Lysins are murein hydrolases produced by bacteriophage that act on the cell wall of host bacteria to release progeny phages. Research indicated that lysins could kill bacteria effectively and specifically in vitro. To prepare recombinant bacteriophage lysin of Streptococcus (PlyC) and analyze its biological activity, we obtained two genes of PlyC named PlyCA and PlyCB by PCR amplification and inserted them into pET-32a(+), then transformed the recombinant expression vectors pET-32a(+)-PlyCA and pET-32a(+)-PlyCB into E. coli BL21(DE3) respectively. After induction with 0.7 mmol/L IPTG at 30 degrees C for 7 h, PlyCA and PlyCB were successfully expressed, SDS-PAGE analysis determined that they all constituted above 30% of the total cell proteins. After Ni(2+)-NTA affinity chromatography, the purity was more than 95%. With the denaturation and protein refolding, we gained the recombinant PlyC. To determine its biological activity, we adopted turbidimetry and plate count method. Before and after lysin treatment, the cell morphology was studied by scanning electron microscopy (SEM). The results showed that the recombinant PlyC could specifically cleavage Streptococcus pyogenes (group A beta-hemolytic streptococci). Under the incubation time of 60 min with 4 microg/mL PlyC in Streptococcus pyogenes dilution which OD600 was 0.56, the germicidal effect was up to 99.6%, while SEM observations showed that cell wall cracked and presented cell debris. This finding laid the foundation for the further study and achieving an effective treatment for streptococcal infection.
Bacteriolysis ; Enzymes ; biosynthesis ; genetics ; isolation & purification ; Escherichia coli ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; isolation & purification ; Streptococcus pyogenes ; drug effects

PURPOSE: Erythromycin-resistant beta-hemolytic streptococci (BHS) has recently emerged and quickly spread between and within countries throughout the world. In this study, we evaluate the antimicrobial susceptibility patterns and erythromycin resistance mechanisms of BHS during 2003-2004. MATERIALS AND METHODS: The MICs of seven antimicrobials were determined for 204 clinical isolates of BHS from 2003 to 2004. Resistance mechanisms of erythromycin-resistant BHS were studied by the double disk test as well as by polymerase chain reaction (PCR). RESULTS: Compared with our previous study, resistance among Streptococcus pyogenes isolates to a variety of drugs decreased strikingly: from 25.7% to 4.8% in erythromycin; 15.8% to 0% in clindamycin; and 47.1% to 19.0% in tetracycline. The prevalent phenotypes and genotypes of macrolide-lincosamide-streptograminB (MLSB) resistance in Streptococcus pyogenes isolates have been changed from the constitutive MLSB phenotype carrying erm(B) to the M phenotype with mef(A) gene. In contrast with Streptococcus pyogenes, resistance rates to erythromycin (36.7%), clindamycin (43.1%), and tetracycline (95.4%) in Streptococcus agalactiae isolates did not show decreasing trends. Among the Streptococcus dysgalactiae subsp. equisimilis isolates (Lancefield group C, G), resistance rates to erythromycin, clindamycin, tetracycline and chloramphenicol were observed to be 9.4%, 3.1%, 68.8%, and 9.4%, respectively. CONCLUSION: Continual monitoring of antimicrobial resistance among large-colony-forming BHS is needed to provide the medical community with current data regarding the resistance mechanisms that are most common to their local or regional environments.
Anti-Bacterial Agents/*pharmacology ; Drug Resistance, Multiple, Bacterial ; Erythromycin/*pharmacology ; Genes, Bacterial ; Genotype ; Hospitals ; Humans ; Incidence ; Korea ; Microbial Sensitivity Tests ; Streptococcus/*drug effects/genetics/isolation & purification ; Streptococcus agalactiae/drug effects/genetics/isolation & purification ; Streptococcus pyogenes/drug effects/genetics/isolation & purification

OBJECTIVETo investigate the genetic features of drug resistance to group A streptococcus(GAS) and macrolides antibiotics among pediatric patients in Beijing 2012.

METHODSA total of 199 strains of GAS were collected from 36 hospitals in Beijing between May and July, 2012. All strains were isolated from oropharyngeal swabs. The minimum inhibitory concentrations (MICs) of ten antibiotics (penicillin, ampicillin, erythromycin, clindamycin, tetracycline, levofloxacin, tigecycline, vancomycin, linezolid and streptogramin) were detected by VITEK-2 compact with GPS-67 test kit. The genes encoding macrolides resistance (ermA, ermB and mefA ) were amplified and tested by PCR. The macrolides resistant phenotype of group A streptococcus was detected by double disc test (D-test).

RESULTSAmong 199 strains of GAS collected in this study, 101(50.8%) were from suburbs and the other 98(49.2%) were from urban areas. 111(55.8%) strains were collected from scarlet fever patients while the other 88(44.2%) were from oropharyngeal infection cases. All the strains were sensitive to penicillin and ampicillin, and the percentage of resistance to erythromycin, clindamycin and tetracycline were 96.5% (192/199), 95.5% (190/199) and 92.0% (183/199), respectively. All strains were susceptible to levofloxacin, tigecycline, vancomycin, linezolid and streptogramin. The rates of resistance to erythromycin, clindamycin and tetracycline were different in different districts, however, the difference in it between ages and clinical diagnosis did not show statistical significance (P > 0.05) . The detected rate of drug resistance gene ermB was 98.5% (196/199). The gene ermA was only detected out in 5 strains and the gene mefA was not detected out. 199 strains showed A macrolides resistant phenotype cMLS, while the phenotype iMLS was not found in this study.

CONCLUSIONThis study demonstrates the high level of clindamycin resistance in group A streptococcus collected from children in Beijing, 2012. The macrolides resistance of group A streptococcus was highly prevalent in Beijing, and the dominant phenotype was cMLS mediated by gene ermB.

Anti-Bacterial Agents ; pharmacology ; Bacterial Proteins ; genetics ; Child ; Child, Preschool ; China ; epidemiology ; Drug Resistance, Multiple, Bacterial ; genetics ; Genotype ; Humans ; Macrolides ; pharmacology ; Streptococcal Infections ; epidemiology ; microbiology ; Streptococcus pyogenes ; drug effects ; genetics ; isolation & purification

BACKGROUND: Group A streptococcus (GAS) is the most common cause of bacterial pharyngitis in children. Antibiotic resistance rates and emm genotypes of GAS isolated from patients with acute pharyngitis were studied in 2009. METHODS: Throat cultures were taken from 499 children with acute pharyngitis in Jinju, Korea, in 2008-2009. A total of 174 strains (34.9%) of GAS were isolated, and antimicrobial susceptibility testing was performed using the disk diffusion method. The phenotypes of macrolide resistance and macrolide resistance genes were determined. The emm genotypes were identified using PCR and sequencing. The data were compared with those acquired in 2002 in the same region. Data on the annual macrolide production were collected between 1999 and 2008. RESULTS: The resistance rates of GAS to erythromycin, clindamycin, and tetracycline were 4.6%, 2.9%, and 2.3%, respectively. The constitutive resistance rate was 62.5% for the erm(B) gene and 37.5% for the M phenotype of the mef(A) gene. emm4 was most frequently detected (28.2%), followed by emm89 (20.1%). Most of the erythromycin resistant strains had the emm28 genotype. We noted a gradual increase in macrolide production during the study period. CONCLUSIONS: The erythromycin resistance rate of GAS isolated from children with acute pharyngitis was significantly lower in 2009 (4.6%) than in 2002 (44.8%). We observed a remarkable change in the distribution of emm genotypes during the 7-yr period. The significant decline in erythromycin resistance in 2009 might be associated with a prominent decrease in the resistant genotype emm12 (3.4% in 2009 vs. 28.0% in 2002) rather than restriction of macrolide use.
Acute Disease ; Adolescent ; Anti-Bacterial Agents/*pharmacology ; Child ; Child, Preschool ; Disk Diffusion Antimicrobial Tests ; Drug Resistance, Bacterial/genetics ; Erythromycin/*pharmacology ; Female ; Genotype ; Humans ; Male ; Pharyngitis/drug therapy/*microbiology ; Phenotype ; Sequence Analysis, DNA ; Streptococcus pyogenes/*drug effects/*genetics/isolation & purification

OBJECTIVETo investigate molecular characterization of streptococcus pyogenes isolates involved in an outbreak of scarlet fever in China in 2011.

METHODSSeventy-four Streptococcal pyogenes involved in an outbreak of scarlet fever were isolated from pediatric patients in the areas with high incidence in China from May to August of 2011. Emm genotyping, pulsed-field gel electrophoresis (PFGE), superantigen (SAg) genes and antimicrobial susceptibility profiling were analyzed for these isolates.

RESULTSA total of 4 different emm types were identified. Emm12 was the most prevalent type which contained four predominating PFGE patterns corresponding to four different virulence and superantigen profiles. Emm12 (79.7%) and emm1 (14.9%) accounted for approximately 94% of all the isolates. The speA gene was all negative in emm12 isolates and positive in emm1 isolates. All strains were resistant to erythromycin, and 89.4% of them were resistant to erythromycin, tracycline, and clindamycin simultaneously.

CONCLUSIONSeveral highly diversified clones with a high macrolide resistance rate comprise a predominant proportion of circulating strains, though no new emm type was found in this outbreak. The data provide a baseline for further surveillance of scarlet fever, which may contribute to the explanation of the outbreak and development of a GAS vaccine in China.

Anti-Bacterial Agents ; administration & dosage ; therapeutic use ; Child ; China ; epidemiology ; Disease Outbreaks ; Drug Resistance, Bacterial ; Electrophoresis, Gel, Pulsed-Field ; Humans ; Incidence ; Molecular Epidemiology ; Scarlet Fever ; drug therapy ; epidemiology ; microbiology ; Streptococcus pyogenes ; drug effects ; genetics ; isolation & purification ; pathogenicity ; Virulence

BACKGROUND: We determined the epidemiological characteristics of erythromycin (EM)-resistant Streptococcus pyogenes (group A streptococci, GAS) strains isolated from Korea and Japan, using emm genotyping and multilocus sequence typing (MLST). METHODS: Clinical isolates of GAS had been collected from 1992 to 2012 in Korea and from 2004 to 2009 in Japan. EM resistance was determined by the microdilution method, and resistance genotypes were assessed by PCR. The emm genotyping and MLST were performed by DNA sequencing. RESULTS: The emm genotypes and sequence types (STs) were concordant in 143 (85.1%) of 168 EM-resistant GAS strains from Korea. ST36/emm12 (35.1%), ST52/emm28 (22.6%), and ST49/emm75 (16.1%) were the most common types. Most of the ST36 (93.9%) and ST52 (95.8%) strains harbored erm(B), whereas strains ST49, ST42, and ST15 contained mef(A). The concordance between emm genotypes and STs was 41 (93.2%) among 44 EM-resistant GAS strains from Japan. ST36/emm12 (34.1%), ST49/emm75 (18.2%), and ST28/emm1 (15.9%) were the major types. ST36 isolates harbored either erm(B) (56.3%) or mef(A) (37.5%), whereas isolates ST28, ST49, and ST38 carried only mef(A). The proportion of erm(B) and mef(A) was 66.1% and 33.3% in Korea and 22.7% and 68.2% in Japan, respectively. CONCLUSIONS: The common STs in Korea and Japan were ST36 and ST49, whereas ST52 was present only in Korea and ST28 only in Japan. Genotype erm(B) was predominant in Korea, whereas mef(A) was frequent in Japan. There were differences between Korea and Japan regarding the frequencies of emm genotypes, STs, and EM resistance genes among the EM-resistant GAS.
Anti-Bacterial Agents/*pharmacology/therapeutic use ; Bacterial Proteins/*genetics ; Bacterial Typing Techniques ; *Drug Resistance, Bacterial ; Epidemiologic Studies ; Erythromycin/*pharmacology/therapeutic use ; Genotype ; Humans ; Japan/epidemiology ; Microbial Sensitivity Tests ; Multilocus Sequence Typing ; Republic of Korea/epidemiology ; Streptococcal Infections/drug therapy/*microbiology ; Streptococcus pyogenes/drug effects/*genetics/isolation & purification