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Annals of Microbiology, 57 (1) 35-38 (2007) Distribution and antimicrobial multiresistance in Gram-negative bacteria isolated from Turkish sea bass (Dicentrarchus labrax L., 1781) farm Fatih MATYAR* Çukurova University, Faculty of Education, Department of Science and Technology Education, 01330 Balcalı, Adana, Turkey Received 8 September 2006 / Accepted 15 December 2006 Abstract - In this study, the frequencies of 111 antibiotic-resistant Gram-negative bacteria obtained from cultured sea bass were inves- tigated. All the strains were identified and tested whether they were resistive against ten different antibiotics or not. The results showed that most of the bacteria were resistant to trimethoprim-sulphamethoxazole, cephalotin, tetracycline and streptomycin. It was found multiple antibiotic resistance (MAR) index values ranging from 0.3-0.8 for the bacteria isolated from gill. A large number of Pseudomanas putida (25.2%), Moellerella wisconsensis (18%) and Pseudomonas fluorescens (10.81%) were identified. Also, strains of Ralstonia pick- ettii (9%), Vibrio fluvialis (8.1%), Pantoea sp. (7.2%) and Erwinia sp. (5.4%) were found. This study suggested that Turkish sea bass farms have antibacterial multiresistance bacteria and may play as a reservoirs response for disease pathogen bacteria. Key words: sea bass; antibiotics; MAR; Pseudomonas. INTRODUCTION However, in recent years, with the increase in produc- tion, some of the pathological problems like disease control Many types of aquatic organisms are being cultured all over had to be solved. Using antibacterial agent is most prefer- the world, especially a wide variety of fish. These organisms able solution for these problems. Unchecked antibiotic are also being cultured in a range environment from fresh using, especially unlicensed farm became most prevalent in to brackish waters, in response to the increasing market recent years in Turkey. demand. The aims of this study are to research the frequency of Pathogen bacteria can spread rapidly through these antibiotic resistant bacteria present in Turkish farmed sea crowded environments, resulting in the bacterial diseases bass, to identify the representative resistant Gram-negative and loss of production. Antibacterial agents may be used to bacteria and to evaluate MAR index of resistant bacteria mitigate this problem. So, intensive fish and shrimp farming from gills and intestinal contents. has resulted massive use of antibacterial agents to prevent or threat disease outbreaks (Sandaa et al., 1992; Holmström et al., 2003). MATERIALS AND METHODS In EU member states, only four or five antimicrobial agents are licensed for use in finfish culture. But laws for Bacterial counts. One hundred and eleven antibiotic governing the application of antibacterial agents for agricul- resistant Gram-negative bacteria strains were recovered tural purpose in developing countries are generally absent from twenty-two Dicentrarchus labrax samples captured or unheeded (Alderman and Hastings, 1998). This from Bahce Region-Karatas/Adana aquaculture farm. unchecked use could also lead to a wide variety of antibiot- Total bacteria counts were made by the spread Plate ic resistant bacterial strains. Count Agar (PCA, Oxoid). Gram-negative bacteria counts, Dicentrarchus labrax is distributed in the northeast were made using MacConkey Agar (McC, Merck). Triplicates Atlantic from Morocco to Norway, in the Mediterranean Sea plates with PCA and McC were inoculated with appropriate and in the Black Sea. In Turkey cultured fish production in dilutions from the sample homogenates and incubated for 1 coastal areas has been developed rapidly. Three sides of day at 37 °C and 2 days at 35 °C respectively. Media were -1 this country are surrounded by the sea and have 7,833 km supplemented with the antibiotics; ampicillin (50 μg ml ), -1 -1 coast. Marine aquaculture has great importance in econo- streptomycin (25 μg ml ), tetracycline (25 μg ml ) and -1 my. Turkey is the second largest producer of farmed fish in chloramphenicol (25 μg ml ) were used to evaluate the the Mediterranean region. incidence of resistant bacteria. Media sets without any antimicrobial agents were also prepared at the same time. Bacterial strains. A total of 51 bacterial strains from gills * Corresponding author. Phone: +90 322 338 60 84-2789; and 60 strains from intestinal content samples were isolat- Fax: +90 322 338 64 40; E-mail: email@example.com 36 F. Matyar ed. Phenotypic characteristics, Gram stain, oxidase, cata- lase, motility, OF glucose and gelatine liquefaction were determined for selected isolates according to procedures recommended by Cowan (1974) and Lemos et al. (1985). The carbohidrate fermenters and non-fermenters resistant strains were further characterised by using API 20E and API 20NE test kits (bioMerieux, France), respectively. These strains were identified using the API-WEB identification soft- ware (bioMérieux). A number of isolates (20%) was re- examinated to check test reproducibility. Antibacterial susceptibility testing. Antibiotic suscepti- bility of bacterial strains was determined by an agar disk dif- AK T AM CF C G NA NF S SXT fusion test (Bauer et al., 1966), using Mueller-Hinton agar Antibiotics (Difco) supplemented with 2% NaCI (Merck), as recom- mended by the National Committee for Clinical Laboratory FIG. 1 - Antibacterial resistance of bacteria isolated from intestin- al content ( ) and gills ( ) of cultured Dicentrarchus Standards (NCCLS, 1997). All bacteria isolates were tested labrax. for susceptibility to 10 antimicrobial agents. Disks contain- ing the following antibiotics (Bioanalyse, Istanbul, TURKEY) were used: ampicillin (AM, 10 μg), chloramphenicol (C, 30 μg), tetracycline (T, 30 μg), gentamicin (G, 10 μg), Antimicrobial resistant pathogen bacteria are important cephalothin (CF, 30 μg), streptomycin (S, 10 μg), amikacin problem all over the world. So, there are many researches (AK, 30 μg), nalidixic acid (NA, 30 μg), nitrofurantoin (NF, related with this problem and during the last decade the dis- 100 μg) and trimethoprim-sulphamethoxazole (SXT, 1.25 tribution of many antimicrobial-resistant strains in aquatic and 23.75 μg). The plates were incubated at 35 °C for 18- environments has been the subject of most studies 24 h. Isolates were considered sensitive according to the (Dalsgaard, 1998; Williams et al., 2002; Kim et al., 2004; standards suggested by NCCLS (2002). Harakeh et al., 2006). Reference strains Escherichia coli ATCC 25922 and An important ratio of bacteria both of gills and intestin- Pseudomonas aeruginosa ATCC 27853, recommended by al contents exhibited similar resistance against for three NCCLS (1997), were used as control organisms. antibiotics as 23.5% and 21.8% respectively. On the other hand 36.6% of the bacteria isolated from intestinal contents showed resistance to the four antibiotics and 29.4% of the RESULTS AND DISCUSSION bacteria isolated from gills showed resistance to the seven antibiotics. Although the ratio of the isolates from the gills Antimicrobial resistance patterns showed resistance to even eight antibiotics was 7.8%, no As can be seen from Table 1 viable counts of total and bacteria could show resistance to the eight antibiotics from resistant bacteria from gills were higher than those from intestinal contents (Fig. 2). intestinal contents. It is known that the intestinal tract flora, where the genus Lactobacillus plays an important role, is Bacterial flora considered as the protective system of the organism As shown in Table 2 a high frequency of Pseudomonas puti- (McCartney et al., 1996; Tannock, 1998). Significant differ- da (25.2%), Moellerella wisconsensis (18%), Pseudomonas ences were observed in counts of total bacteria between fluorescens (10.8%) were identified. Moellerella wisconsen- PCA and McC obtained from gills and intestinal contents (p sis is first found in human stool specimens at Wisconsin, < 0.05). Significant differences were observed between the USA (Brenner et al., 1984). This facultative anaerobic frequency of tetracycline-resistant bacteria and chloram- microorganism is associated with human diarrhoea. Also, phenicol-resistant bacteria from gills and intestinal contents strains of Ralstonia pickettii (9%), Vibrio fluvialis (8.1%), on McC (p < 0.05). No amikacin resistant bacteria were Pantoea sp. (7.2%) and Erwinia sp. (5.4%) were found found among the strains isolated (Fig. 1). (Table 2). Pseudomonas putida was recovered intensively TABLE 1 - Viable bacterial concentration present in gills and intestinal contents of cultured sea bass Sources Total viable count Resistant viable count (%) -1 Media (CFU g ) Tetracycline Ampicillin Streptomycin Chloramphenicol -1 -1 -1 -1 (25 μg ml ) (50 μg ml ) (25 μg ml ) (25 μg ml ) Gills Plate Count Agar 3.1 x 10 14.8 11.7 13.0 3.5 McConkey Agar 4.0 x 10 38.3 18.4 24.1 6.8 Intestinal contents Plate Count Agar 2.2 x 10 12.8 10.2 10.1 4.1 McConkey Agar 1.1 x 10 19.1 12.3 20.5 3.2 Numberr of strains Ann. Microbiol., 57 (1), 35-38 (2007) 37 from the gill (96.4%). Second prevalent species M. wiscon- sensis was isolated intensively from the intestinal contents (80%). On the other hand 91.6% of P. fluorescens recov- ered from the gills, whereas 100% of V. fluvialis recovered from intestinal content. Only one strain of E. coli was recov- ered from all samples examined. As shown in Table 2, 39.6% of bacteria belong to Pseudomonas sp. The genus Pseudomonas is widely distributed in nature, clinically sig- nificant and responsible for several food-borne diseases, also some species of this genus are pathogenic for human, animals and plants. Multiple antibiotic resistance index 1 2345678 When gill and intestinal content bacteria were compared; Number of antibiotics all of the gill bacteria showed resistance to the trimetho- prim-sulphamethoxazole (100%), whereas 70% of the FIG. 2 - Antibacterial multiresistance of bacteria isolated from intestinal content ( ) and gills ( ) of cultured intestinal content bacteria showed resistance to the same Dicentrarchus labrax. antibiotic. The ratio of the intestinal content bacteria resist- ant to the tetracycline was found as 85% and of the resist- ant gill bacteria against the cephalothin as 94.1%. Both gill and intestinal content isolates showed a low resistance between 0.3 0.8 for the gill bacteria and 0.2-0.7 for the against to the gentamicin (1.9% and 1.6% respectively). intestinal content bacteria. As can be seen from Fig. 3, the (Fig. 1). It was found that MAR index of gill bacteria was highest MAR index value was observed in P. putida (0.65). higher than intestinal content bacteria. As shown Fig. 2, an Multiresistant bacteria are spreading within the aquacultur- important proportion of intestinal content bacteria were al environments and become a significant cause of fish dis- resistant to the four antibiotics (36.6%) and an important eases. The use of large amounts of antibiotics in aquacul- proportion of gill bacteria showed resistance to the seven ture may also facilitate the growth of multiresistance bac- antibiotics (29.4%). MAR index values were determined teria. So, these antibacterial agents cause some health problems for human and animals by accumulating in fish meat or fish products. Countries with developed and devel- oping aquaculture industries need to collaborate to pass TABLE 2 - Frequency of bacterial isolates from Dicentrarchus laws in order to govern, control and restrict the use of labrax antibiotics. Species Sources Total In present study only a part of the Mediterranean Intestinal contents Gills region was focused on. Further researches need to study antibiotic resistant bacteria, in cultured fish, aquatic envi- Aeromonas hydrophila 11 ronments and sediments. Scope of these studies should Alcaligenes xylosoxidans 11 2 include other parts of Turkish coasts of Mediterranean and Enterobacter aerogenes 22 Aegean Seas and studies should be designed at molecular Enterobacter cloacae 22 level to evaluate the presence of plasmid encoded resist- ant genes. Erwinia sp. 6 6 Escherichia coli 11 Klebsiella planticola 11 Klebsiella pneumoniae 11 0,70 Klebsiella terrigena 11 0,60 Moellerella wisconsensis 16 4 20 0,50 Ochrobactrum anthropi 11 Pantoea sp. 8 8 0,40 Pasteurella pneumotropica 11 0,30 Pseudomonas aeruginosa 11 0,20 Pseudomonas fluorescens 111 12 Pseudomonas putida 127 28 0,10 Pseudomonas stutzeri 22 0,00 Pseudomonas sp. 1 1 Ralstonia pickettii 55 10 Vibrio fluvialis 99 Not identified 1 1 Bacteria Total 60 51 111 FIG. 3 - MAR index values of dominant species. Number of strains MAR index Erwinia sp. M. wisconsensis Pantonea sp. P. fluorescens P. putida R. pickettii V. fluvialis 38 F. Matyar REFERENCES Kim S.R., Nonaka L., Suzuki S. (2004). Occurrence of tetracycline resistant genes tet(M) and tet(S) in bacteria from marine aquaculture sites. FEMS. Microbiol. Lett., 237: 47-156. Alderman D.J., Hastings T.S. (1998). Antibiotic use in aquacul- Lemos M.L., Toranzo, A.E., Barja J.L. (1985). 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Annals of Microbiology – Springer Journals
Published: Nov 20, 2009
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