Staphylococcus aureus bacteremia is one of the most frequent severe bacterial infections worldwide, with an associated mortality of about 20–40% in developed countries. In 2013, we noted an increase in this infection in the teaching hospital in Grenoble, France, compared to 2012. The mean incidence of S. aureus bacteremia was 0.28 per 1,000 patient-days in 2012 and 0.35 per 1,000 patient-days in 2013. This trend was confirmed in 2014 (0.35 per 1,000 patient-days). In the present work we aimed to study the population of patients presenting with S. aureus bacteremia in 2013 and to genotype the corresponding S. aureus strains in order to identify a successful and/or virulent genotype to design a specific infection control program. One hundred ninety-one S. aureus isolates (including 9 methicillin-resistant) out of 199 corresponding cases of bacteremia were characterized with the spa typing method. Among 108 spa types, t571, t002, t008 and t084 were the most prevalent. Although not widely prevalent, t571 was the most frequently identified clone (8.4% of all isolates). Spa type t571 has been described in previous studies as belonging to the clonal complex CC398, which is consistent with the recent emergence of methicillin-susceptible S. aureus CC398 reported in blood cultures in Europe.

The MDR/MTB ELITe MGB^® kit (ELITech) carried on the ELITe InGenius^® platform is a new real-time PCR assay allowing automated extraction and detection of DNA of the Mycobacterium tuberculosis complex (MTB) and mutations in the rpoB and katG genes and inhA promoter region (pro-inhA) associated to resistance to rifampicin and isoniazid, the two markers of multidrug-resistant TB (MDR). We assessed the performances of the test on a collection of strains (n = 54) and a set of clinical samples (n = 242) from routine practice, comparatively to TB diagnosis and genotypic drug susceptibility testing (gDST) as references. Regarding the 242 clinical samples, the sensitivity and specificity of MTB detection by ELITe were 90.9% and 97.5%, respectively. For the detection of resistance-conferring mutations on positive clinical samples, we observed perfect agreement with gDST for katG and pro-inhA (κ = 1.0) and two discordant results for rpoB (κ = 0.82). Considering the 54 cultured strains, very good agreement with gDST was observed for the detection of the 25 distinct mutations in rpoB, katG, and pro-inhA, (κ = 0.95, 0.88, and 0.95, respectively). In conclusion, the automated MDR/MTB ELITe MGB^® assay shows great promise and appears to be a valuable tool for rapid detection of pre-MDR- and MDR-TB directly from clinical specimens.

Pulmonary tuberculosis (TB) is the most frequent site of TB and the one leading its spread worldwide. Multiple specimens are commonly collected for TB diagnosis including those requiring invasive procedures. This study aimed to review the sampling strategy for the microbiological diagnosis of pulmonary TB. A retrospective analysis of collected samples from September 1st 2014 to May 1st 2016 in the Bacteriology laboratory of Pitié-Salpêtrière Hospital (Paris, France) was performed. All the samples collected in patients aged over 18 years for the bacteriological diagnosis of pulmonary TB were included. A total of 6267 samples were collected in 2187 patients. One hundred and twenty-six patients (6%) had a culture confirmed pulmonary TB. Among them, multiple sputum collections were sufficient for TB diagnosis in 63.5%, gastric lavages permitted to avoid bronchoscopy in only 7.1%, and bronchoscopy was necessary in 29.4%. The culture positivity of sputa (8.6%) was higher than that of bronchial aspirations (3.1%), bronchiolo-alveolar lavages (BAL) (2.3%) or gastric lavages (4.8%) (P<0.001). From its 70.0% theoretical PPV value, the 46.1% selection in bronchial aspirations allocated to molecular test increased PPV up to 88.9%. Based on our data, we suggest to collect sputum consistently. If smear negative a bronchoscopy should be performed and molecular diagnosis be performed on a subset of bronchial aspirations based on expertise of the bronchoscopist.

The optimal treatment for osteoarticular infection due to multidrug-resistant tuberculosis strains (MDR-OATB) remains unclear. This study aims to evaluate the diagnosis, management and outcome of MDR-OATB in France. We present a case series of MDR-OATB patients reviewed at the French National Reference Center for Mycobacteria between 2007 and 2018. Medical history and clinical, microbiological, treatment and outcome data were collected. Twenty-three MDR-OATB cases were reported, representing 3% of all concurrent MDR-TB cases in France. Overall, 17 were male, and the median age was 32 years. Six patients were previously treated for TB, including four with first-line drugs. The most frequently affected site was the spine (n = 16). Bone and joint surgery were required in 12 patients. Twenty-one patients (91%) successfully completed the treatment with a regimen containing a mean of four drugs (range, 2–6) for a mean duration of 20 months (range, 13–27). Overall, high rates of treatment success were achieved following WHO MDR-TB treatment guidelines and individualized patient management recommendations by the French National TB Consilium. However, the optimal combination of drugs, duration of treatment and role of surgery in the management of MDR-OATB remains to be determined.

Multidrug-resistant (MDR) strains of Mycobacterium tuberculosis represent an obstacle to eradicating tuberculosis (TB) due to the low treatment success rate of MDR TB. Among them, the MDR B0/W148 clone has recently evolved from the M. tuberculosis Beijing lineage 2 and is widely disseminated in Russia and Europe. To get more insights into the genetic factors underlying the evolutionary success of the MDR M. tuberculosis B0/W148 clone in addition to environmental and patient-related features, we focused on two mutations specific to this clone that are found in the transcriptional regulators WhiB6 and KdpDE and investigated in a H37Rv strain background the transcriptional profile associated with these mutations and their impact on the in vitro and in vivo growth characteristics. Through the construction and use of H37Rv∆ whiB6 , H37Rv∆ kdpDE , and complemented strains, neither mutation impaired the in vitro growth of M. tuberculosis in standard mycobacterial growth media. The mutation T51P in whiB6 prevented the upregulation of 9 genes in the esx-1 core region and 44 genes elsewhere in the genome, while the deletion of two nucleotides in kdpD leads to a fusion protein of KdpD with KdpE that inhibits the transcriptional activity of KdpE. Neither mutation led to hypervirulence in a mouse infection model. These results point to the role of other MDR B0/W148 specific mutations in the wide geographic diffusion of this clone and/or put in question a hypothesized hypervirulence as a driving factor for this large dissemination. IMPORTANCE: Human tuberculosis (TB), caused by the bacterium Mycobacterium tuberculosis , remains a global public health issue estimated to have been responsible for 1.25 million deaths in 2023. Multidrug-resistant (MDR) strains of M. tuberculosis , resistant to rifampicin and isoniazid, lead to lower treatment success. Among them, the MDR B0/W148 clone has widely disseminated in Russia and Europe. To get more insights into the genetic factors underlying the evolutionary success of this clone, we investigated two strain-specific mutations found in the transcriptional regulators WhiB6 and KdpDE. By constructing and analyzing laboratory M. tuberculosis strains carrying these specific mutations, we found numerous changes in their transcriptional profiles, whereas we observed only a little impact of these mutations on the virulence of M. tuberculosis in a mouse infection model. Our study provides new insights into the transcriptional landscape of the selected MDR strains, although no direct connection to virulence could be established.

The pyruvate dehydrogenase complex (PDHc) is a universally conserved multienzyme system that converts pyruvate into acetyl-CoA for entry into the tricarboxylic acid cycle and for NADH production. Its central scaffold, the dihydrolipoyl transacetylase (E2p), forms an oligomeric inner core that recruits pyruvate dehydrogenase (E1p) and dihydrolipoyl dehydrogenase (E3). All previously characterized PDHc assemblies adopt either an octahedral 24-mer or an icosahedral 60-mer E2p core, each constructed from trimeric building blocks. We recently showed that the Mycobacterium tuberculosis (Mtb) E2p protein DlaT also functions as the core of the pathogen's peroxynitrite reductase/peroxidase complex. Here, using cryo-EM, we demonstrate that DlaT assembles into discrete hexamers and dodecamers at micromolar concentrations, which approximate intracellular DlaT concentrations in Mtb. Structure-guided mutagenesis combined with in vitro activity assays indicates that the hexamer represents the functional E2p core of the Mtb PDHc. This noncanonical architecture arises from unique interfaces between DlaT trimers that preclude formation of the classic spherical 24- or 60-mer structures. We propose that this specialized E2p organization enables Mtb to regulate metabolic activities and to remodel the E2p core for engagement in the peroxynitrite reductase/peroxidase antioxidant pathway under stress. Our findings reveal an unexpected diversity in PDHc architecture and uncover a distinct organization principle for the core metabolic complex in mycobacteria.