제주도 양식넙치에서 분리된 연쇄구균의 신속동정과 항생제 내성

Abstract

The genus Streptococcus comprises about 48 recognized gram-positive species which are widely distributed in estuarine and marine environments. As some Streptococcus species have long been known as bacterial pathogens to marine organisms and human beings, many researchers have attempted to develop the method for rapid and accurate identification. Traditionally, they have been identified based on morphological, serological and biochemical methods. In recent years, rDNA sequences have been adopted for bacterial taxonomy including Streptococcus to overcome the shortage of traditional methods. However, there are still some problems in identification of closely related bacterial species due to the conservation of rDNA sequences. Thus, we tried to develop the more rapid and accurate identification method than before by using the DNA sequences of aroA gene and multiplex PCR. The aroA gene encodes 5-enolphyluvyshikimate-3-phospate synthase which is a key enzyme in the aromatic amino acid biosynthetic pathway in microorganism. Streptococcus species were isolated from the diseased olive flounders (Paralichthys olivaceus) sampled at the hatcheries in Jeju and standard strains of Streptococcus were purchased from KCCM and KCTC. aroA genes of Streptococcus were then cloned and sequenced for detection primer design and phylogenetic analysis. By using detection primers designed based on multiple alignment and phylogenetic analysis, multiplex PCRs have been performed for the simultaneous detection of major Streptococcus spp. isolated from the farmed olive flounder in Jeju. As a result, two major Streptococcus species among total number of isolated strains were Streptococcus parauberis (81.2%), Streptococcus iniae (11.1%), Listeria sp. (7.4%), Enterococcus sp. (0.3%). In addition, antimicrobial susceptibility, resistant pattern and combined action to the 18 antibiotics were tested with the identified Streptococcus species in order to treat the streptococcosis effectively and rapidly. The results showed that both Streptococcus parauberis and Streptococcus iniae had high degree of antimicrobial resistance to clindamycin, and some degree of resistance to flumequine and oxolinic acid as well, whereas these two species were highly sensitive to chloramphenicol, ciprofloxacin, doxycycline and florfenicol. By the way, the degree of antimicrobial susceptibility was various even between strains of the same Streptococcus species.