Pseudomonas aeruginosa mexR and mexEF Antibiotic Efflux Pump Variants Exhibit Increased Virulence.

Antibiotic-resistant Pseudomonas aeruginosa infections are the primary cause of mortality in people with cystic fibrosis (CF). Yet, it has only recently become appreciated that resistance mutations can also increase P. aeruginosa virulence, even in the absence of antibiotics. Moreover, the mechanisms by which resistance mutations increase virulence are poorly understood. In this study we tested the hypothesis that mutations affecting efflux pumps can directly increase P. aeruginosa virulence. Using genetics, physiological assays, and model infections, we show that efflux pump mutations can increase virulence. Mutations of the mexEF efflux pump system increased swarming, rhamnolipid production, and lethality in a mouse infection model, while mutations in mexR that increased expression of the mexAB-oprM efflux system increased virulence during an acute murine lung infection without affecting swarming or rhamnolipid gene expression. Finally, we show that an efflux pump inhibitor, which represents a proposed novel treatment approach for P. aeruginosa, increased rhamnolipid gene expression in a dose-dependent manner. This finding is important because rhamnolipids are key virulence factors involved in dissemination through epithelial barriers and cause neutrophil necrosis. Together, these data show how current and proposed future anti-Pseudomonal treatments may unintentionally make infections worse by increasing virulence. Therefore, treatments that target efflux should be pursued with caution.

Incubation time and culture media impact success of identification of Nocardia spp. by MALDI-ToF mass spectrometry.

Identification of Nocardia can be challenging, even by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-ToF MS). We examined the impact of incubation duration, culture media and bead-beading on identification success. When isolates were grown for 3 days on SAB (Saboround dextrose agar), then extracted using a bead-beating, ethanol/formic acid (EtOH/FA) procedure, MALDI-ToF MS correctly identified 36.4% to species level. By contrast, 80.9% of isolates were correctly identified to species level when processed with the standard EtOH/FA extraction from Columbia blood agar (CBA) cultures incubated until colonies first appeared. Excluding rare species, the latter approach correctly identified 93.0% of isolates of commonly-encountered Nocardia species when additional database entries were included to expand representation of select species. We demonstrate that while bead-beating has little impact on identification success by MALDI-ToF MS, analysis of early growth is crucial. Additionally, culture media that promotes quick growth of Nocardia (e.g. CBA) is recommended.

Antifungal Susceptibility Testing of Aspergillus spp. by Using a Composite Correlation Index (CCI)-Based Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry Method Appears To Not Offer Benefit over Traditional Broth Microdilution Testing.

Aspergillus spp. cause serious invasive lung infections, and Aspergillus fumigatus is the most commonly encountered clinically significant species. Voriconazole is considered to be the drug of choice for treating A. fumigatus infections; however, rising resistance rates have been reported. We evaluated a matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS)-based method for the differentiation between wild-type and non-wild-type isolates of 20 Aspergillus spp. (including 2 isolates of Aspergillus ustus and 1 of Aspergillus calidoustus that were used as controls due their intrinsic low azole susceptibility with respect to the in vitro response to voriconazole). At 30 and 48 h of incubation, there was complete agreement between Cyp51A sequence analysis, broth microdilution, and MALDI-TOF MS classification of isolates as wild type or non-wild type. In this proof-of-concept study, we demonstrated that MALDI-TOF MS can be used to accurately detect A. fumigatus strains with reduced voriconazole susceptibility. However, rather than proving to be a rapid and simple method for antifungal susceptibility testing, this particular MS-based method showed no benefit over conventional testing methods.