Ann Microbiol (2014) 64:921–934 DOI 10.1007/s13213-014-0911-2

REVIEW ARTICLE

The prevalence of antibiotic resistance genes among species in aquatic environments

Marta Piotrowska & Magdalena Popowska

Received: 10 January 2014 /Accepted: 5 May 2014 /Published online: 23 May 2014 # Springer-Verlag Berlin Heidelberg and the University of Milan 2014

Abstract The global rise in antimicrobial resistance (AMR) Keywords Aeromonas . Antibiotic resistance genes . Natural among causing infectious diseases is well documented, water . Aquacultures . Urban drinking water . Wastewater and the associated risks for human health are well known. There treatment plant is much less research on AMR with regard to environmental strains, both opportunistic and pathogenic ones. The genus Aeromonas is widely distributed in the environment and causes Introduction many variable diseases in fish and humans. Infections in humans are predominantly caused by Aeromonas veronii, A. hydrophila Species belonging to the genus Aeromonas are widely distrib- and A. caviae (A. punctata) in a form of bacteremia, gastroen- uted in the environment. They are able to inhabit natural soil, teritis or even septicaemia in immunocompetent and immuno- food and animals, but they most commonly occur in all kinds compromised individuals. Different groups of antibiotics are of aquatic environments (Janda and Abott 2010). Aeromonas used in the treatment, but studies indicate that fluoroquinolones spp. have been isolated from wastewater (Figueira et al. 2011), and cefotaxime are the most efficient. A disturbing consequence natural water such as rivers, lakes and estuaries (Henriques of antibiotic overuse is an increasing number of detection of et al. 2006; Picão et al. 2008), aquacultures (Schmidt et al. various antibiotic resistance genes (ARG) within this genus. The 2001; Sørum 2006) and urban drinking water (Carvalho et al. water environment is one of the major modes of transmission of 2012). The genus Aeromonas has a complex at the resistant bacteria from animals to humans, and, thus, the dis- species level, but it belongs to the family Aeromondaceae, semination of antibiotic resistance genes, particularly those which is part of the class of Gamma-. The located in mobile genetic elements (MGE) occurs in such as species classification changes dynamically with the emer- plasmids and transposons. This review summarizes recently gence of new genetic analyses, but thus far 28 species have published information on the type, distribution, and transmission been validated or proposed (http://www.bacterio.net). of ARG by MGE, widespread in Aeromonas strains living in Aeromonas species belong to one of the two major groups: various aquatic environments, including wastewater, natural psychrophilic or mesophilic bacteria. The former represents water, aquaculture and urban drinking water. The data available nonmotile bacteria with optimal growth temperature balanced indicate that the opportunistic pathogens like Aeromonas spp. between 22 - 25 °C that cause many variable diseases in fish, might serve as important vectors of ARG for clinically relevant e.g., furunculosis, septicaemia, ulcerative or hemorrhagic dis- pathogens present in such bodies of water . eases (Beaz-Hidalgo and Figueras 2013; Dallaire-Dufresne et al. 2013). In contrast, mesophilic species are motile, their : optimal growth temperature is between 35 - 37 °C, and being M. Piotrowska M. Popowska opportunistic pathogens of humans, they are potentially dan- Department of Applied Microbiology, Institute of Microbiology, Faculty of Biology, University of Warsaw, Miecznikowa 1, gerous. Infections are mostly induced by A. veronii, A. 02-096 Warsaw, Poland hydrophila and A. caviae (A. punctata) and result in bacter- emia, gastroenteritis or even septicaemia in immunocompe- * M. Piotrowska ( ) tent and immunocompromised individuals (Lai et al. 2007; Institute of Microbiology, Faculty of Biology, University of Warsaw, Miecznikowa 1, 02-096 Warsaw, Poland Parker and Shaw 2010). The number of infectious cases e-mail: [email protected] increases in the warmer months and the most common 922 Ann Microbiol (2014) 64:921–934 manifestations are and skin infections after contact conditions for the proliferation of bacteria (richness of nutri- with contaminated water. Treatment of these infections varies ents, aeration, optimal temperature). Particularly favorable and depends on the part of the world, but studies indicate that conditions exist in the activated sludge, which is characterized fluoroquinolones and cefotaxime are the most successful ther- by a high density and variety of microorganisms. Moreover, apies (Parker and Shaw 2010). Aeromonas infections are not WWTP receive large amounts of antibiotics with sewage an important public health problem so epidemiology is not water from hospitals, livestock manure and private house- very well known. However, previous studies carried out in holds, where bacteria are exposed to selection pressure different countries suggested that these infections should not (Bouki et al. 2013). These conditions promote the prolifera- be underestimated (Ghenghesh et al. 2008). Given the fact that tion of antibiotic resistant bacteria (ARB) and increase the Aeromonas spp. are opportunistic pathogens of humans and probability of the horizontal gene transfer (HGT) of ARG the causative agent of fish diseases, it is disturbing that more (Tennstedt et al. 2003). and more different antibiotic resistance genes (ARG) are being The study on sewage samples using high-throughput se- detected within this genus (Zhang et al. 2009a). The main quencing of 16S rRNA gene has detected over ten bacterial targets of Aeromonas are fish, which are exposed to these phyla with the highest contribution being from Proteobacteria, natural pathogens. To study the prevalence and horizontal Actinobactera, Chloroflexi, Firmicutes, Bacteriodetes and transfer of resistance genes within this genus, it is important Nitrospirae (Ye et al. 2012) as well as a large number of to examine the aquatic environment as a whole, including fish Aeromonas spp. (Moura et al. 2007; Figueira et al. 2011; and sediments. Igbinosa and Okoh 2012; Moura et al. 2012;Igbinosaand ARG are ubiquitous both in clinical pathogens and envi- Okoh 2013). Treatment processes reduce bacterial count in the ronmental bacterial species. This is in part due to the wide sewage, including the ARB (Guardabassi et al. 2002). How- usage of antibiotics in clinical practice, veterinary medicine ever, even in effluent water, ARG can be taken up by trans- and agriculture (Cantas et al. 2013), resulting in the release of formation and ARB are still detected (Zhang et al. 2009a). large amounts of these pollutants to the environment. Antibi- In the literature there is little information about ARG in otics, in addition to being chemical pollutants, exert a selective Aeromonas spp. present in WWTP. A variety of different pressure retaining and spreading the ARG among microbiota, species that has been detected so far include which poses a risk to human health (Kemper 2008). Addition- A. allosaccharophila, A. aquariorum, A. hydrophila, A. me- ally, it has been demonstrated that heavy metals can co-select dia, A. veronii, A. caviae, A. sanarellii and A. taiwanesis, the spread of ARG in the environment (Seiler and Berendonk (Figueira et al. 2011) (Table 1). The ARG recently found in 2012). Consequently, natural environments, especially water Aeromonas strains from wastewater encoded resistance to four bodies, constitute important reservoirs of ARG that may pro- major groups of antibiotics, i.e., the quinolones, aminoglyco- mote the spread of resistance to different groups of antibiotics sides, β-lactams and . In contrast to previous across different bacterial species (Lupo et al. 2012). Hence, studies, Figueira et al. (2011) who isolated bacteria from opportunistic pathogens such as Aeromonas spp. might be municipal sewage, argued that the vast majority of quinolone dangerous vectors of ARG for clinically relevant pathogens, resistance was the result of chromosomal mutations. These which also occur in the water (Rhodes et al. 2000;Sørum authors found many mutations in gyrA and parC genes, which 2006). are genetic determinants of quinolone resistance. The majority In this review we strive to present the latest information of these strains was resistant to nalidixic acid and belonged to from the literature on the prevalence of ARG in Aeromonas A. media and A. caviae species. This high frequency of muta- spp. from various water environments endowed with different tions could be the effect of contact with a high concentration anthropogenic impact. The main focus is on the differences of mutagenic substances in wastewater (Miyahara et al. 2011). between natural water, drinking water and more polluted These authors also found two plasmid-mediated quinolone environments such as wastewater and aquacultures. Further, resistance genes: qnrS and accA-cr, but only in A. media our intent is to highlight the problem of the horizontal transfer strains. In addition, this study investigated the occurrence of of these genes, which is related to the presence of integrons the cphA chromosomal gene, which is the most common and mobile genetic elements such as plasmids, transposons or metallo-β-lactamase in Aeromonas spp. (Janda and Abott insertion sequences, and the serious risks for human health 2010). This gene was detected in 18 % of strains in WWTP. related to this process. The results of the study by Balsalobre et al. (2009)indicated that the cphA gene is a relevant ARG in the water environ- ment, including wastewater. The gene was found in nearly Wastewater treatment plants 100 % of A. hydrophila and A. jandaei isolates. This confirms previous observations that many Aeromonas species can nat- Wastewater treatment plants (WWTP) constitute a specific urally produce chromosomal β-lactamases and also suggests kind of water environment because of the prevailing good that this resistance could be species-specific (Chen et al. Ann Microbiol (2014) 64:921–934 923

2012). Moreover, different genes encoding β-lactamases of- rivers, lakes, estuaries and groundwater (Zhang et al. 2009a). ten associated with mobile genetic elements (MGE) have been It is hardly possible to find an aquatic environment without recently detected in Aeromonas species from wastewater, i.e., ARG. Literature reports showed that ARG were detected even blaTEM, on the plasmid (Figueira et al. 2011;Igbinosaand in the pristine sources of rivers, deep groundwater and the Okoh 2012),blaKPC, as a part of transposone Tn4401 (Picão ocean (Batt et al. 2006; Storteboom et al. 2010;Chenetal. et al. 2013), and blaPSE1/CARB1, in integron class 1 gene 2013). According to recent studies on ancient DNA from cassette (Igbinosa and Okoh 2012). These β-lactamases were 30,000-year-old permafrost sediments, ARG have existed in found in previous studies in WWTP in various bacterial the natural environment as equally long as antibiotic pro- genera, confirming at the same time the spread of these genes ducers (D'Costa et al. 2012). However, the anthropogenic through HGT (Li et al. 2009; Pignato et al. 2010; Chagas et al. impact, in the form of large quantities of exogenous antibi- 2011; Zhang et al. 2012). Most alarming is the occurrence of otics, has increased the dissemination of ARG in the natural carbapenemases genes blaKPC identified in Aeromonas spp. water environment (Chen et al. 2013;Khanetal.2013). inhabiting hospital effluent wastewater (Picão et al. 2013). The diverse ARG belonging to different groups that are This observation definitely requires following-up with more ubiquitous in various natural waters, were also found in detailed studies. Aeromonas spp. (Henriques et al. 2006; Picão et al. 2008; Moreover, Moura et al. (2007) detected several aminogly- Girlich et al. 2011). Quinolone resistance genes were observed coside resistance genes in Aeromonas spp. that were isolated in lakes and rivers where mutations were found in the quino- from a slaughterhouse WWTP. All ARG were a part of gene lone resistance-determining regions (QRDR) and plasmid- cassettes, found in class 1 and class 2 integrons. Researchers mediated transferable quinolone resistance (PMQR) (Cattoir have identified aadA1, aadA2 and sat1 genes, encoding the et al. 2008; Alcaide et al. 2010). Mutations in the gyrA- resistance to streptomycin, spectinomycin and streptothricin, encoding subunit were identified with greater frequency than respectively. The aadA1 and aadA2 genes were also detected in the parC gene, suggesting that topoisomerase IV is a earlier in wastewater bacteria on mobile genetic elements secondary target for quinolones in Aeromonas spp. (Goñi- (MGE) (Szczepanowski et al. 2004; Tennstedt et al. 2005). Urriza et al. 2002; Han et al. 2012a). The most frequent point Additionally, the trimethoprim resistance gene (dfrA1) has mutation in the gyrA gene is at codon 83, while in the parC been also identified in a number of gene cassettes. This gene it is at codons 80 and 84, which is common among other ARG commonly occurs in integron gene cassettes and has gram-negative bacteria (Alcaide et al. 2010). Two types of recently been found in Vibrio strains isolated from urban PMQR genes have been identified, i.e., qnrS2, encoding surface water, and species from urban proteins that protect DNA gyrase from quinolones, and wastewater (Taviani et al. 2008;Pellegrinietal.2010). aac(6’)-ib-cr (accA4-cr) (Cattoir et al. 2008; Picão et al. Literature reports show that among resistance 2008;Majumdaretal.2011), encoding aminoglycoside ace- genes identified in wastewater, only tetA and tetE have been tyltransferase with the amino acid substitutions (Trp102Arg found in Aeromonas spp. thus far. It should be emphasized that and Asp179Tyr) necessary for the ability to acetylate cipro- both genes code for an efflux pump that removes the drug from floxacin (Park et al. 2006). Both genes were previously de- the cell (Chopra and Roberts 2001) and are located on plasmids tected in Aeromonas spp. in wastewater (discussed above), (Szczepanowski et al. 2009). Han et al. (2012c) found the tetA indicating that quinolone resistance genes are widespread in gene in two isolates of A. veronii in South Korea. The study of the water environment. This could be explained by the use of Kim et al. (2011) identified the tetE gene in A. salmonicida quinolones as the drug of first choice in the treatment of isolated from sewage also in Korea. Igbinosa and Okoh (2012) Aeromonas infections in clinical practice (Goñi-Urriza et al. searched for the tetC gene in their isolates but without success. 2000). Even then, the results of phenotypic studies demonstrated a The most numerous group of ARG among Aeromonas higher level of resistance to tetracycline, compared to those species in natural water are β-lactam ARG (Table 1). This is obtained in the molecular studies, by more than 70 % in Fort associated with the increasing resistance to β-lactam antibi- Beaufort WWTP, and more than 15 % in Alice WWTP. As a otics among this genus, which is also observed in clinical result, further studies should be carried out to search for other isolates (Aravena-Román et al. 2012). A chromosomally tet resistance genes commonly found in wastewater (Auerbach encoded β-lactamase cphA has been found in A. hydrophila et al. 2007; Zhang et al. 2009b; Marti et al. 2013). isolate from estuarine water, as has already been observed in Aeromonas strains from wastewater (Henriques et al. 2006). Moreover, recent studies have also shown the occurrence of

Natural water plasmid-carried blaFOX genes, enconding AmpC enzyme, in natural water (Maravić et al. 2013;Voolaidetal.2013). A variety of intrinsic and acquired ARG have been identified Maravić et al. (2013)studiedA. caviae strains isolated from in the microorganisms living in natural water bodies, such as a marine mussel and found only a few plasmid-encoded 924 Ann Microbiol (2014) 64:921–934

Table 1 Antibiotic resistance genes in different types of environment

Resistance Resistance Biological source Enviromental source* Reference gene phenotype accA4-cr quinolones A. media, A. allosaccharophila WWTP (TWW, RWW), Picão et al. 2008;Figueiraetal.2011 WTP, NW (L) qnrS2 quinolones A. media, A. allosaccharophila, WWTP (TWW, RWW), Cattoir et al. 2008; Picão et al. 2008; A. caviae, A. veronii, A. hydrophila, WTP, NW (L, R, F), AQ Verner-Jeffreys et al. 2009;Ishida A. sobria et al. 2010;Figueiraetal.2011; Majumdar et al. 2011; Han et al. 2012b gyrA quinolones A. allosaccharophila, A. aquariorum, WWTP (TWW, RWW), WTP, Goñi-Urriza et al. 2002; Alcaide et al. 2010; (mutation) A. hydrophila, A. media, A. caviae, NW (L, R), F Figueira et al. 2011; Kim et al. 2011; A. sanarellii, A. taiwanesis, Han et al. 2012a A. veronii, A. sobria, A. salmonicida, A. popoffii parC quinolones A. allosaccharophila, A. aquariorum, WWTP (TWW, RWW), Goñi-Urriza et al. 2002; Alcaide (mutation) A. hydrophila, A. media, A. veronii, WTP, NW (R, F) et al. 2010;Figueiraetal.2011 A. caviae, A. taiwanesis, A. salmonicida aadA1 aminoglycosides Aeromonas sp., A. caviae, A. media WWTP (TWW, EW), Henriques et al. 2006; Moura et al. 2007; A. hydrophila NW (M, F), HC, AQ Verner-Jeffreys et al. 2009;Ishida et al. 2010; Carvalho et al. 2012 aadA2 aminoglycosides Aeromonas sp.,A.caviae, WWTP (TWW), NW Henriques et al. 2006; Moura et al. 2007; A. hydrophila, A. eucrenophila, (M, R, F), HC, AQ (S, F) Verner-Jeffreys et al. 2009; Ndi and A. media, A. veronii, A. salmonicida Barton 2011;Carvalhoetal.2012 aadA7 aminoglycosides A. salmonicida AQ McIntosh et al. 2008 sat2 aminoglycosides Aeromonas sp. WWTP (EW) Moura et al. 2007 aadA1a aminoglycosides A. hydrophila, A. salmonicida, AQ (F,S) Schmidt et al. 2001;Jacobsand A. sobria, A. veronii, A. isthiosomia, Chenia 2007 A. encheleia, A. bestiarum accA4 aminoglycosides A. caviae, A. hydrophila AQ (F) Verner-Jeffreys et al. 2009 strA-strB aminoglycosides A. salmonicida, A. caviae, AQ, NW (R, S, F) Gordon et al. 2008;McIntoshetal. A. bestiarum 2008; Verner-Jeffreys et al. 2009 aacA aminoglycosides A. hydrophila, A. salmonicida, AQ Jacobs and Chenia 2007 A. isthiosomia, A. sobria, A. encheleia cphA/imiS β-lactams A. hydrophila, A. veronii, A. jandei, WWTP (TWW), WTP, Figueira et al. 2011; A. allosaccharophila, A. aquariorum, NW (M), HC Henriques et al. 2006; Balsalobre A. media, A. bestiarum, et al. 2009; Carvalho et al. 2012 A. eucrenophila, A. salmonicida blaKPC-2 β-lactams Aeromonas spp. WWTP, SW Picão et al. 2013 blaVEB-1a β-lactams A. caviae, A. allosaccharophila, NW (R) Girlich et al. 2011 A. veronii, A. media blaSHV-12 β-lactams A. allosaccharophila, A. veronii, NW (R, M) Girlich et al. 2011; Maravić et al. 2013 A. media, A. caviae, A. hydrophila blaPER (1,6) β-lactams A. allosaccharophila, NW (R, M) Girlich et al. 2010; Girlich et al. 2011; A. veronii, A. media, A. caviae Maravić et al. 2013 blaGES-7 β-lactams A. veronii NW (R) Girlich et al. 2011 blaTLA-2 β-lactams A. allosaccharophila NW (R) Girlich et al. 2011 blaOXA (2,7) β-lactams A. hydrophila, A. sobria, NW (M, F), AQ Henriques et al. 2006; A. salmonicida, A. caviae, Jacobs and Chenia 2007; Verner-Jeffreys et al. 2009 blaTEM β-lactams Aeromonas sp., A. media, A. veronii, WWTP (RWW), Henriques et al. 2006; Pontes et al. 2009; A. hydrophila, A. caviae NW (M, R, L, F), HC Verner-Jeffreys et al. 2009; Figueira et al. 2011; Carvalho et al. 2012; Igbinosa and Okoh 2012; Tacão et al. 2012 blaCTX-M β-lactams A. hydrophila, A. caviae NW (R, M) Tacão et al. 2012;Maravić et al. 2013 blaPSE-1/CARB-1 β-lactams Aeromonas sp., A. isthiosomia, WWTP, AQ Jacobs and Chenia 2007; Igbinosa A. sobria, A. encheleia, A. hydrophila and Okoh 2012 blaCMY-2 β-lactams A. salmonicida AQ McIntosh et al. 2008 blaFOX β-lactams Aeromonas sp., A. caviae NW (R, M) Maravić et al. 2013; Voolaid et al. 2013 Ann Microbiol (2014) 64:921–934 925

Table 1 (continued)

Resistance Resistance Biological source Enviromental source* Reference gene phenotype dfrA1/7 trimethoprim Aeromonas sp., A. media, A. sobria, WWTP (TWW, EW), Jacobs and Chenia 2007; Moura et al. 2007; A. salmonicida, A. hydrophila HC, AQ, NW (F) Verner-Jeffreys et al. 2009;Ishida et al. 2010; Carvalho et al. 2012 dfrA12 trimethoprim A. hydrophila, NW (estuarine, F), Henriques et al. 2006;Ishidaetal.2010; A. media, A. veronii HC, AQ Carvalho et al. 2012; Verner-Jeffreys A. eucrenophila, A. caviae et al. 2009 dfr13 trimethoprim A. hydrophila AQ (F) Verner-Jeffreys et al. 2009 dhfr2a trimethoprim A. hydrophila AQ, S Schmidt et al. 2001 dhfr1 trimethoprim A. hydrophila, A. sobria, A. veronii F, AQ, S Schmidt et al. 2001 A. bestiarum sul1 sulphonamides A. salmonicida, A. veronii, A. caviae, AQ (S, F) McIntosh et al. 2008; Verner-Jeffreys A. hydrophila et al. 2009; Ndi and Barton 2011 sul2 sulphonamides A. salmonicida, A. bestiarum AQ, NW (R,S) Gordon et al. 2008;McIntoshetal.2008 estX streptothricin Aeromonas sp. WWTP (EW) Moura et al. 2007 cat chloramphenicol Aeromonas sp. NW (M) Dang et al. 2008 catB2 chloramphenicol A. hydrophila, A. sobria, A. veronii, F, AQ (S) Schmidt et al. 2001 A. bestiarum catB3 chloramphenicol A. allasaccharophila, A. hydrophila NW (L) AQ Picão et al. 2008;Ishidaetal.2010 catB8 chloramphenicol A. hydrophila, A. caviae NW (M), HC, AQ (F) Henriques et al. 2006;Verner-Jeffreys et al. 2009; Carvalho et al. 2012 floR chloramphenicol A. salmonicida, A. hydrophila, AQ (F), NW (R, S) Gordon et al. 2008;McIntoshetal.2008; A. caviae, A. bestiarum Verner-Jeffreys et al. 2009 tetA tetracycline A. hydrophila, A. allosaccharophila, WWTP, HC, AQ (F), Schmidt et al. 2001; Nawaz et al. 2006; A. veronii, A. sobria, A. salmonicida, F, S, NW (R) Akinbowale et al. 2007;Jacobsand A. encheleia, A. media, A. icthiosomia, Chenia 2007; McIntosh et al. 2008; A. caviae Verner-Jeffreys et al. 2009;Girlich et al. 2010; Ishida et al. 2010; Girlich et al. 2011; Kim et al. 2011; Carvalho et al. 2012; Han et al. 2012c tetB tetracycline A. hydrophila, AQ Nawaz et al. 2006;Jacobsand A. encheleia, A. veronii Chenia 2007 tetC tetracycline A. hydrophila, HC, AQ, S, F Nawaz et al. 2006;Jacobsand A. veronii, A. caviae Chenia 2007; Verner-Jeffreys et al. 2009;Ishidaetal.2010; Ndi and Barton 2011;Carvalhoetal.2012; Han et al. 2012c tetD tetracycline Aeromonas sp.,A.hydrophila, HC, AQ, S, Schmidt et al. 2001; Nawaz et al. 2006; A. encheleia, A. veronii, A. sobria F(AQ) Akinbowale et al. 2007;Jacobsand A. salmonicida, A. caviae Chenia 2007; Verner-Jeffreys et al. 2009; Carvalho et al. 2012; Han et al. 2012c tetE tetracycline A. eucrenophila, A. hydrophila, WWTP, HC, AQ, S, F, Schmidt et al. 2001; Nawaz et al. 2006; A. icthiosomia, A. sobria, A. encheleia, F(AQ) Akinbowale et al. 2007;Jacobsand A. veronii, A. caviae, A. salmonicida Chenia 2007; Verner-Jeffreys et al. 2009; Ishida et al. 2010; Kim et al. 2011;Carvalhoetal.2012; Han et al. 2012c tetG tetracycline A. caviae AQ (F) Verner-Jeffreys et al. 2009 tetH tetracycline A. hydrophila, A. encheleia AQ Jacobs and Chenia 2007 tetM tetracycline A. hydrophila, A. sobria, AQ (F) Akinbowale et al. 2007 A. hydrophila tetY tetracycline A. bestiarum NW Gordon et al. 2008 vatE streptogramin A. caviae AQ (F) Verner-Jeffreys et al. 2009

Footnotes: *WWTP- wastewater treatment plant; RWW- raw wastewater; TWW- treated wastewater; WTP- water treatment plant; EW – effluent water; SW- special wastewater from hospital; NW- natural water; HC – human consumption; AQ- aquacultures; F- fishes; S- sediments; L- lakes, R-rivers; M- marine waters, estuaries 926 Ann Microbiol (2014) 64:921–934

blaFOX-2 genes. However, the study of Voolaid et al. (2013) According to the current literature, the resistance to chlor- suggested that the dissemination of blaFOX genes might be amphenicol and florfenicol among Aeromonas spp. has been significantly more widespread in environmental bacteria, with identified quite often. The cat genes (cat, catB3 and catB8), a great contribution of Aeromonas. Previous studies demon- encoding chloramphenicol resistance, were found in seawa- strated that Aeromonas were natural producers of chromosom- ters and lakes (Henriques et al. 2006;Dangetal.2008;Picão al AmpC and MBL β-lactamases (Chen et al. 2012). et al. 2008). Florfenicol/chloramphenicol resistance gene (floR) It is suspected that some of these chromosomal AmpC was found in isolates from river sediments (Gordon et al. 2008). enzymes are ancestors of FOX β-lactamases (Janda and Moreover, the resistance to chloramphenicol was observed Abott 2010). In addition to those listed above, numerous among various environmental bacteria (particularly in

Extended-Spectrum β-lactamases (ESBL) such as blaVEB, ), especially in marine water (Dang et al. 2008). blaSHV, blaPER, blaGES, blaTLA, blaOXA, blaTEM and blaCTX-M In the environment, both groups of the tet genes have been (Henriques et al. 2006; Girlich et al. 2010; Girlich et al. 2011; identified in a number of bacterial species. They code for Maravić et al. 2013) have been found in Aeromonas species. ribosomal protection proteins and efflux pumps (Zhang et al. ESBL do not belong to intrinsic determinants of Aeromonas 2009b), often associated with conjugative and mobilizable spp. but bacteria acquire them from the environment where elements, such as plasmids, transposons and integrons other ARB, e.g., representatives of the Enterobacteriaceae, (Chopra and Roberts 2001; Schmidt et al. 2001). Aeromonas among which ESBL-mediated resistance is widespread, are spp. isolated from natural waters frequently demonstrated a ubiquitous (Blaak et al. 2013). Depending on the gene, the tetracycline resistance phenotype (Goñi-Urriza et al. 2000; location might be different, but generally bla genes were iden- Tacão et al. 2012). However, until now only two molecular tified within MGE even when they were found in chromosomes. determinants of this resistance have been found, i.e., the tetA,

For example, blaSHV-12, blaPER-1 and blaPER-6 were localized and tetY genes that encode efflux pump proteins and are nearby insertion sequences and transposons (Girlich et al. 2011). located on the transposon and plasmid, respectively (Gordon

The blaVEB-1a genes were identified on plasmids and chromo- et al. 2008; Girlich et al. 2011). Additionally, the tetracycline somes, even though certain ESBL were solely attributed to resistance genes occur together with the tetR gene that en- plasmids, e.g., blaGES-7 and blaTLA-2. The majority of these codes the tetracycline repressor protein, located on Tn10 genes such as blaOXA, are also part of the class 1 integron (Meier et al. 1988). The study of Gordon et al. (2008)revealed cassettes (Henriques et al. 2006). According to recent studies, for the first time an association of the tetY and tetR genes, the most widespread ESBL genes among Aeromonas spp. are which was identified on a plasmid of A. bestiarum isolated blaTEM and blaCTX-M, which are detected in lakes, rivers and from freshwater sediments in France. More recently, the tetA- estuarine water (Henriques et al. 2006; Pontes et al. 2009;Lu tetR genes were identified by Girlich et al. (2010, 2011)who et al. 2010; Tacão et al. 2012). Both of them are plasmid- found them in A. allosaccharophila and A. veronii,isolated mediated, and in the study of Tacão et al. (2012)theywere from another river in France. The prevalence of these genes found more frequently in polluted sites. The researchers suggest should definitely be investigated in the future, because that these findings sustain the hypothesis concerning anthropo- Aeromonas can be a unique vector spreading them in the water genic modulation of resistance in environmental bacteria. environment. Aminoglycoside resistance genes from natural waters have been identified thus far in Aeromonas spp. in two studies. According to the literature, aadA1, aadA2 and strA-strB were Aquacultures detected in A. caviae, A. hydrophila and A. bestiarum in estuarine and river environments (Henriques et al. 2006; Farming of aquatic organisms (aquacultures) creates another Gordon et al. 2008). All these ARG were found on MGE- specific water environment, due to the great anthropogenic like plasmids and very often were a part of integron cassettes. impact. Large amounts of antibiotics are added into the water Sulfonamides and trimethoprim resistance genes, sul and dfr, with feed as prophylactic and therapeutic agents. The most are a commonly occurring type of ARG in natural water (Stoll widely used drugs are fluoroquinolones, florfenicol, oxytetra- et al. 2012; Suzuki et al. 2013). Similarly, as in the case of cyclines (OTC), amoxicillin and sulfonamides (Gräslund et al. aminoglycoside resistance, a couple of ARG have been iden- 2003; Holmström et al. 2003;Cabello2006; Primavera 2006; tified in Aeromonas spp. only in the studies of Henriques et al. Soonthornchaikul and Garelick 2009). Consequently, resis- (2006) and Gordon et al. (2008).Theonlyidentifiedgenes tance to antibiotics has been widely spread in aquaculture were sul2anddfrA12. Trimethoprim resistance genes (dfr), environments, which has been reported in many studies encoding dihydrofolate reductases and sulfonamide ARG (Petersen et al. 2002; Colquhoun et al. 2007; Newaj-Fyzul sul1, were frequently found as part of integron cassettes. The et al. 2008; Sørum 2008;Shahetal.2012). As a result of these sul2 gene has so far been found only once in A. bestiarum as a concerns, the usage of antibiotics as growth promoters has plasmid-mediated gene (Gordon et al. 2008). been strictly regulated or even banned in many countries, e.g., Ann Microbiol (2014) 64:921–934 927 in European Union from January 1, 2006 (Regulation (Ec) No predominant agent in all tetracycline resistant Aeromonas 1831/2003). Nevertheless, uncontrolled and continuous long- isolates. In the same study, two or even three genes encoding term use of antibiotics for therapeutic and prophylactic rea- tetracycline resistance were identified in a single Aeromonas sons also had a major influence on increased transfer of ARG isolate but it did not correlate with higher MIC. The study of between microorganisms (Tennstedt et al. 2003; Akinbowale et al. (2007) also investigated Australian aqua- Szczepanowski et al. 2009; Cheng et al. 2012; Moura et al. cultures and found that the tetM genes were the most preva- 2012). Among ARB in aquacultures are also fish pathogens lent, and in all cases were found in combination with more and human opportunistic pathogens, thus, it poses a particu- than one tet gene. These results suggest that the tet genes are larly serious threat to human health. Resistant bacteria can be common in aquacultures, but depending on the source of transferred to humans with infected fish or by direct contact isolation, various combinations of these genes can be found. with aquaculture ecosystems such as in the case of fish farm Certain studies also indicated that the tet determinants are workers (Petersen and Dalsgaard 2003). Antimicrobial resis- associated with conjugative plasmids (Schmidt et al. 2001). tance has long been reported in widespread Aeromonas spe- Resistance to aminoglycosides in Aeromonas spp. isolates cies in aquacultures, and recently an upward trend in the from aquacultures is diverse and additionally depends on the resistance has been observed (Rhodes et al. 2000;Schmidt origin of probes. Dias et al. (2012) who studied samples of et al. 2001; Jacobs and Chenia 2007). The study of Navarrete water and skin of ornamental fish obtained from a national et al. 2008 showed loss of diversity for the microbiota of fish importer, reported the resistance to aminoglycosides in 6-30 % treated with OTC, due to eradication of competing microor- of isolates depending on the antibiotic. However, Hatha et al. ganisms, and included only Aeromonas spp. Disturbing epi- (2005), in their study on samples from freshwater fish farms, demiological and molecular data indicate that Aeromonas observed this resistance at a much lower level (0-7 %). Nev- strains are able to transmit and share AMR determinants with ertheless, many aminoglycoside resistance genes have been such bacteria as isolated from humans found thus far in Aeromonas spp. isolated from this environ- (Rhodes et al. 2000;Sørum2006). ment. Furthermore, genes encoding aminoglycoside- The largest group of ARG identified in aquaculture isolates modifying enzymes, such as aadA1a, aadA2, aadA7, aacA4, of Aeromonas species is the tet genes. Among many tetracy- aacA and streptothricin phosphoryltransferase strA and strB cline resistance genes, eight classes of the tet genes (tetA, tetB, have also been detected (Jacobs and Chenia 2007; McIntosh tetC, tetD, tetE, tetH, tetG and tetM) have been found in et al. 2008; Verner-Jeffreys et al. 2009; Ndi and Barton 2011). Aeromonas spp. isolated from water, sediments and diseased According to the literature, the aad family of genes was fish in aquaculture systems (Table 1)(Schmidtetal.2001; detected most frequently and was localized within potentially Nawaz et al. 2006;Akinbowaleetal.2007; Jacobs and Chenia mobile gene cassettes (Verner-Jeffreys et al. 2009; Ndi and 2007; Verner-Jeffreys et al. 2009). Five of these genes are Barton 2011). located on the plasmid (tetA, tetB tetC, tetD, tetE), one in the The occurrence of antibiotic resistance genes against dif- transposon (tetA)andtwo(tetC and tetE) are adjacent to the ferent groups of antibiotics among Aeromonas spp. derived integrons as part of the same transposons. Most of them from aquacultures, is generally at the similar level and is less encode efflux pump proteins but the gene tetM,encodinga common. Quinolone resistance determinants were identified ribosomal protein has been also found (Akinbowale et al. in diseased fish from aquacultures (Ishida et al. 2010). Han 2007). The results in the literature indicate that the tetA and et al. (2012a), observed in their study both QRDR mutations tetE determinants are the predominant tetracycline resistance in the gyrA or parC genes as well as plasmid-encoded qnrS genes (Nawaz et al. 2006; Han et al. 2012c). The study by genes. The same authors identified qnrS2 genes in a different Schmidtetal.(2001),performedonacollectionfroma work (2012b), as part of the mobile insertion cassette on ColE- Danish farm, showed that the tetE gene was like plasmids, which were isolated from A. sobria and the most frequently identified after the tetA and tetD genes. A. hydrophila strains. Verner-Jeffreys et al. (2009)alsofound What is more, in some instances, more than one tet gene from qnrS2 genes, and using PCR and microarray techniques, different classes was observed in a single isolate. More recent detected the occurrence of these genes in association with studies of Nawaz et al. (2006)andHanetal.(2012c), who Inc A/C plasmids. isolated Aeromonas spp. from commercial catfish ponds in Β-lactam resistance within this genus in aquacultures is Texas and Korean fish farms and aquariums, respectively, also mainly limited to the widespread ampicillin and amoxicillin confirmed the tetE gene as the predominant agent. However, resistance. To date, only a few β-lactamases determinants

Kim et al. (2011), who isolated A. salmonicida strains from have been found and they include: blaOXA,blaPSE-1/CARB-1 salmonid farms and private fish tanks in Korea, reported that and blaCMY-2 (Jacobs and Chenia 2007; McIntosh et al. 2008). tetA was the most frequent gene. Ndi and Barton (2011)who The study by Jacobs and Chenia (2007) found the blaOXA-2a conducted their study on rainbow trout farms in Australia and blaPSE-1 genes within the gene cassettes of class 1 found the tetA genes in some isolates, but tetC was the integrons in almost 6 % and 30 % of isolates, respectively. 928 Ann Microbiol (2014) 64:921–934

In addition, the blaCMY-2 gene, which encodes β-lactamase the following sources of urban water for human consumption: from the AmpC group, was identified on a plasmid isolated fountains, mines, wells and drilled wells. A. media, A. from A. salmonicida (McIntosh et al. 2008). bestiarum and A. hydrophila were the most frequently isolated The determinants of chloramphenicol (catB)and species in that study. As expected, all strains were resistant to florfenicol (floR) resistance identified thus far, encode amoxicillin and the majority of them to the first generation acetylotransferase and efflux proteins. To date, several types cephalosporin, cefalotin, and the carboxypenicillin and of catB have been reported, i.e., catB2, catB3 and catB8 ticarcillin. Searching for β-lactam resistance genes resulted (Schmidt et al. 2001; Verner-Jeffreys et al. 2009; Ishida et al. in the identification of chromosomal cphA/imiS genes in 21

2010). McIntosh et al. (2008) found the floR gene within an strains, mostly in A. hydrophila. Despite the existence of many antibiotic resistance cassette identified in multidrug-resistant bla genes and their broad distribution, only in one strain of

(MDR) A. salmonicida. A. veronii the blaTEM gene was found. The study by Figueira The resistance to trimethoprim and sulphonamide was also et al. (2011)alsoshowedthatthecphA genes were prevalent in observed in this environment among Aeromonas species. This 76 % of strains from a drinking water treatment plant and were led to the identification of trimethoprim and sulphonamide predominant in A. hydrophila, A.veronii and A. jandei.The resistance genes, i.e., dihydrofolate reductase encoding differ- same study identified the determinants of resistance to quino- ent types of dfrA genes such as dfrA1 or dfrA7 (Table 1) lones, i.e., the accA4 and qnrS genes as well as a mutation in (Jacobs and Chenia 2007; Verner-Jeffreys et al. 2009)aswell the gyrA and parC genes that was observed in the previously as a dihydropteroate synthase, encoding the sul1 and sul2 discussed environments. However, the frequency of gyrA genes (McIntosh et al. 2008; Ndi and Barton 2011). Schmidt mutations in the water treatment plant was significantly et al. (2001) showed a strong association between the resis- lower than in the wastewater treatment plant. In addition, the tance to sulphaziadine/trimethoprim (S/T) and the occurrence study of Emekdas et al. (2006), who investigated the tap water of class 1 integrons. Moreover, all S/T resistance isolates of environment, showed that all Aeromonas isolates were sus- Aeromonas (mostly A. hydrophila) contained an integron with ceptible to ciprofloxacin. a sul1 gene and dfr gene cassette insert. The follow-up study Carvalho et al. (2012) identified the tet genes in 10 % of by Verner-Jeffreys et al. (2009) confirmed this observation in isolates, which corresponded with a low level of tetracycline Aeromonas isolates, in which the sul1 genes and various gene resistance. All of the genes observed encoded efflux pump cassettes containing dfr determinants were found. The sul2 proteins (tetA, tetC, tetD and tetE) and were located on the gene is less prevalent but was also identified as part of a plasmid. The most frequent was the tetE gene and the majority plasmid-located sequence containing genes floR, tetA/R and of them were found in A. hydrophila. The study by Scoaris strA/B from MDR A. salmonicida (McIntosh et al. 2008). Dde et al. (2008) observed a higher level of resistance to tetracycline (26 %) but did not prove the presence of ARG using molecular procedures. The same level of resistance was Urban water for human consumption observed in the case of trimethopim-sulfamethoxazole and . However, Carvalho et al. (2012) found several Aeromonas species have been also detected in sources of aminoglycoside and trimethoprim resistance genes (aadA1, treated urban water for human consumption, such as water aadA2, dfrA1 and dfrA12). They were identified in different treatment plants, wells, tap water or even drinking mineral configurations in gene cassettes of group I and II integrons. water (Scoaris Dde et al. 2008;Figueiraetal.2011;Kivanc Additionally, the catB8 gene has been found that encoded et al. 2011). Some species, e.g., A. hydrophila, A. caviae and resistance to chloramphenicol. Surprisingly, all of these po- A. sobria are agents of diarrheal episodes, particularly in tentially mobile genes were found in A. hydrophila. immunocompromised people (Ali et al. 2005; Janda and Abott 2010). For this reason, the occurrence of Aeromonas spp. in water for human consumption and the levels of resis- Resistance genes among Aeromonas spp. localized tance and virulence of these strains should be investigated. on mobile genetic elements There are only a few studies on the occurrence of antibiotic resistance genes in Aeromonas species isolated from urban MGE such as plasmids and transposons are vectors carrying water, thus, our knowledge regarding the occurrence of ARG resistance genes, which are widespread in many different in this environment is poor. Moreover, according to the United environments. They allow the exchange of antibiotic and States Environmental Protection Agency, the number of metal resistance genes among microorganisms through Aeromonas spp. in treated water is typically below 10 cfu/ conjugative transfer, transformation or transduction. As a ml. Therefore, the isolation of bacteria is problematic result, they play a significant role in the HGT between various (Aeromonas: United States Environmental Protection Agency bacterial species, including those phylogenetically distant 2006). Carvalho et al. (2012)isolatedAeromonas spp. from (Martinez et al. 2009). Of most concern, is the dissemination Ann Microbiol (2014) 64:921–934 929 of the broad-host range (BHR) and conjugative plasmids. type of transposon, Tn4401.TheseMGEhavebeenidentified These MGE can transfer ARG from environmental species within conjugative plasmids isolated from Aeromonas spp. to clinically relevant pathogens (Rhodes et al. 2000;Sørum inhabiting hospital sewage (Picão et al. 2013). Additionally, 2006). A water environment is a significant reservoir of mi- many ARG among Aeromonas spp., mostly encoding β- croorganisms that possess mobile resistant genes. MGE car- lactamases, were identified within various IS, e.g., IS26, rying resistance genes are also widespread among Aeromonas ISpa12,ISpa13,IS6100 or ISEcp1 (Table 2). Moreover, two spp. (Table 2). BHR are the most frequently identified plas- insertion sequence common region elements (ISCR), called mids in Aeromonas spp., and they belong to two different ISCR2, were found on the conjugative plasmid pAB5S9 incompatibility groups, i.e., Inc A/C and IncU. The majority (Gordon et al. 2008). These MGE are closely related to the of them are also MDR and are capable of self-conjugative family of IS91 insertion sequences and are considered to play transfer (tra genes) or a transfer by mobilization (mob genes). an important role in the acquisition of ARG (Toleman et al. Del Castillo et al. (2013) recently identified a 165 kb pR148 2006). However, participation of ISCR in gene transfer among plasmid from A. hydrophila. This large plasmid belongs to the Aeromonas spp. at this time seems to be secondary and should IncA/C group, distinguished by MDR, conserved backbone be fully explored in the future. and association with enteric human pathogens (Johnson and Bacteriophages also have to be taken into consideration in Lang 2012). The plasmid identified consisted of resistance the HGT of ARG among Aeromonas spp. Until now, over a genes to seven different pollutants within two transposon dozen of Aeromonas spp. phages have been isolated from types, Tn21 and Tn1721. In addition to antibiotic resistance different environments, e.g., A. hydrophila phages Aeh1 and to β-lactams, sulfonamides, tetracycline, chloramphenicol Aeh2 from sewage (Chow and Rouf 1983), A. hydrophila and aminoglycosides, pR148 carried genes encoding resis- phage CC2 from sewage in China (Shen et al. 2012), tance to mercury and quaternary ammonium compounds. A. salmonicida phage phiAS4 from a river in Korea (Kim More importantly, phylogenetic comparative studies revealed et al. 2012a)andA. salmonicida phage PAS-1 from aquacul- that pR148 is most closely related to human pathogenic E. coli ture in Korea (Kim et al. 2012b). Prophages have also been and Acinetobacter baumanii. This similarity indicates that the found that can contribute to HGT following excision and IncA/C group of plasmids was transferred between different transduction. Five putative prophages associated with viru- genera. Another study also identified several plasmids from lence genes were detected in A. hydrophila isolated from an this group carrying MDR and capable of conjugative transfer epidemic outbreak of catfish in the USA (Hossain et al. 2013). (McIntosh et al. 2008; Moura et al. 2012). One of them, the prophage AH2, shared a significant homol- The second type of plasmids that belongs to the IncU group ogy with ΦO18P and ΦO18P-like prophages of A. caviae is probably even more widespread in the environment. These (Ae398) and A. salmonicida (A449) (Hossain et al. 2013). BHR R-plasmids also have a highly conserved backbone and Beilstein and Dreiseikelmann 2008 described the prophage carry various antibiotic gene cassettes. For instance, closely ΦO18P (Myoviridae)inA. media isolated from a pond in related plasmids pRAS1, pFBOAT4 and pASOT, which were Germany. Despite these reports, the participation of bacterio- isolated from different Aeromonas species, all contained tetA phages in the development of antibiotic resistance has never genes and were capable of conjugative transfer between been demonstrated in Aeromonas spp. thus far. humans and aquaculture environments (Rhodes et al. 2000). Finally, resistance genes in Aeromonas spp. are often adja- Moreover, MDR plasmids such as pRAS1 and pPAR-32 that cent to integrons or even exist within integron cassettes, and share the same core genes but have different integron cassettes they can be located either on chromosomes or MGE. Many (In4-like and In6-like), carrying various resistance genes, also different ARG cassettes have been found among Aeromonas belong to the IncU group (Sørum et al. 2003). Furthermore, spp. in various water environments, including integron classes the IncU plasmids are also associated with the qnrS genes, 1and2(Table2). Class 1 from integrons was identified with which have been found in different Aeromonas species all higher frequency. around the world (Cattoir et al. 2008; Picão et al. 2008). Transposons (Tn) and insertion sequences (IS) carrying ARG have been described in many studies (Table 2). The Conclusions fragments of Tn1721 were identified also in chromosome- like plasmids that carried the tetA and tetR genes (Sørum Resistance to various pollutants, and antibiotics in particular, et al. 2003; Girlich et al. 2011). This transposon has been is broadly distributed among environmental bacteria, includ- previously found in various bacteria also belonging to human ing Aeromonas spp. In this review, we focused on the preva- pathogens, e.g., and hospital isolates of lence of ARG in natural water ecosystems as well as in those E. coli (Popowska and Krawczyk-Balska 2013). What is also with anthropogenic influence. Recent literature has reported worrying from the point of view of public health is the the identification of genes encoding resistance to many differ- prevalence of blaKPC resistance genes harbored on another ent groups of antibiotics in aquatic environments that include 930 Ann Microbiol (2014) 64:921–934

Table 2 Aeromonas spp. resistance genes on motile genetic elements and integrons find in literature data

Motile genetic Antibiotic resistance genes Enviromental Others References elements source ** pR148 MDR*: − Tn21: qacH, blaOXA-10, AQ (F) 165 kb; IncA/C; intI Castillo et al. 2013 aadA1, sul1 -Tn1721: tetA, tetR - catA2, sul1, mer p34 -IS: qnrS2, NW (L) 80 kb; IncU Picão et al. 2008 -intI: aac(6’)-Ib-cr, blaOXA-1,catB3 pAS37, P42 qnrS2 NW (R) 55/20 kb; IncU Cattoir et al. 2008 pTf28 blaGES-7 NW (R) 60-kb; intI;IRfromTn402; mobA Girlich et al. 2011 R-plasmids Tet, dhfr, catB, aadA1a AQ Large plasmids (>30 kb), different Schmidt et al. 2001 resistance profiles, conjugative tranfser; intI pAB5S9 MDR: floR, sul2, NW (R, S) 24,7 kb; ISCR2; conjugative transfer Gordon et al. 2008 strA-strB, tetR-tet(Y) pAG2 qnrS2 AQ 6,9 kb; type ColE; mobA; mobile IS Han et al. 2012b pBRST7.6 qnrS2 F7,6kb;IncQ;mobC, mobB Majumdar et al. 2011

Plasmid IncFIB blaCTX-M-15 NW 40 kb Maravić et al. 2013

Plasmid IncA/C aadA7, floR, sul 2, NW ~ 11 kb; intIMcIntoshetal.2008 tetA, strA-B, blaCMY-2 pRAS1 MDR: dfrA16,sulI, tetA F 45-kb; IncU; conjugative transfer; Sørum et al. 2003 Tn1721; intI; In4-like integron, IS6100 pRAS3 tetC AQ (F) 11 kb; non-conjugative L’Abée-Lund et al. 2002 pAR-32 MDR: aadA2, sul1, FincU;intI; In6-like integron Sørum et al. 2003 catAII pAHH01 tetE, tetR(E) NW (R,L,F), 8,9 kb; mob genes; ColE-like replication Han et al. 2012c WWTP system; relE/B (toxin-antitoxin) pFBOAT1-17 Tn1721: tetA, tetR(A) AQ, SW ~85 kb; IncU; conjugative transfer; intI, Rhodes et al. 2000; (pFBOAT4) IS61100,Tn1721-like region, In4-like Rhodes et al. 2004 pASOT tet A-E genes AQ Conjugative transfer; (Tn1721 – 5.4 kb Adams et al. 1998 EcoRI fragment) Tn1721 blaPER-6 NW (R) Chromosomal location, previously Sørum et al. 2003; (fragments) reported Girlich et al. 2011

Tn4401 blaKPC-2 WWTP (SW) Putative plasmid location Picão et al. 2013 (conjugative or mobilizable) IS26 blaSHV-12 NW (R) Previously reported Girlich et al. 2011 blaVEB-1 ISpa12,ISpa13 blaPER-1 NW (R) Chromosomal location Girlich et al. 2011 (Tn1213) ISCR2,IS6100 blaVEB-1 NW (R) Plasmid location Gordon et al. 2008; Girlich et al. 2011

ISEcp1 bleCTX-M NW (R) - Tacão et al. 2012 Integrons integron class 1 aadA2; dfrA1-aadA1; aadA1; WWTP, NW Putative location: chromosomal, plasmid; Schmidt et al. 2001; Henriques dfrA12-aadA2; catB8-aadA1; (M), HC, et al. 2006;JacobsandChenia2007; aadA1-blaOXA-2;dfrA12; NW (R), Moura et al. 2007;Verner-Jeffreys blaTLA-2;blaVEB-1; dfrA5- dfrA1- AQ et al. 2009;Ishidaetal.2010;Girlich dfrA27-qacE2; aadA1-aadA2; et al. 2011; Ndi and Barton 2011; dfrA22- dfrA1; dfrA21-dfrA22; Carvalho et al. 2012;Igbinosaand dfrA23; tetC/E/catB3; qnrS; Okoh 2012; Tacão et al. 2012;Maravić blaTEM1;dfrA7;tetC; et al. 2013 catB3-aadA1 integron class 2 estX-sat2-aadA1; WWTP, HC, Putative location: chromosomal Jacobs and Chenia 2007; dfrA1-aadA1; AQ, NW Moura et al. 2007; Carvalho et al. 2012; Maravić et al. 2013

Footnotes: * MDR-multidrug resistance; **WWTP- wastewater treatment plant; RWW- raw wastewater; TWW- treated wastewater; WTP- water treatment plant; EW – effluent water; SW- special wastewater from hospital; NW- natural water; HC – human consumption; AQ- aquacultures; F- fishes; S- sediments; L- lakes, R-rivers; M-marine waters, estuaries Ann Microbiol (2014) 64:921–934 931 quinolones, aminoglycosides, β-lactams, tetracyclines, sul- Infections caused by Aeromonas spp. do not directly pose a fonamides, trimethoprim and chloramphenicol. significant threat to public health. However, the transfer of There are differences in the contribution of resistance to ARG causes ineffectiveness of antibiotic treatment of fish antibiotics among Aeromonas spp. derived from particular diseases, and leads to significant economic losses, particularly environments. As a result, certain ARG have been identified in aquacultures. In conclusion, given the role of Aeromonas with a higher frequency than others. The most diverse envi- spp. as ARG vectors between environmental bacteria and ronments in terms of antibiotic resistance are natural water and clinical human pathogens, as well as the existence of MDR aquacultures. However, an aquaculture was the only environ- Aeromonas strains, and the location of ARG on MGE, the ment where no Aeromonas strains were detected that would presence of resistant Aeromonas strains in the environment carry mutations in the gyrA and parC genes, responsible for poses a serious threat to human health. the resistance to quinolones. In addition, these strains were also lacking the chromosomal gene cphA,encodingthe Acknowledgments The authors’ research was financed by a grant from metallo-beta-lactamase. Therefore, it seems that the three the National Center of Science, Poland (741/N-COST/2010/0). The re- aquatic environments, i.e., WWTP, natural and urban waters, search was conducted as part of the European Co-operation in the field of “ possess common features such as a low level of antibiotic Scientific and Technical (COST) Research Action TD0803 Detecting evolutionary hot spots of antibiotic resistances in Europe (DARE)” pressure on Aeromonas spp. In contrast, aquacultures utilize (2009–2013). therapeutically or preventively high concentrations of antibi- otics (particularly OTC), which favors the process of HTG rather than the introduction of chromosomal mutations. References The most numerous group of ARG in natural water are β- lactamase genes, especially those belonging to the type ESBL. The most dominant in aquacultures are tetracycline resistance Akinbowale OL, Peng H, Barton MD (2007) Diversity of tetracycline resistance genes in bacteria from aquaculture sources in Australia. J genes, which are generally widespread in this environment. 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