ANTIBIOTIC RESISTANCE OF PATHOGENIC FOR FISH ISOLATES OF AEROMONAS SPP.

376 Bulgarian Journal of Agricultural Science, 16 (No 3) 2010, 376-386 Agricultural Academy ANTIBIOTIC RESISTANCE OF PATHOGENIC FOR FISH ISOLATES OF AEROMONAS SPP. P. OROZOVA 1, V. CHIKOVA 1 and H. NAJDENSKI 2 1 National Diagnostic Research Institute of Veterinary Medicine, National Referent Laboratory Diseases of Fish and Sea Mollusca, BG - 1606 Sofia, Bulgaria 2 Bulgarian Academy of Sciences, The Stephan Angeloff Institute of Microbiology, Department Pathogenic Bacteria, BG - 1113 Sofia, Bulgaria Abstract OROZOVA, P., V. CHIKOVA and H. NAJDENSKI, 2010. Antibiotic resistance of pathogenic for fish isolates of Aeromonas spp. Bulg. J. Agric. Sci., 16: 376-386 This study has determined and compared the antibiotic resistance towards various groups of antibiotics of 25 isolates from Aeromonas spp. with different origin. The strains have been isolated from drinking water and fish samples in Bulgaria, as well as from intensively bred trout fish in France, Sweden, Denmark, Spain, Scotland and England. The hemolytic activity of the isolates as regards sheep erythrocytes has been investigated in parallel. The Index of Resistance MAR has been calculated for each separate isolate. A wide antibiotic resistance of all clinic isolates has been determined. 60% of water isolates and 90% of clinical isolates possess a hemolytic activity. The presence in the environment of strains of Aeromonas spp. resistant to a wide group of antibiotics is a potential risk for the fishery farms. On the other hand, the results obtained give us grounds to accept the motile representatives of Aeromonas spp. as potential enteropathogens not only in our geographical region but also in Europe, as a whole. Key words: Aeromonas spp., antibiotic resistance, MAR index, hemolysis Introduction E-mail: petyorozova@gmail.com The antibiotics have been used in clinical practice for more than 50 years and nowadays they have been supplied for a wide application a number of substances having different mechanisms of antimicrobial action. Antibiotics have been applied widely not only in the human and veterinary medicine, but as growth promoters in animals breeding, as well. Soon after Penicillium discovery it has been established that the bacteria are able to develop very quickly resistance. During the last decade this process has turned into a constantly increasing problem of a worldwide scale. The therapeutic complications have been determined nowadays by strains of saprophytic and widely spread bacterial species like: pneumococci, staphylococci, enterobacteria, etc., which can acquire resistance to the most widely used and probably to all antimicrobial agents used. A number of investigations have indicated that the wide and irrelevant antibiotics usage leads to genes selection encoding the resistance. The resistance can be also the result of a bacterial modifi-

Antibiotic Resistance of Pathogenic for Fish Isolates of Aeromonas spp. 377 cation, enzymatic inactivation, efflux or impermeability. A danger of resistance exists among 5-10% of infections treated, which leads to a lack of success or a long treatment (Milatovic and Braveny, 1987; Fish et al., 1995). According to a number of authors, the infections caused by resistant bacteria increase the risk of a higher degree of death and a wider spreading as a result of their adaptation towards different aquamedia (Mateos et al., 1993; Kollef et al., 1999; Crowcroft and Catchpole, 2002; Helms et al., 2002; Cosgrove et al., 2003). Increasing cases of multiresistance among isolates of Aeromonas spp., which include not only bacteria pathogenic for the fish but also dangerous agents responsible for opportunistic infections in men have been determined in many regions worldwide (Holliman, 1993). This has been related to the horizontal transfer of genetic elements like plasmids and class 1 integrones (Jacobs and Chenia, 2006). It is known that the representatives of the Aeromonas spp. are especially susceptible towards all antibiotics active against Gram-negative bacteria, with the exception of many β-lactames, because of the production of multiinductive, chromosome determined β-lactamases (Jones and Wilcox, 1995; Rossolini et al., 1996). The motile representatives of Aeromonas spp. cause diseases in fish, like the hemorrhagic septicemia (Bullock et al., 1971; Egusa, 1978; Schaperclaus et al., 1992) and the ulcer (Karunasagar et al., 1995), leading to high economic losses in the fishery farms. The wide spreading of aeromonades is a result of their adaptation towards a different aqua-medium (Mateos et al., 1993). In relation to the increasing proofs in support of the important role of aeromonades as causing diseases among the artificially bred fish species, we have traced the antibiotic resistance towards a wide group of antibiotics of 25 strains from the Aeromonas spp. isolated from waters and clinically diseased fish. Materials and Methods Bacterial strains and growth conditions Isolation and identification The identification of Aeromonas spp. has been done by using routine methods including motility test (+), Gram coloring (-), Voges-Proskauer (VP) test (+), cytochrome oxidase activity (+), D-glucose fermentation (37 o C/24 h) (+), catalyse activity (+) etc. The species belonging has been determined by using concentional specific reactions, described by Popoff (1984), like esculine hydrolysis, L-arabinose utilization, glucose gas production, indol, acetyl methyl carbinol production, gelatinase activity, ornithine-decarboxylase activity, H 2 S separation from cystein, ability for aerobic and anaerobic growth, etc. The identification has been confirmed by means of the automatic system for quick identification Micronaut (Merlin Diagnostics, Bornheim-Hersel, Germany), plates E and NF and by means of API 20E test (BioMerieux, Marcy-l Etoile, France). Antibiotic susceptibility The susceptibility of Bulgarian isolates has been investigated towards Ampicillin (A) 10 μg, Bacitracin (B) 10 UI, Chloramphenicol (C) 30 μg, Ciprofloxacin (Cp) 5 μg, Dalacin C (Clindamicin) (Da) 10 μg, Erythromycin (E) 15 μg, Gentamycin (G) 10 μg, Kanamycine (K) 30 μg, Nalidix acid (Nx) 30 μg, Novobiocin (Nb) 30 μg, Oxacillin (O) 30 μg, Penicillin (P) 10 μg, Cephalotin (Cph) 5 μg, Streptomycin (S) 30 μg, Tetracycline (T) 30 μg, Trimethoprim (ST) 1.25 μg and Vancomycin (V) 30 μg and respectively of foreign isolates towards Ampicillin (A), Sulphatriard (ST), Penicillin (G), (PG), Nitroifurantoin (NI), Chloramphenicol (C), Tetracycline (T), Gentamycin (GM), Carbenicillin (PY), Cephalothin (KF), Nalidix acid (NA), Streptomycin (S), Polymixin E (CO), Cotrimoxazole (TS), Sulphamethizole (SM) and Novobiocin (NB) 30 μg. The antibiotic susceptibility has been checked by means of disk-diffusion method (Bauer et al., 1966) according to the recommendations of CLSI (Clinical Laboratory Standards Institute). The disks have been positioned upon Muller-Hinton agar, inoculated in advance with 100 μl of bacterial suspensions of the isolated strains cultivated in meat-peptone broth for 24 h at 28 o C (Costa et al., 1998). After additional incubation for 24 h at 28 o C, the inhibition zone has been measured (mm)

378 P. Orozova, V. Chikova and H. Najdenski and interpreted according to standards of CLSI (2007) and NCCLS (2003). Multi-resistance index (MAR index) The MAR index can be calculated for each isolate separately and can be determined as a/b, where a is the number of antibiotics, towards which the isolate is resistant, and b is the number of antibiotics, towards which the isolate susceptibility has been checked. The MAR index, which is higher than 0.2 (>0.2) identifies bacteria isolated from objects with higher risk of contamination, where antibiotics has been often used. The MAR index d 0.2 identifies strains from the environment, where antibiotics are rarely used or are not used at all (Krumperman, 1985). Hemolytic activity The hemolytic activity has been determined as a transparent zone of â-hemolysis around the colonies grown on a blood agar containing 5% (v/v) of sheep erythrocytes after incubation of 24 h at 37 o C (Gerhardt et al., 1981). Results and Discussion Out of drinking water and clinically diseased intensively bred fish in Bulgaria, 13 strains of Aeromonas hydrophila have been isolated. 12 clinical isolates from Aeromonas spp. isolated from rainbow trout in France, Italy, Sweden, Denmark, Spain, England and Scotland have been a part of Prof. B. Austin s collection (Heriot-Watt University, Edinburgh, Scotland) and with his kind co-operation they have been biochemically identified and determined by using 16S RNA sequense analysis (Orozova et al., 2009). The biochemical identification by using the automatic system Micronaut has shown that all Bulgarian isolates of Aeromonas hydrophila have been ornitine - negative and only 2 of them have not produced acetoin (Table 1). All strains from the Scottish collection have fermented glucose and has not possessed gelatinase, arginine dehydrolase and oxidase activity. In 2 isolates the acetoine production has not been established. According to Burke et al. (1982, 1983) the acetoine negative strains of Aeromonas Table 1 More important biochemical characteristics of 25 isolates of Aeromonas spp. Biochemical Aeromonas Aeromonas Aeromonas Aeromonas characteristics hydrophila salmonicida sobria bestiarum VP 13 (3*) 2 5 (1) 1 Esculin hydrolysis 6 (7) - - 1 Glucose fermentation 16 2 6 1 Lysine decarboxilase 12 (4) 0 (2) 6 1 Arabinose fermentation 13 (3) 2-5 1 Cytochrome oxidase 16 2 6 1 Gram (-) 16 2 6 1 H 2 S production 0 (16) 0 (2) 0 (6) 0 (1) β-hemolysis 12 (4) 2 6 1 Motility 16 0 (2) 6 1 Gelatinase - 2 6 1 Sorbitol fermentation 0 (16) 2 0 (6) 0 (1) Ornitine decarboxilase 0 (16) 0 (2) 0 (6) 0 (1) (*) Number of Negative Isolates

Antibiotic Resistance of Pathogenic for Fish Isolates of Aeromonas spp. 379 have been accepted as non-enterotoxigenic. A greater part of Aeromonas hydrophila strains have not hydrolyzed esculin, all of them being hemolytic. Among the different phenotype signs traced, usually the esculin hydrolysis can be associated with Aeromonas hydrophila and lysine decarboxylase production with Aeromonas hydrophila and A. sobria. Most of the strains investigated can produce lysine decarboxylase. According to some authors (Kirov et al., 1986) the possession of three and more than three characteristics like the production of acetylmethyl-carbinol, lysine decarboxylase, high hemolytic titer, disability for arabinose fermentation have been associated with enterotoxin production. The production of hemolytic toxins can be accepted as a significant proof for the pathogenic potential in aeromonades. Despite this, non-hemolytic aeromonades have been considered as a proof for human pathogens (Namdari and Bottone, 1990). Our investigations have shown that a small part of clinical isolates have not shown a hemolytic activity as regards sheep erythrocytes. According to some authors the sheep erythrocytes are less susceptible as compared to the erythrocytes of other mammals (Albert et al., 2000). 80% of water isolates are hemolytic as regards sheep erythrocytes, which confirm the observations of other authors, according to whom hemolytic aeromonades Aeromonas spp. have been isolated from uncontaminated waters (Gibboti et al., 2000). Notermans et al. (1986), have determined that all strains investigated by them: 26 of Aeromonas hydrophila strains, isolated from drinking waters and 9 from 22 of A. sobria isolates possess hemolytic activity, enterotoxigenity and cytotoxicity for Vero cells. All 25 strains of Aeromonas spp., investigated by us have been multi-resistant (Table 2). In all Bulgarian isolates Aeromonas hydrophila, as well as those from the alien collection, 100% of the resistance towards Ampiciline, Oxacillin, Penicilline, Bacitracine, Sulphametizole and Sulphatriard has been observed. The index of multi-resistance calculated has been within the limits of to 0.47 for the Bulgarian isolates and within the limits of 0.35 to 0.53 for the alien isolates. In most of the strains investigated the MAR index has been with 0.2 higher, which has been indicative for the wide antibiotics usage. MAR has been determined for a number of Aeromonas hydrophila strains (Pettibone et al., 1996; Son et al., 1997). Despite this, the cumulative chemiotherapeutics effect upon the environment and upon the bacterial ecology for intensively bred fish has obtained a small attention (DePaola et al., 1995). The wide usage of antibiotics and chemiotherapeutics in the fish-breeding farms for prevention or treatment of diseased fish, as well as their usage as food additives applied through the fodder or their solution in water directly has increased the antibiotic resistance among the pathogenic bacteria. Wide range of resistance of aeromonades have been established by a number of authors (Freitas- Almeida et al., 1993; Goni-Urrisa et al., 2000; Kampfer et al., 1999; Vila et al., 2002). They have confirmed that resistance is a characteristic feature for these bacteria having in mind their ability to receive and transmit effectively genes for antibiotic resistance from and to other enterobacteria (Marchandin et al., 2003). All isolates from A. hydrophila, A. sobria and A. bestiarum have been resistant to Ampicillin (10 to 25 μg) (Table 3). The isolates of A. salmonicida have been susceptible to Ampicillin and Carbenicillin. Dilsgaard et al. (1994) have established that the strains of A. salmonicida isolated from various geographic regions, have been 100% susceptible to Ampicillin, Cephalotin, Chloramphenicol, Neomycin, Polymyxin B and Rifampicin. The strains of A. salmonicida investigated by us have been resistant to Cephalotin, Penicillin G and Co-trimoxazole, but have been 100% susceptible to Ampicillin, Carbenicilin, Chloramphenicol, Streptomycin, Polymixine E, Nalidixic acid, Nitrofurantoin and Tetracycline. All isolates are resistant to Penicillin and to sulphonamides, like Sulphatriard and Sulphamethiozole. All Bulgarian isolates A. hydrophila are resistant to Oxacillin (Table 4). 42% of the alien isolates are resistant to Cotrimoxazole, and 8% of the

380 P. Orozova, V. Chikova and H. Najdenski Table 2 Susceptibility to 15 Antibiotics of 12 Clinical Aeromonas spp. Isolates in Europe and England Type Isolate code AP AP GM Nb PY NA NI SM T T TS KF CO S ST C PG MAR 10 5 10 30 μg 100 30 50 200 25 100 25 5 25 10 200 25 1U индекс* A. sobria BACC3 R R S R R R S R I S R I S S R S R 0.53 BACC4 R R S S S S S R S S S S S S R S R 0.26 BACC5 R R S R R R S R S S S I S S R S R 0.46 BACC6 R R S R R S S R S S S R S S R S R 0.47 BACC7 R R S S R S S R S S S I S S R S R 0.33 BACC8 R R S R R S S R S S I I S S R S R 0.40 A. bestiarum BACC9 R R S S R S S R S S I R S S R S R 0.40 A. hydrophila BACC1 R R S R R S S R S S R R S S R S R 0.53 BACC2 R R S S R S S R S S R R S S R S R 0.46 BAORN2 R R S R R R S R S S S R S S R S R 0.53 A. BACC235 S S S S S S S R S S R R S S R S R 0.33 salmonicida BACC237 S S S S S S S R S S R R S S R S R 0.33 * Index of multiresistance S, susceptible; R, resistanrt I, changingly-resistant; Ampicillin, (AP), Sulphatriard, (ST), Penicillin G, (PG), Nitrofurantoin, (NI), Chloramphenicol, (C), Tetracycline, (T), Gentamicin, (GM), Carbenicillin, (PY), Cephalothin, (KF), Nalidixic acid, (NA), Streptomycin, (S), Polymixin E, (CO), Cotrimoxazole, (TS), Suphamethizole, (SM), Novobiocin (Nb)

Antibiotic Resistance of Pathogenic for Fish Isolates of Aeromonas spp. 381 Table 3 Origin, MAR index and resistance limits of Bulgarian isolates A. hydrophila AH 31 AH 32 AH58 AH59 AH40 AH604 AH 385 AH 7 AH390 AH 3851 AHRt AH Rt2 AHca Code Source water water water water water Sturgeon Trout Paddlefish Trout Trout Trout Trout Crucian Carp Resistance limits A, B, O, P, Cph, Nx A, B, O, Cph, P A, B, O, P, T, S, Nb A, B, O, P, Nb, Cph, V A, B, O, P, Da, Nb, St, Cph A, B, O, P, T, Nb, Cph A, B, O, P, S, Nb, Cph A, B, O, P, Cph, Nb A, B, O, P, Da, Nb, Cph A, B, O, P, Nx, Nb, Cph A, B, O, P, T, Nb, Cph A, B, O, P, T, Nb, Cph A, B, O, P, T, Nb, Cph MAR Index 0.35 0.29 0.47 0.35 A, Ampicillin (10μg); B, Bacitracin (10 units); C, Chloramphenicol (30μg); Cp, Ciprofloxacin (5μg); D, Dalacin C (clindamicin) (10 μg); E, Erythromycin (15 μg); G, Gentamycin, (10μg); K, Kanamycin (30μg); Nx, Nalidixic acid (30μg); Nb, Novobiocin, (30μg); O, Oxacillin, (30μg); P, Penicillin (10μg); Cph, Cephalotin, (5μg); S, Streptomycin (30 μg); T, Tetracyclin (30μg); ST, Trimethoprim, (1.25μg); V, Vancomycin, (30μg); MAR Multiresistance Index Bulgarian isolates demonstrate resistance to Trimetoprim. Besides for A. trota, the resistance to Ampicillin has been characteristic for the genus Aeromonas (Carnahan et al., 1991). The antibiotic resistance has been partially connected with the pathogenic Aeromonas spp., in which besides the classical resistance to â-lactame antibiotics, multi-resistance has been often established (Goñi-Urriza et al., 2000; Kampferu et al., 1999; Villa et al., 2002). These bacteria can receive and give out genes of antibiotic resistance from and to other Gram-negative bacteria (Marchandin et al., 2003). All isolates investigated by Castro-Escarpully et al. (2003), show resistance to Ampicillin, Cephalotin, Cephazolin and Ticarcillin/ clavulanic acid and most of them are resistant also to Amoxillin/Subactam (92.6%), Piperacillin (81.5%), Imipenem (77.8%), and Carbenicillin (74%). According to the same authors, the strains Aeromonas investigated by them are 100% resistant to â-lactame antibiotics, with the exception of Imipenem (10.3%), Piperacillin (19.4%) and the second and third generation of cephalosporines (Cephuroxime and Cephotaxime). These strains also show 100% susceptibility to Nitrofurantoin. The strains investigated by us show 100% susceptibility to Nitrofurantoin (Table 2). 100% susceptibility has been established also to all aminoglucosides tested (Streptomycin, Kanamycine and Gentamycin) and macrolides (Erythromycin). To the poly-peptide antibiotic Polymixin E (Colistin sulphate), the alien isolates have shown 100% susceptibility while among the Bulgarian isolates, a 100% resistance to Bacitracin has been observed. Most of the aeromonades from the alien collection (67%) have shown a resistance to Cephalothin (Table 5). Among them, a great part of the A. sobria strains have a changing susceptibility, while two of them are

382 P. Orozova, V. Chikova and H. Najdenski Table 4 Antibiotic Resistance (% ) of Bulgarian Aeromonas hydrophila Isolates Aeromonas hydrophila (n=13) Strain Origin A T O P G K B C E S Da Cp Nx Nb Cp h Water (n=5) 5 1 5 5 0 0 5 0 0 1 1 0 1 2 4 1 1 Fish (n=8) 8 4 8 8 0 0 8 0 0 2 2 0 1 8 8 0 0 Total (%) 100 38 100 100 0 0 100 0 0 23 23 0 15 77 92 8 8 Ampicillin (A) 10 μg, Bacitracin (B) 10 UI, Chloramphenicol (C) 30 μg,ciprofloxacin (Cp) 5 μg, Dalacin C (clindamicin) (Da) 10 μg, Erythromycin (E) 15 μg, Gentamycin(G) 10 μg, Kanamycin (K) 30 μg, Nalidixic acid (Nx) 30 μg, Novobiocin (Nb) 30 μg, Oxacillin (O) 30 μg, Penicillin (P) 10 μg, Cephalotin (Cph) 5 μg, treptomycin (S) 30 μg, Tetracyclin (T) 30 μg, Trimethoprim (ST) 1.25 μg, Vancomycin (V) 30 μg. St V resistant (Tables 2 and 5). Only one clinical isolate from the Bulgarian collection has shown a changing resistance to Cephalothin, while all the rest have demonstrated resistance (Table 5). According to Janda and Motyl (1985) the susceptibility to Cephalothin can serve as a phenotype marker for the identification of A. sobria. The variations in the resistance to Cephalothin can affect upon aeromonades identification by Aerokey II (Carnahan and Joseph, 1991), because the resistance to Cephalothin is used as a differentiation between A. sobria and A. hydrophila. As a result of this, the increasing resistance to this cephalosporine can lead to the increasing of isolates, identified as A. hydrophila and this on its side can lead to taxonomic inaccuracies. In his investigation upon 43 isolates A. hydrophila, A. cavitae and A. veronii, between 93% and 100% have demonstrated susceptibility to Gentamycin and 100% of them to Ciprofloxacin (Vilau et al., 2002). Our results have been similar to the investigated isolates of 25. All of them are susceptible to Gentamycin and Ciprofloxacin. Almost all aeromonades are susceptible to hinolones (Ñiprofloxacin, Norfloxacin, Ofloxacin, Levofloxacin, Sparfloxacin, Moxifloxacin è Gatifloxacin (Zong and Gao, 2002). Resistance to Nalidixic acid has been established between the isolates A. sobria and A. hydrophila (25%) (Tables 2 and 5). The Bulgarian isolates A. hydrophila (15%) have also shown resistance to Nalidixic acid (Table 5). Belem-Costa et al. (2006) has established that most of the widely spread in Brazil fish-breeding farms antibiotics lead to the selection of resistant strains of Aeromonas spp. According to the results obtained, the aminoglucosides Chloramphenicol, Nitrofurantoin and Polymixin but not the first generation of hinulones and β-lactames are the groups of antibiotics with the best antimicrobial activity as regards the clinical isolates of Aeromonas spp. The fluorohinolons have been the first choice in treating of aeromonades infections. Strains resistant to Nalidixic acid and susceptible to Ciprofloxacin possess a mutation in gyra gene. That is why, infections caused by resistant to Nalidixic acid strains should not be treated with fluorohinolons. The resistance to Nalidixic acid is a function from a mutation in the gyra, gyrb, parc and pares genes, which form the hinolon resistant regions (Goni-Urriza et al., 2002). The results obtained have confirmed that â- lactame antibiotics should be escaped when treating infections caused by Aeromonas spp. The Aeromonas spp. has typically been susceptible to Tetracycline, to aminoglucosides, Trimethroprim, Sulphamethoxazole, third generation of cephalosporines and hinolones (Koehler and Ashdown, 1993; Janda and Abbott, 1996). Co et al. (1996) have informed about the increasing resistance to tetracyclines in some strains from Thailand. 38% of the Bulgaria isolates A. hydrophila are resistant to Tetracycline, while 92% of the European isolates have

Antibiotic Resistance of Pathogenic for Fish Isolates of Aeromonas spp. 383 Table 5 Limits of isolates resistance of Aeromonas spp. from Bulgaria (*) and abroad Class Antibiotic been susceptible to this antibiotic. In accordance with our results is the statement that some Aeromonas spp. usually preserves their aminoglucoside susceptibility (Jones and Wilcox, 1995). Some authors have established (Co et al., 1996) that there are more than 49% of Tetracycline resistant Aeromonas spp., in comparison with the established 8-22% by Jones and Wilcox (1995). From previous investigations we have established that 12.5% of the clinical isolates investigated, A. hydrophila and 20% of aqua-isolates of A. hydrophila have been resistant to Gentamicin Susceptibility, % Changing, % Resistant, % β-lactames: - Penicillium Ampicillin 17/0* 0/0* 83 / 100* Carbenicillin 25 0 75 Penicillin G 0/0* 0/0* 100 / 100* Oxacillin 0* 0* 100* Aminoglucosides: Streptomicin 100 0 0 Gentamicin 100/100* 0/0* 0 /0* Kanamycin 100* 0* 0* Tetracyclines: Tetracicline 92/62* 8/0* 0 / 38* Hinulones: - First Generation Nalidixic acid 75/ 85* 0/0* 25/15* - Second Generation Ciprofloxacin 0* 0* 0* Cephalosporines: Cephalotin 0/0* 33/1* 67/ 92* Sulphonamides: Sulphametizole 0 0 100 Sulphatriard 0 0 100 Co-trimoxazole 42 17 42 Trimetoprim 92* 0* 8* Polypeptides: Polymixin E 100 0 0 Bacitracin 0* 0* 100* Macrolides: Erythromycin 100* 0* 0* Others: Chloramphenicol: Chloramphenicol 100/100* 0/0* 0/0* Lincosamides: Dalacin (Clindamicin) 72* 5* 23* Nitroifurantoines: Nitrofurantoin 100 0 0 Aminicumarines: Novobiocin 50/ 20* 0/ 3* 50/ 77* (Orozova et al., 2008) having in mind that these results are similar to those obtained by Ansary et al. (1992), according to whom 23.5% of A. hydrophila strains have been isolated from fish and are resistant to Gentamicin. According to Yucel et al. (2004), 10-54% of Aeromonas spp. strains have been isolated from fish resistant to Gentamicin. The isolates, which have been tested by us have not shown resistance to Polymixin, Chloramphenicol, Nitrofurantoin, Streptomycin and Gentamicin. Most of the isolates can be accepted as multi-resistant because they are resistant

384 P. Orozova, V. Chikova and H. Najdenski to more than 4 of the antibiotics investigated, different from â-lactames (Tables 2 and 3). Conclusion The availability of the hemolytic activity as a marker of virulence, as well as the development of multi-resistance among clinical isolates of the genus Aeromonas underline the necessity of additional routine identification of these bacteria and of a constant monitoring of their range of resistance. Having in mind plasmids spreading among aeromonads it would have had a practical significance to investigate antibiotics usage in aquacultures in various places, the associated resistance as regards other bacteria and the widening of this investigation for the whole country, including other pathogens for the fish. Regulated and profound investigations should be made in order to determine the effect of antibacterial therapy upon microbial ecology of intensively bred fish in Bulgaria. Acknowledgements We would like to thank to UNESCO The Program Keiso Obuchi 2008 for the financial support as well as to Prof. Brian Austin for his co-operation and consultation. References Albert, M. J., M. Ansaruzzaman, K. A. Talukderet al., 2000. Prevalence of enterotoxin genes in Aeromonas spp. isolated from children with diarrhea, healthy controls, and the environment. J. Clin. Microbiol., 38: 3785-3790. Ansary, A., R. M. Haneef, J. L. Torres and M. Yadav, 1992. Plasmids and antibiotic resistance in Aeromonas hydrophila. J. Fish Biol., 15: 191 196. Bauer A. W., W. M. M. Kirby, J. C. Sherris and M. Turck, 1966. Antibiotic susceptibility testing by a standardized single disk method. Am. J. Clin. Pathol., 45: 493-6. Belém-Costa, A., J. E. Possebon Cyrino, 2006. Antibiotic Resistance of Aeromonas hydrophila Isolated From Piaractus mesopotamicus (Holmberg, 1887) and Oreochromis niloticus (Linnaeus, 1758). Sci. Agric. (Piracicaba, Braz.), 63 (3): 281-284. Bullock, G. L., D. A. Conroy and S. F. Sniesko, 1971. Septicemic diseases caused by motile aeromonads and pseudomonads. In: SNIESKO, S. F.; AXELROD, H. R. (Ed.) Diseases of fishes. Bacterial diseases of fishes. Neptune: T. F. H. Publ., 2A: 21-41. Burke, V., J. Robinson, H. M. Atkinson and M. Gracey, 1982. Biochemical characteristics of enterotoxigenic Aeromonas spp. J. Clin. Microbiol., 15: 48-52. Burke, V., J. Robinson, J. Beaman, M. Gracey, M. Lesmana, R. Rockhill, P. Echeverria and J. M. Janda, 1983. Correlation of enterotoxicity with biotype in Aeromonas spp. J. Clin. Microbiol., 18: 1196-1200. Clinical and Laboratory Standards Institute, 2007. M100-S17. Performance standards for antimicrobial susceptibility testing; 16th informational supplement. Clinical and Laboratory Standards Institute, Wayne, PA. Carnahan, A. M., S. Behram, S. W. Joseph, 1991. Aerokey II: a flexible key for identifying clinical Aeromonas species. J. Clin. Microbiol., 29: 2843 2849. Castro-Escarpulli, G., M. J. Figueras, G. Aguilera- Arreola, L. Soler, E. Fernandez-Rendon, G. O. Aparicio, J. Guarro and M. R. Chacon, 2003. Characterization of Aeromonas spp. isolated from frozen fish intended for human consumption in Mexico. International Journal of Food Microbiology, 84: 41-49. Cosgrove, S. E., G. Sakoulas, E. N. Perencevich, M. J. Schwaber, A. W. Karchmer and Y. Carmeli, 2003. Comparison of mortality associated with methicillin-resistant and methicillin-susceptible Staphylococcus aureus bacteremia: a meta-analysis. Clin. Infect. Dis., 36: 53-9. Crowcroft, N. S. and M. Catchpole, 2002. Mortality from methicillin resistant Staphylococcus aureus in England and Wales: analysis of death certificates.

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