가덕도 연안 해수에서 분리된 Vibrio vulnificus의 세균학적 및 유전체 특성

Abstract

Vibrio vulnificus is a Gram-negative aquatic bacterium causing septicemia and wound infection in humans. This study was done to evaluate the distribution, molecular characteristics, and development of antimicrobial resistance in Vibrio vulnificus isolated from Gadeok Island coastal seawater in South Korea from 2019 through 2021, and to analyze the genome of the isolated strain. The monthly average water temperature and average salinity of the seawater ranged from 9.1°C to 28.2°C and 7.8 psu to 33.8 psu in that period, respectively. V. vulnificus was detected every year from June to October when average water temperature was above 19.8°C and was sometimes detected in March and November as well. The detection rate was higher from June to September as compared to other months. This high detection rate coincided with the period of high rainfall and low salinity. The trend of detection values appeared to be different every year. In 2019, most probable number (MPN) value of V. vulnificus soared to 2,100 MPN/100mL after heavy rainfall, but in 2020, MPN values of V. vulnificus were 36-240 MPN/100mL, when salinity levels were low after long periods of rainfall. In 2021, average water salinity was relatively high, with low MPNs of V. vulnificus. Pearson correlation analysis showed that occurrence of V. vulnificus was affected more by salinity than by water temperature. Comparing sampling stations, at nine stations on Gadeok Island’s east coast, which is close to the Nakdong River, the detection rate of V. vulnificus was 41.7%, which was higher than that of the station on Gadeok Island’s west coast, at 13.9%. PCR analysis confirmed the presence of vvhA gene in all strains, and all of the strains were found to possess β-hemolytic activity. viuB and vcgC genes were confirmed in 44.8% and 53.0% of the isolated strains and were mainly detected in strains isolated during the June-October period when the detection rates and Vibrio sepsis incidence rates were high. Antimicrobial disk susceptibility test for the isolated strains confirmed resistance (2.6%) and intermediate resistance (58.1%) to cefoxitin and confirmed intermediate resistances to amikacin (0.6%), cefepime (20.6%), cefotaxime (0.6%), and erythromycin (35.5%). The minimum inhibitory concentration (MIC) of cefoxitin in isolated strains was determined to be less than or equal to 32 μg/mL. Whole-genome sequencing and analysis of V. vulnificus 1908-10, which possesses virulence gene (vvhA, viuB and vcgC) and resistance to cefoxitin antibiotics, showed that it consists of two circular contigs and no plasmid. The total genome size was estimated at 5,018,425 bp, with a GC content of 46.9%. We found 119 tRNA and 34 rRNA genes respectively in the genome, along with 4,352 predicted protein sequences. Virulence factor analysis using VFDB showed that V. vulnificus 1908-10 had similar virulence factors to that of the C-genotype strains except for capsular polysaccharides, and showed differences from the E-genotype strain in the presence of several genes, viz., mannose-sensitive hemagglutinin type Ⅳ pilus, type Ⅳ pilus, flagella, vibriobactin, autoinducer-2 of quorum sensing, and RTX toxin. Antimicrobial resistance gene analysis via the CARD tool identified the presence of PBP3 (ftsI) gene, which corresponds to the variant model of protein binding protein 3 conferring resistance to beta-lactam antibiotics, including cefoxitin. In conclusion, Gadeok Island coast provides favorable conditions for the growth of V. vulnificus due to inflow of fresh water through the Nakdong River, making it necessary to monitor for detection and development of antimicrobial resistance in this strain. Additionally, further studies are required to confirm whether ftsI gene is involved in conferring resistance to cefoxitin in this strain.