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

*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 of this pathogen has increased.

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

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

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

Application of Molecular Typing Methods to the

respective spa types (Harmsen et al. 2003).

SCCmec typing (Gardella et al. 2005).

of prevalent clones in Argentina.

bacterial meningitis (von Specht et al. 2006), (Figure 7).

Study of Medically Relevant Gram-Positive Cocci 125

(Enright, 2000, 2002). Another useful technique is the Staphylococcal cassette chromosome *mec* (SCC*mec*) typing, based on the molecular characterization by multiplex PCR of the mobile genetic element carrying the methicillin-resistant gene (*mecA*) (Oliveira and de Lencastre 2002). The combination of the MLST type and the SCC*mec* type, dened as the "clonal type," is now

Moreover, single-locus DNA sequencing of repeat regions of the *coa* (coagulase) gene and the *spa* gene (protein A), respectively, could be used for reliable and accurate MRSA typing (Shopsin, 1999 2000; Tang et al. 2000; Shopsin & Kreiswirth 2001; Harmsen et al. 2003). Spa typing is especially interesting for rapid typing of MRSA in a hospital setting since it offers higher resolution than coa typing (Shopsin et al. 2000). The repeat region of the *spa* gene is subject to spontaneous mutations, as well as to loss and gain of repeats. Repeats are assigned an alpha-numerical code, and the spa type is deduced from the order of specic repeats. There is a good correlation between clonal groupings determined by MLST and the

We performed different studies to characterize MRSA clones within diverse scenarios of Argentina. In 2005, we demonstrated the replacement of the multiresistant MRSA "Brazilian" clone (SCC*mec* III, ST239) by the "Cordobes" clone (SCC*mec* I, ST5), a MRSA clone susceptible to rifampin, minocycline and trimethoprim/sulfamethoxazole in two university hospitals. Isolates were characterized by using RAPD-PCR and PFGE and

Later, we analyzed community-associated methicillin-resistant *Staphylococcus aureus* (CA-MRSA) isolates recovered from patients suffering from different types of infections. All CA-MRSA isolates carried SCC*mec* type IV. Four major clones were detected in Argentina by PFGE. The largest cluster was named CAA clone: Pulsotype A, spa type 311, ST 5, LPV (+) (Gardella et al. 2008) and two isolates of this clone were recovered from two cases of acute

Fig. 7. (A) Dendrogram of pulsed-field electrophoresis banding pattern of CA-MRSA

isolates and the 3 clonal types most prevalent in Argentinean hospitals: Brazilian clone (Bra), Cordobes clone (Cor), Pediatric clone (Ped). Similarity coefficient was calculated by using Dice coefficient, and cluster analysis was performed by the unweighted pair-group method. Four major pulsotypes were coded from A, B, C and D and representative HA-MRSA strains

used for the international nomenclature of MRSA clones (Enright et al. 2002).

The Panton-Valentine leukocidin (PVL) toxin has been described as a genetic marker of CA-MRSA isolates, rarely identified in HA-MRSA isolates (Ma et al. 2002; Naimi et al. 2003). Several studies have demonstrated that the presence of PVL genes is associated with *S. aureus* recovered from patients suffering from primary skin infections (Lina et al. 1999), severe necrotizing pneumonia, and increased complications of hematogenous osteomyelitis; however, the role of PVL in the pathogenesis of *S. aureus* infections has not yet been fully elucidated.

The spectrum of disease caused by CA-MRSA appears to be similar to that of methicillinsusceptible *Staphylococcus aureus* (MSSA) in the community. Skin and soft tissue infections (SSTIs), specifically furuncles (abscessed hair follicles or "boils"), carbuncles (coalesced masses of furuncles), and abscesses, are the most frequently reported clinical manifestations (Fergie & Purcell 2001; Baggett et al. 2003; Fridkin et al. 2005). Less commonly, MRSA has been associated with severe and invasive staphylococcal infections in the community, including necrotizing pneumonia, bacteremia, osteomyelitis, toxic shock syndrome, and meningitis (Deurenberg & Stobberingh 2008).The rapid emergence of these infections has been one of the most unexpected events in bacterial infectious diseases in the recent years.

Distinct genetic lineages associated with CA-MRSA infections have been determined by typing and their geographic dissemination evaluated in different countries. In Latin America, CA-MRSA has been described several times (Ma et al. 2005; Ribeiro et al. 2005; Alvarez et al. 2006; Gardella et al. 2008).

Typing of MRSA strains is necessary for proper epidemiological investigations of sources and modes of transmission of these strains in hospitals, and the design of appropriate control measures and the application of different typing methods have contributed to understanding the emergence of MRSA in the community. Phenotyping methods generally have limited discriminatory power and poor typeability; therefore, a number of molecular techniques have been developed for *S. aureus* typing, namely restriction fragment length polymorphism (RFLP) analysis techniques, including ribotyping and Southern blot analysis with probes for mobile elements present in multiple copies in the staphylococcal genome, like insertion sequences (IS*256*,IS*257*, IS*431* and IS*1181*) and transposons (Tn*554* and Tn*4001*) (Wei et al. 1992; Tenover et al. 1994; Kreiswirth et al. 1995).

Among PCR methods, rep-PCR and RAPD-PCR analysis were found to be epidemiologically useful, but interlaboratory studies showed that reproducibility is an important drawback for these techniques (Saulnier et al. 1993; van Belkum et al. 1995; Deplano et al. 1997; van der Zee et al. 1999). Pulsed-field gel electrophoresis (PFGE) analysis is an accurate and discriminating method which is now used as the reference method for *S. aureus* typing in some reference centers (Bannerman et al. 1995). However, PFGE analysis is costly and technically demanding and still requires interlaboratory standardization (Cookson et al. 1996). PFGE proved to be a highly discriminatory and sensitive technique in microepidemiological (local or short term) and macroepidemiological (national, continental, or long term) surveys (Struelens et al. 1993; McDougal et al. 2003). Nevertheless, some authors have argued that the stabilities of PFGE markers may be insufcient for the reliable application of PFGE to long-term or macroepidemiological studies (Blanc et al. 2002).

Sequence-based methods such as multilocus sequence typing (MLST) has proved to be adequate for long-term global epidemiology and the study of the recent evolution of *S. aureus*

The Panton-Valentine leukocidin (PVL) toxin has been described as a genetic marker of CA-MRSA isolates, rarely identified in HA-MRSA isolates (Ma et al. 2002; Naimi et al. 2003). Several studies have demonstrated that the presence of PVL genes is associated with *S. aureus* recovered from patients suffering from primary skin infections (Lina et al. 1999), severe necrotizing pneumonia, and increased complications of hematogenous osteomyelitis; however, the role of PVL in the pathogenesis of *S. aureus* infections has not yet been fully

The spectrum of disease caused by CA-MRSA appears to be similar to that of methicillinsusceptible *Staphylococcus aureus* (MSSA) in the community. Skin and soft tissue infections (SSTIs), specifically furuncles (abscessed hair follicles or "boils"), carbuncles (coalesced masses of furuncles), and abscesses, are the most frequently reported clinical manifestations (Fergie & Purcell 2001; Baggett et al. 2003; Fridkin et al. 2005). Less commonly, MRSA has been associated with severe and invasive staphylococcal infections in the community, including necrotizing pneumonia, bacteremia, osteomyelitis, toxic shock syndrome, and meningitis (Deurenberg & Stobberingh 2008).The rapid emergence of these infections has been one of the most unexpected events in bacterial infectious diseases in the recent years. Distinct genetic lineages associated with CA-MRSA infections have been determined by typing and their geographic dissemination evaluated in different countries. In Latin America, CA-MRSA has been described several times (Ma et al. 2005; Ribeiro et al. 2005;

Typing of MRSA strains is necessary for proper epidemiological investigations of sources and modes of transmission of these strains in hospitals, and the design of appropriate control measures and the application of different typing methods have contributed to understanding the emergence of MRSA in the community. Phenotyping methods generally have limited discriminatory power and poor typeability; therefore, a number of molecular techniques have been developed for *S. aureus* typing, namely restriction fragment length polymorphism (RFLP) analysis techniques, including ribotyping and Southern blot analysis with probes for mobile elements present in multiple copies in the staphylococcal genome, like insertion sequences (IS*256*,IS*257*, IS*431* and IS*1181*) and transposons (Tn*554* and Tn*4001*)

Among PCR methods, rep-PCR and RAPD-PCR analysis were found to be epidemiologically useful, but interlaboratory studies showed that reproducibility is an important drawback for these techniques (Saulnier et al. 1993; van Belkum et al. 1995; Deplano et al. 1997; van der Zee et al. 1999). Pulsed-field gel electrophoresis (PFGE) analysis is an accurate and discriminating method which is now used as the reference method for *S. aureus* typing in some reference centers (Bannerman et al. 1995). However, PFGE analysis is costly and technically demanding and still requires interlaboratory standardization (Cookson et al. 1996). PFGE proved to be a highly discriminatory and sensitive technique in microepidemiological (local or short term) and macroepidemiological (national, continental, or long term) surveys (Struelens et al. 1993; McDougal et al. 2003). Nevertheless, some authors have argued that the stabilities of PFGE markers may be insufcient for the reliable

application of PFGE to long-term or macroepidemiological studies (Blanc et al. 2002).

Sequence-based methods such as multilocus sequence typing (MLST) has proved to be adequate for long-term global epidemiology and the study of the recent evolution of *S. aureus*

elucidated.

Alvarez et al. 2006; Gardella et al. 2008).

(Wei et al. 1992; Tenover et al. 1994; Kreiswirth et al. 1995).

(Enright, 2000, 2002). Another useful technique is the Staphylococcal cassette chromosome *mec* (SCC*mec*) typing, based on the molecular characterization by multiplex PCR of the mobile genetic element carrying the methicillin-resistant gene (*mecA*) (Oliveira and de Lencastre 2002). The combination of the MLST type and the SCC*mec* type, dened as the "clonal type," is now used for the international nomenclature of MRSA clones (Enright et al. 2002).

Moreover, single-locus DNA sequencing of repeat regions of the *coa* (coagulase) gene and the *spa* gene (protein A), respectively, could be used for reliable and accurate MRSA typing (Shopsin, 1999 2000; Tang et al. 2000; Shopsin & Kreiswirth 2001; Harmsen et al. 2003). Spa typing is especially interesting for rapid typing of MRSA in a hospital setting since it offers higher resolution than coa typing (Shopsin et al. 2000). The repeat region of the *spa* gene is subject to spontaneous mutations, as well as to loss and gain of repeats. Repeats are assigned an alpha-numerical code, and the spa type is deduced from the order of specic repeats. There is a good correlation between clonal groupings determined by MLST and the respective spa types (Harmsen et al. 2003).

We performed different studies to characterize MRSA clones within diverse scenarios of Argentina. In 2005, we demonstrated the replacement of the multiresistant MRSA "Brazilian" clone (SCC*mec* III, ST239) by the "Cordobes" clone (SCC*mec* I, ST5), a MRSA clone susceptible to rifampin, minocycline and trimethoprim/sulfamethoxazole in two university hospitals. Isolates were characterized by using RAPD-PCR and PFGE and SCCmec typing (Gardella et al. 2005).

Later, we analyzed community-associated methicillin-resistant *Staphylococcus aureus* (CA-MRSA) isolates recovered from patients suffering from different types of infections. All CA-MRSA isolates carried SCC*mec* type IV. Four major clones were detected in Argentina by PFGE. The largest cluster was named CAA clone: Pulsotype A, spa type 311, ST 5, LPV (+) (Gardella et al. 2008) and two isolates of this clone were recovered from two cases of acute bacterial meningitis (von Specht et al. 2006), (Figure 7).

Fig. 7. (A) Dendrogram of pulsed-field electrophoresis banding pattern of CA-MRSA isolates and the 3 clonal types most prevalent in Argentinean hospitals: Brazilian clone (Bra), Cordobes clone (Cor), Pediatric clone (Ped). Similarity coefficient was calculated by using Dice coefficient, and cluster analysis was performed by the unweighted pair-group method. Four major pulsotypes were coded from A, B, C and D and representative HA-MRSA strains of prevalent clones in Argentina.

Application of Molecular Typing Methods to the

equivalent in terms of antibiotic resistance.

al. 1999; Descheemaeker et al. 2000; McGee et al. 2001).

Study of Medically Relevant Gram-Positive Cocci 127

epidemiology of *S. pneumoniae* resistance to β-lactams (Zhang et al. 1990; Munoz et al. 1991). For macrolide-resistant strains, tests for *erm* and *mef* genes have to be performed (http://www.sph.emory.edu/PMEN). The PMEN network has included BOX-PCR typing

In addition, the use of modern typing methods, mainly MLST, has greatly helped track the geographical spread of specic *S. pneumoniae* strains and follow the dynamics of microbial populations over time. The application of all these techniques have shown that the spread of multiresistant international clones dened by the PMEN is the main cause of increase in pneumococcal resistance to β-lactams and other drugs (Munoz et al. 1991; Klugman 2002; Smith et al. 2006; Sadowy et al. 2007; Soriano et al. 2008). In pneumococcus, each serotype may typically be made up of a number of clones, which are not closely related and are not

Furthermore, molecular methods showed that the evolution of penicillin-resistance and multiresistance is a phenomenon in which the acquisition and/or alteration of molecular targets is mainly a consequence of intergenic change and that *S. pneumoniae* diversity has largely been driven by recombination (Hermans et al. 1997; Enright & Spratt 1998; Enright et

As multiresistance has increased the difficulties of treating this serious bacterial infection, prevention through vaccination has become even more important. There are at least 91 known pneumococcus capsular types, with 23 capsular types included in the current pneumococcal polysaccharide (adult) vaccine and 13 types included in the current conjugate (child) vaccine. To overcome serotype specificity of actual vaccines, upcoming pneumococcal vaccines should offer a different approach to the prevention of pneumococcal disease and the decrease in carriage. Several proteins have been identified as possible candidates to develop more appropriate vaccines. One of them , the pneumococcal surface protein A (PspA), is a surface virulence factor, antigenically variable yet cross-reactive that interferes with complement-mediated clearance of pneumococci (McDaniel et al. 1991; Tu et al. 1999).Since 1993, six Latin-American countries have been participating in an epidemiological surveillance study conducted by the Pan American Health Organization (PAHO) in order to determine the relative prevalence of capsular types and antimicrobial resistant patterns of *S. pneumoniae* causing invasive infections in children <5 years of age. One of these studies showed that, the prevalence of penicillin resistant *S. pneumoniae* (PRSP) in Argentina was 24.4%, which was significantly associated with the expansion of serotype 14 clone that had been previously described in Europe expressing serotype 9V (Rossi et al. 1998). A similar situation was encountered in Uruguay in the same period (Camou et al. 1998).

Ongoing surveillance programs for invasive pneumococcal disease also monitor the appropriateness of existing vaccine formulations and provide valuable information on which to base the formulation and application of new vaccines that are currently under development. In this framework, from 1993 to 2000, with the participation of the Argentinean *Streptococcus pneumoniae* Working Group, 1293 invasive isolates were studied to determine capsular type distribution and antimicrobial susceptibility. We selected a sample of 149 strains, having the same serotype distribution as in the total collection, in order to characterize the distribution of PspA variants among Argentinean invasive isolates recovered from children less than 6 years of age. The genetic relatedness among the isolates of the major serotypes was also evaluated by BOX-PCR because it is a quick molecular

in the guidelines for the recognition of pneumococcal clones (McGee et al. 2001).

We also characterized CA-MRSA strains isolated from skin and soft-tissue infections in isolates recovered from Uruguay in 2005. In that study, we identified three major groups of CA-MRSA strains (1, 2, and 4) that were defined according to phenotypic and genotypic characteristics. The most frequent group, G1, showed a PFGE pattern identical to that of CA-MRSA strains previously isolated in Uruguay and Brazil; these strains are still producing SSTI, illustrating the stability of this emergent pathogen over time, as well as its excellent adaptation to the community environment (Pardo et al. 2009).

During the 2008 school- year period we conducted the first epidemiological study of *S. aureus* carriage in Argentina. Carriage was investigated in all children attending the last year of kindergarten in a city of Buenos Aires province, Argentina. Of 316 healthy children, 31.0% carried *S. aureus*, including 14 MRSA carriers (4.4%). All MRSA isolates carried the SCC*mec* type IV cassette. Eight of those 14 carriers were closely related to the CAA clone, which was responsible for the most severe community-acquired MRSA infections caused in our country (PFGE A, SCC*mec* IV, *spa* t311, ST5), (Gardella et al.).

Our results should serve as a warning for the health system since the main clone circulating in the community presents epidemic characteristics and also possesses a genetic background (ST5) of demonstrated plasticity and efficiency to be established as prevalent in the hospital environment.

#### **4.2 Molecular epidemiology of** *Streptococcus pneumoniae*

*Streptococcus pneumoniae* is a human pathogen of increasing clinical relevance causing important diseases such as meningitis, pneumonia, bacteremia and otitis media. Surveillance has become progressively more important because of the worldwide distribution of penicillin-resistant and multidrug-resistant pneumococci clones in the last 15 years.

*S. pneumoniae* with resistance to one or more antibiotics has been isolated since 1990. Penicillin is the drug of choice for the treatment of pneumococcal infections. The resistance mechanism includes the modification of penicillin-binding proteins (PBP), which in general is associated to cephalosporin resistance.

The clinical relevance of multiple antibiotic-resistant pneumococcal strains has led to the creation of a network. The Pneumococcal Molecular Epidemiology Network (PMEN) was established in 1997 with the aim of global surveillance of antibiotic-resistant *S.pneumoniae* and the standardization of nomenclature and classification of resistant clones (http://www.sph.emory.edu/PMEN/index.html). The PMEN also includes major invasive antibiotic-susceptible clones that have a wide geographic spread. Up to date, there are currently 43 clones described by the PMEN. Of those, three penicillin- resistant and two penicillin- susceptible PMEN clones have been detected in Argentina http://www.sph.emory.edu/PMEN/(Zemlickova et al. 2005). Clones to be included in the Network have to be subjected to PFGE, MLST and PBP fingerprinting to confirm that they differ from previously accepted ones. PBP fingerprinting is a typing technique that includes PCR amplification of the *pbp1a*, *pbp2b* and *pbp2x* genes with previously described primers (Gherardi et al. 2000). Amplified genes are digested with *HaeIII*+*DdeI* (*pbp1a*) and *HaeIII*+*RsaI* (*pbp2b* and *pbp2x*) restriction enzymes and electrophoresed on 3% gels. This technique has been used in many reports over the last 20 years to study the molecular

We also characterized CA-MRSA strains isolated from skin and soft-tissue infections in isolates recovered from Uruguay in 2005. In that study, we identified three major groups of CA-MRSA strains (1, 2, and 4) that were defined according to phenotypic and genotypic characteristics. The most frequent group, G1, showed a PFGE pattern identical to that of CA-MRSA strains previously isolated in Uruguay and Brazil; these strains are still producing SSTI, illustrating the stability of this emergent pathogen over time, as well as its excellent

During the 2008 school- year period we conducted the first epidemiological study of *S. aureus* carriage in Argentina. Carriage was investigated in all children attending the last year of kindergarten in a city of Buenos Aires province, Argentina. Of 316 healthy children, 31.0% carried *S. aureus*, including 14 MRSA carriers (4.4%). All MRSA isolates carried the SCC*mec* type IV cassette. Eight of those 14 carriers were closely related to the CAA clone, which was responsible for the most severe community-acquired MRSA infections caused in

Our results should serve as a warning for the health system since the main clone circulating in the community presents epidemic characteristics and also possesses a genetic background (ST5) of demonstrated plasticity and efficiency to be established as prevalent in the hospital

*Streptococcus pneumoniae* is a human pathogen of increasing clinical relevance causing important diseases such as meningitis, pneumonia, bacteremia and otitis media. Surveillance has become progressively more important because of the worldwide distribution of penicillin-resistant and multidrug-resistant pneumococci clones in the last 15

*S. pneumoniae* with resistance to one or more antibiotics has been isolated since 1990. Penicillin is the drug of choice for the treatment of pneumococcal infections. The resistance mechanism includes the modification of penicillin-binding proteins (PBP), which in general

The clinical relevance of multiple antibiotic-resistant pneumococcal strains has led to the creation of a network. The Pneumococcal Molecular Epidemiology Network (PMEN) was established in 1997 with the aim of global surveillance of antibiotic-resistant *S.pneumoniae* and the standardization of nomenclature and classification of resistant clones (http://www.sph.emory.edu/PMEN/index.html). The PMEN also includes major invasive antibiotic-susceptible clones that have a wide geographic spread. Up to date, there are currently 43 clones described by the PMEN. Of those, three penicillin- resistant and two penicillin- susceptible PMEN clones have been detected in Argentina http://www.sph.emory.edu/PMEN/(Zemlickova et al. 2005). Clones to be included in the Network have to be subjected to PFGE, MLST and PBP fingerprinting to confirm that they differ from previously accepted ones. PBP fingerprinting is a typing technique that includes PCR amplification of the *pbp1a*, *pbp2b* and *pbp2x* genes with previously described primers (Gherardi et al. 2000). Amplified genes are digested with *HaeIII*+*DdeI* (*pbp1a*) and *HaeIII*+*RsaI* (*pbp2b* and *pbp2x*) restriction enzymes and electrophoresed on 3% gels. This technique has been used in many reports over the last 20 years to study the molecular

adaptation to the community environment (Pardo et al. 2009).

our country (PFGE A, SCC*mec* IV, *spa* t311, ST5), (Gardella et al.).

**4.2 Molecular epidemiology of** *Streptococcus pneumoniae*

is associated to cephalosporin resistance.

environment.

years.

epidemiology of *S. pneumoniae* resistance to β-lactams (Zhang et al. 1990; Munoz et al. 1991). For macrolide-resistant strains, tests for *erm* and *mef* genes have to be performed (http://www.sph.emory.edu/PMEN). The PMEN network has included BOX-PCR typing in the guidelines for the recognition of pneumococcal clones (McGee et al. 2001).

In addition, the use of modern typing methods, mainly MLST, has greatly helped track the geographical spread of specic *S. pneumoniae* strains and follow the dynamics of microbial populations over time. The application of all these techniques have shown that the spread of multiresistant international clones dened by the PMEN is the main cause of increase in pneumococcal resistance to β-lactams and other drugs (Munoz et al. 1991; Klugman 2002; Smith et al. 2006; Sadowy et al. 2007; Soriano et al. 2008). In pneumococcus, each serotype may typically be made up of a number of clones, which are not closely related and are not equivalent in terms of antibiotic resistance.

Furthermore, molecular methods showed that the evolution of penicillin-resistance and multiresistance is a phenomenon in which the acquisition and/or alteration of molecular targets is mainly a consequence of intergenic change and that *S. pneumoniae* diversity has largely been driven by recombination (Hermans et al. 1997; Enright & Spratt 1998; Enright et al. 1999; Descheemaeker et al. 2000; McGee et al. 2001).

As multiresistance has increased the difficulties of treating this serious bacterial infection, prevention through vaccination has become even more important. There are at least 91 known pneumococcus capsular types, with 23 capsular types included in the current pneumococcal polysaccharide (adult) vaccine and 13 types included in the current conjugate (child) vaccine. To overcome serotype specificity of actual vaccines, upcoming pneumococcal vaccines should offer a different approach to the prevention of pneumococcal disease and the decrease in carriage. Several proteins have been identified as possible candidates to develop more appropriate vaccines. One of them , the pneumococcal surface protein A (PspA), is a surface virulence factor, antigenically variable yet cross-reactive that interferes with complement-mediated clearance of pneumococci (McDaniel et al. 1991; Tu et al. 1999).Since 1993, six Latin-American countries have been participating in an epidemiological surveillance study conducted by the Pan American Health Organization (PAHO) in order to determine the relative prevalence of capsular types and antimicrobial resistant patterns of *S. pneumoniae* causing invasive infections in children <5 years of age. One of these studies showed that, the prevalence of penicillin resistant *S. pneumoniae* (PRSP) in Argentina was 24.4%, which was significantly associated with the expansion of serotype 14 clone that had been previously described in Europe expressing serotype 9V (Rossi et al. 1998). A similar situation was encountered in Uruguay in the same period (Camou et al. 1998).

Ongoing surveillance programs for invasive pneumococcal disease also monitor the appropriateness of existing vaccine formulations and provide valuable information on which to base the formulation and application of new vaccines that are currently under development. In this framework, from 1993 to 2000, with the participation of the Argentinean *Streptococcus pneumoniae* Working Group, 1293 invasive isolates were studied to determine capsular type distribution and antimicrobial susceptibility. We selected a sample of 149 strains, having the same serotype distribution as in the total collection, in order to characterize the distribution of PspA variants among Argentinean invasive isolates recovered from children less than 6 years of age. The genetic relatedness among the isolates of the major serotypes was also evaluated by BOX-PCR because it is a quick molecular

Application of Molecular Typing Methods to the

clone recovered in Argentina (2000).

**5. Conclusion** 

*pneumoniae*.

same ST always presented the same PspA family.

Study of Medically Relevant Gram-Positive Cocci 129

international multiresistant clones whereas, the Spain 9V -ST156 clonal complex being the most prevalent. Moreover, this clone has evolved rapidly, as demonstrated by the observed number of STs , the use of another approach of MLST (multiple locus variable-numbertandem repeat analysis) and the polymorphism of *pbp* and *pspA* genes (coding for penicillinbinding proteins and the pneumococcal surface protein A, respectively) (Sadowy et al.).

Fig. 8. A) BOX-PCR patterns of 4 Argentinean isolates belonging to Poland6B-20 clone. B). Pulse field gel electrophoresis of *Sma*I digested DNA of *S. pneumoniae.* Lane1: *S.pneumoniae*  R6, lane2: *S.pneumoniae* 6B (1994), lanes3-6: *S.pneumoniae* isolates belonging to Poland6B-20

The relationship between PspA and ST was also explored (Qian et al.). This author analyzed 171 invasive *S. pneumoniae* isolates from Chinese children in 11 hospitals between 2006 and 2008. He found that Family 1 and family 2 PspAs were prevalent and that strains with the

This chapter has reviewed some of the most popular molecular methods for the epidemiological typing of two medically relevant gram-positive cocci, discussing their principles, strengths and weaknesses. We have described several examples of our recent work showing the application of molecular typing techniques to the study of two relevant pathogens. The examples we have considered herein include a relative clonal species such as *S.aureus*, and on the other hand, a pathogen showing a high recombination rate, such as *S.* 

method that is suitable for investigation of genetic relatedness of pneumococcal strains and provides results whose interpretation is relatively unambiguous (Hermans et al. 1995; van Belkum et al. 1996). This study provided epidemiological information about the PspA family distribution and the genetic diversity of Argentinean *S. pneumoniae* isolates and informed of the potential coverage of a PspA- based vaccine. It was the first insight into diversity of PspA within strains circulating in Argentina (Mollerach et al. 2004). Family 1 PspA was detected in 54.4% of the isolates, 41.6% of which were family 2 and 4.0% expressed both family 1 and family 2 PspAs. This observation indicates that a PspA vaccine containing only family 1 and family 2 PspAs should be able to cover the bulk of the strains in this region. Box typing revealed the Argentinian strains were from at least 10 clonally related groups.

In some cases, a strong association between one PspA type and a certain capsular type was found. For example, serotype 1 and 5 and the majority of isolates of penicillin-susceptible serotype 14 isolates exhibited PspA family 1. On the other hand, serotypes 7 F, serotype14 PRSP and the majority of type 9V isolates were assigned to PspA family 2. BOX-PCR analysis revealed genetic homogeneity of serotype 14 PRSP and serotype 5 isolates. Antibiotype suggests correlation with the Spain9V-3 clone and Colombia5-19 clone, respectively. These clones had been previously described in the region (Gamboa et al. 2002; Brandileone et al. 2004; Zemlickova et al. 2005).

Nowadays, the sequencing of DNA allows to compare the results between laboratories and to obtain a global look for the situation of the circulating multidrug-resistant clones. This effort includes a database that contains information concerning the clones that are currently widespread in different parts of the world (http://spneumoniae.mlst.net).

In the framework of PAHO in Latin American countries, surveillance data revealed that penicillin-nonsusceptible *S. pneumoniae* (PNSP) type 6B increased from 15.8 % in the period between 1993-1997, to 67.3 % in 1998-2002 (p<0.001). Serotype 6 ranks fourth among capsular types causing invasive diseases in Argentinean patients under 6 years of age, and it has been included in the heptavalent conjugate polysaccharide vaccine licensed in Argentina in 2001 and also in the 13-valent introduced in 2010 (Organización Panamericana de la Salud 2007; Ruvinsky et al. 2008 ). This serotype is a frequent cause of invasive diseases (Riedel et al. 2007; Gabastou et al. 2008; Darabi et al.). We characterized the population of penicillin non-susceptible *S. pneumoniae* type 6B strains isolated from pediatric patients in Argentina between 1993-2002 with the use of molecular typing methods including BOX-PCR, PFGE and MLST (Bonofiglio et al. 2011) (Figure 8). The results of the study showed that the increase in penicillin resistance in serotype 6B may be partly explained by the entrance of the Poland6B-20 clone, which is a PMEN clone not previously described in Argentina. Our findings showed that the Poland6B-20 clone established in 1999; and the use of BOX-PCR and PFGE subtypes suggested that horizontal transfer or differentiation events had occurred after the common lineage became established. Dissemination of this clone could be traced through demographic data, as isolates representative of the clone had been recovered in different regions of Argentina and its expansion is also responsible for the emergence of erythromycin-resistance in *S. pneumoniae* serotype 6B. The pneumococcal MLST database currently contains information of 81 strains of the Poland6B-20 clone.

Other similar studies were carried out by Sadowy et al, who analyzed isolates recovered in Poland in the period 2003-2005 using serotyping, MLST and sequencing of *murM* and *pspA* alleles. They demonstrated that the vast majority of the isolates (90.7%) belong to

method that is suitable for investigation of genetic relatedness of pneumococcal strains and provides results whose interpretation is relatively unambiguous (Hermans et al. 1995; van Belkum et al. 1996). This study provided epidemiological information about the PspA family distribution and the genetic diversity of Argentinean *S. pneumoniae* isolates and informed of the potential coverage of a PspA- based vaccine. It was the first insight into diversity of PspA within strains circulating in Argentina (Mollerach et al. 2004). Family 1 PspA was detected in 54.4% of the isolates, 41.6% of which were family 2 and 4.0% expressed both family 1 and family 2 PspAs. This observation indicates that a PspA vaccine containing only family 1 and family 2 PspAs should be able to cover the bulk of the strains in this region. Box typing revealed the Argentinian strains were from at least 10 clonally related groups. In some cases, a strong association between one PspA type and a certain capsular type was found. For example, serotype 1 and 5 and the majority of isolates of penicillin-susceptible serotype 14 isolates exhibited PspA family 1. On the other hand, serotypes 7 F, serotype14 PRSP and the majority of type 9V isolates were assigned to PspA family 2. BOX-PCR analysis revealed genetic homogeneity of serotype 14 PRSP and serotype 5 isolates. Antibiotype suggests correlation with the Spain9V-3 clone and Colombia5-19 clone, respectively. These clones had been previously described in the region (Gamboa et al. 2002;

Nowadays, the sequencing of DNA allows to compare the results between laboratories and to obtain a global look for the situation of the circulating multidrug-resistant clones. This effort includes a database that contains information concerning the clones that are currently

In the framework of PAHO in Latin American countries, surveillance data revealed that penicillin-nonsusceptible *S. pneumoniae* (PNSP) type 6B increased from 15.8 % in the period between 1993-1997, to 67.3 % in 1998-2002 (p<0.001). Serotype 6 ranks fourth among capsular types causing invasive diseases in Argentinean patients under 6 years of age, and it has been included in the heptavalent conjugate polysaccharide vaccine licensed in Argentina in 2001 and also in the 13-valent introduced in 2010 (Organización Panamericana de la Salud 2007; Ruvinsky et al. 2008 ). This serotype is a frequent cause of invasive diseases (Riedel et al. 2007; Gabastou et al. 2008; Darabi et al.). We characterized the population of penicillin non-susceptible *S. pneumoniae* type 6B strains isolated from pediatric patients in Argentina between 1993-2002 with the use of molecular typing methods including BOX-PCR, PFGE and MLST (Bonofiglio et al. 2011) (Figure 8). The results of the study showed that the increase in penicillin resistance in serotype 6B may be partly explained by the entrance of the Poland6B-20 clone, which is a PMEN clone not previously described in Argentina. Our findings showed that the Poland6B-20 clone established in 1999; and the use of BOX-PCR and PFGE subtypes suggested that horizontal transfer or differentiation events had occurred after the common lineage became established. Dissemination of this clone could be traced through demographic data, as isolates representative of the clone had been recovered in different regions of Argentina and its expansion is also responsible for the emergence of erythromycin-resistance in *S. pneumoniae* serotype 6B. The pneumococcal

MLST database currently contains information of 81 strains of the Poland6B-20 clone.

Other similar studies were carried out by Sadowy et al, who analyzed isolates recovered in Poland in the period 2003-2005 using serotyping, MLST and sequencing of *murM* and *pspA* alleles. They demonstrated that the vast majority of the isolates (90.7%) belong to

widespread in different parts of the world (http://spneumoniae.mlst.net).

Brandileone et al. 2004; Zemlickova et al. 2005).

international multiresistant clones whereas, the Spain 9V -ST156 clonal complex being the most prevalent. Moreover, this clone has evolved rapidly, as demonstrated by the observed number of STs , the use of another approach of MLST (multiple locus variable-numbertandem repeat analysis) and the polymorphism of *pbp* and *pspA* genes (coding for penicillinbinding proteins and the pneumococcal surface protein A, respectively) (Sadowy et al.).

Fig. 8. A) BOX-PCR patterns of 4 Argentinean isolates belonging to Poland6B-20 clone. B). Pulse field gel electrophoresis of *Sma*I digested DNA of *S. pneumoniae.* Lane1: *S.pneumoniae*  R6, lane2: *S.pneumoniae* 6B (1994), lanes3-6: *S.pneumoniae* isolates belonging to Poland6B-20 clone recovered in Argentina (2000).

The relationship between PspA and ST was also explored (Qian et al.). This author analyzed 171 invasive *S. pneumoniae* isolates from Chinese children in 11 hospitals between 2006 and 2008. He found that Family 1 and family 2 PspAs were prevalent and that strains with the same ST always presented the same PspA family.
