**3. Epidemiologic applications of bacterial typing techniques**

A more comprehensive knowledge of the evolution and the epidemiology of bacterial pathogens had been obtained by combination of genetic, phenotypic, spatial and temporal data.

Application of Molecular Typing Methods to the

pulsed-field gel electrophoresis (Struelens 1996).

**4.1 Methicillin-resistant** *Staphylococcus aureus* 

**pathogens** 

of this pathogen has increased.

(Ma et al. 2002; Naimi et al. 2003).

Study of Medically Relevant Gram-Positive Cocci 123

obtained during real outbreaks with common features (e.g. multiple antibiotic-resistant

The threshold of marker similarity used for definition of a clone need to be adjusted to the species studied, the typing system used, the environmental selective pressure and the time and space scale of the study (Tibayrenc 1995; Struelens 1996). Mutation rate and gene flux vary between species, pathovars and environments. In vivo micro-evolution of most pathogens remains poorly understood. Subclonal evolution and emergence of variants that occur in individual hosts or during prolonged transmission can be recognized by several high resolution molecular typing systems, like, for instance, macrorestriction analysis by

**4. Strategies applied for surveillance and typing of relevant gram-positive** 

*Staphylococcus aureus* is recognized as one of the most important human pathogens. *It* has shown great ability to acquire resistance to different antimicrobial agents. The first isolation of methicillin-resistant *S. aureus* (MRSA) was reported in 1960 and since then, the prevalence

Methicillin resistance is conferred by the *mecA* gene which codes for an additional penicillinbinding protein named PBP 2a; this protein has reduced affinity to β-lactam agents. This gene is located in a mobile genetic element of variable size known as staphylococcal cassette chromosome *mec* (SCC*mec*). So far, eight types and several subtypes of SCC*mec* have been

The incidence of MRSA varies geographically throughout the world. MRSA has emerged as an important pathogen among hospitalized patients. Most hospital-acquired infections caused by methicillin-resistant *Staphylococcus aureus* (HA-MRSA) are associated with a relatively small number of epidemic clones that spread over different continents. According to the Sistema Informático de Resistencia (Asociación Argentina de Microbiología, Buenos Aires, Argentina), MRSA strains are among the most prevalent nosocomial pathogens (http://www.aam.org.ar) in Argentina, whereas the Brazilian clone, the pediatric clone and the Cordobés clone have been found to be the main clones associated with HA-MRSA

However, since 1990, MRSA has been recognized as a cause of infections in people without established risk factors for HA-MRSA, such as recent hospitalization, surgery, residence in a long-term care facility, receipt of dialysis, or presence of invasive medical devices (Fridkin et al. 2005; Chambers & Deleo 2009). These infections are thought to be acquired in the community and are referred to as community-associated MRSA infections (CA-MRSA). This term has also been used to refer to MRSA strains with bacteriological characteristics considered typical of isolates recovered from patients with CA-MRSA infections (Salgado et al. 2003). HA-MRSA strains are generally resistant to antibiotics other than β-lactams, whereas typical CA-MRSA strains are only resistant to methicillin. HA-MRSA isolates frequently harbor SSC*mec* types-I, II and III whereas CA-MRSA strains carry types IV and V

characterized (Deurenberg & Stobberingh 2008; Chambers & Deleo 2009).

infections (Corso et al. 1998; Sola et al. 2002; Gardella et al. 2005).

isolates) from different geographic locations, the so-called epidemic clones.

Multiple techniques have been developed to assess genomic differences among different isolates or clones of the same species, such as PCR-based methods and PFGE. These methods present portability problems and limited comprehension of the processes by which variation occurs. However, DNA sequence-based techniques generate portable differentiation in bacterial populations that can be used to understand their phylogenetic history. Extensive genomic and phenotypic diversity exists within populations of microbial pathogens of the same species. This diversity reflects the evolutionary divergence arising from mutations and gene flux. These distinctive characters are scored by typing systems which are designed to optimize discrimination between epidemiologically related and unrelated isolates of the pathogen of interest (Maslow and Mulligan 1996; Struelens 1996).

Epidemiologic typing systems can be used for outbreak investigations to confirm and delineate the transmission patterns of one or more epidemic clone(s), to test hypotheses about the sources and transmission vehicles of these clones and to monitor the reservoirs of epidemic organisms. Typing also contributes to epidemiologic surveillance and evaluation of control measures by documenting the prevalence over time and the circulation of epidemic clones in infected populations. Clearly, different requirements will be needed for these distinct applications (Maslow and Mulligan 1996; Struelens 1996).

Typing can be undertaken at two different levels, depending on the situation: i) short term or local epidemiology, when organisms are recovered in a defined setting over a short period of time, which is used to study nosocomial outbreaks, local transmission and carriage, and the relationship between isolates associated with carriage and infection in a given geographic area, ii) long term or global epidemiology, when strains are recovered from one geographic area related to those isolated worldwide or strains recovered at different times.

Local epidemiology is applied to study outbreaks with the aim to characterize that the increase in incidence of infection is caused by enhanced transmission of a specific strain. In this framework, typing methods are applied to investigate the sources of contamination and the route of transmission. Accurate application of bacterial typing will support appropriate control measures designed to contain or interrupt the outbreak and prevent further spread of disease. Typing may also be used for isolates cultured from the same patient over time to help define whether a second episode of infection is due to relapse or reinfection. PCR fingerprinting is the simplest and most rapid genotypic method for local application; however, PCR typing is very susceptible to minor variations in experimental conditions and reagents. Therefore, the method is more appropriate for the comparison of a limited number of samples processed simultaneously and run on one gel. By contrast, PFGE has good reproducibility and is highly discriminatory. Therefore, PFGE is considered the current gold standard for outbreak and local epidemiology studies.

At a different level, collaborative studies have been performed to define major internationally disseminated bacterial clones of important human pathogens. Currently, MLST in combination with PFGE is the most appropriate strategy for long term epidemiology and have reached useful conclusions from infectious disease surveillance data. The evaluation of global population genetic structure, genetic evolution, genetic diversity and pathogenicity has been successfully developed within this framework.

For eukaryotes, clones are genetically identical organisms. However, in bacterial epidemiology, the clone concept is of an even more pragmatic nature, denoting isolates

Multiple techniques have been developed to assess genomic differences among different isolates or clones of the same species, such as PCR-based methods and PFGE. These methods present portability problems and limited comprehension of the processes by which variation occurs. However, DNA sequence-based techniques generate portable differentiation in bacterial populations that can be used to understand their phylogenetic history. Extensive genomic and phenotypic diversity exists within populations of microbial pathogens of the same species. This diversity reflects the evolutionary divergence arising from mutations and gene flux. These distinctive characters are scored by typing systems which are designed to optimize discrimination between epidemiologically related and unrelated isolates of the pathogen of interest (Maslow and Mulligan 1996; Struelens 1996). Epidemiologic typing systems can be used for outbreak investigations to confirm and delineate the transmission patterns of one or more epidemic clone(s), to test hypotheses about the sources and transmission vehicles of these clones and to monitor the reservoirs of epidemic organisms. Typing also contributes to epidemiologic surveillance and evaluation of control measures by documenting the prevalence over time and the circulation of epidemic clones in infected populations. Clearly, different requirements will be needed for

Typing can be undertaken at two different levels, depending on the situation: i) short term or local epidemiology, when organisms are recovered in a defined setting over a short period of time, which is used to study nosocomial outbreaks, local transmission and carriage, and the relationship between isolates associated with carriage and infection in a given geographic area, ii) long term or global epidemiology, when strains are recovered from one geographic area related to those isolated worldwide or strains recovered at

Local epidemiology is applied to study outbreaks with the aim to characterize that the increase in incidence of infection is caused by enhanced transmission of a specific strain. In this framework, typing methods are applied to investigate the sources of contamination and the route of transmission. Accurate application of bacterial typing will support appropriate control measures designed to contain or interrupt the outbreak and prevent further spread of disease. Typing may also be used for isolates cultured from the same patient over time to help define whether a second episode of infection is due to relapse or reinfection. PCR fingerprinting is the simplest and most rapid genotypic method for local application; however, PCR typing is very susceptible to minor variations in experimental conditions and reagents. Therefore, the method is more appropriate for the comparison of a limited number of samples processed simultaneously and run on one gel. By contrast, PFGE has good reproducibility and is highly discriminatory. Therefore, PFGE is considered the current gold

At a different level, collaborative studies have been performed to define major internationally disseminated bacterial clones of important human pathogens. Currently, MLST in combination with PFGE is the most appropriate strategy for long term epidemiology and have reached useful conclusions from infectious disease surveillance data. The evaluation of global population genetic structure, genetic evolution, genetic

For eukaryotes, clones are genetically identical organisms. However, in bacterial epidemiology, the clone concept is of an even more pragmatic nature, denoting isolates

diversity and pathogenicity has been successfully developed within this framework.

these distinct applications (Maslow and Mulligan 1996; Struelens 1996).

standard for outbreak and local epidemiology studies.

different times.

obtained during real outbreaks with common features (e.g. multiple antibiotic-resistant isolates) from different geographic locations, the so-called epidemic clones.

The threshold of marker similarity used for definition of a clone need to be adjusted to the species studied, the typing system used, the environmental selective pressure and the time and space scale of the study (Tibayrenc 1995; Struelens 1996). Mutation rate and gene flux vary between species, pathovars and environments. In vivo micro-evolution of most pathogens remains poorly understood. Subclonal evolution and emergence of variants that occur in individual hosts or during prolonged transmission can be recognized by several high resolution molecular typing systems, like, for instance, macrorestriction analysis by pulsed-field gel electrophoresis (Struelens 1996).
