Ciprofloxacin-Mediated Mutagenesis Is Suppressed by Subinhibitory Concentrations of Amikacin in Pseudomonas aeruginosa.
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ABSTRACT:
SUBMITTER: Valencia EY
PROVIDER: S-EPMC5328548 | biostudies-other | 2017 Mar
REPOSITORIES: biostudies-other
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Project description:Biofilm infections caused by Pseudomonas aeruginosa are frequently treated with ciprofloxacin (CIP); however, resistance rapidly develops. One of the primary resistance mechanisms is the overexpression of the MexCD-OprJ pump due to a mutation in nfxB, encoding the transcriptional repressor of this pump. The aim of this study was to investigate the effect of subinhibitory concentrations of CIP on the occurrence of nfxB mutants in the wild-type PAO1 flow cell biofilm model. For this purpose, we constructed fluorescent reporter strains (PAO1 background) with an mCherry tag for constitutive red fluorescence and chromosomal transcriptional fusion between the P mexCD promoter and gfp leading to green fluorescence upon mutation of nfxB We observed a rapid development of nfxB mutants by live confocal laser scanning microscopy (CLSM) imaging of the flow cell biofilm (reaching 80 to 90% of the whole population) when treated with 1/10 minimal biofilm inhibitory concentration of CIP for 24 h and 96 h. Based on the observed developmental stages, we propose that nfxB mutants emerged de novo in the biofilm during CIP treatment from filamentous cells, which might have arisen due to the stress responses induced by CIP. Identical nfxB mutations were found in fluorescent colonies from the same flow cell biofilm, especially in 24-h biofilms, suggesting selection and clonal expansion of the mutants during biofilm growth. Our findings point at the significant role of high-enough antibiotic dosages or appropriate combination therapy to avoid the emergence of resistant mutants in biofilms.
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Project description:The opportunistic Gram-negative pathogen Pseudomonas aeruginosa, known for its intrinsic and acquired antibiotic resistance, has a notorious ability to form biofilms, which often facilitate chronic infections. The evolutionary paths to antibiotic resistance have mainly been investigated in planktonic cultures and are less studied in biofilms. We experimentally evolved P. aeruginosa PAO1 colony biofilms and stationary-phase planktonic cultures for seven passages in the presence of subinhibitory levels (0.1 mg/liter) of ciprofloxacin (CIP) and performed a genotypic (whole-bacterial population sequencing) and phenotypic assessment of the populations. We observed a higher proportion of CIP resistance in the CIP-evolved biofilm populations than in planktonic populations exposed to the same drug concentrations. However, the MICs of ciprofloxacin were lower in CIP-resistant isolates selected from the biofilm population than the MICs of CIP-resistant isolates from the planktonic cultures. We found common evolutionary trajectories between the different lineages, with mutations in known CIP resistance determinants as well as growth condition-dependent adaptations. We observed a general trend toward a reduction in type IV-pilus-dependent motility (twitching) in CIP-evolved populations and a loss of virulence-associated traits in the populations evolved in the absence of antibiotic. In conclusion, our data indicate that biofilms facilitate the development of low-level mutational resistance, probably due to the lower effective drug exposure than in planktonic cultures. These results provide a framework for the selection process of resistant variants and the evolutionary mechanisms involved under the two different growth conditions.
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Project description:ObjectivesTo investigate how the SOS response, an error-prone DNA repair pathway, is expressed following subinhibitory quinolone treatment of Mycobacterium tuberculosis.MethodsGenome-wide expression profiling followed by quantitative RT (qRT)-PCR was used to study the effect of ciprofloxacin on M. tuberculosis gene expression.ResultsMicroarray analysis showed that 16/110 genes involved in DNA protection, repair and recombination were up-regulated. There appeared to be a lack of downstream genes involved in the SOS response. qRT-PCR detected an induction of lexA and recA after 4 h and of dnaE2 after 24 h of subinhibitory treatment.ConclusionsThe pattern of gene expression observed following subinhibitory quinolone treatment differed from that induced after other DNA-damaging agents (e.g. mitomycin C). The expression of the DnaE2 polymerase response was significantly delayed following subinhibitory quinolone exposure.
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Project description:Enterococcus faecium has emerged as a major opportunistic pathogen for 2 decades with the spread of hospital-adapted multidrug-resistant clones. As members of the intestinal microbiota, they are subjected to numerous bacterial stresses, including antibiotics at subinhibitory concentrations (SICs). Since fluoroquinolones are extensively prescribed, SICs are very likely to occur in vivo, with potential effects on bacterial metabolism with subsequent modulation of opportunistic traits. The aim of this study was to evaluate globally the impact of SICs of ciprofloxacin on antimicrobial resistance and pathogenicity of E. faecium Transcriptomic analysis was performed by RNA sequencing (RNA-seq) (HiSeq 2500; Illumina) using the vanB-positive reference strain E. faecium Aus0004 in the absence or presence of ciprofloxacin SIC (0.38 mg/liter, i.e., 1/8 of the MIC). Several genetic and phenotypic tests were used for validation. In the presence of ciprofloxacin SIC, 196 genes were significantly induced, whereas 286 genes were significantly repressed, meaning that 16.8% of the E. faecium genome was altered. Among upregulated genes, EFAU004_02294 (fold change, 14.3) encoded a protein (Qnr of E. faecium [EfmQnr]) homologue of Qnr proteins involved in quinolone resistance in Gram-negative bacilli. Its implication in intrinsic and adaptive fluoroquinolone (FQ) resistance in E. faecium was experimentally ascertained. Moreover, EFAU004_02292, coding for the collagen adhesin Acm, was also induced by the SIC of ciprofloxacin (fold change, 8.2), and higher adhesion capabilities were demonstrated phenotypically. Both EfmQnr and Acm determinants may play an important role in the transition from a commensal to a pathogenic state of E. faecium that resides in the gut of patients receiving fluoroquinolone therapy.
| S-EPMC5404537 | biostudies-literature
Project description:Enterococcus faecium has emerged as a major opportunistic pathogen for two decades, with the spread of hospital-adapted multidrug-resistant clones. Members of the intestinal microbiota, they are subjected to numerous bacterial stresses, including antibiotics at subinhibitory concentrations (SICs). Since fluoroquinolones are extensively prescribed, SICs are very likely to occur in vivo with potential effects on bacterial metabolism with subsequent modulation of opportunistic traits. The aim of the study was to evaluate globally the impact of subinhibitory concentrations (SICs) of ciprofloxacin on antimicrobial resistance and pathogenicity of E. faecium. Transcriptomic analysis was performed by RNA-seq (HiSeq 2500, Illumina) using the vanB-positive reference strain E. faecium Aus0004 in the absence or presence of ciprofloxacin SIC (0.38 mg/L, i.e. MIC 1/8). Several genetic and phenotypic tests were used for validation. In the presence of ciprofloxacin SIC, 196 genes were significantly induced whereas 286 were significantly repressed, meaning that 16.8% of the E. faecium genome was altered. Amongst upregulated genes, EFAU004_02294 (fold change of 14.3) encoded a protein (EfmQnr) homologue of Qnr proteins involved in quinolone resistance in Gram-negative bacilli. Its implication in intrinsic and adaptive FQ resistance in E. faecium was experimentally ascertained. Moreover, EFAU004_02292 coding for the collagen adhesin Acm was also induced by SIC of ciprofloxacin (fold change of 8.2), and higher adhesion capabilities were demonstrated phenotypically. Both Efmqnr and Acm determinants may play an important role in the transition from a commensal to a pathogenic state of E. faecium that resides in the gut of patients receiving a fluoroquinolone therapy.
2017-02-04 | GSE94507 | GEO
Project description:Emergence and selection of antibiotic resistance following exposure to high antibiotic concentrations have been repeatedly shown in clinical and agricultural settings, whereas the role of the weak selective pressures exerted by antibiotic levels below the MIC (sub-MIC) in aquatic environments due to anthropogenic contamination remains unclear. Here, we studied how exposure to sub-MIC levels of ciprofloxacin enriches for Escherichia coli with reduced susceptibility to ciprofloxacin using a mallard colonization model. Mallards were inoculated with two isogenic extended-spectrum-β-lactamase (ESBL)-encoding E. coli strains, differing only by a gyrA mutation that results in increased MICs of ciprofloxacin, and exposed to different levels of ciprofloxacin in their swimming water. Changes in the ratios of mutant to parental strains excreted in feces over time and ESBL plasmid spread within the gut microbiota from individual birds were investigated. Results show that in vivo selection of gyrA mutants occurred in mallards during exposure to ciprofloxacin at concentrations previously found in aquatic environments. During colonization, resistance plasmids were readily transferred between strains in the intestines of the mallards, but conjugation frequencies were not affected by ciprofloxacin exposure. Our results highlight the potential for enrichment of resistant bacteria in wildlife and underline the importance of reducing antibiotic pollution in the environment.
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Project description:With few novel antimicrobials in development, resistance to the current selection of antibiotics increasingly encroaches on our ability to control microbial infections. One limitation in our understanding of the basis of the constraints on current therapies is our poor understanding of antibiotic interactions with bacteria on a global scale. Custom DNA microarrays were used to characterize the response of Pseudomonas aeruginosa to ciprofloxacin, a fluoroquinolone commonly used in therapy against chronic infections by this intrinsically resistant bacterium. Of the approximately 5,300 open reading frames (ORFs) on the array, 941 genes showed statistically significant (P </= 0.05) differential expression in response to 0.3x MIC of ciprofloxacin; 554 were promoted and 387 were repressed. Most striking among the responsive genes was the region between PA0613 and PA0648, which codes for the bacteriophage-like R2/F2 pyocins. In this region, virtually every ORF was increased by 0.3x MIC of ciprofloxacin and even more dramatically up-regulated (7- to 19-fold) following treatment with 1x MIC of ciprofloxacin. Pyocin gene expression was confirmed with lux reporter mutants and real-time PCR studies; pyocin-like particles were also present in transmission electron micrographs of supernatants from cells treated with 1x MIC of ciprofloxacin. Interestingly, mutants in this region exhibited >/=8-fold-increased resistance to ciprofloxacin and other fluoroquinolones, demonstrating that this region is a susceptibility determinant. Since this region is known to be variably present in the genomes of clinical isolates of P. aeruginosa (R. K. Ernst et al., Environ. Microbiol. 5:1341-1349, 2003, and M. C. Wolfgang et al., Proc. Natl. Acad. Sci. USA 100:8484-8489, 2003), these findings demonstrate that the R2/F2 pyocin region is a "loaded gun" that can mediate fluoroquinolone susceptibility in P. aeruginosa.
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Project description:Identification of mutants under negative selection mediated by a non-lethal concentration of CTX
2015-07-23 | GSE61988 | GEO
Project description:Pseudomonas aeruginosa is an opportunistic nosocomial pathogen associated with high morbidity and mortality rates. Combination of antibiotics has been found to combat multi-drug resistant or extensively drug resistance P. aeruginosa. In this study we investigate the in vitro and in vivo effect of amikacin and imipenem combination against resistant P. aeruginosa. The checkerboard technique and time-killing curve have been performed for in vitro studies showed synergistic effect for combination. A peritonitis mouse model has been used for evaluation of the therapeutic efficacy of this combination which confirmed this synergistic effect. The in vitro and in vivo techniques showed synergistic interaction between tested drugs with fractional inhibitory concentration indices (FICIs) of ≤0.5. Conventional PCR and quantitative real-time PCR techniques were used in molecular detection of bla IMP and aac(6')-Ib as 35.5% and 42.2% of P. aeruginosa harbored bla IMP and aac(6')-Ib respectively. Drug combination viewed statistically significant reduction in bacterial counts (p value < 0.5). The lowest bla IMP and aac(6')-Ib expression was observed after treatment with 0.25 MIC of imipenem + 0.5 MIC of amikacin. Morphological changes in P. aeruginosa isolates were detected by scanning electron microscope (SEM) showing cell shrinkage and disruption in the outer membrane of P. aeruginosa that were more prominent with combination therapy than with monotherapy.
| S-EPMC8615098 | biostudies-literature
Project description:BackgroundShortcomings of inhaled antibiotic treatments for Pseudomonas aeruginosa infection in patients with cystic fibrosis (CF) include poor drug penetration, inactivation by sputum, poor efficiency due to protective biofilm, and short residence in the lung.MethodsEligible patients with forced expiratory volume in 1 s (FEV1) ≥25% of predicted value at screening and CF with chronic P. aeruginosa infection were randomly assigned to receive 3 treatment cycles (28 days on, 28 days off) of amikacin liposome inhalation suspension (ALIS, 590 mg QD) or tobramycin inhalation solution (TIS, 300 mg BID). The primary endpoint was noninferiority of ALIS vs TIS in change from baseline to day 168 in FEV1 (per-protocol population). Secondary endpoints included change in respiratory symptoms by Cystic Fibrosis Questionnaire-Revised (CFQ-R).ResultsThe study was conducted February 2012 to September 2013. ALIS was noninferior to TIS (95% CI, -4.95 to 2.34) for relative change in FEV1 (L) from baseline. The mean increases in CFQ-R score from baseline on the Respiratory Symptoms scale suggested clinically meaningful improvement in both arms at the end of treatment in cycle 1 and in the ALIS arm at the end of treatment in cycles 2 and 3; however, the changes were not statistically significant between the 2 treatment arms. Treatment-emergent adverse events (TEAEs) were reported in most patients (ALIS, 84.5%; TIS, 78.8%). Serious TEAEs occurred in 17.6% and 19.9% of patients, respectively; most were hospitalisations for infective pulmonary exacerbation of CF.ConclusionsCyclical dosing of once-daily ALIS was noninferior to cyclical twice-daily TIS in improving lung function. ClinicalTrials.gov Identifier: NCT01315678.
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