Eun-Jeong Yoon

Translational Attenuation and mRNA Stabilization as Mechanisms of erm(B) Induction by Erythromycin

ABSTRACT Translational attenuation has been proposed to be the mechanism by which the erm(B) gene is induced. Here, we report genetic and biochemical evidence, obtained by using erythromycin as the inducing antibiotic, that supports this hypothesis. We also show that erythromycin increases the level of the erm(B) transcript by stalling the ribosome on the leader mRNA and thereby facilitating the stabilization and processing of the mRNA. Erythromycin-induced mRNA stabilization and processing were observed with an ochre stop at codons 11 to 13 of the leader but not with an ochre stop at codon 10. This suggests that erythromycin does not stall the ribosome before codon 11 of the leader reaches the aminoacyl site. Secondary structure analyses of the erm(B) transcripts by in vitro and in vivo chemical probing techniques identified conformational changes in the transcripts that result from induction by erythromycin. These findings demonstrate that stalling of erythromycin-bound ribosomes at leader codon 11 causes the refolding of mRNA into a conformation in which the translational initiation site for the structural gene is unmasked and renders erm(B) translationally active.

Clonal Dissemination of Pseudomonas aeruginosa Sequence Type 235 Isolates Carrying blaIMP-6 and Emergence of blaGES-24 and blaIMP-10 on Novel Genomic Islands PAGI-15 and -16 in South Korea

ABSTRACT A total of 431 Pseudomonas aeruginosa clinical isolates were collected from 29 general hospitals in South Korea in 2015. Antimicrobial susceptibility was tested by the disk diffusion method, and MICs of carbapenems were determined by the agar dilution method. Carbapenemase genes were amplified by PCR and sequenced, and the structures of class 1 integrons surrounding the carbapenemase gene cassettes were analyzed by PCR mapping. Multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) were performed for strain typing. Whole-genome sequencing was carried out to analyze P. aeruginosa genomic islands (PAGIs) carrying the blaIMP-6, blaIMP-10, and blaGES-24 genes. The rates of carbapenem-nonsusceptible and carbapenemase-producing P. aeruginosa isolates were 34.3% (148/431) and 9.5% (41/431), respectively. IMP-6 was the most prevalent carbapenemase type, followed by VIM-2, IMP-10, and GES-24. All carbapenemase genes were located on class 1 integrons of 6 different types on the chromosome. All isolates harboring carbapenemase genes exhibited genetic relatedness by PFGE (similarity > 80%); moreover, all isolates were identified as sequence type 235 (ST235), with the exception of two ST244 isolates by MLST. The blaIMP-6, blaIMP-10, and blaGES-24 genes were found to be located on two novel PAGIs, designated PAGI-15 and PAGI-16. Our data support the clonal spread of an IMP-6-producing P. aeruginosa ST235 strain, and the emergence of IMP-10 and GES-24 demonstrates the diversification of carbapenemases in P. aeruginosa in Korea.

The blaOXA-23-associated transposons in the genome of Acinetobacter spp. represent an epidemiological situation of the species encountering carbapenems

Objectives High rates of carbapenem resistance in the human pathogen Acinetobacter baumannii threaten public health and need to be scrutinized. Methods A total of 356 A. baumannii and 50 non-baumannii Acinetobacter spp. (NBA) strains collected in 2013 throughout South Korea were studied. The type of blaOXA-23 transposon was determined by PCR mapping and molecular epidemiology was assessed by MLST. Twelve representative strains and two comparative A. baumannii were entirely sequenced by single-molecule real-time sequencing. Results The carbapenem resistance rate was 88% in A. baumannii, mainly due to blaOXA-23, with five exceptional cases associated with ISAba1-blaOXA-51-like. The blaOXA-23 gene in A. baumannii was carried either by Tn2006 (44%) or Tn2009 (54%), with a few exceptions carried by Tn2008 (1.6%). Of the NBA strains, 14% were resistant to carbapenems, two with blaOXA-58 and five with blaOXA-23 associated with Tn2006. The Tn2006-possessing strains belonged to various STs, whereas Tn2008- and Tn2009-possessing strains were limited to ST208 and ST191, respectively. The three transposons were often multiplied in the chromosome, and the gene copy number and the carbapenem MICs presented linear relationships either very strongly for Tn2008 or moderately for Tn2006 and Tn2009. Conclusions The dissemination of Tn2006 was facilitated by its capability for intercellular transfer and that of Tn2009 was attributable to successful dissemination of the ST191 bacterial host carrying the transposon. Tn2008 was infrequent because of its insufficient ability to undergo intercellular transfer and the scarce bacterial host A. baumannii ST208. Gene amplification is an adaptive mechanism for bacteria that encounter antimicrobial drugs.

Carbapenemase-producing Enterobacteriaceae in South Korea: a report from the National Laboratory Surveillance System

AIM To assess the epidemiology of carbapenemase-producing Enterobacteriaceae (CPE) in South Korea. MATERIALS & METHODS From 2011 to 2015, 2487 carbapenem-nonsusceptible Enterobacteriaceae were collected through the Korean National Laboratory Surveillance System. Disk-diffusion for antimicrobial susceptibility, PCR/sequencing to detect carbapenemase genes and multilocus sequence typing for molecular epidemiology were carried out. RESULTS The number of carbapenem-nonsusceptible Enterobacteriaceae was increasing approximately 1.5-fold per year and the proportion of CPEs was exponentially confirmed from 2014. KPC was the most dominant, mostly associated with Klebsiella pneumoniae ST11 and ST307, NDM was the second and OXA-48-like was the third dominant carbapenemases. The IMP, VIM and GES-5 CPEs were identified sporadically. CONCLUSION The nation-wide spreads of KPC, NDM and OXA-48-like CPEs were in an alarming epidemiological stage.

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.

Detection of mcr-1 Plasmids in Enterobacteriaceae Isolates From Human Specimens: Comparison With Those in Escherichia coli Isolates From Livestock in Korea

Background The emerging mobile colistin resistance gene, mcr-1, is an ongoing worldwide concern and an evaluation of clinical isolates harboring this gene is required in Korea. We investigated mcr-1-possessing Enterobacteriaceae among Enterobacteriaceae strains isolated in Korea, and compared the genetic details of the plasmids with those in Escherichia coli isolates from livestock. Methods Among 9,396 Enterobacteriaceae clinical isolates collected between 2010 and 2015, 1,347 (14.3%) strains were resistant to colistin and those were screened for mcr-1 by PCR. Colistin minimum inhibitory concentrations (MICs) were determined by microdilution, and conjugal transfer of the mcr-1-harboring plasmids was assessed by direct mating. Whole genomes of three mcr-1-positive Enterobacteriaceae clinical isolates and 11 livestock-origin mcr-1-positive E. coli isolates were sequenced. Results Two E. coli and one Enterobacter aerogenes clinical isolates carried carried IncI2 plasmids harboring mcr-1, which conferred colistin resistance (E. coli MIC, 4 mg/L; E. aerogenes MIC, 32 mg/L). The strains possessed the complete conjugal machinery except for E. aerogenes harboring a truncated prepilin peptidase. The E. coli plasmid transferred more efficiently to E. coli than to Klebsiella pneumoniae or Enterobacter cloacae recipients. Among the three bacterial hosts, the colistin MIC was the highest for E. coli owing to the higher mcr-1-plasmid copy number and mcr-1 expression levels. Ten mcr-1-positive chicken-origin E. coli strains also possessed mcr-1-harboring IncI2 plasmids closely related to that in the clinical E. aerogenes isolate, and the remaining one porcine-origin E. coli possessed an mcr-1-harboring IncX4 plasmid. Conclusions mcr-1-harboring IncI2 plasmids were identified in clinical Enterobacteriaceae isolates. These plasmids were closely associated with those in chicken-origin E. coli strains in Korea, supporting the concept of mcr-1 dissemination between humans and livestock.

Antimicrobial resistance and virulence factors of Klebsiella pneumoniae affecting 30 day mortality in patients with bloodstream infection

Objectives To investigate the risk factors of patients with Klebsiella pneumoniae (KP) bloodstream infection (BSI) with a focus on antimicrobial resistance and virulence factors. Methods All KP BSI patients (n = 579) from six general hospitals during a 1 year period were included in this study. The risk factors of hosts and causative KP isolates were assessed to determine associations with the 30 day mortality of KP BSI patients by multivariate Cox hazards modelling. Results The 30 day mortality rate of KP BSI patients was 16.9% (98/579). Among the host-associated factors, increased SOFA score and leucopenia status exhibited strong associations with increased 30 day mortality. Among the pathogenic factors, carriage of the pks gene cluster (adjusted HR 1.80; 95% CI 1.16-2.79) was a risk factor, especially when accompanied by MDR. In this regard, KP isolates of the wzi50 capsular type (n = 22) frequently harboured pks (63.6%, 14/22) and ybtA (68.2%, n = 15) and mostly exhibited MDR (63.6%, n = 14), resulting in increased 30 day mortality. In contrast, hypermucoviscous KP isolates showed an inverse association with 30 day mortality (adjusted HR 0.55; 95% CI 0.33-0.90). Conclusions Despite the reported virulence of hypermucoviscous KP strains, they were associated with good prognoses in KP BSI patients. Importantly, carriage of the pks gene cluster, which is responsible for the synthesis of colibactin, was a relevant marker of early mortality.