Prior in vitro exposure to voriconazole confers resistance to amphotericin B in aspergillus fumigatus biofilms

Triazoles are the mainstay of treatment for aspergillosis, although resistance to these antifungal agents may be associated with treatment failure. Refractory infections often necessitate a switch to other antifungal agents, including amphotericin B (AmB), although these infections may not resolve. The aim of this study was to investigate the effect of prior azole exposure on AmB sensitivity in Aspergillus fumigatus biofilms. It was hypothesised that sequential antifungal therapy has the potential to impact adaptive resistance mechanisms. Antifungal sensitivity was determined for each isolate against AmB ± voriconazole (VRZ) exposure by a broth microdilution method and an XTT metabolic assay. To analyse the role of extracellular DNA (eDNA) and Hsp90 activation, sensitivity to AmB ± DNA-digesting enzyme (DNase) or Hsp90 inhibitor [geldanamycin (GDA)] was also tested. Finally, scanning electron microscopy was performed to assess phenotypic changes. The in vitro data revealed that A. fumigatus sensitivity to AmB was decreased when it was tested in combination with VRZ. In addition, a two- to four-fold decreased sensitivity to AmB was recorded against VRZ-exposed germlings compared with controls. It was also shown that depletion of eDNA by DNase treatment enhanced AmB activity against VRZ-exposed cells by eight-fold, which visually could be explained by destabilisation of the biofilm when examined microscopically. Pharmacological inhibition of Hsp90 by GDA significantly improved biofilm susceptibility to AmB by four- to eight-fold. In conclusion, A. fumigatus pre-exposure to VRZ concomitantly induces eDNA release and activates the stress response, which collectively confers AmB resistance in vitro

In vitro modelling of chronic Aspergillus fumigatus infections

Recent studies have demonstrated the potential of Aspergillus fumigatus to form biofilm structures similar to those identified with Candida albicans. In contrast to the wide berth of knowledge relating to the biology of C. albicans biofilms, relatively little is known regarding the development and morphology of multicellular communities of A. fumigatus. This alternate lifestyle may have profound clinical implications with regard to the pathogenic potential of the organism and the likelihood of successful detection and treatment. A reproducible biofilm model of A. fumigatus was developed using several different substrates, which were used to macroscopically and microscopically investigate the phenotypic attributes and general characteristics of biofilm formation, and to investigate antifungal susceptibility profiles.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Extensive diversification is a common feature of Pseudomonas aeruginosa populations during respiratory infections in cystic fibrosis

AbstractBackgroundPopulations of the Liverpool Epidemic Strain (LES) of Pseudomonas aeruginosa undergo extensive diversification in the cystic fibrosis (CF) lung during long-term chronic infections.MethodsWe analyzed sets of 40 isolates from the sputa of five CF patients, each chronically infected with a different non-LES strain of P. aeruginosa. For each sample (two per patient), diversity was assessed by characterizing nine phenotypic traits.ResultsAll P. aeruginosa populations were highly diverse, with the majority of phenotypic variation being due to within-sample diversity.ConclusionsMaintenance of diverse populations in the CF lung is a common feature of P. aeruginosa infections

Effect of Antibiotic Treatment on Bacteriophage Production by a Cystic Fibrosis Epidemic Strain of Pseudomonas aeruginosa▿

Phage production in response to antibiotics varied among four isolates of a Pseudomonas aeruginosa cystic fibrosis (CF) epidemic strain. Whereas ciprofloxacin induced higher levels of phage production, other CF-relevant antibiotics led to reduced production. We detected free phages directly in CF patient sputum samples by both plaque (40% positive) and PCR (76% positive) assays. Our observations suggest that the choice of antibiotics could influence the number of free phages within the CF lung environment

Azole Resistance of Aspergillus fumigatus Biofilms Is Partly Associated with Efflux Pump Activity▿

This study investigated the phase-dependent expression and activity of efflux pumps in Aspergillus fumigatus treated with voriconazole. Fourteen strains were shown to become increasingly resistant in the 12-h (16- to 128-fold) and 24-h (>512-fold) phases compared to 8-h germlings. An Ala-Nap uptake assay demonstrated a significant increase in efflux pump activity in the 12-h and 24-h phases (P < 0.0001). The efflux pump activity of the 8-h germling cells was also significantly induced by voriconazole (P < 0.001) after 24 h of treatment. Inhibition of efflux pump activity with the competitive substrate MC-207,110 reduced the voriconazole MIC values for the A. fumigatus germling cells by 2- to 8-fold. Quantitative expression analysis of AfuMDR4 mRNA transcripts showed a phase-dependent increase as the mycelial complexity increased, which was coincidental with a strain-dependent increase in azole resistance. Voriconazole also significantly induced this in a time-dependent manner (P < 0.001). Finally, an in vivo mouse biofilm model was used to evaluate efflux pump expression, and it was shown that AfuMDR4 was constitutively expressed and significantly induced by treatment with voriconazole after 24 h (P < 0.01). Our results demonstrate that efflux pumps are expressed in complex A. fumigatus biofilm populations and that this contributes to azole resistance. Moreover, voriconazole treatment induces efflux pump expression. Collectively, these data may provide evidence for azole treatment failures in clinical cases of aspergillosis

Identification and molecular characterisation of New Delhi metallo-β-lactamase-1 (NDM-1)- and NDM-6-producing Enterobacteriaceae from New Zealand hospitals

The global spread of New Delhi metallo-beta-lactamase (NDM) is of significant public health concern. This study sought to determine whether bla(NDM) was present in Enterobacteriaceae isolates displaying resistance to carbapenems that were submitted to the National Antibiotic Reference Laboratory, Institute of Environmental Science and Research (Porirua, New Zealand) during 2009 and 2010. Isolates were tested for the presence of beta-lactamase genes and 16S rRNA methylase genes by polymerase chain reaction (PCR) and sequencing. Plasmid transfer studies were undertaken on isolates found to be harbouring bla(NDM). Molecular typing was performed by multilocus sequence typing (MLST). The bla(NDM-1) gene was identified in four Enterobacteriaceae isolates (two Escherichia coli, one Klebsiella pneumoniae and one Proteus mirabilis) from four patients in New Zealand hospitals in 2009 and 2010. In addition, the bla(NDM-6) gene, which differed from bla(NDM-1) by a point mutation at position 698 (C -> T), was also identified in an E. coli isolate from the same patient who harboured the bla(NDM-1)-positive P. mirabilis. All four patients had recently been hospitalised or received health care in India. Four of the isolates also produced a CTX-M-15 extended-spectrum beta-lactamase and/or plasmid-mediated AmpC beta-lactamase, and all five isolates harboured the plasmid-mediated 16S rRNA methylase rmtC gene. The E. coli types were diverse by MLST, and the K. pneumoniae isolate belonged to the internationally disseminated sequence type 11 (ST11) clone. These findings further illustrate the diversity of phenotypic and genotypic features found in association with bla(NDM), in addition to documenting the international spread of this resistance mechanism, notably into a country with historically low rates of antimicrobial resistance. (c) 2012 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved

Pseudomonas aeruginosa and their small diffusible extracellular molecules inhibit Aspergillus fumigatus biofilm formation

Aspergillus fumigatus is often isolated from the lungs of cystic fibrosis (CF) patients, but unlike in severely immunocompromised individuals, the mortality rates are low. This suggests that competition from bacteria within the CF lung may be inhibitory. The purpose of this study was to investigate how Pseudomonas aeruginosa influences A. fumigatus conidial germination and biofilm formation. Aspergillus fumigatus biofilm formation was inhibited by direct contact with P. aeruginosa, but had no effect on preformed biofilm. A secreted heat-stable soluble factor was also shown to exhibit biofilm inhibition. Coculture of P. aeruginosa quorum-sensing mutants (PAO1:delta LasI, PAO1:delta LasR) did not significantly inhibit A. fumigatus biofilms (52.6-58.8%) to the same extent as that of the PA01 wild type (22.9-30.1%), both by direct and by indirect interaction (P &lt; 0.001). Planktonic and sessile inhibition assays with a series of short carbon chain molecules (decanol, decanoic acid and dodecanol) demonstrated that these molecules could both inhibit and disrupt biofilms in a concentration-dependent manner. Overall, this suggests that small diffusible and heat-stable molecules may be responsible for the competitive inhibition of filamentous fungal growth in polymicrobial environments such as the CF lung