Francisella tularensis is a Gram-negative, intracellular bacterium causing the zoonosis tularemia. This highly infectious microorganism is considered a potential biological threat agent. Humans are usually infected through direct contact with the animal reservoir and tick bites. However, tularemia cases also occur after contact with a contaminated hydro-telluric environment. Water-borne tularemia outbreaks and sporadic cases have occurred worldwide in the last decades, with specific clinical and epidemiological traits. These infections represent a major public health and military challenge. Human contaminations have occurred through consumption or use of F. tularensis-contaminated water, and various aquatic activities such as swimming, canyoning and fishing. In addition, in Sweden and Finland, mosquitoes are primary vectors of tularemia due to infection of mosquito larvae in contaminated aquatic environments. The mechanisms of F. tularensis survival in water may include the formation of biofilms, interactions with free-living amoebae, and the transition to a ‘viable but nonculturable' state, but the relative contribution of these possible mechanisms remains unknown. Many new aquatic species of Francisella have been characterized in recent years. F. tularensis likely shares with these species an ability of long-term survival in the aquatic environment, which has to be considered in terms of tularemia surveillance and control. GRAPHICAL ABSTRACT

The implementation of MALDI-TOF MS in medical microbiology laboratories has revolutionized practices and significantly reduced turnaround times of identification processes. However, although bacteriology quickly benefited from the contributions of this technique, adjustments were necessary to accommodate the specific characteristics of fungi. MALDI-TOF MS is now an indispensable tool in clinical mycology laboratories, both for the identification of yeasts and filamentous fungi, and other innovative uses are gradually emerging. Based on the practical experience of our medical mycology laboratory, this review will present the current uses of MALDI-TOF MS and the adaptations we implemented, to allow their practical execution in a daily routine. We will also introduce some less mainstream applications, like those for fungemia, or even still under development, as is the case for the determination of sensitivity to antifungal agents or typing methods.

To determine the in vitro susceptibility to 18 antibiotics of human strains of Francisella tularensis isolated in France between 2006 and 2016, to check the absence of acquired resistance and to evaluate potential therapeutic alternatives. Fifty-nine clinically unrelated F. tularensis subsp. holarctica strains identified at the French National Reference Centre for Francisella as belonging to the phylogenetic subclade B.FTNF002-00 were used. MICs were determined in CAMHB medium supplemented with 2% PolyViteX^®, using the CLSI broth microdilution method. All strains were susceptible to fluoroquinolones (ofloxacin, ciprofloxacin, levofloxacin and moxifloxacin; MIC range: 0.016–0.25 mg/L), aminoglycosides (gentamicin and tobramycin; MIC range: ≤0.03–0.25 mg/L), doxycycline (MIC range: 0.125–0.25 mg/L) and chloramphenicol (MIC range: 0.5–2 mg/L). The erythromycin MIC range (0.5–2 mg/L) confirmed that all isolates belonged to biovar I of F. tularensis subsp. holarctica. Azithromycin and telithromycin displayed lower MIC ranges (0.25–1 and 0.03–0.5 mg/L, respectively). The tigecycline MIC range (0.25–1 mg/L) was slightly higher than that of doxycycline. All strains were resistant to ampicillin, meropenem, daptomycin, clindamycin and linezolid. F. tularensis strains isolated in France remain susceptible to antibiotic classes recommended for tularaemia treatment. Because fluoroquinolones display the lowest MIC₉₀, have bactericidal activity and have lower therapeutic failure rates compared with doxycycline, they may be advocated as first-line treatment of mild cases of tularaemia, predominant in Europe. MIC data also indicate that azithromycin or telithromycin may be possible therapeutic options against biovar I strains from Western Europe in case of contraindication to first-line antibiotics.

We developed an in-house assay for the direct identification, by matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry, of yeasts in blood culture. Sixty-one representative strains from 12 species were analyzed in spiked blood cultures. Our assay accurately identified 95 of 107 (88.8%) positive blood cultures and outperformed the commercial Sepsityper kit (81.7% identification).

The emergence of fluoroquinolone (FQ)-resistant mutants of Legionella pneumophila in infected humans was previously reported using a next-generation DNA sequencing (NGS) approach. This finding could explain part of the therapeutic failures observed in legionellosis patients treated with these antibiotics. The aim of this study was to develop digital PCR (dPCR) assays allowing rapid and accurate detection and quantification of these resistant mutants in respiratory samples, especially when the proportion of mutants in a wild-type background is low. We designed three dPCRgyrA assays to detect and differentiate the wild-type and one of the three gyrA mutations previously described as associated with FQ resistance in L. pneumophila : at positions 248C→T (T83I), 259G→A (D87N), and 259G→C (D87H). To assess the performance of these assays, mixtures of FQ-resistant and -susceptible strains of L. pneumophila were analyzed, and the results were compared with those obtained with Sanger DNA sequencing and real-time quantitative PCR (qPCR) technologies. The dPCRgyrA assays were able to detect mutated gyrA sequences in the presence of wild-type sequences at up to 1:1,000 resistant/susceptible allele ratios. By comparison, Sanger DNA sequencing and qPCR were less sensitive, allowing the detection of gyrA mutants at up to 1:1 and 1:10 ratios, respectively. When testing 38 respiratory samples from 23 legionellosis patients (69.6% treated with an FQ), dPCRgyrA detected small amounts of gyrA mutants in four (10.5%) samples from three (13.0%) patients. These results demonstrate that dPCR is a highly sensitive alternative to quantify FQ resistance in L. pneumophila , and it could be used in clinical practice to detect patients that could be at higher risk of therapeutic failure.

Tularemia is a zoonosis caused by the bacterium Francisella tularensis. Its specific diagnosis remains based on serological methods, while detection of F. tularensis in clinical samples by culture or PCR is rarely obtained. The aim of the present study was to evaluate the performance of the SERION ELISA classic Francisella tularensis IgG and IgM tests (Virion/Serion GmbH Institute, Würzburg, Germany) and the VIRapid® Tularemia immunochromatographic (ICT) test (VIRCELL, Granada, Spain), compared to the in-house microagglutination (MAT) and indirect immunofluorescence (IFA) tests currently used at the French National Reference Centre for Francisella.