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Indian Journal of Medical Microbiology, Vol. 30, No. 1, January-March, 2012, pp. 16-23 Review Article Typing of Methicillin resistant Staphylococcus aureus: A technical review PL Mehndiratta, P Bhalla Department of Microbiology, Maulana Azad Medical College, New Delhi - 110 002, India Correspondence Address: Date of Acceptance: 11-Dec-2011 Code Number: mb12004 PMID: 22361755 DOI: 10.4103/0255-0857.93015 Abstract Increasing prevalence of Methicillin-resistant Staphylococcus aureus (MRSA) worldwide is a growing public health concern. MRSA typing is an essential component of an effective surveillance system to describe epidemiological trends and infection control strategies. Current challenges for MRSA typing are focused on selecting the most appropriate technique in terms of efficiency, reliability, ease of performance and cost involved. This review summarises the available information on application, potential and problems of various typing techniques in discriminating the strains and understanding the epidemiology of MRSA strains. The phenotypic methods in general are easier to perform, easier to interpret, cost effective and are widely available, however less discriminatory. The genotypic methods are expensive and technically demanding, however more discriminatory. Newer technologies involving sequencing of various genes are coming up as broadly applicable and high throughput typing systems. Still there is no consensus regarding the single best method for typing of MRSA strains. Phage typing is recommended as first line approach in epidemiological investigation of MRSA strains. PFGE remains the gold standard for characterisation of outbreak strains. DNA sequencing methods including MLST, spa typing, SCCmec typing and toxin gene profile typing are more practical methods for detecting evolutionary changes and transmission events. The choice of typing technique further depends on the purpose of the study, the facilities available and the utility of data generated to answer a desirable research question. A need for harmonisation of typing techniques by following standard protocols is emphasised to establish surveillance networks and facilitate global MRSA control. Keywords: Epidemiology of Methicillin-resistant Staphylococcus aureus (MRSA), MRSA typing, surveillance of MRSA, typing techniques Introduction Methicillin-resistant Staphylococcus aureus (MRSA) strains were identified as early as 1961 soon after the introduction of methicillin in clinical settings. [1] Subsequently, MRSA have emerged world wide as a major cause of nosocomial infections. [2],[3] . A considerable increase in the prevalence of MRSA has been observed globally during the last decade. [4],[5] Infections due to MRSA are difficult to treat because of the restricted spectrum of antimicrobials of proven efficacy. In view of this and the consequent implications for mortality and morbidity, it is important to control the spread of MRSA. Effective control measures are dependent on a thorough knowledge of organism′s epidemiology. A prerequisite for a successful epidemiological investigation is a reliable indicator of the relationship between the organisms isolated, in other words a typing scheme. [6] The typing techniques are used in tracking sources, pathways of spreading infections and studying the population genetics. MRSA typing is essential for the establishment of national and international surveillance networks. In MRSA typing both phenotypic and genotypic methods are used. These methods vary greatly in their discriminatory power, reproducibility of the results, and the cost and efforts required. A typing technique is considered successful when it is simple, inexpensive, reproducible, with sufficient discriminatory power and is widely available. [6] A survey of the literature shows that there is no consensus regarding the best method to use for typing of MRSA strains. Application of any typing method requires careful assessment of its suitability and an individual approach depending upon the purpose of the study. The aim of the review is to discuss the currently available techniques for typing of MRSA, their utility in discrimination of strains and their role in clinical application aiding epidemiologists, researchers and infection control professionals to understand the epidemiology of MRSA strains and implementing the infection control measures to contain spread of infections due to these organisms. Typing Methods A wide variety of typing systems including phenotypic and genotypic methods are available for typing of MRSA strains.Phenotypic Methods Antibiogram typing Antibiogram typing involves comparison of susceptibilities of isolates to a range of antibiotics. Isolates which differ in their susceptibilities to antibiotics are considered as different strains. The identification of an unusual pattern of antibiotic resistance among isolates from multiple patients is considered as an indication of an outbreak. Standard methods for antibiotic susceptibility testing and for recording and analysing antimicrobial susceptibility test data are published in the literature. [7] The technique is easy to perform, inexpensive, gives rapid results and is readily available in routine microbiology laboratory; however, in most circumstances, antibiogram typing cannot be used as the only typing method for MRSA because of its poor discriminatory power. Antibiotic resistance patterns are also, to some extent influenced by the local environment, selective antibiotic pressure, acquisition and loss of plasmids carrying resistance genes and various other genetic mechanisms. Antibiogram typing has been successfully used for screening of epidemic strains. Sensitivity to gentamicin was used as one of the important epidemiological markers in identification of epidemic methicillin-resistant Staphylococcus aureus -15 (EMRSA-15) clones. [8] Studies have also reported a correlation between community acquired MRSA (CA-MRSA) and sensitivity to clindamycin. [9] Phage typing Phage typing technique was standardised by the International Subcommittee on Phage Typing of Staphylococci. [10] Strains are classified according to their susceptibility to a set of phages selected. An internationally accepted set of 23 phages is used for typing human strains of Staph. aureus. It is important to follow internationally recommended protocols for propagation and maintenance of phages, and also for interpretation of results to achieve internationally comparable results. [11] The technique requires maintenance of biologically active phages and is available only at reference centres. This technique has a fair amount of reproducibility, discriminatory power, ease of interpretation and is cost effective, though it is time consuming and technically demanding. The main disadvantage of phage typing is that a high proportion of MRSA strains are not typeable. [12],[13] Additional locally isolated phages have been tried by various centres to overcome the problem of non-typeability. Specific phages for MRSA were found to be useful in increasing the typeability and discrimination of MRSA strains. [13] Phage typing has been reported to be valuable in the identification of known epidemic strains among endemic strains and is preferred as first line approach in epidemiological investigation of MRSA strains. [14],[15] Serotyping Serotyping is based on the fact that the strains of same species can differ in the antigenic determinants expressed on the cell surface such as lipopolysaccharides, membrane proteins and capsular polysaccharides. Serotyping is performed using several serological tests such as bacterial agglutination, latex agglutination, co-agglutination, and enzyme labeling assays. The technique is reproducible but has poor discriminatory power. Reports show that 11 S. aureus capsular types have been identified but almost every strain of MRSA is capsular type 5 and 8. [16] This method has a limited role in epidemiological investigation of MRSA. Biotyping Biotyping makes use of the patterns of metabolic characteristics expressed by an isolate. Biotyping methods mostly used in epidemiological studies of MRSA are - production of urease, hydrolysis of tween 80, and tolerance to chemicals and dyes. The techniques are simple, reproducible with relative ease of performance and interpretation. Most strains are typeable by this method but the discriminatory power is not so good to be used for broader based collection of MRSA strains. [12] Urease production has been reported as one of the important epidemiological markers in identification of EMRSA-15 and EMRSA -16 clones. [8] Protein electrophoretic typing Various electrophoretic methods such as whole cell protein typing, immunoblotting, multilocus enzyme electrophoresis (MLEE) and zymotyping has been used for typing of MRSA strains. Whole cell protein typing In this method the proteins are extracted from the culture of a strain, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and stained to compare with those of other strains. A large number of bands are produced in whole cell protein electrophoresis of MRSA strains but the differences, even between unrelated strains are small leading to poor discrimination of MRSA strains. [17] Immunoblotting In immunoblotting the electrophoresed products of SDS-PAGE are transferred to nitrocellulose membrane and then exposed to antisera raised against specific strains. The bound antibodies are then detected by enzyme-labelled anti-immunoglobulins. By this method though the number of bands is less as compared to whole cell protein electrophoresis, discriminatory power is still quite low. [12],[17] Multilocus enzyme electrophoresis MLEE involves extraction of enzymes from the strain, their separation by electrophoresis and examination by selective staining. The technique is labour intensive and the discriminatory power depends upon the number of enzymes extracted. [6] Zymotyping Zymotyping is based on the electrophoretic properties of esterase enzymes. MRSA strains have been shown to possess three esterase enzymes namely A, B and C. Each of them varies in its electrophoretic mobility. The enzymes are extracted as for MLEE and electrophoressed on polyacrylamide gel. Results are analysed according to the differences observed in the mobility of esterases in different strains. The technique is labour intensive, and shows poor discriminatory power for MRSA. [16],[17] All the above electrophoretic methods are labour intensive and have a poor reproducibility and discriminatory power. [17] They are rarely being used currently for typing of MRSA strains. Genotypic Methods Plasmid DNA analysis Plasmid analysis was the first molecular technique used for epidemiological investigation of MRSA. In this technique the isolates are differentiated according to the number and sizes of plasmids carried by an isolate. The method is easy to perform and interpret but poor in discrimination. [12],[17] Reproducibility of this method suffers due to the existence of plasmids in different molecular forms such as supercoiled, nicked or linear, each of which migrates differently on electrophoresis. Since plasmids can be spontaneously lost or readily acquired, related strains can exhibit different plasmid profiles. Also, certain genes are contained in transposons that can be readily acquired or deleted. Some isolates may lack plasmids and will not be typeable by this method. The technique has not been found to be very useful for the investigation of outbreak infections because of inherent mobility of extra chromosomal DNA and unstable plasmid profile of strains. Restriction endonuclease analysis of plasmid DNA has shown to improve the discriminatory power of this method due to differences in the number and position of restriction sites between two unrelated plasmids of the same size. [18] Chromosomal DNA analysis Restriction Endonuclease Analysis (REA) of Chromosomal DNA - A restriction endonuclease cuts DNA at a specific nucleotide recognition sequence. The number and size of restriction fragments generated depends on the recognition sequence of enzyme and the composition of DNA. These fragments are separated according to their size on agarose gel electrophoresis. The patterns are stained by ethidium bromide and examined under UV light. Different strains of the same species can have different REA profiles because of variations in their DNA sequences. All strains can by typed with good reproducibility. [19] The main disadvantage of this method is that complex profile generated consists of a large number of bands. Overlapping of these bands makes interpretation difficult and reduces the discriminatory power. Southern blot analysis of RFLP In southern blot analysis, the restriction fragments which are generated by the digestion of DNA by endonucleases and separated by gel electrophoresis are transferred on to nitrocellulose membranes. The fragments containing specific sequences are then detected by labeled DNA probes. Variations in the number and sizes of the fragments detected are referred to as restriction fragment length polymorphism (RFLP) and make the basis for discrimination of strains. [6] The method is technically complex and the patterns generated are difficult to interpret. Ribotyping Ribotyping is also based on Southern type hybridisation, which involves the blotting of restriction enzyme digestion of ribosomal RNA and insertion sequences. The probes generally used are either labeled with radioisotopes or are biotinylated. Restriction enzyme Eco R1 has been found to be comparatively more useful than other enzymes in producing a good number of bands. [20] The technique is reproducible but time consuming and technically demanding. When compared to PFGE it showed comparatively lower efficiency in differentiating MRSA strains. [20] Binary typing This technique is also a modification of southern type hybridisation. It is based on detecting the presence or absence of a combination of genetic loci by PCR. Amplified products can be detected by various methods including gel electrophoresis or real time PCR. Strains from diverse clinical and geographical origins can be genotyped by means of binary typing using several strain specific DNA probes. The probes are generated by random amplification of chromosomal DNA followed by cloning. [21],[22] Specificity of the probes is validated by testing against several MRSA isolates. The isolates are assigned a binary code depending on the result of hybridisation reaction. Various binary targets including toxin genes, antibiotic resistance genes, SCCmec loci and phage-derived open reading frames can be used. Almost all MRSA strains are typeable by this method and the results are reproducible. Discriminatory power depends on the number of probes used. [21],[22] Binary typing has also been used for studying the clonal relationship and transmission routes in bovine MRSA strains. [23] Pulsed field gel electrophoresis Pulsed field gel electrophoresis (PFGE) is a widely adopted method for typing of MRSA strains. It is a variation of conventional agarose gel electrophoresis in which the orientation of the electric field across the gel is changed periodically. This modification enables large fragments to be separated according to size, minimising the overlapping of fragments. Selection of restriction enzymes is important in generation of simpler patterns consisting of less number of bands and making interpretation easier. Restriction enzyme Sma I has been widely used for investigating MRSA strains by PFGE. All strains are typeable by this method. The technique is highly discriminatory and reproducible. The major difficulties associated with PFGE are the technical demands of the procedure, cost of the reagents and equipments and the time required to perform the test. [24] Interpretation of the results is little difficult, however the recently published guidelines for the interpretation of bands has made it simpler to correlate the results with the epidemiological data available and to determine the genetic relationship between strains. [25],[26] PFGE has been recommended as a ′gold standard′ for typing of MRSA strains however it is important to follow uniform standard protocols to achieve the internationally comparable results. [27] The technique has been compared to other techniques in various studies and has been found extremely useful in the characterisation of outbreak strains. [16],[20],[24],[27],[28],[29] Some workers have reported that the stability of PFGE is not sufficient for reliable application in long term epidemiological surveillance due to long evolutionary history of pandemic clones. [28],[29] Polymerase chain reaction (PCR)- based typing methods PCR techniques for identification of complex DNA molecules provide rapid methods for the discrimination of MRSA strains. [30] Among the various PCR based typing techniques, arbitrarily primed polymerase chain reaction or random amplified polymorphic DNA (AP-PCR/RAPD) and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) have been reported as more useful for typing of MRSA strains. AP-PCR/RAPD This technique involves random amplification of segments of target DNA using small primers of arbitrary sequences of nucleotides of unknown homology with a target sequence. The number and size of the fragments generated during PCR are the basis for typing of an isolate. The technique does not require digestion of amplified fragments. The technique is simple and rapid. Almost all strains are typeable by this method. Discriminatory power is variable depending upon the number and sequence of the primers used. A multicenter study by van Belkam A et al. for evaluation of AP-PCR for typing of S. aureus strains reported that the technique is useful in studying outbreak strains but not suitable to be used as a reference method for typing of MRSA strains because of its poor inter laboratory reproducibility of results. The technique can be used as a rapid screening test. [31] PCR-RFLP PCR-RFLP relies on the amplification of a defined fragment of DNA and subsequent digestion of the amplified product with a restriction enzyme to generate restriction fragment length polymorphisms. These polymorphisms allow discrimination of strains. Coagulase gene (coa) RFLP and spa gene RFLP have been widely used to discriminate MRSA strains. PCR-RFLP of spa gene has been reported to be useful in differentiating the strains, which were otherwise not typeable by bacteriophages. [32] DNA sequence analysis-based typing methods DNA sequence analysis is an objective genotyping method as the genetic code is highly portable, easily stored and can be analysed in a relational database. Two different strategies have been used to type the isolates by analysing their DNA sequences: Multilocus sequence typing (MLST) and Single-locus sequence typing (SLST). Multilocus sequence typing (MLST) MLST has been reported to be useful for studying clonal evolution of MRSA. It is the genotypic descendant of MLEE and is based on sequence analysis of several housekeeping genes. Seven housekeeping genes studied by various workers are arcC, aroE, glpF, gmk, pta, tpi and yqiL. The different sequences of each house keeping gene are assigned as distinct alleles, and each MRSA strain is defined by the alleles of the seven genes. [33] MLST has been used in conjunction with PCR analysis of Staphylococcal Cassette Chromosome mec (SCC mec) element to define the clonal type of MRSA strains. Microbiological Societies′ Subcommittee on S. aureus typing has accepted an international nomenclature for these clonal types, for example an epidemic clone from UK, EMRSA-15 is referred as MLST22-MRSA SCC mec type IV. [34] HARMONY- the International Union of Microbiology Societies′ European Staphylococcal typing network has recommended that combination of MLST and SCCmec typing can be used as a reference typing system for multicenter surveillance of MRSA. [34] A web-based database for MLST typing is available (www.mlst.net) for comparison of results. The technique is expensive, labour intensive and time consuming due to the involvement of various gene targets. The technique has also been used to identify livestock associated MRSA strains and their transmission between humans and animal species. [35] The technique has been compared with other techniques like PFGE and spa typing. A good degree of concordance between results obtained by MLST, PFGE and spa typing has been reported. [27],[28],[29],[36] Single-locus sequence typing Single-locus sequence typing (SLST) is used to compare sequence variation of a single target gene. The genes selected are usually of short sequence repeat (SSR) regions that are sufficiently polymorphic to provide useful resolution. Genes for protein A (spa) and coagulase (coa) in MRSA strains having 24 bp and 81 bp tandem repeats have been studied extensively and it has been reported that MRSA strains can be discriminated by determining the repeat sequence numbers within the X region of spa gene. [37],[38],[39] The technique is simple, rapid and highly reproducible. An online database is available for electronic submission and assignment of spa types ( http://spaserver.Ridom.de/ ) Frenay et al. compared spa typing with phage typing and showed that spa typing clustered isolates belonged to phage type III. [39] Other workers reported in their study that spa typing could discriminate phage nontypeable as well as typeable MRSA strains into further groups. [32] Studies have found spa typing helpful in classification of isolates into particular lineages. Strommenger et al. reported the spa typing technique as an excellent tool for national and international surveillance as well as for short term local epidemiology, however, they also recommended the use of additional markers such as SCCmec and lineage specific resistance genes to overcome the limitations of a single-locus typing method. [28],[40] Staphylococcal cassette chromosome mec (SCCmec) typing S. aureus acquires methicillin resistance through mobile genetic element Staphylococcal Cassette Chromosome mec (SCCmec) that contains mec A gene complex and ccr gene complex. Several mec and ccr allotypes have been found among SCCmec element. Currently, eight main types of SCCmec (type I to type VIII) alongwith many subtypes have been distinguished among MRSA strains. Each SCCmec type encodes for resistance to different antibiotics. [41],[42] Variation in these SCCmec types has made the basis for differentiation among MRSA strains. Studies have found that healthcare associated MRSA (HA -MRSA) strains contain mainly type I, type II and type III SCCmec cassettes while CA-MRSA strains contain type IV and type V cassettes, although several variants have also been reported. [43] Various methods have been proposed to study the mec gene complex, its′ classification and nomenclature. [44],[45],[46] Most of the methods rely on PCR mapping of cassette elements such as mec complex, ccr complex and j region. Other methods involving sequence determination of internal fragments of recombinant genes are available. A combination of two approaches like SCCmec typing along with MLST is recommended for reliable typing for multicentre surveillance, inter-hospital and international transmission and evolution of MRSA strains. [47] Toxin gene profile typing MRSA strains produce various toxins including toxic shock syndrome toxin (tsst1), enterotoxins and exfoliative toxins. Genes encoding for enterotoxins are carried on Staphylococcal pathogenicity islands. Other toxin genes like gene for Panton Valentine Leucocidin (PVL) are carried on bacteriophages and are easily transferred between lineages. Thus toxin gene profile of the strains can be used as an important epidemiological marker for typing of MRSA strains. [48] Studies have shown that MRSA strains possess more toxin genes as compared to MSSA strains. [48] MRSA strains isolated from different geographical areas have shown to possess distinct toxin gene profiles. [48] Studies on toxin gene profile of MRSA have reported that most of the CA-MRSA possess genes for PVL toxins and may have evolved from the established CA-MSSA (community acquired methicillin sensitive S. aureus) strains. [5] Important CA -MRSA clones including CC5, CC22 and USA 300 are positive for PVL genes however health care associated Brazilian, Irish and Iberian clones and also healthcare associated UK EMRSA strains of a series from EMRSA-1 to EMRSA- 17 do not have PVL genes. These UK EMRSA strains have also been shown to be specific in carriage of genes for enterotoxins A to E (sea to see).[8] Correlation between toxin gene profiles and SCCmec type and also between toxin gene profiles and HA-MRSA strains has been reported. [49] Of the various methods available, multiplex PCR technique is recommended for detection of toxins in MRSA. It is rapid, reproducible relatively inexpensive, easier to interpret and provides a high degree of discrimination. [50] The technique is useful for studying the chromosomal diversity and evolutionary history of MRSA strains. [48],[49],[50] Choosing an appropriate typing method MRSA typing is an essential component of an effective surveillance system to describe epidemiological trends and infection control strategies. Current challenges for MRSA typing are focused on selecting the most appropriate method in terms of efficiency, reliability, ease of performance and cost involved. It has been shown that regardless of the typing methods used, only a limited number of strain types can be differentiated among MRSA strains because of the fact that most of the MRSA are evolved from a relatively small number of clones. Historically, isolates were distinguished by phenotypic methods. Phenotypic methods often cluster isolates into few broad groups and are good for initial screening and for identification of known epidemic strains while molecular methods are capable of differentiating epidemic strains from endemic strains. Initial molecular techniques compared restriction endonuclease patterns of plasmid or chromosomal DNA. The second generation genotypic methods are based on subjective interpretation and comparison of gel patterns and fingerprint images. Image-based methods do not provide biological criteria to evaluate the relatedness between different strains. Also, it is difficult to maintain an international data base and retrieval of patterns for comparison of results of multicentre studies. DNA sequence analysis is an objective genotyping method as the genetic code is highly portable and easily stored in a database. Recent DNA sequencing methods including MLST, SLST involving sequencing of various genes, SCCmec typing and TGP typing are rapid, affordable and high-throughput systems for typing of MRSA strains. The choice of typing technique further depends on the purpose of the study, the facilities available and the utility of data generated to answer a desirable research question. Conclusions Application, potential and problems of various typing techniques have been reviewed critically and the role of each technique amongst established typing methods is highlighted. Every method has been shown to have its own advantages and /or disadvantages. The phenotypic methods in general are easier to perform, easier to interpret, cost effective and are widely available however less discriminatory. The genotypic methods though cumbersome, expensive, technically demanding are more discriminatory to answer desirable research questions. There is no consensus regarding the single best method for typing of MRSA strains. Phage typing is recommended as first line approach in epidemiological investigation of MRSA strains. PFGE remains the gold standard for characterisation of outbreak strains. Newer DNA sequencing methods including MLST, spa typing, SCCmec typing and TGP typing are coming up as more practical methods for detecting evolutionary changes and transmission events. Further, international harmonisation of these typing techniques is important to establish surveillance networks and facilitate global MRSA control. References
Copyright 2012 - Indian Journal of Medical Microbiology |
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