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

Antibiotic resistant genes of the ESBL-producing <i>E</i>. <i>coli</i> human and pig isolates.

<p>a: TEM-1 was determined by sequencing.</p><p>Antibiotic resistant genes of the ESBL-producing <i>E</i>. <i>coli</i> human and pig isolates.</p

Representation of the sequences flanking <i>fosA3</i>.

<p>Genes are shown as arrows with the direction of transcription indicated by the arrowheads.</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. Three major clusters (XXIX, XXXIV, and XLIV) were found in two hospitals.</p

Evaluation of fosfomycin susceptibility in overexpressing wild type and mutant MurA.

<p>MIC, minimal inhibitory concentration.</p><p>^a MICs were determined in the presence of 1mM Isopropyl β-D-1-thiogalactopyranoside (IPTG) to induce MurA expression.</p><p>Evaluation of fosfomycin susceptibility in overexpressing wild type and mutant MurA.</p

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

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

Characteristics of QRDR mutations and efflux pump activity of six fluoroquinolone-resistant <i>B. cepacia</i> complex isolates.

<p>Characteristics of QRDR mutations and efflux pump activity of six fluoroquinolone-resistant <i>B. cepacia</i> complex isolates.</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

Among 42 antibiotic-resistant clinical isolates, the efficacy of efflux pumps was tested via antibiotic susceptibilities: ceftazidime (CAZ), meropenem (MEM), minocycline (MI), trimethoprim/sulfamethoxazole (SxT), levofloxacin (LEVO), and chloramphenicol (C) in the presence or absence of 60 µM CCCP.

<p>Efflux pump activity was defined as present at least 4-fold decrease of MIC in the presence of CCCP. *, <i>p</i><0.05 (statistical analysis using the Mann-Whitney U test).</p