How a PCR Sequencing Strategy Can Bring New Data to Improve the Diagnosis of Ethionamide Resistance.

Ethionamide (ETH) is a second-line antituberculosis drug. ETH resistance (ETH-R) is mainly related to the mutations of the monooxygenase-activating ETH (EthA), the ETH target (InhA), and the inhA promoter. Nonetheless, diagnosing ETH-R is still challenging. We assessed the strategy used for detecting ETH-R at the French National Reference Center for Mycobacteria in 497 MDR-TB isolates received from 2008 to 2016. The genotypic ETH's resistance detection was performed by sequencing ethA, ethR, the ethA-ethR intergenic region, and the inhA promoter in the 497 multidrug-resistant isolates, whereas the phenotypic ETH susceptibility testing (PST) was performed using the reference proportion method. Mutations were found in up to 76% of the 387 resistant isolates and in up to 28% of the 110 susceptible isolates. Our results do not support the role of ethR mutations in ETH resistance. Altogether, the positive predictive value of our genotypic strategy to diagnose ETH-R was improved when only considering the variants included in the WHO catalogue and in other databases, such as TB-Profiler. Therefore, our work will help to update the list of mutations that could be graded as being associated with resistance to improve ETH-R diagnosis.

Rational Choice of Antibiotics and Media for Mycobacterium avium Complex Drug Susceptibility Testing.

The Clinical and Laboratory Standards Institute recommends the use of Mueller Hinton (MH) medium to perform drug susceptibility testing (DST) of Mycobacterium avium complex (MAC) using the microdilution method. For MAC, there has been no study on the impact of media on the determination of minimum inhibitory concentrations (MICs) of antibiotics other than clarithromycin. This study aimed at determining the impact of two media used for DST of MAC and at augmenting the number of pertinent MICs for MAC species encountered in clinical practice. MICs of antibiotics used for the treatment of MAC infections were determined for 158 clinical MAC isolates (80 M. avium, 40 M. intracellulare, 35 M. chimaera, two M. yongonense and one M. timonense) in MH and 7H9 broths using the SLOMYCO SensititreTM system (TREK Diagnostic Systems, East Grinstead, United Kingdom). The modal MICs determined in both media were the same for linezolid, moxifloxacin, rifabutin and amikacin but not for clarithromycin, rifampin and ethambutol. The kappa test for MICs converted to susceptibility categories showed an excellent agreement for clarithromycin, a moderate agreement for linezolid and a weak agreement for moxifloxacin and amikacin. For amikacin, 7H9 allowed a better distinction (fewer intermediate strains) of wild-type populations than MH. Existing breakpoints for linezolid and moxifloxacin are spread through the distribution of MICs for wild-type populations. The only breakpoints that can be used rationally are those for amikacin and clarithromycin. For amikacin, 7H9 performs better than MH, whereas both media perform equally for clarithromycin. Given that testing in 7H9, as opposed to MH, allows easier MIC measurements and yields greater reproducibility, we propose the use of 7H9 medium for DST of MAC.

Erwinia billingiae as Unusual Cause of Septic Arthritis, France, 2017.

In 2017 in France, we treated a patient with knee septic arthritis caused by Erwinia billingiae after trauma involving a palm tree. This rare pathogen could only be identified through 16S rRNA gene sequencing. For bacterial infections after injuries with plants, 16S rRNA gene sequencing might be required for species identification.

Poor Performance of Rapid Molecular Tests to Define Eligibility for the Shortcourse Multidrug-resistant Tuberculosis Regimen.

The World Health Organization recommends shortcourse regimen (SCR) to treat multidrug resistant tuberculosis for patients with strains susceptible by line-probe assays (LPAs) to second-line drugs. Our retrospective study shows LPAs have suboptimal specificity in predicting eligibility for SCR; a quarter of eligible patients would receive inadequate therapy with SCR.

Unexpected Genomic and Phenotypic Diversity of Mycobacterium africanum Lineage 5 Affects Drug Resistance, Protein Secretion, and Immunogenicity.

Mycobacterium africanum consists of Lineages L5 and L6 of the Mycobacterium tuberculosis complex (MTBC) and causes human tuberculosis in specific regions of Western Africa, but is generally not transmitted in other parts of the world. Since M. africanum is evolutionarily closely placed between the globally dispersed Mycobacterium tuberculosis and animal-adapted MTBC-members, these lineages provide valuable insight into M. tuberculosis evolution. Here, we have collected 15 M. africanum L5 strains isolated in France over 4 decades. Illumina sequencing and phylogenomic analysis revealed a previously underappreciated diversity within L5, which consists of distinct sublineages. L5 strains caused relatively high levels of extrapulmonary tuberculosis and included multi- and extensively drug-resistant isolates, especially in the newly defined sublineage L5.2. The specific L5 sublineages also exhibit distinct phenotypic characteristics related to in vitro growth, protein secretion and in vivo immunogenicity. In particular, we identified a PE_PGRS and PPE-MPTR secretion defect specific for sublineage L5.2, which was independent of PPE38. Furthermore, L5 isolates were able to efficiently secrete and induce immune responses against ESX-1 substrates contrary to previous predictions. These phenotypes of Type VII protein secretion and immunogenicity provide valuable information to better link genome sequences to phenotypic traits and thereby understand the evolution of the MTBC.

Multiplexed Quantitation of Intraphagocyte Mycobacterium tuberculosis Secreted Protein Effectors.

The pathogenic potential of Mycobacterium tuberculosis largely depends on ESX secretion systems exporting members of the multigenic Esx, Esp, and PE/PPE protein families. To study the secretion and regulation patterns of these proteins while circumventing immune cross-reactions due to their extensive sequence homologies, we developed an approach that relies on the recognition of their MHC class II epitopes by highly discriminative T cell receptors (TCRs) of a panel of T cell hybridomas. The latter were engineered so that each expresses a unique fluorescent reporter linked to specific antigen recognition. The resulting polychromatic and multiplexed imaging assay enabled us to measure the secretion of mycobacterial effectors inside infected host cells. We applied this novel technology to a large panel of mutants, clinical isolates, and host-cell types to explore the host-mycobacteria interplay and its impact on the intracellular bacterial secretome, which also revealed the unexpected capacity of phagocytes from lung granuloma to present mycobacterial antigens via MHC class II.

Estimation of pyrazinamidase activity using a cell-free In vitro synthesis of pnca and its association with pyrazinamide susceptibility in Mycobacterium tuberculosis.

Background: The main mechanism of resistance to PZA in Mycobacterium tuberculosis relies on mutations on its pyrazinamidase/nicotinamidase. Recently, a rapid colorimetric test relying on the PCR-based in vitro-synthesized-PZase assay has been reported for PZase activity determination from clinical M. tuberculosis isolates but the assay has not been compared with other tests to evaluate PZA susceptibility in M. tuberculosis isolates. Methods: In this study, we have used the PCR-based in vitro-synthesized-PZase assay to analyze the specific pyrazinamidase (PZase) activity of PncA mutants and have correlated the results to the PZA susceptibility phenotype determined by culture in acidic agar medium at pH 6.0. A set of 23 clinical isolates displaying mutated pncA genes (11 PZA-resistant and 12 PZA-susceptible) and 55 PZA-susceptible clinical strains displaying a wild-type pncA gene were tested. Results: Among the 23 mutants tested, 4 corresponded to mutations not reported before (I5T, Y99S, T142R and P77L+V131G). Of the 11 PncA mutants expressed from PZA-resistant clinical isolates, 9 were expressed in vitro at yields > 50% relative to the wild type enzyme. Among them, 6 enzymes (T47P, H51P, H51R, H57D, L85R and T142R) showed no detectable activity, while the relative activities for the 3 others, V9A (27%), G97D (10%) and A146V (28%) were low compared to the wild-type PZase. The remaining two mutants, I5T and V9G, presented very low relative expression (5%) and relative activities values of 12 and 1%, respectively. Twelve mutants were expressed from PZA-susceptible isolates. Their expression was similar to the wild type enzyme and behaved as active pyrazinamidase with specific relative activities ranging from 34 to 314%. Finally, discrepant results were observed for two mutants, V7A and P62T. Conclusion: Thus, this study provides the proof of concept that the PCR-based in vitro-synthesized-PZase assay represents a promising rapid approach for the evaluation of PZA susceptibility based on the estimation of the relative PZase activity from clinical isolates.

Encephalitis in a traveller with typhoid fever: efficacy of corticosteroids.

Typhoid fever is a bacterial infection caused by Salmonella typhi or S. paratyphi, recognized as a classical cause of fever in returning travellers. However, neuropsychiatric presentations are rarely reported in travellers diagnosed in western countries, whereas they are more commonly described in patients treated in endemic areas. We describe such a case and discuss the pathophysiologic mechanisms of this complication.

Severe glandular tularemia in a patient treated with anti-tumour necrosis factor for psoriatic arthritis.

A case of severe glandular tularemia in a patient receiving anti-tumour necrosis factor (TNF) therapy is reported here. The patient required prolonged treatment with doxycycline-ciprofloxacin due to early relapse after ciprofloxacin was stopped. Tularemia may have a more severe course in patients receiving anti-TNF. This may thus be an indication for more aggressive treatment.

Molecular Investigation of Resistance to Second-Line Injectable Drugs in Multidrug-Resistant Clinical Isolates of Mycobacterium tuberculosis in France.

The second-line injectable drugs (SLID, i.e., amikacin, kanamycin, capreomycin) are key drugs for the treatment of multidrug-resistant tuberculosis. Mutations in rrs region 1400, tlyA, and eis promoter are associated with resistance to SLID, to capreomycin, and to kanamycin, respectively. In this study, the sequencing data of SLID resistance-associated genes were compared to the results of phenotypic drug susceptibility testing by the proportion method for the SLID in 206 multidrug-resistant clinical isolates of Mycobacterium tuberculosis collected in France. Among the 153 isolates susceptible to the 3 SLID, 145 showed no mutation, 1 harbored T1404C and G1473A mutations in rrs, and 7 had an eis promoter mutation. Among the 53 strains resistant to at least 1 of the SLID, mutations in rrs accounted for resistance to amikacin, capreomycin, and kanamycin for 81%, 75%, and 44% of the isolates, respectively, while mutations in eis promoter were detected in 44% of the isolates resistant to kanamycin. In contrast, no mutations in tlyA were observed in the isolates resistant to capreomycin. The discrepancies observed between the genotypic (on the primary culture) and phenotypic drug susceptibility testing were explained by (i) resistance to SLID with MICs close to the critical concentration used for routine DST and not detected by phenotypic testing (n = 8, 15% of SLID-resistant strains), (ii) low-frequency heteroresistance not detected by sequencing of drug resistance-associated genes on the primary culture (n = 8, 15% of SLID-resistant strains), and (iii) other resistance mechanisms not yet characterized (n = 7, 13% of SLID-resistant strains).

In vivo Mycobacterium tuberculosis fluoroquinolone resistance emergence: a complex phenomenon poorly detected by current diagnostic tests.

OBJECTIVES: Heteroresistance, described both in terms of various point mutations resulting in different levels of resistance and in terms of a mixture of mutant and WT bacilli, is identified in up to one-third of fluoroquinolone (FQ)-resistant Mycobacterium tuberculosis isolates. Heteroresistance is a challenge for current phenotypic and genotypic susceptibility testing (DST) regimes. We aimed to compare the performances of different phenotypic and genotypic DST in the context of FQ heteroresistance by mimicking, in a murine model, the course of selection of FQ resistance during treatment. METHODS: The capacity of different phenotypic DST [Lowenstein-Jensen (LJ) medium containing either 2 mg/L ofloxacin or 0.5, 1 or 2 mg/L moxifloxacin] and genotypic DST (gyrA/B Sanger sequencing) to detect FQ resistance was analysed. RESULTS: Ninety-seven percent of mice harboured a heterogeneous population. The proportion of mice in which FQ resistance was detected varied according to the medium used (97% for 0.5 mg/L moxifloxacin, 80% for 2 mg/L ofloxacin, 47% for 1 mg/L moxifloxacin and 25% for 2 mg/L moxifloxacin). Compared with phenotypic DST, genotypic DST had a low sensitivity for detection of resistance (33%). CONCLUSIONS: Our study shows the in vivo complexity of FQ resistance emergence and the poor sensitivity of Sanger DNA sequencing for detection of heteroresistance. Our data support the use of 0.5 mg/L moxifloxacin in LJ for detection of FQ resistance, but not the recent increase in the ofloxacin critical concentration from 2 to 4 mg/L given in the WHO recommendations.

Comparative study of enzymatic activities of new KatG mutants from low- and high-level isoniazid-resistant clinical isolates of Mycobacterium tuberculosis.

Resistance to isoniazid (INH-R) in Mycobacterium tuberculosis is mainly due to mutations at position 315 (S315T) of the catalase-peroxidase KatG. We identified 16 mutations (including 13 biochemically uncharacterized mutations) in KatG from INH-R clinical isolates of M. tuberculosis showing mutations other than S315T. The KatG enzymatic activities (catalase, peroxidase, free radical production and isonicotinoyl-NAD formation) of wild-type KatG and the 16 mutants were determined and correlated to their spatial location in a KatG model structure. Of all mutations studied, H270R, which conferred a high level of INH-R and results in the disruption of a coordination bond with the heme, caused complete loss of all enzymatic KatG activities. The mutants generally associated with a very high level of INH-R were all characterized by a drastic reduction in catalase activity and a marked decrease in INH activation activities. One mutant, A162E, displayed a behavior similar to S315T, i.e. a moderate decrease in catalase activity and a drastic decrease in the formation of the radical form of INH. Finally, the mutants associated with a low level of INH-R showed a moderate reduction in the four catalytic activities, likely stemming from an overall alteration of the folding and/or stability of the KatG protein.

Standardized interpretation of antibiotic susceptibility testing and resistance genotyping for Mycobacterium abscessus with regard to subspecies and erm41 sequevar.

OBJECTIVES: The objective of this study was to provide standardized antibiotic susceptibility testing (AST) for Mycobacterium abscessus with regard to subspecies. METHODS: One hundred and sixty-five clinical isolates were tested for susceptibility to 15 antibiotics using a commercial microdilution method, at two reading times: (i) early reading time (ERT), when the growth control was first positive; and (ii) late reading time (LRT), of 14 days, for detecting inducible resistance. In addition, genes or mutations involved in resistance were studied [erm(41), rrl and rrs]. RESULTS: Three patterns were observed for clarithromycin: (i) MIC >16 mg/L at ERT (median 5 days) for 15 isolates [10 subsp. abscessus erm(41) sequevar T28, 3 subsp. bolletii and 2 subsp. massiliense] among which 9 harboured an a2058g/c rrl mutation; (ii) MIC ≤16 mg/L at ERT, but >16 mg/L at LRT, for 106 isolates [84 abscessus erm(41) T28 and 22 bolletii] showing intrinsic inducible resistance; and (iii) MIC ≤4 mg/L at ERT and LRT for 44 isolates [18 abscessus erm(41) C28 and 26 massiliense]. Amikacin MIC was >64 mg/L for eight isolates [five abscessus erm(41) T28, two massiliense and one bolletii] among which seven harboured the a1408g rrs mutation, but ≤64 mg/L for the remaining isolates without mutation. For the other antibiotics, only one WT pattern was observed, with cefoxitin, tigecycline and linezolid showing MIC values compatible with susceptibility. CONCLUSIONS: Standard AST can predict clarithromycin and amikacin resistance using interpretation rules with regard to subspecies. For other antibiotics, since only one pattern is observed, there is no need for systematic phenotypic or genotypic testing.

Performance of the New Version (v2.0) of the GenoType MTBDRsl Test for Detection of Resistance to Second-Line Drugs in Multidrug-Resistant Mycobacterium tuberculosis Complex Strains.

Detecting resistance to fluoroquinolones (FQ) and second-line injectable drugs (amikacin [AMK], kanamycin [KAN], and capreomycin [CAP]) is crucial given the worldwide increase in the incidence of extensively drug-resistant tuberculosis (XDR-TB). A new version of the GenoType MTBDRsl test (v2.0) has been developed to improve the detection of resistance to FQ (involving gyrA and gyrB mutations) and to second-line injectable drugs (involving rrs and eis promoter mutations) in Mycobacterium tuberculosis A collection of 127 multidrug-resistant (MDR) M. tuberculosis complex strains was tested using the first (v1) and second (v2.0) versions of the MTBDRsl test, as well as DNA sequencing. The specificities in resistance detection of v1 and v2.0 were similar throughout, whereas the levels of sensitivity of v2.0 were superior for FQ (94.8% versus 89.6%) and KAN (90.5% versus 59.5%) but similar for AMK (91.3%) and CAP (83.0%). The sensitivity and specificity of v2.0 were superior to those of v1 for the detection of pre-XDR strains (83.3% versus 75.0% and 88.6% versus 67.1%, respectively), whereas the sensitivity of v2.0 was superior to that of v1 only for the detection of XDR strains (83.0% versus 49.1%). In conclusion, MTBDRsl v2.0 is superior to MTBDRsl v1 and efficiently detects the most common mutations involved in resistance to FQ and aminoglycosides/CAP. However, due to mutations not recognized by v2.0 or to the presence of resistance mechanisms not yet characterized (particularly mechanisms related to monoresistance to aminoglycosides or CAP), the results for wild-type strains obtained with MTBDRsl v2.0 should be confirmed by further DNA sequencing and phenotypic drug susceptibility testing.

Multidrug-resistant bacteria transmitted through high-density EEG in ICU.

PURPOSE: Prevention of multidrug resistant (MDR) bacterial contamination remains a major challenge in ICUs. Many hospital outbreaks involving MDR transmitted through environmental contamination have been reported. Bedside high-density EEG allow for dynamic cognitive evaluation in brain-injured patients and is used more and more frequently in clinical practice to evaluate brain function and predict outcome in severely neurologically impaired patients. Unfortunately, the material used for this procedure is not entirely disposable. METHOD: We performed a systematic analysis of MDR bacterial contamination in patients contaminated in our ICU using specific bacteriological methods. RESULTS: We report a proven case of cross-contamination of an extended-spectrum beta-lactamase (ESBL)-producing Klebsiella pneumoniae strain, and a possible case of cross-contamination of a carbapenem-resistant Acinetobacter baumannii strain. CONCLUSION: Cross-contamination of MDR bacteria is possible through high-density EEG material. However, appropriate procedures can decrease this risk.

Could the DiversiLab® semi-automated repetitive-sequence-based PCR be an acceptable technique for typing isolates of Pseudomonas aeruginosa? An answer from our experience and a review of the literature.

Recently the DiversiLab® (DL) system (bioMérieux) was developed as an automated platform that uses repetitive element polymerase chain reaction (rep-PCR) technology for standardized, reproducible DNA fingerprinting of bacteria. The purpose of this study was to evaluate the usefulness of DL rep-PCR for typing Pseudomonas aeruginosa isolates. The performance of DL rep-PCR was compared with that of pulsed-field gel electrophoresis (PFGE) in a prospective multicenter study of patients with ventilator-associated pneumonia due to P. aeruginosa, conducted in 3 intensive care units over a 31-month period. In total, 203 P. aeruginosa isolates from 66 patients, from whom at least 2 consecutive respiratory samples each were collected more than 48 h apart, were typed using DL rep-PCR. Forty isolates (corresponding to 20 patients) were also typed using PFGE of SpeI-digested DNA. The typeability was 100% with DL rep-PCR and 95% with PFGE. The discriminatory power was close for DL rep-PCR and for PFGE (Simpson's diversity indices of 0.901 and 0.947, respectively). Insufficient agreement between DL rep-PCR and PFGE typing results was observed for the 40 selected isolates (adjusted Rand coefficient of 0.419), mostly due to isolates of the same DL rep-PCR type but of different PFGE types (adjusted Wallace coefficients of 0.306 for DL rep-PCR with PFGE, and of 0.667 for PFGE with DL rep-PCR). Considered together with published data, DL rep-PCR results should be interpreted with caution for the investigation of outbreaks caused by P. aeruginosa and evaluated in conjunction with epidemiological data.

New Insights into the Geographic Distribution of Mycobacterium leprae SNP Genotypes Determined for Isolates from Leprosy Cases Diagnosed in Metropolitan France and French Territories.

BACKGROUND: Between 20 and 30 bacteriologically confirmed cases of leprosy are diagnosed each year at the French National Reference Center for mycobacteria. Patients are mainly immigrants from various endemic countries or living in French overseas territories. We aimed at expanding data regarding the geographical distribution of the SNP genotypes of the M. leprae isolates from these patients. METHODOLOGY/PRINCIPAL FINDINGS: Skin biopsies were obtained from 71 leprosy patients diagnosed between January 2009 and December 2013. Data regarding age, sex and place of birth and residence were also collected. Diagnosis of leprosy was confirmed by microscopic detection of acid-fast bacilli and/or amplification by PCR of the M. leprae-specific RLEP region. Single nucleotide polymorphisms (SNP), present in the M. leprae genome at positions 14 676, 1 642 875 and 2 935 685, were determined with an efficiency of 94% (67/71). Almost all patients were from countries other than France where leprosy is still prevalent (n = 31) or from French overseas territories (n = 36) where leprosy is not totally eradicated, while only a minority (n = 4) was born in metropolitan France but have lived in other countries. SNP type 1 was predominant (n = 33), followed by type 3 (n = 17), type 4 (n = 11) and type 2 (n = 6). SNP types were concordant with those previously reported as prevalent in the patients' countries of birth. SNP types found in patients born in countries other than France (Comoros, Haiti, Benin, Congo, Sri Lanka) and French overseas territories (French Polynesia, Mayotte and La Réunion) not covered by previous work correlated well with geographical location and history of human settlements. CONCLUSIONS/SIGNIFICANCE: The phylogenic analysis of M. leprae strains isolated in France strongly suggests that French leprosy cases are caused by SNP types that are (a) concordant with the geographic origin or residence of the patients (non-French countries, French overseas territories, metropolitan France) or (b) more likely random in regions where diverse migration flows occurred.