Antibiotic resistance and plasmidic profiles of some Bacillaceae and Pseudomonaceae isolates from west Algerian wetlands M.A.Ziri (1), B.Cheba (2) (1) Laboratory of Microbiology, Department of Biotechnology, Faculty of Sciences nature and life, University of sciences technology Mohamed Boudiaf Oran, Algeria (2) Laboratory of Microbiology, Department of Biotechnology, Faculty of Sciences nature and life, University of sciences technology Mohamed Boudiaf Oran, Algeria [email protected] (1)/[email protected] (2)/Bp 1505.El Mnaouer Oran Algeria Tel : 21357068545 Abstract: The horizontal transfer of genetic heritage between bacterium to another is done gently aquatic, water of a wetland is adequate space to this phenomenon by its insurance carrier mobility of plasmid genes our study revolves around a comparison of antibiotic resistance and plasmid profile for it was isolated bacteria belonging to two different families and away from three wetlands, these strains were tested against six antibiotics and their plasmid DNA was extracted, the statistical study has shown a correlation between plasmid profile and Registered resistance was finally concluded with certainty that the importance of a wetland is its contribution in bacterial evolution through the water area it contains. Key words: Wetlands, Bacillaceae, Pseudomonaceae, antibiotic, plasmid Résumé: Le transfert horizontal du patrimoine génétique entre bactérie à l'autre se fait en aquatique, l'eau d'une zone humide est un espace adéquat à ce phénomène par la mobilité assurer aux gènes plasmidiques notre étude tourne autour d'une comparaison de la résistance aux antibiotiques et le profil plasmidique pour cela on a isolé des bactéries appartenant à deux familles différentes de trois milieux humides, ces souches ont été testés contre six antibiotiques et leur ADN plasmidique a été extrait, l'étude statistique a montré une corrélation entre le profil plasmidique et la résistance enregistré on a finalement conclu avec certitude que l'importance d'une zone humide est sa contribution à l'évolution des bactéries à travers la zone de l'eau qu'il contient. Mots clés: zones humides, Bacillaceae, Pseudomonaceae, antibiotique, plasmide Introduction: Wetlands are a very special habitat for genetic evolution of some bacterial population because aquatic genetic heritage of bacteria can easily attach another bacterial species away through the horizontal transfer reassured by the mobility of some plasmids phenomenon plays a major role in this process as the effect in conjunction settlor a donor bacterium of certain genetic traits such as antibiotic resistance cede its special resistance to another cohabiting the same medium water from a wetland is an area suitable and adequate to gene transfer, bacterial survival requires some time genetic changes by receiving new characters the most common example and resistance to certain antibiotics, while its recently acquired properties are essential to its persistence in the environment, and the latter can be received only through the plasmids during conjugation ,the acquisition of genes needed to elaborate the various mechanisms is greatly aided by a variety of promiscuous gene transfer systems, such as bacterial conjugative plasmids (Bennett 2008) as bacteriologist we tried to approved statically reported theoretically link between the plasmid profile and profile of some resistors of these aquatic bacteria. MATERIALS AND METHODS: 1. Wetlands studied: Photo 01: horizontal view and Satellite imagery taken by Google earth of the sampling site at the Wetland Dam Djorf torba Wilaya of Bechar Photo 02: View Satellite imagery taken by Google earth and horizontal on the wetland lake Telamin Oran Wilaya Photo 03: horizontal and satellite view taken by Google Earth levies site at the Lagoon Wetland Macta Oran these three wetlands are classified in the international Ramsar convention, the first site is a dam at the Bechar region on the watershed of Guir, the second is the Lake Telamin in a lowland receptacle in the Oran region and the third is a coastal lagoon on the watershed Macta in the same area west of Algeria, these places are characterized by a very large aquatic bacterial diversity while Pseudomonaceae and Bacillaceae prominently in this microflora. 2. Isolation of strains: Isolation Bacillaceae was done on agar medium Mosel, it results in five three strains of them are part of the type species Bacillus.cereus while Pseudomonaceae were isolated on agar king b, which has able to have three strains belonging to the species type Pseudomonas.fluorescent (King et al 1954) 3. Antibiogram: The method used is that of diffusion in agar, this simple method allows the determination of sensitivity vis-à-vis the antibiotic strains tested. Muller Hinton agar (MH), cast in Petri dishes on a thickness of 4mm. From a pure culture 18 hours isolation medium, scraping with a platinum loop few well insulated and perfectly identical colonies; unload the cove in 5 to 10 ml of sterile 0.9% saline; homogenize the bacterial suspension, opacity must be equivalent to 0.5 McFarland (shown in the control of the inoculum) or a 0.8 to 0.10 OD read at 625nm. ; The inoculum can be adjusted by adding or culture if it is too low, or sterile saline if it is too strong; seeding should be done within 15 minutes that follows the preparation of the inoculum. Dip a sterile swab into the bacterial suspension; wringing by pressing firmly (turn) on the inner wall of the tube for discharging the maximum rubbing the swab over the entire agar surface, dry, from top to bottom, in tight streaks; repeat twice, turning the box of 60 ° C each time remembering to rotate the swab on him-even and finish seeding through the swab on the periphery of the agar. We used a disk distributor at 6 antibiotic discs by a Petri dish , Protocol established by us. Lists antibiotics recommended for the Bacillaceae sensitivity test and Pseudomonaceae are reported in Tables 1 and 2, Incubate at 37 ° C for 18 to 24 hours. Table I: The main antibiotics used to Bacillaceae family Sigle Antibiotic Disk load Family P penicillin 06g B-Lactam OX Oxacillin 5g S Streptomycin 10µI GM N Gentamycin Nitroxolin 10µI 20 µg Aminosides Quinolones C Chloramphenicol 30µg Phenicols Table II: The main antibiotics used to Pseudomonaceae family Sigle Antibiotic Disk load Family AMX Amoxilin Aminopenicillin AM 06g 5g B-Lactam S Streptomycin 10µI GM k Gentamycin Kanaycin 10µI 20 µg Aminosides C Chloramphenicol 30µg Phenicols 4. Extraction plasmid DNA from bacterial strains: A mini plasmid preparation powerful strains (which have been more active in antibiotic resistance) was made by the method of with slight modifications as followings: 1. Bacterial -Preparation: 10ml LB medium is inoculated with a colony of bacteria and incubated for 24 hours at 37 ° C with stirring: Recovery and washing the cells, Take 1.5 ml of bacterial suspension placed in sterile eppendorf. Centrifuge at 12000 rev / min for 10 min, remove the supernatant, add 1.5 ml of bacterial suspension, centrifuge at 12,000 rev / min for 10 min, remove the supernatant, resuspend the pellet in 1 ml 50 Mm Tris, 50 mM EDTA, 20% sucrose, centrifuge at 12,000 rev / min for 10 min, resuspend the pellet in 200 .mu.l of 50 mM Tris buffer, EDTA 10Mm 2. Alkaline denaturation: Add 200ul of lytic mixture (0.2 M NaOH, 1% SDS) and mixed without vortex until the solution becomes clear and viscous. 3. Purification of plasmid DNA: Place eppendorfs in ice and add 143 .mu.l of 3M sodium acetate, pH = 5.2 to achieve the denatured chromosomal DNA precipitation by soda and complex proteins by SDS (detergent) and mix by inversion (as a movement come back and vertical) successive 6 to 10 times and leave it for 10 minutes, centrifuge at 12000 rev / min at room T °, collect the supernatant volume V Add V absolute iso propanol at -20 ° C and place to 20C ° for 30 minutes to precipitate the plasmid DNA, centrifuge at 14000 rev / min for 15 min, remove the supernatant, resuspend the pellet in 300 .mu.l of 75% ethanol, centrifuged at 14,000 rev / min for 15 min. remove the supernatant, dry the pellet minutes at 37 ° C to remove ethanol, to 300 .mu.l TE in the pellet that is already prepared. 4. Electrophoresis on agarose gel: was prepared agarose gel at 1%, weigh the amount needed to make the agarose gel 1% (w / v) knowing that the final volume of the gel will 75ml, transfer the agarose powder in a conical flask and add 75 ml of 1X TBE measured specimen, boil beak benzene until all the agarose is melted, let cool (we must be able to hold the flask in his hand without getting burn and add 0.5 ml of BET pour on the horizontal support and put the comb ,allow the agarose to cool and solidify, after it begins the identification plasmid DNA ,one starts, add 500 .mu.l loading buffer to each eppendorf, place gel in the tank with 01 X TBE buffer (the gel should be slightly submerged) gel length is about 80 mm (8cm) and a 50 mm wide (5cm), remove the samples, close the lid of the vessel to electrophoresis and migrate à17-50-80 V, the electro phoretic profile analysis is detected after, stopping migration by cutting the current when the dye (bromothymol blue) of the load buffer has sufficiently migrated, take a picture of the gel under UV radiation, the protocol is updated by us. RESULTS: Table III: diameters antibiotics resistance of Pseudomonaceae Family Strain Pseudomonaceae A3 T3 M3 Antibiotics AMX AM 0 0 0 0 0 0 S 1 0,8 2 K 2 1 0,5 GM 2,5 2,6 2,4 C 5 1,9 3,5 Table IV: diameters antibiotics resistance of Bacillaceae Family Strain Bacillaceae A1 A12 T12 M1 M12 Antibiotics P OX 0 0 0 0 0 0 0 0 0 0 N 2 2,5 1,5 3 4 S 1 2 0,8 1 1,7 GM 3 4 1,8 3 4 C 2 3 2 2 2 4,5 4 3,5 3 2,5 A1 2 A12 1,5 T12 1 M1 0,5 M12 0 Figure 01:Variation in resistance diameters upon Bacillaceae 6 5 4 3 A3 2 T3 1 M3 0 Figure 02:Variation in resistance diameters upon Pseudomonaceae A1 A12 M1 A3 M3 M12 T12 T3 Photo 04: The plasmid profile of some aquatic bacteria isolated from three wetlands (Djorf Torba, Macta, Télamine) belonging to the family of Bacillaceae and Pseudomonaceae. Table V: Plasmid profile of Pseudomonaceae and Bacillaceae Family code Genre/species plasmid band 1band Bacillaceae Pseudomonaceae 2band A1 Bacillus.sp 3 bands + A12 Bacillus.cereus + M1 Bacillus.sp + M12 Bacillus.cereus + T12 Bacillus.cereus + A3 Pseudomonas.f + M3 Pseudomonas.f + T3 Pseudomonas.f + DISCUSSION: Bacillaceae showed deprecated resistance to the penicillin and oxacillin ,the positives bacilli are resistant to beta-lactam family of antibiotics (Louis 2011), while Pseudomonaceae proved a common resistance to the amoxillin and aminopeniciline ,the negative bacilli are resistant strongly to betalactam family of antibiotics a conformality with quote about plasmid profile of some bacilli vibrioneceae (Wang, Leung et al. 2006) , this leads us to say that it is a natural resistance and chromosomal origin (Louis 2011), plasmid remains irresponsible and the exception of antibiotic resistance to these four the rest were recorded different diameters is open probability that it is an unstable intermediate resistance or acquired as measured diameters vary in the same case against a only antibiotic, so the plasmid is probably responsible for the resistance acquired and Bacterial plasmids but costs of plasmids for the bacterial host remain unclear (Ude, Bailey et al. 2007) ,serve as the scaffold on which are assembled arrays of antibiotic resistance genes, by transposition(Bennett 2008) the strongest argument is the change in diameter of the same species on the same tested antibiotic, changes in diameter to the same antibiotic resistances for all three strains of Pseudomonaceae is explained by the different geographical origin of each strain which is confirmed by (Louis 2011), so the expected results are directly dependent on the local bacterial ecology Telamin lake shaped a different habitat from that of Macta lagoon and the other Djorf Toba dam, Bacillaceae resistente strains by producing plasmid transferase enzyme which acetylate the antibiotic, the Pseudomonaceae also exhibit acetylase enzyme and plasmid transferase enzyme for resistance, and the shelf strains induced the loss of the plasmids in vivo and consequently the profile plasmid would be weakened and will not be identical from one strain to another a conformality with quote (Kessie, Ettayebi et al. 1998) , the two tierd of this plasmid content is well known that plasmid conjugative resistance, this loss leads to a weakening which will affect rates Registered resistance which will be reflected in the Rating Decrease of resistors diameters but if that loss will be made in the aquatic environment and not in the storage tube, the plasmid is free and includes aquatic subsequently other surrounding bacteria There is a strong correlation between the number of plasmids harbored by an isolate and resistance to various drugs tested (Sharma, Ray et al. 2010) Most of bacterial strains contain plasmids. There may be a correlation between plasmid and the resistance of strains to gentamycin a conformality with quote(Chen, Xu et al. 2007) , is called horizontal transfer between indigenous bacteria and other transitional, changing the bacterial gene expression and accumulation of this transfer will upset the genetic heritage of descendant population for the same bacterial species and other. the three bands of plasmids, view the agarose gel proves some wealth for the family of plasmids by Bacillaceae against content is very low Pseudomonaceae reflects a plasmid deficiency following a loss, as both family occupies the same pool area the probability of a horizontal transfer is very convincing of Pseudomonaceae to Bacillaceae, because these showed distinct bands of fluorescence’s rich in original plasmids and other plasmids received from neighboring bacteria while the gel photograph clearly shows the difference between the profile of the Bacillaceae, and Pseudomonaceae profile ,these results are in agreement with those of (Pyzik and Marek 2013, Lobova, Yemelyanova et al. 2015) ,the plasmid and the associated antimicrobial resistance could be transferred to another type of bacteria by conjugation with a certain frequency (Son, Rusul et al. 1997) CONCLUSION: This study suggests that these three wetland’s harbors Bacillaceae and Pseudomonaceae of unique characteristics expressed in their profile plasmid and resistance to antibiotic that warrant further investigation for the approved horizontal transfer between two different kinds of bacteria. 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