Prevalence and Molecular Characteristics of Methicillin-resistant Staphylococcus aureus Isolates in a Neonatal Intensive Care Unit

The molecular characteristics of methicillin-resistant Staphylococcus aureus (MRSA) isolated from neonates in a neonatal intensive care unit (NICU) were investigated by multilocus sequence typing (MLST), staphylocoagulase (SC) genotyping, staphylococcal cassette chromosome mec (SCCmec) typing, accessory gene regulator (agr) typing, and the presence of Panton-Valentine leukocidin (PVL). Among the 44 S. aureus isolates from nares in neonates between March and June 2014 at hospital in Busan, 27 (61.4%) were MRSA and 17 (38.6%) were methicillin-susceptible S. aureus (MSSA). The most prevalent clone in MRSA isolates was ST72-SC type Vb-SCCmec IV-agr I (n=26) and the remaining one was ST89-SC type I-SCCmec II-agr II. In MSSA isolates, the prevalent clone was ST121-SC type Va-agr IV (n=13), followed by ST72-SC type Vb-agr I (n=2), ST8-SC type III-agr I (n=1) and ST15-SC type X-agr II (n=1). All isolates did not possess the PVL. The data showed that the neonates in NICU carried high prevalence of ST72 MRSA and remarkably different clones with SC diversity between MRSA and MSSA isolates.

Emergence of Panton-Valentine Leukocidin-Positive ST80 Clone of Community-Associated Methicillin-Resistant Staphylococcus aureus in Busan, Korea

Community-associated methicillin resistant Staphylococcus aureus (CA-MRSA) has become widespread in the community and healthcare settings, and a number of clonal lineages emerged on every country. Sequence type (ST) 80 clone of CA-MRSA was dominant in Europe and has increasingly been isolated from the Middle East but so far never found in Korea. In this study, 48 MRSA isolates recovered from ear infections were characterized by multilocus sequence typing (MLST), staphylococcal cassette chromosome mec (SCCmec) typing, staphylocoagulase (SC) genotyping, staphylococcal protein A gene (spa) typing, accessory gene regulator (agr) typing, and virulence gene profiling. Most MRSA strains belonged to three major clones: ST5-SCCmec II-SC type II (n=19, 39.6%), ST239-SCCmec III-SC type IV (n=15, 31.2%), and ST72-SCCmec IV-SC type Vb (n=11, 22.9%). Among the isolates, one strain was Panton- Valentine leukocidin (PVL)-positive ST80-SCCmec IV-SC type XIa - spa type t044-agr group III, and exfoliative toxin D-positive. This strain was susceptible to most antibiotics, but resistant to tetracycline and fusidic acid. This is the first report on the emergence of European ST80 CA-MRSA clone in Korea.

Genotypes of Clinical and Environmental Isolates of Cryptococcus neoformans and Cryptococcus gattii in Korea

Multilocus sequence typing analysis was applied to determine the genotypes of 147 (137 clinical and 10 environmental) Cryptococcus neoformans and three clinical Cryptococcus gattii isolates from 1993 to 2014 in Korea. Among the 137 clinical isolates of C. neoformans, the most prevalent genotype was ST5 (n = 131), followed by ST31 (n = 5) and ST127 (n = 1). Three C. gattii strains were identified as ST57, ST7, and ST113. All environmental isolates were identified as C. neoformans with two genotypes, ST5 (n = 7) and ST31 (n = 3). Our results show that C. neoformans isolates in Korea are genetically homogeneous, and represent a close genetic relationship between clinical and environmental isolates.

Molecular Characterization of Community-Associated Methicillin-Resistant and Methicillin-Susceptible Staphylococcus aureus Isolates from Children with Skin Infections in Busan, Korea

The prevalence and molecular characteristics of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) and methicillin-susceptible S. aureus (CA-MSSA) from children with skin infection were investigated by staphylocoagulase (SC) typing, multilocus sequence typing (MLST), SCCmec typing and virulent toxins, including Panton-Valentine leucocidin (PVL), and exfoliative toxins (ET). Among 69 cases of CA-S. aureus for a 3 month period from March to June, 2014 at hospital in Busan, 28 (40.6%) were MRSA and 41 (59.4%) were MSSA. Of the 28 CA-MRSA isolates, two major clones were identified as SC type Vb-ST72-SCCmec type IV (53.6%) and SC type l-ST89-SCCmec type II variant (42.8%), and the remaining one (3.6%) was SC type lll-ST8-SCCmec type IV. In CA-MSSA, the prevalent clone was SC type Vb-ST72 (29.3%), followed by SC type Vb-ST188 (21.9%), SC type Va-ST121 (19.5%) and SC type lV-ST30 (9.6%). None was positive for PVL gene, and all of the SC type l-ST89-SCCmec type II variant clones were ETB gene positive. The data suggest that there are significant clonal relatedness with specific SC types, and genetic diversities in both community strains isolated from children with skin infections.

Molecular Epidemiology of Clinical Cryptococcus neoformans Isolates in Seoul, Korea

Cryptococcal infection is primarily caused by two species, Cryptococcus neoformans and C. gattii. Between the two species, C. neoformans var. grubii is the major causative agent of cryptococcosis in Asia. We investigated the molecular characteristics of 46 isolates of C. neoformans from patients with cryptococcosis between 2008 and 2012 in Seoul, Korea. All the isolates were determined to be C. neoformans var. grubii (serotype A), mating type MATalpha, and molecular type VNI by PCR-restriction fragment length polymorphism of the URA5 gene. Multilocus sequencing type (MLST) analysis using the International Society of Human and Animal Mycoses (ISHAM) consensus MLST scheme identified two sequence types (ST). Out of the 46 strains, 44 (95.7%) were identified as ST5, and remaining 2 were identified as ST31. Our study revealed that the clinical strains of C. neoformans in Korea are genetically homogeneous with the VNI/ST5 genotypes, and new appearance of VNI/ST31 genotype may serve as an important indicator of global genetic analysis.

Molecular Typing of Cryptococcus neoformans Isolated from Korean Patients

Cyptococcosis is generally caused by Cryptococcus neoformans, the opportunistic agent which has two species such as C. neoformans and C. gattii. Both C. neoformans and C. gattii species contain a number of genetically diverse subgroups that can be differentiated by various molecular typing methods. We conducted a molecular epidemiological analysis of 30 clinical isolates of the C. neoformans from cryptococcosis patients who had been hospitalized between 2008 and 2010 in medical centers located in Seoul and Busan in Korea. To determine the genetic diversity, 30 strains of C. neoformans were typed using PCR fingerprinting with the microsatellite specific primer of the phage M13 and the restriction fragment length polymorphism (RFLP) of orotidine monophosphosphate pyrophosphorylase (URA5) gene. All isolates were identified as serotype A, mating type MATa and molecular type VNI. The random amplified polymorphic DNA (RAPD) profiles obtained by using two primers revealed a single pattern. Our study shows that 30 strains of clinical C. neoformans are genetically homogeneous, with all of the isolates were molecular type VN1, serotype A, mating type MATa.

Molecular Typing of Clinical Cryptococcus gattii Isolates in Korea

Cryptococcus gattii causes life-threatening yeast infection in the pulmonary and central nervous systems of humans and animals, and traditionally has been considered to restrict into the tropical and subtropical areas. Despite rare incidence of cryptococcosis caused by C. gattii in Korea, three strains of C. gattii isolated from cryptococcosis patients between 1993 and 2010 were identified. To determine the genetic diversity, 3 strains of C. gattii were typed using PCR fingerprinting with primer M13 and the restriction fragment length polymorphism (RFLP) of orotidine monophosphosphate pyrophosphorylase (URA5) gene. All isolates were identified as serotype B and MATalpha mating type. The molecular types of each strain, on the other hand, turned out to be distinct belonging to VGI, VGII or III types, respectively. Although the travel histories of the patients were not available, clinical C. gattii strains isolated in Korea may represent the diverse molecular types existing worldwide.

Correlation Between the Prevalence of Superantigenic Toxin Genes and Coagulase Serotypes of Staphylococcus aureus Isolates

A heterogenic group of staphylococcal exotoxins, including staphylococcal superantigenic toxins, enterotoxin (SE), toxic shock syndrome toxin-1 (TSST-1), and coagulase are the most important virulence factors of Staphylococcus aureus. We analyzed the prevalence of genes encoding five enterotoxins and TSST-1 in S. aureus isolated from clinical ear discharges. The genes were identified by multiplex PCR and we compared the results to references of coagulase serotypes. In 102 isolates of S. aureus, 44 of them were methicillin-resistant S. aureus (MRSA) and the others were methicillin-susceptible S. aureus (MSSA). Among both types of S. aureus, 33 strains were positive for sea, 2 for seb, 23 for sec, 26 for see, and 26 for tst. Overall, 59 (57.8%) isolates were positive for one or more superantigenic toxin genes. From these, 71.2% (42/59) strains harbored more than one toxin gene in different combinations. The major combinations of genes were sea and see, and sec and tst. The degree of possession of superantigenic toxic genes was similar in both MRSA and MSSA isolates (56.8% vs 58.6%, respectively), yet significant differences in toxin gene profiles and coagulase serotypes between two isolates were detected. All of 13 positive strains for sec and tst were MRSA and belonged to coagulase serotype II. On the other hand, 80.0% of 20 positive strains for sea and see were MSSA with coagulase serotype IV and VII, whereas 20.0% of them were MRSA with coagulase serotype IV. This data indicates that the profile of superantigenic toxin genes correlates to coagulase serotype and methicillin resistance in S. aureus isolates.

Molecular Characterization of Clinical and Environmental Strains of Cryptococcus neoformans Isolated from Busan, Korea

Cryptocococcus neoformans is an encapsulated yeast that can cause life-threatening infections in immunocompromised patients. In this study, the genetic variability and epidemiological relationships of clinical and environmental isolates of C. neoformans from Busan, Korea, 2000~2005 were investigated. A total of 12 strains of C. neoformans, 7 clinical and 5 environmental isolates were analyzed by random amplified polymorphic DNA (RAPD) using three different primers and PCR-fingerprinting with a minisatellite-specific core sequence of phage M13. All strains belonged to C. neoformans serotype A and mating type MATa. Two different RAPD profiles (I and II) and a single pattern by M13 PCR-fingerprinting were identified. The major RAPD profile was pattern I (8 of 12 strains) and pattern II was identified from 2 clinical and 2 environmental strains, which clearly distinguished among isolates. Clinical strains with pattern II were isolated from the patients with HIV positive. Taken together, molecular patterns provide a good characterization of strains of C. neoformans as a heterogeneous group and epidemiological relationships in clinical and environmental strains.

Correlation between Sau1 Restriction and Modification Complex Type and Coagulase Serotype or SCCmec Type of Staphylococcus aureus

Staphylococcus aureus coagulase serotype I to VIII isolated from clinical samples could be classified into two groups, methicillin-sensitive S. aurues (MSSA) and methicilln-resistant S. aurues (MRSA), by antibiotics susceptibility and existence of mecA which is a gene related with methicillin resistance. Coagulase serotype I, VI, and VIII were MSSA which showed different antimicrobial susceptibility. Coagluase serotype II-V and VII were MRSA in which mecA and SCCmec were detected. To analyze Sau1 restriction and modification (R-M) complex types by coagulase type and SCCmec type, sau1hsdR, sau1hsdM and sau1hsdS genes involved in Sau1 R-M complex were detected by PCR, we found five complex types such as M1, R2M2, R2M2, R2M2S1, and R2M2S2. Coagulase serotype I, VI, and VIII of MSSA were M1, R2M2 and R2M2, respectively. SCCmec type II and coagulase serotype II, SCCmec type III and coagulase serotype III, SCCmec type IV and coagulase serotype V, and SCCmec type IV and coagulase serotype IV, VII of MRSA were Sau1 R-M complex type R2M2S1, R2M2, R2M2, and R2M2S2, respectively. Taken together, correlation between Sau1 R-M complex types and coagulase or SCCmec types of S. aureus was found.

Characterization of Vancomycin-Resistant Enterococci Isolated from Stools and Their Acquisition of Vancomycin Resistance

We have isolated 6 vancomycin resistant (VR) Enterococcus faecium and 5 VR-E. gallinarum. Vancomycin resistant enterococcus (VRE) isolates were resistant to multi-drugs, but susceptible to linezolid and quinupristin/dalfopristin. VRE isolates showed 10 VanA phenotypes and 1 VanB phenotype (E. gallinarum). However, all of them showed vanA genotype. vanA gene was detected on both genomic and plasmid DNA from all VRE isolates. Almost of VR-E. faecium had IS1216V which is worldwide type and almost of VR-E. gallinarum had IS1542 which is European type. IS1216V and IS1542 genes were not related with antibiotic types of VRE. Copy numbers of vanA were decreased in VRE with IS1216V or IS1542 but not in VRE with both ISs in broth without vancomycin. The copy numbers of vanA were significantly decreased in VanB phenotype of VRE with IS1542 in broth without vancomycin. Copy numbers of vanA were recovered in the presence of vancomycin. Growth time of reference E. faecium is faster than that of reference E. faecalis when cultured in the broth containing vancomycin. Reference strains cultured in the broth containing vancomycin showed intermediate resistance or resistance to antibiotics without acquisition of van genes. Naturally, multidrug-resistant E. faecium might be fast adapted in the presence of vancomycin compared to E. faecalis. Taken together, VanA phenotype E. gallinarum as well as E. feacium have been increasing in nosocomial infection and showed acquired inducible resistance. E. faecium and E. faecalis showed intermediate resistance in long exposure of vancomycin without acquisition of vanA.

Correlation Between Staphylococcal Cassette Chromosome mec Type and Coagulase Serotype of Methicillin-Resistant Staphylococcus aureus

Staphylococcal cassette chromosome mec (SCCmec) type and coagulase serotype are important epidemiologic factors in methicillin-resistant Staphylococcus aureus (MRSA). To investigate correlation between SCCmec type and coagulase serotype of MRSA, we analyzed SCCmec types of MRSA strains isolated from clinical sources and compared the results to coagulase serotypes and antimicrobial susceptibility patterns. A total of 108 MRSA isolates were classified into four SCCmec types: II (55.6%), IV (21.3%) III (13.0%) and IIIA (8.3%), and five coagulase serotypes: II (54.6%), IV (21.3%), V (18.5%) and VII (2.8%). All of coagulase type II, IV and V strains belonged to SCCmec type II, III/IIIA and IV, respectively. SCCmec types II, III and IIIA were multidrug resistant, whereas SCCmec type IV strains were non-multidrug resistant except beta-lactams and erythromycin. The data provide that there is a significant correlation between SCCmec types and phenotypic characteristic of coagulase serotypes.

Genotype, Coagulase Type and Antimicrobial Susceptibility of Methicillin-Resistant Staphylococcus aureus Isolated from Dermatology Patients and Healthy Individuals in Korea

Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most prevalent dermatology pathogens in hospitals and increasingly recognized in communities. We determined PFGE pattern of SmaI-restricted genomic DNA, coagulase type, and antimicrobial susceptibility of MRSA isolated in 2008 from dermatology inpatients and healthy hospital employees in A Hospital and from primary school children in Iksan city, Korea. Overall, the isolation rate of MRSA was 3.8% from the 788 normal persons: 4.9% from hospital employees and 1.1% from primary school children. MRSA was isolated in six of 13 (46.2%) family members of four school children with MRSA. The most prevalent coagulase serotype was II from patients and V from healthy individuals. Ten of twenty and six of twenty MRSA isolates from patients and from healthy personnel, respectively, had identical PFGE patterns, suggesting that these are originated from identical clones. Against MRSA from patients, only vancomycin was the most active (MIC range or =90% to amikacin, clindamycin, ciprofloxacin, erythromycin, fusidic acid, gentamicin and tetracycline. In conclusion, the MRSA carriage rates of healthy hospital workers were relatively high, 2.3~7.7%, depending on groups. Family members of a few primary school children with MRSA showed a high carriage rate, suggesting that intrafamily transmission occurred. MRSAs isolated from dermatology inpatients were relatively more resistant to various antimicrobial agents, including mupirocin, but all isolates were susceptibility to vancomycin.

Isolation and Characterization of Cryptococcus neoformans from Environmental Sources in Busan

Twenty nine samples of pigeon droppings (n = 12) and soil contaminated with avian excreta (n = 19), collected from different sites in Busan, were examined for isolation and characterization of Cryptococcus neoformans. Of these samples, 5 strains of C. neoformans were recovered from pigeon droppings (5/12 : 41.7%). All isolates were belonged to C. neoformans var. grubii (serotype A). The extracellular enzyme activities of the strains by using the API-ZYM system showed two different enzymatic patterns. The genetic variability among C. neoformans isolates was analyzed by random amplified polymorphic DNA (RAPD) using three 10-mer primers. Two different RAPD patterns, which clearly distinguished the isolates, were identified. Analysis of RAPD patterns provided a good characterization of environmental strains of C. neoformans serotype A as a heterogeneous group and were in good agreement with enzymatic profiles.