Sook Jin Jang

Chromosome-encoded AmpC and CTX-M extended-spectrum β-lactamases in clinical isolates of Proteus mirabilis from Korea

ABSTRACT Among 222 Proteus mirabilis clinical isolates collected from 17 hospitals in Korea in 2008, 28 (12.6%) and 8 (3.6%) isolates exhibited extended-spectrum β-lactamase (ESBL) and AmpC phenotypes, respectively. The most common type of ESBL gene identified by PCR and sequencing experiments was bla CTX-M-14a (n = 12). The bla CTX-M-90 (n = 4), bla CTX-M-15 (n = 3), bla CTX-M-12 (n = 3), bla CTX-M-2 (n = 2), bla CTX-M-14b (n = 1), bla TEM-52 (n = 5), and bla SHV-12 (n = 1) genes were also detected. Eight isolates carried an AmpC β-lactamase gene, such as bla CMY-2 (n = 6) or bla DHA-1 (n = 2). All bla genes encoding CTX-M-1- and CTX-M-9-type enzymes and all bla CMY-2 genes were preceded by ISEcp1-like elements. The bla CTX-M-2 gene found in two isolates was located on a complex class 1 integron. The bla DHA-1 gene was preceded by a transcriptional regulator gene and was followed by phage shock protein genes. The bla CTX-M genes were located on the chromosome in 21 isolates. A plasmid location for the bla CTX-M gene was found in only four isolates: the bla CTX-M-14a gene was located on ∼150-kbp IncA/C plasmids in three isolates and on a ∼50-kbp IncN plasmid in one isolate. The bla TEM-52 gene was located on ∼50-kbp IncN plasmids in all five isolates. The AmpC β-lactamase genes were located on the chromosome in seven of eight isolates; one isolate carried the bla CMY-2 gene on a ∼150-kbp IncA/C plasmid. Our results show that a chromosomal location of CTX-M ESBL and AmpC β-lactamase genes in P. mirabilis is no longer an unusual phenomenon in hospital environments.

In Vitro Efficacy of the Combination of Ciprofloxacin and Cefotaxime against Vibrio vulnificus

Objectives The in vivo efficacy of a cefotaxime-ciprofloxacin combination against Vibrio vulnificus and the effects on rtxA1 expression of commonly used antibiotics are unknown. Methods In vitro time-kill studies were performed to evaluate synergism. Female BALB/c mice were injected subcutaneously with 1×107 or 1×108 cfu of V. vulnificus. Antibiotic therapy was initiated at 2 h after inoculation in the following four therapy groups: cefotaxime; ciprofloxacin; cefotaxime-plus-ciprofloxacin; and cefotaxime-plus-minocycline. The cytotoxicity of V. vulnificus for HeLa cells was measured using the lactate dehydrogenase assay; rtxA1 transcription was measured in a transcriptional reporter strain using a β-galactosidase assay. Results In vitro time-kill assays exhibited synergism between cefotaxime and ciprofloxacin. In the animal experiments, the 96-h survival rate for the cefotaxime-plus-ciprofloxacin group (85%; 17/20) was significantly higher than that of the cefotaxime-plus-minocycline (35%; 7/20) and cefotaxime alone (0%; 0/20) groups (P<0.05 for both). Bacterial counts in the liver and spleen were significantly lower in the cefotaxime-plus-ciprofloxacin group 24 and 48 h after treatment, relative to the other groups. At sub-inhibitory concentrations, ciprofloxacin inhibited more effectively rtxA1 transcription and mammalian cell cytotoxicity than either minocycline or cefotaxime (P<0.05 for both). Conclusions Ciprofloxacin is more effective at reducing rtxA1 transcription and subsequent cytotoxicity than either minocycline or cefotaxime, and the combination of ciprofloxacin and cefotaxime was more effective in clearing V. vulnificus in vivo than previously used regimens. These data suggest that the combination of ciprofloxacin and cefotaxime is an effective option for the treatment of V. vulnificus sepsis in humans.

Comparison of rpoB gene sequencing, 16S rRNA gene sequencing, gyrB multiplex PCR, and the VITEK2 system for identification of Acinetobacter clinical isolates

Since accurate identification of species is necessary for proper treatment of Acinetobacter infections, we compared the performances of 4 bacterial identification methods using 167 Acinetobacter clinical isolates to identify the best identification method. To secure more non-baumannii Acinetobacter (NBA) strains as target strains, we first identified Acinetobacter baumannii in a total of 495 Acinetobacter clinical isolates identified using the VITEK 2 system. Because 371 of 495 strains were identified as A. baumannii using gyrB multiplex 1 PCR and blaOXA51-like PCR, we performed rpoB gene sequencing and 16S rRNA gene sequencing on remaining 124 strains belonging to NBA and 52 strains of A. baumannii. For identification of Acinetobacter at the species level, the accuracy rates of rpoB gene sequencing, 16S rRNA gene sequencing, gyrB multiplex PCR, and the VITEK 2 were 98.2%, 93.4%, 77.2%, and 35.9%, respectively. The gyrB multiplex PCR seems to be very useful for the detection of ACB complex because its concordance rates to the final identification of strains of ACB complex were 100%. Both the rpoB gene sequencing and the 16S rRNA gene sequencing may be useful in identifying Acinetobacter.

Interspecies Dissemination of the bla Gene Encoding PER-1 Extended-Spectrum β-Lactamase

ABSTRACT PER-1 extended-spectrum β-lactamase-producing Gram-negative bacilli are resistant to oxyimino-cephalosporins. However, the bla PER-1 gene has never been reported in Klebsiella pneumoniae. Here, we studied interspecies dissemination of the bla PER-1 gene by horizontal transfer of Tn1213 among Acinetobacter baumannii, Pseudomonas aeruginosa, and K. pneumoniae. In a K. pneumoniae clinical isolate, the bla PER-1 gene was located on a 150-kbp incompatibility group A/C plasmid.

Detection of Amphotericin B Resistance in Candida haemulonii and Closely Related Species by Use of the Etest, Vitek-2 Yeast Susceptibility System, and CLSI and EUCAST Broth Microdilution Methods

ABSTRACT The emerging fungal pathogens Candida haemulonii and Candida pseudohaemulonii often show high-level resistance to amphotericin B (AMB). We compared the utilities of five antifungal susceptibility testing methods, i.e., the Etest using Mueller-Hinton agar supplemented with glucose and methylene blue (Etest-MH), the Etest using RPMI agar supplemented with glucose (Etest-RPG), the Vitek-2 yeast susceptibility system, and the Clinical and Laboratory Standards Institute (CLSI) and European Committee on Antimicrobial Susceptibility Testing (EUCAST) broth microdilution methods, for the detection of AMB-resistant isolates of C. haemulonii and closely related species. Thirty-eight clinical isolates (8 C. haemulonii, 10 C. pseudohaemulonii, and 20 Candida auris isolates) were analyzed. Of the 18 C. haemulonii and C. pseudohaemulonii isolates, 18, 15, 18, 10, and 9 exhibited AMB MICs of >1 μg/ml by the Etest-MH, Etest-RPG, Vitek-2, CLSI, and EUCAST methods, respectively. All 20 C. auris isolates showed AMB MICs of ≤1 μg/ml by all five methods. Of the methods, the Etest-MH generated the broadest distribution of AMB MICs for all 38 isolates and showed the best discrimination between the C. haemulonii and C. pseudohaemulonii isolates (4 to 32 μg/ml) and those of C. auris (0.125 to 0.5 μg/ml). Taking the Etest-MH as the reference method, the essential agreements (within two dilutions) for the Etest-RPG, Vitek-2, CLSI, and EUCAST methods were 84, 92, 55, and 55%, respectively; the categorical agreements were 92, 92, 79, and 76%, respectively. This study provides the first data on the efficacy of the Etest-MH and its excellent agreement with Vitek-2 for discriminating AMB-resistant from AMB-susceptible isolates of these Candida species.

Comparison of Conventional, Nested, and Real-Time PCR Assays for Rapid and Accurate Detection of Vibrio vulnificus

ABSTRACT We conducted a prospective study to target toxR in the blood of patients with skin and soft tissue infections who were admitted to four tertiary hospitals to assess the clinical usefulness of real-time quantitative PCR (Q-PCR) as a diagnostic technique. We performed conventional PCR (C-PCR), nested PCR (N-PCR), and Q-PCR assays and compared the results to those obtained using the “gold standard” of microbiological culture. The lower detection limit for the Q-PCR assay was 5 × 100 copies/μl. By use of blood samples of patients with skin and soft tissue infections, the sensitivities of the C-PCR and N-PCR assays against the target toxR gene of V. vulnificus as diagnostic tools were determined to be 45% and 86%, respectively. The C-PCR and N-PCR assays had specificities of 100% and 73%, respectively. When we adopted a crossing-point (cp) cutoff value of <38 cp as a positive result, the Q-PCR assay had 100% sensitivity and specificity. Q-PCR to detect V. vulnificus-specific genes is not only the most sensitive and specific of the techniques but also the most rapid diagnostic method. Therefore, the appropriate application of the Q-PCR assay using blood is useful for the rapid diagnosis and subsequent treatment of V. vulnificus sepsis.

Frequency of Extended-spectrum β-lactamase (ESBL) and AmpC β-lactamase Genes in Escherichia coli and Klebsiella pneumoniae over a Three-year Period in a University Hospital in Korea

BACKGROUND The aim of this study was to determine the yearly prevalence and genotype distribution of extended-spectrum β-lactamase (ESBL)-producing Escherichia coli and Klebsiella pneumoniae collected over a 3-yr period in Gwangju, Korea. METHODS Clinical isolates of E. coli and K. pneumoniae collected at Chosun University Hospital from September 15, 2005 to September 14, 2008 were evaluated. Antimicrobial susceptibility testing was performed using the Vitek II system (bioMérieux, USA) and agar dilution methods. Screening for ESBL and AmpC β-lactamase genes was performed using PCR amplification of plasmid DNA followed by direct sequencing of the PCR products. RESULTS The percentage of ESBL-producing isolates was 12.6% (196/1,550) for E. coli and 26.2% (294/1,121) for K. pneumoniae. The ESBL gene sequencing results showed that the most prevalent ESBL types were CTX-M (93.5%) and SHV (12.9%) in E. coli, and SHV (73.2%) and CTX-M (46.3%) in K. pneumoniae. The most common ESBL in E. coli was CTX-M-15-like, followed by CTX-M-14-like, SHV-2a-like, and SHV-12-like. The most prevalent ESBL type in K. pneumoniae was SHV-12, followed by CTX-M-14-like and CTX-M-15-like. Fifty-one percent (21/41) of ESBL-producing K. pneumoniae with ESBL types verified by sequencing also had DHA-1-like AmpC β-lactamases. However, none of the ESBL-producing E. coli was positive in the AmpC β-lactamase PCR analysis. CONCLUSIONS In this study, the most common types of class A ESBLs identified were CTX-M-15-like in E. coli and SHV-12-like in K. pneumoniae.

Increasing Resistance to Extended-Spectrum Cephalosporins, Fluoroquinolone, and Carbapenem in Gram-Negative Bacilli and the Emergence of Carbapenem Non-Susceptibility in Klebsiella pneumoniae: Analysis of Korean Antimicrobial Resistance Monitoring System (KARMS) Data From 2013 to 2015

Background National surveillance of antimicrobial resistance becomes more important for the control of antimicrobial resistance and determination of treatment guidelines. We analyzed Korean Antimicrobial Resistance Monitoring System (KARMS) data collected from 2013 to 2015. Methods Of the KARMS participants, 16 secondary or tertiary hospitals consecutively reported antimicrobial resistance rates from 2013 to 2015. Data from duplicate isolates and institutions with fewer than 20 isolates were excluded. To determine the long-term trends, previous KARMS data from 2004 to 2012 were also considered. Results The prevalence of methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus faecium from 2013 to 2015 was 66–72% and 29–31%, respectively. The resistance rates of Escherichia coli to cefotaxime and cefepime gradually increased to 35% and 31%, respectively, and fluoroquinolone resistance reached 48% in 2015. The resistance rates of Klebsiella pneumoniae to cefotaxime, cefepime, and carbapenem were 38–41%, 33–41%, and <0.1–2%, respectively, from 2013 to 2015. The carbapenem susceptibility rates of E. coli and K. pneumoniae decreased from 100% and 99.3% in 2011 to 99.0% and 97.0% in 2015, respectively. The resistance rate of Pseudomonas aeruginosa to carbapenem increased to 35% and the prevalence of carbapenem-resistant Acinetobacter baumannii increased from 77% in 2013 to 85% in 2015. Conclusions Between 2013 and 2015, the resistance rates of E. coli to third- and fourth-generation cephalosporins increased continuously, while carbapenem-susceptibility gradually decreased, particularly in K. pneumoniae. The prevalence of carbapenem-resistant P. aeruginosa and A. baumannii increased significantly; therefore, few treatment options remain for these resistant strains.

In vitro efficacy of the combination of ciprofloxacin and cefotaxime against nalidixic acid-resistant Salmonella enterica serotype Typhi.

Typhoid fever is a systemic intracellular infection caused by Salmonellaenterica serotype Typhi. The emergence and spread of nalidixic acid-resistant S. Typhi (NARST) is challenging for clinicians in many countries owing to the lack of suitable treatment options. The aim of this study was to identify in vitro synergistic combinations of antibiotics against S. Typhi. In vitro time-kill studies were performed on three clinical NARST isolates and one type strain of nalidixic acid-susceptible S. Typhi (NASST) ATCC 9992 with ciprofloxacin, cefotaxime and azithromycin in various combinations. The combination of ciprofloxacin (0.012-0.375 microg/mL) and cefotaxime (0.063-0.125 microg/mL) against all three NARST strains and the NASST strain was significantly more effective in vitro in reducing bacterial counts by >or=3log(10) colony-forming units at 24h and showed synergistic effects. Combination therapy with ciprofloxacin and cefotaxime might be the treatment of choice for patients with typhoid fever. The combination of a fluoroquinolone and a beta-lactam, which are directed against different targets, may improve efficacy compared with a fluoroquinolone alone and may reduce the chance of fluoroquinolone-resistant mutants emerging in patients with severe typhoid fever.

Evaluation of matrix-assisted laser desorption/ionization time-of-fight mass spectrometry for identification of 345 clinical isolates of Aspergillus species from 11 Korean hospitals: comparison with molecular identification

We evaluated the ability of the Filamentous Fungi Library 1.0 of the MALDI-TOF MS Biotyper system to identify 345 clinical Aspergillus isolates from 11 Korean hospitals. Compared with results of the internal transcribed spacer region sequencing, the frequencies of correct identification at the species-complex level were 94.5% and 98.8% with cutoff values of 2.0 and 1.7, respectively. Compared with results of β-tubulin gene sequencing, the frequencies of correct identification at the species level were 96.0% (cutoff 2.0) and 100% (cutoff 1.7) for 303 Aspergillus isolates of five common, non-cryptic species, but only 4.8% (cutoff 1.7) and 0% (cutoff 2.0) for 42 Aspergillus isolates of six cryptic species (identifiable by β-tubulin or calmodulin sequencing). These results show that the MALDI Biotyper using the Filamentous Fungi Library version 1.0 enables reliable identification of the majority of common clinical Aspergillus isolates, although the database should be expanded to facilitate identification of cryptic species.