Molecular Characterization of Enterococci Isolates from Northeastern and Southern Regions of Thailand

Abstract

Enterococci have been becoming one of the most important pathogens due to resistance to common-used antibiotics and prolonged persistence in hospital settings. In addition, biofilm formation and various virulence factors activities are supported for colonization and pathogenicity of enterococci. Therefore, the study aimed to determine the occurrence of antibiotic resistance, biofilm formation and virulence determinants in clinical enterococcal isolates from two tertiary care hospitals in Northeastern and Southern Thailand. Information of antibiotic resistance and virulence factors activities in enterococcal infections are required to develop the control plan for emerging antibiotic resistance. However, data related with enterococcal infection in Thailand is limited. Seven hundred eighty-four enterococcal clinical isolates were obtained from 2 tertiary care hospitals in Thailand; Sunpasitthiprasong Hospital (SS), Northeastern Thailand and Hat Yai Hospital (HTY), Southern Thailand between 2015 and 2018. Species and vancomycin-resistant genes identification were achieved by multiplex PCR. E. faecalis, E. faecium, E. casseliflavus, E. gallinarum, and Enterococcus spp. from urine, pus, blood, body fluid, and sputum found at 73.2%, 25.5%, 0.1%, 0.1% and 1.0%, respectively. Kirby-Bauer disc diffusion method for susceptibility testing was carried out using 10 antibiotics in common enterococci isolates. Five additional antibiotics were tested for VRE isolates. MIC of linezolid, tigecycline and vancomycin were determined for VRE isolates by broth microdilution method. MDR revealed in 79.9% and 80.5% of isolates from SS and HTY, exhibiting greater resistance to erythromycin (85.2% and 75.0%), tetracycline (76.8% and 77.6%), and ciprofloxacin (71.9% and 72.8%), respectively. High-level aminoglycoside resistance (HLAR) and VRE were demonstrated in 60% and 4.7% of isolates from SS, and 59.6% and 5.1% of isolates from HTY, respectively. High-level vancomycin resistance gene, vanA gene was revealed in 32 out of 38 VRE isolates and no van gene was revealed in 4 VRE isolates from two hospitals. vanB, vanD and vanM genes were not detected. Low-level vancomycin resistance gene, vanC1/2 gene was carried in E. casseliflavus and E. gallinarum isolates. All VRE isolates were susceptible to linezolid and tigecycline. Biofilm formation, hemolysin, gelatinase, caseinase and lipase were detected. Biofilm, hemolysin, gelatinase, caseinase, and lipase were produced in 40.8%, 50.5%, 55.1%, 46.3%, and 1.6% of isolates from SS and 34.6%, 86.1%, 44.9%, 25.7%, and 14.7% of isolates from HTY, respectively. Moreover, correlation between antibiotic resistance and virulence factors activities were explored using Chi-square test in SPSS software. Species distribution and antibiotic-resistant patterns were similar in two hospitals. However, gelatinase and caseinase enzymes were more produced in isolates from SS, hemolysin and lipase were more produced in isolates from HTY. Enterococci isolated in urinary tract were significantly more produced biofilm (p<0.05). Resistance to ciprofloxacin, chloramphenicol, high-level aminoglycosides, and tetracycline were statistically associated with biofilm formation and secreted virulence factors activities in isolates from two hospitals (p<0.05). When the clonal relationship of all VRE isolates were evaluated using random amplified polymorphic DNA (RAPD) with four primers sets such as AB1-15, AP4 plus ERIC1R, AP4 and M13, AP4 plus ERIC1R primer gave rise the informative clonal clusters among VRE isolates from two hospitals. The clusters based on 80% similarity in RAPD dendrogram using AP4 plus ERICIR primer revealed VRE infection was clonally disseminated in an individual hospital especially between June 2017 and January 2018 in SS and between January 2015 and August 2015 in HTY.