METHICILLIN-RESISTANT STAPHYLOCOCCUS AUREUS (MRSA)

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Methicillin resistant Staphylococcus aureus (MRSA) is defined as a pathogenic Staphylococcus aureus strain that is resistant to methicillin and other beta-lactam antibiotics such as oxacillin. MRSA is a highly contagious strain of the staphylococci bacteria, which cause a number of infections in human population. MRSA strain cause community-acquired methicillin-resistant S. aureus (CA-MRSA) infection and hospital-acquired methicillin-resistant S. aureus (HA-MRSA) infections. Their occurrence has been reported in many parts of the world. MRSA cause a wide variety of deep tissue infections, including osteomyelitis, arthritis, endocarditis, skin infections, and pneumonia. Methicillin resistance was found in the strains of Staphylococcus aureus, soon after the introduction of methicillin into clinical medicine in the 1960s. Methicillin is a beta-lactam antibiotic that bears similar structure with the penicillins because they both contain beta-lactam ring in their structure. Irrespective of their potent antimicrobial activity, methicillin resistance was found in some strains of Staphylococcus aureus, and MRSA cause significant infections in both the hospital and community settings. The resistance of Staphylococcus aureusto the antibiotic methicillin has been associated to a chromosomal mecA gene that specifies the production of an abnormal penicillin-binding-protein (PBP) in the organism.

Penicillin-binding-proteins (PBPs) are membrane-bound enzymes that catalyze the transpeptidation reaction that is necessary for cross-linkage of peptidoglycan layer in both Gram-positive and Gram-negative bacteria. This transpeptidation reaction is critical for the formation of a solid cell wall in bacteria. MRSA also include S. aureus strains that are oxacillin resistant and not only methicillin resistant. MRSA and/or oxacillin-resistant S. aureus strains are multiply resistant to some commonly available antibiotics including erythromycin, clindamycin, tetracycline, cephalosporins and carbapenems. However, MRSA strains are usually susceptible to glycopeptides, vancomycin and teicoplanin. These antibiotics such as teicoplanin, glycopeptides and vancomycin are the only drug of choice for the treatment of severe infections due to MRSA. Some strains of MRSA also remain susceptible to some non-beta-lactam agents including fluoroquinolones, sulphamethoxazole-trimethoprim, and gentamicin. But reports of MRSA strains with decreased susceptibility to vancomycin (minimum inhibitory concentration [MIC], >8 mg/ml) have been observed in some MRSA strains.

The Clinical and Laboratory Standard Institute (CLSI), formerly National Committee for Clinical Laboratory Standards (NCCLS) recommends the use of oxacillin disk (1 µg or 5 µg) or methicillin disk for the screening of S. aureus isolates for the detection of resistance to methicillin/oxacillin (Table 1). Table 9.4 shows the antibiotic breakpoints for the phenotypic screening of S. aureus isolates for the presence of mecA genes. Nevertheless, the accurate detection of oxacillin/methicillin resistance in potential pathogenic S. aureus strains suspected to be MRSA strains can be difficult due to the presence of two subpopulations of MRSA isolates (i.e. MRSA heterosusceptible strains and MRSA heteroresistant strain). While heterosusceptible MRSA strains are susceptible to methicillin/oxacillin, the later (heteroresistant strains) are resistant; and both strains can coexist within a particular culture plate (Figure 1).

Table 1. MIC and inhibition zone diameter breakpoints for disk diffusion test for MRSA detection

            MICsOxacillin susceptibleOxacillin intermediateOxacillin resistant
S. aureus< 2 mg/mlno intermediate MICMIC > 4 mg /ml
CoNS*< 0.25 mg /mlNo intermediate MICMIC > 0.5 mg /ml
    
Zone sizesOxacillin SusceptibleOxacillin intermediateOxacillin Resistant
    
S. aureus> 13 mm11-12 mm< 10 mm
CoNS*> 18 mmno intermediate zone< 17 mm
*CoNS = Coagulase negative Staphylococcus aureus
Figure 1. Illustration of disk diffusion test used for the detection of MRSA. In this image, the surface of a Mueller-Hinton agar plate was inoculated with a strain of S. aureus suspected of being methicillin-resistant. Though the zone of inhibition is at the borderline for resistance (18 mm), the presence of small colonies within the zone of inhibition (arrows) indicates the presence of heteroresistant strains of S. aureus. The interpretation here, therefore, is “methicillin-resistant” staphylococci, even though the zone diameter appears to be adequate. The detection of the heteroresistant strains in disk diffusion test indicates that minimum inhibitory concentration (MIC) studies are required to confirm the presence of MRSA strains.

All S. aureus cells in a culture plate may harbour the genetic information that is responsible for S. aureus resistance to oxacillin/methicillin but only a small number of these cells can actually express the resistance in vitro. This phenomenon is termed “heteroresistance” and it only occurs in staphylococci that are resistant to penicillinase-stable penicillins such as oxacillin. However, amplification tests like those based on the polymerase chain reaction (PCR) technique can detect the mecA gene that is responsible for S. aureus resistance to oxacillin/methicillin. PCR gene amplification test and other genotypic tests for MRSA detection helps to confirm oxacillin/methicillin resistance caused by mecA gene in pathogenic Staphylococcus species.

Heteroresistance is a problem for clinical microbiology laboratory personnel who look out for MRSA strains from clinical specimens. This is because S. aureus cells expressing heteroresistance may grow more slowly than the susceptible S. aureus population in a culture. They are not easily inhibited by oxacillin. S. aureus isolates being tested against oxacillin, methicillin, or nafcillin should be incubated at 37°C for complete 24 hours incubation before taking the antibiogram and/or culture reading. The breakpoints for S. aureus are different from those for coagulase-negative staphylococci (CoNS) such as S. epidermidis which is usually the most common CoNS isolated from clinical samples (Table 1). It is also noteworthy that methicillin-susceptible S. aureus (MSSA) can become MRSA through the acquisition of staphylococcal chromosome cassette mec (SCCmec), which contains mecA gene (the methicillin resistance determinant gene). Gene acquisition of SCCmec gene is usually through genetic transfer mechanisms such as conjugation, transduction or transformation.

FURTHER READING

Ashutosh Kar (2008). Pharmaceutical Microbiology, 1st edition. New Age International Publishers: New Delhi, India. 

Block S.S (2001). Disinfection, sterilization and preservation. 5th edition. Lippincott Williams & Wilkins, Philadelphia and London.

Courvalin P, Leclercq R and Rice L.B (2010). Antibiogram. ESKA Publishing, ASM Press, Canada.

Denyer S.P., Hodges N.A and Gorman S.P (2004). Hugo & Russell’s Pharmaceutical Microbiology. 7th ed. Blackwell Publishing Company, USA. Pp.152-172.

Ejikeugwu Chika, Iroha Ifeanyichukwu, Adikwu Michael and Esimone Charles (2013). Susceptibility and Detection of Extended Spectrum β-Lactamase Enzymes from Otitis Media Pathogens. American Journal of Infectious Diseases. 9(1):24-29.

Finch R.G, Greenwood D, Norrby R and Whitley R (2002). Antibiotic and chemotherapy, 8th edition. Churchill Livingstone, London and Edinburg.

Russell A.D and Chopra I (1996). Understanding antibacterial action and resistance. 2nd edition. Ellis Horwood Publishers, New York, USA.

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