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.

Adjustment of Modified Carbapenem Inactivation Method Conditions for Rapid Detection of Carbapenemase-Producing Acinetobacter baumannii.

BACKGROUND: The existing modified carbapenem inactivation methods (mCIMs) recommended by the CLSI for detecting carbapenemase production have not been applicable for Acinetobacter baumannii. We evaluated the influence of matrices used in mCIMs and CIMTris on the stability of the disks for detecting carbapenemase producers and suggested optimal mCIM conditions for detecting carbapenemase-producing A. baumannii. METHODS: Seventy-three A. baumannii isolates characterized for antimicrobial susceptibility and carbapenemase encoding genes were tested for carbapenemase production using mCIM and CIMTris. The influence of the matrices (Tryptic soy broth [TSB] and Tris-HCl) used in these methods on the stability of the meropenem (MEM) disk was also evaluated. The mCIM conditions were adjusted to enhance screening sensitivity and specificity for detecting carbapenemase-producing A. baumannii. RESULTS: The matrices had an impact on the stability of the MEM disk after the incubation period (two or four hrs). TSB nutrient broth is an appropriate matrix for mCIM compared with Tris-HCl pH 7.6, which leads to the loss of MEM activity in CIMTris. The sensitivity and the specificity of the optimal mCIM were both 100%. CONCLUSIONS: We established optimal mCIM conditions for simple, accurate, and reproducible detection of carbapenemase-producing A. baumannii.

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.

Modification and evaluation of the Triton Hodge test for screening carbapenemase-producing Enterobacteriaceae.

Detection of carbapenemase-producing Enterobacteriaceae (CPE) has become critical for appropriate antimicrobial therapy and for controlling the spread of infection. We evaluated Triton Hodge test (THT) for screening CPE. A spreader can be used to apply more constant volume of Triton on whole surface of Mueller-Hinton agar (MHA), or alternatively, a 10-µL inoculating loop can be used to apply a 20% Triton solution lineally. The THT procedure can be simplified by eliminating the 1/10 dilution step of indicator bacteria from the McFarland 0.5 turbidity suspension. The presence of Triton in the MHA plates significantly increased the enhanced growth size of not only Enterobacteriaceae producing NDM-1-like enzymes but also those producing the most prevalent KPC-2-like enzyme, resulting in 100% sensitivity of the test.

Antimicrobial Susceptibility Patterns of Anaerobic Bacterial Clinical Isolates From 2014 to 2016, Including Recently Named or Renamed Species.

BACKGROUND: Anaerobic bacterial resistance trends may vary across regions or institutions. Regional susceptibility patterns are pivotal in the empirical treatment of anaerobic infections. We determined the antimicrobial resistance patterns of clinically important anaerobic bacteria, including recently named or renamed anaerobes. METHODS: A total of 521 non-duplicated clinical isolates of anaerobic bacteria were collected from a tertiary-care hospital in Korea between 2014 and 2016. Anaerobes were isolated from blood, body fluids, and abscess specimens. Each isolate was identified by conventional methods and by Bruker biotyper mass spectrometry (Bruker Daltonics, Leipzig, Germany) or VITEK matrix-assisted laser desorption ionization time-of-flight mass spectrometry (bioMérieux, Marcy-l'Étoile, France). Antimicrobial susceptibility was tested using the agar dilution method according to the CLSI guidelines. The following antimicrobials were tested: piperacillin-tazobactam, cefoxitin, cefotetan, imipenem, meropenem, clindamycin, moxifloxacin, chloramphenicol, tetracycline, and metronidazole. RESULTS: Most Bacteroides fragilis isolates were susceptible to piperacillin-tazobactam, imipenem, and meropenem. The non-fragilis Bacteroides group (including B. intestinalis, B. nordii, B. pyogenes, B. stercoris, B. salyersiae, and B. cellulosilyticus) was resistant to meropenem (14%) and cefotetan (71%), and Parabacteroides distasonis was resistant to imipenem (11%) and cefotetan (95%). Overall, the Prevotella and Fusobacterium isolates were more susceptible to antimicrobial agents than the B. fragilis group isolates. Anaerobic gram-positive cocci exhibited various resistance rates to tetracycline (6-86%). Clostridioides difficile was highly resistant to penicillin, cefoxitin, imipenem, clindamycin, and moxifloxacin. CONCLUSIONS: Piperacillin-tazobactam, cefoxitin, and carbapenems are highly active ß-lactam agents against most anaerobes, including recently named or renamed species.

Genetic and biochemical characterisation of CTX-M-37 extended-spectrum ß-lactamase from an Enterobacter cloacae clinical isolate from Mongolia.

OBJECTIVES: The aims of this study were to determine the resistance level of a blaCTX-M-37-carrying Enterobacter cloacae isolate from Mongolia, to analyse kinetic parameters of the purified enzyme and to compare the genetic environment of the gene. METHODS: Minimum inhibitory concentrations (MICs) were determined using the Clinical and Laboratory Standards Institute (CLSI) agar dilution method. Purified CTX-M-37 enzyme was used to determined kinetic parameters. The genetic environment of the blaCTX-M-37 gene in E. cloacae was compared with a Kluyvera cryocrescens isolate. RESULTS: The E. cloacae isolate showed relatively low-level resistance to cefotaxime (MIC=16mg/L) compared with a CTX-M-3-producing strain (MIC=256mg/L), and CTX-M-37 had a lower kcat/Km value for cefotaxime (2.0µM-1s-1) compared with CTX-M-3 (3.5µM-1s-1), possibly due to Asn114Asp substitution. The blaCTX-M-37 gene in the E. cloacae isolate was carried on a conjugative plasmid and was associated with an ISEcp1 element containing the -35 and -10 putative promoter sequences TTGAAA and TACAAT, respectively, unlike in the K. cryocrescens isolate. CONCLUSIONS: The CTX-M-37-producing E. cloacae isolate showed relatively low-level resistance to cefotaxime and the purified enzyme had lower kinetic parameters as the result of Asn114Asp substitution. Presence of an ISEcp1 element and putative promoters upstream of the blaCTX-M-37 gene in E. cloacae, but not in the K. cryocrescens isolate, indicated their roles in mobilisation and expression of the gene.

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.

Panel strain of Klebsiella pneumoniae for beta-lactam antibiotic evaluation: their phenotypic and genotypic characterization.

Klebsiella pneumoniae is responsible for numerous infections caused in hospitals, leading to mortality and morbidity. It has been evolving as a multi-drug resistant pathogen, acquiring multiple resistances such as such as horizontal gene transfer, transposon-mediated insertions or change in outer membrane permeability. Therefore, constant efforts are being carried out to control the infections using various antibiotic therapies. Considering the severity of the acquired resistance, we developed a panel of strains of K. pneumoniae expressing different resistance profiles such as high-level penicillinase and AmpC production, extended spectrum beta-lactamases and carbapenemases. Bacterial strains expressing different resistance phenotypes were collected and examined for resistance genes, mutations and porin alterations contributing to the detected phenotypes. Using the Massive parallel sequencing (MPS) technology we have constructed and genotypically characterized the panel strains to elucidate the multidrug resistance. These panel strains can be used in the clinical laboratory as standard reference strains. In addition, these strains could be significant in the field of pharmaceuticals for the antibiotic drug testing to verify its efficiency on pathogens expressing various  resistances.

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.

Levofloxacin Efflux and smeD in Clinical Isolates of Stenotrophomonas maltophilia.

Trimethoprim-sulfamethoxazole is the first-line antimicrobial combination for Stenotrophomonas maltophilia infections. However, allergy or intolerance and increasing resistance limit the use of trimethoprim-sulfamethoxazole. Quinolones can be used as an alternative therapeutic option, but resistance can emerge rapidly during therapy. We analyzed the contribution of SmeABC and SmeDEF efflux pumps to levofloxacin resistance in clinical isolates of S. maltophilia. Nonduplicate clinical isolates of S. maltophilia were collected in 2010 from 11 university hospitals (n = 102). Fifty-five levofloxacin nonsusceptible (minimum inhibitory concentration [MIC] ≥4 µg/ml) and 47 susceptible (MIC ≤2 µg/ml) isolates were tested for efflux pump overexpression. Real-time reverse transcription-PCR was performed for amplification and quantification of smeB, smeC, smeD, and smeF mRNA. To determine which antimicrobials were affected by smeD overexpression, the growth rates of a levofloxacin-susceptible S. maltophilia isolate were compared by measuring absorbance of antimicrobial-supplemented Luria-Bertani broth (LB) cultures with or without triclosan. Significant relationships between sme gene overexpression and resistance were observed for smeD against levofloxacin, smeC and smeF against ceftazidime, and smeC against ticarcillin-clavulanate. The mean MICs of moxifloxacin and tigecycline did not significantly differ for isolates with or without overexpression of smeB, smeC, and smeF, but were significantly higher for isolates with smeD overexpression. The mean MICs of amikacin were significantly higher for smeC or smeF overexpressing isolates. Increased growth of a levofloxacin-susceptible isolate was observed in LB with 1/2 MIC levofloxacin in the presence of triclosan. These data suggest that the expression of smeD plays a role in levofloxacin resistance in S. maltophilia.

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.

Disk Carbapenemase Test for the Rapid Detection of KPC-, NDM-, and Other Metallo-ß-Lactamase-Producing Gram-Negative Bacilli.

BACKGROUND: Rapid detection of carbapenemase-producing gram-negative bacilli (GNB) is required for optimal treatment of infected patients. We developed and assessed a new disk carbapenemase test (DCT). METHODS: Paper disks containing 0.3 mg of imipenem and bromothymol blue indicator were developed, and the performance of the DCT were evaluated by using 742 strains of GNB with or without carbapenemases. RESULTS: The paper disks were simple to prepare, and the dried disks were stable at -20°C and at 4°C. The DCT detected 212 of 215 strains (98.6% sensitivity with 95% confidence interval [CI] 96.0-99.5%) of GNB with known class A (KPC and Sme) and class B (NDM, IMP, VIM, and SIM) carbapenemases within 60 min, but failed to detect GES-5 carbapenemase. The DCT also detected all two Escherichia coli isolates with OXA-48, but failed to detect GNB with OXA-232, and other OXA carbapenemases. The DCT showed 100% specificity (95% CI, 99.2-100%) in the test of 448 imipenem-nonsusceptible, but carbapenemase genes not tested, clinical isolates of GNB. CONCLUSIONS: The DCT is simple and can be easily performed, even in small laboratories, for the rapid detection of GNB with KPC, NDM and the majority of IMP, VIM, and SIM carbapenemases.

Increasing Incidence of High-Level Tetracycline-Resistant Neisseria gonorrhoeae due to Clonal Spread and Foreign Import.

PURPOSE: The detection of high-level tetracycline-resistant strains of Neisseria gonorrhoeae (TRNG) can make important epidemiological contributions that are relevant to controlling infections from this pathogen. In this study, we aimed to determine the incidence of TRNG isolates over time and also to investigate the characteristics and genetic epidemiology of these TRNG isolates in Korea. MATERIALS AND METHODS: The antimicrobial susceptibilities of 601 isolates of N. gonorrhoeae from 2004 to 2011 were tested by standard Clinical and Laboratory Standards Institute methods. To determine the molecular epidemiological relatedness, N. gonorrhoeae multi-antigen sequence typing was performed. RESULTS: The incidence of TRNG increased from 2% in 2004 to 21% in 2011. The minimum inhibitory concentration distributions of ceftriaxone and susceptibility of ciprofloxacin in TRNG were different from non-TRNG and varied according to the year of isolation. Most of the TRNG isolates collected from 2004 to 2007 exhibited genetic relatedness, with sequence type (ST) 1798 being the most common. From 2008 to 2011, the STs of the isolates became more variable and introduction of genetically unrelated TRNG were noted. CONCLUSION: The increased incidence of TRNG strains until 2007 appears to be due, at least in part, to clonal spread. However, we propose that the emergence of various STs since 2008 could be associated with foreign import.

New treatment options for infections caused by increasingly antimicrobial-resistant Neisseria gonorrhoeae.

The emergence of high-level resistance to ceftriaxone is giving rise to serious concern about absence of effective treatment options to cure gonococcal infections. Increasing the dosage regimen can be applied to ceftriaxone and azithromycin, but the emergence of high-level resistance has already been reported. Spectinomycin is another active drug but has low efficacy in the treatment of pharyngeal gonorrhoea. Conventional antibiotics could be introduced for gonococcal treatment, but they have some limitations, such as the absence of clinical trials and breakpoint. Combining antibiotics is another promising method to cure patients and to prevent the emergence of resistance. The most important strategy to maintain the efficacy of antibiotics is rapid detection and dissemination control of novel resistant isolate.

Establishing quality control ranges for antimicrobial susceptibility testing of Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus: a cornerstone to develop reference strains for Korean clinical microbiology laboratories.

Quality control (QC) processes are being performed in the majority of clinical microbiology laboratories to ensure the performance of microbial identification and antimicrobial susceptibility testing by using ATCC strains. To obtain these ATCC strains, some inconveniences are encountered concerning the purchase cost of the strains and the shipping time required. This study was focused on constructing a database of reference strains for QC processes using domestic bacterial strains, concentrating primarily on antimicrobial susceptibility testing. Three strains (Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus) that showed legible results in preliminary testing were selected. The minimal inhibitory concentrations (MICs) and zone diameters (ZDs) of eight antimicrobials for each strain were determined according to the CLSI M23. All resulting MIC and ZD ranges included at least 95% of the data. The ZD QC ranges obtained by using the CLSI method were less than 12 mm, and the MIC QC ranges extended no more than five dilutions. This study is a preliminary attempt to construct a bank of Korean QC strains. With further studies, a positive outcome toward cost and time reduction can be anticipated.

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.

The sul1 gene in Stenotrophomonas maltophilia with high-level resistance to trimethoprim/sulfamethoxazole.

Emerging resistance to trimethoprim/sulfamethoxazole (SXT) poses a serious threat to the treatment of Stenotrophomonas maltophilia infections. We determined the prevalence and molecular characteristics of acquired SXT resistance in recent clinical S. maltophilia isolates obtained from Korea. A total of 252 clinical isolates of S. maltophilia were collected from 10 university hospitals in Korea between 2009 and 2010. Antimicrobial susceptibility was determined by using the CLSI agar dilution method. The sul1, sul2, and sul3 genes, integrons, insertion sequence common region (ISCR) elements, and dfrA genes were detected using PCR. The presence of the sul1 gene and integrons was confirmed through sequence analysis. Among the 32 SXT-resistant isolates, sul1 was detected in 23 isolates (72%), all of which demonstrated high-level resistance (≥64 mg/L) to SXT. The sul1 gene (varying in size and structure) was linked to class 1 integrons in 15 of the 23 isolates (65%) harboring this gene. None of the SXT-susceptible isolates or the SXT-resistant isolates with a minimum inhibitory concentration of 4 and 8 mg/L were positive for sul1. Moreover, the sul2, sul3, and dfrA genes or the ISCR elements were not detected. The sul1 gene may play an important role in the high-level SXT resistance observed in S. maltophilia.