**2.2.2 Pulsed-field gel electrophoresis (PFGE) of chromosomal DNA**

Pulsed-field gel electrophoresis is based on the digestion of bacterial DNA with restriction endonucleases that recognize few sites along the chromosome, generating large DNA fragments (30-800 Kb) that cannot be effectively separated by conventional electrophoresis. The basis for PFGE separation is the size-dependent time-associated reorientation of DNA migration achieved by periodic switching of the electric field in different directions. The DNA fragments will form a distinctive pattern of bands in the gel, which can be analyzed visually and electronically (Figure 4 A). Bacterial isolates with identical or very similar band patterns are more likely to be related genetically than bacterial isolates with more divergent band patterns.

This technique is laborious and includes several steps, requires good standardization and takes at least two days for obtention of results. Procedures will differ to some extent depending on the organism that is being analyzed.

Regarding DNA preparation, PFGE requires intact DNA for restriction endonuclease treatment. The risk of mechanical breakage to DNA molecules during the extraction procedure is avoided by embedding intact organisms into agarose plugs where cells are enzymatically lysed and cellular proteins digested. After endonuclease treatment, the agarose plugs containing the digested DNA are then submitted to PFGE (Figure 4B). The choice of the restriction enzyme for DNA digestion and pulse-time switching parameters for PFGE are critical variables for the obtention of restriction profiles to show well- resolved fragments.

Recent protocols can be completed in as little as two days through shortcuts such as the direct addition of lytic enzymes to the agarose mixture before the blocks are cast and also high temperature short-term washes which facilitate the extraction of unwanted compounds (Goering 2010; Halpin et al. 2010).

Isolates with identical PFGE patterns were considered to represent the same epidemiological type. Isolates differing by one genetic event were considered epidemiologically-related subtypes, expecting that a single genetic event could occur in the chromosome of an

Application of Molecular Typing Methods to the

**2.3 DNA sequencing-based methods** 

**2.3.1 Single-locus sequence typing** 

Kreiswirth 2001; Harmsen et al. 2003), (Figure 5).

Fig. 5. Sequence of steps involved in *spa* typing.

and electrophoresis conditions.

Study of Medically Relevant Gram-Positive Cocci 119

This success is due to an emphasis on standardized quality control especially in major areas of potential PFGE variability such as DNA sample preparation, choice of restriction enzyme,

PFGE has been applied to a wide range of microorganisms and has remarkable discriminatory power and reproducibility. It is currently considered the strain typing method of choice for many commonly encountered pathogens. However, one of the main

Genotyping methods based on DNA sequencing discriminate among bacterial strains directly from polymorphisms in their DNA considering the original sequence of nucleotides.

Sequencing of a single genetic locus has been used for epidemiological studies of many bacterial species, yielding valuable typing results. In this approach, it is essential to select highly variable gene sequences. Valuable typing results have been obtained for *S. pyogenes* by DNA sequencing of 150 nucleotides coding for the N-terminal end of M protein (*emm* typing) (Beall et al. 2000). Another example is spa typing for *S.aureus* that consists in sequencing of the X region of the protein A gene (*spa typing*). This technique is widely used for subtyping methicillin-resistant *S. aureus* (MRSA) strains (Shopsin, 1999, 2000; Shopsin &

notable limitations is the need for specialized and relatively expensive equipment.

organism as it moved from patient to patient. Isolates differing by two genetic events were also deemed to be potentially related, while three or more chromosomal differences were thought to represent an epidemiologically-significant difference (unrelated isolates). Van Belkum suggested a more conservative approach where only nosocomial isolates differing by a single genetic event (up to four differences in the PFGE restriction fragment pattern) were considered related subtypes. The terminology within both proposed formats was left intentionally vague, understanding that molecular typing is only one component of epidemiological evaluation which must include other available clinical data for accurate analysis (Tenover et al. 1995; van Belkum et al. 2007; Goering 2010).

Fig. 4. A. Schematic representation of pulse field gel electrophoresis. On the right PFGE of *Sma*I-digested genomic DNA of *S. aureus* isolates. B. Sequence of steps involved in PFGE.

Furthermore, isolates with more uniform PFGE profiles require more conservative interpretation. The fact that two strains share the same pattern does not prove that they are epidemiologically related. The establishment of an epidemiologic relationship depends on the frequency with which the "indistinguishable" pattern is seen among epidemiologicallyunrelated isolates and correlation with clinical and epidemiological information. If common contact between two patients with strains having the same pulsed-field gel electrophoresis (PFGE) type can be established, the chances are greater that an epidemiologic link could be ascribed. Thus, the greatest power of PFGE typing lies in showing strain dissimilarity rather than in proving similarity or relatedness. These considerations must be taken into account for banding pattern analysis from other molecular typing methods.

In some instances, initial unsatisfactory PFGE results may be aided by the use of an alternative restriction enzyme (Kam et al. 2008; Bosch et al.) or, in more difficult situations, the use of one or more additional typing methods (van Belkum et al. 2007).

The intra- and interlaboratory reproducibility of this method depends on understanding and controlling variables (Cookson et al. 1996; van Belkum et al. 1998; te Witt et al. 2010).

organism as it moved from patient to patient. Isolates differing by two genetic events were also deemed to be potentially related, while three or more chromosomal differences were thought to represent an epidemiologically-significant difference (unrelated isolates). Van Belkum suggested a more conservative approach where only nosocomial isolates differing by a single genetic event (up to four differences in the PFGE restriction fragment pattern) were considered related subtypes. The terminology within both proposed formats was left intentionally vague, understanding that molecular typing is only one component of epidemiological evaluation which must include other available clinical data for accurate

Fig. 4. A. Schematic representation of pulse field gel electrophoresis. On the right PFGE of *Sma*I-digested genomic DNA of *S. aureus* isolates. B. Sequence of steps involved in PFGE.

Furthermore, isolates with more uniform PFGE profiles require more conservative interpretation. The fact that two strains share the same pattern does not prove that they are epidemiologically related. The establishment of an epidemiologic relationship depends on the frequency with which the "indistinguishable" pattern is seen among epidemiologicallyunrelated isolates and correlation with clinical and epidemiological information. If common contact between two patients with strains having the same pulsed-field gel electrophoresis (PFGE) type can be established, the chances are greater that an epidemiologic link could be ascribed. Thus, the greatest power of PFGE typing lies in showing strain dissimilarity rather than in proving similarity or relatedness. These considerations must be taken into account

In some instances, initial unsatisfactory PFGE results may be aided by the use of an alternative restriction enzyme (Kam et al. 2008; Bosch et al.) or, in more difficult situations,

The intra- and interlaboratory reproducibility of this method depends on understanding and controlling variables (Cookson et al. 1996; van Belkum et al. 1998; te Witt et al. 2010).

for banding pattern analysis from other molecular typing methods.

the use of one or more additional typing methods (van Belkum et al. 2007).

analysis (Tenover et al. 1995; van Belkum et al. 2007; Goering 2010).

This success is due to an emphasis on standardized quality control especially in major areas of potential PFGE variability such as DNA sample preparation, choice of restriction enzyme, and electrophoresis conditions.

PFGE has been applied to a wide range of microorganisms and has remarkable discriminatory power and reproducibility. It is currently considered the strain typing method of choice for many commonly encountered pathogens. However, one of the main notable limitations is the need for specialized and relatively expensive equipment.
