Development of a Two Triplex Real-Time Polymerase Chain Reaction for Rapid Detection of Six Carbapenemase Genes in Enterobacteriaceae

ObjectivesCarbapenem resistance is a serious clinical and public health threat. Carbapenemase can confer carbapenem resistance, and most carbapenemase genes are plasmid encoded so resistance can easily spread. In this study, we aimed to develop a novel system based on the TaqMan platform for the rapid detection of 6 clinically prevalent carbapenemase genes: Klebsiella pneumoniae carbapenemase, New Delhi metallo-β-lactamase, oxacillinase, imipenem-hydrolyzing, Verona integron-encoded metallo-β-lactamase, and Guiana extended-spectrum β-lactamase.MethodsThe triplex assay was verified by testing genomic DNA of 6 carbapenemase-producing Klebsiella pneumoniae. It was validated with a blinded panel of 310 Enterobacteriaceae isolates, including 225 carbapenemase-producers and 85 non-producers, by direct colony triplex real-time polymerase chain reaction (PCR). The real-time PCR was performed using the ABI 7500 fast instrument (Applied Biosystems, CA, USA) and specific primers for each carbapenemase target were designed to include modified peptide-nucleic acid oligonucleotides.ResultsNo amplification was detected among the negative samples. The result showed 100% concordance with the genotypes previously identified. The entire assay, including DNA extraction and real-time PCR, was completed within 2 hours.ConclusionThe newly developed triplex real-time PCR assay was useful for the rapid, accurate and simultaneous detection of 6 carbapenemase genes in Enterobacteriaceae, suggesting its potential to allow an early decision on the appropriate treatment, management, and prevention of the spread of resistant infections in hospitals.

MCR1 and KPC2 Co-producing Klebsiella pneumoniae Bacteremia: First Case in Korea

Klebsiella pneumoniae carbapenemase-producing K. pneumoniae (KPC-KP) has been disseminating nationwide due to clonal spread and is taking a serious action at the national level in Korea. The mobilized colistin resistance (MCR1) gene confers plasmid-mediated resistance to colistin and is known to be capable of horizontal transfer between different strains of a bacterial species. We have experienced a fatal case of the patient who developed MCR1-possessing, ST307/Tn4401a[blaKPC2] K. pneumonia bacteremia in the community of non-capital region after being diagnosed as pancreatic cancer with multiple liver metastases and treated in the capital region. The ST307/Tn4401a[blaKPC2] K. pneumonia was the most commonly disseminated clone in Korea. Our strain is the first MCR1 and KPC2 co-producing K. pneumonia in Korea and our case is the critical example that the multi-drug resistant clone can cause inter-regional spread and the community-onset fatal infections. Fortunately, our patient was admitted to the intensive care unit on the day of visit, and the contact precaution was well maintained throughout and KPC-KP was not spread to other patients. The high risk patients for KPC-KP need to be screened actively, detected rapidly and preemptively isolated to prevent outbreak of KPC-KP. Inter-facility communications are essential and the nationwide epidemiologic data of KPC-KP should be analyzed and reported regularly to prevent spread of KPC-KP. The prompt identification of species and antimicrobial susceptibilities for successful treatment against KPC-KP should be emphasized as well.

Detection of mcr-1 Plasmids in Enterobacteriaceae Isolates From Human Specimens: Comparison With Those in Escherichia coli Isolates From Livestock in Korea

BACKGROUND: The emerging mobile colistin resistance gene, mcr-1, is an ongoing worldwide concern and an evaluation of clinical isolates harboring this gene is required in Korea. We investigated mcr-1-possessing Enterobacteriaceae among Enterobacteriaceae strains isolated in Korea, and compared the genetic details of the plasmids with those in Escherichia coli isolates from livestock. METHODS: Among 9,396 Enterobacteriaceae clinical isolates collected between 2010 and 2015, 1,347 (14.3%) strains were resistant to colistin and those were screened for mcr-1 by PCR. Colistin minimum inhibitory concentrations (MICs) were determined by microdilution, and conjugal transfer of the mcr-1-harboring plasmids was assessed by direct mating. Whole genomes of three mcr-1-positive Enterobacteriaceae clinical isolates and 11 livestock-origin mcr-1-positive E. coli isolates were sequenced. RESULTS: Two E. coli and one Enterobacter aerogenes clinical isolates carried carried IncI2 plasmids harboring mcr-1, which conferred colistin resistance (E. coli MIC, 4 mg/L; E. aerogenes MIC, 32 mg/L). The strains possessed the complete conjugal machinery except for E. aerogenes harboring a truncated prepilin peptidase. The E. coli plasmid transferred more efficiently to E. coli than to Klebsiella pneumoniae or Enterobacter cloacae recipients. Among the three bacterial hosts, the colistin MIC was the highest for E. coli owing to the higher mcr-1-plasmid copy number and mcr-1 expression levels. Ten mcr-1-positive chicken-origin E. coli strains also possessed mcr-1-harboring IncI2 plasmids closely related to that in the clinical E. aerogenes isolate, and the remaining one porcine-origin E. coli possessed an mcr-1-harboring IncX4 plasmid. CONCLUSIONS: mcr-1-harboring IncI2 plasmids were identified in clinical Enterobacteriaceae isolates. These plasmids were closely associated with those in chicken-origin E. coli strains in Korea, supporting the concept of mcr-1 dissemination between humans and livestock.

Molecular Typing and Resistance Profiles of Vancomycin-Intermediate Staphylococcus aureus in Korea: Results from a National Surveillance Study, 2007-2013 / 대한임상미생물학회지

BACKGROUND: To investigate the national molecular epidemiology and resistance profiles of vancomycin-intermediate Staphylococcus aureus (VISA), we analyzed the characteristics of methicillin-resistant Staphylococcus aureus (MRSA) collected from clinical samples at tertiary or general hospitals participating in a nationwide surveillance program for VISA and vancomycin-resistant Staphylococcus aureus (VRSA) in Korea during an 12-week period in each year from 2007 to 2013. METHODS: VISA was defined by agar dilution, broth dilution and E-test methods with vancomycin minimum inhibitory concentrations of >2 μg/mL. All VISA isolates were characterized by multilocus sequence typing, staphylococcal cassette chromosome mec typing, spa typing, accessory gene regulator typing, Diversilab analysis, and antibiogram analysis. RESULTS: Of 109,345 MRSA isolates, 87,354 were screened and 426 isolates were identified as positive on brain heart infusion agar containing 4 μg/mL vancomycin (BHI-V4). Of 426 isolates, 76 isolates were identified as VISA. No VRSA isolates were detected among the isolates. Overall, a total of 6 genotypes were identified among VISA strains and the predominant clones were ST5-II-t2460, ST72-IV-t324, and ST239-III-t037 (44.7%, 15.8%, and 10.5%, respectively). Of note, ST72-IV-t324 clones are known to be a typical community-associated MRSA. ST239-III-t037 strains were more resistant to trimethoprim-sulfamethoxazole than any other type of strain. ST72-IV-t324 strains were susceptible to all of the antimicrobial agents tested except erythromycin and daptomycin. All of the VISA isolates were susceptible to linezolid and quinupristin-dalfopristin. CONCLUSION: Although VRSA is still rare, continuous monitoring of VRSA occurrence is needed, as well as VISA prevalence, epidemic clonal shift, and antimicrobial resistance.

Prevalence of Major Methicillin-Resistant Staphylococcus aureus Clones in Korea Between 2001 and 2008

BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) are important pathogens causing nosocomial infections in Korean hospitals. This study aimed to investigate the epidemiological and genetic diversity of clinical S. aureus isolates in healthcare settings from 2001 to 2008. METHODS: Samples and data were obtained from 986 individuals as part of the National Antimicrobial Surveillance Project, involving 10 regions nationwide. Molecular typing studies, including multilocus sequence typing (MLST) and staphylococcal cassette chromosome mec (SCCmec) typing were performed, and a representative clone of Korean MRSA was classified by combinational grouping using a DiversiLab (DL; bioMérieux, France) repetitive element polymerase chain reaction (rep-PCR) system. RESULTS: Nine Korean MRSA clones (KMRSA-1 to -9) were identified by analysis of genetic backgrounds and molecular characteristics. KMRSA-1 to -3, expressing clonal complex (CC) 5 (carrying SCCmec II), CC8 (carrying SCCmec III), and CC72 (carrying SCCmec IV) were spread nationwide. In contrast, KMRSA-6 was highly prevalent in Gyeongsangnam-do, and KMRSA-4 was highly prevalent in Jeollanam-do and Jeollabuk-do. CONCLUSIONS: Epidemic KMRSA clones were genetically similar to major clones identified from the USA, with the exception of KMRSA-2, which had the SCCmec III type. Our results provide important insights into the distribution and molecular genetics of MRSA strains in Korea and may aid in the monitoring of MRSA spread throughout the country.

Ultrasonic Device Part I: Overview / 대한피부과학회지

Ultrasound is over 20 khz, which represents the upper frequency limit of human hearing. Acoustic vibrations are generated when piezoelectric materials on the thin disc-shaped transducers expand and contract. Although low frequency ultrasound devices have been used widely in the dermatologic area for a long time, the mechanism and side effects have been overlooked. A low-frequency ultrasound device has many benefits on the cosmetic dermatology area by thermal effect, vibration effect, and increase of transdermal delivery of lipophilic drugs or cosmetics. However, there have been reports of dermatitis, dyspnea, dizziness, and burns after treatment with ultrasound. Therefore, the use of this device should be under a doctor's supervision.

A Case of SAHA Syndrome / 대한피부과학회지

The SAHA syndrome is an acronym which stands for seborrhea, acne, hirsutism and androgenic alopecia. The SAHA syndrome generally occurs in young to middle-aged women and may be caused by elevated blood levels of androgens or increased androgen-driven peripheral response with normal circulating androgen levels. In SAHA syndrome, careful diagnostic and clinical evaluation is necessary in order to identify the cause of peripheral hyperandrogenism, and to exclude androgen-producing tumors. SAHA can be classified into 5 subtypes: familial, ovarian, adrenal, hyperprolactinemic SAHA and HAIRAN (hyperandrogenism, insulin resistance, acanthosis nigricans) syndrome. Among them, ovarian SAHA syndrome is associated with polycystic ovarian syndrome. We report a case of ovarian SAHA syndrome in 15-year-old girl who showed seborrea, acne, hirsutism and androgenic alopecia associated with polycystic ovarian syndrome.

A Case of Reconstruction of Upper Cheek with Subcutaneous Island Pedicle Flap / 대한피부과학회지

Subcutaneous island pedicle flap is a kind of advancement flap and provides a blood supply by the vascular pedicle. It guarantees a small wound, less tension, but sometimes leaves cosmetic problems like a triangular or trap-door scar. Generally, reconstruction of the upper cheek with subcutaneous island flap is not a good choice. A rotation flap or simple elliptical excision is relatively easy and gives a better cosmetic result. However, our patient had a long history of taking blood thinning medication and the location of cancer was close to the eyes and nose. Therefore, we chose subcutaneous island pedicle flap and got relatively good cosmetic results without severe bleeding or distortion of the eyes and nose.

A Case of Acneiform Eruption Occurring after Injection of Cetuximab (Erbitux(R), IMC-C225) / 대한피부과학회지

We report a case of acneiform skin eruption induced by cetuximab in a 58-year-old man. Cetuximab is a new anticancer agent, which acts as a selective epidermal growth factor receptor monoclonal antibody. Our patient had taken cetuximab for the treatment of metastatic rectal cancer and visited our clinic with multiple erythematous papules and pustules on the face, postauricular area and anterior chest. A skin biopsy from his forehead revealed destruction of the follicular structure and perifollicular inflammatory cellular infiltrations composed of neutrophils and lymphocytes.

A Case of Cutaneous Scedosporium apiospermum Infection / 대한피부과학회지

Scedosporium (S.) apiospermum is the asexual stage of Pseudallescheria (P.) boydi. The organism is ubiquitous in nature, and has a world-wide distribution. It has been isolated from soil, plant debris, polluted water and sewage. It is an opportunistic organism with low virulence. Infection may occur via direct inoculation and usually affects the extremities. We report a case of cutaneous S. apiospermum infection which occurred in a 58-year-old male during immunosuppressive therapy, 3 months after a kidney transplantation. He presented with an one-month history of cutaneous nodules on the dorsum of the right foot. Cultural isolation showed S. apiospermum and we treated him daily with itraconazole and drainage.

A Retrospective Analysis of Efficacy and Safety of Cyclosporine in the Treatment of Mild to Moderate Psoriasis / 대한피부과학회지

No abstract available.

A Case of Skin Infection Caused by Mycobacterium abscessus / 대한피부과학회지

We report a case of cutaneous Mycobacterium (M.) abscessus infection in a 57-year-old healthy woman who presented with an erythematous plaque on her cheek. No previous trauma history was reported. A skin biopsy specimen revealed an abscess and granuloma as well as some acid-fast bacilli in the dermis. The microorganism was identified as M. abscessus by culture and PCR-restriction fragment length polymorphism analysis. The patient was treated with clarithromycin and ciprofloxacin. After 6 months, the skin lesion had been resolved.

Genotype Analysis of Pyrogenic Exotoxin and emm Genes of Streptococcus pyogenes Clinical Isolates

A variety of proteins produced by Streptococcus pyogenes contribute to the virulence of the pathogen. Among the proteins, the M protein and streptococcal pyrogenic exotoxins (Spe) are considered the major S. pyogenes virulence factors. To better characterize the correlation of M protein type and pyrogenic exotoxins with clinical diseases, we tested 269 S. pyogenes clinical isolates from patients with scarlet fever, pharyngitis, skin infection, otitis media, or other invasive streptococcal infections that provided appropriate clinical data. The strains were genotyped (M type) and assayed for speA, speB, and speC genes. The speB gene was detected in all isolates. Also, speA and speC genes were detected in 54 strains (18.2%) and 140 strains (47.3%), respectively. The strains isolated from invasive disease patients showed the highest frequency of speA gene (40.5%). The correlation among emm genotype, speA gene, and clinical patterns was analyzed. Genotypes emm1 (55.6%) and emm3 (22.2%) were predominant in stains with speA gene. The distribution of emm genotypes did not significantly associate with clinical patterns. These data suggest that SpeA is significantly associated with specific emm genotypes, and the exotoxin serve a dominant virulence factor.

Biochemical Characteristics and Antimicrobial Susceptibility Patterns of Vibrio vulnificus Isolated from the Environment of Korea in 2001

Vibrio vulnificus is a pathogen causing two types of severe illness, septicemia and wound infections, and is continually detected in marine environments. To investigate the biochemical characteristics and the antimicrobial susceptibility of V. vulnificus isolated from environment of Korea in 2001, the API 20E kit test, PCR, and antibiotic disk diffusion method were performed. A total of 210 V. vulnificus strains was isolated from seawater, shell-fish, sediments, coastal water, aquarium water, sewage, and others. All of the isolates could be divided into 15 groups on the basis of their API 20E profiles, and were positive in Indole test. Only 173 isolates (82.4%) were positive by the PCR amplifying the cytolysinhemolysin gene. Almost all isolates were susceptible to chloramphenicol (99.5%), tetracycline (90.0%), ciprofloxacin (92.4%), trimethoprim/sulfamethoxazole (89.0%), nalidixic acid (87.6%). Some isolates were resistant to cephalothin (57.6%), amikacin (33.3%), cefoxitin (31.9%). One hundred and forty three isolates (68.1%) were resistant to one or more antimicrobial agents. These results show that V. vulnificus environmental isolates possessed various biochemical characteristics, and some isolates were not detected of the cytolysin-hemolysin gene by PCR, and a part of isolates were resistant to multiple antibiotics.

Molecular Characterization and Sequence Analysis of Pneumococcal Surface Protein A ( PspA ) from Invasive Streptococcus pneumoniae Isolated in Korea

To investigate the genetic variation within pspA from 17 clinical isolates of Streptococcus pneumoniae representing 12 capsular serotypes, we used specific PCR primers LSM12 and LSM2 derived from the DNA sequence of pspA of S. pneumoniae Rxl (type 2). We have found that all 17 isolates of S. pneumoniae have a pspA gene whose size ranges from 1.8 to 2.3 kb. RFLP analysis of the PCR-amplified pspA genes of the isolates exhibited distinct restriction patterns. Even within the same capsular type, the individual isolates of S. pneumoniae generally differed in PspA molecular masses and showed variabilities in the pspA gene locus. The nucleotide sequence of the pspA gene of S. pneumonaie KNIH1156 (type 19F) isolated from a blood specimen was determined. The sequence revealed an open reading frame of 1,827 bp nucleotides. Predicted size of the mature PspA was approximately 63 kDa. Deduced amino acid sequence of PspA of S. pneumonaie KNIH1156 revealed 57.0% identity with that of S. pneumonaie Rxl. Comparison of the nucleotide and amino acid sequences of PspA S. pneumoniae KNIH1156 (type 19F) with those of Rxl (type 2) showed considerable differences in the a-helical coiled-coil region of the two PspAs. These results suggest that the PspA of S. pneumoniae KNIH1156 has antigenic variations distinguished from those of Rxl strains.

Antigenic Analysis of Haemophilus influenzae Based on Biotypes and Patterns of OMP P2

No abstract available.

Genotypic and Phenotypic Analysis Among Clinical Isolate of Streptococcus pyogenes in Seoul , Korea

A total of 152 strains of Streptococcus pyogenes were isolated from patients with pharyngitis, scarlet fever, skin infection, or invasive streptococcal infections in Seoul, Korea from January 1988 to December 1999. All isolates were epidemiologically characterized to decide phenotypes by T protein serotype and serum opacity factor (OF) detection. Genetic diversity of the isolates were analyzed by emm genotyping and pulsed-field gel electrophoresis (PFGE). T protein serotype showed 17 kinds in distribution and T12 (40.1% of study strains), T4 (19.1%), and T1 (7.9%) were the prevalent ones. When sources of S. pyogenes isolates were analyzed by T serotype distribution, T12 type was predominant in pharyngitis and skin infection isolates which contributed to 30 strains (49.2%) and 11 strains (18.0%), respectively. When T serotype of S. pyogenes isolates were analyzed by emm genotype distribution, of the 61 isolates of T12 type, 48 strains (78.7%) belonged to the emm type 12 (M12) and of the 29 isolates of T4 type, 27 strains (93.1%) belonged to the emm genotype 4 (M4). PFGE of genomic DNA of different emm genotype (emm12, emm4 and emm1) showed distinctive patterns. When the DNA of same emm gene type isolates were analyzed genetic relatedness by PFGE pattern, emm4, emm1, and emm12 types showed over 90%, 75%, and 70% of genetic similarity, respectively. Therefore, it was suggested that these emm genotype isolates were closely related genetically whereas among the isolates of other emm genotypes showed less than 30% of genetic similarity. Show genotypes are more diverse in comparison with phenotypes. In even epidemiologically unrealated isolates, genetic subtypes appeared correlated. The phenotypic and genotypic analysis used in the study were discriminative and appropriate for epidemiological study of S. pyogenes.

Pulsed field gel electrophoresis profile of erythromycin-clindamycin resistant Streptococcus pyogenes isolated in Korea

Ninety two strains of Streptococcus pyogenes were isolated from patients with pharyngitis, scarlet fever, skin infection, and invasive streptococcal infections in Seoul, Korea from January to December, 1998. All isolates were epidemiologically characterized by T protein serotype, and serum opacity factor (OF) detection to phenotypes. To analyze the genetic relationship, fifty two isolates including 32 erythromycin-clindamycin (Em-Cm) resistant strains, 20 antimicrobial susceptible strains were attempted to the pulsed-field gel electrophoresis (PFGE). T protein serotype showed 16 kinds in distribution including T12 and T4. Among the total isolates, 40 strains (43.5%) belonged to the T12 serotype and twenty strains (21.7%) to T4 serotype. On the other hand, when infection aspect of S. pyogenes isolates were analysed by T serotype distribution, T12 type was predominant for pharyngitidis which contributed to 21 strains (53%) and for skin infection isolates which contributed to 11 strains (28%), respectively. In case of T4 type, it was the most predominant pharyngitidis isolates which contributed to 8 strains (40%). In T serotype distribution of Em-Cm resistant strains, 27 strains (84%) of the thirty two showed T12 serotype. In minimum inhibitory concentration (MIC) values of Em-Cm resistance isolates, thirty two isolates showed resistant to erythromycin 27 strains (84%), had high MIC of >128 mug/ml. And also to clindamycin, twenty two strains (69%) had high MIC of >128 mug/ml. When OF detection of Em-Cm resistance of S. pyogenes isolates were analyzed by T serotype distribution, T12 serotype isolates revealed that all of the isolates except one strain were OF negative. In PFGE profile analysis to Em-Cm resistance isolates, of the twenty seven, Em-Cm resistance of T12 serotype isolates, 26 strains showed identical PFGE profile and all of these isolates revealed that OF negative. Eighty four percent of Em-Cm resistance S. pyogenes isolates had identical phenotype and PFGE profile. These results strongly suggested that the Em-Cm resistant S. pyogenes isolates from Seoul area showed close genetic correlation and PFGE could be available tool for molecular epidemiology.

Cloning of the Form I-antigen Genes from Shigella sonnei KNIH104

Shigella sonnei KNIH104S, which was selected by Korean National Institute of Health, expresses form I-antigen as a somatic antigen. In this study, we cloned the genes responsible for form I-antigen synthesis from S. sonnei KNIH104S. A Sau3AI-generated cosmid library of S. sonnei KNIH104S plasmids were transfected into E. coli LE392 and transfectants were tested for agglutination with antiserum against S. sonnei form I-antigen. A clone, JH222, showing the strongest agglutination activity was chosen for further analysis. A recombinant cosmid, pJH222, was isolated from the strain JH222 and retransfected into E. coli LE392. All of the transfectants agglutinated with antiserum against form I-antigen, indicating that pJH222 carried the genes required for S. sonnei form I-antigen synthesis. Restriction analysis of pJH222 revealed a 38 kb insert, which was confirmed by Southern hybridization analysis to be present on a large plasmid of S. sonnei KNIH104S.

Two cases of ovarian pregnancy / 대한산부인과학회잡지

No abstract available.