The Contribution of Antibiotic Resistance Mechanisms in Clinical <i>Burkholderia cepacia</i> Complex Isolates: An Emphasis on Efflux Pump Activity

<div><p>Due to the limited information of the contribution of various antibiotic resistance mechanisms in clinical <i>Burkholderia cepacia</i> complex isolates, Antibiotic resistance mechanisms, including integron analysis, identification of quinolone resistance-determining region mutations, measurement of efflux pump activity, and sequence analysis of efflux pump regulators, were investigated in 66 clinical <i>B. cepacia</i> complex isolates. Species were identified via <i>recA</i>-RFLP and MALDI-TOF. Four genomovars were identified by <i>recA</i>-RFLP. <i>B. cenocepacia</i> (genomovar III) was the most prevalent genomovar (90.1%). Most isolates (60/66, 90.9%) were correctly identified by MALDI-TOF analysis. Clonal relatedness determined by PFGE analysis revealed 30 pulsotypes, including two major pulsotypes that comprised 22.7% and 18.2% of the isolates, respectively. Seventeen (25.8%) isolates harboured class 1 integron with various combinations of resistance genes. Among six levofloxacin-resistant isolates, five had single-base substitutions in the <i>gyrA</i> gene and three demonstrated efflux pump activities. Among the 42 isolates exhibiting resistance to at least one antimicrobial agent, 94.4% ceftazidime-resistant isolates (17/18) and 72.7% chloramphenicol-resistant isolates (16/22) demonstrated efflux pump activity. Quantitation of efflux pump RNA level and sequence analysis revealed that over-expression of the RND-3 efflux pump was attributable to specific mutations in the RND-3 efflux pump regulator gene. In conclusion, high-level expression of efflux pumps is prevalent in <i>B. cepacia</i> complex isolates. Mutations in the RND-3 efflux pump regulator gene are the major cause of efflux pump activity, resulting in the resistance to antibiotics in clinical <i>B. cepacia</i> complex isolates.</p></div

Association of bacterial genotypes and epidemiological features with treatment failure in hemodialysis patients with methicillin-resistant <i>Staphylococcus aureus</i> bacteremia

<div><p>Objectives</p><p>Methicillin-resistant <i>Staphylococcus aureus</i> (MRSA) infections in the hemodialysis (HD) population are epidemiologically classified as healthcare-associated infections. The data about the clinical impact and bacterial characteristics of hospital-onset (HO)- and community-onset (CO)-MRSA in HD patients are scarce. The current study analyzed the difference in the clinical and molecular characteristics of HO-MRSA and CO-MRSA.</p><p>Methods</p><p>We performed a retrospective review and molecular analysis of clinical isolates from 106 HD patients with MRSA bacteremia from 2009 to 2014. CA genotypes were defined as isolates carrying the SCC<i>mec</i> type IV or V, and HA genotypes were defined as isolates harboring SCC<i>mec</i> type I, II, or III.</p><p>Results</p><p>CO-MRSA infections occurred in 76 patients, and 30 patients had HO-MRSA infections. There was no significant difference in the treatment failure rates between patients with CO-MRSA infections and those with HO-MRSA infections. CA genotypes were associated with less treatment failure (odds ratio [OR]: 0.18; 95% confidence interval [95% CI], 0.07–0.49; <i>p</i> = 0.001). For isolates with a vancomycin minimum inhibitory concentration (MIC) < 1.5 mg/L, the multivariate analysis revealed that HA genotypes and cuffed tunneled catheter use were associated with treatment failure. For isolates with a vancomycin MIC ≥1.5 mg/L, the only risk factor for treatment failure was a higher Pitt score (OR: 1.76; 95% CI, 1.02–3.05; <i>p</i> = 0.043).</p><p>Conclusion</p><p>CA genotypes, but not the epidemiological classification of CO-MRSA, impacted the clinical outcome of MRSA bacteremia in the HD population.</p></div

Molecular Epidemiology and Phylogenetic Analysis of Human Adenovirus Caused an Outbreak in Taiwan during 2011

<div><p>An outbreak of adenovirus has been surveyed in Taiwan in 2011. To better understand the evolution and epidemiology of adenovirus in Taiwan, full-length sequence of hexon and fiber coapsid protein was analyzed using series of phylogenetic and dynamic evolution tools. Six different serotypes were identified in this outbreak and the species B was predominant (HAdV-3, 71.50%; HAdV-7, 15.46%). The most frequent diagnosis was acute tonsillitis (54.59%) and bronchitis (47.83%). Phylogenetic analysis revealed that hexon protein gene sequences were highly conserved for HAdV-3 and HAdV-7 circulation in Taiwan. However, comparison of restriction fragment length polymorphism (RFLP) analysis and phylogenetic trees of fiber gene in HAdV-7 clearly indicated that the predominant genotype in Taiwan has shifted from 7b to 7d. Several positive selection sites were observed in hexon protein. The estimated nucleotide substitution rates of hexon protein of HAdV-3 and HAdV-7 were 0.234×10^-3 substitutions/site/year (95% HPD: 0.387~0.095×10^-3) and 1.107×10^-3 (95% HPD: 0. 541~1.604) respectively; those of the fiber protein of HAdV-3 and HAdV-7 were 1.085×10^-3 (95% HPD: 1.767~0.486) and 0.132×10^-3 (95% HPD: 0.283~0.014) respectively. Phylodynamic analysis by Bayesian skyline plot (BSP) suggested that using individual gene to evaluate the effective population size might possibly cause miscalculation. In summary, the virus evolution is ongoing, and continuous surveillance of this virus evolution will contribute to the control of the epidemic.</p></div

Class 1 integron cassette analysis.

<p>Genes are shown as arrows with the direction of transcription indicated by the arrowheads. <i>Int1</i>: class 1 integrase, <i>qacF</i>: quaternary ammonium compound-resistance protein, <i>qacEΔ1</i>: remnants of quaternary ammonium compound resistance protein, <i>sul1</i>: sulphonamides resistance gene, <i>aacA4</i>: aminoglycoside 6′-acetyltransferase, <i>aacA7</i>: aminoglycoside 6′-acetyltransferase, and <i>catB3</i>: chloramphenicol acetyltransferase.</p

Univariate analyses of the association between potential predictor variables and treatment failure in patients with methicillin-resistant <i>Staphylococcus aureus</i> (MRSA) bacteremia.

<p>Univariate analyses of the association between potential predictor variables and treatment failure in patients with methicillin-resistant <i>Staphylococcus aureus</i> (MRSA) bacteremia.</p

A dendrogram of pulsotype relationships developed via the unweighted pair group method using arithmetic averages (UPGMA) with BioNumerics software version 6.5 (Applied Maths).

<p>Pulsotypes were assigned to the same clusters if they exhibited 80% similarity in the dendrogram. Species identification was performed by <i>recA</i>-RFLP and MALDI-TOF analysis for 66 <i>B. cepacia</i> complex isolates.</p

Genetic features of four <i>bla</i>_OXA-48<i>Klebsiella pneumoniae</i>.

<p>Genetic features of four <i>bla</i>_OXA-48<i>Klebsiella pneumoniae</i>.</p

Multivariate analyses of the association between potential predictor variables and treatment failure in patients with methicillin-resistant <i>Staphylococcus aureus</i> (MRSA) bacteremia.

<p>Multivariate analyses of the association between potential predictor variables and treatment failure in patients with methicillin-resistant <i>Staphylococcus aureus</i> (MRSA) bacteremia.</p

Characterization of Fosfomycin Resistant Extended-Spectrum β-Lactamase-Producing <i>Escherichia coli</i> Isolates from Human and Pig in Taiwan

<div><p>To investigate the efficacy of fosfomycin against extended-spectrum β-lactamases (ESBL) producing <i>Escherichia coli</i> in Taiwan and the resistance mechanisms and characterization of human and pig isolates, we analyzed 145 ESBL-producing isolates collected from two hospitals (n = 123) and five farms (n = 22) in Taiwan from February to May, 2013. Antimicrobial susceptibilities were determined. Clonal relatedness was determined by PFGE and multi-locus sequence typing. ESBLs, <i>ampC</i>, and fosfomycin resistant genes were detected by PCR, and their flanking regions were determined by PCR mapping and sequencing. The fosfomycin resistant mechanisms, including modification of the antibiotic target (MurA), functionless transporters (GlpT and UhpT) and their regulating genes such as <i>uhpA</i>, <i>cyaA</i>, and <i>ptsI</i>, and antibiotic inactivation by enzymes (FosA and FosC), were examined. The size and replicon type of plasmids carrying fosfomycin resistant genes were analyzed. Our results revealed the susceptibility rates of fosfomycin were 94% for human ESBL-producing <i>E</i>. <i>coli</i> isolates and 77% for pig isolates. The PFGE analysis revealed 79 pulsotypes. No pulsotype was found existing in both human and pig isolates. Three pulsotypes were distributed among isolates from two hospitals. IS<i>Ecp1</i> carrying <i>bla</i>_{CTX-M-group 9} was the predominant transposable elements of the ESBL genes. Among the thirteen fosfomycin resistant isolates, functionless transporters were identified in 9 isolates. Three isolates contained novel amino acid substitutions (Asn67Ile, Phe151Ser and Trp164Ser, Val146Ala and His159Tyr, respectively) in MurA (the target of fosfomycin). Four isolates had fosfomycin modified enzyme (<i>fosA3</i>) in their plasmids. The <i>fosA3</i> gene was harboured in an IncN-type plasmid (101 kbp) in the three pig isolates and an IncB/O-type plasmid (113 kbp) in the human isolate. In conclusion, we identified that 6% and 23% of the ESBL-producing <i>E</i>. <i>coli</i> from human and pigs were resistant to fosfomycin, respectively, in Taiwan. No clonal spread was found between human and pig isolates. Functionless transporters were the major cause of fosfomycin resistance, and the <i>fosA3</i>-transferring plasmid between isolates warrants further monitoring.</p></div

EcoRI-digested plasmid digestion profile of four OXA-48-producing <i>Klebsiella pneumoniae</i> isolates.

<p>M, marker.</p