Fitness costs of Tn1546-type transposons harboring the vanA operon by plasmid type and structural diversity in Enterococcus faecium.

BACKGROUND: This study analyzed the genetic traits and fitness costs of vancomycin-resistant Enterococcus faecium (VREfm) blood isolates carrying Tn1546-type transposons harboring the vanA operon. METHODS: All E. faecium blood isolates were collected from eight general hospitals in South Korea during one-year study period. Antimicrobial susceptibility testing and vanA and vanB PCR were performed. Growth rates of E. faecium isolates were determined. The vanA-positive isolates were subjected to whole genome sequencing and conjugation experiments. RESULTS: Among 308 E. faecium isolates, 132 (42.9%) were positive for vanA. All Tn1546-type transposons harboring the vanA operon located on the plasmids, but on the chromosome in seven isolates. The plasmids harboring the vanA operon were grouped into four types; two types of circular, nonconjugative plasmids (Type A, n = 50; Type B, n = 46), and two types of putative linear, conjugative plasmids (Type C, n = 16; Type D, n = 5). Growth rates of vanA-positive E. faecium isolates were significantly lower than those of vanA-negative isolates (P < 0.001), and reduction in growth rate under vancomycin pressure was significantly larger in isolates harboring putative linear plasmids than in those harboring circular plasmids (P = 0.020). CONCLUSIONS: The possession of vanA operon was costly to bacterial hosts in antimicrobial-free environment, which provide evidence for the importance of reducing vancomycin pressure for prevention of VREfm dissemination. Fitness burden to bacterial hosts was varied by type and size of the vanA operon-harboring plasmid.

The Discordance between G-Banding Karyotyping and Microarray in Structural Abnormality.

BACKGROUND: Cytomolecular genetic laboratory techniques have developed from conventional G-banding karyotyping to whole genome sequencing. Although resolution has greatly increased, various cytogenetic techniques have their advantages and limitations in detecting genomic variations. METHODS: We compared the chromosomal abnormalities detected by G-banding karyotyping and SNP-based microarray testing in 62 patients from July 2020 to December 2022. We analyzed their difference according to chromosomal abnormalities, including numerical and structural and others. RESULTS: Of the 62 patients, 28 patients showed chromosomal aberration detected in one or more of the two test methods. Aneuploidy was detected in both methods, while gain and loss less than 3 Mb were only detectable by the microarray. G-banding karyotyping is fundamental to detect structural chromosome rearrangement such as inversions, ring chromosomes, and translocations, but additional breakpoint or unknown origin materials informa-tion obtained from microarray. Loss of heterozygosity was only detectable in microarray, and mosaicism had limitations in both G-banding karyotyping and microarray. CONCLUSIONS: Various disease cause genomic structural variants, it is very important to detect this. We showed discordance between G-banding karyotyping and SNP based microarray in clinical laboratory. It can be helpful to clinical physicians to decide which diagnostic tool to use.

Disseminated nontuberculous mycobacteria infection in an immunocompetent host: A case report.

RATIONALE: Disseminated nontuberculous mycobacterial (NTM) infections are rare and occur primarily in immunocompromised hosts. Mycobacterium abscessus complex (MABC), including M abscessus subsp. massiliense (hereafter M massiliense) is a complex of rapidly growing mycobacterial (RGM) species of NTM. Here, we present a rare case of disseminated NTM infection with RGM bacteremia caused by M massiliense in an immunocompetent host. PATIENT CONCERNS: A 64-year-old woman with a recent history of spine fracture and septic pneumonia was transferred to our emergency room for dyspnea and fever. A peripherally inserted central catheter (PICC) had been placed over 6 months prior. DIAGNOSES: Chest computed tomography (CT) showed multifocal patchy consolidations and ground-glass opacity in both lungs. NTM suspected of RGM was isolated from the blood cultures. During hospitalization, multiple erythematous and hemorrhagic crusted nodules developed on the patient's upper and lower extremities, which were confirmed as disseminated NTM infection on skin biopsy. INTERVENTIONS: After NTM suspected of RGM was isolated from the blood cultures, the patient was empirically treated with antibiotics used for NTM infection, and the PICC was removed. Thereafter, the subspecies of NTM was reported as M massiliense and she was treated according to the antibiotic susceptibility testing results. OUTCOME: Although skin lesions and inflammatory markers improved gradually during antibiotic treatment over 10 weeks, NTM could still be isolated from the blood culture. LESSONS: Disseminated NTM infections with RGM bacteremia in an immunocompetent host have rarely been reported. In this case, PICC placement for more than 6 months was suspected to be an important risk factor for RGM bacteremia in an immunocompetent patient. To date, there are only insufficient case reports, moreover no clear guidelines regarding the optimal choice of antibiotics or length of treatment for disseminated NTM infection. Therefore, it is necessary to establish treatment guidelines for patients with disseminated NTM infection and bacteremia.

Emergence of NDM-1-producing Pseudomonas aeruginosa Sequence Type 773 Clone: Shift of Carbapenemase Molecular Epidemiology and Spread of 16S rRNA Methylase Genes in Korea.

Imipenemase (IMP)-6-producing Pseudomonas aeruginosa sequence type (ST) 235 is a dominant clone of carbapenemase-producing P. aeruginosa (CPPAE) in Korea. As part of the Antimicrobial Resistance Surveillance System in Korea, we found an increase in the carbapenem resistance rate of P. aeruginosa isolates from blood cultures and a shift in the molecular epidemiology of CPPAE. A total of 212 non-duplicated P. aeruginosa blood isolates were obtained from nine general hospitals and two nursing homes. Twenty-four isolates were identified as CPPAE. We observed the emergence of the NDM-1 P. aeruginosa ST 773 clone (N=10), mostly from Gyeongsang Province. The IMP-6 ST 235 clone (N=11) was detected in all provinces. CPPAE isolates showed very high resistance rates to amikacin, and all NDM-1 P. aeruginosa strains carried rmtB. This is the first nationwide surveillance of the recently emerged NDM-1-producing P. aeruginosa ST773 clone in Korea. Continuous surveillance is necessary to prevent the infection and transmission of carbapenem- and amikacin-resistant P. aeruginosa in Korea.

Serotype Distribution and Antimicrobial Resistance of Streptococcus pneumoniae Causing Invasive Pneumococcal Disease in Korea Between 2017 and 2019 After Introduction of the 13-Valent Pneumococcal Conjugate Vaccine.

Background: Streptococcus pneumoniae is a serious pathogen causing various infections in humans. We evaluated the serotype distribution and antimicrobial resistance of S. pneumoniae causing invasive pneumococcal disease (IPD) after introduction of pneumococcal conjugate vaccine (PCV)13 in Korea and investigated the epidemiological characteristics of multidrug-resistant (MDR) isolates. Methods: S. pneumoniae isolates causing IPD were collected from 16 hospitals in Korea between 2017 and 2019. Serotyping was performed using modified sequential multiplex PCR and the Quellung reaction. Antimicrobial susceptibility tests were performed using the broth microdilution method. Multilocus sequence typing was performed on MDR isolates for epidemiological investigations. Results: Among the 411 S. pneumoniae isolates analyzed, the most prevalent serotype was 3 (12.2%), followed by 10A (9.5%), 34 (7.3%), 19A (6.8%), 23A (6.3%), 22F (6.1%), 35B (5.8%), 11A (5.1%), and others (40.9%). The coverage rates of PCV7, PCV10, PCV13, and pneumococcal polysaccharide vaccine (PPSV)23 were 7.8%, 7.8%, 28.7%, and 59.4%, respectively. Resistance rates to penicillin, ceftriaxone, erythromycin, and levofloxacin were 13.1%, 9.2%, 80.3%, and 4.1%, respectively. MDR isolates accounted for 23.4% of all isolates. Serotypes 23A, 11A, 19A, and 15B accounted for the highest proportions of total isolates at 18.8%, 16.7%, 14.6%, and 8.3%, respectively. Sequence type (ST)166 (43.8%) and ST320 (12.5%) were common among MDR isolates. Conclusions: Non-PCV13 serotypes are increasing among invasive S. pneumoniae strains causing IPD. Differences in antimicrobial resistance were found according to the specific serotype. Continuous monitoring of serotypes and antimicrobial resistance is necessary for the appropriate management of S. pneumoniae infections.

Nationwide Surveillance of Antifungal Resistance of Candida Bloodstream Isolates in South Korean Hospitals: Two Year Report from Kor-GLASS.

We incorporated nationwide Candida antifungal surveillance into the Korea Global Antimicrobial Resistance Surveillance System (Kor-GLASS) for bacterial pathogens. We prospectively collected and analyzed complete non-duplicate blood isolates and information from nine sentinel hospitals during 2020−2021, based on GLASS early implementation protocol for the inclusion of Candida species. Candida species ranked fourth among 10,758 target blood pathogens and second among 4050 hospital-origin blood pathogens. Among 766 Candida blood isolates, 87.6% were of hospital origin, and 41.3% occurred in intensive care unit patients. Adults > 60 years of age accounted for 75.7% of cases. Based on species-specific clinical breakpoints, non-susceptibility to fluconazole, voriconazole, caspofungin, micafungin, and anidulafungin was found in 21.1% (154/729), 4.0% (24/596), 0.1% (1/741), 0.0% (0/741), and 0.1% (1/741) of the isolates, respectively. Fluconazole resistance was determined in 0% (0/348), 2.2% (3/135, 1 Erg11 mutant), 5.3% (7/133, 6 Pdr1 mutants), and 5.6% (6/108, 4 Erg11 and 1 Cdr1 mutants) of C. albicans, C. tropicalis, C. glabrata, and C. parapsilosis isolates, respectively. An echinocandin-resistant C. glabrata isolate harbored an F659Y mutation in Fks2p. The inclusion of Candida species in the Kor-GLASS system generated well-curated surveillance data and may encourage global Candida surveillance efforts using a harmonized GLASS system.

Performance of MALDI-TOF Mass Spectrometry (VITEK MS) in the Identification of Salmonella Species.

Salmonella is a major pathogen causing foodborne infections in humans. Salmonella isolates are identified using biochemical and serological tests, including automated systems such as the VITEK2 system. However, there are few reports on Salmonella identification using VITEK MS. Therefore, we aimed to evaluate the usefulness of MALDI-TOF VITEK MS for Salmonella identification. A total of 1389 Salmonella isolates were identified using VITEK MS ver3.0 or ver3.2. All Salmonella isolates were confirmed by serotyping using the Kauffmann-White scheme, and the results were compared with the VITEK MS results. A total of 1389 Salmonella isolates, including 66 serotypes, were correctly identified at the genus level by VITEK MS. However, these systems failed to correctly identify typhoidal Salmonella. Among the five Salmonella enterica ssp. diarizonae isolates, only one was correctly identified, whereas one and three isolates were partially identified and misidentified, respectively. On the other hand, the VITEK2 system successfully identified all typhoidal Salmonella (Typhi and Paratyphi A) and Salmonella enterica ssp. diarizonae isolates. VITEK MS was useful for identifying Salmonella species isolated from clinical specimens; however, additional biochemical tests, such as the VITEK2 System, should be considered to accurately identify Salmonella ser. Typhi, and Salmonella ser. Paratyphi A.

Characteristics of Escherichia coli Urine Isolates and Risk Factors for Secondary Bloodstream Infections in Patients with Urinary Tract Infections.

Escherichia coli is responsible for more than 80% of all incidences of urinary tract infections (UTIs). We assessed a total of 636 cases of patients with E. coli UTIs occurring in June 2019 in eight tertiary hospitals in South Korea for the traits of patients with E. coli UTIs, UTI-causative E. coli isolates, and risk factors associated with bloodstream infections (BSIs) secondary to UTIs. Antimicrobial susceptibility testing was conducted using the disc diffusion method, and the genes for extended-spectrum beta-lactamases (ESBLs) and plasmid-mediated ampC genes were screened by using PCR and sequencing. Multilocus sequence typing and virulence pheno-/genotyping were carried out. A total of 49 cases developed BSIs. The E. coli urine isolates primarily comprised sequence type 131 (ST131) (30.0%), followed by ST1193, ST95, ST73, and ST69. Three-quarters of the ST131 H30Rx isolates possessed the blaCTX-M-15-like gene, whereas 66% of H30R and 50% of H41 isolates possessed the blaCTX-M-14-like gene. All the ST1193 isolates showed biofilm formation ability, and three-quarters of the ST73 isolates exhibited hemolytic activity with high proportions of papC, focG, and cnf1 positivity. The prevalence of the ST131 H41 sublineage and its abundant CTX-M possession among the E. coli urine isolates were noteworthy; however, no specific STs were associated with bloodstream invasion. For BSIs secondary to UTIs, the papC gene was likely identified as a UTI-causative E. coli-related risk factor and urogenital cancer (odds ratio [OR], 12.328), indwelling catheter (OR, 3.218), and costovertebral angle tenderness (OR, 2.779) were patient-related risk factors. IMPORTANCE Approximately half of the BSIs caused by E. coli are secondary to E. coli UTIs. Since the uropathogenic E. coli causing most of the UTIs is genetically diverse, understanding the risk factors in the E. coli urine isolates causing the BSI is important for pathophysiology. Although the UTIs are some of the most common bacterial infectious diseases, and the BSIs secondary to the UTIs are commonly caused by E. coli, the assessments to find the risk factors are mostly focused on the condition of patients, not on the bacterial pathogens. Molecular epidemiology of the UTI-causative E. coli pathogens, together with the characterization of the E. coli urine isolates associated with the BSI secondary to UTI, was carried out, suggesting treatment options for the prevalent antimicrobial-resistant organisms.

Serotype Distribution and Antimicrobial Resistance of Salmonella Isolates in Korea between 2016 and 2017.

Salmonella is one of the major causes of food-borne infections. We investigated the serotype distribution and antimicrobial resistance of Salmonella isolates collected in Korea between January 2016 and December 2017. In total, 669 Salmonella isolates were collected from clinical specimens at 19 university hospitals. Serotyping was performed according to the Kauffmann-White scheme, and antimicrobial susceptibility was tested using Sensititre EUVSEC plates or disk diffusion. Among the strains, C (39.8%) and B (36.6%) were the most prevalent serogroups. In total, 51 serotypes were identified, and common serotypes were S. enterica serovar I 4,[5],12:i:- (16.7%), S. Enteritidis (16.1%), S. Bareilly (14.6%), S. Typhimurium (9.9%), and S. Infantis (6.9%). The resistance rates to ampicillin, chloramphenicol, and trimethoprim-sulfamethoxazole were 32.6%, 12.1%, and 8.4%, respectively. The resistance rates to cefotaxime and ciprofloxacin were 8.1% and 3.0%, respectively, while 5.4% were multidrug-resistant. S. enterica serovar I 4,[5],12:i:- and S. Enteritidis were highly prevalent, and there was an increase in rare serotypes. Multidrug resistance and ciprofloxacin resistance were highly prevalent. Periodic investigations of Salmonella serotypes and antimicrobial resistance are needed.

A novel likely pathogenic PLCG2 variant in a patient with a recurrent skin blistering disease and B-cell lymphopenia.

Pathogenic variants of PLCG2 encoding phospholipase C gamma 2 (PLCγ2) were first reported in 2012 and their clinical manifestations vary widely. PLCG2-associated antibody deficiency and immune dysregulation (PLAID) and autoinflammation and PLCγ2-associated antibody deficiency and immune dysregulation (APLAID) are representative examples of PLCG2 pathogenic variants. In this report, we describe a 17-year-old male with recurrent blistering skin lesions, B-cell lymphopenia, and asthma. Distinct from the patients in previous reports, this patient had the heterozygous de novo c.2119T > C missense variant (NM_002661.4) resulting in a serine to proline amino acid substitution (p.Ser707Pro). The variant located to the PLCγ2 C-terminal Src homology 2 (cSH2) domain, which is a critical site for the restriction of intrinsic enzyme activity. This variant could be classified as "likely pathogenic" according to American College of Medical Genetics and Genomics guidelines. Laboratory results showed a reduction in circulating B cells without a decrease of serum IgG and IgA. Our findings expand the variety of clinical phenotypes for PLCG2 missense variants.

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.

The Prognostic Ability of RAS Pathway-Related Gene Mutations in Patients with Myeloid Neoplasms Treated with Hypomethylating Agents.

INTRODUCTION: This study aimed to identify genetic predictors of treatment response and survival in patients with myeloid neoplasms treated with hypomethylating agents (HMAs). METHODS: We performed next-generation sequencing on bone marrow aspiration samples of 59 patients diagnosed with acute myeloid leukemia (AML), myelodysplastic syndrome with excess blasts-2, or chronic myelomonocytic leukemia and treated with decitabine or azacitidine as a frontline therapy. RESULTS: A single gene with the most common mutations was TP53 (14 of 59 patients), and mutations in RAS pathway-related genes including KRAS, NRAS, FLT3, PTPN11, CBL, and KIT were found in 28.8% of patients. The overall response rate to HMAs was 33.9%. Predictive factors for a poor response were an age >75 years (p = 0.007), 3 or more gene mutations (p = 0.004), mutations in RAS pathway-related genes (p = 0.033), and a mutated NRAS gene (p = 0.042). An age >75 years (hazard ratio 2.946), diagnosis of AML (hazard ratio 2.915), and mutations in NRAS (hazard ratio 4.440) were identified as poor prognostic factors for survival. CONCLUSION: In conclusion, mutations in RAS pathway-related genes were predictors of a poor response to HMAs. Particularly, mutated NRAS was associated with inferior survival rates.

A rapid quantitative on-site coronavirus disease 19 serological test.

On-site severe acute respiratory syndrome coronavirus 2 (SARS CoV-2) serological assays allow for timely in-field decisions to be made regarding patient status, also enabling population-wide screening to assist in controlling the coronavirus disease 2019 (COVID-19) pandemic. Here we propose a rapid microfluidic serological assay with two unique functions of nanointerstice filling and digitized flow control, which enable the fast/robust filling of the sample fluid as well as precise regulation of duration and volume of immune reaction. Developed microfluidic assay showed enhanced limit of detection, and 91.67% sensitivity and 100% specificity (n = 152) for clinical samples of SARS CoV-2 patients. The assay enables daily monitoring of IgM/IgG titers and patterns, which could be crucial parameters for convalescence from COVID-19 and provide important insight into how the immune system responds to SARS CoV-2. The developed on-site microfluidic assay presented the mean time for IgM and IgG seroconversions, indicating that these titers plateaued days after seroconversion. The mean duration from day 0 to PCR negativity was 19.4 days (median 20 d, IQR 16-21 d), with higher IgM/IgG titres being observed when PCR positive turns into negative. Simple monitoring of these titres promotes rapid on-site detection and comprehensive understanding of the immune response of COVID-19 patients.

Risk Factors for Extended-Spectrum-ß-Lactamase-Producing Escherichia coli in Community-Onset Bloodstream Infection: Impact on Long-Term Care Hospitals in Korea.

BACKGROUND: The prevalence of extended-spectrum ß-lactamase-producing Escherichia coli (ESBL-EC) in the community has increased worldwide due to multifactorial reasons. ESBL-EC bloodstream infection (BSI) complicates the decision for proper antimicrobial administration. In this multicenter study, we investigated the prevalence, risk factors, and molecular background of community-onset (CO) ESBL-EC BSI. METHODS: We included data for all episodes of ESBL-EC BSI of community origin from May 2016 to April 2017 obtained from the Korean national antimicrobial resistance surveillance system, which comprises six sentinel hospitals. Data, including previous history of admission and use of antimicrobials and medical devices before BSI, were collected, along with microbiological analysis results. RESULTS: Among 1,189 patients with CO BSI caused by E. coli, 316 (27%) were identified as ESBL producers. History of admission, especially to a long-term care hospital (LTCH), and previous use of ß-lactams/ß-lactamase inhibitors, carbapenem, lincosamide, aminoglycoside, and extended-spectrum cephalosporin were independent risk factors for CO ESBL-EC BSI; admission to an LTCH showed the highest odds ratio (3.8, 95% confidence interval 2.3-6.1). The most common genotype was CTX-M-15 (N=131, 41%), followed by CTX-M-14 (N=86, 27%). ST131 was the most common sequence type among ESBL-EC groups (57%). CONCLUSIONS: In Korea, 27% of CO E. coli BSI were caused by ESBL producers. From perspectives of empirical treatment and infection control, history of admission to an LTCH and antimicrobial use should be noted.

Major Bloodstream Infection-Causing Bacterial Pathogens and Their Antimicrobial Resistance in South Korea, 2017-2019: Phase I Report From Kor-GLASS.

To monitor national antimicrobial resistance (AMR), the Korea Global AMR Surveillance System (Kor-GLASS) was established. This study analyzed bloodstream infection (BSI) cases from Kor-GLASS phase I from January 2017 to December 2019. Nine non-duplicated Kor-GLASS target pathogens, including Staphylococcus aureus, Enterococcus faecalis, Enterococcus faecium, Streptococcus pneumoniae, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter spp., and Salmonella spp., were isolated from blood specimens from eight sentinel hospitals. Antimicrobial susceptibility testing, AMR genotyping, and strain typing were carried out. Among the 20,041 BSI cases, 15,171 cases were caused by one of the target pathogens, and 12,578 blood isolates were collected for the study. Half (1,059/2,134) of S. aureus isolates were resistant to cefoxitin, and 38.1% (333/873) of E. faecium isolates were resistant to vancomycin. Beta-lactamase-non-producing ampicillin-resistant and penicillin-resistant E. faecalis isolates by disk diffusion method were identified, but the isolates were confirmed as ampicillin-susceptible by broth microdilution method. Among E. coli, an increasing number of isolates carried the bla CTX-M-27 gene, and the ertapenem resistance in 1.4% (30/2,110) of K. pneumoniae isolates was mostly (23/30) conferred by K. pneumoniae carbapenemases. A quarter (108/488) of P. aeruginosa isolates were resistant to meropenem, and 30.5% (33/108) of those carried acquired carbapenemase genes. Over 90% (542/599) of A. baumannii isolates were imipenem-resistant, and all except one harbored the bla OXA-23 gene. Kor-GLASS provided comprehensive AMR surveillance data, and the defined molecular mechanisms of resistance helped us to better understand AMR epidemiology. Comparative analysis with other GLASS-enrolled countries is possible owing to the harmonized system provided by GLASS.