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Available online at ajdhs.com  

Asian Journal of Dental and Health Sciences 
Open Access to Dental and Medical Research 

Copyright  © 2023 The  Author(s): This is an open-access article distributed under the terms of the CC BY-NC 4.0 
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original author and source are credited  

 

 

The Contribution of Curable Plasmid-Mediated Resistance in Isolates of 
Staphylococcus aureus at the University of Benin Teaching Hospital, Benin 
City, Nigeria 

S. O Onemu1, Faith Ademulegun 1, M. O. Onemu-Metitiri 2, *Emmanuel Ifeanyi Obeagu3  and Abdulwasiu 
Oladele Hassan1 

1 Faculty of Medical Laboratory Science, College of Basic Health Sciences, Achievers University, Owo, Nigeria. 

2 School of Sciences, Engineering and Environment. University of Salford, United Kingdom.  

3 Department of Medical Laboratory Science, Kampala International University, Uganda.  

Article Info: 
_______________________________________ 
Article History: 

Received 26 June 2023     
Reviewed  29 July 2023 
Accepted 19 August 2023 
Published 15 September 2023 

_______________________________________ 
Cite this article as:  

Onemu SO, Ademulegun F, Onemu-Metitiri MO, 
Obeagu EI, Hassan AO, The Contribution of 
Curable Plasmid-Mediated Resistance in 
Isolates of Staphylococcus aureus at the 
University of Benin Teaching Hospital, Benin 
City, Nigeria, Asian Journal of Dental and Health 
Sciences. 2023; 3(3):30-32 

DOI: http://dx.doi.org/10.22270/ajdhs.v3i3.50        

_______________________________________
*Address for Correspondence:   

Emmanuel Ifeanyi Obeagu, Department of 
Medical Laboratory Science, Kampala 
International University, Uganda 

Abstract 
___________________________________________________________________________________________________________________ 

S. aureus, is an important human pathogen and commensal that is responsible for infections ranging 
from minor to deep-seated life-threatening conditions. Multi-drug resistant S. aureus or MRSA is a major 
cause of hospital acquired infection (HAIs) or nosocomial infections with consequential reduction in 
treatment options and overtly increased cost of healthcare, morbidity and mortality. The study was 
conceived to determine the contribution of curable transmissible plasmids to the ever-increasing 
proportion of multi-drug resistant S. aureus at the University of Benin Teaching Hospital, Benin City. A 
total of 448 consecutive multi-drug resistant clinical isolates of S. aureus were collected, confirmed by 
SCT and TCT and resistance to commonly used antimicrobial agents. Each isolate was inoculated into 
Mueller-Hinton Broth containing 100 µg/mL acridine orange and incubated at 37oC for 24 h. Each broth 
culture was subsequently sub-cultured onto blood agar plates and incubated at 37oC for 24 h. Sensitivity 
tests were thereafter done on each sub-culture by the Kirby-Bauer disc diffusion method. SCT and TCT 
were re-tested on each sub-culture. Isolates with curable transmissible plasmids were 31/448(6.9%) 
and there was complete reversion to sensitivity in all the cured strains to antimicrobial agents tested 
including ampicillin. The remaining isolates (93.1%) retained their resistance to all the antimicrobial 
agents. The isolates with curable plasmids (6.9%) also lost the coagulase activity of both types. Plasmid-
mediated resistance in S. aureus remain an important route of multi-drug resistance, however this is 
dwarfed by chromosomally-mediated resistance as the major mechanism of resistance in multi-drug 
resistance S. aureus. Additionally, the cure of drug resistance was also concomitantly associated with 
lose of the pathogenicity factor-coagulase in these isolates. 

Keywords: S. aureus, multi-drug resistance, transmissible plasmids.   

 

INTRODUCTION 

Staphylococcus aureus (S. aureus) is a major human pathogen 
and commensal that is responsible for infections that range 
from minor to deep-seated life-threatening conditions 1-3. S. 
aureus colonizes between 30% and 50% of humans which 
increases the tendency to initiate infection 4. After the 
introduction of penicillin into clinical practice in the 1940s, 
the first case of methicillin-resistant strains of S. aureus 
(MRSA) was reported 5. There has been a steady increase in 
the number of methicillin-resistant strains of S. aureus 
worldwide due to antimicrobial agents’ selective pressure 6. 
Rates that range from 61.8 – 80% have been reported in 
Nigeria 7 and 61% in the United States 8. Resistance to 
commonly prescribed antimicrobial agents has been 
recognized as a contributory to morbidity and mortality in 
hospitalized patients 9-11. MRSA strains are some of the 
commonest microorganisms associated with hospital-acquired 
infection (HAIs), possessing improved capabilities for 
resistance to methicillin and other antimicrobial agents have 
been reported 12. Resistance of S. aureus has been largely 
attributed to chromosomal and extra-chromosomal genes 

acquisition 13. Infection with MRSA strains comes with 
increased costs, treatment failure and longer hospital bed-
time 14-15. This study was conceived to determine the relative 
contribution of curable transmissible plasmids to the 
resistance of S. aureus isolates in Benin City. 

MATERIALS AND METHODS 

Consecutive isolates of S. aureus that were resistant to 
gentamycin, amoxycilin-clavulanate, ciprofloxacin, 
sparfloxacin ceftazidime, ceftriaxone azithromycin, and 
ampicillin, were collected. Each isolate coagulase activity was 
confirmed by the SCT and TCT and resistance to the 
antimicrobial agents. 

Slide Coagulase Test (SCT) 

Emulsion of each isolate was done on two separate spots on 
clean grease-free slide with a loopful of normal saline. To one 
emulsion was added a loopful of citrated human plasma and 
mixed with the loop and to the other spot was added a loopful 
of normal saline. The slide was rock-mixed in a figure “8” 
fashion. The presence of clumping or granulation was 

                       Open Access                                                                                                                                                                                                         Research Article                                          

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Onemu et al                                                                                                                                 Asian Journal of Dental and Health Sciences. 2023; 3(3):30-32 

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recorded as positive SCT if the other emulsion with saline only 
renamed smooth. 

Tube Coagulase (TCT)   

Each isolate that was SCT test negative was further tested by 
the TCT as well the SCT positive as confirmation of the SCT. 
Each isolate was inoculated into the bijou bottle containing 
1.0ml of sterile nutrient both of which 0.2 mL of citrated 
plasma was added. A local coagulase-positive strain of S. 
aureus was treated in a similar manner as control. The 
preparations were incubated at 370C and examined for the 
presence of a coagulum in 3 h, 6 h, if no coagulum developed, 
the bottles were re-incubated and examined after 18h using 
the control test to validate the result. 

Sensitivity Test 

A standard inoculum was prepared from each isolate adhering 
to the Clinical Laboratory Standard Institute guidelines (2021) 
to obtain 106 organisms/mL. A plate of Mueller Hinton agar 
(Oxoid CM 0337) previously dried at 370C for 30 minutes was 
inoculated with the prepared inoculum using a sterile swab 
stick. Oxford strain of S. aureus (NCTC6571) was treated in the 
same manner as control. Antimicrobial agent discs were 
placed with a sterile forcep at a minimum distance of 25 mm 
apart. The plates were thereafter incubated at 37oC for 18h. 
The sensitivity tests were read with reference to the control 
strain as sensitive or resistant. 

Plasmid Curing 

Each isolate was inoculated into a bijou bottle containing 2 mL 
Mueller-Horton broth (Oxoid (CM 0405B) containing 100 
µg/mL acridine orange and incubated at 37oC for 18h. This 
was subsequently sub-cultured onto a blood agar plates 
(Oxoid CM 55) and incubated at 37oC for 18h. Sensitivity tests 
were carried out on each sub-cultured growth with the same 
antimicrobial agent discs prior the curing process. The Oxford 
strain of S. aureus was also used as a sensitivity test control 
organism. The results were recorded as sensitive or resistant. 

RESULTS 

Isolates with curable plasmids were 31/448 (6.9%) from the 
curing process as presented in Table 1. All the isolates with 
curable resistance plasmid genes showed complete reversion 
to sensitivity with all the antimicrobial agents tested including 
ampicillin. The cure of resistance plasmids in these multi-drug 
S. aureus isolates also resulted in the loss of the coagulase 
activity of both types. All the other isolates that were not 
cured of resistance genes (93.1%) during the process 
remained resistant to all the antimicrobial agents and tested 
positive by both SCT and TCT.  

Table 1: Tests on isolates after growth in the presence of 
100 µg/mL acridine orange 

Test No. of Cases (%) 

Slide coagulase 448(100%) 

Tube coagulase 448(100%) 

Sensitivity to Ampicillin (10 µg) 31(6.9%) 

 

DISCUSSION 

The study revealed that 6.9% of S. aureus isolates possess 
curable or transmissible plasmids that mediate resistance to 
antimicrobial agents in this study population. This, in turn, 
suggests that the resistance of S. aureus to antimicrobial 
agents is to a large extent chromosomally mediated and less to 
extra-chromosomal or plasmid genes. Chromosomal 

resistance often results from mechanisms that involve target 
site alteration to antimicrobial agents known to bind 
penicillin-binding proteins (PBPs) such as the beta-lactams or 
through the use of specialized exporter proteins (efflux 
pumps) to pump out harmful substances from the cell interior 
16-17. This has also been observed in many bacteria populations 
18. This study further shows that the acquisition resistance in 
S. aureus strains through infection with a virus 
(bacteriophage) or through extra-chromosomal genetic 
elements such as transposons and integrons in bacterial 
populations makes less degree of contribution to resistance in 
S. aureus strains as other studies have similarly observed 18. S. 
aureus resistance to antimicrobial agents as with other 
bacterial species appears to be an evolutionary demand for 
the survival of living systems in an ever-changing 
environmental conditions which is inevitable and unavoidable 
consequence of the continuous use of antimicrobial agents. 
The contribution of curable transmissible plasmids to the 
ever-rising tide of MRSA in hospitals is disproportionately low 
in comparison to the chromosomally-mediated mechanism of 
resistance. The loss of resistance after the curing process also 
resulted in the loss of coagulase activity which may suggest 
that the pathogenicity factor (coagulase) is closely linked to 
the resistance gene. Transmissible plasmids contribute much 
less to antimicrobial resistance in strains of S. aureus than 
chromosomally-mediated resistance. Resistance genes in 
these isolates appear to be co-inherited genes with the 
pathogenicity factor coagulase.  It is therefore, expedient to 
redouble efforts to protect reserved valuable antimicrobial 
agents against unnecessary exposure. A renewed and 
sustained search for new antimicrobial agents must be given 
the priority it demands in attempts to win the battle against 
bacterial infections and curtail the development of resistance. 

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