World J Microbiol Biotechnol DOI 10.1007/s11274-012-1147-6
ORIGINAL PAPER
Comparative study on the antibiotic susceptibility and plasmid profiles of Vibrio alginolyticus strains isolated from four Tunisian marine biotopes Rim Lajnef • Mejdi Snoussi • Jesu´s Lo´pez Romalde Cohen Nozha • Abdennaceur Hassen
•
Received: 10 April 2012 / Accepted: 6 August 2012 Ó Springer Science+Business Media B.V. 2012
Abstract The antibiotic resistance patterns and the plasmids profiles of the predominant etiological agent responsible for vibriosis in Tunisia, V. alginolyticus were studied to contribute to control their spread in some Mediterranean aquaculture farms and seawater. The sixty-nine V. alginolyticus strains isolated from different marine Tunisian biotopes (bathing waters, aquaculture and conchylicole farms and a river connected to the seawater during the cold seasons) were multi-drug resistant with high resistance rate to ampicillin, kanamycin, doxycyclin, erythromycin, imipinem, and nalidixic acid. The multiple resistance index ranged from 0.3 to 0.7 for the isolates of Khenis, from 0.5 to 0.8 for those of Menzel Jmil, from 0.5 to 0.75 (Hergla) and from 0.3 to 0.7 for the isolates of Oued Soltane. The high value of antibiotic resistance index was recorded for the V. alginolyticus population isolated from the fish farm in Hergla (ARI = 0.672) followed by the population isolated from the conchylicole station of Menzel Jmil (ARI = 0.645). The results obtained by the MIC tests confirmed the resistance of the V. alginolyticus to ampicillin, erythromycin, kanamycin, cefotaxime, streptomycin and trimethoprim. Plasmids were found in 79.48 % of the R. Lajnef (&) M. Snoussi A. Hassen Laboratoire de Traitement des Eaux Use´es, Centre de Recherches et des Technologies des Eaux, Technopoˆle de Borj-Ce´dria, BP 901, 2050 Hammam-Lif, Tunisia e-mail:
[email protected] R. Lajnef J. L. Romalde Departamento de Microbiologia y Parasitologia, CIBUS-Facultad de Biologia, Universidad de Santiago, Santiago de Compostela, 15782 Santiago de Compostela, Spain C. Nozha Laboratoire de Microbiologie et d’Hygie`ne des Aliments et de l’Environnement, Institut Pasteur, Casablanca, Morocco
strains analyzed and 30 different plasmid profiles were observed. The strains had a high difference in the size of plasmids varying between 0.5 and 45 kb. Our study reveals that the antibiotic-resistant bacteria are widespread in the aquaculture and conchylicole farm relatively to others strains isolated from seawater. Keywords Vibrio Disk diffusion test Antibiotic resistance Plasmids MICs MBCs MAR index ARI
Introduction Disease outbreaks in marine organisms appear to be escalating worldwide (Harvell et al. 2002) and a growing number of human bacterial infections have been associated with recreational and commercial uses of marine resources (Baffone et al. 2005; Ben Kahla-Nakbi et al. 2007). A surprising number of Vibrio species have been reported from marine environments (Gomez-Le´on et al. 2005; Hidalgo et al. 2008; Balcazar et al. 2010), and the probability of their transmission to humans is correlated with abiotic factors that affect their distribution, especially the temperature of seawater during the summer (Croci et al. 2001; Thompson et al. 2004). In the other hand, Vibrio species have been described as important fish and shellfish pathogens (Woo and Kelly 1995; Nakayama et al. 2006), as in the case of V. harveyi in shrimp (Austin and Zhang 2006) and V. alginolyticus in prawns (Lee et al. 1996) and clams (Gomez-Le´on et al. 2005), accounting for the widespread use of antibiotics in such aquaculture setting (Ferrini et al. 2008). Vibrio alginolyticus is considered as marine fish and shellfish pathogen (Gomez-Le´on et al. 2005). This bacterium is a common inhabitant of the marine environment in both
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temperate and tropical waters (Zanetti et al. 2000) and is associated with high mortality in aquaculture systems through the Tunisian seacoasts causing a several economic losses and high mortality in larvae of many species especially: Sparus aurata and Dicentrarchus labrax (Bakhrouf et al. 1995; Snoussi et al. 2006; Ben Kahla-Nakbi et al. 2007). Vibrio alginolyticus is associated with human infections related to consumption of raw or undercooked sea products (fishes and shellfishes) causing severe gastroenteritis and extra-intestinal diseases (Wounds, intracranial infection in immunocompromised and cirrhotic patients). These illnesses occur frequently during the summer related to an increase in the seawater temperature (Croci et al. 2001; Thompson et al. 2004). This microorganism produces many extracellular proteases responsible for interaction between the bacterium and cell hosts (human and animals) and plays an important role in human infection and fish pathology (Ottaviani et al. 2001; Thompson et al. 2004). The mechanism of pathogenicity induced by Vibrio infections is still complex and related to several factors including cytotoxins, enterotoxins and lytic enzymes (Ottaviani et al. 2001). Antibiotics and other chemotherapeutic agents commonly used in fish farms either as feed additives or immersion baths to achieve either prophylaxis or therapy may result in an increase of drug-resistant bacteria as well as R-plasmids (Son et al. 1997; Saitanu et al. 1994). Marine vibrios have long been recognized as important reservoirs and vehicles of antibiotic resistance because of their importance as potential human/or marine animal pathogens (Thompson et al. 2004), their abundance and diversity in coastal waters, their ability to readily develop and acquire antibiotic resistance in response to selective pressure and their ability to spread resistance by horizontal genetic material exchanges (Aoki 2000). Traditionally, Vibrio is considered highly susceptible to all antimicrobials (Oliver 2006). Tetracycline has been recommended as the antimicrobial of choice to treat severe Vibrio human infections (Morris and Tenney 1985), and alternative treatments are a combination of third-generation cephalosporins (e.g., ceftazidime) and doxycycline, or a Fluoroquinolones alone (Tang et al. 2002). The increase in multi-antibiotics resistance bacteria in recent years is worrisome and the presence of resistance gene in bacteria has further enhanced the transmission and spread of drugs resistance among microbial pathogens. Resistance to antibacterial can, in fact, be reached either with a step wise progression from low to high resistance levels through sequential mutations in chromosomal genes (Wang et al. 2001), or through the acquisition of mobile genetic elements such as bacteriophages, plasmid, naked DNA or transposons (Levy and Marshall 2004), whose transmission between bacteria, even belonging to different taxonomic and
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ecological groups, contributes to the diffusion of antibiotic resistance gene in the environment (Wang et al. 2006). Furthermore, inappropriate use of antibiotics is likely to cause an unnecessary impact on the environment. Therefore, standardisation and safety of drugs used in aquaculture for protection of the environment and human has recently been emphasised (Scholtfeldt 1992). The aim of the present study was to investigate the antibiotic susceptibilities of 69 V. alginolyticus strains isolated from different marine Tunisian biotopes (seawater, aquaculture and conchylicole farms, sediment, river connected to the Mediterranean seawater) using both the disc diffusion assay and the microdilution method. Moreover, the presence of multiple antibiotic resistance of V. alginolyticus in Tunisian biotopes was assessed. In addition, the correlation between antibiotic resistance and presence of plasmids was undertaken.
Materials and methods Sampling sites and strains identification The strains were isolated from four marine biotopes including two fish farms where S. aurata and D. labrax are reared (Khenis and Hergla), from the conchylicole station of Menzel Jmil (M. edulis and C. gigas) and from Oued Soltane which is in connection with Mediterranean seawater during the cold seasons. Seawater samples were filtered through a 0.45 lm membranes, cultured in alkaline peptone water (1 % NaCl, pH 8.6) and incubated at 37 °C for 18–24 h. A loopful of the enrichment culture was streaked onto Thiosulphatecitrate-bile salt-sucrose agar (TCBS, Difco, Spain). Yellow colonies were randomly selected then subcultured on Tryptic soy agar (TSA, Difco, Spain) supplemented with 1 % NaCl. Confirmation of the purity of cultures was obtained for each strain by re-streaking on tryptic soy agar added with 1 % NaCl. The isolated bacteria were frozen at -80 °C with 20 % (v/v) glycerol for further analysis. Seventy-eight Vibrio strains were analyzed in this study including sixty-nine strains of V. alginolyticus, nine reference strains including seven strains of V. alginolyticus (CCM 2575, CCM 2576, CCM 2578T, ATCC 33787, ATCC 17749T, I12, I14), one V. parahaemolyticus type strain (ATCC 43969) and one V. harveyi (CAIM 86). The strains were identified by the following phenotypic tests: cell morphology and motility, Gram staining (KOH method: Fluharty and Packard 1967), oxidase, growth on TCBS, susceptibility to the vibriostatic agent 0/129 (150 lg/disc), production of arginine dihydrolase, lysine and ornithine decarboxylase, glucose fermentation, indole, hydrolysis of gelatin, starch, esculin and Tween 80, reduction of nitrate to nitrite,
World J Microbiol Biotechnol
production of gas from glucose, methyl red, growth at different temperatures (4, 37, 44 °C) and at different salinities (0, 6, 8 and 10 %). These tests were the main assays employed to identify the organisms belonging to Vibrio genus (Thompson et al. 2004). The DNA extraction and molecular identification of V. alginolyticus strains was done according to the protocol described by Di-Pinto et al. (2005) targeting the collagenase gene. Determination of antibiotic susceptibility The antibiotic susceptibility was determined by using the Kirby-Bauer method and Mueller–Hinton agar plates supplemented with 1 % NaCl as described by Ottaviani et al. (2001). Antibiotics tested are as follow: Ampicilline (AMP) 10 lg, Cefotaxime (CTX) 30 lg, Chloramphenicol (C) 30 lg, Fosfomycin (FOS) 200 lg, Gentamycin (CN) 10 lg, Imipenem (IMI) 10 lg, Kanamycin (K) 30 lg, Nalidixic Acid (NA) 30 lg, Norfloxacine (NOR) 10 lg, Streptomycin (S) 10 lg, Sulfamethoxazole (SMX) 50 lg, Trimethoprime (TM) 5 lg, Doxycycline (DXT) 30 lg, Nitrofurantoine (F) 300 lg, Cephalothin (KF) 30 lg, Erythromycin (E) 15 lg, Ticarcilline (TC) 75 lg, Ciprofloxacin (CIP) 5 lg, Co-Trimoxazole Trimethoprime ? Sulfamethoxazole (SXT) 25 lg, Amikacin (AK) 30 lg (Liofilchem s.r.l., Roseto, Italy). After incubation at 37 °C for 18–24 h, the diameter of the inhibition zone was measured with 1 mm flat rule and the diameters were interpreted according to CLSI: Performance Standards for Antimicrobial Disk and Dilution Susceptibility Tests for Bacteria Isolates From animals (2008). For the two antibiotics (Fosfomycin and Doxycycline), results of the diameters of inhibition were interpreted according the diameters indicated by the Liofilchem company. The antibiotic resistance index (ARI) of each bacterial population was determined using the following formula: ARI = y/nx, where y was the actual number of resistance determinants recorded in a population of a size n, and x was the total number of antibacterial tested for in the sensitivity test. Based on the occurrence of resistance to more than three antibiotics the isolates were grouped as multiple antibiotic resistant isolates. The multiple antibiotic resistance (MAR) an d ARI indexes were done as reported by Snoussi et al. (2011) for Vibrio strains. The MAR index was defined as a/b where a represents the number of multiple antibiotics to which the particular isolate is resistant and b as the number of multiple antibiotics to which the particular isolates were exposed. A MAR index value of B0.2 was an indication that the antibiotics were seldom or never been used for animals treatment whereas the MAR index value of [0.2 was considered as an indication that the animals received high exposure to the antibiotics (Sarter et al. 2007).
Minimum inhibitory concentration determination The broth Microdilution method was used to determine the Minimum inhibitory concentration (MIC) and Minimum bactericidal concentration (MBC) of eleven antibiotics. An overnight culture at 37 °C of each strain was diluted tenfold in fresh Mueller–Hinton broth (Biorad, France) supplemented with 1 % NaCl and incubated at 37 °C until they reached exponential phase. Serial twofold dilutions of the tested antibiotics were prepared on 96-wells plate (190 ll per well). Ten microlitres of the inocula (OD600 = 1) were added to each well and the tested antibiotic. In each plate, two wells were reserved to control the sterility of the medium used (no inoculum added) and the viability of the inoculum (no antibiotic added). After 24 h of incubation at 37 °C, bacterial growth was visually evaluated by the presence of turbidity and a pellet on the U-bottom of the 96-wells plate. The MIC value was defined as the lowest concentration of antibiotic that inhibited visible cell growth after 24 h of incubation at 37 °C comparatively to the control well without antibiotic. Minimum bactericidal concentration determination The minimum bactericidal concentration was defined as the lowest concentration of antibiotic able to kill 99 % of bacteria in the well. For this, 10 microtiter of each well medium with no visible growth was plated on MH-1 % NaCl plates and the survived bacteria were estimated after 24 h of incubation at 37 °C. Plasmid profiling Cells were grown on overnight in 3 ml of Luria Broth. The plasmid-DNA extraction was performed as described by the protocol of alkaline exraction method described by Birnboim and Doly (1979) and modified by Sambrook et al. (1989). DNA was electrophoresed on 0.7 % agarose gel. DNA bands were visualized under ultraviolet transillumination and were photographed. V. alginolyticus plasmids sizes were estimated by comparaison with Lambda-DNA-HindIII Marker (Promega, Madison, WI, USA). The analyses were repeated three times.
Result and discussion Biochemical and molecular identification of the isolated strains Sixty-nine V. alginolyticus strains isolated from four marine biotopes including two fish farms where S. aurata and D. labrax are reared (Khenis and Hergla), conchylicole
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Fig. 1 Map of Tunisia showing the different sites of study and the number of V. alginolyticus strains isolated from each sample (seawater, fish and shellfish samples)
station (M. edulis and C. gigas) and from a river ‘‘Soltane’’ which is in connection with Mediterranean seawaters during the cold seasons (Fig. 1). Biochemical and phenotypic identification were based on many specific traits (Table 1). Yellow colonies isolated from TCBS agar were identified as Gram-negative motile fermentative rods, producing enzymes like catalase and oxidase, susceptible to vibriostatic compounds O/129 (150 lg/disk) and swarming colonies on TSA 1 % NaCl at different temperature: 4, 37, 44 °C. Most strains (67/69) were Voges-Proskauer and lysine decarboxylase positive. Only six strains were ornithine decarboxylase positive. However, all strains were negative for arginine dihydrolase. All, the strains grew in peptone water prepared respectively with 3, 8 and 10 % of NaCl (Table 1). All strains tested amplify a 737-pb size fragment showing the characteristic profile of V. alginolyticus (Di-Pinto et al. 2005).
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Antibiotic susceptibility The antibiotic susceptibility of V. alginolyticus strains showed wide resistance to previously tested antibiotics (Fig. 2). In fact, the results showed that most strains (59/ 69; 85.5 %) were resistant to at least six antimicrobials agents (Table 2). Fifteen strains isolated from the seawater in the region of Khenis (strains: 57, 244, k11, k5, 213), from the mussels in Menzel Jmil (strains: A16, A19, A30, A29), from fish in Hergla (strains: S50, S38) and from the Oued sultan in Borj-Cedria (strains: H1, H18, H20) were resistant to all antibiotic tested in this study. The strains tested were resistant to ampicillin (94.2 %), erythromycin (85.5 %), kanamycin (84 %), gentamycin (76.8 %) and cefotaxim (75.3 %). The lowest percentages of resistance were noted for trimethoprim-sulfamethoxazole (20.2 %), ciprofloxacin (26 %), and nalidixic acid (31.8 %).
Seawater (Khenis)
Seawater (Khenis)
Seawater (Khenis)
Seawater (Khenis)
Seawater (Khenis)
225
P8
226
234
Juvenile sea Bream (Khenis farm)
58
P7
Juvenile sea Bream (Khenis farm)
223
Seawater (Khenis)
Sea water Khenis
213
K9
Juvenile sea Bream (Khenis farm)
56
Seawater (Khenis)
Juvenile sea Bream (Khenis farm)
57
K11
Gills of S. aurata (Khenis farm)
118
Seawater (Khenis)
38
Blood of S. aurata (Khenis farm)
126
Seawater (Khenis)
Kidney of S. aurata (Khenis farm)
112
36
Origin
Strains
-
-
-
-
-
-
-
-
-
-
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-
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-
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-
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O/129 (150 lg/ disk)
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4 °C
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37 °C
Growth at
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44 °C
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VP
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?
?
LDC
-
-
-
-
-
-
-
-
-
-
?
-
-
-
-
-
-
ADH
?
?
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GF
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Indole
-
-
-
-
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?
Gelatin
Table 1 Biochemical characteristics of the 78 V. alginolyticus strains tested isolated on TCBS agar medium
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-
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Starch
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-
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-
-
-
-
-
?
Esculin
-
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-
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-
-
-
?
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-
-
-
-
-
T80
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Nitrate
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-
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-
-
Gas
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0%
NaCl
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3%
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8%
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10 %
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Seawater (Khenis)
Seawater (Khenis)
Seawater (Khenis)
Seawater (Khenis)
Seawater (Khenis)
Seawater (Khenis)
Seawater (Khenis)
Seawater (Khenis)
Seawater (Khenis)
Sea water Menzel Jmil (MJ)
Sea water Menzel Jmil (MJ)
Sea water Menzel Jmil (MJ)
Sea water Menzel Jmil (MJ)
M. edulis (conchylicole farm MJ)
M. edulis (conchylicole farm MJ)
M. edulis (conchylicole farm MJ)
M. edulis (conchylicole farm MJ)
K8
K6
K5
244
241
K1
EM2
EM3
K3
A3
A6
A13
A12
A34
A16
A19
A23
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O/129 (150 lg/ disk)
Origin
Strains
Table 1 continued
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37 °C
Growth at
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44 °C
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VP
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-
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LDC
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ADH
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GF
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Indole
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Gelatin
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Starch
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-
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Esculin
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-
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T80
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Nitrate
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Gas
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0%
NaCl
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3%
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8%
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10 %
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M. edulis (conchylicole farm MJ)
M. edulis (conchylicole farm MJ)
M. edulis (conchylicole farm MJ)
M. edulis (conchylicole farm MJ)
M. edulis (conchylicole farm MJ)
M. edulis (conchylicole farm MJ)
C. gigas (conchylicole farm MJ)
C. gigas (conchylicole farm MJ) C. gigas (conchylicole farm MJ)
C. gigas (conchylicole farm MJ)
D. labrax (fish farm of Hergla)
D. labrax (fish farm of Hergla)
A40
A41
A38
A26
A29
A28
A33
A30
A37
S38
S50
A27
?
M. edulis (conchylicole farm MJ)
A24
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-
-
-
-
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-
M. edulis (conchylicole farm MJ)
A25
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4 °C
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37 °C
Growth at
O/129 (150 lg/ disk)
Origin
Strains
Table 1 continued
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44 °C
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VP
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LDC
-
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ADH
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GF
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Indole
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-
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Gelatin
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Starch
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Esculin
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T80
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Nitrate
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Gas
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0%
NaCl
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3%
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8%
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10 %
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Liver of D. labrax (fish farm of Hergla)
Kidney of D. labrax (fish farm of Hergla)
D. labrax (fish farm of Hergla)
D. labrax (fish farm of Hergla)
S. aurata (fish farm of Hergla)
D. labrax (fish farm of Hergla)
S. aurata (fish farm of Hergla)
S. aurata (fish farm of Hergla)
Sediment of Oued Soltane
Sediment of Oued Soltane
Sediment of Oued Soltane
Sediment of Oued Soltane
Sediment of Oued Soltane
Sediment of Oued Soltane
Sediment of Oued Soltane
Sediment of Oued Soltane
Sediment of Oued Soltane
Sediment of Oued Soltane
S37
S49
S55
S57
S32
S56
SL1
S1K
H1
H3
H4
H6
H8
H9
H10
H11
H12
H18
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
O/129 (150 lg/ disk)
Origin
Strains
Table 1 continued
?
?
?
?
?
?
?
?
?
?
?
?
?
?
?
?
?
?
4 °C
?
?
?
?
?
?
?
?
?
?
?
?
?
?
?
?
?
?
37 °C
Growth at
?
?
?
?
?
?
?
?
?
?
?
?
?
?
?
?
?
?
44 °C
?
-
?
?
?
?
?
?
?
?
?
?
?
?
?
?
?
?
VP
?
?
?
?
-
?
?
?
?
?
?
?
?
?
?
?
?
?
LDC
-
?
-
-
-
-
?
-
-
-
-
-
-
-
-
-
-
-
ADH
?
?
?
?
?
?
?
?
?
?
?
?
?
?
?
?
?
?
GF
?
?
?
?
?
?
?
?
?
?
?
?
?
?
?
?
?
?
Indole
?
?
?
?
?
?
?
?
?
?
?
?
?
?
?
?
?
?
Gelatin
?
-
-
-
?
-
-
-
-
?
?
-
-
?
-
?
-
?
Starch
-
-
-
?
?
-
-
?
-
?
-
?
-
?
-
?
-
?
Esculin
-
-
-
-
?
?
-
-
?
-
?
-
?
-
?
-
?
-
T80
?
?
?
?
?
?
?
?
?
?
?
?
?
?
?
?
?
?
Nitrate
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
Gas
-
-
-
-
-
-
-
-
-
-
-
-
-
-
?
?
?
?
0%
NaCl
?
?
?
?
?
?
?
?
?
?
?
?
?
?
?
?
?
?
3%
?
?
?
?
?
?
?
?
?
?
?
?
?
?
?
?
?
?
8%
?
?
?
?
?
?
?
?
?
?
?
?
?
?
?
?
?
?
10 %
?
?
?
?
?
?
?
?
?
?
?
?
?
?
?
?
?
?
(737 pb)
World J Microbiol Biotechnol
-
Sediment of Oued Soltane
Sediment of Oued Soltane
Sediment of Oued Soltane
Type strain
Type strain
Type strain
Type strain Type strain
Type strain
Type strain
Italy
Italy
H20
H21
H22
ATCC 17749
ATCC 33787
ATCC 43969
CAIM 86 CCM2575
CCM2576
CCM2578
I12
I14 ?
?
?
?
? ?
?
?
?
?
?
?
4 °C
?
?
?
?
? ?
?
?
?
?
?
?
37 °C
Growth at
?
?
?
?
? ?
?
?
?
?
?
?
44 °C
?
?
?
?
? ?
?
?
?
?
?
?
VP
?
?
?
?
? ?
?
?
?
?
?
?
LDC
-
-
-
-
-
-
-
-
-
-
-
ADH
?
?
?
?
? ?
?
?
?
?
?
?
GF
?
?
?
?
? ?
?
?
?
?
?
?
Indole
?
?
-
?
? ?
?
?
-
?
?
?
Gelatin
-
?
?
?
-
?
-
?
-
?
?
Starch
?
-
?
-
? ?
?
-
-
-
?
-
Esculin
?
?
-
?
? -
?
?
-
-
-
?
T80
?
?
?
?
? ?
?
?
?
?
?
?
Nitrate
-
-
-
-
-
-
?
-
-
-
-
Gas
-
-
-
-
?
-
-
-
-
-
-
0%
NaCl
?
?
?
?
? ?
?
?
?
?
?
?
3%
?
?
?
?
? ?
?
?
?
?
?
?
8%
?
?
?
?
? ?
?
?
?
?
?
?
10 %
?
?
?
?
? ?
?
?
?
?
?
?
(737 pb)
VP Voges-Proskauer, LDC Lysine Decarboxylase, ADH Arginine Dihydrolase, GF glucose fermentation, T80 Tween 80, Gaz Gas production from Kligler-Hajna, Nitrate Nitrate reduction
-
-
-
-
-
-
-
-
-
-
O/129 (150 lg/ disk)
Origin
Strains
Table 1 continued
World J Microbiol Biotechnol
123
World J Microbiol Biotechnol
Fig. 2 Percentage of resistance to 20 antibiotics of the 78 V. alginolyticus strains isolated from different Tunisian marine biotopes. Antibiotics tested are as follow: AMP Ampicillin (10 lg), CTX Cefotaxim (30 lg), C Chloramphenicol (30 lg), FOS Fosfomycin (200 lg), CN Gentamycin (10 lg), IMI Imipenem (10 lg), K Kanamycin (30 lg), NA Nalidixic Acid (30 lg), NOR Norfloxacin
(10 lg), S Streptomycin (10 lg), SMX Sulfamethoxazole (50 lg), TM Trimethoprim (5 lg), DXT Doxycyclin (30 lg), F Nitrofurantoin (300 lg), KF Cephalothin (30 lg), E Erythromycin (15 lg), TC Ticarcillin (75 lg), CIP Ciprofloxacin (5 lg), SXT Co-Trimoxazole Trimethoprime ? Sulfamethoxazole (25 lg), AK Amikacin (30 lg)
Among the 69 strains isolated from the four marine biotopes tested in our study, the 30 strains isolated from aquaculture and conchylicole farms presented a high resistance to erythromycin (60.8 %), kanamycin (60.8 %), cephalotin (57.9 %), ampicillin (56.5 %), and gentamycin (50.7 %). Similarly, other researches report that V. alginolyticus, V. parahaemolyticus and other Vibrio strains present a high resistance to ampicillin (82–85 %) confirming that this antibiotic is the most common resistance (Vaseeharan et al. 2005). Ferrini et al. (2008) who studied the resistance of 46 V. alginolyticus strains isolated from imported seafood and Italian aquaculture settings founded that these strains present a high resistance to ampicillin (93 %). In this study, 85.5 % of isolates were resistant to erythromycin, gentamicin (76.8 %), and to cefotaxim (75.3 %). These results are in accordance with those reported by Snoussi et al. (2008) who founded high rates of resistance to erythromycin (88 %), gentamicin (84 %) and cefotaxim (86 %) of 43 V. alginolyticus strains isolated from diseased juveniles and older fish of S. aurata reared in a marine hatchery installed along the seacoasts of Monastir (Center of Tunisia). Additionally, more than 70 % of isolates showed a susceptibility to trimethoprim-sulfamethoxazole and more than 60 % to nalidixic acid and ciprofloxacin. Therefore, ciprofloxacin and trimethoprimsulfamethoxazole could be effective to control V. alginolyticus. The present findings are similar to those reported by Ottaviani et al. (2001) who reported that more than 90 % of Vibrio spp. isolates showed a susceptibility to trimethoprim-sulfamethoxazole and more than 80 % to nalidixic acid and ciprofloxacin. According to results, 31.88 % of tested V. alginolyticus strains presented a resistance to chloramphenicol and 49.28 % are resistant to imipenem. Whereas Ottaviani et al. (2001) founded that all ‘non-cholera vibrios’ (NCVs) isolated from seafood were susceptible to imipenem, meropenem and chloramphenicol. Indeed, we can notice
also that the strains isolated from aquaculture and conchylicole farm are more resistant relatively to other isolated from seawater, river and sediment. These findings were in accordance with those reported by Vaseeharan et al. (2005) who concluded that the occurrence of antibiotic-resistant bacteria were more common in aquaculture systems because of the intensive use of antibiotics, also the increasing in multi-antibiotics resistance bacteria in recent years is worrisome and the presence of resistance gene in bacteria has further aided the transmission and spread of drugs resistance among microbial pathogens (Zulkifli et al. 2009). The MAR (Multiple Antibiotic Resistance) index was also calculated in this study for all the strains, this index ranged from 0.3 to 0.7 for the isolates of Khenis and for the isolates of Oued Soltane, from 0.5 to 0.8 for those of Menzel Jmil and from 0.5 to 0.75 for the isolates of the aquaculture farm of Hergla. The high value of antibiotic resistance index was recorded for the V. alginolyticus population isolated from the fish farm in Hergla (ARI = 0.672) followed by the population isolated from the conchylicole station of Menzel Jmil (ARI = 0.645). Wei et al. (2011) indicated that the MAR index value of isolates from cultured freshwater fish was 0.43, where it was much higher than 0.2. Overall, the MAR index value of all present isolates was 0.29. The MAR index in their study indicates that cultured freshwater fish, namely African Catfish and Red Hybrid Tilapia, in Terengganu are under high-risk exposed-antibiotic sources. It is also important to mention that the isolates from the four biotopes (Khenis seawater, conchylicole farm of Menzel Jmil, aquaculture farm of Hergla, sediments of Oued Soltan) showed a MAR index value higher than 0.2. These results indicate that all the isolates are under high-risk exposedantibiotics. In fact the isolates from conchylicole farm (Menzel Jmil) and aquaculture farm (Hergla) present the higher value which confirm the overuse of antimicrobials in the aquaculture settings. These results are in accordance
123
World J Microbiol Biotechnol Table 2 Correlation between resistance patterns and plasmid profiles Strains
Resistance patterns
112
R9: AMP-FOS-CN-S-TM-DXT-E-CIP
Plasmid free
126
R10: AMP-CTX-IMI-K-NOR-DXT-F-E-TC-CIP
P11: 2.3
36
R11: AMP-FOS-CN-K-NA-SMX-F-KF-TC-CIP
P1: 1.5
R11: AMP-FOS-CN-K-NA-SMX-F-KF-TC-CIP
P1: 1.5
R12: AMP-FOS-CN-K-S-TM-DXT-E-CIP
P11: 2.3
38 118
Plasmid profiles
57
R13: AMP-CTX-C-NA-S-TM-DXT-F-TC-CIP-SXT
P18: 9.4; 3; 2.3
56
R14: AMP-IMI-K-NOR-DXT-KF-SXT
P10: 1; 0.8; 0.6; 0.5
213
R15: AMP-CTX-FOS-K-NOR-S-DXT-F-E-SXT-AK
P3: 45; 1.5
223
R16: AMP-FOS-K-S-SMX-TC-CIP-AK
P18: 9.4; 3; 2.3
R17: AMP-C-K-NA-NOR-DXT-F-E-CIP-AK
P13: 9.4
R18:AMP-CTX-C-CN-IMI-S-DXT-F-E-TC-CIP-AK R18:AMP-CTX-C-CN-IMI-S-DXT-F-E-TC-CIP-AK
P20: 23.2; 3 P4: 4.3; 1.5
58 K11 K9 P7
R19: AMP-CTX-C-IMI-K-S-SMX-DXT-KF-E-TC-CIP
P8: 0.8; 0.6; 0.5
225
R20: AMP-K-NA-NOR-S-DXT-TC-CIP-AK
P12: 6.5
P8
R21: AMP-CTX-K-NA-DXT-F-TC-AK
P19: 9.4; 3
226
R15: AMP-CTX-FOS-K-NOR-S-DXT-F-E-SXT-AK
P3: 45; 1.5
234
R11: AMP-FOS-CN-K-NA-SMX-F-KF-TC-CIP
P1: 1.5
K8
R22: AMP-CTX-C-FOS-CN-K-TM-DXT-KF-CIP-AK
P19: 9.4; 3
K6
R23: AMP-CTX-C-FOS-CN-IMI-K-S-SMX-DXT
Plasmid free Plasmid free
K5
R23: AMP-CTX-C-FOS-CN-IMI-K-S-SMX-DXT
244
R24: AMP-CTX-K-NA-K-S-TM-DXT-F-KF-E-TC-CIP
P12: 6.5
241
R22: AMP-CTX-C-FOS-CN-K-TM-DXT-KF-CIP-AK
Plasmid free
K1
R24: AMP-CTX-K-NA-K-S-TM-DXT-F-KF-E-TC-CIP
P6: 23.2; 2.3
EM2
R25: AMP-CTX-CN-K-S-SMX-TC
P21: 9.4; 2.3
EM3
R25: AMP-CTX-CN-K-S-SMX-TC
P21: 9.4; 2.3
K3
R26: AMP-CTX-C-FOS-K-NOR-TM-DXT-E-TC
P7: 0.8; 0.6
A3 A6
R27: AMP-CN-IMI-K-TM-F-E-TC-AK R28: AMP-CTX-FOS-IMI-K-KF-E-TC
Plasmid free P2: 45
A13
R28: AMP-CTX-FOS-IMI-K-KF-E-TC
P2: 45
A12
R29: AMP-CTX-FOS-IMI-K-NA-SMX-TM-DXT-TC-CIP
P5: 23.2
A34
R29: AMP-CTX-FOS-IMI-K-NA-SMX-TM-DXT-TC-CIP
P5: 23.2
A16
R30: CTX-CN-IMI-K-NA-DXT-F-KF-E-TC-CIP
Plasmid free
A19
R30: CTX-CN-IMI-K-NA-DXT-F-KF-E-TC-CIP
P29: 45; 15
A23
R31: AMP-FOS-IMI-K-TM-DXT-F-KF-E-TC-CIP-SXT
P23: 23.2; 9.4; 2.3
A25
R31: AMP-FOS-IMI-K-TM-DXT-F-KF-E-TC-CIP-SXT
P5: 23.2
A24
R32: AMP-CTX-C-FOS-CN-IMI-K-NA-SMX-TM-F-KF-E-TC-AK
P2: 45
A40
R33: AMP-FOS-IMI-K-NA-S-DXT-KF-E-TC
Plasmid free
A41
R34: AMP-CN-IMI-K-S-SMX-TM-DXT-F-E-TC-CIP-SXT
P5: 23.2
A38
R35: AMP-CN-IMI-K-NA-S-TM-DXT-F-E-TC-AK
Plasmid free
A26
R36: AMP-FOS-CN-IMI-K-TM-DXT-F-KF-E-TC-AK
P29: 45; 15
A29
R36: AMP-FOS-CN-IMI-K-TM-DXT-F-KF-E-TC-AK
P28: 15
A28 A33
R37: C-FOS-IMI-K-S-SMX-TM-KF-E-SXT R38: AMP-FOS-IMI-K-NA-TM-DXT-F-KF-E-TC-CIP-AK
Plasmid free P28: 15
A30
R39:CN-IMI-K-NA-S-SMX-F-TC- CIP-AK
Plasmid free
A27
R40: AMP-C-IMI-K-NA-S-SMX-TM-F-E-TC-CIP-SXT
Plasmid free
A37
R41: AMP-CTX-CN-IMI-K-DXT-F-KF-E-TC-CIP
P22: 45; 30; 23; 6.5; 1.5
S38
R42: AMP-C-FOS-IMI-K-NA-TM-DXT-F-KF-E-TC-CIP-SXT
P6: 23.2; 2.3
S50
R42: AMP-C-FOS-IMI-K-NA-TM-DXT-F-KF-E-TC-CIP-SXT
P6: 23.2; 2.3
123
World J Microbiol Biotechnol Table 2 continued Strains
Resistance patterns
Plasmid profiles
S37
R42: AMP-C-FOS-IMI-K-NA-TM-DXT-F-KF-E-TC-CIP-SXT
P13: 9.4
S49
R42: AMP-C-FOS-IMI-K-NA-TM-DXT-F-KF-E-TC-CIP-SXT
P13: 9.4
S55
R43: AMP-FOS-IMI-K- S-DXT-KF-E-TC
Plasmid free
S57
R44: AMP-CTX-FOS-CN-IMI-K-NA-SMX-TM-DXT-K-KF-E-TC-CIP
P26: 3
S32
R41: AMP-CTX-CN-IMI-K-DXT-F-KF-E-TC-CIP
P22: 45; 30; 23; 6.5; 1.5
S56
R43: AMP-FOS-IMI-K-S-DXT-KF-E-TC
Plasmid free
SL1
R9: AMP-FOS-CN-S-TM-DXT-E-CIP
Plasmid free
S1K
R9: AMP-FOS-CN-S-TM-DXT-E-CIP
Plasmid free
H1
R45: AMP-CTX-K-DXT-F-TC-AK
Plasmid free
H3
R46: AMP-CTX-FOS-CN-NA-TM-F
P6: 23.2; 2.3
H4
R47: AMP-FOS-CN-K-NOR-S-DXT-E
P9: 1; 0.8; 0.6
H6
R47: AMP-FOS-CN-K-NOR-S-DXT-E
P9: 1; 0.8; 0.6
H8 H9
R48: AMP-FOS-NA-F-E-TC- CIP-AK R49: AMP-CTX-C-IMI-K-S-SMX-F-E-TC
P11: 2.3 P12: 6.5
H10
R50: AMP-CTX-FOS-NOR-S-F-E-AK
P11: 2.3
H11
R51: AMP-FOS-K-S-SMX-TC-CIP-AK
P25: 4.3; 2.3
H12
R51: AMP-FOS-K-S-SMX-TC-CIP-AK
P25: 4.3; 2.3
H18
R52:AMP-K-NA-NOR-S-DXT-TC-CIP-AK
P24: 4.3
H20
R22: AMP-CTX-C-FOS-CN-K-TM-DXT-KF-CIP-AK
P24: 4.3
H21
R22: AMP-CTX-C-FOS-CN-K-TM-DXT-KF-CIP-AK
P24: 4.3
H22
R22: AMP-CTX-C-FOS-CN-K-TM-DXT-KF-CIP-AK
P24: 4.3
ATCC 17749
R4: AMP-FOS-K-S-SMX-TC-CIP
P13: 9.4
ATCC 33787
R5: AMP-K-NA-NOR-S-DXT-TC-AK
P14: 6.5; 5.7; 4.3
ATCC 43969
R7: AMP-CTX-C-FOS-CN-IMI-TM-DXT-E-TC- AK
P27: 3; 2.3; 2; 1.5
CAIM 86
R3: AMP-CTX-FOS-K-NA-NOR-S-TM-DXT-F-E-TC-CIP
P30: 30; 23.2; 15; 3; 2.3; 1.5
CCM 2575
R1: AMP-FOS-CN-K-S-TM-DXT-E-CIP
P16: 9.4; 2.3
CCM 2576
R1: AMP-FOS-CN-K-S-TM-DXT-E-CIP
P16: 9.4; 2.3
CCM 2578 I12
R2: AMP-FOS-NA-S-SMX-F-E-TC R6: AMP-CTX-C-FOS-CN-K-NOR-TM-DXT-CIP-AK
P16: 9.4; 2.3 P17: 23.2; 9.4
I14
R6: AMP-CTX-C-FOS-CN-K-NOR-TM-DXT-CIP-AK
P15: 6.5; 2.3
AMP Ampicillin (10 lg), CTX Cefotaxim (30 lg), C Chloramphenicol (30 lg), FOS Fosfomycin (200 lg), CN Gentamycin (10 lg), IMI Imipenem (10 lg), K Kanamycin (30 lg), NA Nalidixic Acid (30 lg), NOR Norfloxacin (10 lg), S Streptomycin (10 lg), SMX Sulfamethoxazole (50 lg), TM Trimethoprime (5 lg), DXT Doxycycline (30 lg), F Nitrofurantoine (300 lg), KF Cephalothin (30 lg), E Erythromycin (15 lg), TC Ticarcillin (75 lg), CIP Ciprofloxacin (5 lg), SXT Co-Trimoxazole Trimethoprime ? Sulfamethoxazole (25 lg), AK Amikacin (30 lg)
with those of Manjusha et al. (2005) who reported that strains isolated from various tissue samples collected from site of highest antibiotic resistance emphasizes the fact that antibiotic resistance in fish and tissue samples augment at alarming levels as compared to water samples. According to Mukherji et al. (2000), V. alginolyticus has been etiologically associated with cellulitis and acute otitis media or externa. As a whole, these infections have responded well to appropriate antibiotics. Seven Korean cases of V. alginolyticus infection have previously been reported. Four cases were related with otitis media (Doh et al. 1997) or myringitis (Lee and Choi 1995) two with soft tissue infection (Cho et al. 1995) and the last one was related with gastroenteritis (Kim et al. 2000).
123
Lee et al. (2008) present a case of septic shock due to V. alginolyticus in a patient in whom the clinical presentation did not suggest the presence of this organism. He was treated with the appropriate antibiotics, but his late visit to the hospital and failure to achieve surgical debridement may have caused his death. V. alginolyticus was susceptible to a variety of antibiotics including ampicillin, amoxillin-clavulanate, cephalothin, cefuroxime, gentamicin, ciprofloxacin, and trimethoprim sulfamethoxazole. Immunocompromised hosts should be careful about eating raw fish, especially during the warm seasons. At present, thorough cooking of seafood is the only effective means of prevention. Early administration of antibiotics and surgical intervention, if needed, is critical for controlling these invasive vibrios infections.
World J Microbiol Biotechnol
MICs of antibiotics The results of the MICs and MBCs values tested using test 11 antimicrobials agents are listed in (Table 3). The results obtained in this study are in accordance with those reported by Ferrini et al. (2008) who found that MBC of ampicillin, streptomycin, kanamycin, and ciprofloxacin are respectively (C256, C128, 128 and 1 mg/l). In contrast, we noted some differences between MIC90 reported by these Authors for some antimicrobials such as tetracycline, chloramphenicol, oxytetracycline and doxycycline. We can notice that the results of the MIC values confirmed the resistance of the V. alginolyticus tested to ampicillin, to erythromycin, to kanamycin, to cefotaxime, to streptomycin, to trimethoprim. According to the results founded in the present work, there is no difference in the MIC and MBC values of ampicillin: 256 and [256 mg/l, respectively, which are in accordance with those reported by Zanetti et al. (2001), but a significant difference was observed between our MBC for cefotaxime and doxycycline which were higher (64 and 8 mg/l respectively) than those reported by Zanetti et al. (2001) in V. alginolyticus strains (0.12 and 0.25 mg/l, respectively). Furthermore, the MIC range of oxytetracycline to control V. alginolyticus was 0.12–1 mg/l, this range is lower than that found in the study of Vaseeharan et al. (2005) (22.8–33.5 mg/l). Roque et al. (2001), founded that the MIC value of oxytetracycline was 301.0 mg/l which is an extremely high value compared with other reported results. This worldwide resistance to oxytetracycline of Vibrio isolates from shrimp is probably due to its frequent use; resistance is plasmid mediated and inducible, allowing horizontal transfer of resistance (Towner. 1995). Whereas, in the present study the majority of strains were sensitive to the oxytetracycline and the MIC range of this antibiotic was 0.12–1 mg/l. In 2005, Vaseeharan and colleagues founded the MIC range of ciprofloxacin of 0.32–0.43 mg/l was able to control effectively the Vibrio and Aeromonas species. This result is in accordance with that reported by Zanetti et al. (2001) who reported that the MIC of ciprofloxacin to control Vibrio spp. isolated from the environment was 0.38 mg/l. Our result of the MIC of ciprofloxacin (\0.06–1) was near to those founded in previous studies (Zanetti et al. 2001; Vaseeharan et al. 2005) which confirm that the ciprofloxacin is the most active of quinolones and could be effective in the case of environmental V. alginolyticus. Additionally, we founded that the majority of strains were sensitive to the oxytetracycline and the MIC range of this antibiotic: 0.12–1 mg/l. In a recent study on Vibrio spp. and Photobacterium damsela ssp. picicida isolated from Italian aquaculture farms (fish, shellfish and crustaceans), Lagana` et al. (2011)
founded that the tested strains were resistant to ampicillin, carbenicillin, cephalotin, kanamycin, while they were sensitive to chloramphenicol, nitrofurantoin and to tobramycin. As a conclusion, the results obtained by the MIC tests confirmed the resistance of the V. alginolyticus tested to ampicillin (256 and [256 mg/l), erythromycin (64 and [128 mg/l), kanamycin ([32 and 128), cefotaxime (1 and 2 mg/l), streptomycin (64 and [128 mg/l) and trimethoprim (16 and 32 mg/l). Plasmid profiling The plasmids profiles in vibrios have been studied in some species such as Vibrio ordalii (Tianen et al. 1995), Vibrio vulnificus (Radu et al. 1998) and Vibrio salmonicida (Sorum et al. 1990), and most extensively in V. anguillarum (Pedersen 1999) where a high diversity of profiles was observed (Pedersen et al. 1996). Plasmid profiling has proven to be useful proven to differentiate between V. salmonicida, but their discriminatory power has also been questioned (Pedersen 1999). Our result (Table 2) showed 30 plasmid profiles among them 12 profiles with the same resistance patterns. Additionally, sixty-two V. alginolyticus strains (79.5 %) harbored one to six plasmids with molecular sizes ranging from 0.5 to 45 kb. According to plasmid content, strains were classified into 30 clusters. We showed the presence of fifteen plasmid with different sizes and only 16 strains among 78 tested were plasmideless. Zanetti et al. (2001) showed that only 24 of 48 strains resistant to ampicillin harbored plasmids. The molecular weight of these plasmids ranged from 1.5 to 26 kb. There are also strains which had the same resistance patterns but different plasmids profiles. Zorilla et al. (2003) revealed the presence of twelve different sized plasmids which were detected in V. alginolyticus strains, originating eight different plasmid patterns. A high percentage of the isolates tested (53 %) did not carry plasmids and among the strains harboured plasmids, eight different plasmid profiles were recorded. The plasmid content of V. alginolyticus was heterogeneous in number and size, varying from five to one, and lower than 2.1–53.78 kb. The same authors showed that some V. alginolyticus harbored the plasmid responsible for virulence, and others strains possessed plasmid which had no relationship with pathogenicity and this plasmid was supposed to be responsible to antibiotic-resistance. The results obtained showed that among 78 strains, 62 had one or more plasmids and there is a high difference in the size of this plasmids variance between 0.5 and 45 kb. These results are in accordance with those of Molina-Aja et al. (2002) who found 24 strains (80 %) among 30 strains with one or more plasmid and showed that there is a high
123
World J Microbiol Biotechnol Table 3 Distribution of MICs and MBCs values in the V. alginolyticus strains tested against eleven antibiotics Strains
Antibiotics tested 1 MIC
2 MBC
3
MIC
MBC
CMI
4 CMB
MIC
5 MBC
MIC
6 MBC
MIC
MBC
112
256
512
32
128
64
128
0.48
2
0.48
2
0.96
2
126
256
[512
32
128
64
128
0.48
[2
0.48
[2
0.96
[2 [2
36
256
[512
32
128
64
[128
0.48
[2
0.48
[2
0.96
38
256
512
32
128
64
[128
0.48
2
0.48
2
0.96
2
118
256
512
32
128
64
[128
0.48
[2
0.48
[2
0.96
[2
57
256
512
32
128
64
[128
0.48
2
0.48
2
0.96
2
56 213
128 128
[512 [512
64 64
128 128
32 64
[128 [128
0.48 0.48
2 2
0.48 0.48
2 2
0.96 0.96
2 2
223
128
[512
64
128
32
[128
0.48
2
0.48
2
0.96
2
58
256
[512
64
128
32
[128
0.48
2
0.48
2
0.96
2
K11
128
[512
64
128
16
[128
0.48
2
0.48
2
0.96
2
K9
256
[512
64
128
64
[128
0.48
2
0.48
2
0.96
2
P7
256
[512
64
128
32
[128
0.48
2
0.48
2
0.96
2
225
128
[512
32
128
32
128
0.48
[2
0.48
[2
0.96
[2
P8
128
512
64
128
64
[128
0.48
[2
0.48
[2
0.96
[2
226
256
512
64
128
64
[128
0.48
[2
0.48
[2
0.96
[2
234
256
[512
64
128
64
[128
0.48
2
0.48
2
0.96
2
K8
256
[512
64
128
32
[128
0.48
[2
0.48
[2
0.96
[2
K6
128
[512
64
128
64
[128
0.48
2
0.48
[2
0.96
[2
K5
256
512
64
128
64
128
0.48
2
0.48
[2
0.96
[2
244
128
512
64
128
64
128
0.48
2
0.48
2
0.96
2
241 EM3
256 256
[512 512
64 64
128 128
32 64
[128 128
0.48 0.96
[2 [2
0.48 0.96
[2 [2
0.96 0.96
[2 [2
K1
128
512
32
128
64
128
0.48
2
0.48
2
0.48
2
EM2
128
[512
64
128
64
[128
0.48
2
0.48
[2
0.48
[2
K3
256
512
64
128
64
128
0.96
[2
0.96
[2
0.96
[2
A3
256
[512
64
128
64
128
0.48
[2
0.48
[2
0.48
[2
A6
128
512
32
128
32
128
0.48
2
0.48
2
0.48
2
A13
128
512
32
128
64
128
0.48
2
0.48
2
0.48
2
A12
256
[512
32
128
64
[128
0.48
2
0.48
2
0.96
2
A34
256
[512
32
128
64
[128
0.48
[2
0.48
[2
0.96
[2
A16
256
[512
32
128
64
[128
0.48
[2
0.48
[2
0.96
[2
A19
256
512
32
128
64
[128
0.48
2
0.48
2
0.96
2
A23
256
[512
32
128
64
[128
0.48
[2
0.48
[2
0.96
[2
A25
256
[512
32
128
64
[128
0.48
2
0.48
2
0.96
2
A24
128
[512
64
128
32
[128
0.48
2
0.48
2
0.96
2
A40 A41
128 256
[512 [512
64 64
128 128
64 32
[128 [128
0.48 0.48
2 2
0.48 0.48
2 2
0.96 0.96
2 2
A38
128
[512
64
128
32
[128
0.48
2
0.48
2
0.96
2
A26
256
[512
64
128
16
[128
0.48
2
0.48
2
0.96
2
A29
128
[512
64
128
64
[128
0.48
2
0.48
2
0.96
2
A28
128
512
64
128
32
[128
0.48
2
0.48
2
0.96
2
A33
128
512
32
128
32
128
0.48
[2
0.48
[2
0.96
[2
A30
128
512
64
128
64
[128
0.48
[2
0.48
[2
0.96
[2
A27
256
[512
64
128
64
[128
0.48
[2
0.48
[2
0.96
[2
A37
256
[512
64
128
64
[128
0.48
2
0.48
2
0.96
2
123
World J Microbiol Biotechnol Table 3 continued Strains
Antibiotics tested 1
2
MIC
MBC
3
4
5
6
MIC
MBC
CMI
CMB
MIC
MBC
MIC
MBC
MIC
MBC
S38
256
512
64
128
32
[128
0.48
[2
0.48
[2
0.96
[2
S50
128
[512
64
128
64
[128
0.48
[2
0.48
[2
0.96
[2
Strains
Antibiotics tested 7
8
9
MIC
MBC
MIC
MBC
MIC
112 126
0.48 0.48
0.96 0.96
0.24 1
4 4
36
0.48
0.96
0.48
38
0.48
0.96
0.48
118
0.48
0.96
57
0.48
56
0.48
213
10
11
MBC
MIC
MBC
MIC
MBC
64 32
128 128
0.96 0.96
4 4
16 16
32 32
4
64
[128
0.96
4
16
32
4
64
[128
0.96
4
16
32
1
8
64
[128
0.48
2
16
32
0.96
0.48
4
64
[128
0.48
2
16
32
0.96
1
8
32
[128
0.96
4
16
64
0.48
0.96
1
8
64
[128
0.48
2
16
64
223
0.48
0.96
1
8
32
[128
0.48
2
16
64
58
0.48
0.96
1
8
32
[128
0.48
2
16
64
K11
0.48
0.96
1
8
16
[128
0.96
2
16
64
K9
0.48
0.96
1
8
64
[128
0.96
2
16
64
P7
0.48
0.96
0.48
4
32
[128
0.96
4
16
64
225
0.48
0.96
0.48
4
32
128
0.96
4
16
32
P8
0.48
0.96
0.48
4
64
[128
0.96
4
16
64
226 234
0.48 0.48
0.96 0.96
0.24 0.48
4 4
64 64
[128 [128
0.96 0.96
4 4
16 16
64 64 64
K8
0.48
0.96
0.48
4
32
[128
0.96
4
16
K6
0.48
0.96
0.48
8
64
[128
0.96
4
16
64
K5
0.48
0.96
1
8
64
128
0.96
4
16
64
244
0.48
0.96
1
8
64
128
0.48
4
16
64
241
0.48
0.96
1
4
32
[128
0.48
2
16
64
EM3
0.48
0.96
0.48
4
32
128
0.96
4
16
64
K1
0.48
0.96
1
8
32
128
0.96
4
16
32
EM2
0.48
0.96
0.48
4
32
[128
0.96
2
16
64
K3
0.48
0.96
0.48
4
16
128
0.96
4
16
64
A3
0.48
0.96
0.48
4
16
128
0.96
4
16
64
A6
0.48
[0.96
1
8
32
128
0.96
4
16
32
A13
0.48
0.96
0.24
4
32
128
0.96
4
16
32
A12
0.48
0.96
0.24
4
64
128
0.96
4
16
32
A34
0.48
0.96
1
8
32
128
0.96
4
16
32
A16 A19
0.48 0.48
0.96 0.96
0.48 0.48
4 4
64 64
[128 [128
0.96 0.96
4 4
16 16
32 32
A23
0.48
0.96
1
8
64
[128
0.48
2
16
32
A25
0.48
0.96
0.48
4
64
[128
0.48
2
16
32
A24
0.48
0.96
1
8
32
[128
0.96
4
16
64
A40
0.48
0.96
1
8
64
[128
0.48
2
16
64
A41
0.48
0.96
1
8
32
[128
0.48
2
16
64
A38
0.48
0.96
1
8
32
[128
0.48
2
16
64
123
World J Microbiol Biotechnol Table 3 continued Strains
Antibiotics tested 7
8
MIC
MBC
A26
0.48
A29 A28
9
10
11
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
0.96
1
8
16
[128
0.96
2
16
64
0.48
0.96
1
8
64
[128
0.96
2
16
64
0.48
0.96
0.48
4
32
[128
0.96
4
16
64
A33 A30
0.48 0.48
0.96 0.96
0.48 0.48
4 8
32 64
128 [128
0.96 0.96
4 4
16 16
32 64
A27
0.48
0.96
0.24
4
64
[128
0.96
4
16
64
A37
0.48
0.96
0.48
4
64
[128
0.96
4
16
64
S38
0.48
0.96
0.48
4
32
[128
0.96
4
16
64
S50
0.48
0.96
0.48
8
64
[128
0.96
4
16
64
Strains
Antibiotics tested 1
2
MIC
MBC
3
MIC
MBC
CMI
4
5
6
CMB
MIC
MBC
MIC
MBC
MIC
MBC [2
S37
128
512
64
128
64
128
0.48
[2
0.48
[2
0.96
S49
128
512
64
128
64
128
0.48
2
0.48
2
0.96
2
S55
256
[512
64
128
32
[128
0.48
[2
0.48
[2
0.96
[2
S57
128
[512
32
128
64
128
0.48
2
0.48
2
0.48
2
S32
128
[512
64
128
64
[128
0.48
[2
0.48
[2
0.48
[2
S56
128
512
64
128
64
128
0.96
[2
0.96
[2
0.96
[2
SL1
256
512
64
128
64
128
0.96
[2
0.96
[2
0.96
[2
S1K
128
[512
64
128
64
128
0.48
[2
0.48
[2
0.48
[2
H1 H4
128 256
512 [512
32 32
128 128
32 64
128 128
0.48 0.48
2 2
0.48 0.48
2 2
0.48 0.96
2 2
H6
256
[512
32
128
64
128
0.48
[2
0.48
[2
0.96
[2 [2
H8
256
[512
32
128
64
[128
0.48
[2
0.48
[2
0.96
H9
256
512
32
128
64
[128
0.48
2
0.48
2
0.96
2
H10
64
512
32
128
64
[128
0.48
[2
0.48
[2
0.96
[2
H11
256
[512
32
128
64
[128
0.48
2
0.48
2
0.96
2
H12
128
512
64
128
32
[128
0.48
2
0.48
2
0.96
2
H18
256
[512
64
128
64
[128
0.48
2
0.48
2
0.96
2
H20
256
[512
64
128
32
[128
0.48
2
0.48
2
0.96
2
21
128
512
64
128
32
[128
0.48
2
0.48
2
0.96
2
H22
256
[512
64
128
16
[128
0.48
2
0.48
2
0.96
2
Strains
Antibiotics tested 7
8
MIC
MBC
9
MIC
MBC
MIC
10
11
MBC
MIC
MBC
MIC
MBC
S37
0.48
0.96
1
4
64
128
0.96
4
16
64
S49 S55
0.48 0.48
0.96 0.96
1 1
4 4
64 32
128 [128
0.48 0.48
4 2
16 16
64 64
S57
0.48
0.96
1
8
32
128
0.96
4
16
32
S32
0.48
0.96
0.48
4
32
[128
0.96
2
16
64
S56
0.48
0.96
0.48
4
32
128
0.96
4
16
64
SL1
0.48
0.96
0.48
4
16
128
0.96
4
16
64
123
World J Microbiol Biotechnol Table 3 continued Strains
Antibiotics tested 7
8
9
10
MIC
MBC
MIC
MBC
MIC
S1K
0.48
0.96
0.48
4
H1
0.48
[0.96
1
H4
0.48
0.96
1
H6 H8
0.48 0.48
0.96 0.96
1 0.48
H9
0.48
0.96
H10
0.48
0.96
H11
0.48
H12
0.48
H18
11
MBC
MIC
MBC
MIC
MBC
16
128
0.96
4
16
64
4
32
128
0.96
4
16
32
4
64
128
0.96
4
16
32
4 4
32 64
128 [128
0.96 0.96
4 4
16 16
32 32
0.48
4
64
[128
0.96
4
16
32
1
8
64
[128
0.48
2
16
32
0.96
1
4
64
[128
0.48
2
16
32
0.96
1
8
32
[128
0.96
4
16
64
0.48
0.96
1
8
64
[128
0.96
4
16
64
H20
0.48
0.96
1
8
32
[128
0.48
2
16
32
21
0.48
0.96
1
8
32
[128
0.48
2
16
32
H22
0.48
0.96
1
8
16
[128
0.96
4
16
32
1: Ampicillin; 2: Kanamycin; 3: Streptomycin; 4: Tetracycline; 5: Chloramphenicol; 6: Cefotaxime; 7: Oxytetracycline; 8: Doxycyclin; 9: Erythromycin; 10: Nalidixic acid; 11: Trimethoprim
difference in the size of these plasmids which varied between 816 and 84,299 pb. According to these authors, a significant correlation was found between resistance to carbenicillin and the presence of a 21,223-bp plasmid, 65.5 % of the strains had the plasmid and were resistant to carbenicillin, and three strains were resistant but did not have the plasmid. Interestingly, we founded that some strains had the same resistance patterns but some of these were plasmideless and the others presented different plasmid profiles. Since some exceptions were found, it is suggested that resistance can be encoded in some strains in plasmids and in others in the chromosomes (Aoki et al. 1984). Other studies will be done in the future in the purpose to know the origin of the antibiotic resistance. Conclusion To our knowledge, there are a few reports available on the multiple antibiotic resistance in V. alginolyticus isolated in Tunisia. Our results can serve as a baseline data for future research on the extent of antibiotic resistant. The restriction of overuse of antimicrobials in the aquaculture field should be respected.
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