recommended by the WHO as the first-line treatment for Mycobacterium ulcerans infection (Buruli ulcer). To gain better insight into the mode of action of these antibiotics against established M. ulcerans infection foci and to characterize recovery of local immune responses during chemotherapy, we conducted a detailed histopathological study of M. ulcerans-infected and RIF-STR-treated mice. Mice were inoculated with M. ulcerans in the footpad and 11 weeks later treated with RIF-STR. Development of lesions during the first 11 weeks after infection and subsequent differences in disease progression between RIF-STR-treated and untreated mice were studied. Changes in histopathological features, footpad swelling, and number of CFU were analyzed. After inoculation with M. ulcerans, massive infiltrates dominated by polymorphonuclear leukocytes developed at the inoculation site but did not prevent bacterial multiplication. Huge clusters of extracellular bacteria located in large necrotic areas and surrounded by dead leukocytes developed in the untreated mice. Chemotherapy with RIF-STR led to a rapid drop in CFU associated with loss of solid Ziehl-Neelsen staining of acid-fast bacilli. Development of B-lymphocyte clusters and of macrophage accumulations surrounding the mycobacteria demonstrated the resolution of local immune suppression. Results demonstrate that the experimental M. ulcerans mouse infection model will be a valuable tool to investigate efficacy of new treatment regimens and of candidate vaccines.

Background

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.

Conclusions/Significance

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.

The main mechanism of fluoroquinolone (FQ) resistance in Mycobacterium tuberculosis is mutation in DNA gyrase (GyrA2GyrB2), especially in gyrA. However, the discovery of unknown mutations in gyrB whose implication in FQ resistance is unclear has become more frequent. We investigated the impact on FQ susceptibility of eight gyrB mutations in M. tuberculosis clinical strains, three of which were previously identified in an FQ-resistant strain. We measured FQ MICs and also DNA gyrase inhibition by FQs in order to clarify the role of these mutations in FQ resistance. Wild-type GyrA, wild-type GyrB, and mutant GyrB subunits produced from engineered gyrB alleles by mutagenesis were overexpressed in Escherichia coli, purified to homogeneity, and used to reconstitute highly active gyrase complexes. MICs and DNA gyrase inhibition were determined for moxifloxacin, gatifloxacin, ofloxacin, levofloxacin, and enoxacin. We demonstrated that the eight substitutions in GyrB (D473N, P478A, R485H, S486F, A506G, A547V, G551R, and G559A), recently identified in FQ-resistant clinical strains or encountered in M. tuberculosis strains isolated in France, are not implicated in FQ resistance. These results underline that, as opposed to phenotypic FQ susceptibility testing, the DNA gyrase inhibition assay is the only way to prove the role of a DNA gyrase mutation in FQ resistance. Therefore, the use of FQ in the treatment of tuberculosis (TB) patients should not be ruled out only on the basis of the presence of mutations in gyrB.

Resistance to antibiotics has increased dramatically over the past few years and has now reached a level that places future patients in real danger. Microorganisms such as Escherichia coli and Klebsiella pneumoniae, which are commensals and pathogens for humans and animals, have become increasingly resistant to third-generation cephalosporins. Moreover, in certain countries, they are also resistant to carbapenems and therefore susceptible only to tigecycline and colistin. Resistance is primarily attributed to the production of beta-lactamase genes located on mobile genetic elements, which facilitate their transfer between different species. In some rare cases, Gram-negative rods are resistant to virtually all known antibiotics. The causes are numerous, but the role of the overuse of antibiotics in both humans and animals is essential, as well as the transmission of these bacteria in both the hospital and the community, notably via the food chain, contaminated hands, and between animals and humans. In addition, there are very few new antibiotics in the pipeline, particularly for Gram-negative bacilli. The situation is slightly better for Gram-positive cocci as some potent and novel antibiotics have been made available in recent years. A strong and coordinated international programme is urgently needed. To meet this challenge, 70 internationally recognized experts met for a two-day meeting in June 2011 in Annecy (France) and endorsed a global call to action ("The Pensières Antibiotic Resistance Call to Action"). Bundles of measures that must be implemented simultaneously and worldwide are presented in this document. In particular, antibiotics, which represent a treasure for humanity, must be protected and considered as a special class of drugs.

Background

Assistance Publique-Hôpitaux de Paris (APHP), the largest public health care institution in France (38 hospitals, 23,000 beds, serving 11.6 millions inhabitants) launched in 1993 a long term programme to control and survey multidrug resistant bacteria (MDR).

Findings

AP-HP MDR programme consisted in successive waves of actions: bundle measures to survey and control cross transmission of MRSA and extended-spectrum betalactamase producing enterobacteria (ESBL) in 1993, large campaign to promote the use of alcohol-based hand rub solution (ABHRS) in 2001, specific strategy to quickly control the spread of emerging MDR (vancomycin resistant Enterococcus, VRE; carbapenemase producing enterobacteria, CPE) in 2006, large campaign to decrease antibiotics consumption in 2006.

Following this programme, the ABHRS consumption dramatically increased, the antibiotic consumption decreased by 10%, the incidence of MRSA, including MRSA bacteraemia, decreased by 2/3, all VRE and CPE events were rapidly controlled. However, the incidence of ESBL, mainly Klebsiella pneumoniae and Escherichia coli, that remained low and stable until 2003 increased markedly afterwards, justifying adapting our programme in the future.

Conclusion

A sustained and coordinated strategy can lead to control multidrug resistant bacteria at the level of a large multihospital institution.

Background

Global dissemination of Escherichia coli producing CTX-M extended-spectrum β-lactamases (ESBL) is a public health concern. The aim of the study was to determine factors associated with CTX-M- producing E. coli infections among patients hospitalised in the Assistance Publique-Hôpitaux de Paris, the largest hospital system in France (23 000 beds), through a prospective case-control-control study.

Methods/Principal Findings

From November 2008 to June 2009, 152 inpatients with a clinical sample positive for CTX-M-producing E. coli (cases), 152 inpatients with a clinical sample positive for non ESBL-producing E. coli on the day or within the three days following case detection (controls C1), and 152 inpatients with culture-negative clinical samples since the beginning of hospitalisation and until three days after case detection (controls C2) were included in ten hospitals of the Paris area. Factors studied were related to patient's origin, lifestyle and medical history as well as care during hospitalisation. Those independently associated with CTX-M-producing E. coli were determined. Three independent factors were common to the two case-control comparisons: birth outside of Europe (cases vs C1: OR1 = 2.4; 95%CI = [1.3–4.5] and cases vs C2: OR2 = 3.1; 95%CI = [1.4–7.0]), chronic infections (OR1 = 2.9; 95%CI = [1.3–6.9] and OR2 = 8.7; 95%CI = [2.0–39.7]), and antibiotic treatment between hospital admission and inclusion (OR1 = 2.0; 95%CI = [1.0–3.8] and OR2 = 3.3; 95%CI = [1.5–7.2]). Cases were also more likely to be (i) functionally dependent before hospitalisation than C2 (OR2 = 7.0; 95%CI = [2.1–23.5]) and (ii) living in collective housing before hospitalisation than C2 (OR2 = 15.2; 95%CI = [1.8–130.7]) when CTX-M-producing E. coli was present at admission.

Conclusion

For the first time, patient's origin and lifestyle were demonstrated to be independently associated with isolation of CTX-M-producing E. coli, in addition to health care-related factors.

Although beta-lactam antibiotics are not considered as antituberculous drugs, it has been recently shown that the combination of carbapenems and clavulanate is bactericidal in vitro. We evaluated in a murine model of tuberculosis the activity of carbapenems alone and combined with clavulanate against Mycobacterium tuberculosis. Swiss mice infected intravenously with 3 × 105 M. tuberculosis H37Rv were treated for 4 weeks with clavulanate alone or imipenem, meropenem, and ertapenem alone or combined with clavulanate, whereas a positive control group was treated with isoniazid, and a negative control group was held without treatment. The combination of imipenem or meropenem plus clavulanate significantly improved survival. Among groups of mice with 100% survival, only isoniazid reduced lung CFU counts; the carbapenem-clavulanate combinations did not prevent bacterial growth. Although less active than isoniazid, the combinations of imipenem or meropenem plus clavulanate improved the survival of mice infected with M. tuberculosis and should be further evaluated.

The breakage-reunion domain of M. tuberculosis DNA gyrase was crystallized using the hanging-drop vapour-diffusion method. One of the four crystal forms obtained belonged to space group C2 and diffraction data were collected to a resolution of 2.7 Å.

Mycobacterium tuberculosis DNA gyrase, a nanomachine that is involved in the regulation of DNA topology, is the only type II topoisomerase present in this organism and hence is the sole target for fluoroquinolone action. The breakage-reunion domain of the A subunit plays an essential role in DNA binding during the catalytic cycle. Two constructs of 53 and 57 kDa (termed GA53BK and GA57BK) corresponding to this domain have been overproduced, purified and crystallized. Diffraction data were collected from four crystal forms. The resolution limits ranged from 4.6 to 2.7 Å depending on the crystal form. The best diffracting crystals belonged to space group C2, with a biological dimer in the asymmetric unit. This is the first report of the crystallization and preliminary X-ray diffraction analysis of the breakage-reunion domain of DNA gyrase from a species containing one unique type II topoisomerase.

The detection of extended-spectrum β-lactamase-producing (ESBL) bacteria is of importance for infection control and epidemiological surveillance. We aimed to compare phenotypic methods available in the routine laboratory and to evaluate two-step strategies using these methods for the detection of ESBL-positive Enterobacteriaceae. Two methods used for routine susceptibility testing (Vitek2 and disk diffusion methods) and seven methods designed for the detection of ESBL production (ESBL Etests, combination disks, double-disk synergy [DDS] methods on Mueller-Hinton [MH] agar and cloxacillin-containing MH agar, and the Cica-Beta test) were tested against 107 strains of Enterobacteriaceae not susceptible to extended-spectrum cephalosporins. All strains were screened for the presence of acquired ESBL-encoding genes by PCR, and the PCR result was considered the gold standard for evaluation of the other test methods. Among the 107 strains, 52 (49%) were ESBL positive. With Vitek2, sensitivities were the highest when using extended cards (73% to 79%), but 25% to 31% of the strains yielded indeterminate results. For the disk diffusion method, sensitivities were the highest (96%) when testing at least cefotaxime, cefepime, and a third compound (ceftazidime, cefpodoxime, or aztreonam). For the specific methods, specificities ranged from 62% (ceftazidime ESBL Etest) to 100% (DDS using a disk spacing of 20 mm). When a method designed for ESBL detection was used on strains considered ESBL negative or with an indeterminate result by a first routine susceptibility method, sensitivities reached 100% for a majority of combinations. In conclusion, two-step strategies using phenotypic methods available in most clinical laboratories may reach a sensitivity of 100% for ESBL detection among a large panel of species, including AmpC producers, providing a sensible choice of tests.

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.

Mycobacterial identification is based on several methods: conventional biochemical tests that require several weeks for accurate identification, and molecular tools that are now routinely used. However, these techniques are expensive and time-consuming. In this study, an alternative method was developed using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). This approach allows a characteristic mass spectral fingerprint to be obtained from whole inactivated mycobacterial cells. We engineered a strategy based on specific profiles in order to identify the most clinically relevant species of mycobacteria. To validate the mycobacterial database, a total of 311 strains belonging to 31 distinct species and 4 species complexes grown in Löwenstein-Jensen (LJ) and liquid (mycobacterium growth indicator tube [MGIT]) media were analyzed. No extraction step was required. Correct identifications were obtained for 97% of strains from LJ and 77% from MGIT media. No misidentification was noted. Our results, based on a very simple protocol, suggest that this system may represent a serious alternative for clinical laboratories to identify mycobacterial species.

The prevalence of extensively drug-resistant tuberculosis (XDR-TB), defined as TB that is resistant to isoniazid, rifampin, fluoroquinolones, and aminoglycosides, is rising worldwide. The extent of Mycobacterium tuberculosis resistance to fluoroquinolones depends on the mutation in the DNA gyrase, the only target of fluoroquinolones. The MIC of moxifloxacin, the most active fluoroquinolone against M. tuberculosis, may be lower than its peak serum level for some ofloxacin-resistant strains of Mycobacterium tuberculosis. Therefore, if the MIC of moxifloxacin is lower than its peak serum level, it may be effective against XDR-TB. Our objective was to determine the efficacy of moxifloxacin in treating ofloxacin-resistant TB. We selected isogenic fluoroquinolone-resistant mutants of M. tuberculosis H37Rv in vivo. We infected Swiss mice with either wild-type H37Rv or one of three mutant strains with different MICs that are commonly seen in clinical practice. The MICs of the mutant strains ranged from below to above the peak moxifloxacin level seen in humans (3 μg/ml). Each mouse was treated with one of four moxifloxacin doses for 1 month. Moxifloxacin was effective against mutant strain GyrB D500N, with the lowest MIC (0.5 μg/ml), when the standard dose was doubled. Moxifloxacin reduced mortality in mice infected with mutant strain GyrA A90V with an intermediate MIC (2 μg/ml). However, it had no impact on the mutant strain GyrA D94G with the highest MIC (4 μg/ml). Our study underscores current WHO recommendations to use moxifloxacin when there is resistance to early-generation fluoroquinolones such as ofloxacin, restricting this recommendation to strains with moxifloxacin MICs of less than or equal to 2 μg/ml.

Rationale

The sterilizing activity of the regimen used to treat multidrug resistant tuberculosis (MDR TB) has not been studied in a mouse model.

Objective and Methods

Swiss mice were intravenously inoculated with 6 log10 of Mycobacterium tuberculosis (TB) strain H37Rv, treated with second-line drug combinations with or without the diarylquinoline TMC207, and then followed without treatment for 3 more months to determine relapse rates (modified Cornell model).

Measurements

Bactericidal efficacy was assessed by quantitative lung colony-forming unit (CFU) counts. Sterilizing efficacy was assessed by measuring bacteriological relapse rates 3 months after the end of treatment.

Main Results

The relapse rate observed after 12 months treatment with the WHO recommended MDR TB regimen (amikacin, ethionamide, pyrazinamide and moxifloxacin) was equivalent to the relapse rate observed after 6 months treatment with the recommended drug susceptible TB regimen (rifampin, isoniazid and pyrazinamide). When TMC207 was added to this MDR TB regimen, the treatment duration needed to reach the same relapse rate dropped to 6 months. A similar relapse rate was also obtained with a 6-month completely oral regimen including TMC207, moxifloxacin and pyrazinamide but excluding both amikacin and ethionamide.

Conclusions

In this murine model the duration of the WHO MDR TB treatment could be reduced to 12 months instead of the recommended 18–24 months. The inclusion of TMC207 in the WHO MDR TB treatment regimen has the potential to further shorten the treatment duration and at the same time to simplify treatment by eliminating the need to include an injectable aminoglycoside.

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

Mycobacterium tuberculosis DNA gyrase, an indispensable nanomachine involved in the regulation of DNA topology, is the only type II topoisomerase present in this organism and is hence the sole target for quinolone action, a crucial drug active against multidrug-resistant tuberculosis. To understand at an atomic level the quinolone resistance mechanism, which emerges in extensively drug resistant tuberculosis, we performed combined functional, biophysical and structural studies of the two individual domains constituting the catalytic DNA gyrase reaction core, namely the Toprim and the breakage-reunion domains. This allowed us to produce a model of the catalytic reaction core in complex with DNA and a quinolone molecule, identifying original mechanistic properties of quinolone binding and clarifying the relationships between amino acid mutations and resistance phenotype of M. tuberculosis DNA gyrase. These results are compatible with our previous studies on quinolone resistance. Interestingly, the structure of the entire breakage-reunion domain revealed a new interaction, in which the Quinolone-Binding Pocket (QBP) is blocked by the N-terminal helix of a symmetry-related molecule. This interaction provides useful starting points for designing peptide based inhibitors that target DNA gyrase to prevent its binding to DNA.

Because of operational limitations, a significant proportion of the health centers at the peripheral level are able to provide treatment to Buruli ulcer patients with the combination rifampin (rifampicin)-streptomycin (RIF-STR) only five times weekly (5/7) instead of seven times weekly (7/7), as recommended. The objective of this experiment is to assess the impacts of various dosing frequencies of the combination on its bactericidal and sterilizing activities against Mycobacterium ulcerans in mice. The results demonstrate that the bactericidal activities did not differ significantly among five dosing frequencies of the combination, ranging from seven times to twice weekly, whereas the sterilizing activities differed widely. RIF-STR 7/7 was the only regimen that was able to sterilize the infection after 4 to 8 weeks of treatment; the sterilizing activities associated with reduced dosing frequencies were significantly diminished, and 8 weeks of 5/7 administration yielded a relapse rate greater than the generally accepted level of 5%. We recommend that the duration of treatment with 5/7 administration be prolonged beyond 8 weeks and that additional experiments with mice be carried out, with sufficient statistical power to compare the relapse rates of M. ulcerans infection between 8 weeks of 7/7 administration and 10 and 12 weeks of 5/7 administration of RIF-STR.

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.

We report a case of endocarditis due to the rod-shaped Neisseria species Neisseria bacilliformis. The phenotypic characterization of this recently characterized bacteria is difficult, and the identification requires the sequencing of the 16S rRNA gene. The resolution of the disease was complete after appropriate antibiotic therapy, and surgery was not required.

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.

Two clinical strains of Escherichia coli (2138) and Enterobacter cloacae (7506) isolated from the same patient in France and showing resistance to extended-spectrum cephalosporins and low susceptibility to imipenem were investigated. Both strains harbored the plasmid-contained blaTEM-1 and blaKPC-2 genes. blaKLUC-2, encoding a mutant of the chromosomal β-lactamase of Kluyvera cryocrescens, was also identified at a plasmid location in E. cloacae 7506, suggesting the ISEcp1-assisted escape of blaKLUC from the chromosome. Determination of the KPC-2 structure at 1.6 Å revealed that the binding site was occupied by the C-terminal (C-ter) residues coming from a symmetric KPC-2 monomer, with the ultimate C-ter Glu interacting with Ser130, Lys234, Thr235, and Thr237 in the active site. This mode of binding can be paralleled to the inhibition of the TEM-1 β-lactamase by the inhibitory protein BLIP. Determination of the 1.23-Å structure of a KPC-2 mutant in which the five C-ter residues were deleted revealed that the catalytic site was filled by a citrate molecule. Structure analysis and docking simulations with cefotaxime and imipenem provided further insights into the molecular basis of the extremely broad spectrum of KPC-2, which behaves as a cefotaximase with significant activity against carbapenems. In particular, residues 104, 105, 132, and 167 draw a binding cavity capable of accommodating both the aminothiazole moiety of cefotaxime and the 6α-hydroxyethyl group of imipenem, with the binding of the former drug being also favored by a significant degree of freedom at the level of the loop at positions 96 to 105 and by an enlargement of the binding site at the end of strand β3.

We conducted a prospective multicenter study of methicillin-resistant Staphylococcus aureus (MRSA) isolates, including the first five consecutive clinical isolates, collected between September 2006 and February 2007 in 23 hospitals located throughout France (Fig. 1). The 111 isolates were tested for their antibiotic susceptibility patterns and were extensively characterized by screening for drug resistance and agr alleles, multilocus sequence typing (ST), staphylococcal cassette chromosome mec (SCCmec) typing, spa typing, and PCR profiling of 21 toxin genes. Clones were designated by their ST followed by their SCCmec type (I to VI). The Lyon clone ST8-IV or ST8-IVvariant (n = 77; 69.4%) was widely distributed. Four minor clones were also detected, namely, the “classical” Pediatric clone ST5-IV (n = 9; 8.1%), the “new” Pediatric clone ST5-VI (n = 8; 7.2%), the clone Geraldine ST5-Itruncated (n = 7; 6.3%), and the European clone ST80-IV (n = 4; 3.6%). The six other isolates were related to five rare clones. Relative to that of other European countries, the situation in France is marked by the predominance of a specific major clone and the worrying emergence of minor clones with enhanced virulence and new antibiotic susceptibility profiles.