**3. Results**

#### **3.1 Antimicrobial susceptibility**

The 90 isolates showed resistance to penicillin (100%), oxacillin (99.3%), amoxicillin (100%) , cefotaxime (100%), cephalothin (100%), cefazolin (100%), chloramphenicol (100%), imipenem (99.3%), ciprofloxacin (87.7%); eleven strains (12.2%) showed low susceptibility for clindamycin, erythromycin, clarithromycin and were susceptible to ciprofloxacin; two strains (2.2%) showed low susceptibility for oxacillin (MIC 4µg/mL) and imipenem. All strains were susceptible to rifampin, tetracycline, gentamicin, trimethoprim-sulfamethoxazole and vancomycin.

Pulsed Field Gel Electrophoresis in Molecular Typing and

Epidemiological Detection of Methicillin Resistant *Staphylococcus aureus* (MRSA) 183

Fig. 2. Dendrogram comparing MRSA clones A, B and C from the Instituto Nacional de Cardiología "Dr. Ignacio Chávez", Mexico with different international MRSA clones: BK2464-New York/Japan-USA clone; JP27-New York/Japan-Japan clone; HDE288-Pediatric clone; HU25-Brazilian-clone; HPV107-Iberian clone; EMRSA-16-U.K. clone. For cluster analysis, Dice coefficients were calculated to compute matrix similarity a transformed into

an agglomerative cluster with the unweighted pair group method with arithmetic

Number of subtypes 5 3 6 SSC*mec* type3 IV II II

ST 30 305/36 5 1Antibiotic abbreviations: CLA- clarithromycin; CD – clindamycin; ERY – erythromycin GEN gentamicin; CIP - ciprofloxacin. 2Intermediate resistance pattern. 3Staphylococcal cassette chromosome

Table 1. Antibiotype and Genotypic characterization of the MRSA clones presented in the

(CLA,CD,ERY)2

*mec*. 4Except B1 and B2; 5Sequence typing (MLST), only the patterns B1 and B2.

Instituto Nacional de Cardiología "Dr. Ignacio Chávez", Mexico (2002-2009).

**Property Clone A Clone B Clone C** 

ß-lactams, (CIP, CLA,CD, ERY)

˜180 ˜2114 ˜211

˜180 ˜211-640 ˜211-640

ß-lactams, (CIP, CLA,CD, ERY)

average.

*SmaI-mecA* 

*Sma-Tn554*

Antibiotype1 (Resistance) ß-lactams,

Hybridization bands(Kb)

Hybridization bands(Kb)

#### **3.2 Molecular typing**

#### **3.2.1 PFGE analysis**

The PFGE analysis separated the MRSA strains into three types, A (5 subtypes), B (3 subtypes) and C (6 subtypes) (Figure 1). PFGE pattern C and subtypes were predominant in this isolates n=72 (80%), Clone A, n=11 (12.2%) and B, n=7 (7.8%) were only found in the isolates of 2002, and these two clones (A and B) were totally replaced by clone C in 2004 and continue until 2009. The results produced by a computer analysis of the banding patterns show clearly the division of the three clone groups (A, B and C); interestingly, the A and B clone isolates have very similar PFGE patterns (coefficient similarity 95%). Nevertheless, the three clones A, B and C could easily be distinguished by antibiograms and other molecular properties as well (Table 1). The three clones were multiresistant, however, each one of them showed a characteristic resistance pattern; clone A was resistant to ß-lactams and showed a low susceptibility to clarithromycin, clindamycin, erythromycin and was susceptible to ciprofloxacin; while clones B and C were resistant to ß-lactams, clarithromycin, clindamycin, erytromycin and ciprofloxacin; only the strains with subtypes B1 and B2 showed low susceptibility for oxacillin and imipenem.

Fig. 1. Pulsed field gel electrophoresis profiles of MRSA clinical isolates from the Instituto Nacional de Cardiología "Dr. Ignacio Chávez", Mexico and representatives of international clones. Lanes: 1-14 lambda ladder used a molecular size (MW) markers; 2 and 13 reference strain NCTC8325; 3-4 (44CAR and 47CAR) pattern C; 5 (2CAR) pattern A; 6 (20CAR) pattern B; 7-12 (HDE288, BK2464, JP27, EMRSA16, HPV107 and HU25) control strains representative of Pediatric, New York/Japan-USA, New York/Japan-Japan, EMRSA16-U.K, Iberian and Brazilian clones.

The PFGE analysis separated the MRSA strains into three types, A (5 subtypes), B (3 subtypes) and C (6 subtypes) (Figure 1). PFGE pattern C and subtypes were predominant in this isolates n=72 (80%), Clone A, n=11 (12.2%) and B, n=7 (7.8%) were only found in the isolates of 2002, and these two clones (A and B) were totally replaced by clone C in 2004 and continue until 2009. The results produced by a computer analysis of the banding patterns show clearly the division of the three clone groups (A, B and C); interestingly, the A and B clone isolates have very similar PFGE patterns (coefficient similarity 95%). Nevertheless, the three clones A, B and C could easily be distinguished by antibiograms and other molecular properties as well (Table 1). The three clones were multiresistant, however, each one of them showed a characteristic resistance pattern; clone A was resistant to ß-lactams and showed a low susceptibility to clarithromycin, clindamycin, erythromycin and was susceptible to ciprofloxacin; while clones B and C were resistant to ß-lactams, clarithromycin, clindamycin, erytromycin and ciprofloxacin; only the strains with subtypes B1 and B2 showed low

Fig. 1. Pulsed field gel electrophoresis profiles of MRSA clinical isolates from the Instituto Nacional de Cardiología "Dr. Ignacio Chávez", Mexico and representatives of international clones. Lanes: 1-14 lambda ladder used a molecular size (MW) markers; 2 and 13 reference strain NCTC8325; 3-4 (44CAR and 47CAR) pattern C; 5 (2CAR) pattern A; 6 (20CAR) pattern

representative of Pediatric, New York/Japan-USA, New York/Japan-Japan, EMRSA16-U.K,

B; 7-12 (HDE288, BK2464, JP27, EMRSA16, HPV107 and HU25) control strains

**3.2 Molecular typing 3.2.1 PFGE analysis** 

susceptibility for oxacillin and imipenem.

Iberian and Brazilian clones.

Fig. 2. Dendrogram comparing MRSA clones A, B and C from the Instituto Nacional de Cardiología "Dr. Ignacio Chávez", Mexico with different international MRSA clones: BK2464-New York/Japan-USA clone; JP27-New York/Japan-Japan clone; HDE288-Pediatric clone; HU25-Brazilian-clone; HPV107-Iberian clone; EMRSA-16-U.K. clone. For cluster analysis, Dice coefficients were calculated to compute matrix similarity a transformed into an agglomerative cluster with the unweighted pair group method with arithmetic average.


1Antibiotic abbreviations: CLA- clarithromycin; CD – clindamycin; ERY – erythromycin GEN gentamicin; CIP - ciprofloxacin. 2Intermediate resistance pattern. 3Staphylococcal cassette chromosome *mec*. 4Except B1 and B2; 5Sequence typing (MLST), only the patterns B1 and B2.

Table 1. Antibiotype and Genotypic characterization of the MRSA clones presented in the Instituto Nacional de Cardiología "Dr. Ignacio Chávez", Mexico (2002-2009).

Pulsed Field Gel Electrophoresis in Molecular Typing and

type 30 (ST30) .

(pattern B2).

(Figure 2).

**3.2.3 Homology pattern** 

Epidemiological Detection of Methicillin Resistant *Staphylococcus aureus* (MRSA) 185

MRSA strains of clones B and C had SCC*mec* type II, sequences type 36 and 5 (ST36 and ST5) respectively, except B1 and B2 which did not amplify SCC*mec*, this isolates showed sequence

Fig. 4. (A) PFGE patterns of MRSA strains from the Instituto Nacional de Cardiología "Dr. Ignacio Chávez", Mexico. Lane 1, 3CAR, (pattern B1); lane 2, control strain and lane 3, 8CAR

One isolate belonging to each type of clones (A, B and C) were compared to strains belonging to previously characterized MRSA clones, i. e., representatives of the pediatric clone and isolates belonging to the New York-Japan clone and also to other international pandemic clones, namely, the Iberian, Brazilian and EMRSA-16 clones (Figure 1). Clone C showed a high degree of similarity to the pediatric (85.5%) and the New York-Japan (89.5%) clones. Clones A and B showed a high degree of similarity to the EMRSA-16 (80%) clone

#### **3.2.2 Hybridization pattern**

The hybridization patterns with *mecA* and Tn*554* probes indicated that the MRSA strains accompanying clone A carried the *mecA* gene on a *Sma*I fragment of approximately 180 Kb, while the *mecA* gene of the clones B and C were found on a fragment of approximately 211 Kb (Figure 3-A). One Tn*554* copy was identified usually on the fragment approximately 180 Kb between the isolates of clone A; while the MRSA strains accompanying clones B and C usually carried two identified Tn*554* copies between the *SmaI* fragments of approximately 211 and 640 Kb; only the strains 3CAR, (B1) and 8CAR, (B2), carried the transposon Tn*554* in a fragment of 640 Kb (Figure 3-B).

Fig. 3. (A) *SmaI*-*mecA* and (B) *SmaI* Tn*554* patterns identified among the clones A, B and C of the Instituto Nacional de Cardiología "Dr. Ignacio Chavéz, Mexico. Lane 1, molecular weight markers, lambda ladder; lane 2, 2CAR (pattern A); lane 3, 20CAR (pattern B) and lane 4, 44CAR (pattern C).

Two strains collected in this hospital 3CAR, (pattern B1) and 8CAR, (pattern B2) did not hybridize with the *mecA* DNA probe, interestingly both presented a low susceptibility to oxacillin and these isolates were subtypes of pattern B. The only difference was found in the *SmaI* hybridization fragment, which contains the *mecA* gene: in the two isolates, this fragment had a smaller molecular size (145 instead of 180 Kb) and did not react with the *mecA* probe, indicating a deletion of approximately 35 Kb, which must have included both the *mecA* gene and part of the *mec* element (Figure 4A and 4B). All the isolates accompanying clone A presented SCC*mec* type IV and sequence type 30 (ST30) whereas the MRSA strains of clones B and C had SCC*mec* type II, sequences type 36 and 5 (ST36 and ST5) respectively, except B1 and B2 which did not amplify SCC*mec*, this isolates showed sequence type 30 (ST30) .

Fig. 4. (A) PFGE patterns of MRSA strains from the Instituto Nacional de Cardiología "Dr. Ignacio Chávez", Mexico. Lane 1, 3CAR, (pattern B1); lane 2, control strain and lane 3, 8CAR (pattern B2).

## **3.2.3 Homology pattern**

184 Gel Electrophoresis – Advanced Techniques

The hybridization patterns with *mecA* and Tn*554* probes indicated that the MRSA strains accompanying clone A carried the *mecA* gene on a *Sma*I fragment of approximately 180 Kb, while the *mecA* gene of the clones B and C were found on a fragment of approximately 211 Kb (Figure 3-A). One Tn*554* copy was identified usually on the fragment approximately 180 Kb between the isolates of clone A; while the MRSA strains accompanying clones B and C usually carried two identified Tn*554* copies between the *SmaI* fragments of approximately 211 and 640 Kb; only the strains 3CAR, (B1) and 8CAR, (B2), carried the transposon Tn*554*

 Fig. 3. (A) *SmaI*-*mecA* and (B) *SmaI* Tn*554* patterns identified among the clones A, B and C of the Instituto Nacional de Cardiología "Dr. Ignacio Chavéz, Mexico. Lane 1, molecular weight markers, lambda ladder; lane 2, 2CAR (pattern A); lane 3, 20CAR (pattern B) and

Two strains collected in this hospital 3CAR, (pattern B1) and 8CAR, (pattern B2) did not hybridize with the *mecA* DNA probe, interestingly both presented a low susceptibility to oxacillin and these isolates were subtypes of pattern B. The only difference was found in the *SmaI* hybridization fragment, which contains the *mecA* gene: in the two isolates, this fragment had a smaller molecular size (145 instead of 180 Kb) and did not react with the *mecA* probe, indicating a deletion of approximately 35 Kb, which must have included both the *mecA* gene and part of the *mec* element (Figure 4A and 4B). All the isolates accompanying clone A presented SCC*mec* type IV and sequence type 30 (ST30) whereas the

**3.2.2 Hybridization pattern** 

in a fragment of 640 Kb (Figure 3-B).

lane 4, 44CAR (pattern C).

One isolate belonging to each type of clones (A, B and C) were compared to strains belonging to previously characterized MRSA clones, i. e., representatives of the pediatric clone and isolates belonging to the New York-Japan clone and also to other international pandemic clones, namely, the Iberian, Brazilian and EMRSA-16 clones (Figure 1). Clone C showed a high degree of similarity to the pediatric (85.5%) and the New York-Japan (89.5%) clones. Clones A and B showed a high degree of similarity to the EMRSA-16 (80%) clone (Figure 2).

Pulsed Field Gel Electrophoresis in Molecular Typing and

**5. Conclusion** 

MRSA clones.

**7. References**

**6. Acknowledgment** 

We thank PhD. Lilia Chihu for style review of the paper

Epidemiological Detection of Methicillin Resistant *Staphylococcus aureus* (MRSA) 187

population, together with the few means of antibiotic restriction it could represent a potential short term risk for the VRSA appearance in the hospitals of our country. Clone A and B were only found in the isolates in 2002, these clones showed a high degree of similarity to the EMRSA-16 clone, this clone is one of the dominant types of MRSA found in a UK hospital (Moore & Lindsay, 2002) and was widely disseminated in Canada (Simor et al., 2002), Greece (Aires de Sousa et al., 2003) and Mexico (Aires de Sousa et al., 2001). Interestingly, both clones (A and B) are very similar (95%) (Figure 2), nevertheless, clone A showed a reduced resistance profile as clone B, and this is because of the existence of the SCC*mec* IV in these isolates, this chromosomal cassette was found in relation to isolated MRSA strains in the community (CA-MRSA) (Coombs et al., 2011). Different reports of several infections caused by CA-MRSA in Latin America (Uruguay, Rio de Janeiro, Colombia, Argentina and Mexico) have been published (Alvaréz et al., 2006; Ma et al., 2005; Reyes et al., 2009; Ribeiro et al., 2005; Velazquez et al., 2011). All pattern of PFGE of the clones A, B and C showed subtypes. Probably the PFGE subtypes indicate the continued

evolutionary divergence of these clones during its massive geographic expansion.

Relative genetic instability of the *mecA* element was observed in two strains and this was associated with an apparent deletion of the *mec* element, these isolates were very similar to profile B (B1 and B2) and presented a low susceptibility to oxacillin. In the literature there are reports of *S. aureus* strains with low-level methicillin resistance (MIC 2-4 µg/mL) which are not associated to the presence of *mecA* gene, Tomasz et al. reported one class of borderline methicillin-resistant strains having PBP1 and PBP2 with altered methicillinbinding affinities and overproduction of PBP4 (Tomasz et al., 1989). Another class of lowsusceptibility has been reported and was attributed to overproduction of penicillinase (McDougal & Thornsberry, 1986). Hackbarth et al. studied the nucleotide sequence of the PBP2 gene and identified a point mutation near the penicillin-binding motive of transpeptidase (Hackbarth et al., 1995). An MRSA clinical strain with significant methicillin resistance (MIC 64µg/mL) despite absence of *mec A* was reported (Yoshida et al., 2003).

The combination of molecular typing methods (PFGE, *mecA*, Tn*554* probes, SCC*mec*, and MLST) with epidemiologic and clinical information allows the detection of MRSA clusters and outbreaks and therefore provides a rationale for appropriate infection control intervention. Our study emphasizes the need of national and international collaborations to monitor the spread of current epidemic strains as well as the emergence of new ones in our country. The mechanisms of spread in different areas are poorly understood and further studies are necessary to understand the dynamics involved in the predominance of unique

Aires de Sousa, M. Miragaia, M. Santos, I. Avila, S. Adamson, I. Casagrande, S. Brandileone,

C. Palacio, R. Dell'Acqua, L. Hortal, M. Camou, T. Rossi, A. Velazquez, ME.
