THE PRESENCE OF PUTATIVE VIRULENCE DETERMINANTS, TETRACYCLINE AND β-LACTAMS RESISTANCE GENES OF Aeromonas SPECIES ISOLATED FROM PET TURTLES AND THEIR ENVIRONMENT
DOI:
https://doi.org/10.26873/SVR-1144-2020Abstract
This study aimed to characterize Aeromonas spp. isolated from ten popular species of pet turtles and their environment 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 (blaTEM, blaSHV, blaOXA and blaCTX-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. dharkensis, A. veronii and A. caviae were carried seven tested virulence genes except for exu while only four virulence genes were detected 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, blaOXA and blaTEM genes were detected in 54% and 36% of β-lactam resistant isolates, respectively. No blaCTX-M and blaSHV genes were detected. Our results indicate that pet turtle-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
PRISOTNOST DETERMINANT ZA DOLOČITEV DOMNEVNE VIRULENCE TER GENOV ZA ODPORNOST NA TETRACIKLIN IN β-LAKTAM VRST Aeromonas IZOLIRANIH IZ LJUBITELJSKIH VRST ŽELV IN IZ NJIHOVEGA OKOLJA
Izvleček: Namen študije je bil določiti bakterije Aeromonas spp., izolirane iz desetih priljubljenih vrst hišnih želv in njihovega okolja, 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 odpornost na tetracikline (tetA, tetB in tetE) in β-laktame (blaTEM, blaSHV, blaOXA in blaCTX-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 blaOXAin blaTEM odkriti pri 54 % oziroma 36 % izolatov, odpornih proti β-laktamu. V nobenem vzorcu nista bila zaznana gena blaCTX-M in blaSHV. 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
References
(1.) Janda JM, Abbott SL. The genus Aeromonas: taxonomy, pathogenicity, and infection. Clin Microbiol Rev 2010; 23: 35–73. https://doi.org/10.1128/CMR.00039-09.
(2.) Kim KT, Kwak D. A case of Aeromonas hydrophila infection due to captivity-induced stress in a spectacled caiman (Caiman crocodilus). J Anim Plant Sci 2013; 23 (6): 1761–3.
(3.) Chen J, Zhu N, Kong L, Bei Y, Zheng T, Ding X, He Z. First case of soft shell disease in chinese soft-shelled turtle (Trionyx sinens) associated with Aeromonas sobria–A. veronii complex. Aquaculture 2013; 406–407, 62–7. https://doi.org/10.1016/j.aquaculture.2013.05.006.
(4.) Evangelista-Barreto NS, Vieira RH, Carvalho FCT, et al. Aeromonas spp. isolated from oysters (Crassostrea rhizophorea) from a natural oyster bed, Ceará, Brazil. Rev Inst Med Trop São Paulo 2006; 48: 129–33.
(5.) Shakir Z, Khan S, Sung K, et al. Molecular characterization of fluoroquinolone-resistant Aeromonas spp. isolated from imported shrimp. Appl Environ Microbiol 2012; 78: 8137–41. https://doi.org/10.1128/AEM.02081-12.
(6.) Lupescu I, Baraitareanu S. Emerging diseases associated with" New companion animals": review in zoonoses transmitted by reptiles. Sci Works Ser C Vet Med 2015; 61: 135–8.
(7.) Wimalasena SHMP, Shin GW, Hossain S, Heo GJ. Potential enterotoxicity and antimicrobial resistance pattern of Aeromonas species isolated from pet turtles and their environment. J Vet Med Sci 2017; 79: 921–6. https://doi.org/10.1292/jvms.16-0493.
(8.) Khajanchi BK, Fadl AA, Borchardt MA, et al. Distribution of virulence factors and molecular fingerprinting of Aeromonas species isolates from water and clinical samples: suggestive evidence of water-to-human transmission. Appl Environ Microbiol 2010; 76: 2313–25. https://doi.org/10.1128/AEM.02535-09.
(9.) Ottaviani D, Parlani C, Citterio B, et al. Putative virulence properties of Aeromonas strains isolated from food, environmental and clinical sources in Italy: a comparative study. Int J Food Microbiol 2011; 144: 538–45. https://doi.org/10.1016/j.ijfoodmicro.2010.11.020.
(10.) Yi SW, You MJ, Cho HS, Lee CS, Kwon JK, Shin GW. Molecular characterization of Aeromonas species isolated from farmed eels (Anguilla japonica). Vet Microbiol 2013; 164: 195–200. https://doi.org/10.1016/j.vetmic.2013.02.006.
(11.) Deng YT, Wu Y, Tan AP, et al. Analysis of antimicrobial resistance genes in Aeromonas spp. isolated from cultured freshwater animals in China. Microb Drug Resist 2014; 20: 350–6. https://doi.org/10.1089/mdr.2013.0068.
(12.) Jacobs L, Chenia HY. Characterization of integrons and tetracycline resistance determinants in Aeromonas spp. isolated from South African aquaculture systems. Int J Food Microbiol 2007; 114: 295–306. https://doi.org/10.1016/j.ijfoodmicro.2006.09.030.
(13.) Nawaz M, Khan SA, Khan AA, et al. Detection and characterization of virulence genes and integrons in Aeromonas veronii isolated from catfish. Food Microbiol 2010; 27: 327–31. https://doi.org/10.1016/j.fm.2009.11.007.
(14.) Yi SW, Chung TH, Joh SJ, Park C, Park BY, Shin GW. High prevalence of blaCTX-M group genes in Aeromonas dhakensis isolated from aquaculture fish species in South Korea. J Vet Med Sci 2014; 76: 1589–93. https://doi.org/10.1292/jvms.14-0274.
(15.) Akinbowale Ol, Peng H, Barton MD. Diversity of tetracycline resistance genes in bacteria from aquaculture sources in Australia. J Appl Microbiol 2007; 103: 2016–25. https://doi.org/10.1111/j.1365-2672.2007.03445.x.
(16.) Nawaz M, Sung K, Khan SA, Khan AA, Steele R. Biochemical and molecular characterization of tetracycline-resistant Aeromonas veronii isolates from catfish. Appl Environ Microbiol 2006; 72: 6461–6. https://doi.org/10.1128/AEM.00271-06.
(17.) Henriques IS, Fonseca F, Alves A, Saavedra MJ, Correia A. Occurrence and diversity of integrons and β-Lactamase genes among ampicillin-resistant isolates from estuarine waters. Res Microbiol 2006; 157: 938–47. https://doi.org/10.1016/j.resmic.2006.09.003.
(18.) Maravić A, Skočibušić M, Šamanić I, et al. Aeromonas spp. simultaneously harboring bla(CTX-M-15), bla(SHV-12), bla(PER-1) and bla(FOX-2), in wild-growing mediterranean mussel (Mytilus galloprovincialis) from Adriatic sea, Croatia. Int J Food Microbiol 2013; 166: 301–8. https://doi.org/10.1016/j.ijfoodmicro.2013.07.010.
(19.) Tacão M, Correia A, Henriques I. Resistance to broad-spectrum antibiotics in aquatic systems: anthropogenic activities modulate the dissemination of blaCTX-M-like genes. Appl Environ Microbiol 2012; 78: 4134–40. https://doi.org/10.1128/AEM.00359-12.
(20.) Piotrowska M, Popowska M. The Prevalence of antibiotic resistance genes among Aeromonas species in aquatic environments. Ann Microbiol 2014; 64: 921–34. https://doi.org/10.1007/s13213-014-0911-2.
(21.) Wimalasena SHMP, De Silva BCJ, Hossain S, Pathirana HNKS, Heo GJ. Prevalence and characterization of quinolone resistance genes in Aeromonas species isolated from pet turtle in Korea. J Glob Antimicrob Resist 2017; 11: 34–8 https://doi.org/10.1016/j.jgar.2017.06.001.
(22.) Chung T, Yi S, Kim B, Kim W, Shin GW. Identification and antibiotic resistance profiling of bacterial isolates from septicaemic soft-shelled turtles (Pelodiscus Sinensis). Vet Med 2017; 62: 169–77. https://doi.org/10.17221/65/2016-VETMED.
(23.) Bhaskar M, Dinoop KP, Mandal J. Characterization of ceftriaxone-resistant Aeromonas spp. isolates from stool samples of both children and adults in southern India. J Health Popul Nutr 2015; 33: e26. https://doi.org/10.1186/s41043-015-0036-7.
(24.) Figueira V, Vaz-Moreira I, Silva M, Manaia CM. Diversity and antibiotic resistance of Aeromonas spp. in drinking and waste water treatment plants. Water Res 2011; 45: 5599–611. https://doi.org/10.1016/j.watres.2011.08.021.
(25.) Piotrowska M, Przygodzińska D, Matyjewicz K, Popowska M. Occurrence and variety of β-lactamase genes among Aeromonas spp. isolated from urban wastewater treatment plant. Front Microbiol 2017; 8: e863 https://doi.org/10.3389/fmicb.2017.00863.
(26.) Puah SM, Puthucheary SD, Liew FY, Chua KH. Aeromonas aquariorum clinical isolates: antimicrobial profiles, plasmids and genetic determinants. Int J Antimicrob Agents 2013; 41: 281–4. https://doi.org/10.1016/j.ijantimicag.2012.11.012.
(27.) Han JE, Kim JH, Choresca CH, et al. Prevalence of tet gene and complete genome sequencing of tet gene-encoded plasmid (PAHH01) isolated from Aeromonas species in South Korea. J Appl Microbiol 2012; 112: 631–8. https://doi.org/10.1111/j.1365-2672.2012.05237.x.
(28.) Chopra I, Roberts M. Tetracycline antibiotics: mode of action, applications, molecular biology, and epidemiology of bacterial resistance. Microbiol Mol Biol Rev 2001; 65: 232–60. https://doi.org/10.1128/MMBR.65.2.232-260.2001.
(29.) Kim JH, Hwang SY, Son JS, et al. Molecular characterization of tetracycline- and quinolone-resistant Aeromonas salmonicida isolated in Korea. J Vet Sci 2011; 12: 41–8. https://doi.org/10.4142/jvs.2011.12.1.41.
(30.) Chacón MR, Figueras MJ, Castro-Escarpulli G, Soler L, Guarro J. Distribution of virulence genes in clinical and environmental isolates of Aeromonas spp. Antonie Van Leeuwenhoek 2003; 84: 269–78.
(31.) Sha J. Role of various enterotoxins in Aeromonas hydrophila-induced gastroenteritis: Generation of enterotoxin gene-deficient mutants and evaluation of their enterotoxic activity. Infect Immun 2002;70:1924–35. https://doi.org/10.1128/IAI.70.4.1924-1935.2002.
(32.) Ghenghesh KS, Ahmed SF, Cappuccinelli P, Klena JD. Genospecies and virulence factors of Aeromonas species in different sources in a north African country. Libyan J Med 2014; 9 (1): e25497. https://doi.org/10.3402/ljm.v9.25497.
(33.) Kirov SM, Castrisios M, Shaw JG. Aeromonas flagella (polar and lateral) are enterocyte adhesins that contribute to biofilm formation on surfaces. Infect Immun 2004; 72: 1939–45. https://doi.org/10.1128/IAI.72.4.1939-1945.2004.
(34.) Umelo E. Identification and molecular characterization of two tandemly located flagellin genes from Aeromonas salmonicida A449. J Bacteriol 1997; 179: 5292–9.
(35.) Pemberton JM, Kidd SP, Schmidt R. Secreted enzymes of Aeromonas. FEMS Microbiol Lett 1997; 152: 1–10. https://doi.org/10.1111/j.1574-6968.1997.tb10401.x.
(36.) Sen K, Rodgers M. Distribution of six virulence factors in Aeromonas species isolated from US drinking water utilities: A PCR identification. J Appl Microbiol 2004; 97: 1077–86. https://doi.org/10.1111/j.1365-2672.2004.02398.x.
(37.) Vilches S, Urgell C, Merino S, et al. Complete type III secretion system of a mesophilic Aeromonas hydrophila strain. Appl Environ Microbiol 2004; 70: 6914–9. https://doi.org/10.1128/AEM.70.11.6914-6919.2004.
(38.) Carvalho-Castro GA, Lopes CO, Leal CAG, Cardoso PG, Leite RC, Figueiredo HCP. Detection of type III secretion system genes in Aeromonas hydrophila and their relationship with virulence in nile tilapia. Vet Microbiol 2010; 144: 371–6. https://doi.org/10.1016/j.vetmic.2010.01.021.
(39.) Khor WC, Puah SM. Tan JAMA, Puthucheary S, Chua KH. Phenotypic and genetic diversity of Aeromonas species isolated from fresh water lakes in Malaysia. PLOS ONE 2015; 10 (12): e0145933. https://doi.org/10.1371/journal.pone.0145933.