Rapid Identification of OXA-48-like, KPC, NDM, and VIM Carbapenemase-Producing Enterobacteriaceae From Culture: Evaluation of the RESIST-4 O.K.N.V. Multiplex Lateral Flow Assay

There is an urgent need for accurate and rapid diagnostic assays capable of identifying carbapenemase-producing Enterobacteriaceae (CPE). We assessed the performance of the RESIST-4 O.K.N.V. (OKNV) assay (Coris BioConcept, Gembloux, Belgium) for the identification of oxacillinase (OXA)-48-like-, Klebsiella pneumoniae carbapenemase (KPC)-, New Delhi metallo-β-lactamase (NDM)-, and Verona integron-encoded metallo-β-lactamase (VIM)-producing Enterobacteriaceae grown on sheep blood agar (SBA) and the CHROMagar KPC medium. Sixty-five carbapenem-resistant Enterobacteriaceae (CRE) isolates with characterized carbapenemase content were used to evaluate the OKNV assay. The assay correctly identified all 30 isolates that produced one of the four targeted carbapenemase families. Additionally, it correctly identified 15 isolates that co-produced KPC and NDM, VIM and NDM or OXA-48-like and NDM, but failed to identify an NDM-1 and OXA-232 co-producing Klebsiella pneumoniae isolate. All 16 non-carbapenemase-producing CRE and four CPE isolates exhibited negative results, and no cross-reaction was observed. Overall, the sensitivity and specificity of the assay were 97.8% and 100%, respectively. The OKNV assay is an accurate and rapid assay for identifying OXA-48-like, KPC, NDM, and VIM carbapenemases produced by Enterobacteriaceae isolates cultured on both SBA and the CHROMagar KPC media in the clinical microbiology laboratory.ope

Emergence of multidrug‑resistant Providencia rettgeri isolates co‑producing NDM‑1 carbapenemase and PER‑1 extended‑spectrum β‑lactamase causing a first outbreak in Korea

BACKGROUND: Nosocomial outbreak due to carbapenem-resistant Enterobacteriaceae has become serious challenge to patient treatment and infection control. We describe an outbreak due to a multidrug-resistant Providencia rettgeri from January 2016 to January 2017 at a University Hospital in Seoul, Korea. METHODS: A total of eight non-duplicate P. rettgeri isolates were discovered from urine samples from eight patients having a urinary catheter and admitted in a surgical intensive care unit. The β-lactamase genes were identified using polymerase chain reaction and direct sequencing, and strain typing was done with pulsed-field gel electrophoresis (PFGE). RESULTS: All isolates showed high-level resistance to extended-spectrum cephalosporins, aztreonam, meropenem, ertapenem, ciprofloxacin, and amikacin. They harbored the blaNDM-1 carbapenemase and the blaPER-1 type extended-spectrum β-lactamases genes. PFGE revealed that all isolates from eight patients were closely related strains. CONCLUSIONS: The 13-month outbreak ended following reinforcement of infection control measures, including contact isolation precautions and environmental disinfection. This is the first report of an outbreak of a P. rettgeri clinical isolates co-producing NDM-1 and PER-1 β-lactamase.ope

국내에서 분리된 인체분리균주를 이용한 MicroScan KSCM 패널의 항균제 감수성 시험 분석능 평가

Background: Antimicrobial resistant continues to pose a threat to public health. Therefore, rapid and accurate antimicrobial susceptibility testing is very important. The objectives of this study were to evaluate the performance of the MicroScan system (Beckman Coulter, USA) with newly developed Korean Antimicrobial Susceptibility Testing Panels (KSCM panels) for antimicrobial susceptibility testing (AST) against clinical isolates in South Korea. Methods: Three KSCM panels were designed in this study. For the performance evaluation, a total of 1,325 clinical isolates including 1,027 of Gram-negative bacilli and 298 Gram-positive cocci collected from eight general hospitals in South Korea were used. The results by KSCM panels were compared with those by conventional methods. Results: By KSCM-1 panel for Gram-positive cocci, the rates of categorical agreement (CA) were ＞90% in all the antimicrobials tested in this study. The rates of major error (ME) were also ＜3%, and only three very major error (VME) were identified; each of ampicillin, tetracycline, and quinupristin-dalfopristin in enterococcal isolates. By KSCM-2 panel for Enterobacteriaceae, the rates of CA were also above 90%, and those of ME and VME were less than 3% and 1.5%, respectively. KSCM-3 panels for glucose- non-fermenting Gram-negative bacilli, also showed good agreement rates, i.e., CA rates ＞90%, ME rates ＜3%, and VME rates ＜1.5%. Conclusion: The newly developed three KSCM panels for MicroScan system (Beckman Coulter) showed excellent performance in AST against a large number of clinical isolates, and they are applicable to clinical microbiology laboratories.ope

Counter Clinical Prognoses of Patients With Bloodstream Infections Between Causative Acinetobacter baumannii Clones ST191 and ST451 Belonging to the International Clonal Lineage II

This study was conducted to evaluate the possible clinical and bacteriologic features associated with 30-day mortality from Acinetobacter baumannii (A. baumannii) bloodstream infections (BSIs). We conducted a prospective, multicenter, observational study of 181 entire episodes of A. baumannii BSI from six general hospitals between May 2016 and April 2017 in South Korea. Cox proportional-hazards regression model was used to estimate risks of the primary endpoint, i.e., all-cause mortality within 30 days from the initial blood culture. Most (84.5%) of the A. baumannii blood isolates belonged to the international clonal lineage II (ICLII) and 89.5% of the isolates were either multidrug- or extensively-drug resistant. We identified three risk factors including the old age of patient {hazard ratio, 1.033; [95% Confidential Interval (CI), 1.010-1.056]}, the sequential organ failure assessment score [1.133 (1.041-1.233)], and causative A. baumannii sequence type (ST) 191 belonging to ICLII [1.918 (1.073-3.430)], and three protective factors including causative A. baumannii ST451 belonging to ICLII [0.228 (0.078-0.672)], platelet count [0.996 (0.993-0.999)], and definitive therapy within 72 h [0.255 (0.125-0.519)]. Differing 30-day mortality rate in the dominant ICLII was observed by ST, which was much high in ST191 and low in ST451 and it was likely associated with the molecular traits, rather than the drug resistance.ope

MALDI-TOF Mass Spectrometry Technology as a Tool for the Rapid Diagnosis of Antimicrobial Resistance in Bacteria

Species identification by using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a routine diagnostic process for infectious diseases in current clinical settings. The rapid, low-cost, and simple to conduct methodology is expanding its application in clinical microbiology laboratories to diagnose the antimicrobial resistance (AMR) in microorganisms. Primarily, antimicrobial susceptibility testing is able to be carried out either by comparing the area under curve of MALDI spectra of bacteria grown in media with antimicrobial drugs or by identifying the shift peaks of bacteria grown in media including 13C isotope with antimicrobial drugs. Secondly, the antimicrobial resistance is able to be determined through identifying (i) the antimicrobial-resistant clonal groups based on the fingerprints of the clone, (ii) the shift peak of the modified antimicrobial drug, which is inactivated by the resistance determinant, (iii) the shift peak of the modified antimicrobial target, (iv) the peak specific for the antimicrobial determinant, and (v) the biomarkers that are coproduced proteins with AMR determinants. This review aims to present the current usage of the MALDI-TOF MS technique for diagnosing antimicrobial resistance in bacteria, varied approaches for AMR diagnostics using the methodology, and the future applications of the methods for the accurate and rapid identification of AMR in infection-causing bacterial pathogens.ope

Identification of Acinetobacter Species Using Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry

BACKGROUND: Acinetobacter baumannii has a greater clinical impact and exhibits higher antimicrobial resistance rates than the non-baumannii Acinetobacter species. Therefore, the correct identification of Acinetobacter species is clinically important. Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) has recently become the method of choice for identifying bacterial species. The purpose of this study was to evaluate the ability of MALDI-TOF MS (Bruker Daltonics GmbH, Germany) in combination with an improved database to identify various Acinetobacter species. METHODS: A total of 729 Acinetobacter clinical isolates were investigated, including 447 A. baumannii, 146 A. nosocomialis, 78 A. pittii, 18 A. ursingii, 9 A. bereziniae, 9 A. soli, 4 A. johnsonii, 4 A. radioresistens, 3 A. gyllenbergii, 3 A. haemolyticus, 2 A. lwoffii, 2 A. junii, 2 A. venetianus, and 2 A. genomospecies 14TU. After 212 isolates were tested with the default Bruker database, the profiles of 63 additional Acinetobacter strains were added to the default database, and 517 isolates from 32 hospitals were assayed for validation. All strains in this study were confirmed by rpoB sequencing. RESULTS: The addition of the 63 Acinetobacter strains' profiles to the default Bruker database increased the overall concordance rate between MALDI-TOF MS and rpoB sequencing from 69.8% (148/212) to 100.0% (517/517). Moreover, after library modification, all previously mismatched 64 Acinetobacter strains were correctly identified. CONCLUSIONS: MALDI-TOF MS enables the prompt and accurate identification of clinically significant Acinetobacter species when used with the improved database.ope

First Case Report of Human Infection With Ochrobactrum tritici Causing Bacteremia and Cholecystitis

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Killing Dynamics of Carbapenems against Pseudomonas aeruginosa Harboring Varied Determinants of Carbapenem Resistance

Background: Ideal dose of the antimicrobials should be decided by considering their killing dynamics since sufficient elimination of the causative microorganisms is critical for proper antimicrobial treatment. In this study, the bactericidal activities of carbapenems by resistance mechanisms were assessed for carbapenem-resistant Pseudomonas aeruginosa. Methods: Minimal inhibition concentrations (MICs) of carbapenems were determined by broth dilution method and the resistance mechanisms were identified by PCR and DNA sequencing. The expression levels of efflux pumps were determined by reverse transcriptase real-time PCR. Time-kill curves were plotted by time-course numeration of the viable cells grown under imipenem and meropenem at 1× and 4× MICs, respectively. Results: One P. aeruginosa strain was susceptible, whereas three were resistant to carbapenems by defective OprD, efflux pump overproduction, and/or IMP-6 production. The susceptible strain had imipenem and meropenem MICs of 2 and 1 mg/L, respectively. The MICs were elevated by eight-fold by defective OprD, 16- and 32-fold by the pump overproduction, and four- and <64-fold by the combination of two determinants and the IMP-6 carbapenemase. While both the carbapenems showed time-dependent bactericidal activity to the susceptible isolate, either of the carbapenem-resistant determinants, such as decreased membrane permeability, carbapenemase production, or the defective OprD,presented concentration-dependent bacteriostatic activity.Conclusion: Different killing dynamics of the carbapenems were observed depending on the resistance determinants, and the results would guide a proper treatment strategy for the patients using these drugs.ope

A Multicenter Study of Antifungal Use and Species Distribution and Antifungal Susceptibilities of Candida Isolates in South Korea

Background: Candidiasis control should include monitoring the epidemiology and resistance to various antifungal agents. In this study, the researchers investigated the Candida species recovered from clinical specimens at particular geographic areas or hospitals. Objective: The present study is geared toward the evaluation of antifungal drug usage at Korean hospitals in 2016. It is also essential that species distribution and antifungal susceptibilities of Candida isolates should be looked into to provide important data that can help devise therapeutic strategies to control the disease. Methods: Systemic antifungal agent usage over a one-year period was investigated at 10 Korean hospitals. Identification and antifungal susceptibility tests were performed on clinical isolates of the Candida species, which were collected over a three-month period. Results: The total antifungal usage in each hospital ranged from 7.7 to 158.9 defined daily doses (DDDs) per 1,000 patient days. Fluconazole was most commonly used (37.1%), followed by amphotericin B (30.6%), itraconazole (9.7%), echinocandins (8.8%), voriconazole (7.5%), and posaconazole (6.3%), respectively. Among 274 Candida isolates, C. albicans was the most frequently recovered (51.1%), followed by C. glabrata (15.7%), C. tropicalis (15.0%), and C. parapsilosis (13.5%), respectively. Through the application of either species-specific clinical breakpoints or epidemiological cutoff values to Candida isolates, the non-susceptibility rates to fluconazole, voriconazole, amphotericin B, and micafungin were found in 20.7%, 5.6%, 0%, and 0% of isolates, respectively. Conclusion: This nationwide multicenter study showed that total antifungal use varied considerably according to each hospital. Non-susceptibility to fluconazole should be further monitored, considering the drug's frequent use in Korea.ope

Dynamics and Predictors of Mortality Due to Candidemia Caused by Different Candida Species: Comparison of Intensive Care Unit-Associated Candidemia (ICUAC) and Non-ICUAC

We investigated mortality and predictors of mortality due to intensive care unit-associated candidemia (ICUAC) versus non-ICUAC by Candida species. This study included all candidemia cases in 11 hospitals from 2017 to 2018 in South Korea. The all-cause mortality rates in all 370 patients with ICUAC were approximately twofold higher than those in all 437 patients with non-ICUAC at 7 days (2.3-fold, 31.1%/13.3%), 30 days (1.9-fold, 49.5%/25.4%), and 90 days (1.9-fold, 57.8%/30.9%). Significant species-specific associations with 7- and 30-day ICUAC-associated mortality were not observed. Multivariate analysis revealed that ICU admission was an independent predictor of Candida glabrata (OR, 2.07-2.48) and Candida parapsilosis-associated mortality (OR, 6.06-11.54). Fluconazole resistance was a predictor of C. glabrata-associated mortality (OR, 2.80-5.14). Lack (less than 3 days) of antifungal therapy was the strongest predictor of 7-day mortality due to ICUAC caused by Candida albicans (OR, 18.33), Candida tropicalis (OR, 10.52), and C. glabrata (OR, 21.30) compared with 30- and 90-day mortality (OR, 2.72-6.90). C. glabrata ICUAC had a stronger association with lack of antifungal therapy (55.2%) than ICUAC caused by other species (30.6-36.7%, all p < 0.05). Most predictors of mortality associated with ICUAC were distinct from those associated with non-ICUAC and were mediated by Candida species.ope