Comparative Study of the Genetic Diversity,Antimicrobial Resistance,and Pathogenicity of Aeromonas lsolates from Clinical Patients and Healthy lndividuals＊

MENG Shuang , DU Xiao Li , WANG Yong Lu , QU Feng Tian, , XIE Gui Lin , ZHOU Hai Jian ,HU Jin Rui, QIN Zheng,4, WANG Yue, KAN Biao, and CUI Zhi Gang,#

1.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;2.Ma’anshan Center for Disease Control and Prevention,Ma’anshan 243000,Anhui,China;3.College of Life Science,Northeast Agricultural University,Ha’erbin 150000,Heilongjiang,China;4.School of Public Health,Jiamusi University,Jiamusi 154000,Heilongjiang,China

Abstract Objective This study was performed to compare the genetic diversity,virulence,and antimicrobial resistance of Aeromonas strains isolated from patients and healthy individuals.Methods A total of 38 clinical strains and 19 strains from healthy individuals were isolated from the samples collected in Ma’anshan City,Anhui Province.Their taxonomy was investigated using concatenated gyrB-cpn60 sequences,and their resistance to 12 antibiotics was evaluated.The pathogenicity of these strains was examined through beta-hemolysis,protease activity,and virulence gene assays.Results The 57 Aeromonas strains were divided into 55 sequence types.Of these types,21 were novel,suggesting that their genetic diversity was high.These Aeromonas isolates could be divided into 7 species,and the positive rates of beta-hemolysis and protease activity were 49.1% and 73.7%,respectively.The detection rate of clinical patients in terms of beta-hemolysis and protease activity was higher than that of healthy individuals.Among the four most common Aeromonas strains,A.dhakensis had the highest detection rate of virulence genes.The multidrug resistance rate of the clinical isolates was much higher than that of the strains isolated from healthy individuals.Conclusions The taxonomy,virulence properties,and antibiotic resistance of Aeromonas isolates from patients differ from those of the isolates from healthy individuals.

Key words:Aeromonas;Genetic diversity;Multidrug resistance;Virulence gene;Antimicrobial resistance gene

INTRODUCTION

Aeromonas,which belongs to Aeromonadaceae,is a Gram-negative facultative anaerobe positive for oxidase and catalase.Aeromonasspp.are widely distributed in aquatic environments[1],and more than 28Aeromonasspp.have been identified[2].At least 19 of these species are associated with human diseases,and they can cause widespread infection[3].The common clinical manifestations ofAeromonasare diarrhea,localized soft tissue infection,and bacteremia[4]. Bacteremia primarily occurs in patients with underlying diseases,such as hepatobiliary diseases,cancer,and diabetes[5].

Phenotypically identifyingAeromonasspecies is considered reasonably difficult because of the limitations of commercially available identification systems forAeromonas,e.g.,API20E,API20NE,Vitek,and BioMerienx[6].Therefore,molecular methods are widely used to identifyAeromonasspp.correctly.The nucleotide sequences of housekeeping genes,such asgyrB,cpn60,andrpoB,have been regarded as reliable markers that can be used to identifyAeromonasspecies[7-9].Some reports have shown that strains initially identified asA.hydrophilaviaphenotypic methods can be recognized asA.dhakensisthroughcpn60orgyrBsequencing[8,10].A.dhakensiswas initially described as anA.hydrophilasubspecies in 2002 and asA.aquariorum;in 2013,it was described as a novel species,namelyAeromonas dhakensis[11].A.dhakensishas been shown to be a pathogenic bacterium linked to numerous human diseases[5].Previous studies onA.aquariorumrecognized the virulence ofA.dhakensis,and subsequent evaluations have shown that this strain carries several important virulence genes[6].This strain also has the most potent cytotoxic activities against human blood cell lines[10].However,the prevalence ofA.dhakensisinfection is likely underestimated because phenotypic identification systems often misidentifyA. dhakensisasA.hydrophila.

Aeromonassp.pathogenesis involves a series of virulence factors[12],including hemolytic toxins,such as aerolysin-related cytotoxic enterotoxin (act)[13],heat-labile cytotoxic enterotoxin (alt),hemolysin(hlyA),heat-stable cytotonic toxins (ast)[14],and aerolysin (aerA)[15].In addition,the type III secretion system (TTSS),lateral flagella (laf),polar flagellum(fla)[16],elastase (ela),and lipase (lip) contribute toAeromonaspathogenicity[17].With these virulence factors,bacteria can colonize,invade,and overcome their hosts’ immune response,leading to infection and disease development.

Diarrhea caused byAeromonasis self-limiting,and it is effectively treated with the oral or intravenous antibiotic infusion.However,patients with severe diarrhea or extraintestinal infection should receive antibacterial treatments[18].Aeromonasis resistant to ampicillin,but it is susceptible to third-generation cephalosporins and aminoglycosides that exhibit potent antibacterial effects against clinicalAeromonasisolates[19].However,the extensive use of antibiotics in aquaculture and clinical treatment has caused an increase in antimicrobial resistance[20]. The development of resistance inAeromonasspp.against several different classes of antibiotics threatens human health because resistant isolates can be transmitted from the aquatic environment to humans via the food chain or direct contact[21].Therefore,the antimicrobial resistance ofAeromonasshould be monitored to guide treatments.

In this study,the diversity and distribution ofAeromonasspecies isolated from healthy individuals and clinical patients in Ma’anshan,Anhui Province,China,were evaluated.Their genetic diversity,pathogenicity,and antimicrobial resistance profiles were compared.

MATERIALS AND METHODS

Aeromonas Isolates

A total of 57Aeromonasisolates (38 from clinical patients and 19 from healthy individuals) were obtained from Ma’anshan,Anhui Province,China,between 2018 and 2019 (Table 1).They were identified using an automatic bacteriological analyzer (Vitek 2 Compact,BioMèrieux) and cultured on Luria-Bertani (LB) broth or brain heart infusion agar plates overnight at 37 °C.

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

TheAeromonasMLST scheme (http://pubmlst.org/Aeromonas/),which relies on the amplification and sequencing of six housekeeping genes,namely,gyrB,groL,gltA,metG,ppsA,andrecA,was applied to identify the subtype of eachAeromonasisolate[22].The total chromosomal DNA from theAeromonassamples was prepared using a DNA purification kit(Promega,Madison,USA) in accordance with the manufacturer’s instructions.PCR was performed using previously described primers and protocols[23],and the sequences of the six loci were compared with those hosted on theAeromonasMLST database and STs.New alleles and STs were submitted to theAeromonasMLST database for naming.

With the limitations related to molecular identification involving16S rRNAsequencing[24],the sequences of the housekeeping genesgyrBandcpn60were included in our species identification assays.Concatenated two-gene phylogenetic trees were constructed and compared with representative species as previously described[25].A phylogenetic tree was also constructed using the neighbor-joining method in Clustal-W[24]with Bootstrap values calculated using 1,000 replicates.

Beta-hemolysis and Exoprotease Assays

The degree of beta-hemolysis was assessed in isolates cultured in LB agar containing 5% (vol/vol)sheep blood agar (KeMaJia,Shanghai,China).Qualitative assays were performed with LB agar containing 2% (wt/vol) skim milk (KeMaJia,Shanghai,China) to investigate the exoprotease activity.The presence of clear zones surrounding the streaks indicated positive reactions in both tests[26].

Detection of Virulence-associated Genes

An assay was designed to evaluate the presence of the genes encoding exotoxins or secretion system components,structural components,and extracellular enzymes associated withAeromonasvirulence.In this assay,aerolysin (aerA),hemolysin(hlyA),heat-labile enterotoxin (act),heat-stable cytotoxin (ast),heat-labile cytotoxin (alt),type III secretion system components (ascV),ADP-ribosyl transferase toxin (aexT),lipase (lip),elastase (ela),and flagellin (fla) were detected using previously described primers and protocols[25]. PCR was performed in a 50 μL reaction mixture containing 25 μL of Taq PCR MasterMix (Takara Bio,Inc.,Japan),1 μL of the relevant primers (10 μmol/L),21 μL of ddH2O,and 2 μL of DNA template under the following cycling conditions:pre-denaturation at 95 °C for 5 min;30 cycles of denaturation at 95 °C for 30 s,annealing at 55-60 °C for 30 s,and extension at 72 °C for 1 min;and a final cycle at 72 °C for 5 min.Sequencing was conducted to confirm the positive PCR products.

Antimicrobial Susceptibility

The antimicrobial susceptibility of eachAeromonasisolate was evaluated using the microbroth dilution method in accordance with the current guidelines of the Clinical and Laboratory Standards Institute. The minimum inhibitory concentrations of 12 antibiotics were measured: amoxicillin/clavulanate,cefepime,ceftriaxone,ceftazidime,imipenem,aztreonam,gentamycin,tetracycline,ciprofloxacin,trimethoprim/sulfamethoxazole (SXT),chloramphenicol,and colistin.Escherichia coliATCC 25922 was the strain used for the quality control strain of all the susceptibility tests.

Evaluation of Antimicrobial Resistance Genes

The strains were screened for the presence of various antimicrobial resistance genes,including those for tetracycline resistance (tetA,tetB,andtetE),extended-spectrum b-lactamase (ESBL)resistance (blaTEM,blaSHV,andblaCTX)[27],aminoglycoside resistance (armA,aphAI-IAB,aac(6′)-Ib,andaac(3)-IIa)[28],sulfonamide resistance (sul1andsul2;)[29],mobile colistin resistance (mcr-1,mcr-2,mcr-3,andmcr-4),and PMQR (qnrA,qnrB,andqnrS)[27],by using previously described primers and protocols[25].Sequencing was conducted to confirm the positive PCR products.

RESULTS

MLST of Aeromonas Isolates

The 57Aeromonasisolates were divided into 55 STs.Of these isolates,21 were novel (ST722-ST742),indicating that the degree of genetic diversity was high (Figure 1).No dominant STs were found in either the patient or healthy groups.

Figure 1. Phylogenetic relationships were established using the concatenated sequences of the six genes included in this study.The source,species,virulence genes,and antibiotic resistance are shown on the right.A phylogenetic tree was determined using a neighbor-joining algorithm.ST:sequence type.

Diversity and Distribution of the Aeromonas Species Isolated in This Study

gyrBandcpn60were sequenced to evaluate the phylogeny of the 57Aeromonasisolates identified in this study (Figure 2).This analysis revealed that these isolates could be divided into seven different species.The most common species wereA.caviae(36.8%),A.veronii(33.3%),A.dhakensis(10.5%),andA.jandaei(10.5%).Notably,the distribution of theAeromonasisolates from the patients and the healthy individuals varied (Table 1).A.caviae(47.3%)andA.veronii(26.3%) were the most prevalent in the patient samples,whereasA.veronii(47.4%) andA.jandaei(26.3%) were the most common species in the healthy samples.In addition,the abundance ofA.dhakensiswas higher in the patients than in the healthy individuals.

Table 1. Distribution of Aeromonas spp.in isolates collected from clinical patients and healthy individuals

Figure 2. Neighbor-joining phylogenetic tree was constructed using the concatenated sequences of gyrB and cpn60 to reveal the relationships between 57 Aeromonas isolates from clinical patients and healthy individuals from Ma’anshan,Anhui Province,China.Isolates were designated as either P or H to indicate the strains isolated from clinical patients or healthy individuals,respectively.

Beta-hemolysis and Exoprotease Assays

A total of 28 (49.1%) isolates demonstrated betahemolysis,and 42 (73.7%) isolates possessed a proteolytic activity (Table 2).A.mediaisolates lacked hemolytic and proteolytic activities.A. caviaeisolates (19.0% and 66.7%,respectively) had less hemolytic and exoprotease activities than those ofA.dhakensisisolates (83.3% and 100%,respectively) orA.hydrophilaisolates (50% and 100%,respectively).Of the strains isolated from healthy individuals,only 36.8% processed any beta-hemolytic activity,whereas 63.2% exhibited some proteolytic activities.However,55.2% of the clinical isolates exhibited a hemolytic activity,and 84.2% had a proteolytic activity (Table 2).

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

Distribution of Virulence Genes

The distribution of the virulence genes in theseAeromonasisolates is summarized in Table 3.In particular,flawas the most common virulence gene identified in 87.7% of the 57 isolates,andelawas the second-most prevalent virulence gene in these isolates (n=41,71.9%).Laf,ast,andaerAwere identified in less than 10% of the isolates.The prevalence oflipdiffered significantly between patient-and healthy-derived strains (P＜ 0.05,Fisher's exact test).Onlylip,alt,andaerAwere more prevalent in patient-derived isolates than in healthyderived isolates.

Table 3. Distribution of virulence-associated genes in Aeromonas strains isolated from clinical patients and healthy individuals

In the four most commonAeromonasspecies,the detection rates ofela,lip,alt,hlyA,andaerAinA.dhakensisisolates were significantly higher than those inA.caviae,A.veronii,andA.jandaei(P＜0.05,Fisher’s exact test;Table 4).Although the total number ofA.jandaeiisolates was low,few of them were positive for any of the evaluated virulence genes.The extracellular protease geneselaandlipwere abundant inA.caviaeandA.dhakensis,and the hemolytic geneactwas abundant inA.veronii.The enterotoxin genealtand hemolytic geneshlyAandaerAwere most commonly identified inA.dhakensis.Their rates of occurrence in other species identified in this study were relatively low.

Table 4. Distribution of virulence genes in the four most common Aeromonas spp.

Antimicrobial Susceptibility

The antimicrobial susceptibility rates of 12 common antimicrobials in theAeromonasisolates identified in this study are presented in Table 5.Almost all theAeromonasstrains had high amoxicillin/clavulanic acid (96.4%) resistance;however,most of the isolates (≥ 90%) were susceptible to cefepime,imipenem,chloramphenicol,gentamicin,and tetracycline.In general,clinicalA.dhakensis,A.hydrophila,A.jandaei,andA.caviaeisolates were more resistant than their healthy individual counterparts to the commonly prescribed antibiotics,including ceftriaxone,ceftazidime,ciprofloxacin,and amoxicillin/clavulanic acid. The healthy sample isolates had an increased rate of resistance to colistin and imipenem only (Supplementary Table S1 available in www.besjournal.com).A.enteropelogenesandA.media,identified only in the patient samples,were susceptible to most of the tested antibiotics except amoxicillin/clavulanic acid,aztreonam,ceftriaxone,and ceftazidime(Supplementary Table S1).

Table 5. Prevalence of resistance to different antibiotics

Of the 57 strains identified in this study,15(26.3%) were found to have multidrug resistance(MDR),displaying resistance to at least 3 of the antibiotics tested in this assay.The MDR rates of the healthy-individual-derived strains (5.3%) were significantly lower than those of the patient isolates(36.8%).Therefore,these MDR phenotypes were likely acquired after treatment and hospitalization.

Detection of Antimicrobial Resistance Genes

The tetracycline resistance genestetAandtetEwere found in 4 (6.9%) isolates and 1 (1.7%) isolate,respectively.ThePMQRgeneqnrSwas present in 7(12.1%) isolates,and the aminoglycoside resistance genesaac (6′)-IbandarmAwere detected in 3 (5.2%)isolates and 1 (1.7%) isolate,respectively.TheESBLgenesblaCTXandbla-TEMwere observed in 2 (3.4%)isolates and 1 (1.7%) isolate,and the sulfonamide genessul1andsul2were found in 7 (12.1%) isolates and 1 (1.7%) isolate,respectively (Supplementary Table S2 available in www.besjournal.com).The mobile colistin resistance genemcr-3was detected in 1 (1.7%) isolate.The following genes were not detected in any isolate:theESBLgeneblaSHV;the aminoglycoside resistance genesaphAI-IABandaac(3)-IIa;the tetracycline resistance genetetB;the colistin resistance genesmcr-1,mcr-2,andmcr-4;and thePMQRgenesqnrAandqnrB.

The detection rate of resistance genes in the clinical strains was higher than that in their healthy individual counterparts.The resistance genes ofA.caviaewere higher than that of any of the other species identified in this study (Supplementary Table S2).

DISCUSSION

Aeromonasspp.are ubiquitous in most aquatic environments,and certain species have been reported to be an important cause of acute diarrhea in children and adults[2].Aeromonasstrains were found in 7.6% of patients with diarrhea and 3.8% of a healthy population[30,31].These rates may reflect actual differences or may be influenced by seasonal or environmental factors.

In this study,57Aeromonasisolates were described in detail in terms of species distribution,ST type,pathogenicity,and antimicrobial susceptibility to evaluate the differences in the epidemiology and etiology ofAeromonasstrains isolated from clinical patients and healthy individual samples.The results of these analyses were then used to evaluate the significance ofAeromonasinfections in clinical settings in China.They could be considered as a basis for developing the clinical protocols of treatment and surveillance.

These 57 isolates were separated into 55 STs.Of these isolates,21 were novel,indicating that the degree of genetic diversity within these sample populations was high.Aeromonasis often isolated from patients with diarrhea.However,the role ofAeromonasspp. as potential human enteric pathogens remains unclear,possibly because of the lack of systematic analysis of these strains and the inherent difficulty in classifying them.

In this study,Aeromonasstrains were identified to the species level viagyrB-cpn60concatemer sequencing,and their species distribution was compared between the clinical patients and healthy individuals.The distribution ofAeromonasspecies isolated from the clinical patients and healthy individuals varied.Among them,A.caviae(47.3%)andA.veronii(26.3%) were the most prevalent clinical isolates,andA.veronii(47.4%) andA.jandaei(26.3%) were the most common isolates from the healthy individual samples.Wu et al.[32]reported that the most prevalent species in clinical patients areA.caviaeandA.dhakensis,which account for 29.8%;they also found thatA.veroniiis the second-most prevalent species,which account for 23.4%.In the present study,the isolates were identified as follows:21 (36.8%) asA.caviae,19(33.3%) asA.veronii,and 6 (10.5%) asA.dhakensis.Studies in other countries have shown a slightly different distribution; in particular,the most prevalent clinical strain in India isA.hydrophila,while the most prevalent one in France,Italy,and the Netherlands isA.caviae[30].In addition,mostA.veroniistrains are isolated from human samples,whereas mostA.hydrophilastrains are obtained from water samples.These differences may also depend on sampling season and geographical location.A.dhakensis,the third-most prevalent species in our study,is a novelAeromonasspecies[33]that has been identified as a potential pathogen in humans[5].A.dhakensisisolates have been collected from stool,blood,wound,and other extraintestinal samples from humans globally;skin and soft tissue infections (SSTIs) caused byA.dhakensishave been reported in Taiwan,China[6].In a set of 80Aeromonaswound isolates,37 (46.3%)samples containA.dhakensis,which can cause severe SSTIs[34].Chang et al.[8]demonstrated thatA.dhakensisis the most frequently isolated aeromonad in Australia,accounting for 30.7%.In clinical specimens,A. dhakensisis the most prevalent in wounds but is less frequently isolated from fecal strains and blood samples.Salas et al.[35]believed thatA.dhakensisis the second-most prevalent species in clinical isolates,accounting for 25.5%.Zhou et al.[31]revealed that the four most prevalent species ofAeromonasin clinical isolates areA.caviae(41.7%),A.veronii(31.3%),A.dhakensis(13.9%),andA. hydrophila(5.2%).Senderovich et al.[36]showed that severalAeromonasspecies are recovered from Israeli patients suffering from diarrhea; among the isolates,A.caviae(65.0%) andA.veronii(29.0%)are the most prevalent species.Therefore,previous studies supported our findings on the most prevalent strains in our samples.

The pathogenic mechanism ofAeromonasspp.is complex and multifactorial.In this study,the pathogenic potential was evaluated using betahemolytic and proteolytic activity assays,and several virulence genes were detected.A total of 28 (49.1%)isolates showed beta-hemolysis activities,and 42(73.7%) had some proteolytic activities.Consistent with previous findings[32],our results indicated thatA.dhakensisandA.hydrophilaisolates were more likely to exhibit hemolytic and proteolytic activities thanA.caviaeisolates.The beta-hemolytic and proteolytic activities of the clinical isolates differed from those of the healthy individual isolates.In particular,36.8% of the healthy sample strains demonstrated any beta-hemolytic activity,and 63.2% of these strains exhibited a proteolytic activity.However,55.2% and 84.2% of the clinical isolates had hemolytic and proteolytic activities,respectively.Therefore,the strains from clinical patients were more likely to exhibit beta-hemolytic and proteolytic activities than their healthy sample counterparts (Table 2).

The distribution of the virulence-associated genes in these isolates was determined.The results revealed thatfla,ela,andlipwere identified in most of the strains,i.e.,50 (87.7%),41 (71.9%),and 34(59.6%) of the 57 isolates,respectively.In addition,flawas present in most species,whileelaandlipwere more prevalent inA.dhakensisandA.caviaeisolates than in other isolates (Table 4).The enterotoxin and hemolysin genesact,aerA,alt,andastwere detected in 25 (43.9%),2 (3.5%),19(33.3%),and 2 (3.5%) of the 57 isolates,respectively.actwas observed in 89.4% ofA.veroniiisolates,andaerAwas found in 16.7% ofA.dhakensisisolates.altwas detected in 100% ofA.dhakensisand 38.1% ofA.caviaeisolates.Conversely,astwas not present in any of theA.dhakensis,A.caviae,andA.veroniistrains (Table 4).TheTTSSgenesascVandaexTwere present in 18 (31.6%) and 16 (28.1%) of the 57 isolates,respectively,andascVandaexTwere both present in 63.2% ofA.veroniistrains and 16.7% ofA.dhakensisstrains.The detection rate oflipwas significantly higher in the clinical isolates than in the healthy individual isolates.By comparison,ascVandlafwere detected more frequently in the strains from the healthy samples than from the clinical samples.Enterotoxins and hemolysins are important virulence factors inAeromonasspp.,and many studies have shown that the number of toxin genes harbored by any isolate is positively correlated with its potential virulence[37,38].Among the four most commonAeromonasstrains,A.dhakensishad the highest prevalence of virulence genes.This finding was also consistent with previous reports,which described thatA.dhakensisstrains harbor high cytotoxicity and liquid toxicity[34].In our study,fla,ela,lip,alt,andhlyAwere detected in all theA.dhakensisisolates,butaerAandhlyAwere detected inA.dhakensisisolates only.The abundance of these virulence genes also varies among studies.For example,astwas not detected inA.dhakensisisolates in our study;however,some researchers have reported that 50.0%-83.3%astis found inA.dhakensisisolates from wound and blood infections[6,10].

Consistent with previous findings[39],our results suggested that 96.4% of theAeromonasstrains exhibited resistance against amoxicillin/clavulanic acid.The resistance rates to most antibiotics were higher in the clinical isolates than in the healthy individual isolates,but the resistance to colistin and imipenem was higher in the strains identified in the healthy individual samples than in the clinical samples.All theA.dhakensisisolates exhibited resistance to colistin.This result suggested thatA.dhakensisshould be the focus of future research because it harbors a large number of virulence genes,high rates of drug resistance,and a high degree of beta-hemolytic and proteolytic activities.Some scholars indicated thatAeromonashas a high MDR,which is 38.7%,because of the different sources of strains[40].In our study,15 strains (26.3%)exhibited MDR to 12 antimicrobial agents,and this value was consistent with 28.7% as previously reported[31].The MDR rates of the healthy individualderived strains were significantly lower than those of the clinical isolates.Therefore,the extensive use of antimicrobial agents increases the selective pressure on nosocomial infectious bacterial strains to develop resistance. This selective pressure should be addressed in future studies.

Resistance to SXT and quinolone,which are antimicrobials used to treatAeromonasinfection,has been widely described.Deng et al.[40]reported that at least 18.86% ofAeromonasisolates collected from cultured freshwater animals are resistant to SXT;they also detectedsul1andqnrSin 18.9% and 4.7% of these samples,respectively.Our study obtained an SXT resistance rate of 22.8%,and 12.1%of these strains were positive for bothsul1andqnrS.The detection rate of antimicrobial resistance genes in the clinical isolates was higher than that in the strains identified in healthy individuals. This observation was consistent with the higher antimicrobial resistance rate in clinical isolates.This increased abundance of antimicrobial resistance genes inA.caviaewas also consistent with their resistance phenotype,which may explain the increased prevalence ofA.caviaeisolates in clinical samples.However,this finding should be evaluated in future studies.

CONCLUSIONS

We analyzed 57Aeromonasstrains isolated from patients and healthy individuals in Ma’anshan City,Anhui Province,China.These samples exhibited a high degree of genetic diversity with the 57 isolates that produced 55 independent STs.The constructed phylogenetic tree with concatenatedgyrb-cpn60sequences divided the 57 isolates into 7 species.Beta-hemolysis,proteolytic activity,and virulence gene analyses revealed that these properties were more common in the clinical isolates than in the isolates from the healthy individuals.Furthermore,A.dhakensisharbored the highest number of virulence genes.Our results suggested that 26.3% of the strains were MDR (≥ 3) positive,and the MDR status was significantly more common in the clinical isolates than in the isolates from the healthy individuals.PMQR,tetA,tetE,ESBL,aminoglycoside resistance,sulfanilamide,andmcr-3genes were detected in some of the isolates.Their prevalence increased in the clinical isolates.Therefore,the genetic diversity,antimicrobial resistance,and pathogenicity ofAeromonasstrains isolated from clinical patients significantly differed from those of the isolates from healthy individuals.

CONFLICT OF INTEREST

The authors have declared that no competing interests exist.