Genotyping methods are vital epidemiological tools for discriminating different bacterial isolates within same species, which in turn provide useful data in tracing source of infection and disease management. There have been a revolutionary efforts in ways to distinguish between bacterial types and subtypes at molecular level utilizing DNA in the genomes. Notably, the growth of various DNA typing methods has provided innovative apparatuses for improved surveillance and outbreak investigation. Thus, early identification and genotyping are indispensable as resources for managing therapeutic treatment and the control of rapid expansion of clinically important bacteria. Methicillin-resistant Staphylococcus aureus (MRSA) has been in a great attention due to its contagious nature and subjected to various typing analyses. Thus, in this chapter, we aimed to review the contribution of various genotyping methods of commonly used as well as those unique to staphylococci in understanding its epidemiology, infection and dissemination pattern, and to provide an impression of specific advantages and disadvantages of each tool.
- Staphylococcus aureus
Typing is a process to characterize the species and properties of organisms, in particular the discrimination at the strain level both phenotypically and genetically. Conventional typing such as serotypes, biotype, and phage type has been in practice for many years. Nevertheless, typing at molecular level is nowadays very essential due to its specificity, which is often used to support the associated phenotypic characteristics. For example, one species may comprise many subtypes or a subpopulation which one might be more pathogenic than the others. Thus, genotyping plays an important role to identify potential differences at genetic level as well as for epidemiological traceability of all the presented isolates .
A good typing method must have the discriminatory power to differentiate all unrelated isolates epidemiologically to facilitate any outbreak investigation. This will allow investigation to demonstrate person-to-person strain transmission, subsequently, allowing preventive measures to be designed to inhibit further dispersion of the pathogens. Additionally, genotyping method must be inexpensive, rapid, easy to interpret and highly reproducible . For a continuous surveillance, genotyping methods must produce results with a sufficient stability over time. Also, it should produce portable data and can be easily accessed through open source web-based database or a client-server database connected via the internet, facilitating global comparison of the isolates.
Genotyping methods are basically based on phenotyping, PCR/sequence typing and genome typing approaches. Remarkably, a great effort has been put up in epidemiological investigations of
2. Phenotypic detection and identification of MRSA
Numerous conventional or molecular methods can be applied for detection and identification of
Upon identification of
Currently, these tests are considered as the most popular methods of choice that can support genotyping data . Nevertheless, previous reports suggested that phenotypic methods of identification have drawbacks due to the variability in expression of phenotypic characterization by isolates belonging to the same species and their reliance on subjective interpretation of test results . As a result, phenotypic detection and identification was heavily burdened with several issues; including low reproducibility, reliability, sensitivity and specificity as well as lack of resolution in epidemiology investigation. Therefore, several reports have suggested that genotypic identification and detection methods offered a higher discriminatory power, reliability, reproducibility and typeablity . Genotypic identification can be done with the phenotypic approach together for a better comparative analysis. Furthermore, the introduction of molecular screening for MRSA detection as well as identification directly from clinical specimens has been developed to enhance and identify common
3. Genotyping of MRSA
The ultimate goals for bacterial typing are to further clarify the population dynamicity and also to track the spread of the microorganisms. As mentioned earlier, traditional bacterial typing of phenotypic-based alone, does not provide the prudent resolution for identifying and tracking an infection-causing pathogen, and also does not clearly describe the transmission pattern of an outbreak. However, molecular typing has been an invaluable tool for molecular epidemiologist as well as clinical researchers for tracing the spread of particular strains, discovering the route of dissemination and the potential reservoirs. Usually, the outcomes of epidemiological investigations are often used to guide and assist the clinical treatment of the patients by selecting the appropriate antimicrobial agents. Furthermore, molecular typing contributes to the comprehensive understanding of the epidemiology of infection and facilitates infection control measures as well as management .
It is well known that
3.1. Polymerase chain reaction (PCR)-based identification
Polymerase chain reaction (PCR) is known as enzymatic method used to exponentially amplify a specific preselected fragment of DNA. It is well known that PCR uses a thermostable DNA polymerase
3.1.1. Amplified fragment length polymorphism (AFLP)
AFLP is a PCR-based method applied in DNA fingerprinting and genetic research. In this method, restriction enzymes (e.g. endonuclease) are used to cut the genomic DNA of the typed species, subsequently, double-stranded oligonucleotide adaptors which are comprised of a core sequence and an enzyme-specific sequence, are bound to one of the sticky ends of the restricted fragments. After that, a PCR thermocycler is used to amplify those restricted fragments ending with the adapter selectively, using primers complementary to the adapter sequence. Then, the restriction site sequence and a number of additional nucleotides from the end of the unknown DNA are designed. Usually, the restriction fragments (50–100) are amplified using florescent dye-labeled PCR primers, to detect those separated fragments by size using automated DNA sequencer. Likewise, gel electrophoresis can also be used to visualize and analyze the amplified fragments of typed DNA. Upon analysis, a high-resolution banding is generated via computer reflecting the genetic relatedness among bacterial isolates .
This kind of technique has a higher discriminatory power in comparison to PFGE, where it was shown in a study that AFLP analysis provided greater genetic resolution and was less sensitive to DNA quality during genetic typing of bacterial pathogens
3.1.2. 16s ribosomal RNA (16s rRNA)
16S rRNA comprises ∼1500 pair nucleotide sequence coding for catalytic RNA that is part of the 30S ribosomal subunit. 16S rRNA gene is comprised of nine variable regions (V1–V9/30–100 base pairs long), that show sequence diversity among different bacterial species, subsequently enable for identification purposes. V1–2-3 regions are located at the 50 end of the 16s rRNA gene which is shown to be appropriate and more sensitive than other regions for identification of different types of bacteria . This gene is constant in function, promising a valid molecular chronometer, where it exists in all prokaryotic cells. Therefore, it is used to elucidate both close and distant phylogenetic relationships at the genus and at the species level  based on the differences in the nucleotide sequence of 16s RNA gene. Additionally, dedicated 16S databases  that include near full-length sequences for a large number of strains and their taxonomic placements are available. The sequence from an unknown strain can be compared against these available sequences in the database. This approach is considered as a common substitute for traditional methods using (rRNA) gene sequencing . It is less time-consuming and labor intensive, where DNA sequencing can offer more absolute taxonomic classification than culture-based approaches for numerous organisms . However, there are also limitations with this approach associated with the short read lengths, variances ascending from the diverse regions selected, sequencing errors and difficulties in evaluating operational taxonomic units (OTUs) [23, 24]. Additionally, single marker (16S rRNA) usage is considered challenging to assess the bacterial diversity, subsequently difficult to identify bacterial species , as well as the resolution of 16S rRNA that is very limited among closely related species. A previous study has shown that 16S rRNA combined with
3.1.3. Staphylococcal cassette chromosome mec typing (SCC
Also, two different multiplex PCR methods were developed by Zhang et al.  and Milheirico et al.  for specific characterization of SCC
3.1.4. Multiple-locus variable-number tandem repeat assay (MLVA)
Multiple-locus variable-number tandem repeat analysis (MLVA), was previously known as a variable-number tandem repeat (VNTR)  by making use the VNTR polymorphism. In 2008, after the introduction of spa typing as a standard molecular typing method in the Germany MRSA surveillance, MLVA was added as a supportive typing technique. This method involves PCR amplification of five specific loci (
It was shown that this approach has a reproducibility as good as PFGE technique . The main drawback of this approach is that in highly conserved genomes, there may not be sufficient DNA polymorphisms in these limited sequence targets to exhibit alleles. Another limitation was, small deletions and insertion in the regions flanking the repeat units may lead to misinterpretations, making the MLVA results slightly more ambiguous than sequenced-based methods. However, to overcome this limitation, the DNA sequence of each new allele is determined to confirm the deduced number of repeats . However, the level of discrimination can be increased by adding more loci and repeating the assay with different restriction enzymes .
3.1.5. Repetitive element polymerase chain reaction (rep-PCR)
rep-PCR is a DNA-based technique that discriminates microbes at subspecies or strain level by observing genomic DNA fingerprint patterns . In this approach, the hybridization of primers to noncoding intergenic repetitive sequences takes place across the genome. The amplicons are produced during DNA amplification of the repetitive elements. Depending on the distribution of the repeat elements across the genome, the genetic relatedness between the bacterial isolates can be inferred by comparing the banding pattern of the amplicons. Enterobacterial repetitive intergenic consensus’ (ERIC 124–127 bp), ‘the repetitive extragenic palindromic’ (REP 35–40 bp), and the ‘BOX 154 bp’ sequences are examples of conserved repeat sequences that have been used successfully in rep-PCR typing . This kind of approach is considered as highly discriminatory tool for different bacterial organisms such as
Alternatively, rep-PCR approach is developed and used by a semi-automated method using DiversiLab system (bioMérieux, Marcy l’Etoile, France), where clinically important organisms can be detected by commercial PCR kits . Then, high-resolution chip-based microfluidic capillary electrophoresis is used to separate amplified genomic DNA within repetitive elements, where chip-based microfluidic capillary electrophoresis can increase the determination and reproducibility of the rep-PCR method compared to traditional gel. In the next step, DiversiLab software is used to normalize and analyze the data automatically. Several reports have evaluated the usefulness of this approach (DiversiLab) in outbreak-related and epidemiology unrelated bacterial isolates . It was shown that this approach is rapid, reproducible and easy for typing microorganisms. Hospital outbreaks of MRSA have been identified using this useful DiversiLab tool by Fluit et al., . In contrast, other study found that this tool is not highly discriminative tool for MRSA typing particularly in outbreak setting . The main limitation of this approach is the DiversiLab databases are stored only on manufacturer server, resulting in some users not allowed to use this typing system due to security purposes.
3.1.6. Restriction fragment length polymorphism (RFLP)
In PCR-RFLP approach, restricted enzymes are used to detect the variations in homologous DNA fragments. Then, the DNA fragments are amplified using regular PCR, subsequently these fragments are separated by gel electrophoresis based on length of the fragments. For example, coagulase gene (coa) and
3.2. Sequence typing method for bacterial identification
3.2.1. Staphylococcal protein A (spa) typing
Subsequently, Based Upon Repeat Pattern (BURP) algorithm is used to analyze the diverse spa types associated to each other. The analysis shows a good consistency with MLST-CCs, where ST that shares at least five of seven identical alleles are grouped into a single CC . The advantages of this typing method are the results generated are easy to interpret, less time-consuming, highly reproducible, less laborious and highly comparable between laboratories via ridom.spa.server compared with PFGE. Besides, spa typing is impressive for its ease of interpretation and suitability for international comparison. It is also able to detect both slowly and rapidly accumulated molecular variations as well as to investigate outbreaks in epidemiological studies and molecular evolutions of population structure . However, non-typeable (NT) isolates are increasingly found in the Dutch MRSA surveillance as well as globally. Thus, to overcome the issue of NT strains, other typing method should be concurrently used to be a supportive method . Malachowa et al.  found that spa typing was more approximate to MLST approach upon comparing four genotyping methods (PFGE, MLST, MLVA and spa typing) in 59
3.2.2. Multilocus sequences typing (MLST)
MLST has been invented to overcome the poor or insufficient portability of traditional and older molecular typing application. The main idea of this tool is based on MLEE  which depends on the differences in electrophoretic mobility of various enzymes exist in a bacterial species.
In the subsequent analysis, MRSA isolates are grouped within a single CC when five out of seven housekeeping genes (400–500 bases) in that particular MRSA isolates having identical sequences and isolates with the seven same allelic profiles may be descended from a common ancestor [66, 67]. If there are various alleles at each of the seven loci, the isolates are unlikely to have the identical allelic profiles by chance, while isolates that have similar allelic profile can be considered members of the same clone . The variations found among these genes are mostly synonymous and neutral. Since these genes accumulate variations in a slow manner, they are considered to be reliable indicator of evolutionary history . The main advantage of this tool is that whole produced data are obvious due to standardized nomenclature internationally and reproducible. Additionally, ST profile as well as alleles sequences are available in huge central databases (http://pubmlst.organdwww.mlst.net)  that are freely accessible online. Moreover, the genetic relatedness between bacterial strains within a species can also be identified via the databases.
Thus, it is a useful tool to compare the data with other laboratories via web-based electronic data. Furthermore, it allows the exchange of data collected over internet through the MLST database. BURST software package can analyze the evolutionary events within
3.3. Genomics-based typing tool
3.3.1. Pulsed-field gel electrophoresis (PFGE)
PFGE is an approach used to detect the dispersion of large segments of DNA using gel with high electrical fields that facilitate changing in DNA direction periodically . In brief, molecular sieve of gel is used to transform DNA from cathode to anode using common electrophoresis method. Two electric fields are used in PFGE technique, where it allows to change the directions of the DNA as mentioned above. Subsequently, ethidium bromide dye is used to differentiate the DNA band spectrum as a typing result. Clinically, various types of bacteria can be genotyped by PFGE which is considered as the “gold standard” genotyping method. It is assumed as an epidemiological tool for most bacterial species since 1990s . Currently, PFGE is used worldwide to identify and characterize isolates of bacteria in outbreak investigations [19, 71, 72]. It is also considered as prototype tool to analyze center to center transmission events .
In term of
PFGE has been found to show a higher discriminatory power than PCR-RFLP of coa gene and other PCR-based fingerprinting methods as it enables the entire chromosome to be analyzed, whereas the PCR-based fingerprinting methods explore only selected (random) portions of it . Previous studies stated the reproducibility of PFGE is considered high due to the standardization of protocols , allowing national and international surveillance systems , and standard interpretation guidelines to investigate the emergence of bacterial species particularly
3.3.2. DNA microarray
DNA microarray typing method uses a collection of DNA probes that are attached to a solid surface in ordered manner. Ideally, complementary nucleotide sequences for specific bacterial isolates are detected by DNA probes. This approach is specific tools to identify several genes for specific bacterial strains. It can also be used to identify allelic variations of a gene which exists in all strains for particular species. Usually, target DNA could be labeled by chemical, enzymatic reaction and DNA microarray hybridization. Then, labeled target DNA and an immobilized probe create signal due to successful hybridization, giving measurement automatically using scanner. Currently, this approach is extensively used to analyze genomic mutations such as single-nucleotide polymorphisms (SNPs). It is also found that this approach is an excellent application to identify exceptional antibiotic resistance and virulence genes simultaneously to represent epidemiological markers of certain isolates of interest . Whole genome microarray approach is the alternative tool for whole genome sequencing (WGS) for saving time, expenses and efforts, where it has ability to investigate genetic features of isolates involved in outbreak. For example, 31 chromosomes and 46 plasmids were identified from a various set of
3.4. Whole genome sequencing (WGS)
To investigate genome variations, cost-effective way has been invented for genetic investigations, which is second generation sequencing (NGS) or high-throughput sequencing. This technique is named second generation to differentiate it from first generation sequencing based on the Sanger method. The main advantage of this approach over several traditional sequencing methods is the ability to create millions of reads (35–700 bp length) in one shot, which also leads to a reduction in cost. The nucleotide sequence of the genome is constructed by gathering numerous short sequences reads from overlapping regions, or comparison with previous reference sequences genomes (re-sequencing).
Currently, WGS is considered as a high attractive tool for epidemiological studies , and it is believed that this method has the potential as routine tool for bacterial identification and characterization in the near future. Nevertheless, the main challenge for this approach is the interpretation and computation of the huge set of data. This approach is currently used to determine the genetic relatedness between bacterial isolates based on sequence analysis of the whole genome. Additionally, WGS has the ability to distinguish various genomes within an SNP, which cannot be achieved in conventional molecular typing approaches. Thus, characterization of transmission events and outbreaks will be accurate. However, extensive studies must be conducted to translate this prospective tool into a routine practice. It is well known that the methods based on SNPs permit detailed and targeted analysis of variations among related organisms. Thus, WGS using SNPs analysis can identify the isolates related to an outbreak from non-outbreak isolates. Moreover, various phenotypic characteristics such as virulence and antibiotic resistance of particular pathogen can also be inferred by WGS technique. Finally, this approach enables the search for genetic markers, such as the presence or absence of a gene or an amino acid substitution in a protein, facilitating the linkage with the occurrence, severity and virulence of the disease.
Clinically, SNPs analysis on MRSA isolates recovered from an outbreak in a unit care for neonates using WGS sequencing approach was able to offer relevant data within a time frame that can stimulate patient care . Additionally, through WGS, data can also reliably predict antibiotic susceptibility phenotype of MRSA from an outbreak scene , leading to development of hospital infection management and patient outcomes in routine clinical practice. Some previous studies took benefits from WGS by investigating CA-MRSA in USA including USA300-0114 , where genetic variation was found. Considering the fact that the isolates were recovered and originated from a confined geographical area, the WGS analysis suggested the continuous evolution of this clone within the limited region. These results offer additional support for the use of WGS as a first-line screening method, which is comparable with those gained by phenotypic methods . Furthermore, additional genes may be added to the panel to increase the coverage and sensitivity, where sequenced isolates can be screened to recognize new resistance genes.
As a result, WGS is considered as a rapid prediction of resistance which contributes to effective clinical management, particularly for
Higher rates of morbidity and excessive healthcare costs are the two main reasons that can be caused by the growing number of HA, CA and LA-MRSA infections. The management of these infections must be conducted through the screening of individuals as well as infection control program. Currently, MRSA can be reliably detected within hours using rapid screening methods. However, the continuous evolution of SCC
For instance, AFLP has a higher discriminatory power in comparison to PFGE, where it provided greater genetic resolution and is less sensitive to DNA quality during genetic typing of bacterial pathogens in epidemiological investigation. Additionally, AFLP can be automated and has portable results, as well as reproducible approach to facilitate the analysis. Moreover, AFLP it is more suitable for inter-laboratory data exchange using sequence-based data. 16s rRNA analysis is considered a good discrimination approach among unrelated isolates, particularly in
Finally, WGS is considered as a very useful tool in epidemiological investigations to discriminate MRSA, and it may assist to trace person-to-person transmission in health care systems. Additionally, this technique is considered suitable for MRSA epidemics surveillance at national and international levels as well as reproducible approach, which is essential as baseline resources for managing therapeutic treatment and the control of rapid expansion of these strains.