Extremely low prevalence of erythromycin-resistant Streptococcus pyogenes isolates and their molecular characteristics by M protein gene and multilocus sequence typing methods

Background: Group A streptococci (GAS) are notorious bacteria causing a wide variety of clinical manifestations ranging from mild, acute streptococcal pharyngitis to chronic non-suppurative diseases and immunological sequelae. They are further complicated by the global rise on the emergence of macrolide resistance among these bacteria in which several M protein gene (emm) and sequence types are associated with invasive diseases. Objectives: The current study aimed at determining the erythromycin resistance patterns and molecular characteristics of GAS clinical strains by emm and multilocus sequence typing (MLST) methods. Methods: Thirty-five GAS clinical isolates were subjected to antibiotic susceptibility testing by disk diffusion method. The minimum inhibitory concentration (MIC) of erythromycin against GAS by E-test was determined. Clinical and laboratory standards institute (CLSI) guideline was used for the interpretation of results. Detection of ermA, ermB, and mefA genes by polymerase chain reaction (PCR) was performed and emm typing was done by amplification and sequencing of emm genes per standard protocol. Allele and sequence type (ST) of GAS were obtained using the S. pyogenes MLST database. Results: All the isolates were sensitive to erythromycin, penicillin, clindamycin, chloramphenicol, and vancomycin (100%). Resistance to tetracycline was 54.3%. The mefA gene was found in one erythromycin susceptible isolate. No other erythromycin resistance genes were detected in the isolates. Twenty different emm types were found and the most frequent emm types/subtypes detected were emm1, emm18.21, emm28.5, emm97.4, and emm102.2 (each 8.6%). However, no new emm type was detected. A total of 15 sequence types (STs), eight clonal clusters (CCs), and eight singletons were identified among 21 representative isolates. Three isolates exhibited CC1 (ST28/emm1). Conclusions: High susceptibility of GAS isolates against erythromycin could be due to low antibiotic selective pressure in Malaysian clinical settings. High diversity of emm and ST types revealed the heterogenic nature of the strains circulating in Malaysian hospitals. Continuous epidemiological monitoring by molecular typing methods is warranted to improve the management strategies of GAS infections in future

Distribution of virulence genes and the molecular epidemiology of Streptococcus pyogenes clinical isolates by emm and multilocus sequence typing methods

Background: Streptococcus pyogenes has a variety of virulence factors and the predominant invasive strains differ according to specific emm types and geographical orientation. Although emm typing is commonly used as the gold standard method for the molecular characterisation, multilocus sequence typing (MLST) has become an important tool for comparing the genetic profiles globally. This study aimed to screen selected virulence genes from invasive and non-invasive clinical samples and to characterise the molecular epidemiology by emm typing and MLST methods. Materials and methods: A total of 42 S. pyogenes isolates from invasive and non-invasive samples collected from two different tertiary hospitals were investigated for the distribution of virulence factors and their molecular epidemiology by emm and multilocus sequence typing methods. Detection of five virulence genes (speA, speB, speJ, ssa and sdaB) was performed using multiplex polymerase chain reaction (PCR) using the standard primers and established protocol. Phylogenetic tree branches were constructed from sequence analysis utilised by neighbour joining method generated from seven housekeeping genes using MEGA X software. Results: Multiplex PCR analysis revealed that sdaB/speF (78.6%) and speB (61.9%) were the predominant virulence genes. Regardless of the type of invasiveness, diverse distribution of emm types/subtypes was noted which comprised of 27 different emm types/subtypes. The predominant emm types/subtypes were emm63 and emm18 with each gene accounted for 11.8% whereas 12% for each gene was noted for emm28, emm97.4 and emm91. The MLST revealed that the main sequence type (ST) in invasive samples was ST402 (17.7%) while ST473 and ST318 (12% for each ST) were the major types in non-invasive samples. Out of 18 virulotypes, Virulotype A (five genes, 55.6%) and Virulotype B (two genes, 27.8%) were the major virulotypes found in this study. Phylogenetic analysis indicated the presence of seven different clusters of S. pyogenes. Interestingly, Cluster VI showed that selected emm/ST types such as emm71/ST318 (n=2), emm70.1/ST318 (n=1), emm44/ST31 (n=1) and emm18/ST442 (n=1) have clustered within a common group (Virulotype A) for both hospitals studied. Conclusion: The present study showed that group A streptococcci (GAS) are genetically diverse and possess virulence genes regardless of their invasiveness. Majority of the GAS exhibited no restricted pattern of virulotypes except for a few distinct clusters. Therefore, it can be concluded that virulotyping is partially useful for characterising a heterogeneous population of GAS in hospitals

Virulence factor and genetic characterization of Streptococcus pyogenes from clinical isolates

Streptococcus pyogenes or group A streptococcus (GAS) is commonly known as a flesh-eating bacterium. Several virulence genes are responsible for this phenomenon and other clinical diseases ranging from mild to life threatening infections. Extracellular toxins such as superantigen streptococcal pyrogenic exotoxins are commonly associated with tissue invasion and initiate the release of cytokinesin S. pyogenes pathogenesis. This study aimed to determine the virulence characteristics of S. pyogenes by haemolysin test and DNase test and the detection of selected toxin genes (speA, speB, speJ, ssa and sdaB) in S. pyogenes isolates. Its genetic relatedness was determined by multilocus sequence typing (MLST). A total of 42 S. pyogenes clinical isolates were collected from Hospital Kuala Lumpur and Hospital Serdang. The isolates were obtained from five different sources which were blood (23.8%), pus (52.4%), tissue (16.7%), wound (4.8%) and throat (2.4%). Regardless of the type of invasiveness, all S. pyogenes produced positive result in haemolysin test. A total of 81.0% of GAS hydrolyzed DNA while only 19.0% negative toward DNase test. Among those with positive DNase, all GAS isolates from throat, tissue and wound contributed positive detection. The highest virulence gene detected was sdaB(83.3%), followed by speB(64.3%), ssa(54.8%), speA(52.4%) and speJ(52.4%). In blood isolates, sdaB gene was highly detected with (80.0%) while in wound isolates, (100.0%) of sdaB, speA, and speB genes were detected as positive findings. All five different virulent genes were detected in all types of samples except speA gene which not been detected in an isolate from throat sample. MLST analysis showed that the most predominant STs were in descending order as follows: ST28, ST473, ST402, ST13 and ST318 (7.14% for each), ST60, ST313, ST205, ST101, ST55 (4.76% for each) and ST300, ST599, ST36, ST25, ST168, ST5, ST426, ST408, ST156, ST31, ST442, ST306, ST147, ST114, ST549, ST89, ST83 (2.38% for each). Based on the phylogenetic tree analysis, eleven pairs of S. pyogenes isolates shared the same branches with the current ancestor such as (HKL14 and HKL23), (HKL1 and HKL22), (HKL17 and HS2), (HKL18 and HS15), (HKL20 and HS18), (HKL2 and HKL6), (HKL5 and HS4), (HS6 and HKL4), (HKL9 and HS5), (HKL10 and HS9) and (HKL19 and HS21).Diverse genetic heterogeneity of GAS isolates in the present study was observed with high distribution of virulence genes. Similarities and differences in ST numbers among GAS isolates shared one common ancestor despite they were isolated from two different locations were also observed. The presence of emm1/(ST28) which is associated with hospital outbreaks worldwide deserves continuous surveillance in Malaysian hospitals