Evaluation of Disk carbapenemase test using improved disks for rapid detection and differentiation of clinical isolates of carbapenemase-producing Enterobacterales.

OBJECTIVES: Rapid detection of carbapenemase-producing Enterobacterales (CPE) is important to control spread of the resistance. We previously reported that imipenem disks prepared from injectable imipenem-cilastatin could rapidly detect KPC- and NDM-type carbapenemases. In the present study, we evaluated performance of disks of IPM and combined disks of imipenem-tazobactam and imipenem-EDTA, which were prepared from powders of imipenem and inhibitors. METHODS: Isolates of Enterobacterales were recovered from specimens of patients at a tertiary care hospital in Korea during January 2017 and March 2018. Routine CPE detection was performed by the CPE surveillance personnel whereas evaluation of the Disk carbapenemase test (DCT) was performed by the other personnel without knowing the results of surveillance. The DCT was carried out by pressing disks on to colonies and rehydrating in Petri plates and observing color change. RESULTS: The DCT differentiated 688 of 694 (sensitivity 99.1%) carbapenemase-producing isolates in 2.5-20 min: 630 with KPC, 51 with NDM, three with IMP, one with VIM, two with KPC and IMP, and one with NDM and OXA-181. The DCT failed to detect six OXA- 48-like enzyme-producing isolates, but the modified method using 96-well flat-bottom microplates with mineral oil cover detected all 29 OXA-48-like enzyme-producing isolates in 20-120 min. The DCT was negative for all 440 ertapenem-nonsusceptible, carbapenemase gene-negative isolates (specificity 100%). CONCLUSION: The procedure of DCT is simple and can differentiate isolates of Enterobacterales with KPC-, NDM-, IMP- and VIM-type carbapenemases rapidly, and the modified DCT can detect isolates with OXA-48-like enzymes rapidly.

First Report of the Carbapenemase Gene blaOXA-499 in Acinetobacter pittii.

We identified the carbapenemase gene blaOXA-499, a variant of blaOXA-143, from a clinical isolate of Acinetobacter pittii for the first time. OXA-499 shared 93.1% amino acid identity with OXA-143, and the gene was located on the chromosome. By cloning the OXA-499-encoding gene into the pWH1266 vector and transforming it into susceptible Acinetobacter spp., we were able to show that OXA-499 confers resistance to carbapenems.

Relative Prevalence and Antimicrobial Susceptibility of Clinical Isolates of Elizabethkingia Species Based on 16S rRNA Gene Sequencing.

Some of the previously reported clinical isolates of Elizabethkingia meningoseptica may be later named species of Elizabethkingia We determined the accuracy of species identification (with two matrix-assisted laser desorption ionization-time of flight mass spectrometry [MALDI-TOF MS] systems and the Vitek 2 GN card), relative prevalence of three Elizabethkingia spp. in clinical specimens, and antimicrobial susceptibility of the species identified by 16S rRNA gene sequencing. Specimens for culture were collected from patients in a university hospital in Seoul, South Korea, between 2009 and 2015. All 3 Elizabethkingia spp. were detected in patients; among the 86 isolates identified by 16S rRNA gene sequencing, 17 (19.8%) were E. meningoseptica, 18 (20.9%) were Elizabethkingia miricola, and 51 (59.3%) were Elizabethkingia anophelis Only the MALDI-TOF Vitek MS system with an amended database correctly identified all of the isolates. The majority (76.7%) of the isolates were from the lower respiratory tract, and 8 (9.3%) were from blood. Over 90% of E. meningoseptica and E. anophelis isolates were susceptible to piperacillin-tazobactam and rifampin. In contrast, all E. miricola isolates were susceptible to fluoroquinolones except ciprofloxacin. Further studies are urgently needed to determine the optimal antimicrobial agents for the treatment of infections due to each individual Elizabethkingia species.

Molecular epidemiology and resistome analysis of multidrug-resistant ST11 Klebsiella pneumoniae strain containing multiple copies of extended-spectrum ß-lactamase genes using whole-genome sequencing.

The aim of this work was to investigate the mechanism responsible for multidrug resistance in ST11 Klebsiella pneumoniae YMC 2013/7/B3993 containing multiple copies of ESBL genes using multiple parallel sequencing technology. In-depth analysis of the strain revealed multiple copies of ESBL genes, 2 copies of blaSHV-12 and 1 copy of blaCTX-M-15. Furthermore, 1 copy of blaOXA-9 and 3 copies of blaTEM-1 were found. The insertion of Tn1331 was detected, which consisted of blaOXA-9, blaTEM-1, aac(6')-lb-cr, and aadA1 genes. The acquisition of multiple copies of resistance genes was due to the insertion of transposons in the bacterial genome and plasmid. The genotypic analysis revealed that the isolates belonging to ST11 showed severe resistance phenotypes and greater dissemination potential. To the best of our knowledge, this is the first report demonstrating multiple copies of same ESBL genes in K. pneumoniae ST11 isolate. Furthermore, massive parallel sequencing studies of genetic factors to enhance the fitness of this type strain would be warranted to determine whether ST11 K. pneumoniae can spread the KPC-type gene.

Biochemical characterization of the POM-1 metallo-ß-lactamase from Pseudomonas otitidis.

The POM-1 metallo-ß-lactamase is a subclass B3 resident enzyme produced by Pseudomonas otitidis, a pathogen causing otic infections. The enzyme was overproduced in Escherichia coli BL21(DE3), purified by chromatography, and subjected to structural and functional analysis. The purified POM-1 is a tetrameric enzyme of broad substrate specificity with higher catalytic activities with penicillins and carbapenems than with cephalosporins.

Emergence of decreased susceptibility and resistance to extended-spectrum cephalosporins in Neisseria gonorrhoeae in Korea.

OBJECTIVES: Antimicrobial resistance (AMR) in Neisseria gonorrhoeae is a major concern globally; however, no comprehensive AMR data for gonococcal isolates cultured after 2006 in Korea have been published internationally. We determined the susceptibility of N. gonorrhoeae isolates cultured in 2011-13, the mechanism of extended-spectrum cephalosporin (ESC) resistance and the molecular epidemiology of gonococcal strains in Korea. METHODS: In 2011-13, 210 gonococcal isolates were collected in Korea and their AMR profiles were examined by the agar dilution method. The penA, mtrR, penB, ponA and pilQ genes were sequenced in 25 isolates that were resistant to ESCs and 70 randomly selected isolates stratified by year. For molecular epidemiology, N. gonorrhoeae multiantigen sequence typing and MLST were performed. RESULTS: None of the N. gonorrhoeae isolates was susceptible to penicillin G and most were resistant to tetracycline (50%) and ciprofloxacin (97%). The rates of resistance to ceftriaxone, azithromycin, cefpodoxime and cefixime were 3%, 5%, 8% and 9%, respectively. However, all isolates were susceptible to spectinomycin. Twenty-one (84%) of the 25 ESC-resistant isolates contained the non-mosaic PBP2 XIII allele; however, the remaining 4 (16%) possessed the mosaic PBP2 X allele, which has been previously associated with ESC resistance including treatment failures. CONCLUSIONS: In Korea, susceptibility to spectinomycin remains high. However, the recent emergence of ESC-resistant N. gonorrhoeae strains, including strains possessing the PBP2 mosaic X and non-mosaic XIII alleles, is a major concern and enhanced AMR surveillance is necessary to prevent transmission of these strains.

First outbreak of KPC-2-producing Klebsiella pneumoniae sequence type 258 in a hospital in South Korea.

In this study, we report the first outbreak of KPC-2-producing Klebsiella pneumoniae isolates from three patients admitted to a neurosurgery department in a South Korean teaching hospital. Multilocus sequence typing showed that the isolates were identical to the previous KPC producers in South Korea and other countries, suggesting clonal spread.

Complete genome sequence of the bacteriophage YMC01/01/P52 PAE BP, which causes lysis of verona integron-encoded metallo-ß-lactamase-producing, carbapenem-resistant Pseudomonas aeruginosa.

Multidrug-resistant Pseudomonas aeruginosa commonly causes serious nosocomial infections. In this study, a novel lytic bacteriophage belonging to a member of the family Podoviridae, YMC01/01/P52 PAE BP, which infects carbapenem-resistant Pseudomonas aeruginosa, was isolated and characterized. YMC01/01/P52 PAE BP genome was analyzed by whole-genome sequencing and putative function identification. The bacteriophage genome consists of a double-stranded linear DNA genome of 49,381 bp with a GC content of 62.16%.

Complete genome sequence of the podoviral bacteriophage YMC/09/02/B1251 ABA BP, which causes the lysis of an OXA-23-producing carbapenem-resistant Acinetobacter baumannii isolate from a septic patient.

The emergence of carbapenem-resistant Acinetobacter baumannii, responsible for causing nosocomial infections, has been becoming a significant global health issue. In this article, we report the complete genome sequence of bacteriophage B-B1251 (YMC/09/02/B1251 ABA BP), which causes lysis of a carbapenem-resistant A. baumannii strain. The bacteriophage belongs to the family Podoviridae and has a double-stranded circular DNA genome with a length of 45,364 bp and a 39.05% G+C content. Genome analysis showed that it had no similarity to other previously reported bacteriophages capable of infecting A. baumannii.

Antimicrobial susceptibility of stenotrophomonas maltophilia isolates from Korea, and the activity of antimicrobial combinations against the isolates.

The aim of this study was to determine antimicrobial susceptibility of recent clinical Stenotrophomonas maltophilia isolates from Korea, and to compare the activity levels of several combinations of antimicrobials. A total of 206 non-duplicate clinical isolates of S. maltophilia was collected in 2010 from 11 university hospitals. Antimicrobial susceptibility testing was performed using the Clinical Laboratory Standards Institute agar dilution method. In vitro activity of antimicrobial combinations was tested using the checkerboard method. The susceptibility rates to trimethoprim-sulfamethoxazole and minocycline were 96% and 99%, respectively. The susceptibility rate to levofloxacin was 64%. All of four antimicrobial combinations showed synergy against many S. maltophilia isolates. A combination of trimethoprim-sulfamethoxazole plus ticarcillin-clavulanate was most synergistic among the combinations. None of the combinations showed antagonistic activity. Therefore, some of the combinations may be more useful than individual drugs in the treatment of S. maltophilia infection. Further clinical studies are warranted to validate our in vitro test results.

Evaluation of double-disk potentiation and disk potentiation tests using dipicolinic acid for detection of metallo-ß-lactamase-producing pseudomonas spp. and Acinetobacter spp.

Accurate detection of metallo-ß-lactamase (MBL)-producing Pseudomonas spp. and Acinetobacter spp. became very important with the increasing prevalence of carbapenem-nonsusceptible clinical isolates. The performance of phenotypic MBL detection methods may depend on the types of MBL and the characteristics of the isolates. A high false-positive rate is a problem with EDTA-based MBL detection methods. We evaluated the performance of double-disk potentiation tests (DDPTs) and disk potentiation tests (DPTs) with dipicolinic acid (DPA) using 44 isolates of Pseudomonas spp. and Acinetobacter spp. producing IMP-1-like, VIM-2-like, and SIM-1 type MBLs. Also, we characterized P. aeruginosa isolates with positive imipenem (IPM)-DPA DDPT, but negative meropenem (MEM)-DPA DDPT, and determined possibility of improving a DDPT by using MacConkey agar. Among five different DDPT methods, the IPM-DPA 250-µg method showed the highest sensitivity (97.7%) and specificity (100%). Among four DPT tests, the highest sensitivity (100%) was shown by the IPM-EDTA 1,900-µg disk method, but the specificity was very low (11.4%). Five of six P. aeruginosa isolates with false-negative DDPTs with MEM-DPA 250-µg disks carried bla(IMP-6,) and the high level resistance to MEM (MIC ≥ 512 µg/ml) was reduced by the presence of phenylalanine arginine ß-naphtylamide. Improvement of DDPTs was observed when MacConkey agar was used instead of Mueller-Hinton agar. In conclusion, DPA is a better MBL inhibitor than EDTA for detection of Pseudomonas spp. and Acinetobacter spp. with IMP-1-like, VIM-2-like, and SIM-1-type MBLs. In DPA DDPTs, IPM disks perform better than MEM disks when the isolates are highly resistant to MEM due to the overexpression of efflux pumps.

Nosocomial clustering of NDM-1-producing Klebsiella pneumoniae sequence type 340 strains in four patients at a South Korean tertiary care hospital.

In November 2010, NDM-1-producing Klebsiella pneumoniae (NDMKP) was identified for the first time in South Korea from four patients with no history of traveling abroad who stayed for 21 to 205 days in a tertiary care hospital. All were sequence type (ST) 340 and had nearly identical XbaI pulsed-field gel electrophoresis (PFGE) patterns. The bla(NDM-1)-carrying plasmids were in the IncN group, with sizes ranging from 50 to 200 kb. These findings suggest that NDMKP had already been introduced into South Korea before this clustering was found.

Metallo-beta-lactamase production by Pseudomonas otitidis: a species-related trait.

Susceptibility to several ß-lactams and ß-lactamase production was investigated in a collection of 20 strains of Pseudomonas otitidis, a new Pseudomonas species that has been recently recognized in association with otic infections in humans. All strains appeared to be susceptible to piperacillin, cefotaxime, ceftazidime, and aztreonam, while resistance or decreased susceptibility to carbapenems was occasionally observed. All strains were found to express metallo-ß-lactamase (MBL) activity and to carry a new subclass B3 MBL gene, named bla(POM), that appeared to be highly conserved in this species. P. otitidis, therefore, is the first example of a pathogenic Pseudomonas species endowed with a resident MBL. The POM-1 protein from P. otitidis type strain MCC10330 exhibits the closest similarity (60 to 64%) to the L1 MBL of Stenotrophomonas maltophilia. Expression in Escherichia coli and Pseudomonas aeruginosa revealed that, similar to L1 and other subclass B3 MBLs, POM-1 confers decreased susceptibility or resistance to carbapenems, penicillins, and cephalosporins but not to aztreonam. Expression of the POM MBL in P. otitidis is apparently constitutive and, in most strains, does not confer a carbapenem-resistant phenotype. However, a strong inoculum size effect was observed for carbapenem MICs, and carbapenem-resistant mutants could be readily selected upon exposure to imipenem, suggesting that carbapenem-based regimens should be considered with caution for P. otitidis infections.

Biochemical characterization of the TEM-107 extended-spectrum ß-lactamase in a Klebsiella pneumoniae isolate from South Korea.

The TEM-107 extended-spectrum ß-lactamase detected in a Klebsiella pneumoniae clinical isolate had a Gly238Ser substitution compared to the TEM-43 ß-lactamase. The MIC of ceftazidime was higher (64 µg/ml) than that of cefotaxime (2 µg/ml) for the isolate. Clavulanic acid reduced the MIC of ceftazidime 64-fold.

Trends in antimicrobial resistance of Neisseria gonorrhoeae isolated from Korean patients from 2000 to 2006.

BACKGROUND: The recent emergence and spread of antimicrobial-resistant Neisseria gonorrhoeae has compromised treatment and control of gonorrhea. We determined recent trends in antimicrobial susceptibility of the isolates, analyzed recent use of antigonococcal agents, and investigated the relationship between fluoroquinolone nonsusceptibility and amino acid substitutions within the fluoroquinolone resistance-determining regions in Korea. METHODS: The antimicrobial susceptibilities of 977 isolates of N. gonorrhoeae collected from 2000 to 2006 in Korea were determined with penicillin, ceftriaxone, spectinomycin, tetracycline, and ciprofloxacin disks. Some of the randomly selected isolates were tested by the Clinical and Laboratory Standards Institute agar dilution method, to determine subtle changes in susceptibility to the above antibiotics and cefixime. ß-lactamase was detected using a cefinase disk. RESULTS: All of the isolates exhibited plasmid- or chromosomally mediated resistance to penicillin; however, the proportions of penicillinase-producing N. gonorrhoeae decreased rapidly from 64% in 2000 to 21% in 2006. All isolates were susceptible to third-generation cephalosporins, except for 1 isolate that was not susceptible to cefixime. The proportion of ciprofloxacin-resistant isolates increased from 26% in 2000 to 83% in 2006. Of 7 substitution types, 5 (Ser-91-Phe in Gyrase A (GyrA), Ser-87-Arg in ParC subunit of topoisomerase IV (ParC); Ser-91-Phe and Asp-95-Ala in GyrA, and Ser-87-Asn in ParC; Ser-91-Phe and Asp-95-Gly in GyrA, and Asp-86-Asn in ParC; Ser-91-Tyr in GyrA; Ser-91-Phe in GyrA, and Asp-86-Asn in ParC) were new ones not identified in our 2004 study. All isolates were susceptible to spectinomycin. About half of the patients in our current study (52.6%-58.1%, depending on the year) received spectinomycin treatment. Majorities were resistant to tetracycline, and the rate of highly tetracycline-resistant N. gonorrhoeae increased from 3% in 2000 to 9% in 2006. CONCLUSIONS: The incidence of penicillinase-producing N. gonorrhoeae declined significantly, but none of the isolates were susceptible to penicillin G. All isolates were susceptible to spectinomycin, in contrast majority were resistant to tetracycline. Inappropriate use of fluoroquinolone was frequent. The minimum inhibitory concentrations of ceftriaxone were within the susceptible range for all isolates, but those of cefixime were slightly higher, and it was 0.5 µg/mL (nonsusceptible) for 1 isolate.

Comparative in vitro activities of torezolid (DA-7157) against clinical isolates of aerobic and anaerobic bacteria in South Korea.

Resistance of Gram-positive pathogens to first-line antimicrobial agents has been increasing in many parts of the world. We compared the in vitro activities of torezolid with those of other antimicrobial agents, including linezolid, against clinical isolates of major aerobic and anaerobic bacteria. Torezolid had an MIC(90) of ≤0.5 µg/ml for the Gram-positive bacterial isolates tested and was more potent than either linezolid or vancomycin.

Antimicrobial susceptibility patterns for recent clinical isolates of anaerobic bacteria in South Korea.

We determined the antimicrobial susceptibilities of 255 clinical isolates of anaerobic bacteria collected in 2007 and 2008 at a tertiary-care hospital in South Korea. Piperacillin-tazobactam, cefoxitin, imipenem, and meropenem were highly active beta-lactam agents against most of the isolates tested. The rates of resistance of Bacteroides fragilis group organisms and anaerobic gram-positive cocci to moxifloxacin were 11 to 18% and 0 to 27%, respectively.

Various penA mutations together with mtrR, porB and ponA mutations in Neisseria gonorrhoeae isolates with reduced susceptibility to cefixime or ceftriaxone.

OBJECTIVES: To examine mutations within the penA, mtrR, porB, ponA and pilQ genes of Neisseria gonorrhoeae to determine their contribution to cephalosporin resistance. METHODS: A total of 46 N. gonorrhoeae isolates with reduced susceptibility to cefixime or ceftriaxone (MICs > or = 0.12 mg/L) and two susceptible isolates were selected. The full sequence of penA and partial sequences previously reported as hot mutation sites of the other genes were analysed. Genotyping by N. gonorrhoeae multiantigen sequence typing (NG-MAST) was also performed. RESULTS: A mosaic penicillin-binding protein 2 (PBP 2) was found in a single isolate that exhibited the highest cefixime MIC (0.5 mg/L). The majority of the isolates with reduced susceptibility to cephalosporins contained non-mosaic PBP 2 sequences, of which PBP 2 pattern XIII was most common (28/46). All isolates with reduced susceptibility to cephalosporins also had mtrR and porB mutations. Two susceptible isolates had the PBP 2 pattern XIV and an incomplete MtrR protein, which was a new mutation. Isolates with identical PBP 2 patterns comprised multiple NG-MAST sequence types. CONCLUSIONS: Reduced susceptibility of N. gonorrhoeae to ceftriaxone and cefixime was associated with diverse penA mutations, particularly PBP 2 pattern XIII containing an Ala-501-->Val substitution, together with mtrR and porB mutations. The existence of only one strain having the mosaic penA sequence indicated that ceftriaxone and cefixime resistance in Korea is mostly not associated with a mosaic penA sequence. Highly heterogeneous NG-MAST sequence types excluded the clonal expansion of a particular subtype.

Three cases of Moraxella osloensis meningitis: a difficult experience in species identification and determination of clinical significance.

We had three cases of Moraxella osloensis meningitis. The species identification was impossible by conventional and commercial phenotypic tests. However, we could identify the species using the 16S rRNA gene sequencing. Determination of clinical significance was difficult in one patient. All three patients recovered by appropriate antimicrobial therapy.

An agar plate-based modified carbapenem inactivation method (p-mCIM) for detection of carbapenemase-producing Enterobacteriaceae.

Detecting carbapenemase-producing Enterobacteriaceae (CPE) has become increasingly difficult due to the emergence of diverse enzymes. The aim of the study was to evaluate an agar plate-based modified carbapenem inactivation method (p-mCIM) for detection of CPE. Stock strains and clinical isolates of CPE were used to evaluate the p-mCIM. The p-mCIM was performed as described for the mCIM, except that meropenem disks were placed on the lawn of test organisms on Mueller-Hinton agar (MHA) plates. Among 17 stock strains of CPE, six of eight KPC-2-like- and all six NDM-1-like carbapenemase-producing strains were positive by the p-mCIM without incubation in the carbapenem inactivation (CI) step. Among 380 CPE clinical isolates detected, 308 and 38 were KPC-2-like and NDM-1-like enzyme producers, respectively. The required incubation time in the CI step to show all isolates were positive by p-mCIM was 3 h for isolates with KPC-2-like enzyme and 1 h for isolates with metallo-ß-lactamases. Twenty-eight of 30 isolates with OXA-48-like enzymes were p-mCIM positive. Sensitivities of both the p-mCIM and the mCIM (based on inhibition zone of ≤15 mm) for detection of CPE were 100%. All 70 ertapenem-nonsusceptible, but carbapenemase gene-negative isolates tested were both p-mCIM (based on inhibition zone of ≥21 mm) and mCIM negative. In conclusion, performance of the p-mCIM, which uses a lawn of bacterial colonies on MHA plate instead of a bacteria-suspended Tryptic soy broth tube in the CI step, is essentially identical to that of the CLSI-recommended mCIM in the detection of clinical isolates of Enterobacteriaceae producing carbapenemases including difficult to detect blaOXA-48-like enzymes.