Studies On Antibiotic-resistant Bacteria And Antibiotic Resistance Genes In Marine Fish Cage-culture Areas In South China

In the past ten years,with the frequent occurrence of antibiotic resistance around the world,bacterial resistance has attracted widespread attention,as well as become one of the most popular scientific research.As one of the important aquaculture methods in China,marine fish cage-culture provides delicious and nutritious seafood products,and it has made great contributions to China's economic development.The coastal area of South China,a large scale,a long history,a wide variety,a complex disease situation,a high incidence rate and a high mortality rate,is a populated area for marine fish cultured.The abuse and misuse of antibiotics in aquaculture is serious,due to lack of the effective supervision and guidance for veterinary antibiotics use.This not only leads to serious antibiotic residues in aquatic products and the deterioration of the seawater environment,but more serious problem is the emergence of antibiotic-resistant bacteria?ARB?.Antibiotic resistance is transmitted with horizontally and vertically by antibiotic resistance genes?ARGs?,which poses a huge potential for environmental safety and human health.In this study,the antibiotic resistance distribution of Hainan Province and Guangdong Province were investigated in the seedling period?April?,rearing period?July?and harvesting period?October?.Hainan Province consists of six main marine fish cage-culture areas,namely Huiwen?HW?,Wanning?HL?,Lingshui?LS?,Tielugang?TLG?,Haitougang?HTG?and Lingao?LG?;Guangdong Province consists of seven main marine fish cage-culture areas,namely Raoping?RP?,Yanzhoudao?YZD?,Dayawan?DYW?,Guishandao?GSD?,Yangjiang?YJ?,Zhanjiang?ZJ?and Liusha?LS?.The abundance and diversity of ARB and ARGs in marine fish cage-culture areas were characterized by medium screening and quantitative PCR techniques,respectively.The regularition of ARB and ARGs abundances with temporal and spatial variation was revealed.By combining environmental factors,ARB,and ARGs for redundancy analysis?RDA analysis?,the contribution of environmental factors to antibiotic resistance changes was determined.Based on this investigation,the resistant strains of Vibrio harveyi to ciprofloxacin?CIP?were successfully obtained by induced in vitro.Comparative genomic analysis of different resistant strains found possible mutation sites that caused V.harveyi resistance to quinolones,which laid a theoretical foundation for exploring the resistance mechanism of V.harveyi to quinolones.The main findings of this study are as follows:?1?ARB and ARGs are widely distributed in marine fish cage-culture areas in Hainan Province.The ARB concentration in each sampling period was between 5.5×10¹².6×10⁴ CFU/mL.The resistance rates of amoxicillin?AMO?and gentamicin?GEN?are high in the seedling period,and reached 100%in HW.The resistance rate of oxytetracycline?OT?bacteria is high during the rearing period and harvesting period.The resistance rates of bacteria in different marine fish cage-culture areas are significantly different?p<0.05?.In general,the resistance rate of bacteria in HW is higher,while the resistance rate of bacteria in TLG,HTG and LG are generally low.From the perspective of ARB diversity,327 strains of ARB were identified as 32genera in the seedling period,while Alteromonas and Vibrio accounted for 32.72%and24.77%,respectively.A total of 715 strains of ARB were identified as 24 genera and Vibrio?76.64%?was dominate ARB.At the phylum level,Proteobacteria was the dominant phylum in each sampling period.In addition,the isolated dominant ARB,such as Vibrio,Acinetobacter,and Pseudoalteromonas are all opportunistic pathogens.13 ARGs,except tetS,ermA and ermB,were all detected.In general,the abundance of sul family gene is higher than the abundance of tet family gene,and sul2,sul1,sul3 are detected at each sampling site.However,in different sampling periods,the dominant ARGs showed obvious replacement phenomena.From the perspective of spatial and temporal variation,the abundance of ARGs in harvesting period was significantly higher than that in rearing period?p<0.05?;the total relative abundance of ARGs in HW and HTG was significantly higher than that in others sampling sites?p<0.05?.RDA analysis showed that most ARGs and ARB were positively correlated with dissolved oxygen?DO?and pH,and negatively correlated with salinity.Temperature had no effect on ARGs and ARB.?2?The concentration of ARB in the marine fish cage-culture areas in Guangdong Province ranges from 0 CFU/mL to 3.94×10⁴ CFU/mL,except for the concentration of OT resistant bacteria ranges from 10² CFU/mL to 10⁴ CFU/mL,and the concentration of other antibiotic ARBs ranges from 0 CFU/mL to 10² CFU/mL.From the perspective of bacterial resistance rate,except for OT resistance rate?about34.15%?,the bacterial resistance rate of other types of antibiotics was low in each culture area?about 1.15%?.Overall,there was no significant difference in the concentration of ARBs and antibiotic resistance rates with temporal and spatial variation?p>0.05?.From the perspective of ARB diversity,577 strains of ARB were identified as 40 genera,and the diversity index of rearing period>seedling period>harvesting period.Vibrio?50.95%?is the dominant ARB in three sampling periods,and Proteobacteria is the dominant phylum.Among the 13 ARGs,except for tetS and tetA,all 11 other ARGs were detected,sul1 was the dominant ARGs in the sul family,tetB was the dominant ARGs in the tet family,and ermB was the dominant ARGs in the erm family.The order of total ARGs abundance is:sul>tet>erm,which is consistent with the abundance distribution of ARGs in Hainan Province.With the temporal and spatial variation,the ARGs abundance in the harvesting period was significantly higher than that in the seedling and rearing period?p<0.01?,and the ARGs abundance in the RP was significantly higher than that in the DYW and LS?p<0.01?,and the ARGs abundance in GSD were significantly higher than that in DYW?p<0.05?.RDA analysis showed that temperature?WT?was positively correlated with most ARGs and ARB,while DO and pH were negatively correlated with most ARGs,only positively correlated with sul2 and tetM;salinity was positively correlated with most ARGs.Overall,WT,pH,and salinity have contributed greatly to the abundance changes of ARGs and ARBs.?3?Four strains with different antibiotic resistance were successfully obtained by CIP induction in vitro,which were 345-CIP₅₀,345-CIP₁₈₀,345-CIP_20g,and 345-CIP₃₀₀.Compared with the original strain F₀,each resistant strain minimum inhibitory concentration?MIC?was increased by 64,256,256,and 128 times,respectively.Significant cross-resistance?>4 MIC?of norfloxacin?NOR?and enrofloxacin?ENR?was induced by CIP-induced antibiotic-resistant strains,as well as in NOR.In addition,it is worth noting that the obtained MIC values of the antibiotic-resistant strains of different degrees have a different times decrease.A mild response was obtained by passage of the resistant strain 345-CIP_20g resistant phenotype of the 20th generation in the anti-free medium,but it was still a high-level resistant strain.The growth rate of antibiotic-resistant strains with different drug resistance levels was found to be significantly negatively correlated with the growth rate?p<0.01?.?4?Using the original strain V.harveyi 345 as the reference genome,the345-CIP₅₀ and 345-CIP₃₀₀ strains were re-sequenced by genome.The comparative genomic analysis found that resistance-related mutations occur in the zinc-absorbing system membrane protein ZnuB in the ATP-binding cassette?ABC?,the quinolone resistance-determining region GyrA,and the OmpR family in the two-component system.The histidine protein kinase EnvZ,LuxR family encodes the histidine kinase TtrS and the transmembrane protein YscT,SctT,HrcT in the quorum sensing system,indicating that these proteins play an important role in bacterial resistance.Overall,the SNP variants of the two different levels of resistant bacteria were basically the same,and no specific resistance-related SNPs were found in 345-CIP₃₀₀.InDel analysis revealed that the variability of the AcrA protein genome in the outer membrane pore proteins OmpA and OmpF,the chemotactic protein CheW and the AcrAB-TolC efflux system were all related to antibiotic resistance.It is speculated that these mutations may lead to high levels of bacterial resistance.