**1. Introduction**

The development of molecular genotyping methods has been a landmark in the possibility of classifying microorganisms below the species level. The ability to differentiate efficiently related bacterial isolates is essential for the control of infectious diseases and has become a necessary technology for clinical microbiology laboratories.

Strain typing is an integral part of epidemiological investigations of bacterial infections. Typing methods fall into two broad categories: phenotypic and genotypic methods. Phenotypic methods are those that characterize the products of gene expression in order to differentiate strains. Properties such as biochemical profiles, antimicrobial susceptibility profiles, bacteriophage types, and antigens present on the cell surface are examples of phenotypic methods that can be used for typing isolates. Since they involve gene expressions, these properties have a tendency to vary, based on changes in growth conditions and growth phase, being often difficult to detect.

Methods for distinguishing among bacterial strains have profoundly changed over the last years mainly due to the introduction of molecular technology. Genotypic strain typing methods are based on the analysis of differences in the chromosomal and extrachromosomal nucleic acid sequences between strains. Molecular epidemiology of infectious diseases integrates practices and principles of molecular biology with those of epidemiology (Tenover et al. 1997).

Investigations of presumed outbreaks of bacterial infections in hospitals often require strain typing data to identify outbreak-related strains and to distinguish epidemic from endemic or sporadic isolates.

All typing systems can be characterized in terms of typeability, reproducibility, discriminatory power, ease of performance, and ease of interpretation. For each isolate, the system should provide an interpretable result, preferably based on objective criteria. Ideally, results should be reproducible from day to day and from laboratory to laboratory and should allow differentiation of unrelated strains. Additionally, the method should be standardized and if possible should be technically simple, cost-effective, and rapid (van Belkum, et al. 2007).

Application of Molecular Typing Methods to the

*pneumoniae* isolates using BOXAR1 primer.

elucidated.

**PCR (AP-PCR)** 

fragments vary depending on the strain (Figure 1).

Study of Medically Relevant Gram-Positive Cocci 115

fragments between those sites (interrepeat fragments) are amplified. Since the number and location of the repetitive elements are variable, the sizes and number of effectively amplified

Fig. 1. Schematic representation of REP-PCR assay. On the right BOX-PCR patterns of *S.* 

Two different Rep-PCR have been used for typing enteric bacteria: a 38-bp repetitive extragenic palindromic element (REP) and a 126-bp enterobacterial repetitive intergenic consensus (ERIC) sequence (Versalovic et al. 1991), whose function has not yet been

A BOX repetitive element is a highly conserved repeated DNA element that has been identified in the *Streptococcus pneumoniae (*pneumococcus) chromosome. Although the function of this element has not yet been completely understood, it has been demonstrated that the presence of a BOX element is associated with variation in colony opacity of the pneumococcus (Saluja & Weiser 1995). BOX-PCR has been effectively used for typing *S.* 

Several genetic elements have been used for developing Rep-PCR to type *Staphylococcus aureus*. The element IS*256* occurs in the genome either independently or as part of the composite transposon Tn*4001*, IS*256* insertion position is strain-specific and spaced close enough to allow amplification of polymorphic inter-IS*256* element sequences (Deplano et al. 1997). Another element used for this methodology is RW3A, a repetitive sequence initially found in *Mycoplasma pneumoniae*, which also generates strain-specific DNA fragments when

**2.1.2 Randomly Amplified Polymorphic DNA-PCR (RAPD-PCR) or Arbitrarily-Primed** 

Randomly Amplified Polymorphic DNA-PCR (RAPD-PCR), also referred to as Arbitrarily-Primed PCR (AP-PCR), is a variation of the PCR technique employing a single, generally short primer, that is not targeted to amplify a specific bacterial DNA sequence. Low annealing temperatures are used during amplification, allowing imperfect hybridization at multiple chromosomal locations. When the primer binds in two sites on opposite strands, at the proper orientation and with sufficient affinity to allow the initiation of polymerization,

*pneumoniae* as well as other bacterial species (van Belkum et al. 1996).

*S. aureus* DNA is used as template (van der Zee et al. 1999).

The results of bacterial strain typing have many different applications including outbreak investigation and surveillance in clinical care settings and public health investigations and also within other contexts such as food and pharmaceutical industries and environmental analysis.

The aim of this chapter is to provide an overview of the methods available for analyzing bacterial isolates, focusing on those methods employed for typing *Streptococcus pneumoniae* and *Staphylococcus aureus*. Different molecular approaches have been used to better understand the epidemiology of these medically relevant gram-positive cocci (Willems. et al. 2011).
