Annals of Microbiology, 57 (3) 443-447 (2007) Distribution and antibacterial drug resistance of Aeromonas spp. from fresh and brackish waters in Southern Turkey Fatih MATYAR1*, Ays¸enur KAYA2, Sadık DINÇER2 1Department of Science and Technology Education, Faculty of Education, 2Department of Biology, Faculty of Science and Letters, Çukurova University, 01330 Balcalı, Adana, Turkey Received 8 March 2007 / Accepted 15 June 2007 Abstract - The frequency of antibiotic resistance was compared in Aeromonas spp. isolated from fresh and brackish water in Southern Turkey. A total of 97 Aeromonas spp. strains were isolated from four zones (three from fresh and one from brackish water). Most of the strains isolated from all samples were Aeromonas hydrophila (79.4%), while the amount of Aeromonas sobria and Aeromonas cavi- ae, were rather lower in the samples examined (17.5% and 3.1% respectively). A high proportion of isolates from all water sources showed resistance to cephalotin (86.6%) and trimethoprim-sulphamethoxazole (69%). On the other hand, a low proportion of bacte- ria showed resistance to tetracycline (14.4%), chloramphenicol (11.3%), gentamicin (7.2%) and nitrofurantoin (6.8%). Only one strain showing resistance to amikacin was found. Multiple Antibiotic Resistance Index (MARI) to at least two antibiotics was highest in brack- ish water (zone 4), followed by fresh water (zone 3). MARI values ranging from 0.2 to 0.8 for the bacteria isolated from brackish water. This study suggest that, multiple antibiotic resistant Aeromonas spp., especially A. hydrophila, can be easily recovered from fresh and brackish water sources in Turkey and these sources may play as a reservoirs responsible for disease pathogen aeromonads. Key words: Aeromonas, antibiotics, MARI, antimicrobial resistance. INTRODUCTION Members of the genus Aeromonas can be divided into the non-motile psychrophiles and the motile mesophiles. Aeromonas spp. are Gram-negative, rod shaped, non Members of the non-motile group, commonly referred to as spore forming, facultative anaerobic bacteria. Members Aeromonas salmonicida, appear to be obligate fish para- of this genus are found wide spread in natural habitats sites (Austin and Austin, 1985). The motile mesophilic such as soil, fresh and brackish water, sewage and group represents a more diverse collection, most frequent- wastewater (Araoju et al., 1991). Aeromonas can be ly classified as Aeromonas hydrophila, Aeromonas sobria, found in soil, fresh and saline waters, drinking water and and Aeromonas caviae. Of the 14 currently described animal faeces (Fiorentini et al., 1998). Water and food species of Aeromonas, the A. hydrophila, A. caviae, are important sources of transmission of Aeromonas spp. Aeromonas veronii biovar sobria, Aeromonas jandaei and infections to humans (Kirov, 2001). Members of the Aeromonas schubertii have been suggested as human genus Aeromonas have also been found moreover in dif- pathogens, though other species. Pathogenicity is associat- ferent foods (Fricker and Tompsett, 1989; Pin et al., ed with the liberation of virulence factors and cell associat- 1997) such as vegetables (Callister and Agger, 1987), ed endotoxin (Vadivelu et al., 1995). meat (Okrend et al., 1987), chicken (Hanninen, 1993) Antibiotic sensitivity of food and clinical isolates of and fish (Lee et al., 2000). Aeromonas spp. have been extensively studied (Demerta Aeromonas spp. is occasionally recovered from aquatic et al., 2004; Monteil et al., 2004; Palú et al., 2006; Wu et environment. This genus can easily found in chlorinated al., 2007) but little is known about the antibiotic resistance and non-chlorinated drinking water supplies (Kersters et profiles of this microorganism from environmental waters. al., 1995; Chauret et al., 2001). Furthermore, Aeromonas Fluvial waters receive human and animal wastewater dis- spp. is able to survive and multiply in a variety of food charges, which are expected to contain antimicrobial products stored between -2 and +10 °C (Mano et al., agents likely to exert a selective pressure, and commen- 2000). These organisms have also emerged as opportunis- sally resistant bacteria, capable of transferring their resist- tic pathogens responsible for gastroenteritis, skin and soft ances to autochthonous bacteria (Urriza et al., 2000). Many tissue infections and a variety of clinical syndromes in com- hospitals, discharge antibiotic wastes in large quantities to promised patients (Jones and Wilcox, 1995). the environment. This has lead to an increase in bacteria, having multiple antibiotic resistances and to an increase in * Corresponding author. Phone: +90 322 338 60 84-2789; more virulent pathogens. Fax: +90 322 338 64 40; E-mail: [email protected] Antibiotic multi-resistance bacteria in water have great 444 F. Matyar et al. importance all over the world. Since pathogen bacteria mended by the National Committee for Clinical Laboratory responsible of several diseases, many studies have been Standards (NCCLS, 1997). Antibiotic susceptibility of bac- focused on these bacteria (Calomiris et al., 1984; Messi et terial strains was determined by an agar disk diffusion test al., 2005; Lobova et al., 2006). Three sides of Turkey are (Bauer et al., 1966). Isolates were screened for resistance surrounded by the seas and the country is quite rich in to imipenem (IMP, 10 µg), chloramphenicol (C, 30 µg), freshwater sources. Water sources, near the disposals of tetracycline (T, 30 µg), gentamicin (G, 10 µg), cephalothin urban or hospital sewages might play a role as carriers of (CF, 30 µg), streptomycin (S, 10 µg), amikacin (AK, 30 antibiotic resistant bacteria. Public drinking water supplies µg), nalidixic acid (NA, 30 µg), nitrofurantoin (NF, 100 µg) sometimes could be contaminating chlorine-resistant and trimethoprim-sulphamethoxazole (SXT, 1.25 and pathogen Aeromonas spp. This might result in serious pub- 23.75 µg). The plates were incubated at 30 °C for 24 h. lic health problems. Characterisation of strains whether they are sensitive, The aims of this study were to identify of Aeromonas intermediate or resistant was based on the size of the inhi- spp. strains recovered from fresh and brackish water in bition zones around each disc according to the manufac- Southern Turkey and determine the level of antibiotic turer’s instructions. resistance rates against widely used antimicrobial agents in The strains Escherichia coli ATCC 25922 and Turkey. Pseudomonas aeruginosa ATCC 27853 were used as con- trol microorganisms for verification of antibacterial effect of the discs on Mueller-Hinton agar plates. All disks were pur- MATERIALS AND METHODS chased from Oxoid (Istanbul, TURKEY). Sampling. Fourteen stations were selected from the four Calculating of the Multiple Antibiotic Resistance zones along Seyhan River, Southern Turkey. Water sam- Index (MARI). The MAR index when applied to a single ples (250 mL) were collected from May 2005 to April 2006 isolate is defined as a/b, where ‘a’ represents the number from four selected zones (Zone 1: Seyhan Dam Lake, Zone of antibiotics to which the isolate was resistant and ‘b’ rep- 2: Seyhan River, Zone 3: Just before Seyhan River Estuary, resents the number of antibiotics to which the isolate was Zone 4: Seyhan River Estuary). Samples were taken using exposed. MAR index higher than 0.2 identifies organisms sterile bacteriological sample bottles and brought to the that originate from high-risk sources of contamination, laboratory in an ice chest (APHA, 1992). The samples were where antibiotics are often used. MAR indices less than, or processed within 4 hours of collection. A total of 58 sam- equal to 0.2, identify strains from environments where ples (14, 12, 16 and 16 from zones 1, 2, 3 and 4 respec- antibiotics are seldom or never used (Krumperman, 1985). tively) were examined for presence of Aeromonas spp. Bacterial characterisation. Samples were filtered RESULTS AND DISCUSSION through gridded, cellulose acetate, 0.45 µm, 47 mm diam- eter filter, and filters were placed on Ampicillin-Dextrin Presence and antibiotic resistance patterns of Aeromonas agar (ADA) medium which is suitable to isolate and count strains in fresh and brackish water sources in Southern Aeromonas spp. in freshwater (Kersters et al., 1996). ADA Turkey were investigated. Studies were carried out approx- medium is freshly prepared (g/l in deionised water): tryp- imately 70 km along Seyhan River including Dam Lake and tose (Difco), 5; Dextrin (Merck), 10; yeast extract (Merck), its estuary. Species characterisation revealed that A. 2; sodium chloride (Merck), 3; potassium chloride (Merck), hydrophila was the dominant species (79.4%) in the all 2; magnesium sulphate·7H2O (Merck), 0.2; ferric chlo- zones studied. A total of 77 strains of A. hydrophila, 17 ride·6H2O (Merck), 0.1; sodium desoxycholate (Merck), strains of A. sobria and 3 strains of A. caviae were found. 0.1; bromothymol blue (Merck), 0.04; agar (Oxoid), 15; Our results are in agreement with the findings of Havelaar ampicillin (ICN Biomedicals), 0.01. Presumptive et al. (1990) who reported that A. hydrophila was the most Aeromonas spp. isolates were obtained from ADA medium, frequently isolated species from distribution waters, where- after 24 h incubation at 30 ºC, by colony morphologies as A. caviae and A. sobria were predominant in a few water (yellow, circular, convex, 1-3 mm in diameter colonies systems in Netherlands. Aeromonas hydrophila is known as were selected). The genus Aeromonas was identified based an important fish pathogen