Bacteriological and Molecular Detection of Staphylococcus Aureus and its Resistance to Methicillin among Specimens from Kirkuk Community
Sarah Ahmed Hasan *1, Prof. Dr. Ibrahim Saleh 2, Prof. Dr. Hager Ali3 1 Department of Biology, College of Pure Science, Kirkuk University, IRAQ
2 Dean of College of Education AL-Hawija, Kirkuk University, IRAQ 3 Department of Biology, College of Science, Kirkuk University, IRAQ
*Corresponding author: [email protected] Abstract
Background: The nasal cavity is the main colonization site of Staphylococcus aureus (S. aureus) in human body. Nasal carriage may be a strong risk factor for some serious infection.
Methods: One thousand anterior nasal swabs were collected,700 from healthy adult individuals in Kirkuk university which include 100 swabs from science staff , 300 from science students , 150 swabs from medical and 150 swabs from nursing students and 300 from health care workers with other 50 clinical samples from burn and surgery wound patients in Azadi teaching hospital in Kirkuk city\Iraq the samples were collected by cotton swabs and diagnosed by standard tests in addition to molecular diagnosis and mecA gene detection for the isolates.
Results :)%22.7( 159of isolates from community students and 91 )%30.3( from HCWs were recorded as S.aureus and 3(1.9%) with 4(4.4%) were identified as CA-MRSA and HA-MRSA respectively depending on the standard tests of S.aureus identification, According to the age (18-28) and (51-60) age group were demonstrated the higher rate of S.aureus and MRSA carriage 181 )72.4( and 3 )1.2( respectively. According to sex the male 131 )52.4( and 3 )1.2( were recorded the higher range of S.aureus and MRSA carriage respectively. Also 50 clinical samples were collected by which 25 samples were from burn patients and 25 samples from surgery wound patients : the higher rate of S.aureus and MRSA isolates were from burn patients 13 )%54.2( and 11)%45.8( respectively.all S.aureus and MRSA isolates were demonstrated high resistant towards beta-lactam antibiotics except oxacillin because in present study 10mg of oxacillin was used, while the isolates were recorded low resistant towards non beta-lactam antibiotics and all of them were sensitive towards ciprofloxacin and vancomycin. All the isolates (n = 20) molecularly diagnosed as S.aureus, also all of them expressed specific sequence gene of mecA gene that confirmed all the isolates were MRSA
Conclusions: The continuous monitoring of nasal S.aureus is needed to control MRSA-related infections
Keywords: Staphylococcus aureus, Nasal carriage, Antimicrobial susceptibility, MRSA ,Community , Healthcare workers , PCR , mecA gene.
Staphylococcus aureus is a frequent cause of dangerous health complications increasing rate of morbidity and mortality. It is a part of normal flora colonizes the nasal cavity and skin. About 25–
30% of healthy persons in community are nasal carriers for S.aureus [1, 2]. S.aureus invades tissues in case of epithelial barriers damaging by trauma or surgical interventions [3, 4].
In recent years, S.aureus gradually increased its resistance to various antibiotics. misuse and/or overuse of antimicrobial drugs increased the resistant strains of S.aureus .
It causes both community and hospital infections. The Methicillin resistant S.aureus (MRSA) is considered as commonest causes of hospital acquired infection and considered as common factor in causing failure of management . MRSA strains have appeared in community, not only in hospitals, called as community acquired MRSA (CA-MRSA),these strains have spread among healthy persons
in communities and entered the hospitals .
Cross contamination of Community acquired-MRSA and Hospital acquired MRSA may happened by Healthcare workers (HCWs). In developing countries, HCWs considered as the source of nosocomial infections [9, 10].
The mecA gene is carried by cassette chromosome mec(SCCmec) causes methicillin resistance.
This study done to determine the nasal carriage rate of S.aureus and MRSA among community persons’ and HCWs in addition to clinical samples and to determine antibiotic susceptibility pattern of the isolates and molecular diagnosis with mecA gene detection of the isolates.
Materials and methods Samples
From January 2012 to May 2012,one thousand anterior nasal swabs were collected,700 from healthy adult individuals in Kirkuk university which include 100 swabs from science staff , 300 from science students , 150 swabs from medical and 150 swabs from nursing students and 300 from health care workers with other 50 clinical samples from burn and surgery wound patients in Azadi teaching hospital in Kirkuk city\Iraq the samples were collected by cotton swabs and then transferred to laboratory ,cultured on mannitol salt agar and blood agar media , all culture plates incubated at 37c for 24hr.
Diagnosis of Staph. aureus
Staph. aureus isolates were identified by using standard microbiological methods which include colony morphology on blood and mannitol salt agar , Gram stain , Catalase , DNAase and Coagulase test(slide and tube method).
The collected isolates were stored at 20c in brian-heart infusion containing 15% glycerol utile use [12,13].
Detection of MRSA
Detection of MRSA isolates was carried out by using disk diffusion test . A sterile swab was dipped in an Staph. aureus suspension (McFarland standard 0.5) and plated on to muller – hinton agar, methicillin/ oxacillin disks (10mg from Bioanalyes company )
Drug Susceptibility Tests. The antimicrobial susceptibility of S. aureus isolates was assessed by disk-diffusion tests, according to the Clinical Laboratory Standards Institute guidelines . The following antimicrobial discs were used: Antibiotic susceptibility test was done on Mueller-Hinton agar (Oxoid) by Kirby– Bauer method. 19 antibiotic discs, Ampicillin, Penicillin, Oxacillin (10mg), Amoxicillin, Cephalothin ,Cefoxitin ,Cloxacillin ,Cefotaxime ,Cefexime, Erythromycin, Lincomycin ,Trimethoprim , Chloramphenicol, Tetracycline Amikacin, Rifampicin, Vancomycin, Gentamycin and Ciprofloxacin
S. aureus ATCC 25923 was used as the internal control in each run of the test.
It made by version 6 of Graph Pad Prism software.
MRSA chromosomal DNA isolation
Total genomic DNA was extracted from only S.aureus isolates were identified as ethecillin/oxacillin resistant by disk diffusion using Genomic DNA Mini kit (Geneaid;USA).The DNA concentration has
been determined by measuring absorbance of the sample at 260 nm using spectrophotometer ,then resolved by gel electrophoresis in 0.8% agarose gel in 1xTBE buffer at 60 volt for 30 min. ,visualized under Uv light and photographed using a high resolution digital camera .
PCR amplification Primer selection
Table -1 list the primers were selected to detect the 16SrRNA gene and mecA gene (Bioneer: USA), according to .
Table-1 : primers used in this study
Size(bp) Target gene
1267 Staphylococcus aureus
16SrRNA 5-CGA TTC CCT TAG TAG CGG CG-
3-CCA ATC GCA CGC TTC GCC.5 Sauf 234
1339 mec A gene
5-GTG GAA TTG GCC AATACA GG- 3
3-TGA GTT CTG CAG TAC CGG AI- 5
PCR protocol (Bioneer:USA) PCR premix kit: Table-2 list the PCR premix component Table-2: PCR component volume
Reaction size components
1U 250mM 10mM 30mM 1.5mM Taq DNA Polymerase
( dNTPs )
(pH9.0) Tris- Hcl Kcl
PCR mixture reaction
According to the kit procedure a mixture of a total of 20 uL reaction volume was prepared as in table- 3
Table-3:PCR reaction mixture
Final concentration Volume ul
Final reaction content
50 pmole 50 pmole
50 ng 3ul
2ul 2ul 5ul 8u Master mix
Forward primer Reverse primer Template DNA Sterile de ionized water
The amplification of DNA were performed in thermocycler programmed as in table-4 Table-4:Steps of thermo cycler program
No.of cycles Time
94 c Initial denaturation
30 30 sec
94 c Denaturation
30 sec 55 c
45 sec 72 c
1 5 min
72 c Extra incubation
The amplification products were resolved by electrophoresis in 1.5% agarose gel at 90 volt for 50 min , gels stained with ethidium bromide visualized under UV light which appeared as light fluorescent band and photographed using a high resolution digital camera.
700 samples were collected from community students and 300 samples from healthcare workers at Azadi teaching hospital in Kirkuk city, Iraq.)%22.7( 159of isolates from community students and 91
)%30.3 from hospital staff were identified as S.aureus and 3(1.9%) with 4(4.4%) were identified as CA-MRSA and HA-MRSA respectively depending on standard tests of S.aureus identification (Table 5).
Table5 : Distribution of Nasal carriage S.aureus and MRSA isolates among community and HCWs samples
NO. of nasal carriage MRSA(%) NO. of nasal
carriage S.aureus(%) Total number
( 3 )%1.9
( 159 )%22.7
Community persons’ 700
( 4 )%4.4
( 91 )%30.3
300 Health care workers
( 7 )%2.8
( 250 )%25 1000
The higher rate of nasal carriage of S.aureus were recorded among medical students40 ( 26.6% ) and the CA-MRSA were higher among science college staff 2( 9.5% ) (Table 6)
Table 6 : Distribution of Nasal carriage S.aureus and MRSA isolates among community groups NO. of nasal
carriage MRSA(%) NO. of nasal
carriage S.aureus(%) SEX
0 0 36
%59 25 %41 150
male 150 female Science college students
1 0 21
%52.5 19 %47.5 71
male 79 female Medical college students
40 %26.6 150
0 0 12
%31.6 26 %68.4 51
male 99 female Nursing college students
1 1 11
10 %47.6 54
male 46 female Science college staff
374 female Total Number
159 %22.7 700
while the nurses were recorded the higher carriage rate of S.aureus among health care workers 47 ( 31.5% ) and the laboratory technicians were demonstrated the higher carriage of HA-MRSA 2 ( 10% ) (Table 7).
Table 7 : Distribution of Nasal carriage S.aureus and MRSA among HCWs
NO. of nasal carriage MRSA(%) NO. of nasal
carriage S.aureus(%) Health Care Workers SEX
- - 1
5 female Doctors
1 1 11
38 female Laboratory technicians
1 1 26
71 female Nurses
- - 13
33 female Clean workers
2 2 51
147 female Total Number
91 30.33 % 300
According to the age (18-28) and (51-60) age group were demonstrated the higher rate of S.aureus
and MRSA carriage 181 )72.4( and 3 )1.2( respectively.(Table 8)
Table8 : Prevalence of Nasal carriage S.aureus and MRSA isolates according to their age NO. of nasal
carriage MRSA(%) NO. of nasal
carriage S.aureus(%) Total
number Age groups
( 2 )0.8
( 181 )72.4
( 2 )0.8
( 24 )9.6
0 ( 29
( 3 )1.2
( 16 )6.4
- 60 51
( 7 )2.2
( 250 )25
According to sex the male 131 )52.4( and 3 )1.2( were recorded the higher range of S.aureus and MRSA carriage respectively. (Table 9)
Table9 : Prevalence of Nasal carriage S.aureus and MRSA isolates according to their sex NO. of nasal
carriage MRSA(%) NO. of nasal
carriage S.aureus(%) Total
( 4 )1.6
( 131 )52.4
( 3 )1.2
( 119 )47.6
( 7 )2.8
( 250 )25
In the present study, also 50 clinical samples were collected by which 25 samples were from burn patients and 25 samples from surgery wound patients : the higher rate of S.aureus and MRSA isolates were from burn patients 13 )%54.2( and 11)%45.8( respectively. (Table 10)
Table 10 : Distribution of Nasal carriage S.aureus and MRSA isolates among clinical samples NO. of nasal
carriage MRSA(%) NO. of nasal
carriage S.aureus(%) Total
number Clinical samples
( 8 )%33.3
( 13 )%54.2
25 Burn patients
( 5 )%20.8
( 11 )%45.8
25 Surgery wound patients
( 13 )%54.1
( 24 )%48
all S.aureus and MRSA isolates were demonstrated high resistant towards beta-lactam antibiotics except oxacillin because in present study 10mg of oxacillin was used, while the isolates were recorded low resistant towards non beta-lactam antibiotics and all of them were sensitive towards
ciprofloxacin and vancomycin. (Table 11,12).
Table11 : Nasal carriage S.aureus resistance towards antibiotics according to their source Source of
ANTIBIOTICS NASAL ISOLATES
=159 n HCW
=91 n Burn & surgery
(10 )%10,9 (5
Chloram phenicol (9
Table 12: Nasal carriage MRSA resistance towards antibiotics according to their source
ANTIBIOTICS NASAL ISOLATES
=3 n HCW
=4 n Community
Beta lactam antibiotics (3
(1 )%25 (4
(1 )%25 (6
(1 )%25 (4
All the isolates (n = 20) molecularly diagnosed as S.aureus, also all of them expressed specific sequence gene of mecA gene that confirmed all the isolates were MRSA(Fig.1,2).
Figure 1- PCR product of ((Sauf 236 , SauR 1501) gene for S.aureus identification by gel electrophoresis. Lane M: 100bp DNA ladder. Lane 1-3: CA-MRSA 4-7: HA-MRSA 8-14:
Figure 2- PCR product of (MR2 , MR1) gene for mecA gene detection by gel electrophoresis.
Lane M: 100bp DNA ladder. Lane 1-3: CA-MRSA 4-7: HA-MRSA 8-14: Clinical isolates.
The distribution of S.aureus and MRSA among community persons’ and HCWs were ( 22.7% , 30.3%) and (1.9% , 4.4%) respectively.
These findings agreed with many studies ; in a study in China ,prevalence of S.aureus and MRSA carriage in community were 24.7%and (1.4%) respectively.
In another study in Nepal ,prevalence of MRSA nasal carriage was 4.6 % among HCWs . Other studies in Nepal have demonstrated 20.37 – 43.8 % nasal carriage rate of S.aurues among HCWs [18–21] and agreed with study in Iraq in which the prevalence of MRSA was 4.2% . In contrast ,some studies didn’t agree with the present study , in Jordan ,rate of nasal MRSA was 10.1% among HCWs , 73% among healthcare workers from Saudi Arabia . in Northern China (16.5%), of which 0.3% were MRSA and in adults in community settings in Taiwan (22.1%) [25, 26]. These variations in the rate of S.aureus nasal carriage among countries may be because of the differences in culturing, geographical distribution, diagnostic techniques and sampling used in studies.[27-30]
The prevalence of S. aureus and MRSA were higher in male than in female and in 18-28 , 51-60 age group respectively , these findings agreed with many studies [31, 32].
The rate of S. aureus and MRSA were higher in burn patients than in surgery wound patients ,this agreed with many studies .[33,34].
In present study , S. aureus and MRSA were demonstrated higher resistance towards “Beta-lactam antibiotics” group than “non Beta-lactam” group, by β-lactamase and penicillin-binding proteins
production and mutations effect on the permeability of bacterial outer membrane toward these antibiotics also through alterations in the targets of drugs.[27-29],with exception in oxacillin because in present study oxacillin 10mg was used,this agreed with study of Norfarid et al , ciprofloxacin and vancomycin were the most affective antibiotics in present study ,no resistance demonstrated towards both of them , many studies demonstrated various rate of bacterial resistant like Olayemi etal , Norfarid et al ,Nagi et al and Carvalho et al [35-38] ,the differentiations among the studies may be because of hygienic culture of studied population and their practices with having these antibiotics and kind of clinical samples that were taken in each studies [27-29].
All the isolates (n = 20) molecularly diagnosed as S.aureus, also all of them expressed specific sequence gene of mecA gene that confirmed all the isolates were MRSA ,molecular detections consider as most sensitive techniques at both genus and species level of S.aureus detection with 100% accuracy in MRSA detection, when compared with the classical tests in S.aureus identification.
To control the nasal colonization of S.aureus and MRSA among healthcare workers, continuous screening program ,infection control measures and treatment of MRSA positive healthcare workers in addition to control in the frequency of their exposure with the vulnerable patients.
Such studies can provide information on the spread or rate of nasal colonization of S.aureus and MRSA and detect new strains in hospitals and communities. Active surveillance of MRSA in health care settings is highly recommended.
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