Epidemiology and Molecular Characterization ofStreptococcus pyogenesRecovered from Scarlet Fever Patients in Central Taiwan from 1996 to 1999

Abstract

One hundred seventy-nine Streptococcus pyogenes isolates recovered from scarlet fever patients from 1996 to 1999 in central Taiwan were characterized by emm, Vir, and pulsed-field gel electrophoresis (PFGE) typing methods. The protocols for Vir and PFGE typing were standardized. A database of the DNA fingerprints for the isolates was established. Nine emm or emm-like genes, 19 Vir patterns, and 26 SmaI PFGE patterns were detected among the isolates. Among the three typing methods, PFGE was the most discriminatory. However, it could not completely replace Vir typing because some isolates with identical PFGE patterns could be further differentiated into several Vir patterns. The prevalent emm types were emm4 (n ‫؍‬ 81 isolates [45%]), emm12 (n ‫؍‬ 64 [36%]), emm1 (n ‫؍‬ 14 [8%]), and emm22 (n ‫؍‬ 13 [7%]). Some emm type isolates could be further differentiated into several emm-Vir-PFGE genotypes; however, only one genotype in each emm group was usually predominant. DNA from nine isolates was resistant to SmaI digestion. Further PFGE analysis with SgrAI showed that the SmaI digestion-resistant strains could be derived from indigenous strains by horizontal transfer of exogenous genetic material. The emergence of the new strains could have resulted in an increase in scarlet fever cases in central Taiwan since 2000. The emm sequences, Vir, and PFGE pattern database will serve as a basis for information for the long-term evolutionary study of local S. pyogenes strains.Streptococcus pyogenes causes a variety of human diseases ranging from relatively mild skin infections to severe invasive diseases, such as acute rheumatic fever, glomerulonephritis, puerperal sepsis, necrotizing fasciitis, meningitis, and streptococcal toxic shock syndrome (9,29). Among the diseases caused by this pathogen, scarlet fever, characterized by a strawberry tongue, skin rash, and sore throat, is most prevalent in schoolchildren aged 4 to 7 years. In Taiwan, scarlet fever is a notifiable disease, with 150 to 230 confirmed cases per year from 1996 to 1999. Since only 9% of the medical centers, regional hospitals, and district hospitals in central Taiwan had ever reported cases to the health authorities from 1996 to 1999, the number of scarlet fever cases should be severely underreported. Scarlet fever outbreaks are not uncommon at daycare centers, kindergartens, and elementary schools (5, 16). However, the molecular epidemiology of this disease has not been well studied in Taiwan.Analysis of the clonal relationships between clinical isolates from patients by various typing methods is a practical approach to elucidation of the epidemiology of a disease. To date, a number of phenotyping and genotyping methods have been described for S. pyogenes (1, 3, 10, 13, 18-20, 22, 26, 27, 30, 33). Among these methods, M serotyping has been taken as the "gold standard" for the characterization of S. pyogenes strains, in light of its importance to streptococcal virulence. However, application of M serotyping is restricted due to the lack of a comprehensive set...

Cost-effective procedure for Streptococcus pyogenes immobilized DNA preparation and miniPFGE subtyping

“…It is noticeably shorter than the oneto three-day minimum previously reported [25][26][27][28][29][30]. The procedure reported here was also useful to prepare, in 6hours, immobilized DNA from S. pyogenes in the sample format of the CHEFMapper (BioRad) and to obtain well-resolved patterns.…”

“…There are other rapid PFGE protocols reported for subtyping Gram-positive bacteria [19,[23][24][25]34,[43][44][45]. Matushek et al reported a rapid procedure for DNA preparation of Gram-positive bacteria for pulsed field, reducing the incubation times to 4 or 5 hours in some cases.…”

“…Only the protocol developed by McEllistrem et al eliminated the cell lysis step and performed DNA release and deproteinization without the addition of complex buffers or enzymes [34]. However, this procedure is only applicable to S. pneumoniae, due to the unique ability of this bacterium to undergo autolysis [46][47] On the basis of PulseNet's Listeria monocytogenes PFGE protocol established in 1998, Chiou et al fingerprinted 179 S. pyogenes clinical isolates in approximately 24hours by incubating the cell suspension with lysozyme, treating the immobilized bacteria with proteinase K, and applying 20hours of electrophoresis for separating the DNA restriction fragments [25] (Table 2). Unlike to this rapid enzymatic protocol to prepare GAS DNA, where the lysis time is only 10 minutes, our procedure required more time because all the treatments were performed in situ (Table 2).…”

“…As well, the separation of DNA macrorestriction fragments takes between 18 and 24 hours in the CHEF chamber [18][19][20][21][22][23][24]. Procedures for GAS subtyping have been reported based on these optimized protocols, but in general, they have the same mentioned drawbacks in relation to the DNA preparation and the long electrophoresis times [25][26][27][28][29][30].…”

Changing Epidemiology of Streptococcus pyogenes emm Types and Associated Invasive and Noninvasive Infections in Southern Taiwan

“…2). This clonal pattern has also been identified in northern and central Taiwan (4,5). The reemergence of emm12 strains throughout Taiwan may explain the reported increase in cases of scarlet fever noted since 2005.…”