Antibiotic resistance and plasmidic profiles of some Bacillaceae and Pseudomonaceae isolates from west Algerian wetlands

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
06g
B-Lactam
OX
Oxacillin
5g
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
06g
5g
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.
ACKNOWLEDGEMENT: I thank my supervisor Dr. Cheba Ben Amar and my translator Mr.
Kamal Ziri
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