Characteristics And Distribution Of Quinolone-Resistant Bacteria And Their Resistance Genes In Tilapia And Hybrid Snakehead Aquaculture

Antimicrobial resistance is one of the most serious public health problems in the world.In recent years,it has been paid more and more attention in aquaculture.Quinolones are commonly used in aquaculture,which contribute to resistance problem and has become rapid developed.Tilapia and hybrid snakehead（Channa maculata♀×Channa argus♂）are both important freshwater economic fish species in China,and they are both breeding in high density farms and with high productivity.The occurrence of quinolones resistance is also increasing in tilapia and hybrid snakehead aquaculture.Most domestic and foreign researches focus only on certain species of bacteria isolated from aquatic animals or their aquatic environments.It would not reveal the occurrence and characteristics of bacterial resistance in aquaculture comprehensively.In this study,in order to determine the quantity and composition of resistant bacteria from the intestine and aquatic environment（pond water and sediment）of tilapia and hybrid snakehead,resistant bacteria were screened by nalidixic acid or enrofloxacin selected medium.Type and abundance analysis of the plasmid-mediated quinolone resistance（PMQR）genes were also performed to evaluate the resistance risk in tilapia and hybrid snakehead at different breeding stages.This study will help establishing the control methods of bacterial resistance,help understanding the rational application of antimicrobial and guarantee the aquatic product safety.Plate count method was performed to investigate the total viable counts of bacteria in intestine and environmental samples from tilapia and hybrid snakehead.The results showed that the number of bacteria from fish intestine and environmental samples was stable at the different breeding stages,maintained between 10⁵ CFU·g^-1 to 10⁷ CFU·g^-1.Resistance risk of fish intestine was higher than which of environmental samples.The resistance rates of nalidixic acid and enrofloxacin in bacteria from tilapia intestine were26.7%and 18.3%,respectively,while higher incidences were found in hybrid snakehead,the ratios of which were 37.7%and 21.8%,respectively.There was no significant difference of quinolone resistance occurred at different breeding stages,whereas different incidences were observed in different fish species and farming districts.16S rDNA gene high throughput sequencing was performed to reveal the bacterial diversity of the microbiota in fish intestine and the aquatic environment.High diversity was observed in sediment samples from tilapia,followed by intestine and pond water samples.High diversity was observed in intestine of hybrid snakehead,followed by sediment and pond water samples.Escherichia spp.,Enterobacter spp.and Aeromonas spp.were the predominant genus of quinolones resistant microbiota in tilapia intestine,while Escherichia spp.,Aeromonas spp.and Plesiomonas spp.were dominant in hybrid snakehead intestine.Escherichia spp.and Aeromonas spp.were the predominant genus of quinolones resistant microbiota in environmental samples from tilapila and hybrid snakehead,the total abundance of both genuse was significant higher than which in intestine samples.No significant regularity was observed in the composition and community changes of microbiota at different sample sites and at different breeding stages.PCR and qPCR were launched to determine the PMQR genes.A total of 4 PMQR genes were detected in tilapia intestine and the environmental samples,including qnr A（qnr A1）,qnr B（qnr B10）,qnr S（qnr S1、qnr S5）and oqx AB（oqx A）,and their positive rates were 2.5%,7.4%,42.0%and 34.7%,respectively.Four genes of qnr B（qnr B10）,qnr S（qnr S1）,oqx AB（oqx A）and aac（6’）-Ib-cr were detected in hybrid snakehead intestine and the environmental samples,their positive rates were 3.7%、29.6%、22.2%and 7.4%.PMQR gens were prevalent in tilapia and hybrid snakehead aquaculture.The total abundance of PMQR genes were the highest in sediment samples,followed by intestine and pond water samples.The absolute abundance of PMQR gens in samples from tilapia aquaculture was in the range of 10³ to 10⁷ copies g^-1(copies·m L^-1),while the relative abundance of which was between 0.02%to 0.96%.The absolute abundance of PMQR gens in samples from hybrid snakehead aquaculture was in the range of 10³ to 10⁸ copies g^-1(copies·m L^-1),while the relative abundance of which was between0.004%to 41.17%.The abundances of oqx AB,qnr C and qnr B genes in samples from hybrid snakehead aquaculture were significant higher than those from tilapia aquaculture,whereas the abundances of qnr S and qnr D genes were significant lower.No significant regularity was observed at different breeding stages in all kinds of samples.In conclusion,quinolones resistant bacteria and resistance gens were prevalent in tilapia and hybrid snakehead aquaculture.It revealed that the resistance risk was significant higher in fish intestines than that in aquacuatic environment.No significant difference of resistance risk was observed at different breeding stages.Aeromonas spp.and Escherichia spp.were the predominant genus of quinolone resistant bacteria in aquaculture.It’s suggested that antimicrobial resistance surveillance of these two genuses should be carried out,as well as support researches on the mechanism of quinolone resistance.In addition,breeding management should be improved throughout the stages.Only if incidence of diseases decreased should the resistance risk reduce brought by the application of antimicrobial agents and ensure the quality and safety of aquatic products.