Objective: To isolate and characterise multidrug resistant strains of Staphylococcus aureus from healthcare workers who are at potential risk of nosocomial infections.
Methods: The observational, cross-sectional study was conducted from November 2014 to April 2015 at different hospitals of Haripur and Abbottabad, Pakistan, and comprised ward and operation theatre staff. The isolates were identified on the basis of microbiological and biochemical tests and further confirmed by polymerase chain reaction. Disc diffusion method was used for antibiotic sensitivity testing, and panton valentine leukocidin and methicillin resistance mecA genes were detected using polymerase chain reaction.
Results: Of 208 isolates, 108(52%) were from the ward staff and 100(48%) were from the operation theatre staff. Overall, 167(80.3%) isolates were positive for Staphylococcus aureus, and 75(36%) were methicillin-resistant Staphylococcus aureus. The number of antibiotic-resistant isolates was 75(45%) cefoxitin, 60(36%) ofloxacin, 152(91%) erythromycin, 52(31%) doxycycline, 127(76%) lincomycin, 53(32%) amoxicillin-clavulanate, 67(40%) ciprofloxacin, and 89(53%) ceftriaxone.
Conclusion: A high number of hospital staff, including those working in operation theatres, were found to be carrying methicillin-resistant Staphylococcus aureus and multidrug resistant strains in their nasal passage that may be a source of infection to patients.
Key Words: Methicillin resistance, Nosocomial infections, Vancomycin, MecA gene, Pvl gene. (JPMA 71: .47; 2021)
Staphylococcus (S.) aureus is one of the most common causes of healthcare-associated bacterial infections among humans, causing a range of illnesses, including skin and soft tissue, vaginal, urethral and intestinal as well as gastrointestinal (GI) infections.1,2 S. aureus hemolyses blood, coagulates plasma and produces extracellular enzymes and toxins that contribute to their virulence.3 S. aureus may also cause many epidemic and endemic infections and result in a high rate of mortality and morbidity.4,5 S. aureus is coagulase-positive which is considered the distinguishing characteristic for this bacterial species from other staphylococci.2
They are normal human commensal, present on skin and nasal passage of a large population. It was found that the number of colonies isolated from nasal samples were higher in number than colonies isolated from skin swab samples, as nose is the main ecological niche of S. aureus.6
One of the leading causes of healthcare-associated infections is methicillin-resistant Staphylococcus aureus (MRSA).7 S. aureus nasal carriage has been identified as a risk factor for the nosocomial infections in general hospital population, surgical patients, general and haemodialysis patients among others.8 MRSA emerged for the first time in the United Kingdom in the early 1960s and since then it is one of the major health problem globally.9
Penicillin was introduced for the first time in the 1940s and at the time of introduction, 95% isolates were susceptible to it.10 Beta lactamase-resistant type of penicillin, called methicillin, was introduced in the 1960s with the hope that it would work better, but soon after its introduction, methicillin-resistant strains emerged and disseminated globally. Nosocomial acquisition of MRSA has been a worldwide problem and a leading cause of healthcare-associated as well as postsurgical infections.10,11
Studies have reported that the MRSA carriage by hospital staff may be a risk of transmission to those receiving treatment.8 During any surgery or injury, skin and mucous membrane barrier may be broken, and act as an entry point of skin bacteria into the body.2 MRSA infections have become more difficult to treat nowadays due to high levels of antibiotic resistance against almost all conventionally used antimicrobial agents.9-11
The current study was planned to isolate, identify and test antibiotic sensitivity of S. aureus strains from those working in operation theatre (OT) or hospitals wards.
Materials and Methods
The observational, cross-sectional study was conducted from November 2014 to April 2015 at three hospitals of Haripur and two of Abbottabad, in the Hazara Division, Pakistan, and comprised full-time ward and OT staff working for at least a year, including physicians, surgeons, nurses, ward boys, cleaners, technicians etc. who volunteered to participate. Those working for less than a year or those not willing to participate were excluded, and so were those who suffered from any sort of skin infection. The study was carried out after approval from the ethics review committee of The University of Haripur and the identities of both the participants and the hospitals were kept confidential.
Nasal samples were collected using sterile cotton swabs. Data such as age, gender, employment and medical history was also recorded. The samples were transported to the microbiology lab of the University of Haripur for further processing.
For the identification of S. aureus, different microbiological and biochemical tests were performed including microscopy, growth and cultural characteristics on selective medium i.e. mannitol salt agar (Oxoid, UK). Biochemical tests included catalase, coagulase, mannitol fermentation tests as well deoxyribonucleic acid-ase (DNAse) test.
All the isolates identified as S. aureus were subjected to disc diffusion assay using different antibiotics, according to the Kirby Bauer method.12 The antibiotics included cefoxitin (FOX), erythromycin (E), ofloxacin (OFX), doxocyclin (DOX), lincomycin (LCM), amoxicillin-clavulanate (AMC), ciprofloxacin (CIP) and ceftriaxone (CRO). Strains resistant to FOX were considered MRSA. Clinical & Laboratory Standards Institute (CLSI) breakpoints were used to determine antibiotic resistance.13
In order to perform molecular characterisation of S. aureus isolates, polymerase chain reaction (PCR) was performed for panton valentine leukocidin (pvl) and methicillin resistance mecA genes, as they are antibiotic resistance and virulence genes respectively. Each PCR reaction consisted of 25µl reaction mixture. Each reaction contained 2µL DNA, 1µl of forward and reverse primers for pvl and mecA genes, 2.5µl magnesium chloride (MgCl2), 2.5µl PCR buffer, 0.5µl deoxyribonucleotide triphosphate (dNTPs), 0.5µL taq polymerase and 13µL ultrapure deionised water. The annealing temperature for both primers was 55o. All samples were run on gel with 100bp ladder; mecA gene product size was 310bp and pvl gene amplification product size was 433bp. Detail of all primers used in the study is given in Table-1.14-16
Of 208 isolates, 108(52%) were from the ward staff and 100(48%) from the OT staff. Overall, 167(80.3%) isolates were positive for S. aureus and 75(36%) were MRSA (Table 2).
The number of antibiotic-resistant isolates were FOX 75(45%), OFX 60(36.%), E 152(91%), DO 52(31%), LCM 127(76%), AMC 53(32%), CIP 67(40%) and CRO 89(53%) (Figure 1).
The number of isolated strains positive for mecA gene was 75 (45%), while 48(29%) were positive for pvl gene (Figure 2).
The findings of the present study are alarming with an overall detection of 80.3% S. aureus and 36.1% MRSA.
S. aureus is one of the most common causes of nosocomial infections worldwide. Penicillin was considered a drug of choice for the treatment of staphylococcal infections at the time of introduction in the 1940s, but, presently, over 90% strains show resistance to pencillin.17
Multidrug resistance (MDR) has been increasing in S. aureus since the introduction of antibiotics in the 1940s. According to the National Nosocomial Infection Surveillance (NNIS), a unit of the Centre for Disease Control and Prevention (CDC), methicillin resistance of S. aureus among hospitals in the United States increased from 2.4% in 1975 to 29% in 1991. MRSA strains were reported for the first time in England in the 1960s.18 Since then its prevalence is on rise and in almost all countries of the world.
Nasal carriage of MDR S. aureus has previously been reported.16 Of concern is their carriage by healthcare professionals who are in direct and close contact with patients receiving treatment at the hospitals. A study in India reported 8% nasal carriage by healthcare professionals.19 Another study performed on healthcare ward staff of a hospital in Rawalpindi, Pakistan, reported 18% detection of S. aureus whereas 1.5% of them were MRSA.20 A recent study conducted in Lahore, Pakistan, reported 23.4% S. aureus with 8% MRSA.21 As such, the current study’s findings represent the highest frequency of S. aureus and MRSA carriage by healthcare professionals in the region.
It is reported in studies that death toll and risk of nosocomial infection and sepsis is higher with MRSA compared to methicillin-sensitive Staphylococcus aureus (MSSA).22,23 Further, it is also reported that antibiotic resistance profiles of MRSA is higher compared to MSSA.24 In our case, 29% strains were carrying the pvl gene, which is also a risk for healthcare workers themselves.
S. aureus amd MRSA prevalence was found to be alarming, as hospital staff may be a cause of both healthcare-associated infections as well as an outbreak by highly resistant strains of S. aureus. Appropriate infection control measures must be ensured at both public and provate sector healthcare facilities.
Acknowledgements: We are grateful to Vice-Chancellor of the University of Haripur for approving financial assistance as well as to the staff and administration of the participating hospitals.
Conflict of Interest: None.
Source of Funding: The University of Haripur and COMSATS University, Islamabad.
1. Melzer M, Eykyn.JS, Gransden RW, Chinn S. Is Methicillin resistant Staphyloccocus aureus more virulent than methicillin susceptible S.aureus? A comparative cohort study of British patients with nosocomial infection and bacteremia. Clin Infect Dis. 2003; 37:1453-60.
2. Harris GL, Foster JS, Richards GR. An introduction to Staphylococcus aureus and techniques for identifying and quantifying S. aureus adhesions in relation to adhesion to biometerials. Eur Cell Mater. 2002; 4:1473-2262.
3. Cox RA. Strategies for the management of health Care staff colonized with epidemic methicillin-resistant Staphylococcus aureus. J Hosp Infect.1998; 35:117-27.
4. Eiff V C, Becker K, Machka K, Stammer H, Peters G. Nasal carriage as a source of Staphylococcus aureus bacteremia. N Engl J Med. 2001; 344:11-6.
5. Coello R, Glynn JR, Gaspar C, Picazo JJ, Fereres J. Risk factors for developing clinical infection with methicillin-resistant Staphylococcus aureus (MRSA) amongst hospital patients initially only colonized with MRSA. J Hosp Infect. 1999; 37:39-46.
6. Williams REO. Healthy carriage of Staphylococcus aureus: its prevalance and importance. Bacteriol Rev.1963; 27:56-71.
7. Huang SS, Yokoe SD, Hinrichsen VL, Spurchise LS, Datta R, Miroshnik I, et al. Impact of routine intensive care unit surveillance cultures and resultant barrier precautions on hospital- wide methicillin-Resistant Staphylococcus aureus bacteremia. Clin Infect Dis. 2006; 43:971-8.
8. Qureshi AH, Rafi S, Qureshi SM, Ali AM. The current susceptibility patterns of MRSA to conventional antistaphylococcal antimicrobials at Rawalpindi. Pak J Med Sci. 2005; 20:361-4.
9. Panlilio AL, Culver DH, Gaynes RP, Banerjee S, Henderson TS, Tolson JS, et al. Methicillin-resistant Staphylococcus aureus in U.S. Hospitals, 1975-1991. Infect Cont Hospit Epidemiol. 1992; 13:582-6.
10. Nwankwo EOK, Sale A, Magagi A, Gabriel I. Methicillin resistant S. aureus (MRSA) and their antibiotic sensitivity pattern in Kano, Nigeria. Afr J Clin Exp Microbiol. 2010; 11:129-36.
11. Kluytmans J, Belkum VA, Verbrugh H. Nasal carriage of Staphylococcus aureus: Epidemiology, underlying mechanisms, and associated risks. Clin Microbiol Rev. 1997; 10:505-20.
12. Bauer AW, Perry DM, Kirby WMM. Single disc antibiotic sensitivity testing of Staphylococci. AMA Arch Intern Med. 1959; 104:208-16.
13. Clinical Laboratory Standards Institute Document Performance Standards for Antimicrobial Susceptibility Testing (2017). 27th Edition.
14. Lina G, Piémont Y, Godail-Gamot F, Bes M, Peter MO, Gauduchon V, etal. Involvement of Panton-Valentine Leukocidin-producing Staphylococcus aureus in primary skin infections and pneumonia. Clin Infect Dis. 1999; 29:1128-32.
15. Zhang K, Sparling J, Chow BL, El-Sayed S, Hussain Z, Church DL, et al. New quadriplex PCR assay for detection of methicillin and mupirocin resistance and simultaneous discrimination of Staphylococcus aureus from coagulase-negative staphylococci. J Clin Microbiol. 2004; 42:4947-55.
16. Poulsen AB, Skov R, Pallesen LV. Detection of methicillin resistance in coagulase-negative staphylococci and in staphylococci directly from simulated blood cultures using the EVIGENE MRSA Detection Kit. J Antimicrob Chemother. 2003; 51:419-21.
17. Ubukata K, Nonoguchi R, Matsuhashi M, Konno M. Expression and inducibility in Staphylococcus aureus of the mecA gene, which encodes a methicillin- resistant S. aureus -specific penicillin-binding protein. J Bacteriol. 1989; 171:2882-5.
18. Adler A, Temper V, Block SC, Abramson N, Moses EA. Panton- valentine leukocdin-producing Staphylococcus aureus. Emerg Infect Dis. 2006; 12:1789-90.
19. Malini J, Harle A S, Padmavathy M, Umapathy B L. Methicillin-resistant Staphylococcus aureus carriage among the health care workers in a tertiary care hospital. J Clinl Diag Res. 2005; 6:791-3.
20. Akhtar NC. Staphylococcal nasal carriage of health care workers. J Coll Phys Surg Pak. 2005; 20:439-43.
21. Asif A, Javed I, Mushtaq S, Anwar S. Nasal carriage of methillin resistant Staphylococcus aureus among tertiary care hospital employees in a non-outbreak setting. Pak J Pathol. 2017; 28:171-5.
22. Melzer M, Eykyn SJ, Grandsden WR, Chinn S. Is methicillin-resistant Staphylococcus aureus more virulent than methicillin-susceptible S. aureus? A comparative cohort study of British patients with nosocomial infection and bacteremia. Clin Infect Dis. 2003; 37:1453-60.
23. F Rozgonyi, E Kocsis, K Kristóf, K Nagy. Is MRSA more virulent than MSSA? Is MRSA more virulent than MSSA? Clin Microbiol Infect. 2007; 13:843-5.
24. Sharma N, Garg KR, Baliga S, Bhat GK. Nosocomial infections and drug susceptibility patterns in methicillin sensitive and methicillin resistant Staphylococcus aureus. J Clin Diagn Res. 2013; 7: 2178-80.