Fabrice Compain

Multiplex PCR for Detection of Seven Virulence Factors and K1/K2 Capsular Serotypes of Klebsiella pneumoniae

ABSTRACT A single multiplex PCR assay targeting seven virulence factors and the wzi gene specific for the K1 and K2 capsular serotypes of Klebsiella pneumoniae was developed and tested on 65 clinical isolates, which included 45 isolates responsible for community-acquired severe human infections. The assay is useful for the surveillance of emerging highly virulent strains.

Inhibition of the β-lactamase BlaMab by avibactam improves the in vitro and in vivo efficacy of imipenem against Mycobacterium abscessus

ABSTRACT Mycobacterium abscessus pulmonary infections are treated with a macrolide (clarithromycin or azithromycin), an aminoglycoside (amikacin), and a β-lactam (cefoxitin or imipenem). The triple combination is used without any β-lactamase inhibitor, even though M. abscessus produces the broad-spectrum β-lactamase BlaMab. We determine whether inhibition of BlaMab by avibactam improves the activity of imipenem against M. abscessus. The bactericidal activity of drug combinations was assayed in broth and in human macrophages. The in vivo efficacy of the drugs was tested by monitoring the survival of infected zebrafish embryos. The level of BlaMab production in broth and in macrophages was compared by quantitative reverse transcription-PCR and Western blotting. The triple combination of imipenem (8 or 32 μg/ml), amikacin (32 μg/ml), and avibactam (4 μg/ml) was bactericidal in broth (<0.1% survival), with 3.2- and 4.3-log10 reductions in the number of CFU being achieved at 72 h when imipenem was used at 8 and 32 μg/ml, respectively. The triple combination achieved significant intracellular killing, with the bacterial survival rates being 54% and 7% with the low (8 μg/ml) and high (32 μg/ml) dosages of imipenem, respectively. In vivo inhibition of BlaMab by avibactam improved the survival of zebrafish embryos treated with imipenem. Expression of the gene encoding BlaMab was induced (20-fold) in the infected macrophages. Inhibition of BlaMab by avibactam improved the efficacy of imipenem against M. abscessusin vitro, in macrophages, and in zebrafish embryos, indicating that this β-lactamase inhibitor should be clinically evaluated. The in vitro evaluation of imipenem may underestimate the impact of BlaMab, since the production of the β-lactamase is inducible in macrophages.

Central venous catheters and biofilms: where do we stand in 2017?

The use of central venous catheters (CVC) is associated with a risk of microbial colonization and subsequent potentially severe infection. Microbial contamination of the catheter leads to the development of a microbial consortia associated with the CVC surface and embedded in an extracellular matrix, named biofilm. This biofilm provides bacterial cells the ability to survive antimicrobial agents and the host immune system and to disseminate to other sites of the body. The best preventive strategy is to avoid any unnecessary catheterization or to reduce indwelling duration when a CVC is required. Beside aseptic care and antibiotic‐impregnated catheters (like minocycline/rifampin), preventive locks can be proposed in some cases, whereas non‐biocidal approaches are under active research like anti‐adhesive or competitive interactions strategies. When the diagnosis of catheter‐related bloodstream infection (CRBSI) is suspected on clinical symptoms, it requires a microbiological confirmation by paired blood cultures in order to avoid unnecessary catheter removal. The treatment of CRBSI relies on catheter removal and systemic antimicrobials. However, antibiotic lock technique (ALT) can be used as an attempt to eradicate biofilm formed on the inside lumen of the catheter in case of uncomplicated long‐term catheter‐related BSI caused by coagulase‐negative staphylococci (CoNS) or Enterobacteriaceae. Recently, promising strategies have been developed to improve biofilm eradication; they rely on matrix degradation or destabilization or the development of anti‐persister compounds, targeting the most tolerant bacterial cells inside the biofilm. Understanding biofilm formation at the molecular level may help us to develop new approaches to prevent or treat these frequent infections.

In vitro activity of miltefosine in combination with voriconazole or amphotericin B against clinical isolates of Scedosporium spp.

Scedosporium spp. are significant fungal pathogens in both immunocompromised and immunocompetent individuals. The effectiveness of antifungal therapy against Scedosporium spp. is limited due to their multidrug resistance to most widely used antifungal chemotherapies such as azoles, polyenes and echinocandins (Tortorano et al., 2014). Voriconazole, sometimes combined with surgery, appears to be the only drug significantly active against scedosporiosis. However, the mortality due to scedosporiosis remains high and finding more effective antifungal strategies is necessary. Antifungal combination treatment of drugs belonging to different classes may be a useful approach. So far, azoles in combination with echinocandins or polyenes or terbinafine, as well as polyenes in combination with other drugs, have been tested in vitro with various results (Afeltra et al., 2002; CuencaEstrella et al., 2008; Lackner et al., 2014; Meletiadis et al., 2003; Rodrı́guez et al., 2009; Troke et al., 2008; Yustes & Guarro, 2005).

β LACTA test for rapid detection of Enterobacteriaceae resistant to third-generation cephalosporins from positive blood cultures using briefly incubated solid medium cultures

Inappropriate empirical antibiotic treatment results in poor outcomes in patients with severe sepsis and septic shock (Angus et al., 2001; Kumar et al., 2009). The administration of broad-spectrum antibiotics is therefore recommended in these critically ill patients, with a reassessment of antimicrobial therapy according to microbiological data for deescalation when appropriate (Dellinger et al., 2013). The emergence of multidrugresistant Enterobacteriaceae over the last 20 years represents a serious issue when it comes to the introduction of appropriate empirical antibiotic therapy. As b-lactams remain the drugs of choice in the treatment of Enterobacteriaceae, rapid detection of Enterobacteriaceae resistant to broadspectrum cephalosporins, especially resistant to third-generation cephalosporins (3GCs), is becoming a major challenge in everyday practice.

Molecular Characterization of Streptococcus agalactiae Isolates Harboring Small erm(T)-Carrying Plasmids

ABSTRACT Among 1,827 group B Streptococcus (GBS) strains collected between 2006 and 2013 by the French National Reference Center for Streptococci, 490 (26.8%) strains were erythromycin resistant. The erm(T) resistance gene was found in six strains belonging to capsular polysaccharides Ia, III, and V and was carried by the same mobilizable plasmid, which could be efficiently transferred by mobilization to GBS and Enterococcus faecalis recipients, thus promoting a broad dissemination of erm(T).

Impaired Inhibition by Avibactam and Resistance to the Ceftazidime-Avibactam Combination Due to the D179Y Substitution in the KPC-2 β-Lactamase

ABSTRACT The ceftazidime-avibactam antibiotic combination was recently shown to be at risk for the emergence of resistance under treatment. To gain insight into the underlying mechanism, we have analyzed the catalytic properties of a Klebsiella pneumoniae carbapenemase type 2 (KPC-2) β-lactamase harboring the D179Y substitution. We show that impaired inhibition by avibactam combined with significant residual activity for ceftazidime hydrolysis accounts for the resistance. In contrast, the D179Y substitution abolished the hydrolysis of aztreonam and imipenem, indicating that these drugs might provide therapeutic alternatives.

Non-O1/Non-O139 Vibrio cholerae Avian Isolate from France Cocarrying the blaVIM-1 and blaVIM-4 Genes

ABSTRACT We describe here a non-O1/non-O139 Vibrio cholerae isolate producing both VIM-1 and VIM-4 carbapenemases. It was isolated from a yellow-legged gull in southern France. The blaVIM genes were part of a class 1 integron structure located in an IncA/C plasmid. This study emphasizes the presence of carbapenemase genes in wildlife microbiota.

Hydrolysis of Clavulanate by Mycobacterium tuberculosis β-Lactamase BlaC Harboring a Canonical SDN Motif

ABSTRACT Combinations of β-lactams with clavulanate are currently being investigated for tuberculosis treatment. Since Mycobacterium tuberculosis produces a broad spectrum β-lactamase, BlaC, the success of this approach could be compromised by the emergence of clavulanate-resistant variants, as observed for inhibitor-resistant TEM variants in enterobacteria. Previous analyses based on site-directed mutagenesis of BlaC have led to the conclusion that this risk was limited. Here, we used a different approach based on determination of the crystal structure of β-lactamase BlaMAb of Mycobacterium abscessus, which efficiently hydrolyzes clavulanate. Comparison of BlaMAb and BlaC allowed for structure-assisted site-directed mutagenesis of BlaC and identification of the G132N substitution that was sufficient to switch the interaction of BlaC with clavulanate from irreversible inactivation to efficient hydrolysis. The substitution, which restored the canonical SDN motif (SDG→SDN), allowed for efficient hydrolysis of clavulanate, with a more than 104-fold increase in kcat (0.41 s−1), without affecting the hydrolysis of other β-lactams. Mass spectrometry revealed that acylation of BlaC and of its G132N variant by clavulanate follows similar paths, involving sequential formation of two acylenzymes. Decarboxylation of the first acylenzyme results in a stable secondary acylenzyme in BlaC, whereas hydrolysis occurs in the G132N variant. The SDN/SDG polymorphism defines two mycobacterial lineages comprising rapidly and slowly growing species, respectively. Together, these results suggest that the efficacy of β-lactam–clavulanate combinations may be limited by the emergence of resistance. β-Lactams active without clavulanate, such as faropenem, should be prioritized for the development of new therapies.

In Vitro Activity of Ceftolozane-Tazobactam against Multidrug-Resistant Nonfermenting Gram-Negative Bacilli Isolated from Patients with Cystic Fibrosis

ABSTRACT Ceftolozane-tazobactam was tested against 58 multidrug-resistant nonfermenting Gram-negative bacilli (35 Pseudomonas aeruginosa, 11 Achromobacter xylosoxydans, and 12 Stenotrophomonas maltophilia isolates) isolated from cystic fibrosis patients and was compared to ceftolozane alone, ceftazidime, meropenem, and piperacillin-tazobactam. Ceftolozane-tazobactam was the most active agent against P. aeruginosa but was inactive against A. xylosoxydans and S. maltophilia. In time-kill experiments, ceftolozane-tazobactam had complete bactericidal activity against 2/6 clinical isolates (33%).