Slov Vet Res 2021: 58 (1): 25 – 33 Original Research Article DOI 10.26873/SVR-1144-2020 UDC 57.083.1:579.842:615.015.8 THE PRESENCE OF PUTATIVE VIRULENCE DETERMINANTS, TETRACYCLINE AND β - LACTAMS RESISTANCE GENES OF Aeromonas SPECIES ISOLATED FROM PET TURTLES AND THEIR ENVIRONMENT S.H.M.P Wimalasena, Gang-Joon Heo* Laboratory of Aquatic Animal Medicine, Veterinary Medical Center and College of Veterinary Medicine, Chungbuk National University, Chung- dae-ro 1, Seowon-gu, Cheongju 28644, Republic of Korea *Corresponding author, E-mail: gjheo@cbu.ac.kr Abstract: This study aimed to characterize Aeromonas spp. isolated from ten popular species of pet turtles and their envi- ronment to evaluate the potential risk of pet turtles as a source of virulence-associated genes, and tetracycline and β-lactams resistance determinants. Presence of eight virulence genes (ser, aer, exu, lip, fla, ascV, ahyB and gcat), and tetracycline (tetA, tetB and tetE) and β-lactams (bla TEM , bla SHV , bla OXA and bla CTX-M ) resistance genes were evaluated by conventional PCR assays. The aerA gene showed the highest frequency of occurrence (92%), followed by fla (75%), gcaT (68%), ahyB (59%), ser (39%), lip (37%) and ascV (25%) genes. None of the isolates carried amplicon of DNase-associated exu gene. A. hydrophila, A. dharken- sis, A. veronii and A. caviae were carried seven tested virulence genes except for exu while only four virulence genes were de- tected in A. enteropelogenes. Among the 75 tetracycline-resistant isolates, tetA, tetE and tetB genes were detected in 38, 26 and 6 isolates, respectively. Among the tested β-lactam resistance genes, bla OXA and bla TEM genes were detected in 54% and 36% of β-lactam resistant isolates, respectively. No bla CTX-M and bla SHV genes were detected. Our results indicate that pet tur- tle-associated aeromonads, exhibiting potential virulence and antimicrobial (tetracycline and β-lactams) resistance genes, may pose a serious health risk to pet turtle owners, particularly to immunocompromised individuals. Key words: Aeromonas spp.; virulence-associated genes; tetracycline resistance; β-lactams resistance; pet turtle Introduction Mesophilic aeromonads are ubiquitous bacteria that are a component of the normal microbiota of many aquatic animals such as fish, amphibians, and reptiles (1). They can cause ulcerative stoma- titis, pneumonia, dermatitis, and septicemia in rep- tiles under stressful conditions such as trapping, handling and temperature variations of rearing en- vironment (2, 3). Over the years, many studies have been investigated to evaluate the prevalence of Aero- monas species in aquatic animals, mainly food-pro- ducing animals (4, 5). However, a limited number of Received: 6 June 2020 Accepted for publication: 28 September 2020 studies evaluating the distribution of aeromonads in pet turtles have been published up to date (6, 7). The pathogenesis of Aeromonas species in- volves various virulence factors including cytotoxic heat-labile enterotoxin (act), cytotonic heat-labile enterotoxin (alt) and cytotonic heat-stable entero- toxin (ast), aerolysin (aer), lipase (lip), serine pro- tease (ser), elastase (ahyB), DNase (exu), glycero- phospholipid-cholesterol acyltransferase (gcaT), flagellar system (fla) and Type III secretion system (TTSS) effector (ascV). These genes encoding viru- lence factors have been broadly used in determining the potential pathogenicity of Aeromonas species isolated from the environment, foodstuffs, and hu- man clinical samples (1, 8-10). 26 S. H. M. P. Wimalasena, G-J. Heo Recently, antibiotic-resistant aeromonads have been recognized as a serious concern due to their potential health risks to animals and humans (11, 12). Especially, the dissemination of tetracycline and β-lactams resistance aeromonads in the aquatic environment has been widely documented (12, 13, 14). Among many tetracycline resistance genes, the tetE, tetA and tetB genes were frequently identified from Aeromonas species in the aquatic environment (12, 15, 16). Aeromonas species can produce nu- merous β-lactamases for conferring resistance to β-lactams. According to isolation sources, the pre- vious studies have shown the different prevalence of genes encoding β-lactamases in Aeromonas spe- cies. In the aquatic environment, the blaTEM, blaSHV, blaOXA and blaCTX-M β-lactams genes were frequently detected from Aeromonas species (17-19). These resistance genes containing plasmids and transposons are known as mobile genetic elements that can be transferred horizontally among distantly related lineages. Particularly, The aquatic environ- ment is more favorable for the transmission of resis- tant bacteria, thus, Aeromonas species as opportu- nistic pathogens might be dangerous vectors for the spreading of antibiotic resistance genes through the aquatic environment (18, 20). Hence, the present study was conducted to determine the occurrence of antimicrobial resistance genes (tetracyclines and β-lactams) and virulence-associated genes of Aer- omonas species isolated from pet turtles and their environment. Materials and methods Bacterial isolates One hundred and two Aeromonas species iso- lates obtained from ten commercially popular pet turtles species (Chinese stripe-necked turtles Ocadia sinensis, yellow belly sliders Trachemys scripta scripta, river cooters Pseudemys concinna concinna, northern Chinese softshell turtles Pelo- discus maackii, western painted turtles Chrysemys picta belli, peninsula cooters Pseudemys peninsu- laris, African sideneck turtles Pelusios castaneus, common musk turtles Sternotherus odoratus, red belly cooters Pseudemys rubriventris and alliga- tor snapping turtles Macroclemys Temminckii) and their rearing environment was screened to investi- gate the presence of putative virulence, and β-lact- ams and tetracycline resistance genes. These iso- lates have been previously characterized for their antimicrobial susceptibilities, enterotoxin (act, alt and ast) genes and quinolone resistance determi- nants (7, 21). Detection of antibiotic resistance genes Twenty-eight and seventy-five isolates were selected (21) for the detection of β-lactams and tetracycline resistance determinants, respectively. These isolates were tested by PCR assays to detect the genetic determinants associated with resistance to β-lactams (blaTEM, blaSHV, blaOXA and blaCTX-M), and tetracyclines (tetA, tetB and tetE). The primer sets used in PCR amplification are summarized in table 1. PCR amplifications were conducted in 20 µL volumes consisting of 10 µL of Quick Taq® HS DyeMix (Toyobo, Japan), 1 µL of 10 pmol/µL each primer and 1 µL of the template under standard conditions. The PCR products were analyzed by electrophoresis on 2% (wt/vol) agarose gels. Positive controls were implemented with previously characterized enterobacterial strains that harbored the corresponding genes (21, 22). Detection of virulence-associated genes All isolates were subjected to PCR assays to detect the 8 tested virulence genes including ser, aer, exu, lip, fla, ascV, ahyB and gcat. The PCR amplification of the virulence-associated genes was carried out according to the PCR primers and conditions reported previously (Table 1). The PCR mixture of 20 µL contained 10 µL Quick Taq HS DyeMix (Toyobo, Japan), 7 µL PCR water, 1 µL template and 1 µL of each primer. The PCR products were examined by electrophoresis on 1.5% (W/V) agarose gel. Results Bacterial isolates One hundred and two Aeromonas species isolates were isolated from the feces, skin and rearing environments of pet turtles and identified by biochemical and gyrB sequence analyses. Aeromonas enteropelogenes was the predominant species among the isolates (52.9%) followed by A. hydrophila (32.4%), A. dharkensis (5.9%), A. veronii (4.9%) and A. caviae (3.9%) 7. 27The presence of putative virulence determinants, tetracycline and β-lactams resistance genes of Aeromonas species ... Gene Target Nucleotide Sequence (5’-3’) Size (bp) Annealing temperature (ºC) Reference aerA Aerolysin F: CTATGGCCTGAGCGAGAAG 431 62 30 R: CAGTTCCAGTCCCACCACT ser Serine protease F: ACCGAAGTATTGGGTCAGG 350 55 13 R: GCTCATGCGTAACTCTGGT fla Flagella F: CCAACCGTYTGACCTC 608 56 36 R: MYTGGTTGCGRATGGT ahyB Elastase F: CACGGTCAAGGAGATCAAC 513 58 13 R: GCTGGTGTTGGCCAGCAGG lip Lipase F: ATCTTCTCCGACTGGTTCGG 382 62 36 R: CCGTGCCAGGACTGGGTCTT exu DNase F: AGACATGCACAACCTCTTCC 323 59 13 R: GATTGGTATTGCCTTGCAAG gcaT Glycerophospholipid- cholesterol acyltransferase F: TCCTGGAATCCCAAGTATCAG 237 65 13 R: GCAGGTTGAACAGCAGTATCT ascV Type III Secretion System F: AGCAGATGAGTATCGACGG 891 58 38 R: AGGCATTCTCCTGTACCAG blaTEM β - lactams resistance F: ATAAAATTCTTGAAGACGAAA 1080 60 22 R: GACAGTTACCAATGCTTAATC blaSHV F: TTATCTCCCTGTTAGCCACC 795 60 R: GATTTGCTGATTTCGCTCGG blaCTX-M F: CGCTTTGCGATGTGCAG 550 52 R: ACCGCGATATCGTTGGT blaOXA F: TCAACTTTCAAGATCGCA 591 60 R: GTGTGTTTAGAATGGTGA tetA Tetracycline resistance F: GTAATTCTGAGCACTGTCGC 1000 62 22 R: CTGCCTGGACAACATTGCTT tetB F: CTCAGTATTCCAAGCCTTTG 400 57 R: CTAAGCACTTGTCTCCTGTT tetE F: GTGATGATGGCACTGGTCAT 1100 62 R: CTCTGCTGTACATCGCTCTT Table 1: Oligonucleotide primers and PCR conditions a used to amplify virulence and antibiotic resistance genes of Aeromonas spp. a PCR thermocycle conditions for each reaction; initial denaturation of 94 ºC for 2 min followed by a total of 35 cycles of amplification. Each cycle consisted of 94 ºC denaturation for 30 s, annealing for 50 s and 72 ºC extension for 10 min. Presence of resistance genes Among the tested β-lactam resistance genes, blaOXA and blaTEM genes were detected in 54% and 36% of β-lactam resistant isolates, respectively. No blaCTX-M and blaSHV genes were detected (Table 2). Among the 75 tetracycline-resistant isolates, tetA, tetE and tetB genes were detected in 38, 26 and 6 isolates, respectively (Table 3). 28 S. H. M. P. Wimalasena, G-J. Heo Isolate Host a β-lactam resistance b β-lactam resistance genes Aeromonas caviae AD14 CSN AMP, AMX, CEP, FOX blaTEM AC50 RC AMP, AMX, CEP, CRO, FOX, IMI blaOXA, blaTEM A. dharkensis AD17 RC AMP, AMX, CEP, CRO, FOX, CTX blaOXA, blaTEM AD18 RC AMP, AMX, CEP, CRO, FOX, CTX blaOXA, blaTEM AD19 RC AMP, AMX, CEP, CRO, FOX blaOXA AD15 CSN AMP, AMX, CEP, FOX, CTX blaOXA A. enteropelogenes AC2 RC AMP, AMX, CEP blaOXA AC6 RC CEP, FOX - AC15 NCS AMP, AMX, CEP, FOX blaOXA AC30 CM CEP, CTX, ATM - AC31 WP CEP, CTX, ATM - AC32 WP CEP, CTX, ATM - AC35 RC CEP, CTX, ATM - AC44 YB AMP, AMX, CRO blaTEM AC45 RC CEP, CRO, ATM - AC53 WP AMP, AMX, CEP, FOX blaOXA AV4 RC AMP, AMX, CEP, FOX blaOXA AD1 CM AMP, AMX, CEP, FOX blaOXA A. hydrophila AH1 RC AMP, CEP - AH11 CSN AMP, AMX, CEP, CRO blaOXA, blaTEM AH13 CSN CEP, CRO, FOX, IMI - AH19 NCS AMP, AMX, CEP, CRO blaOXA AH20 NCS AMP, AMX, CEP blaTEM AH22 YB AMP, AMX, CEP - AH23 YB AMP, AMX, CEP, CRO blaOXA AH25 CM AMP, AMX, CEP, FOX blaTEM AD10 AF AMP, AMX, CEP, FOX blaOXA, blaTEM A. veronii AC52 SN AMP, AMX, CEP, FOX blaOXA, blaTEM Species Number of positive isolates (Subtotal %) tetA tetB tetE Aeromonas enteropelogenes (n = 50) 32 (64) - 8 (2) A. hydrophila (n = 17) 6 (35) - 12 (71) A. dharkensis (n = 4) - 2 (50) 3 (75) A. veronii (n = 3) - 3 (100) 2 (66) A. caviae (n = 1) - 1 (100) 1 (100) Total (%) (n = 75) 38 (51) 6 (1) 26 (35) Table 2: β-lactams resistance profiles of turtle-associated Aeromonas spp. Table 3: Distribution of tetracycline resistance genes among tetracycline resistant Aeromonas species isolated from pet turtles and their environment aHost: CSN= Chinese stripe-necked turtle, YB= yellow belly slider, RC= river cooter, PC= peninsula cooter, NCS= northern Chinese softshell turtle, CM= common musk turtle, WP= western painted turtle, AF= African sideneck turtle, SN= Alligator snapping turtle. bβ-lactams resistance: AMX=Amoxicillin (10 µg), AMP=Ampicillin (10 µg), CEP=Cephalothin (30 µg), CRO=Ceftriaxone (30 µg), FOX=Cefoxitin (30 µg), CTX=Cefotaxime (30 µg), IMI=Imipenem (10 µg) 29The presence of putative virulence determinants, tetracycline and β-lactams resistance genes of Aeromonas species ... Species Number of positive isolates (Subtotal %) aerA lip ahyB ser exu fla gcat ascV Aeromonas enteropelogenes (n = 54) 46 (85) 0 21 (39) 0 0 51 (94) 23 (43) 0 A. hydrophila (n = 33) 33 (100) 30 (91) 28 (85) 31 (94) 0 19 (58) 33 (100) 15 (47) A. dharkensis (n = 6) 5 (83) 3 (50) 4 (67) 4 (67) 0 2 (33) 6 (100) 4 (67) A. veronii (n = 5) 5 (100) 3 (60) 5 (100) 3 (60) 0 2 (40) 4 (80) 3 (60) A. caviae (n = 4) 4 (100) 2 (50) 2 (50) 2 (50) 0 3 (75) 3 (75) 4 (100) Total (%) (n = 102) 93 (92) 38 (37) 60 (59) 40 (39) - 77 (75) 69 (68) 26 (25) Distribution of virulence-associated genes The occurrence and frequencies of virulence genes are shown in Table 4. The aerA gene showed the highest frequency of occurrence (92%), followed by fla (75%), gcaT (68%), ahyB (59%), ser (39%), lip (37%) and ascV (25%) genes. None of the isolates carried amplicon of the DNase-associated exu gene. Discussion The Aeromonas spp. under study were multi- drug-resistant turtle-associated bacteria which carried quinolone resistance determinants, as well as enterotoxin genes (7, 21). The isolates were highly resistant to β-lactams especially amoxicil- lin, ampicillin and cephalothin. β-lactam antibiot- ics have used for the treatment of Aeromonas infec- tion during the last decade. However, their efficacy has significantly declined due to the production of β-lactamases by resistant bacterial strains (14, 17, 23). The Aeromonas spp. are naturally resistant to β-lactams because of the expression of chromoso- mal β-lactamases (24). In this study, twenty-eight aeromonads isolates were resistant to the more than one β-lactam an- tibiotics. Among them, 54% and 36% of isolates harbored blaOXA and blaTEM genes. Several previ- ous studies have documented the detection of the blaOXA and blaTEM genes in Aeromonas isolates re- covered from the environment (14, 25) and clinical samples (26) and the prevalence of gene detection varies according to the isolation sources. In Korea, a previous study reported that the blaOXA and blaTEM genes were detected in 3% and 100% of Aeromonas isolates from aquaculture fish [14]. However, a dif- ferent trend was observed in this study which the blaOXA and blaTEM genes were detected in Aeromonas Table 4: Prevalence of virulence-associated genes in Aeromonas species isolates from pet turtles and their environment isolates from pet turtles that suggest a wide distri- bution of β-lactamase genes in Aeromonas isolates from various sources. A much higher level of tetracycline resistance was observed amongst aeromonads in our previous study (7) and 78 of tetracycline-resistant isolates were selected to detect their tetracycline resistance determinants (tetA, tetB and tetE). A. enteropel- ogenes and A. hydrophila harbored tetA and tetE genes while other Aeromonas species harbored tetB and tetE genes. Previous reports indicate that the tetA and tetE determinants are the predominant tetracycline resistance genes in the aquatic envi- ronment (16, 27) and both genes code for an efflux pump that eliminates the drug from the cell 28. The tetA, tetB and tetE genes are located on the plas- mid as well as tetA in the transposon (Tn1721) and tetE is adjacent to the integrons (15). Han et al. (27) has reported that tetE gene was the predom- inant tetracycline determinant in Aeromonas spp. isolated from Korean fish farms and aquariums. However, Kim et al. (29) reported that tetA was the most frequent gene in A. salmonicida strains iso- lated from salmonid farms and private aquariums in Korea. The tetB gene was detected at a low fre- quency, while Jacobs and Chenia. (12) reported a lower prevalence of tetB genes among Aeromonas spp. isolated from the South African aquaculture system. Detection of virulence encoding genes of Aero- monas spp. have been widely applied for evaluat- ing their potential pathogenicity (30, 31). However, the prevalence of virulence-associated genes has rarely been reported in Aeromonas strains from pet turtles (7). In the current study, Aeromonas iso- lates were found to possess genes aerA, lip, ahyB, ser, fla, gcat and ascV, while genes for DNase (exu) was not identified. Especially, none of A. entero- pelogenes isolates harbored lip, ser, exu and ascV 30 S. H. M. P. Wimalasena, G-J. Heo genes. Previous studies have revealed that multi- ple virulence-associated genes are present in Aero- monas isolates and having high heterogeneity in the distribution of virulence-associated genes (10, 30, 31). The pore-forming aerolysin/hemolysin encoded aer gene was the most prevalent in this study which was detected in 92% of the total isolates representing all species of the genus. Several studies have reported the high prevalence of the aer gene in clinical and environ- mental Aeromonas isolates (30, 32). The three enterotoxins act, alt, and ast have been implicated as major virulence factors in diarrhoeal disease which had been investigated in our previous study (7). However, the presence of these toxins might not be enough for virulence (31). The temperature- stable metalloprotease with elastolytic activity (ahyB) and serine protease (ser) play an important role in the invasiveness and establishment of infection (1). In the current study, the ahyB and ser genes were detected in 59% and 39% of isolates, respectively. None of the A. enteropelogenes isolates harbored ser gene. The flagella are important appendages for the initial attachment of bacteria to the gastrointestinal epithelium and involve in the subsequent adherence process and biofilm formation (33, 34). The fla gene-encoded polar flagella were common among the Aeromonas isolates from the aquatic environment. The fla gene was detected in 99% of Aeromonas isolates from diseased eel in Korea (10). The gcaT gene plays a coherent, integrated role in the establishment of pathogenicity of Aeromonas spp. by involving in the regulation and secretion of extracellular glycerophospholipid-cholesterol acyltransferase (13). The gcaT gene was detected in 68% of Aeromonas isolates. Lipases play a role as virulence factors by interacting with leukocytes or by disturbing several immune system functions through free fatty acids produced by the lipolytic activity. Extracellular lipases secreted by Aeromonas spp. actively involve in the alteration of the host plasma membrane and thus increase the severity of infection (35). Among Aeromonas strains isolated in the present study, 91% of A. hydrophila, 60% of A. veronii, 50% of A. dharkensis and 50% of A. caviae isolates were found to have lip gene. Several previous studies reported a high prevalence of lip gene among the Aeromonas isolates from the aquatic environment (10, 36). Type III secretion system (T3SS) plays a crucial role in host- pathogen interactions by injecting effector toxins directly into the cytosol of host cells (37). The acsV gene encodes the T3SS and which was detected in 59% of Aeromonas spp. except for A. enteropelogenes isolates. The presence of ascV gene was previously detected in 68% of Aeromonas spp. isolated from diseased farmed fish and farm environment (38). Besides, the high frequency of ascV gene was reported in human clinical isolates (37). The exu gene is responsible for DNA hydrolysis which was not detected in this study. The absence of exu gene was also reported by Nawaz et al. (13) in A. veronii isolated from catfish in the USA. In contrast, the high prevalence of exu gene was observed in Aeromonas spp. isolated from freshwater lakes in Malaysia (39) and diseased eel in South Korea (10). The specificity of the host or environmental source could be the possible reasons for the absence of exu gene in this study. According to the available literature, this is the first description of these virulence-associated genes in Aeromonas of pet turtle origin. 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Heo Izvleček: Namen študije je bil določiti bakterije Aeromonas spp., izolirane iz desetih priljubljenih vrst hišnih želv in njihovega okol- ja, z namenom ocenjevanja potencialnega tveganje hišnih želv kot vira genov, povezanih z virulenco, ter determinante odpornosti proti tetraciklinom in β-laktamom. Prisotnost osmih virulentnih genov (ser, aer, exu, lip, fla, ascV, ahyB in gcat) ter genov za odpor- nost na tetracikline (tetA, tetB in tetE) in β-laktame (bla TEM , bla SHV , bla OXA in bla CTX-M ) je bila ocenjena s konvencionalnimi testi PCR. Najbolj pogost je bil Gen aerA (92 %), sledili so geni fla (75 %), gcaT (68 %), ahyB (59 %), ser (39 %), lip (37 %) in ascV (25 %). Nobeden od izolatov ni imel pomnoženega gena exu, povezanega z DNAzo. A. hydrophila, A. dharkensis, A. veronii in A. caviae so vsebovali sedem testiranih genov virulence, razen exu, medtem ko so bili v A. enteropelogenih odkriti le štirje virulenčni geni. Med 75 izolati, odpornimi na tetracikline, so bili geni tetA, tetE in tetB odkriti v 38, 26 oziroma 6 izolatih. Med preizkušenimi geni za odpornost proti β-laktamu so bili geni bla OXA in bla TEM odkriti pri 54 % oziroma 36 % izolatov, odpornih proti β-laktamu. V nobenem vzorcu nista bila zaznana gena bla CTX-M in bla SHV . Rezultati študije kažejo, da bakterije Aeromonas spp. iz hišnih želv lahko imajo potencialne virulenčne gene in gene za odpornost proti tetraciklinu in β-laktamom, in lahko potencialno ogrožajo zdravje lastnikov hišnih želv, zlasti imunsko oslabljenih posameznikov. Ključne besede: Aeromonas spp.; geni povezani z virulenco; odpornost na tetracikline; rezistenca na β-laktami; ljubiteljske vrste želv