Differences in Colistin-resistant Acinetobacter baumannii Clinical Isolates Between Patients With and Without Prior Colistin Treatment.

BACKGROUND: The increasing morbidity and mortality rates associated with Acinetobacter baumannii are due to the emergence of drug resistance and the limited treatment options. We compared characteristics of colistin-resistant Acinetobacter baumannii (CR-AB) clinical isolates recovered from patients with and without prior colistin treatment. We assessed whether prior colistin treatment affects the resistance mechanism of CR-AB isolates, mortality rates, and clinical characteristics. Additionally, a proper method for identifying CR-AB was determined. METHODS: We collected 36 non-duplicate CR-AB clinical isolates resistant to colistin. Antimicrobial susceptibility testing, Sanger sequencing analysis, molecular typing, lipid A structure analysis, and in vitro synergy testing were performed. Eleven colistin-susceptible AB isolates were used as controls. RESULTS: Despite no differences in clinical characteristics between patients with and without prior colistin treatment, resistance-causing genetic mutations were more frequent in isolates from colistin-treated patients. Distinct mutations were overlooked via the Sanger sequencing method, perhaps because of a masking effect by the colistin-susceptible AB subpopulation of CR-AB isolates lacking genetic mutations. However, modified lipid A analysis revealed colistin resistance peaks, despite the population heterogeneity, and peak levels were significantly different between the groups. CONCLUSIONS: Although prior colistin use did not induce clinical or susceptibility differences, we demonstrated that identification of CR-AB by sequencing is insufficient. We propose that population heterogeneity has a masking effect, especially in colistin non-treated patients; therefore, accurate testing methods reflecting physiological alterations of the bacteria, such as phosphoethanolamine-modified lipid A identification by matrix-assisted laser desorption ionization-time of flight, should be employed.

Combination therapy with polymyxin B and netropsin against clinical isolates of multidrug-resistant Acinetobacter baumannii.

Polymyxins are last-resort antibiotics for treating infections of Gram-negative bacteria. The recent emergence of polymyxin-resistant bacteria, however, urgently demands clinical optimisation of polymyxin use to minimise further evolution of resistance. In this study we developed a novel combination therapy using minimal concentrations of polymyxin B. After large-scale screening of Streptomyces secondary metabolites, we identified a reliable polymixin synergist and confirmed as netropsin using high-pressure liquid chromatography, nuclear magnetic resonance, and mass spectrometry followed by in vitro assays using various Gram-negative pathogenic bacteria. To evaluate the effectiveness of combining polymixin B and netropsin in vivo, we performed survival analysis on greater wax moth Galleria mellonella infected with colistin-resistant clinical Acinetobacter baumannii isolates as well as Escherichia coli, Shigella flexineri, Salmonella typhimuruim, and Pseudomonas aeruginosa. The survival of infected G. mellonella was significantly higher when treated with polymyxin B and netropsin in combination than when treated with polymyxin B or netropsin alone. We propose a netropsin combination therapy that minimises the use of polymyxin B when treating infections with multidrug resistant Gram-negative bacteria.

Prediction of Putative Resistance Islands in a Carbapenem-Resistant Acinetobacter baumannii Global Clone 2 Clinical Isolate.

BACKGROUND: We investigated the whole genome sequence (WGS) of a carbapenem-resistant Acinetobacter baumannii isolate belonging to the global clone 2 (GC2) and predicted resistance islands using a software tool. METHODS: A. baumannii strain YU-R612 was isolated from the sputum of a 61-yr-old man with sepsis. The WGS of the YU-R612 strain was obtained by using the PacBio RS II Sequencing System (Pacific Biosciences Inc., USA). Antimicrobial resistance genes and resistance islands were analyzed by using ResFinder and Genomic Island Prediction software (GIPSy), respectively. RESULTS: The YU-R612 genome consisted of a circular chromosome (ca. 4,075 kb) and two plasmids (ca. 74 kb and 5 kb). Its sequence type (ST) under the Oxford scheme was ST191, consistent with assignment to GC2. ResFinder analysis showed that YU-R612 possessed the following resistance genes: four ß-lactamase genes bla(ADC-30), bla(OXA-66), bla(OXA-23), and bla(TEM-1); armA, aadA1, and aacA4 as aminoglycoside resistance-encoding genes; aac(6')Ib-cr for fluoroquinolone resistance; msr(E) for macrolide, lincosamide, and streptogramin B resistance; catB8 for phenicol resistance; and sul1 for sulfonamide resistance. By GIPSy analysis, six putative resistant islands (PRIs) were determined on the YU-R612 chromosome. Among them, PRI1 possessed two copies of Tn2009 carrying bla(OXA-23), and PRI5 carried two copies of a class I integron carrying sul1 and armA genes. CONCLUSIONS: By prediction of resistance islands in the carbapenem-resistant A. baumannii YU-R612 GC2 strain isolated in Korea, PRIs were detected on the chromosome that possessed Tn2009 and class I integrons. The prediction of resistance islands using software tools was useful for analysis of the WGS.

In Vivo Selection of Pan-Drug Resistant Acinetobacter baumannii during Antibiotic Treatment.

PURPOSE: Colistin resistance in Acinetobacter baumannii (A. baumannii) is mediated by a complete loss of lipopolysaccharide production via mutations in lpxA, lpxC, and lpxD gene or lipid A modifications via mutations in the pmrA and pmrB genes. However, the exact mechanism of therapy-induced colistin resistance in A. baumannii is not well understood. MATERIALS AND METHODS: We investigated the genotypic and phenotypic changes that underlie pan-drug resistance mechanisms by determining differences between the alterations in extensively drug-resistant (XDR) A. baumannii (AB001 and AB002) isolates and a pan-drug resistant (PDR) counterpart (AB003) recovered from one patient before and after antibiotic treatment, respectively. RESULTS: All three clinical isolates shared an identical sequence type (ST138), belonging to the global epidemic clone, clonal complex 92, and all produced OXA-23 carbapenemase. The PDR AB003 showed two genetic differences, acquisition of armA gene and an amino acid substitution (Glu229Asp) in pmrB gene, relative to XDR isolates. No mutations were detected in the pmrA, pmrC, lpxA, lpxC, or lpxD genes in all three isolates. In matrix-assisted laser desorption ionization-time of flight analysis, the three isolates commonly showed two major peaks at 1728 m/z and 1912 m/z, but peaks at 2034 m/z, 2157 m/z, 2261 m/z, and 2384 m/z were detected only in the PDR A. baumannii AB003 isolate. CONCLUSION: Our results show that changes in lipid A structure via a mutation in the pmrB gene and acquisition of armA gene might confer resistance to colistin and aminoglycosides to XDR A. baumannii strains, resulting in appearance of a PDR A. baumannii strain of ST138.

Clinical factors associated with acquisition of resistance to levofloxacin in Stenotrophomonas maltophilia.

PURPOSE: Fluoroquinolones, rapidly gaining prominence in treatment of Stenotrophomonas maltophilia (SMP), are noted for their potency and tolerability. However, SMP may rapidly acquire resistance to fluoroquinolones. We evaluated associations of clinical factors with acquisition of levofloxacin resistance (LFr) in SMP. MATERIALS AND METHODS: Our retrospective cohort study was based on patient data collected between January 2008 and June 2010. Through screening of 1275 patients, we identified 122 patients with data for SMP antibiotic susceptibility testing in ≥3 serial SMP isolates. RESULTS: We assigned the 122 patients to either the SS group (n=54) in which levofloxacin susceptibility was maintained or the SR group (n=31) in which susceptible SMP acquired resistance. In multivariate regression analysis, exposure to levofloxacin for more than 3 weeks [odds ratio (OR) 15.39, 95% confidential interval (CI) 3.08-76.93, p=0.001] and co-infection or co-colonization with Klebsiella pneumoniae resistant to levofloxacin (OR 4.85, 95% CI 1.16-20.24, p=0.030) were independently associated with LFr acquisition in SMP. CONCLUSION: Acquisition of LFr during serial sampling of SMP was related to the levofloxacin exposure.

In vitro anticandidal activity of xanthorrhizol isolated from Curcuma xanthorrhiza Roxb.

OBJECTIVES: Xanthorrhizol, isolated from the methanol extract of Curcuma xanthorrhiza Roxb., was investigated for its anticandidal activity using six Candida species. METHODS: The in vitro susceptibility tests for xanthorrhizol were carried out in terms of MIC and minimal fungicidal concentration (MFC) using the NCCLS M27-A2 broth microdilution method. Time-kill curves were determined to assess the correlation between MIC and fungicidal activity of xanthorrhizol at concentrations ranging from 0 MIC to 4x MIC. RESULTS: All Candida species showed susceptibility to xanthorrhizol in the MIC range 1.0-15.0 mg/L for Candida albicans, 1.0-10 mg/L for Candida glabrata, 2.0-8.0 mg/L for Candida guilliermondii, 2.5-7.5 mg/L for Candida krusei, 2.5-25 mg/L for Candida parapsilosis and 2.0-8.0 mg/L for Candida tropicalis. Time-kill curves demonstrated that xanthorrhizol was able to kill the Candida strains with MFCs of 20 mg/mL, 15 mg/mL, 12.5 mg/mL, 10 mg/L, 30 mg/mL and 10 mg/L for C. albicans, C. glabrata, C. guilliermondii, C. krusei, C. parapsilosis and C. tropicalis, respectively. CONCLUSIONS: The potent anticandidal activity of xanthorrhizol may support the use of C. xanthorriza for the treatment of candidiasis.