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.

Cluster of Serogroup W-135 Meningococcal Disease in 3 Military Recruits

We describe a group of 3 cases of invasive meningococcal disease that occurred in a military training camp in April 2011. All three patients were hospitalized. Ultimately, two patients recovered and one died. One patient had meningitis, one patient had septicemia and meningitis, and the other had no definite septicemia or meningitis. Neisseria meningitidis serogroup W-135 was detected in the serum and cerebrospinal fluid (CSF) of all patients by real-time polymerase chain reaction. In the one case of mortality, two strains were isolated from the patient's blood and CSF. Using multilocus sequence typing analysis, these strains were identified as a novel sequence type, ST-8912. Special attention is required for the meningococcal disease in military camp because the military personnels are in high risk of contact transmission.

Carriage Rates and Serogroups of Neisseria meningitidis among Freshmen in a University Dormitory in Korea

PURPOSE: Neisseria meningitidis is a leading cause of bacterial meningitis in young adults. University students, especially those living in dormitories, have been known to be at increased risk of meningococcal disease. We performed a longitudinal study to determine the carriage rates of N. meningitidis and the changes thereof. MATERIALS AND METHODS: We recruited Inha University freshmen who were, at that time, admitted to a student dormitory. A pharyngeal swab was taken from all participant who were also asked to complete a questionnaire. This was repeated four weeks later. RESULTS: A total of 136 students were enrolled at the first culture. After four weeks, 128 students were enrolled, including 106 re-participants. The overall carriage rates changed from 11.8% to 14.1%. In analysis of the 106 re-participants, "visiting to pubs" was associated with carriage of N. meningitis for both the first (p=0.047) and second cultures (p=0.026). Serogroup C was found to be the most frequent serogroup (5 isolates), while 3 isolates were found from serogroup B. The most prevalent PorA types were P1.22,14-6 (4 isolates) and P1.19,15 (3 isolates). The DNA sequences of PorA VR2 were changed in 2 students during prolonged carriage. CONCLUSION: The meningococcal carriage rate among first year university students who resided in a dormitory did not significantly increase over 4-week interval between cultures, which is markedly different from those reported in Western studies. Close social contact appeared to be related with carriage. Our data also revealed diversity in PorA types, suggesting the possibility of rapid mutation of the PorA gene during the 4-week interval.

Prevalence of Mycoplasma pneumoniae Antibodies in Healthy Residents of Jeonnam Province / 대한임상미생물학회지

BACKGROUND: Mycoplasma pneumoniae is the most frequent cause of respiratory tract infections in schoolaged children and adolescents. For appropriate use of antibiotics, diagnosis of M. pneumoniae infection in routine clinical practice has been based on serology using a single serum sample. We evaluated the seroprevalence of anti-M. pneumoniae-specific antibodies in 500 asymptomatic, healthy persons in Jeonnam Province. METHODS: Sera were collected from 500 healthy persons in Jeonnam Province. Anti-M. pneumoniae antibody titer was measured using a microparticle agglutination assay Serodia Myco II (Fujirebio, Japan) and VIRCELL IgM Mycoplasma ELISA kits (Vircell, Granada, Spain). RESULTS: Anti-M. pneumoniae antibody titers in 500 healthy individuals were 1:20 in 344 (68.8%), 1:40 in 16 (3.2%), 1:80 in 71 (14.2%), 1:160 in 45 (9.0%), 1:320 in 14 (2.8%), and 1:160) and positive IgM, and an assessment of current infection with single serum serology has its limitation for the diagnosis of M. pneumoniae infections.

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.

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.

Characterization of Haemophilus influenzae by SDS-PAGE, Restriction Enzyme Analysis and rRNA Gene Restriction Patterns

Among the fifty-three clinical isolates of Haemophilus influenzae, nineteen isolates including eight isolates of each biotype I-VIII, six of serotype b (Hib) strains and five of nontypeable strains were characterized by SDS-PAGE about outer membrane protein (OMP), restriction enzyme analysis (REA) and rRNA gene restriction pattems. OMP patterns showed to common band patterns in each H. influenzae isolate. Based on the two major proteins, 31KDa-38KDa, isolated strains were classified into 7 subtypes. In the OMP patterns about biotype and serotype, the specific pattern of each biotype was not distinguishable, but all of the serotype b strains were shown identical unique pattern, therefore it made distinctive difference with nontypeable strains. The digested genomic DNAs with EcoRI were identical result with rRNA gene restriction. It was more subdivided into 10 ribotypes. The most common ribotype I and serotype 1 accounted for 6 strains (31.6%) and 7 strains (36.8%) of the 19 clinical isolates, respectively. Hib isolates that were both OMP subtype 1 and ribotype I accounted for 2 strains (10.5%). In the epidemiologically unrelated strains, the putative association between the subtypes could not be confirmed. According to these results, the three methods were discriminatory and appropriate techniques for epidemiological studies of H. influenzae.