Prevalence of Methicillin Resistance in Staphylococcus species Isolated from Various Pus Samples

Introduction

Pus is a yellow-to-white fluid comprised of dead WBCs, cellular debris and necrotic tissue that is produced following trauma, burn injuries, surgical procedures, by the human skin and soft tissue infections (SSTI).¹ Antibacterial resistance has become a fast emerging process with the resistance being transferred from bacteria to bacteria.² Staphylococcus aureus is a Gram positive coccus that accounts for 20-40% of the infections, causing opportunistic infections like toxic shock syndrome, Staphylococcal scalded skin syndrome. It is known to be resistant to the drug methicillin by producing altered penicillin binding protein PbP2a.² Methicillin resistance, first reported in 1960, has become a devastating cause of infection in hospital and intensive care units, that is now being observed even in healthy individuals through nosocomial and community acquired routes.³ Coagulase negative Staphylococci (CoNS) strains such as S. epidermidis, S. haemolyticus are some of the opportunistic pathogens that are also responsible for nosocomial and community acquired infections worldwide.⁴ CoNS being a skin colonizer has been frequently known to cause cardiovascular, joint, blood stream and various other infections with a high proportion of isolates being resistant to methicillin.⁵

Originally most of the Staphylococcal strains were sensitive to Penicillin, a β-Lactam antibiotic and was extensively used for a variety of infections up to 1940s.^6,7 Following its continuous usage, the Staphylococcal strains started developing resistance to Penicillin due to the production of the enzyme Penicillinase.⁷ The enzyme penicillinase is also called ‘β Lactamase’ and acts by inactivating the β Lactam ring of the antibiotic penicillin.7 Four types of β Lactamases (Type A- Type D) are produced by Staphylococcal species.⁷ The β Lactamase enzyme is plasmid coded, transmitted by transduction or conjugation among the different species of Staphylococci, leading to emergence of resistant strains.⁷

To counter the effect of β Lactamase, a semi-synthetic penicillin variant called Methicillin was developed.⁶ Methicillin, Oxacilin, Dicloxicillin, Nafcicillin were classified under Penicillinase resistant penicillins.⁶ The mode of action for Methicillin is through the Penicillin Binding Protein (PBP) that is present on the cell wall of Staphylococcal species.⁷

However, Staphylococcal strains developed resistance to methicillin as well by producing altered Penicillin Binding Protein (PBP2a).⁷ This new protein is encoded by the Staphylococcal species due to the presence of gene by name Mec A. The strains that possess Mec A gene are considered as resistant strains and are known as Methicillin Resistant Staphylococcus aureus (MRSA).⁷ The mec-A gene is transmitted chromosomally and can result in outbreaks of MRSA hospital infection.⁷

MRSA strains after acquiring Mec A gene are rendered resistant to all class of β Lactam antibiotics, aminoglycosides and fluoroquinolones. Vancomycin or Teicoplanin are used in the treatment of MRSA infections.⁷ Methicillin resistant S. epidermidis has also been detected in various infections and is more common among patients who have undergone prosthetic heart valve surgery.⁷

Methicillin resistant strains are an important cause of wound infections, hospital acquired infections and may result in epidemics of hospital cross infections. These strains are named ‘Community Acquired’ (CA-MRSA) and ‘Hospital Acquired’ (HA-MRSA).⁷ The strains restricted to hospital are known as HA-MRSA which is one of the cause for nosocomial infections.

Hospital personnel harbouring MRSA are the chief source of nosocomial infection. These strains can cause both minor and systemic Staphylococcal infections.⁷

Coagulase Negative Staphylococci (CoNS) are known to be colonizers of the human skin, but are true pathogens in cases of infections involving bloodstream, joints, intravascular catheters and cardiovascular system.⁵ The common CoNs species among whom Methicillin has been detected include S. epidermidis, S. haemolyticus, S. warneri, S. lugdunensis, S. hominis and a few other un-typeable strains.⁸ MR CoNS have simultaneously shown resistance to various other antibiotics like Gentamycin, Chloramphenicol, Clindamycin and Trimethoprim - sulphamethoxazole. CoNS have the ability to form slime on the surfaces of medical devices, which assists multi drug resistant strains to colonize in the hospital, thus making it a reservoir for the spread of antimicrobial resistance.⁹ Long term usage of indwelling catheter, patient illness like burns, admission to intensive care unit may result in CoNS bacteraemia.⁹

Materials and Methods

Sample collection

The sample was collected prior to institution of antibiotic therapy. The infected wound was debrided, wound was cleansed with normal saline. The skin /mucosal surface was debrided for proper wound preparation. Viable tissue was sampled rather than superficial debris using a sterile swab.

Aspirates or biopsy samples were collected as alternative to swabs. Preferably two samples (aspirates/swabs/ biopsy specimen) were collected at the time of sample collection.

Closed abscess

Disinfection process followed for blood culture collection was performed. The material was aspirated with needle & syringe. If any difficulty in aspiration was encountered, non-bacteriostatic saline was injected subcutaneously and aspiration was attempted. The contents were collected and placed in a sterile container.

If the wound was dry, the specimen was collected with cotton tipped swabs moistened with sterile non bacteriostatic saline.

The wound margins and superficial areas were thoroughly cleansed with sterile normal saline. Superficial exudates and debris were removed using a scalpel. The wound was swabbed at the base covering the exudates and necrotic areas as well.

Sample processing

Gross examination: The sample was inspected for colour, consistency, blood tinge and other details.

Microscopy: Gram’s stain and ZN stain were performed and the smear was inspected for the presence of pus cells, epithelial cells and the type of organism was noted.

Culture: The second swab was used for aerobic culture on Blood agar and MacConkey agar. The plates were incubated at 37o C for 24-48 hrs.

At the end of incubation period, the plates were inspected for the colonies. The colony count was noted and Gram’s stain was performed to confirm if the organism was same as that observed in direct smear. β haemolytic colonies for Staphylococcal species were noted. Coagulase test was performed to identify Staphylococcal species up to species level.

The colonies were further inoculated on various biochemical reactions and the organism(s) were identified up to the genus level.

The identified colonies were further subjected to Antibiotic susceptibility testing (AST) by Kirby Bauer’s method.

Methicillin resistance was detected using Cefoxitin (CX) antibiotic disc. Staphylococcal strains resistant to Cefoxitin were recorded as Methicillin resistant strains.

Results

A total of 50 samples were processed aerobically. Out of 50 samples, 48 showed growth which constituted 96% while 4%, i.e. two samples showed no growth (Table 1).

Out of the 48 samples that showed growth, 46% , i.e. 22 Staphylococcus species isolated were S. aureus and 23%, i.e. 11 were Coagulase Negative Staphylococcal species (CoNS) (Table 2)

Table 3 shows that 77% of the S. aureus isolates are Methicillin resistant and 9% of Coagulase Negative species are Methicillin resistant. On conducting further studies on the 22 S. aureus species isolated, 17 isolates were resistant to methicillin, which were counted as MRSA & one strain out of 11 CoNS were methicillin resistant which was about 9% (Table 3, Figure 1).

Regarding the prevalence of MRSA based on age, 40% prevalence was seen among males and females in the age category of 25-45 years & 45-65 years. Among patients aged above 65 years, a prevalence of 29% was noted (Table 4).

Discussion

In the present study, 96% of the pus samples showed growth whereas 4% of the samples did not show growth of any pathogen. A study by Trojan et al. in Punjab, India, showed 60% bacterial growth and 57% of the pus samples were negative for growth. The same study by Trojan et al. also revealed 21% of the isolates to be S. aureus which is consistent with the present study results (17%).¹

A study by Mita Wadekar et al. isolated 48% MRSA strains and 10 CoNS strains with no resistance to methicillin.² The present study isolated 45% Staphylococcus aureus and 11 strains of CoNS, out of which only one strain was resistant to methicillin.

Local and systemic infections can result in pus formation which could have polymicrobial or monomicrobial etiology.² S. aureus was the most common Gram positive organism isolated in a study by Mita D Wadekar et al. which is consistent with the present study findings.²

A study by Olson et al. reported 47% methicillin resistance in S. epidermidis and 29% in S. aureus isolates which has been responsible to the raising trend of ocular infections.³

In the present study, 77% of S. aureus isolates were MRSA and 11% of CoNS were MRSA isolates. This is in coherence with the study by Olson R et al. pointing to the fact that CoNS being a part of the skin flora can contribute to the raising trend of multi drug resistant infections.³ The chances of MRSA infection showed an increasing trend among patients aged 60 years and above, whereas the present study showed higher incidence of MRSA infection among ages ranging between 25-45 years, which is quite conflicting with the results reported by Olson R et al. ³

A study by Koksal et al. in Turkey isolated 67% MRCoNS and found that that methicillin resistance was higher among strains which had slime production.⁹ The present study isolated only 11% out of the total CoNS strains isolated which does not match with the findings of the study conducted in Turkey.

Conclusion

Staphylococcus aureus has been demonstrating an increasing trend towards becoming a multidrug resistant strain as majority of the strains isolated in this study were MRSA. Pyogenic wound infections in a tertiary care hospital requires strict monitoring of the patients infected with drug resistant strains like MRSA and strict patient care should be followed till the patient recovers completely. Prior to starting treatment in pyogenic infections, it is important to screen the isolated organisms for the prevalent multi drug resistant mechanisms and then consider the empirical therapy. This reduces the cost of treatment, duration of stay for the patient. Irrational use of antibiotics in tertiary care hospitals should be avoided, and antibiotic stewardship should be implemented strictly in various departments to prevent the spread of infection.

Conflict of interest

None

Funding

Rajiv Gandhi University of Health Sciences Short Term Research Project for Undergraduates.

Acknowledgements

Very thankful to RGUHS Research wing for encouraging Undergraduate Short term projects. We are also thankful to St. Alphonsa Institute of Allied Health sciences