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ORIGINAL ARTICLE
Year : 2019  |  Volume : 7  |  Issue : 1  |  Page : 1-6

Phenotypic determination of methicillin-resistant Staphylococcus aureus in Aminu Kano Teaching Hospital, Kano, Nigeria


1 Department of Medical Microbiology and Parasitology, Faculty of Clinical Sciences, Bayero University, Kano, Nigeria
2 Department of Medical Laboratory Sciences, Faculty of Allied Health Sciences, Bayero University, Kano, Nigeria

Date of Web Publication13-Sep-2019

Correspondence Address:
Mr. Abdulrazak Muhammad Idris
Department of Medical Microbiology and Parasitology, Faculty of Clinical Sciences, Bayero University, Kano
Nigeria
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/njecp.njecp_20_18

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  Abstract 


Background: Phenotypic detection of methicillin-resistant Staphylococcus aureus(MRSA) has been problematic ever since its discovery in the early 1960s. The emergence of low-level resistant MRSA clones acquired in the community has only added to these difficulties. In 2005, the Clinical and Laboratory Standards Institute (CLSI) published zone diameter (10), breakpoint guidelines for cefoxitin. However, a number of technical issues remain regarding the use of cefoxitin as a predictor for methicillin resistance. Materials and Methods: In these studies, 252 nonduplicate samples of staphylococcal isolates were collected from various clinical samples obtained from patients attending Aminu Kano Teaching Hospital. The isolates were subcultured and identified using standard bacteriological procedure according to CLSI (13). Antibiotic susceptibility testing was done using a modified form of the Kirby–Bauer method. Methicillin resistance was screened using disk-diffusion method with cefoxitin 30 μg and oxacillin 1 μg. Results: High percentage of the isolates were recovered from patients of age groups of 1–9 years and <1 year with 45.2% and 23.4%, respectively. About 77% isolates were obtained from blood culture followed by wound (11.5%) and ear swab (6.7%). MRSA prevalence of 20.6% and 25.8% was obtained in this study using cefoxitin (30 μg) and oxacillin (1 μg), respectively. High prevalence of MRSA was obtained from people of the old age group which may be due to used and misused of antibiotics. From the 252 isolates obtained in this study, 84.1%, 77.4%, and 77.0% were found to be susceptible to ciprofloxacin, gentamicin, and clindamycin, respectively. The least susceptible was found 49.2%, 52.0%, and 62.7% in erythromycin, co-trimoxazole, and tetracycline, respectively. Conclusion: The study revealed that routine phenotypic screening of MRSA gives a better result when both oxacillin and cefoxitin were used, especially in resource-limited areas where molecular analysis is not available.

Keywords: Antibiotics, disk diffusion, methicillin-resistant Staphylococcus aureus, phenotypic


How to cite this article:
Idris AM, Kumurya AS, Mohammed Y, Mustapha HM. Phenotypic determination of methicillin-resistant Staphylococcus aureus in Aminu Kano Teaching Hospital, Kano, Nigeria. Niger J Exp Clin Biosci 2019;7:1-6

How to cite this URL:
Idris AM, Kumurya AS, Mohammed Y, Mustapha HM. Phenotypic determination of methicillin-resistant Staphylococcus aureus in Aminu Kano Teaching Hospital, Kano, Nigeria. Niger J Exp Clin Biosci [serial online] 2019 [cited 2019 Nov 12];7:1-6. Available from: http://www.njecbonline.org/text.asp?2019/7/1/1/266836



Staphylococcus aureus is an important pathogen in human infections and is implicated in a wide variety of infections, from mild skin infections to more serious and invasive infections, including sepsis, pneumonia, endocarditis, deep-seated abscesses, and toxinoses including food poisoning and toxic shock syndrome.[1] Its impact is enhanced by the development of antibiotic resistance, most notably methicillin-resistant S. aureus (MRSA) that is resistant to virtually all β-lactam antibiotics.[2] MRSA is any strain of staphylococci that can develop resistance to β-lactam antibiotics, which include the penicillins (methicillin, dicloxacillin, nafcillin, oxacillin, etc., and the cephalosporins) through the process of natural selection.[3] Strains unable to resist these antibiotics are classified as methicillin-sensitive S. aureus (MSSA). The mechanism of such resistance is based on the acquisition of staphylococcal cassette chromosome mec elements containing a mec gene (mec A, more rarely mec C), which codes for an additional penicillin-binding protein (PBP) that has low affinity for β-lactam antibiotics and therefore mediates resistance to nearly all compounds from this antibiotic class (besides ceftobiprole and ceftaroline). So far, at least 11 different structural types of SCCmec are known.[4] Soon after the discovery of methicillin resistance S. aureus in 1961.[5],[6] The unusual behavior of the strains in susceptibility tests was noted. Early reports indicated that MRSA was heterogeneous in their expression of resistance to β-lactam agents, in that large differences in the degree of resistance were seen among the individual cells in a population.[6] The expression of resistance is markedly affected by test conditions although the UK “epidemic” strains of MRSA appear less affected than some others. Optimal conditions for phenotypic susceptibility testing vary between strains, and no single test will detect all resistant strains.[7]

Phenotypic detection of MRSA has been problematic ever since its discovery in the early 1960s. The emergence of low level-resistant MRSA clones acquired in the community has only added to these difficulties.

The detection of the mec A gene or its product, PBP2a, is considered the gold standard,[8] for MRSA confirmation. Recent investigations suggest that disk diffusion using cefoxitin is superior to most previously recommended phenotypic methods, including oxacillin disk-diffusion and oxacillin screen agar testing because Mec A MRSA strains show resistance to both cefoxitin and oxacillin, by contrast, to the majority of mec C MRSA which show resistance to cefoxitin and however show susceptibility to oxacillin.[9] In 2005, the Clinical and Laboratory Standards Institute (CLSI) published zone diameter,[10] breakpoint guidelines for cefoxitin. However, a number of technical issues remain regarding the use of cefoxitin as a predictor for methicillin resistance.


  Materials and Methods Top


Study area

The study was carried out in the Medical Microbiology Laboratory of Aminu Kano Teaching Hospital (AKTH) located in Kano metropolis. Kano State is located in northwest geopolitical zone of Nigeria. It comprises 44 Local Government Area with an estimated population of 9,383,682 and 20,760 km2 in the area.[11] It lies between latitudes 10° 33N to 11° 15N and longitudes 34°CE to 8° 20CE.[11]

Sample collection

A total of 252 nonduplicate samples of staphylococcal isolates were collected from various clinical samples obtained from patients attending AKTH and subcultured onto chocolate agar (Oxoid, UK), MacConkey agar (TM, media, India), and Mannitol Salt Agar (BD BBL™, France).

Bacterial isolation and identification

Staphylococcal isolates from various clinical samples were identified and confirmed using standard bacteriological procedures which Gram stain, catalase test, coagulase test, hemolysis (5% sheep blood) activities, mannitol, and lactose fermentation.[12]

Antibiotic susceptibility test

The susceptibility testing of isolates to six antibiotics was carried out by the disk-diffusion method according to CLSI.[13] Briefly, a bacterial suspension adjusting to 0.5 McFarland was inoculated onto Muller–Hinton agar (Lib-Biotech). The antibiotics (Oxoid, UK) used include gentamicin (10 μg), ciprofloxacin (10 μg), erythromycin (15 μg), tetracycline (30 μg), co-trimoxazole (25 μg), and clindamycin (2 μg). All the plates were incubated at 37°C for overnight, after which the plates were held a few inches above a black, nonreflective surface illuminated with reflected light, and a ruler was used to measure each zone with the unaided eye while viewing the back of the  Petri dish More Details. The result was recorded and was compared with the zone diameter interpretive standard of the CLSI.[13]

S. aureus (ATCC 25923) was used as control strain in every test carried out.

Screening for methicillin-resistant Staphylococcus aureus

Preparation of 0.5 McFarland standard (turbidity standard)

Sulfuric sulfate (1% v/v) standard suspension was used as turbidity standard which was prepared in accordance with the procedure explained by Cheesbrough.[8]

Cefoxitin disk diffusion

Cefoxitin (Oxoid, UK) disk (30 μg) susceptibility testing was performed according to CLSI.[13] Briefly, a bacterial suspension adjusting to 0.5 McFarland was inoculated onto Muller–Hinton agar (Lib-Biotech). A filter paper disk containing 30 μg cefoxitin was placed on the inoculated Muller–Hinton agar. All the plates were incubated at 37°C for overnight, after which the plates were held a few inches above a black, nonreflective surface illuminated with reflected light, and a ruler was used to measure each zone with the unaided eye while viewing the back of the Petri dish. The result was recorded and was compared with the zone diameter interpretive standard of the CLSI.[13]

Oxacillin disk diffusion

Oxacillin (Oxoid, UK) disk (1 μg) susceptibility testing was performed according to CLSI.[13] Briefly, a bacterial suspension adjusted to 0.5 McFarland was inoculated onto Muller–Hinton agar (Lib-Biotech). A filter paper disk containing 1 μg oxacillin was placed on the inoculated Muller–Hinton agar. All the plates were incubated at 37°C for overnight, after which the plates were held a few inches above a black, nonreflective surface illuminated with reflected light, and a ruler was used to measure each zone with the unaided eye while viewing the back of the Petri dish. The result was recorded and was compared with the zone diameter interpretive standard of the CLSI.[13]


  Results Top


The result showed that of the 252 samples collected, high percentage 114 (45.2%) were obtained from patients from the age group of 1–9 years followed by the age groups of < 1 year 59 (23.4%), the least were in the age groups of 50–59 years and >60 years with 2 (0.8%) each. One hundred and twenty-six (50.0%) participants were collected from both males and females. High percentage were obtained from blood culture sample 195 (77.4%) followed by wound swab 29 (11.5%), the least were from semen sample 2 (0.8%) as shown in [Table 1].
Table 1: Demographical distribution of the participants

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All the 252 (100.0%) isolates were found to be Gram-positive cocci in clusters exhibiting the typical grape-like shape, and few pairs were also seen. All isolates were catalase positive and ferment lactose, 151 (59.9%) were coagulase positive to both slide and tube, whereas 38 (15.1%) isolates only were found to be beta hemolytic [Table 2].
Table 2: Biochemical reaction of the positive staphylococcal isolates

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The isolated organisms showed that high sensitivity was found in ciprofloxacin (10 μg) 212 (84.1%) followed by gentamicin (10 μg) and least sensitivity was found in erythromycin (25 μg) [Table 3].
Table 3: Antibiotics susceptibility pattern of the isolated staphylococcal using conventional antibiotics

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The result of the methicillin resistance screening test showed that 52 (20.6%) isolates were resistance to oxacillin (1 μg) while 65 (25.8%) isolates showed resistance to cefoxitin (30 μg) disk-diffusion method [Table 4].
Table 4: Screening of methicillin-resistant Staphylococcus aureus from isolated organisms (n=252)

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The relationship between the participants' demographical factors and MRSA using oxacillin (1 μg) and cefoxitin (30 μg) disk diffusion showed no relationship between any of the variables as P > 0.05 [Table 5] and [Table 6].
Table 5: Relationship between the participant demographical factors and methicillin-resistant Staphylococcus aureus using oxacillin (1 μg) disk-diffusion method

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Table 6: Relationship between the participant demographical factors and methicillin-resistant Staphylococcus aureus using cefoxitin (30 μg) disk-diffusion method

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  Discussion Top


Phenotypic detection of MRSA has been problematic ever since its discovery in the early 1960s. The emergence of low level-resistant MRSA clones acquired in the community has only added to these difficulties.

In this study, high percentage of the staphylococcal isolates were obtained in the age groups of 1–9 and <1 year with 45.2% and 23.4%, respectively, which compressed both neonate and infants that have undeveloped immunity which makes them vulnerable to must bacterial infection, this agree with the finding of Kumurya et al.[14] in the same study area who report the high percentage of 44.0% in aged group 0–10 years, but this disagree with the finding of Abdullahi and Iregbu [15] in their study in Central Nigeria who report the high percentage of 54.7% in those of age groups ≥25 years. The present study reported that both sexes have an equal percentage of 50.0%, this disagree with the finding of Kumurya et al.[14] who reported that males are more infected with these organisms than females. Blood culture 195 (77.4%) yielded the highest proportion staphylococcal isolates in study following by wound swab 29 (11.5), these are in line with finding of Kumurya et al.,[14] who reported that high percentage of these organisms were isolated from blood culture followed by wound swabs but in contrasts with the finding of Abdullahi and Iregbu [15] from Central Nigeria in their study who reported that the high percentage were obtained from wound swab followed by urine and blood culture.

In this study, 59.9% isolates were found to be both bound and free coagulase which are in contrast with the result of Kumurya [16] from previous studies conducted in eight health institutions in six states in Northwestern Nigeria who reported that 100% of the isolates are positive to free coagulase while 96% and 94% isolates are bound coagulase positive to MSSA and MRSA, respectively. In this study, 100% of the isolates showed a positive result in lactose fermentation test, this result is in strong agreement with the finding of Kumurya [16] who reported that 100% of the isolates are ferment lactose by developed pink color colony on the MacConkey agar plates due to its crystal violet and bile salt contains. It was also observed that 61.9% isolates showed ferment mannitol and only 15.1% showed to be beta-hemolysis on blood agar by complete hemolysin the red blood cell on the medium. This is in contrast to previous studies conducted in Zaria by Nneoma and Walter.[17]

The phenotype-based detection of MRSA in the present study involved the cefoxitin (30 μg) and oxacillin (1 μg) disk-diffusion method. Results obtained from this study show that the prevalence of MRSA isolates is 20.6% with cefoxitin (30 μg) and 25.8% with oxacillin (1 μg) using disk-diffusion method. This prevalence is similar to the report of Kesah et al.[18] which recorded 21%–30% in eight African countries including Nigeria. It is also in slighty agreement with the finding of Kumurya [16] which recorded the prevalence of 25.0% in Northwestern Nigeria. However, this prevalence is lower than the previous study of Taiwo et al.[1] in Nigeria where they reported 34.7% prevalence. The prevalence of MRSA using both screened drugs (i.e., cefoxitin 30 μg and oxacillin 1 μg) in this present study was found to be high in age groups ≥60 years (100%) followed by age group 50–59 years (50.5%), 40–49 and 30–39 years (40.0%) each, this may be due to used and misused of antibiotics, this agree with finding of Abdullahi and Iregbu [15] in their study conducted at National Hospital Abuja, Nigeria, who reported the prevalence of 68.0% in the age group of ≥25 years. High prevalence was obtained in female 29.4% using oxacillin disk 1 μg, but using cefoxitin 30 μg, male participants have high prevalence of 25.5%, the reason of this is not clearly understood but is in contrast with the finding of Abdullahi and Iregbu.[15] Throat swab yields high prevalence of MRSA in this study followed by semen and ear swab using cefoxitin 30 μg and oxacillin 1 μg, respectively.

The choice of antibiotics for bacterial treatment is becoming a therapeutic problem. Susceptibility pattern is very essential to determine the future challenges of effective therapy. In this study, antibiotics susceptibility pattern of staphylococci isolates showed that 84.1% were sensitive to ciprofloxacin while 77.4% and 77.0% were susceptible to gentamicin and clindamycin, respectively. The isolates were less sensitive to erythromycin. This study is in agreement with the finding of Nwankwo and Nasiru [19] which recorded high sensitivity of ciprofloxacin (78.9%) and erythromycin (52.4%). This study further agrees with the work done by Kumurya [16] which recorded the high sensitivity of the isolates to ciprofloxacin (96.7%) and clindamycin (80.9%). The susceptibility patterns of S. aureus in this study were compared with data from an international multicenter study in which 21 laboratories in 19 countries (including Nigeria) participated.[20]


  Conclusion Top


This study revealed that routine phenotypic screening of MRSA can still be used for the identification of MRSA, especially in resource-limited areas. However, no single test can guarantee reliable results, and hence, there is a need for using both cefoxitin (30 μg) and oxacillin (1 μg) disk when screening of MRSA phenotypically.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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Taiwo SS, Bamidele M, Omonigbehin EA, Akinsinde KA, Smith SI, Onile BA, et al. Molecular epidemiology of methicillin-resistant Staphylococcus aureus in Ilorin, Nigeria. West Afr J Med 2005;24:100-6.  Back to cited text no. 1
    
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Paterson GK, Harrison EM, Holmes MA. The emergence of mecC methicillin-resistant Staphylococcus aureus. Trends Microbiol 2014;22:42-7.  Back to cited text no. 2
    
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Gurusamy KS, Koti R, Toon CD, Wilson P, Davidson BR. Antibiotic therapy for the treatment of methicillinresistant Staphylococcus aureus (MRSA) infections in surgical wounds. Cochrane Database Syst Rev 2013;19:CD009726.  Back to cited text no. 3
    
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Knox R. Celbenin-resistant staphylococci. Br Med J 1961;1:12-6.  Back to cited text no. 6
    
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Brown DF. Detection of methicillin/oxacillin resistance in staphylococci. J Antimicrob Chemother 2001;48 Suppl 1:65-70.  Back to cited text no. 7
    
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Chambers HF. Methicillin resistance in staphylococci: Molecular and biochemical basis and clinical implications. Clin Microbiol Rev 1997;10:781-91.  Back to cited text no. 8
    
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Swenson JM, Tenover FC; Cefoxitin Disk Study Group. Results of disk diffusion testing with cefoxitin correlate with presence of mecA in Staphylococcus spp. J Clin Microbiol 2005;43:3818-23.  Back to cited text no. 9
    
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Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing. 15th Informational Supplement M100-S15. Wayne, PA: Clinical and Laboratory Standards Institute; 2005.  Back to cited text no. 10
    
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National Population Commission. National Population Commission of Nigeria. Nigerian Demographic and Survey. National Population Commission; 2006.  Back to cited text no. 11
    
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Cheebrough M. District Laboratory Practice in Tropical Countries. Vol. 2. New York, United States of America: Cambridge University Press; 2010. p. 647.  Back to cited text no. 12
    
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Clinical and Laboratory Standard Institute. Performance Standards for Antimicrobial Susceptibility Testing; Twenty – Seven Informational Supplement. M02-A12, M07-A10, and M11-A8. Vol. 27. Wayne, PA: Clinical and Laboratory Standards Institute; 2017. p. 18-22.  Back to cited text no. 13
    
14.
Kumurya AS, Sule H, Abba NA. Phenotypic characters of Staphylococcus aureus isolates from clinical samples in Aminu Kano teaching hospital, Kano, Nigeria. Am J Internal Med 2017;5:74-8.  Back to cited text no. 14
    
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Abdullahi N, Iregbu KC. Methicillin-resistant Staphylococcus aureus in a central Nigeria tertiary hospital. Ann Trop Pathol 2018;9:6-10.  Back to cited text no. 15
  [Full text]  
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Kumurya AS. Characters and antibiotics susceptibility pattern of typical and atypical Staphylococcus aureus isolates from clinical samples in Northwestern Nigeria. Acta Vellit 2015;1:42-9.  Back to cited text no. 16
    
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Nneoma CJ, Walter CJ. Conventional and rapid methods for identification of Staphylococcus aureus from clinical specimens. Am J Biomed Life Sci 2013;1:41-3.  Back to cited text no. 17
    
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Kesah C, Ben Redjeb S, Odugbemi TO, Boye CS, Dosso M, Ndinya Achola JO, et al. Prevalence of methicillin-resistant Staphylococcus aureus in eight African hospitals and Malta. Clin Microbiol Infect 2003;9:153-6.  Back to cited text no. 18
    
19.
Nwankwo EO, Nasiru MS. Antibiotic sensitivity pattern of Staphylococcus aureus from clinical isolates in a tertiary health institution in Kano, Northwestern Nigeria. Pan Afr Med J 2011;8:4-11.  Back to cited text no. 19
    
20.
Zinn CS, Westh H, Rosdahl VT; Sarisa Study Group. An international multicenter study of antimicrobial resistance and typing of hospital Staphylococcus aureus isolates from 21 laboratories in 19 countries or states. Microb Drug Resist 2004;10:160-8.  Back to cited text no. 20
    



 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]



 

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