Analysis Of Antibiotic Resistance Phenotype And Genotype Of Aeromonas From Freshwater Aquaculture In Jiangsu

The genus Aeromonas belongs to Gram-negative bacteria and are one of the important pathogens in freshwater aquaculture.Several species of Aeromonas could cause septicaemia,enteritis and ascites,and so on,which brings great harm and economic loss to aquaculture.At present,antibacterial drugs become an integral part of the prevention and treatment of bacterial infectious diseases in aquaculture and the reduction of economic losses.However,there is a growing concern over the negative effects of antibacterial drugs.The irrational use of antibiotics not only brings hidden dangers to the quality and safety of aquatic products,but also can easily lead to develop antibacterial resistance(ABR),which greatly weakens the ability of antibacterials to control aquatic animal diseases,and is also the potential risk of spreading resistance to human pathogens.Jiangsu province is an important freshwater aquaculture area in China.In order to understand ABR of Aeromonas from freshwater aquaculture in Jiangsu,the Aeromonas strains were isolated and identified,the sensitivity to antibiotics and antibacterial resistant genes were detected,the prevalence of ABR of Aeromonas from aquaculture in Jiangsu were analyzed.The relationship between antibacterial resistance phenotype and genotype,and the correlation between target gene mutation sites and quinolone resistance were also discussed.It provides some basic data for the control of Aeromonas diseases from aquaculture and the further study of ABR mechanism in Aeromonas.1.Identification of Aeromonas strains from freshwater aquaculture sourcesThe bacteria strains isolated from the main freshwater aquaculture areas in Jiangsu province in 2015 and 2020 were screened by RS selective medium,which were from the diseased animals including grass carp(Ctenopharyngodon idellus),silver crucian carp(Carassius auratus gibelio),silver carp(Hypophthalmichthys molitrix),Chinese mitten crab(Eriocheir sinensis)and other aquatic animals.Gyr B and 16 S r RNA genes were used for molecular biological identification in the laboratory.A total of 117 strains of Aeromonas were identified,involving 6 species of the genus Aeromonas.Among them,A.veerinii(61 strains)accounted for 52.1% of the total,A.hydrophila(24 strains)accounted for 20.5%,A.salmonicida(17 strains)accounted for 14.5%,and the other 15 strains were of A.sobria,A.media and A.caviae.The dominant species of Aeromonas isolated from different regions were different.A.veerinii was the main species in Sheyang and Yandu,A.hydrophila was the main species in Yangzhong,and A.salmonicida was the main species in Kunshan and Tongshan,which showed obvious regional characteristics.2.Analysis of antibacterial resistance phenotype of Aeromonas isolates from freshwater aquaculture sourcesTo further study the drug-resistance of Aeromonas isolates in collected samples,the antibacterial resistance phenotypes of 117 strains of Aeromonas to 14 antibiotics were determined by agar dilution method.The results showed that Aeromonas had the highest resistance rate to ampicillin(100%),followed by erythromycin(90.6%)and cotrimoxazole(71.8%),sulfamethoxazole(68.4%)and fluoxetine Benicol(61.5%)with a higher resistance rate.In addition,the ABR rates of other antibiotics were less than 50%.The ABR rates of chloramphenicol,oxytetracycline and rifampicin were 44.4 ～ 46.2%,the ABR rates of enrofloxacin,ciprofloxacin and neomycin were 20.5 ～ 33.3%,and that of doxycycline was the lowest,only 1.7%.The multiple drug resistance(MDR)of Aeromonas is relatively serious.There are 101 strains showed multidrug resistance(that is resistance to 3 or more types of antibiotics),accounting for 86.3%(101/117),6 strains of bacteria are resistant to all 8 types of antibiotics The antibacterial resistance phenotype spectrum was complicated,and 117 strains of Aeromonas had 70 ABR phenotype spectrum.Among the seven antibiotics detected in this study,the ABR rates of enrofloxacin,ciprofloxacin,neomycin and doxycycline were relatively low,which could be used to control bacterial diseases caused by Aeromonas.3.Detection of antibacterial resistance genes in Aeromonas isolates from freshwater aquaculture sourcesTo detect PMQR and QRDR genes and other types of ARGs of Aeromonas isolates,PCR technique was used to detect 34 ARGs and 3 movable gene elements(int1,int SXT,ISCR1)of 117 strains of Aeromonas,the gene mutation sites of gyr A and par C were detected,and the correlation between ABR phenotype and genotype was analyzed.The results showed that the detection rate of quinolone resistance gene qnr S was the highest at 57.3%(67/117),followed by rifamycin resistance gene arr of 54.7%(64/117),and the detection rate of tetracycline resistance gene tet M was 40.2%(47/117).The ARG detection rates of macrolide,sulfonamides and amphenicols were relatively low.There was no correlation between Aeromonas ABR phenotype and genotype,which showed the complexity of Aeromonas antibacterial resistance mechanism.Class I integron(INT1)and insertion sequence common region(ISCR1)of Aeromonas strains had a high detection rate,which increased the risk of horizontal transfer of plasmid mediated quinolone resistance(PMQR)gene in aquaculture or through food animals.Meanwhile,among the 117 Aeromonas strains,91 strains had QRDR mutations,accounting for 77.8% of all strains.There were 84 strains(71.8%)with mutations in the amino acid sequence encoded by the gyr A gene,62 strains with Ser83 Ile mutation,and22 strains with Ser83 Val mutation,accounting for 53.0% and 18.8% respectively;the amino acid sequence encoded by par C gene had mutations in 62 strains.63 strains,accounting for 53.8%,are all Ser87 Ile.The double mutation of gyr A Ser83 Ile + par C Ser87 Ile and the single mutation of gyr A Ser83 Ile are two dominant mutation modes,which are one of the main mechanisms that caused Aeromonas resistant to quinolones.However,the emergence of quinolone ABR was not only related to the mutation of gyr A and par C subunits,but also might have other resistance mechanisms such as active efflux mechanism or plasmid-mediated resistance,and may also be related to the resistance mechanism under the press of other antibacterials.The mechanism of quinolone ABR of Aeromonas remained to be further studied.