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Journal of Advances in Microbiology
20(6): 37-45, 2020; Article no.JAMB.57932
ISSN:
2456-7116
Molecular Characterization and Detection of
Antibiotic Resistance Genes in Pseudomonas
Species Isolated from Tympanotonus fuscatus
T. Sampson
1*
, N. P. Akani
1
and I. O. Hakam
1
1
Department of Microbiology, Rivers State University, P.M.B. 5080, Nkpolu-Oroworukwo,
Port Harcourt, Nigeria.
Authors’ contributions
This work was carried out in collaboration among all authors. Author TS supervised the entire study,
managed the analyses of the study and also provided extensive discussion for the study. Author NPA
designed the study, performed the literature searches and performed the statistical analysis. Author
IOH managed the laboratory proceedings and wrote the first draft of manuscript. All authors read and
approved the final manuscript.
Article Information
DOI: 10.9734/JAMB/2020/v20i630252
Editor(s):
(1)
Dr. C. Graciela Castro Escarpulli, Mexico.
Reviewers:
(1)
Abhishek Mishra, Houston Methodist Research Institute, USA.
(2)
Amal Alsulaiman, Damascus University, Syria.
Complete Peer review History:
http://www.sdiarticle4.com/review-history/57932
Received 20 April 2020
Accepted 25 June 2020
Published 04 July 2020
ABSTRACT
Aim:
This was carried out to characterize Pseudomonas species isolated from the West African
Mud Creeper (Tympanotonus fuscatus) molecularly and as well detect the possible presence of
inducible AmpC gene that mediates resistance to cephalosporins and most penicillins.
Sample: Tympanotonus fuscatus (West African Mud Creeper), a gastropod mollusc found in
brackish waters of West Africa was used for the study.
Place and Duration of Study: This study was carried out between February and August 2019 at
the Department of Microbiology, Rivers State University, Port Harcourt, Nigeria.
Methodology: Thirty two (32) Pseudomonas species were isolated and identified culturally from T.
fuscatus. Pseudomonas species isolates were subjected to a group of ten (10) antibiotics using the
Kirby-Bauer disc diffusion method and resistant isolates were screened molecularly for the
presence of resistance gene (AmpC). AmpC screening was carried out in a step wise process of
DNA extraction, quantification, amplification of ampC gene using appropriate primer and Agarose
Original Research Article
Sampson et al.; JAMB, 20(6): 37-45, 2020; Article no.JAMB.57932
38
gel electrophoresis to reveal which DNA extracts had ampC genes amplified. The two most
resistant isolates had their 16S rRNA sequenced, identified and were also profiled for plasmids by
extracting plasmid DNA.
Results: Results revealed that 96.67% of isolates had MAR index greater than 0.2 indicating high
a risk source of contamination where antibiotics are often used. Results also showed the presence
of ampC gene in seven (7) out of the twelve (12) isolates screened for ampC gene. Molecular
characterization via sequencing of the 16S rRNA gene of the two (2) most resistant isolates
confirmed that both isolates were strains of Pseudomonas aeruginosa. Profiling of plasmids also
revealed the presence of plasmid DNA of about 10 kilo base pairs in both isolates profiled.
Conclusion: This study has revealed the resistance ability of Pseudomonas and some reasons
behind this resistance. Appropriate investigation into antimicrobial resistance is recommended for
the administration of drugs for the treatment of food-mediated Pseudomonas infections.
Keywords: Resistant genes; Pseudomonas species; AmpC; molecular characterization;
Tympanotonus fuscatus.
1. INTRODUCTION
The treatment of bacterial infection is
increasingly complicated by the ability of bacteria
to develop resistance to antimicrobial agents [1].
Bacterial resistance to these antimicrobial agents
is basically as a result of one or more resistance
determinants which reduce the antimicrobial
activities of the drugs [2].
Pseudomonas species are Gram-negative
bacteria belonging to the phylum Proteobacteria,
class, Gammaproteobacteria, family
Pseudomonadaceae and genus Pseudomonas in
which members demonstrate a great deal of
metabolic diversity and consequently are able to
colonize a wide range of niches ranging from
water, soil, plant seeds, air [3].
Pseudomonas aeruginosa represents a classical
phenomenon of antimicrobial resistance since
virtually all known mechanisms of antimicrobial
resistance can be seen in it. These mechanisms
include but not limited to the de-repression of
chromosomal AmpC cephalosporinase,
production of plasmids or integron-mediated β-
lactamases from different molecular classes
(carbenicillinases and extended-spectrum β-
lactamases belonging to class A, class D
oxacillinases and class B carbapenem-
hydrolysing enzymes), diminished outer
membrane permeability (loss of OprD proteins),
overexpression of active efflux systems with wide
substrate profiles, synthesis of aminoglycoside
modifying enzymes (phosphoryltransferases,
acetyl transferases, adenylyltransferases);
and structural alterations of topoisomerases II
and IV determining quinolone resistance
[4].
P. aeruginosa has naturally occurring
chromosomal AmpC b-lactamase or
cephalosporinase which relates to β-lactams
such as Penicillin G; Aminopenicillins including
those combined with β-lactamase inhibitors; first
and second generation cephalosporins. AmpC β-
lactamase is encoded by the ampC gene and it is
the primary cause of the organism’s resistance to
β-lactams. Enzyme production is the major
mechanism of acquired resistance of P.
aeruginosa to β-lactam antibiotics. β-lactamases
rupture the amide bond of the β-lactam ring
leaving obtained products which lack
antimicrobial activity [5]. P. aeruginosa is
naturally susceptible to carboxypenicillins,
ceftazidimes and aztreonams; however, it can
acquire resistance to third generation
cephalosporins. This happens readily through the
constitutive excessive production of AmpC β-
lactamase [5]. The AmpC β-lactamase enzyme
belonging to molecular class C is naturally
produced in low quantities by P. aeruginosa
and determines resistance to aminopenicillins
and some of the early cephalosporins [6].
However, chromosomal cephalosporinase
production may increase from 100 to 1000
times in the presence of inducing β-lactams
(including imipenem) [5]. Pseudomonas species
is highly resistant to cephalexin, cephalosporin,
ampicillin, amoxicillin/Clavulanic acid and
nalidixic acid [7] and also to a variety of
medicinal herbs such as bitter cola seed and
turmeric [8].
This study was carried out to characterize and
detect antibiotic resistant genes in some
Pseudomonas species isolated from the West
African Mud Creeper using molecular
techniques.
Sampson et al.; JAMB, 20(6): 37-45, 2020; Article no.JAMB.57932
39
2. MATERIALS AND METHODS
2.1 Study Period
This study was carried out between February
and August 2019.
2.2 Sample Description, Size and
Collection
A total of 42 edible samples of Tympanotonos
fuscatus were collected between February and
May 2019 from three different locations in Rivers
State Nigeria; Mile 1 market in Port Harcourt City
Local Government Area (4.7918° N, 6.9986° E),
Rumueme Market in Obio/Akor Local
Government Area (4.8273° N, 6.9820° E) and
Mile 3 Market in Port Harcourt City Local
Government Area (4.8042° N, 6.9924° E).
Microbiological examination was carried out at
the Microbiology laboratory, Rivers State
University, Port Harcourt, Nigeria.
2.3 Isolation and Identification of Bacteria
Bacteria was isolated from sample using cultural
means, following the process of serial dilution,
spread plating, incubating over night and sub
culturing to obtain pure cultures. Morphological
and biochemical tests were carried out for
identification of Pseudomonas species.
Biochemical tests such as oxidase test, motility
test, catalase test, starch hydrolysis test, indole
test, methyl red test, Voges Proskaeur test,
citrate utilization test were carried out on isolates
[9]. Molecular characterization was employed to
confirm the identities of some isolates using the
method of Queipo-ortuno et al. [10]; Al-Awadhi et
al. [11].
2.4 Antibiotic Sensitivity Testing
Standardization of isolates was carried out by
adjusting the turbidity of isolates in test tubes to
that of a 0.5 McFarland standard. The
antimicrobial susceptibility profiles of the isolates
to conventional antibiotics were determined using
the Kirby Bauer disk diffusion method [12] on
sterile Mueller-Hinton agar. The surface solid
media plate was inoculated with bacterial
suspension by streaking the swab over the agar
plate surface; being sure that no zone of the
surface is left free of inoculum. This procedure
was repeated several times, rotating the agar
plate 60° each time to ensure even distribution of
the inoculum to the edge of the agar. The plates
were left to dry for 3–5 min to allow absorption of
any moisture prior to applying the antibiotic disks.
Antibiotic disks of ten conventional antibiotics
(Cephalexin (CEP) – 10 µg, Ofloxacin (OFX) -10
µg, Nalidixic acid (NA) – 30 µg, Pefloxacin (PEF)
– 10 µg, Gentamycin (CN) – 10 µg,
Amoxicillin/Clavulanic acid (AU) – 30 µg,
Ciprofloxacin (CPX) – 10 µg, Trimethoprim (SXT)
– 30 µg, Streptomycin (S) – 30 µg and Ampicillin
(PN) – 30 µg) were aseptically placed on the
surface of the inoculated agar plate with sterile
forceps. Each disk was pressed down to ensure
full contact with the surface of the agar. At least
24 mm was left between the centres of the disks,
and not less than 15 mm from the border of the
plate too. The plates were then inverted and
placed in an incubator within 15 min of applying
the disks. Finally, the plates were incubated for
24 hours at 33 to 35°C [12]. After incubation, the
plates (control and test plates) were examined to
ensure growth was confluent or near confluent.
On the underside of the plate, the diameter of
each zone of inhibition for those that had zones
of inhibition were measured in millimetre (mm)
using a meter rule. The measurement included
the diameter of the disc. For interpretation MIC
Analysis and Susceptibility Testing, the criteria
provided by CLSI were followed.
2.5 Molecular Studies
Molecular screening was carried out on the most
resistant strains of Pseudomonas isolated for
identification, plasmid profiling and detection of
antibiotic resistance gene (AmpC).
2.5.1 DNA extraction and quantification
DNA extraction was carried out using the Boiling
method by Bell et al. [13]. 24 hour old pure
cultures of Pseudomonas species were put in
Luria-Bertani (LB) Broth and allowed to incubate
at 37°C. After 24 hours, the cells were washed
thrice in microcentrifuge tubes with normal saline
by centrifuging for 3 minutes at 14,000 xg and
decanting supernatant leaving the DNA at the
base. The DNA was washed with 1 ml of normal
saline and vortexed to mix and then centrifuged
again. The cells were re-suspended in 500 ul of
normal saline and heated at 95°C for 20 min in a
heating block after which it was cooled on ice
and then centrifuged for 3 minutes at 14000 xg.
Using a 1.5 ml microcentrifuge tube, the
supernatant containing the DNA was transferred
and stored at 20°C [13]. The extracted genomic
DNA was quantified by using the Nanodrop 1000
Spectrophotometer as described by Olsen and
Morrow [14].
Sampson et al.; JAMB, 20(6): 37-45, 2020; Article no.JAMB.57932
40
2.5.2 16S rRNA amplification
The method as described by Srinivasan et al.
[15] was adopted. An ABI 9700 Applied
Biosystems Thermal Cycler was used to amplify
the 16S rRNA. The 16S rRNA region of the rRNA
genes of Pseudomonas species isolates were
amplified using the forward primer; 27F: 5’
AGAGTTTGATCMTGGCTCAG-3 and Reverse
Primer 1492R: 5’-CGGTTACCTTGTTACGACTT-
3’ at a final volume of 30 µl for 35cycles. The
PCR cocktail was prepared using the primers at
0.6µM concentration, the Template (the
extracted DNA), Buffer 1X, water, PCR Mix
(15M) which consists of; dNTPs, MgCl and Taq
Polymerase. The conditions for PCR were as
follows; Initial denaturation, 95°C for 5 minutes;
Denaturation, 95°C for 30 seconds; Annealing
52°C for 30 seconds; Extension, 72°C for 5
minutes. The product was fixed in a 1% agarose
gel at 120V for 15 minutes and visualized on a
UV transilluminator.
2.5.3 DNA sequencing
The amplified products were labelled using the
BigDye Terminator Cycle Sequencing kit
(Applied Biosystems). The sequencing was done
at a final volume of 10 µl; the components
included 0.25 µl BigDye terminator v1.1/v3.1,
2.5 µl of 5x BigDye sequencing buffer, 10 µM
Primer PCR primer and 2-10 ng PCR
template per 100 bp. The sequencing conditions
were as follows: 32 cycles of 96°C for 10
seconds, 55°C for 5 seconds and 60°C for 4
minutes [15].
2.5.4 Phylogenetic analysis
Sequences were edited using the bioinformatics
algorithm Trace edit, similar sequences were
downloaded from the National Center for
Biotechnology Information (NCBI) data base
using BLASTN. These sequences were aligned
using MAFFT. The evolutionary history was
inferred using the Neighbor-Joining method in
MEGA 6.0 [16]. The evolutionary distances
were computed using the Jukes-Cantor method
[17].
2.6 Determination of Multiple Antibiotic
Resistance (MAR) Index
Multiple Antibiotic Resistance Index was
determined from Sensitivity testing Results using
the formular, MAR index =a/b, where a, = the
number of resistance to antibiotics displayed and
b = the total number of antibiotics tested, as
described by Osundiya et al. [18].
3. RESULTS
The result shown on Fig. 1 is the agarose gel
electrophoresis of the amplified 16S rRNA gene
of two selected and most resistant Pseudomonas
isolates before sequencing.
The evolutionary distance between the P.
aeruginosa isolates from this study and the
accession numbers of their closest relatives on
the phylogenetic tree is shown on Fig. 2.
The agarose gel electrophoresis showing the
plasmid DNA bands of the most resistant
Pseudomonas isolates is displayed on
Fig. 3.
The agarose gel electrophoresis image showing
the amplified ampC gene of the 12 most resistant
Pseudomonas isolates to antibiotics is shown
Fig. 1. Agarose gel electrophoresis of the 16S rRNA gene of selected most resistant bacterial
isolates
Lanes B1 and B2 represent the 16SrRNA gene bands (1500bp) while lane L represents the 100bp molecular
ladder
Sampson et al.; JAMB, 20(6): 37-45, 2020; Article no.JAMB.57932
41
Fig. 2. Phylogenetic tree showing the evolutionary distance between the bacterial isolates
Fig. 3. Agarose gel electrophoresis showing the plasmid DNA bands of the Pseudomonas
aeruginosa isolates
Lane 1 and 2 showing plasmid DNA bands at >10 kbp while lane L represents the 10 kbp molecular ladder.
Plasmid DNA from both strains bands at regions greater than 10 kilobase pairs. This represents high molecular
weight plasmids
on Fig. 4. Lane L represents the 100 bp
molecular ladder, while Lane 1-3, 6-8 and 10
showing the ampC band at 500 bp. From the
result it was observed that 7 out of the 12
isolates (58.3%) screened for ampC gene had
the gene present in their genetic material.
Table 1 shows the Multiple Antibiotic Resistance
(MAR) Index of Pseudomonas isolates after
subjection to a group of 10 conventional
antibiotics. MAR index values greater than 0.2
indicate high risk source of contamination where
antibiotics are often used.
Sampson et al.; JAMB, 20(6): 37-45, 2020; Article no.JAMB.57932
42
Fig. 4. Agarose gel electrophoresis showing the amplified ampC gene of the 12 most resistant
Pseudomonas isolates to antibiotics
Table 1. Multiple Antibiotic Resistance (MAR)
index of Pseudomonas species
MAR index
Number (%)
0.1 1 (3.33)
0.2 3 (10.0)
0.3 5 (16.67)
0.4 6 (20.0)
0.5 11 (36.67)
0.6 4 (13.33)
4. DISCUSSION
The contributions of bacterial species in the
epidemiology of disease are influenced by the
method used in the identification and
characterization of the bacterial isolates, as
researchers employ various microbiological
techniques to achieve this aim. Molecular
characterization has recently been employed as
a high-throughput approach which enable a
reliable identification of disease causing agents.
PCR has the potential for identifying microbial
species rapidly by amplification of sequences
unique to a particular organism [19]. This
research therefore explored the use of a culture-
based molecular technique in accessing and
probing of Pseudomonas species isolated from
West African Mud Creeper (Tympanotonus
fuscatus), for the presence of genes that confer
antibiotic resistance, such as ampC gene.
Results from the molecular study showed that
the obtained 16S rRNA sequence from the
selected most resistant isolates produced an
exact match during the mega blast search for
highly similar sequences from the NCBI non-
redundant nucleotide (nr/nt) database. Also, the
evolutionary distances computed using the
Jukes-Cantor method were in agreement with
the phylogenetic placement of the 16S rRNA of
the isolates 2SBd and 6SBa within the
Pseudomonas sp. which revealed a close
relatedness to Pseudomonas aeruginosa than
other Pseudomonas sp.
Persistence of antibiotic resistant bacteria,
including multidrug resistant (MDR)
pseudomonads, is an important environmental
health problem associated with food samples
from environmental samples. Beyond the
phenotypic determination of antibiotic resistance
pattern earlier reported [7], the presence of
antibiotic resistance gene (ampC) which could
mediate the antibiotic resistance in the
Pseudomonas sp. was screened through a
molecular technique. It followed that these two
isolates reported in this paper were the most
resistant to almost all the antibiotics tested. The
result further revealed the presence of plasmid
DNA of over 10 kilo base pairs. Several resistant
attributes are borne on the plasmid DNA of
bacteria and the heavier or longer the plasmid,
the more likely it is to carry several genes coding
for extra bacterial attributes [20].
Resistant gene analysis for the detection of
ampC gene in twelve (12) resistant
Pseudomonas species isolates revealed that
seven (7) out of twelve isolates (58.3%)
screened had the ampC gene present in their
genome. ampC is largely responsible for the
resistance of Pseudomonas species isolates to
Sampson et al.; JAMB, 20(6): 37-45, 2020; Article no.JAMB.57932
43
cephalosporin antibiotics used in the study and
its overproduction can even further increase the
ability of isolates to resist these antibiotics
completely [21]. ampC gene codes for the
production of ampC beta-lactamase enzyme
which acts on the beta-lactam ring of antibiotics
to inhibit their activity [22].
There is paucity of information relating to studies
on the molecular characterization of antibiotic
resistance genes among Nigeria environmental
pseudomonads, therefore, making it difficult to
compare with studies from Nigeria [23]. However,
among few of the studies, Chikwendu et al. [24]
reported the presence of bla
SHV
and bla
TEM
among environmental pseudomonads from
Nigeria while Odumosu et al. [25] reported
bla
oxa-10
, ampC β-lactamase in 50 and 70% of
Pseudomonas aeruginosa, respectively from
Nigeria clinical source.
MAR index of Pseudomonas species isolated in
this study revealed that the percentage of
isolates with MAR index ≥ 0.2 was 96.67%. It is
important to note that MAR index values > 0.2
indicate high risk source of contamination where
antibiotics are often used [18,22]. This shows
that 96.67% of the Pseudomonas species
isolated in this study are likely to show multiple
resistance to the antibiotics used in this study.
This finding is very critical as it indicates that
these antibacterial agents may not be potent in
the management of infections caused by these
species of Pseudomonas.
The presence of resistant strains of P.
aeruginosa in T. fuscatus which may have risen
from the market environment due to poor
hygienic practices poses a problem for public
health as it can cause a wide range of infections
such as endocarditis, gastrointestinal infections,
osteomyelitis, septicaemia and meningitis
especially in immunocompromised individuals
[26]. The study of bacterial genomics informs our
understanding of resistance mechanisms and
comparatively analysing these genes can provide
relevant information on the evolution of resistant
strains of organisms and on resistance genes
[27].
5. CONCLUSION
This study has shown that the resistance of most
Pseudomonas species to some antibiotics is
largely due to the presence of the resistance
ampC gene, presence of heavy plasmids
amongst other factors not covered by the current
study. This molecular identification of
Pseudomonas species has shown that genomic
studies are needed to confirm the exact
taxonomic identity of Pseudomonas spp due to
their public health importance. Further studies
are however needed to decipher the factors
influencing their high genetic plasticity. The risks
of resistance of P. aeruginosa to antibiotics are
also very high considering the MAR index values
obtained from the study.
From this study it can also be inferred that West
African Mud Creeper (Tympanotonus fuscatus)
harbours bacterial populations that can lead to
serious foodborne ailments with associated
multiple antibiotic resistant traits. This
observation is not however unconnected with
microbiological quality of its marine habitat. This
further buttresses the fact that the marine
environment serves as a sink for antibiotic
resistant microbial population. Proper waste
disposal/management is therefore recommended
to check and prevent the build-up or proliferation
antibiotic resistant microbial community in the
environment vis-à-vis the invaluable importance
of the aquatic environment to man.
COMPETING INTERESTS
Authors have declared that no competing
interests exist.
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