Genetic Diversity,Antibiotic Resistance,and Pathogenicity of Aeromonas Species from Food Products in Shanghai,China＊

QU Feng Tian ,WANG Wen Qing ,LIU Qian ,ZHOU Hai Jian ,HU Jin Rui ,DU Xiao Li ,WANG Yue,XUE Jia Qi,5,CUI Zhi Gang,#,XIE Gui Lin,and MENG Shuang,#

1.College of Life Science,Northeast Agricultural University,Ha’erbin 150000,Heilongjiang,China;2.State Key Laboratory of Infectious Disease Prevention and Control,National Institute for Communicable Disease Control and Prevention,Chinese Center for Disease Control and Prevention,Beijing 102206,China;3.Shanghai Pudong New Area Center for Disease Control and Prevention,Shanghai 200136,China;4.Laboratory Medicine,Beijing Hospital,National Center of Gerontology;Institute of Geriatric Medicine,Chinese Academy of Medical Sciences,Beijing 100730,China;5.Clinic Medical College,North China University of Science and Technology,Tangshan 063000,Hebei,China

Abstract Objective Aeromonas has recently been recognized as an emerging human pathogen.Aeromonasassociated diarrhea is a phenomenon occurring worldwide.This study was designed to determine the prevalence,genetic diversity,antibiotic resistance,and pathogenicity of Aeromonas strains isolated from food products in Shanghai.Methods Aeromonas isolates (n=79) collected from food samples were analyzed using concatenated gyrB-cpn60 sequencing.The antibiotic resistance of these isolates was determined using antimicrobial susceptibility testing.Pathogenicity was assessed using β-hemolytic,extracellular protease,virulence gene detection,C.elegans liquid toxicity (LT),and cytotoxicity assays.Results Eight different species were identified among the 79 isolates.The most prevalent Aeromonas species were A.veronii [62 (78.5%)],A.caviae [6 (7.6%)],A.dhakensis [3 (3.8%)],and A.salmonicida [3(3.8%)].The Aeromonas isolates were divided into 73 sequence types (STs),of which 65 were novel.The isolates were hemolytic (45.6%) and protease-positive (81.0%).The most prevalent virulence genes were act (73.4%),fla (69.6%),aexT (36.7%),and ascV (30.4%).The results of C.elegans LT and cytotoxicity assays revealed that A.dhakensis and A.hydrophila were more virulent than A.veronii, A.caviae,and A.bivalvium.Antibiotic resistance genes [tetE,blaTEM,tetA,qnrS,aac(6)-Ib,mcr-1,and mcr-3] were detected in the isolates.The multidrug-resistance rate of the Aeromonas isolates was 11.4%,and 93.7% of the Aeromonas isolates were resistant to cefazolin.Conclusion The taxonomy,antibiotic resistance,and pathogenicity of different Aeromonas species varied.The Aeromonas isolates A.dhakensis and A.hydrophila were highly pathogenic,indicating that food-derived Aeromonas isolates are potential risks for public health and food safety.The monitoring of food quality and safety will result in better prevention and treatment strategies to control diarrhea illnesses in China.

Key words: Aeromonas;Genetic diversity;Antibiotic resistance;Virulence gene;Cytotoxicity assay;Multidrug-resistance

INTRODUCTION

Aeromonasspp.are Gram-negative bacteria belonging to theAeromonadaceaefamily and are widely distributed in the aquatic environment.Aeromonashas been isolated from dairy products (4%),vegetables (26%-41%),meat (5%-10%),poultry(3%-70%),and seafood (31%-72%)[1].Aeromonasspp.are opportunistic pathogens that can directly infect or co-infect with other pathogenic bacteria,thus causing sepsis and meningitis in humans and animals[2].Aeromonasspp.cause acute watery diarrhea,dysentery,and chronic gastrointestinal diseases[3],which pose threats to human health and quality of life.China continues to be one of the countries with the highest mortality rate owing to childhood diarrhea.Indeed,＞ 10,000 deaths from diarrheal diseases occur annually[4].

TheAeromonasgenus currently consists of ＞ 26 known species[5].Owing to the limitations of the existing phenotypic identification systems and the complexity of the taxonomy ofAeromonasspp.,phenotypic identification is deemed reasonably difficult to the species level,especially forAeromonasspp.related to human diseases[6].Furthermore,conventional methods,such as matrixassisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS),are timeconsuming,labor-intensive,and error-prone[7].For instance,Morinaga et al.[8]reported that isolates originally recognized asA.hydrophilausing traditional phenotypic analysis have been reidentified asA.dhakensisbased oncpn60andgyrBsequencing methods.David et al.[9]concluded that the universal target (UT) sequence from thecpn60gene can be used for phylogenetic and taxonomic studies of theAeromonasgenus.The type I chaperone gene,cpn60[9],and the single-copy protein-coding gene,gyrB[10],are widespread and have been applied in phylogenetic analysis and species identification ofAeromonas[10].

The mechanism underlying the pathogenesis ofAeromonasis multifactorial[11].Specifically,the pathogenicity is related to virulence factors,including aerolysin (aer),heat-stable cytotoxin (ast),hemolysin(hlyA),lateral flagella (laf),polar flagellum (fla),elastase (ela),lipase (lip),cytotoxic enterotoxin (act),and cytotoxic enterotoxin (alt).TheAeromonasspp.utilize these virulence factors to enable survival within the host,thereby causing extensive cell and tissue destruction,evading the host immune response,and enhancing pathogenic resistance mechanisms,which subsequently help the pathogen to establish an infection[12].Therefore,further elucidation of the correlation betweenAeromonaspathogenicity and the associated virulence factors is warranted.

Owing to the ease-of-culture,low cost,short life cycle,and simple genetic background ofCaenorhabditis elegans[13],wild-typeC.eleganshas been increasingly used as a model to evaluate the virulence of bacteria with pathogenic potential,includingAeromonasspp.[14].Moreover,Wu et al.[15]reported that the virulence manifested in the cytotoxicity assay is correlated with virulence traits demonstrated in a mouse infection model.

Antibiotics are frequently abused in industries to prevent and controlAeromonasinfections,thus resulting in increased antimicrobial resistance[16].It has been reported thatAeromonasisolates from South Korea were 100% resistant to amoxicillin and nalidixic acid,and 98.5% of the isolates were resistant to ampicillin;all isolates showed multiple antimicrobial resistance phenotypes[17].Multidrugresistant(MDR)Aeromonasstrains from food products can bypass the acidic conditions of the digestive tract of humans and animals[18].Hence,antimicrobial resistance ofAeromonasfrom food items should be strictly monitored to reduce the risk of food-borneAeromonasinfections[19].

In the current study we analyzed the distribution,genetic diversity,antimicrobial resistance,and pathogenicity ofAeromonasisolated from food items to assess the risk of disease in humans and animals.Our study provided the basis for clinical treatment of diseases caused byAeromonasspp.

MATERIALS AND METHODS

Aeromonas Isolates

All isolates were obtained from food products,including fish,shrimp,clams,pigs,chickens,cows,and sheep,collected from six supermarkets in Pudong New District,Shanghai in 2019 (Figure 1).An automatic bacteriologic analyzer (Vitek 2 Compact;BioMèrieux,Marcy I'Etoile,France) was used to identify theAeromonasisolates[20],which were then cultured at 28 °C on brain heart infusion agar plates(Oxoid Ltd.,Basingstoke,UK).

Figure 1.Phylogeny of Aeromonas spp.The concatenated sequences of six housekeeping genes were used to analyze the phylogeny of Aeromonas spp.The information of source,species,virulence genes,and antibiotic resistance are shown.MDR,multidrug-resistance.

Multi-locus Sequence Typing (MLST) and Subtyping of Aeromonas Isolates

We extracted total chromosomal DNA from theAeromonasisolates using the QIAamp DNA Mini Kit(Qiagen,Shanghai,China) according to the manufacturer’s instructions.ConcatenatedgyrB-cpn60phylogenetic trees were constructed,and we identified theAeromonasisolates at the species level by comparing two-gene sequences of representative species[21].TheAeromonasMLST scheme (http://pubmlst.org/Aeromonas/),which relies on the amplification and sequencing of six housekeeping genes (gyrB,groL,gltA,metG,ppsA,andrecA),was applied to identify eachAeromonasisolate subtype[22].PCR was performed using previously described primers and protocols and the sequences of the six loci were compared with the loci hosted on theAeromonasMLST database and sequence types(STs)[23].A phylogenetic tree was also constructed using the neighbor-joining method in Clustal-W with bootstrap values calculated using 1,000 replicates.

The β-hemolytic Activity and Extracellular Protease Assay

The hemolytic ability and extracellular protease activity of theAeromonasisolates were determined on Luria Bertani (LB) agar plates containing 5% sheep blood (KeMaJia,Shanghai,China) and LB agar plates containing 2% (w/v) skim milk (KeMaJia),respectively.AnAeromonascolony was inoculated on blood and milk agar plates and incubated at 28 °C[20].The presence of transparent zones surrounding theAeromonascolonies were considered positive reactions for both tests.

C.elegans LT Assay

Aeromonasisolates were selected for theC.elegansLT assay[15].A single colony ofAeromonaswas incubated in 45 mL of LB broth at 28 °C for 24 h.TheAeromonassuspension was adjusted to an OD600of 3.0,which was prepared for the toxicity assay.Nematode growth medium (NGM) agar plates withEscherichia colistrain OP50 were used to cultureC.elegans.The synchronized adult L4worms were washed in M9buffer (http://www.wormbook.org/).After centrifugation,the worms were re-suspended in 5 μL of S-medium (http://www.wormbook.org/).Approximately 40-50 worms were added to a 48-well plate with 5 μL of fluorodeoxyuridine to prevent reproduction.Finally,190 μL of the LB broth withAeromonasisolates was added to each lawn to a final volume of 200 μL.The plate was incubated at 20 °C and the survival rate of worms was observed after 1,2,and 3 days.

Cytotoxicity Assay

The mouse C2C12fibroblast cell line obtained from the American Type Culture Collection(Manassas VA,USA) was used for cytotoxicity assays[20].The cells were cultured in complete medium with Dulbecco’s modified Eagle’s medium(DMEM;Gibco,Grand Island,NY,USA)supplemented with 10% fetal bovine serum (FBS;Gibco) at 37 °C in a 5% CO2incubator for 24 h.The cells were then thrice-washed with DMEM and reacted withAeromonasspp.for 3 h[15].TheAeromonascultures were incubated with cells at a 100 multiplicity of infection (MOI).The CytoTox 96?Assay (Promega,Madison,WI,USA) was used to measure cell death following treatment with a cytotoxic drug or compound[19].The CytoTox 96?Assay kit was used to determine the release of lactate dehydrogenase (LDH) from the cell after interacting withAeromonasisolates at 37 °C for 3 h.A group treated with 10× lysis solution (Promega)was used as the positive control,and an untreated group with DMEM (Gibco) was the negative control[15].Cytotoxicity was expressed as the released LDH level induced byAeromonasisolates compared with that induced by 10× lysis solution(defined as 100% cytotoxicity).

Detection of Virulence-Associated Genes

Virulence genes (aerA,hlyA,act,ast,alt,ascV,aexT,lip,ela,fla,andlaf) were screened as potential toxicity factors ofAeromonas[24].The PCR system (30 μL) contained 15 μL of Taq PCR MasterMix (Takara Bio,Inc.,Kyoto,Japan),1 μL of primer (10 μmol/L),11.5 μL of ddH2O,and 1.5 μL of DNA template.The cycling conditions were as follows: pre-denaturation at 96 °C for 5 min;34 cycles of denaturation at 96 °C for 30 s,annealing at 55-60 °C for 30 s,and extension at 72 °C for 1 min and the final extension was at 72 °C for 5 min.The PCR products were analyzed using sequencing.

Antimicrobial Susceptibility Test

Antimicrobial susceptibility testing forAeromonasspp.was performed following the 2010 Clinical and Laboratory Standard Institute (CLSI) guideline M45-A2[6].The minimal inhibition concentrations (MICs) of 10 antibiotics [amoxicillin/clavulanic acid (AMC),ampicillin/sulbactam (SAM),cefazolin (CZO),ceftazidime (CAZ),ceftriaxone (CRO),aztreonam(ATM),ciprofloxacin (CIP),levofloxacin (LEV),oxazin/sulfamethoxine oxazole (SXT),and colistin (CT)]were measured.The quality control strains for all antimicrobial susceptibility tests wereEscherichia coliATCC 25922[25].

Evaluation of Antimicrobial Resistance Genes

We detected 19 resistance genes (tetA,tetB,tetE;blaTEM,blaSHV,blaCTX;armA,aphAI-IAB,aac(6′)-Ib,aac(3′)-IIa;sul1,sul2;mcr-1,mcr-2,mcr-3,mcr-4;qnrA,qnrB,andqnrS) in theAeromonasisolates.The primer sequences of the antibiotic resistance genes are shown inTable 1.The PCRpositive products were confirmed using sequencing.

Table 1.Primer sequences used to amplify antimicrobial resistance genes

Statistics

Data analysis was performed using SPSS software (version 23.0 for Windows;IBM Corp,Armonk,NY,USA).The 95%CIs of categorical variables for the distribution of virulence genes and the results of theC.elegansLT assay were calculated using Fisher’s exact tests.The cytotoxicity test data were analyzed using a nonparametric test to reveal the toxicity differences among differentAeromonasisolates.

RESULTS

MLST of Aeromonas Isolates

The 79 isolates ofAeromonaswere categorized into 73 STs,65 of which were novel (ST889-ST961 and ST991-ST1023),revealing a high degree of genetic diversity among theAeromonasstrains(Figure 1).There was no dominant ST observed in any of theAeromonasisolates.

Diversity and Distribution of Aeromonas

We identified all theAeromonasisolates based ongyrB-cpn60sequencing,which were distributed as follows:A.veronii(78.5%),A.caviae(7.6%),A.dhakensis(3.8%),A.salmonicida(3.8%),A.bivalvium(2.5%),A.hydrophila(1.3%),A.jandaei(1.3%),andA.media(1.3%),Figure 2.In addition,theAeromonasspp.were isolated from a variety of food products,of which the four most frequent foodstuffs were fish,pork,chicken,and spiral shell(Figure 1).

Figure 2.Subtyping of Aeromonas isolates.A neighbor-joining phylogenetic tree was constructed using the concatenated sequences of gyrB and cpn60 to reveal the relationships between 79 Aeromonas isolates from food products.

The β-hemolytic and Exoprotease Assays

From the 79Aeromonasisolates,36 (45.6%)exhibited β-hemolytic activity and 64 (81%) exhibited proteolytic activity (Table 2).A.caviaelacked hemolytic activity;the positive rate of proteolytic activity was 16.7%.A.dhakensis(66.7% and 100%,respectively) andA.hydrophila(100% and 100%,respectively) had more hemolytic and exoprotease activities thanA.veronii(50.0% and 85.5%,respectively),A.caviae(0 and 16.7%,respectively)andA.bivalvium(0 and 100%,respectively).Moreover,the positive rate of hemolytic and proteolytic activities inA.salmonicidastrains were 33.3% and 66.7%,respectively.A.jandaeishowed no signs of β-hemolysis.

C.elegans LT and Cytotoxicity Tests

The pathogenicity ofAeromonasisolates was assessed using theC.elegansLT test,and the results are shown inFigure 3.The 1,2,and 3-day survival rates ofC.elegansfed withA.dhakensis,A.hydrophila,A.veronii,A.caviae,andA.bivalviumwere as follows: 26.1%,23.2%,and 17.4%;26.7%,11.1%,and 11.1%;41.7%,33.8%,and 29.6%;43.8%,35.8%,and 31.0%;and 47.7%,20.9%,and 15.1%,respectively.Thus,A.dhakensisandA.hydrophilawere more pathogenic than otherAeromonasspp.Overall,theC.eleganssurvival rates when cultured withA.dhakensisandA.hydrophilawere significantly lower thanA.veronii,A.caviae,andA.bivalvium(P＜ 0.05).

Figure 3.C.elegans liquid toxicity assay.Three-day survival of C.elegans co-cultivated with the following five Aeromonas species: A.dhakensis;A.hydrophila;A.veronii;A.caviae;and A.bivalvium in the liquidtoxic assay.***P ＜ 0.05,as compared with A.dhakensis.

The pathogenicity ofAeromonasisolates was assessed using cytotoxicity tests.Compared with LDH release of the 10× lysis solution,the volume of LDH release induced byA.dhakensis,A.hydrophila,A.veronii,A.caviae,andA.bivalviumwere 75.9%,74.5%,46.4%,11.9%,and 5.2%,respectively.Our results indicated thatA.dhakensisandA.hydrophilawere more virulent thanA.veronii,A.caviae,andA.bivalvium,which was in agreement with the results of theC.elegansLT assay.The LDH release level varied betweenAeromonasspp.(P＜ 0.05).

Distribution of Virulence Genes

The distribution of the 11 virulence genes in theAeromonasstrains is summarized in detail (Table 3).In particular,actandflawere the two most frequent virulence genes among all the isolates,accounting for 73.4% and 69.6%,respectively.TheastandaerAgenes were detected in ＜ 10% of all isolates.The detection rates oflip,ela,act,alt,aerA,andhlyAwere significantly different among the eight species ofAeromonas[P＜ 0.05 (Fisher’s exact test)].Apartfrom the three genes (ast,aexT,andascV),all other virulence genes were detected inA.dhakensis.Onlyact,fla,laf,aexT,andascVwere more prevalent inA.veroniithan otherAeromonasspp.

Table 2.Prevalence of hemolytic and proteolytic activities in Aeromonas isolates

In addition to three genes (fla,ascV,andaexT),the prevalence of other genes differed significantly among the fiveAeromonasspp.[P＜ 0.05 (Fisher’s exact test);Table 4].OnlyhlyAwas more frequent inA.dhakensisandA.hydrophilathanA.veronii,A.caviae,andA.bivalvium.The genes,actandaerA,were abundant inA.dhakensisandA.veronii,but neither was detected in the other threeAeromonasspp.The gene,alt,was most commonly identified inA.dhakensis,A.hydrophila,andA.bivalvium.Moreover,the genes,lipandela,were less prevalent inA.veroniithanA.dhakensis,A.hydrophila,A.caviae,andA.bivalvium.

Antimicrobial Resistance

Resistance to CZO was demonstrated in 74(93.7%)Aeromonasisolates.The antibiotic resistance rates ofA.veroniito AMC,CRO,CIP,LEV,trimethoprim-sulfamethoxazole,and CT were 1.6%,3.2%,4.8%,1.6%,9.7%,and 1.6%,respectively.A.veroniiwas susceptible to SAM,CAZ,and ATM.A.dhakensisshowed 100% resistance to CT and CZO,and 33.3% to AMC and CRO.A.caviaewas shown to be 100% resistant to CZO and 66.7% against CAZ,CRO,and ATM.In addition to CZO,A.bivalviumwas susceptible to other antibiotics.The antimicrobial resistance rates ofAeromonasisolates against 10 common antibiotics are presented in (Table 5).

Table 3.Distribution of putative virulence factors in all Aeromonas isolates

Table 4.Distribution of virulence factors among isolates of A.dhakensis,A.hydrophila, A.veronii A.caviae,and A.bivalvium

Table 5.Prevalence of resistance to different antibiotics

In this study,9 (11.4%)Aeromonasisolates were multidrug-resistant,and the high MDR rates ofA.dhakensis,A.hydrophila,A.veronii,andA.caviaewere 33.3%,100%,3.2%,and 66.7%,respectively,which is an enormous challenge for treatment options.

Detection of Antimicrobial Resistance Genes

The tetracycline resistance gene,tetE,was found inA.veronii(35.5%),A.caviae(33.3%),A.salmonicida(66.7%),andA.media(100%).The aminoglycoside resistance gene,aac(6′)-Ib,was detected inA.veronii(16.1%),A.caviae(50.0%),A.bivalvium(50.0%),A.dhakensis(33.3%),A.media(100%),andA.hydrophila(100%).Moreover,the CT resistance gene,mcr-3,accounted for 16.1% ofA.veroniiand 33.3% ofA.dhakensis.ThePMQRgene,qnrS,was present in 3.2% ofA.veroniiand 100% ofA.hydrophila.In addition,blaTEM(1.6%),tetA(1.6%),andmcr-1(1.6%) were present inA.veronii.No drug resistance gene was observed inA.jandaei.The following genes were not detected in anyAeromonasisolates: theESBLgenes,blaSHVandblaCTX-M;the tetracycline resistance gene,tetB;thePMQRgenes,qnrAandqnrB;the aminoglycoside resistance genes,aphAI-IAB,aac (3′)-IIa,andarmA;the CT resistance genes,mcr-2andmcr-4;and the sulphonamide resistance genes,sul1andsul2(Figure 1).

DISCUSSION

Common clinical manifestations ofAeromonasinfections include gastrointestinal infections,diarrhea,bacteremia,and localized soft tissue infections[11].The clinicalAeromonasisolates (96.5%)that have been identified to date belong to one of the following species:A.caviae(29.9%);A.dhakensis(26.3%);A.veronii(24.8%);andA.hydrophila(15.5%)[26].Escarpulli et al.[27]detected 82 species ofAeromonasin 250 frozen edible fish in Mexico City,Mexico and emphasized the risks associated with the consumption of theAeromonasisolated from frozen edible fish.In our study,29 (36.7%) strains ofAeromonaswere isolated from fish samples.Consumption of food infected withAeromonasposes a potential threat to human health.Accordingly,we analyzedAeromonasisolates obtained from food samples from Shanghai Agricultural Market and evaluated the species distribution,antibiotic resistance,and pathogenicity.

Among the 73 STs identified from the 79Aeromonasisolates,65 were novel,which indicated that the isolates obtained from food products had high genetic diversity.Lau et al.[28]also found 36 STs in 47 isolates in Malaysia,34 of which were novel STs,which suggested that the isolates obtained from food products often had high genetic diversity.The phylogenetic relationship of 79Aeromonasisolates was evaluated using the housekeeping gene,gyrBcpn60[28].A.veroniiwas the most dominantAeromonasspecies,comprising 78.5% of all isolates,and was isolated from meat (43.5%) and fish(33.9%).A.caviae(7.6%) was the second most prevalentAeromonasspecies and was distributed in various foods,such as fish and calms.Elala et al.[29]isolatedA.caviaefrom chickens,suggesting that this species could survive in both aquatic and terrestrial environments.The third most abundant species wasA.dhakensis,which has been reported[5]to have a higher virulence potential and is a notable human pathogen.In our study,A.dhakensiswas isolated from fish and shrimp samples at a 2:1 ratio.Similarly,Wu et al.[15]reported bacteria isolated from fish and clinical isolates in Tainan to beAeromonasspp.;the most prevalent species wereA.caviae,A.dhakensis,andA.veronii.

In this study,we analyzed the virulence ofAeromonasisolates using β-hemolysis and proteolytic,C.elegansLT,and cytotoxicity assays.The positivity rates ofA.dhakensisandA.hydrophilabased on the β-hemolysis and proteolytic assays were higher thanA.veronii,A.caviae,andA.bivalvium,which was confirmed by the survival rate ofC.elegansfed withAeromonasisolates,as well as the LDH release from mouse fibroblasts C2C12infected withAeromonasspp.Wu et al.[15]reported that the pathogenicity ofA.veroniiandA.caviaewere less virulent thanA.dhakensisandA.hydrophila,which was consistent with our findings.The virulence ofA.dhakensisagainstC.eleganswas shown to be more evident than that ofA.hydrophila[20],in contrast to the pathogenicity ofA.dhakensis,which differed little fromA.hydrophilain our investigation.

Cody et al.[30]reported that the virulence phenotype ofAeromonaswas the result of a combined effect of its virulence factors.We detected several virulence genes from theAeromonasspp.In allAeromonasisolates,actwas the most prevalent virulence gene,followed byfla,aexT,andascV.The cytolytic enterotoxin gene,act,was the most frequently detected among all isolates,and the result corresponded with the data ofact(63%) andalt(57%),which were higher thanast(6%) inAeromonasfrom water and fish sourced in India[31].Furthermore,actwas mainly distributed inA.veronii(85.5%),andflawas observed in mostAeromonasspp.exceptA.jandaei,A.hydrophila,andA.media.TheaexTgene was present in allA.jandaei,41.9% ofA.veronii,33.3% ofA.salmonicida,and 16.7% ofA.caviae.Another study[15]suggested that compared to otherAeromonaspathogens,the lack ofaerAorhlyAmay be associated with low virulence ofA.caviaeandA.bivalvium.These results represented the distribution of virulence genes in food products and indicated the potential pathogenicity ofAeromonasisolates,which could help enhance food quality and hygiene in this region,and aid in preventing and controlling the outbreak ofAeromonas-related diseases.

The results of this study indicated thatAeromonasisolates displayed 93.7% resistance to the first-generation cephalosporin (CZO) and ＜ 11%resistance to third-generation cephalosporins (10.1%for CRO and 5% for CAZ).Several studies[32,33]reported that the majority of isolates had 100%susceptibility to third-generation cephalosporins(CAZ and cefotaxime),while there was a different degree of resistance to first-generation cephalosporins,ranging from 79.3% to 91.8%.Generally,our findings were in agreement with other studies[32,33].In addition,A.bivalviumhad resistance to CZO (50%),and this finding corroborated the finding that all isolates from clinical and environmental samples were 80% resistant to CZO[33],which indicates that resistance to firstgeneration cephalosporins could be prevalent[34].We found that isolates ofA.dhakensisexhibited resistance to CZO (100%),CT (100%),CRO (33.3%),and AMC (33.3%),confirming multiple drug resistance of this species,which is in agreement with previous findings[6].This study demonstrated thatA.veroniihad various types of drug resistance,with 100% susceptibility to SAM,CAZ,and ATM.Absolute(100%) sensitivity to CIP was reported in a previous study in which severalA.veroniistrains were isolated from clinical patients[33];however,this differed from the CIP resistance rate of 5%determined in the current study.Moreover,we found that the resistance rate ofAeromonasspp.to CT was 7.6%,which was approximately the same as that ofAeromonasspp.isolated from Australian fish.CT is the last line of antibiotic treatment used for clinically severe infections caused by multidrugresistant gram-negative bacteria[33].In the present study,nine strains ofAeromonasshowed a multidrug resistance rate of 11.4%,which was lower than that reported in a previous study[6].

Consistent with the findings of a study conducted by Dahanayake et al.[35],tetEwas detected in 27 strains (34.2%) andtetAwas detected in 1 strain(1.3%);However,tetBwas not detected in the present study.Moreover,thetetEdetection rates varied amongA.veronii,A.caviae,A.media,andA.salmonicida,ranging from 33.3% to 100%.Recently,aminoglycosides have been extensively used for treating severe infections caused byAeromonas,and Dahanayake et al.[35]reported the presence ofaac(6′)-Ibin allAeromonasspp.isolated from cockles in Korea.In the present study,21.5% of the isolates ofAeromonasspp.harboredaac(6′)-Ib,except forA.salmonicidaandA.jandaei.

Our study indicated that approximately 13.9% ofAeromonasspp.harboredmcr-3 (16.1% ofA.veroniiand 33.3% ofA.dhakensis).These data support the discovery ofmcr-3inA.jandaeiisolated from fish sold in Beijing,China[36].Studies have reported the presence ofqnrSnot only inEnterobacteriaceae,but also inAeromonas,suggesting that this gene is distributed across different bacterial species[37].We found thatAeromonasstrains collected from food products had a resistance rate of 8.9% to SXT,and the same strains exhibited a detection rate of 3.8%forqnrS.This gene was detected in 3.2% and 100%ofA.veroniiandA.hydrophilaisolates,respectively.Previous studies have reported different rates of detection ofqnrSin Lake Lugano (Switzerland)[37]and China[6].Moreover,the rate of detection ofblaTEMinA.veroniiwas 1.6%,confirming the presence ofblaTEMinA.veroniiisolated from German water sources[38].Nonetheless,these findings should be further assessed in future studies.

CONCLUSIONS

Aeromonaswas an important zoonotic pathogen that is mainly distributed in fish and other aquatic products.Studies have demonstrated that these isolates had a high degree of genetic diversity;the 79 isolates in the current study were shown to produce 73 independent STs.The isolates were divided into eight species bygyrB-cpn60sequencing.The distribution,taxonomy,drug resistance,and pathogenicity varied among differentAeromonasspp.The percentage ofAeromonasisolates shown to be MDR was 11.4%.Food-derivedAeromonasisolates (93.7%) were resistant to CZO.Aeromonascarried some virulence and antibiotic resistance genes,which posed a threat and challenge to food safety and clinical treatment monitoring.A few isolates,such asA.dhakensisandA.hydrophila,had high pathogenicity and could cause extensive infections and posed risks to human and animal health.

CONFLICTS OF INTEREST

The authors have declared that no competing interests exist.

Received:November 23,2021;

Accepted:April 22,2022