Molecular Typing of Methicillin-Resistant Staphylococcus Aureus - Coursework Example

Molecular Typing of Methicillin Resistant Staphylococcus aureus (MRSA) Staphylococcus aureus (S. aureus) was experimentally characterized and identified in the late 19th century, which epidemiologists have consistently labelled a potential disease causing Gram-positive bacterium (Deurenberg, Kalenic, Friedrich, van Tiel & Stobberingh, 2007). Understanding the genetics of S. aureus is vital because it has been isolated as the causative agent of many diseases that cut across benign infections of the skin to severe infections on wounds after operations (Mehndiratta & Bhalla, 2012). In addition, S. aureus was long renowned for high mortality rate among its victims before the discovery of penicillin in 1940s and the ensuing use of the antibiotics for S. aureus infections (Coombs et al., 2011). Of crucial interest in this context is the dynamic shift in the pathogen’s genetic structure that appeared to create highly pathogenic strains that could no longer be treated by penicillin. Coombs et al. (2011) note that the initial resistant strain of S. aureus was identified in 1940s initially from healthcare institutions and the community later on. This premise has been the core of the challenges in understanding the resistant S. aureus strains in that distinguishing between the community and healthcare strains has increasingly become blurred mainly because of the close genetic similarities. It is further presented by Coombs et al. (2011) that just two years in to the discovery of methicillin in 1962, S. aureus evolved remarkable resistance but through then a scientifically identified gene, the mecA. This gene has been the focal point of MRSA research in terms of genetic typing in that it is believed to promote the methicillin resistance by encoding the 78-kDA penicillin binding protein (PBP) 2a (Deurenberg et al., 2007). The gene is found within the Staphylococcal Cassette Chromosome mec (SCCmec), and research has differentiated SCCmec types (type I-IV) through experiments as presented in the figure below: Figure 1: A Drawing of the Types of SCCmec Source: Adopted from Deurenberg et al. (2007) Of increasingly weighty concern is that the uncontrolled rise in the Methicillin-resistant S. aureus (MRSA) puts more patients at risk. This context has continually called for concerted efforts to innovative MRSA typing techniques to facilitate establishment of reliable surveillance mechanisms that can effectively describe current epidemiological of the pathogen and infection management strategies. As argued by Mehndiratta and Bhalla (2012), the prevailing challenges plaguing MRSA typing revolve around the identification of the most reliable technique that can be effective, and easy to use within affordable means. PVL Polymerase Chain Reaction (PCR) It is imperative that Polymerase chain reaction (PCR) based typing techniques of complex DNA sequencing is a platform for diverse methods of isolating MRSA. The Panton-Valentine leucocin (PVL) has been described as a two-component pore-forming cytotoxin, which research links with skin and soft tissue diseases (Otter, Kearns, French, and Ellington, 2010). The PVL can be present in approximately two percent of S. aureus isolates, and Otter et al. (2010) provides that two genes, lukS-PV and lukF-PV, that are situated on lysogenized bacteriophages encode it and are transcribed together. The PCR phage typing technique follows internationally Staphylococci typing standards that involve classification of the PVL strains (Fey et al., 2003). The PVL-PCR technique involves a careful maintenance of biologically active bateriophages, which can only be maintained in the research reference centres. Considered a fair method of PVL isolation, the PCR based technique bears some degree of reproducibility and strain isolative power (Otter et al., 2010). Additionally, the method is easy to interpret, is affordable, but technically involving and time intensive, but this technique is considered disadvantageous in the context of MRSA because it lacks ability to type many of the resistant strains (Otter et al., 2010) Pulsed-field Gel Electrophoresis (PFGE) The PFGE technique is the the popular typing method of choice in MRSA research (Melin et al., 2009). PFGE is described as a variation of standard agarose gel electrophoresis fundamentally because of the periodic changes on the orientation of the electric fields across the gel (O’Brien, Udo & Grubb, 2006). This premise facilitates modification that enlarges segments that are separable according to size, capability that eliminates possibility of fragments overlapping (O’Brien et al., 2006). Mehndiratta and Bhalla (2012) emphasise the selection of restriction enzymes that they claim is crucial in production of simpler patterns that have reduced band number of bands, which makes the interpretation easy. Epidemiologists and health researchers alike have lauded PFGE technique because of its MRSA typing power for all strains (Melin et al., 2009). Additionally, PFGE has demonstrated high discriminatory capabilities and is considered highly reproducible (Stranden, Frei & Widmer, 2003). However, some of the techniques inherent drawbacks include the high cost of associated reagents and equipment, it technical demands, and the time consumed while conducting the tests (Melin et al., 2009). Despite reservations among some researchers that PFGE is a little challenging in terms of interpreting the results, the publication of conventional guidelines for interpreting bands has typically simplified correlations of epidemiological data (O’Brien et al., 2006). Multi-locus Sequence Typing (MLST) MLST as a typing concept has been largely applied in studies concerning the clonal evolution of MRSA. The technique is a development from the Multilocus Enzyme Electrophoresis (MLEE), and is primarily built on sequence analysis of different housekeeping genes (Deurenberg et al., 2007). The underlying principle underlying MLST is that various sequences of each of the housekeeping gene is differentiated into specified alleles, which are used to define each MRSA strain. (Deurenberg et al., Kalenic, 2007). In practice, MLST is applied together with PCR analysis of SCCmec element that helps in identifying the clonal type of MRSA strains. Various regionally professional bodies have identified standardised MLST protocols, with European Union (EU) Staphylococcal typing network supporting a combined MLST and SCCmec for use as the guiding benchmark for multicenter MRSA surveillance (Mehndiratta & Bhalla, 2012). A notable feature of MLST is that is a substantially expensive approach and considerably labour intensive, and the involvement of many gene targets makes it time consuming (Enright, Day, Davies, Peacock, & Spratt., 2000). Its strength has been identified from its use in isolation of livestock associated MRSA strains and explanations of their transmission between humans and other species (Enright et al., 2000). SCCmec Typing As provided by Tenover, Arbeit and Goering (1997), SCCmec that has mec A and ccr complexes is the factor that has been found to initiate methicillin resistance in S. aureus. This typing technique has been more important in demonstrating the sophistication of the MRSA in that many types of SCCmec and their subtypes have distinguished from MRSA strains (Tenover., 1997). This premise is evinced by the fact that each SCCmec type initiates resistance to different antibiotics, which has however helped characterize the various types of MRSA strains (IWG-SCC, 2009). Proposals have thus been advanced for studies of the mec gene complex, which include its classification and nomenclature (IWG-SCC, 2009). The technique has not demonstrated stand alone reliability and is that proposed that a combination of different approaches like MLST and SCCmec can reliably improve typing for multicenter monitoring the diverse strains of MRSA (Deurenberg et al., 2007). There is an array the current typing techniques favoured by epidemiologists in describing the epidemiological trends MRSA. It has emerged that MRSA typing is a strongly warranted practice towards creation of an effective surveillance for monitory capabilities on the epidemiological patterns of MRSA and disease management planning. The main challenges facing MRSA typing revolve around the selection of the most effective, easy to use, affordable and easily reproducible technique (Mehndiratta & Bhalla, 2012). Although various typing techniques have been discussed, it is apparent that only a limited number of MRSA strains are differentiable. This is considerably so because MRSA evolve from relatively limited clonal size in terms of numbers. Although it has been apparent that every typing method has its advantages and disadvantages, the genotypic techniques appear to be more reliable and desirable in terms of discriminating reliable characteristics for research questions. However, appears that all the methods are cumbersome and technically demanding, and that they are relatively expensive. References Coombs, G.W et al. (2011). Evolution and diversity of community-associated methicillin- resistant Staphylococcus aureus in a geographical region. Biomedical Central Microbiology, 11 (215), 1-12. Deurenberg, R.H., Kalenic, S., Friedrich, A.W., van Tiel, F.H., & Stobberingh, E.E. (2007). Molecular epidemiology of methicillin-resistant Staphylococcus aureus. Communicating Current Research and Educational Topics and Trends in Applied Microbiology, 766-777. Enright, M.C., Day. N.P., Davies, C.E., Peacock, S.J., &Spratt, B.G. (2000). Multilocus sequence typing for characterization of methicillin-resistant and methicillin- susceptable clones of Staphylococcus aureus. Journal of Clinical Microbiology, 38(3), 1008-1015. Fey, et al. (2003). Comparative molecular analysis of community- or hospital-acquired Methicillin-restistant Staphylococcus aureus. Antimicrobial Agents and Chemotherapy, 47 (1), 196-203. International Working Group on the Classification of Staphylococcus Cassette Chromosome Elements (IWG-SCC). (2009). Classification of Staphylococcal cassette chromosome mec (SCCmec): Guidelines for reporting novel SCCmec elements. Antimicrobial Agents and Chemotherapy, 53(12), 4961-4967. Mehndiratta, P.L., & Bhalla, P. (2012). Typing of Methicillin resistant Staphylococcus aureus: A technical review, 30 (1), 16-23. Melin, S et al. (2009). Epidemiological typing of methicillin-resistant Staphylococcus aureus (MRSA): Spa typing versus pulsed-field gel electrophoresis. Scandinavian Journal of Infectious Diseases, 41, 433-439. O’Brien, F.G., Udo, E.E., & Grubb, W.B. (2006). Contour-clamped homogenous electric field electrophoresis of Staphylococcus aureus. National Protocols, 1(6), 3028-3033. Otter, J.A., Kearns, A.M., French, G.L., & Ellington, M.J. (2010). Phanton-Valentine leucocidin-encoding bacteriophage and gene sequence variation in community- associated methicillin-resistant Staphylococcus aureus. Clinical Microbiology and Infection, 16 (1), 68-73. Stranden, A., Frei, R., & Widmer, A. F. (2003). Molecular typing of methicillin-resistant Staphylococcus aureus: Can PCR replace pulsed-field gel electrophoresis. Journal of Clinical Microbiology, 41(7), 3181-3186. Tenover, F.C., Arbeit, R.D., & Goering, R.V. (1997). How to select and interpret molecular strain typing methods for epidemiological studies of bacterial infections: A review of healthcare epidemiologists. Infection Control Hospital Epidemiology, 18, 426-439. Read More