ISAba1-dependent overexpression of eptA in clinical strains of Acinetobacter baumannii resistant to colistin.

BACKGROUND: Colistin resistance in Acinetobacter baumannii often results from mutational activation of the two-component system PmrAB and subsequent addition of phospho-ethanolamine (pEtN) to lipooligosaccharide by up-regulated pEtN transferase PmrC. OBJECTIVES: To characterize mechanisms of colistin resistance independent of PmrCAB in A. baumannii. METHODS: Twenty-seven colistin-resistant A. baumannii were collected from 2012 to 2018. Analysis of operon pmrCAB was performed by PCR and sequencing. Seven strains were investigated further by WGS and whole-genome MLST (wgMLST). RESULTS: Seven out of the 27 selected isolates were found to overexpress eptA, a gene homologous to pmrC, likely as a consequence of upstream insertion of an ISAba1 element. Insertion sites of ISAba1 were mapped 13, 18 and 156 bp ahead of the start codon of eptA in five strains, one strain and one strain, respectively. The finding that the isolates did not cluster together when compared by wgMLST analysis supports the notion that distinct insertion events occurred in close, but different, genetic backgrounds. CONCLUSIONS: Activation of eptA and subsequent addition of pEtN to the cell surface represents a novel mechanism of resistance to colistin in A. baumannii.

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).

Long-term outcome and safety of prolonged bedaquiline treatment for multidrug-resistant tuberculosis.

Bedaquiline, a recently approved drug for the treatment of multidrug-resistant tuberculosis (MDR-TB), is recommended for a duration of 24 weeks. There are scarce data on patients treated with this drug outside clinical trials.All MDR-TB patients who started treatment from January 1, 2011 to December 31, 2013 and received ≥30 days of bedaquiline were included in a multicentre observational cohort.Among 45 MDR-TB patients, 53% harboured isolates resistant to both fluoroquinolones and second-line injectables, and 38% harboured isolates resistant to one of these drug classes. Median bedaquiline treatment duration was 361 days and 33 patients (73%) received prolonged (>190 days) bedaquiline treatment. Overall, 36 patients (80%) had favourable outcome, five were lost to follow-up, three died, and one failed and acquired bedaquiline resistance. No cases of recurrence were reported. Severe and serious adverse events were recorded in 60% and 18% of patients, respectively. Values of Fridericia-corrected QT interval (QTcF) >500 ms were recorded in 11% of patients, but neither arrhythmias nor symptomatic cardiac side-effects occurred. Bedaquiline was discontinued in three patients following QTcF prolongation. No significant differences in outcomes or adverse events rates were observed between patients receiving standard and prolonged bedaquiline treatment.Bedaquiline-containing regimens achieved favourable outcomes in a large proportion of patients. Prolonged bedaquiline treatment was overall well tolerated in this cohort.

Evaluation of the new GenoType NTM-DR kit for the molecular detection of antimicrobial resistance in non-tuberculous mycobacteria.

Objectives: Non-tuberculous mycobacteria (NTM) are emerging pathogens causing difficult-to-treat infections. We tested a new assay (GenoType NTM-DR) that detects natural and acquired resistance mechanisms to macrolides and aminoglycosides in frequently isolated NTM species. Methods: Performance was assessed on 102 isolates including reference strains [16 Mycobacterium avium , 10 Mycobacterium intracellulare , 8 Mycobacterium chimaera , 15 Mycobacterium chelonae and 53 Mycobacterium abscessus (including subsp. abscessus isolates, 18 with a t28 in erm(41) and 10 with a c28, 13 subsp. bolletii isolates and 12 subsp. massiliense isolates)]. Genotypes were determined by PCR sequencing of erm(41) and rrl for clarithromycin resistance and of the 1400-1480 rrs region for aminoglycoside resistance. Phenotypes were determined by MIC microdilution. Results: GenoType NTM-DR yielded results concordant with Sanger sequencing for 100/102 (98%) isolates. The erm(41) genotypic pattern was accurately identified for M. abscessus isolates . Mutations in rrl were detected in 15 isolates (7 M. avium complex, 5 M. abscessus and 3 M. chelonae ) with acquired clarithromycin resistance harbouring rrl mutations (a2057c, a2058g, a2058t or a2059c). Mutations in rrs were detected in five isolates with amikacin resistance harbouring the rrs mutation a1408g. In two isolates, the NTM-DR test revealed an rrl mutation (initial sequencing being WT), which was confirmed by re-sequencing. The test results were concordant with phenotypic susceptibility testing in 96/102 (94.1%) isolates, with four clarithromycin-resistant and two amikacin-resistant isolates not harbouring mutations. Conclusions: The GenoType NTM-DR test is efficient in detecting mutations predictive of antimicrobial resistance in M. avium complex, M. abscessus and M. chelonae.

Are moxifloxacin and levofloxacin equally effective to treat XDR tuberculosis?

Background: Moxifloxacin retains partial activity against some fluoroquinolone-resistant mutants of Mycobacterium tuberculosis . Levofloxacin is presumed to be as active as moxifloxacin against drug-susceptible tuberculosis and to have a better safety profile. Objectives: To compare the in vivo activity of levofloxacin and moxifloxacin against M. tuberculosis strains with various levels of fluoroquinolone resistance. Methods: BALB/c mice were intravenously infected with 10 6 M. tuberculosis H37Rv and three isogenic mutants: GyrA A90V, GyrB E540A and GyrB A543V. Treatment with 50 or 100 mg/kg levofloxacin and 60 or 66 mg/kg moxifloxacin was given orally every 6 h, for 4 weeks. Results: Levofloxacin 50 and 100 mg/kg q6h and moxifloxacin 60 and 66 mg/kg q6h generated AUCs in mice equivalent to those of levofloxacin 750 and 1000 mg/day and moxifloxacin 400 and 800 mg/day, respectively, in humans. Moxifloxacin 60 and 66 mg/kg q6h had bactericidal activity against strain H37Rv (MIC ≤ 0.25 mg/L) and mutants GyrB E540A and GyrB A543V (MIC = 0.5 mg/L). Against mutant GyrA A90V (MIC = 2 mg/L), moxifloxacin 60 mg/kg q6h did not prevent bacillary growth, whereas 66 mg/kg q6h had bacteriostatic activity. Levofloxacin 50 mg/kg q6h had bactericidal activity against H37Rv (MIC ≤ 0.25 mg/L) but not against the mutant strains. Levofloxacin 100 mg/kg q6h had bactericidal activity against H37Rv and mutants GyrB E540A (MIC = 0.5 mg/L) and GyrB A543V (MIC= 1 mg/L) but not against mutant GyrA A90V (MIC = 4 mg/L). Conclusions: All mutations reduced fluoroquinolone activity, even those classified as susceptible according to phenotypic tests. High-dose levofloxacin is less effective than high-dose moxifloxacin against both fluoroquinolone-resistant and -susceptible M. tuberculosis strains in mice.

Preliminary Favorable Outcome for Medically and Surgically Managed Extensively Drug-Resistant Tuberculosis, France, 2009-2014.

We report 20 cases of extensively drug-resistant tuberculosis managed in France. Treatment was individualized and included bedaquiline and linezolid for most patients and surgery in 8 patients. At last follow-up (22 months), 19 patients had achieved conversion from positive to negative on culture testing. These promising results of comprehensive management obtained in a small series deserve confirmation.

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.

Molecular diagnosis of fluoroquinolone resistance in Mycobacterium tuberculosis.

As a consequence of the use of fluoroquinolones (FQ), resistance to FQ has emerged, leading to cases of nearly untreatable and extensively drug-resistant tuberculosis. Mutations in DNA gyrase represent the main mechanism of FQ resistance. A full understanding of the pattern of mutations found in FQ-resistant (FQ(r)) clinical isolates, and of their proportions, is crucial for improving molecular methods for the detection of FQ resistance in Mycobacterium tuberculosis. In this study, we reviewed the detection of FQ resistance in isolates addressed to the French National Reference Center for Mycobacteria from 2007 to 2012, with the aim of evaluating the performance of PCR sequencing in a real-life context. gyrA and gyrB sequencing, performed prospectively on M. tuberculosis clinical isolates, was compared for FQ susceptibility to 2 mg/liter ofloxacin by the reference proportion method. A total of 605 isolates, of which 50% were multidrug resistant, were analyzed. The increase in FQ(r) strains among multidrug-resistant (MDR) strains during the time of the study was alarming (8% to 30%). The majority (78%) of the isolates with gyrA mutations were FQ(r), whereas only 36% of those with gyrB mutations were FQ(r). Only 12% of the FQ(r) isolates had a single mutation in gyrB. Combined gyrA and gyrB sequencing led to >93% sensitivity for detecting resistance. The analysis of the four false-positive and the five false-negative results of gyrA and gyrB sequencing illustrated the actual limitations of the reference proportion method. Our data emphasize the need for combined gyrA and gyrB sequencing in the investigation of FQ susceptibility in M. tuberculosis and challenge the validity of the current phenotype-based approach as the diagnostic gold standard for determining FQ resistance.

Molecular Analysis of the embCAB Locus and embR Gene Involved in Ethambutol Resistance in Clinical Isolates of Mycobacterium tuberculosis in France.

Modification of codon 306 in embB is regarded as the main mechanism leading to ethambutol (ETB) resistance in clinical isolates of Mycobacterium tuberculosis. However, numerous mutations elsewhere in the embCAB locus and in embR, a putative transcriptional activator of this locus, have been reported to be involved in ETB resistance. Here, we investigated the diversity of nucleotide variations observed in embCAB and embR in M. tuberculosis complex isolates from France. These regions were sequenced in 71 ETB-resistant (ETB-R) and 60 ETB-susceptible (ETB-S) clinical isolates of known phylogenetic lineages. The 131 isolates had 12 mutations corresponding to phylogenetic markers. Among the 60 ETB-S isolates, only 3 (5%) had nonsynonymous mutations that were not phylogenetic markers. Among the 71 ETB-R isolates, 98% had mutations in embCAB that likely contribute to ETB resistance: 70% had mutations located in embB codon 306, 406, or 497; 13% had mutations located outside these three positions between codons 296 and 426; and 15% had mutations corresponding to mutations in the embC-embA intergenic region. We found a strong association between resistance to ETB and the presence of mutations in embB and the embC-embA intergenic region (P < 0.001). In contrast, the mutations detected in embC and embA were not involved in ETB resistance, and no mutation was detected in embR. These results strongly suggest that the sensitivity of diagnostic assays for detecting ETB resistance based on testing of embB codon 306 can be increased by testing of the embB region between codons 296 and 497 and by including the embC-embA intergenic region between positions -8 and -21.

Cerebral microcirculation shear stress levels determine Neisseria meningitidis attachment sites along the blood-brain barrier.

Neisseria meningitidis is a commensal bacterium of the human nasopharynx. Occasionally, this bacterium reaches the bloodstream and causes meningitis after crossing the blood-brain barrier by an unknown mechanism. An immunohistological study of a meningococcal sepsis case revealed that neisserial adhesion was restricted to capillaries located in low blood flow regions in the infected organs. This study led to the hypothesis that drag forces encountered by the meningococcus in the bloodstream determine its attachment site in vessels. We therefore investigated the ability of N. meningitidis to bind to endothelial cells in the presence of liquid flow mimicking the bloodstream with a laminar flow chamber. Strikingly, average blood flows reported for various organs strongly inhibited initial adhesion. As cerebral microcirculation is known to be highly heterogeneous, cerebral blood velocity was investigated at the level of individual vessels using intravital imaging of rat brain. In agreement with the histological study, shear stress levels compatible with meningococcal adhesion were only observed in capillaries, which exhibited transient reductions in flow. The flow chamber assay revealed that, after initial attachment, bacteria resisted high blood velocities and even multiplied, forming microcolonies resembling those observed in the septicemia case. These results argue that the combined mechanical properties of neisserial adhesion and blood microcirculation target meningococci to transiently underperfused cerebral capillaries and thus determine disease development.

Portal sensing of intestinal gluconeogenesis is a mechanistic link in the diminution of food intake induced by diet protein.

Protein feeding is known to decrease hunger and subsequent food intake in animals and humans. It has also been suggested that glucose appearance into portal vein, as occurring during meal assimilation, may induce comparable effects. Here, we connect these previous observations by reporting that intestinal gluconeogenesis (i.e., de novo synthesis of glucose) is induced during the postabsorptive time (following food digestion) in rats specifically fed on protein-enriched diet. This results in glucose release into portal blood, counterbalancing the lowering of glycemia resulting from intestinal glucose utilization. Comparable infusions into the portal vein of control postabsorptive rats (fed on starch-enriched diet) decrease food consumption and activate the hypothalamic nuclei regulating food intake. Similar hypothalamic activation occurs on protein feeding. All these effects are absent after denervation of the portal vein. Thus, portal sensing of intestinal gluconeogenesis may be a novel mechanism connecting the macronutrient composition of diet to food intake.

Assessing pain in critically ill brain-injured patients: a psychometric comparison of 3 pain scales and videopupillometry.

Three clinical scales (the Nociception Coma Scale adapted for Intubated patients [NCS-I], its Revised version [NCS-R-I], and the Behavioral Pain Scale [BPS]) and videopupillometry were compared for measuring pain in intubated, noncommunicating, critically ill, brain-injured patients. Pain assessment was performed before, during, just after, and 5 minutes after 3 procedures: the reference non-nociceptive procedure (assessment of the Richmond Agitation Sedation Scale) and 2 nociceptive procedures (turning and tracheal suctioning). The primary endpoint was construct validity (discriminant and criterion validation), determined by comparing pain measurements between different times/procedures. Secondary endpoints were internal consistency, inter-rater reliability, and feasibility. Fifty patients (54% women, median age 63 years [56-68]) were included 13 [7-24] days after brain injury (76% hemorrhagic or ischemic strokes). All tools increased significantly more (P < 0.001) during the nociceptive procedures vs the non-nociceptive procedure. The BPS was the only pain tool that did not increase significantly during the non-nociceptive procedure (P = 0.41), suggesting that it was the most discriminant tool. The BPS, NCS-I, and NCS-R-I were good predictors of nociception with areas under the curves ≥0.96, contrary to videopupillometry (area under the curve = 0.67). The BPS, NCS-I, and NCS-I-R had high inter-rater reliabilities (weighted kappa = 0.86, 0.82 and 0.84, respectively). Internal consistency was moderate (>0.60) for all pain scales. Factor analysis represented a majority of information on a first dimension, with motor domains represented on a second dimension. Scale feasibility was better for the NCS-I and NCS-R-I than for the BPS. In conclusion, the BPS, NCS-I, and NCS-R-I are valid, reliable, and acceptable pain scales for use in intubated critically ill, brain-injured patients, unlike videopupillometry. Future research requires tool design centered on domains of observation adapted to this very specific population.

Relation between menin expression and NF-kappaB activity in an intestinal cell line.

In a previous study, we demonstrated that the Men1 gene is mainly expressed in the proliferative crypt compartment of the small intestine and that a reduction of menin expression in the crypt-like IEC-17 cell line induces an increase in proliferation rate concomitant with an increase in cyclin D1 expression. The aim of the present study was to test the hypothesis that the NF-kappaB pathway may be involved in cyclin D1 overexpression. Transcriptional activity of the cyclin D1 gene promoter was increased upon reduction of menin expression. Blockade of the NF-kappaB pathway restored proliferation, cell cycle, cyclin D1 gene transcription and cyclin D1 expression levels to those observed in the presence of menin. These data support a correlation between cyclin D1 expression, NF-kappaB activity and menin expression in this epithelial cell line and are relevant to the physiological function of menin in regulating proliferation in the intestinal epithelium.

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.