Surveillance of Methicillin-Resistant Staphylococcus aureus in a Pediatric Hospital in Mexico City during a 7-Year Period (1997 to 2003): Clonal Evolution and Impact of Infection Control

Abstract

Between 1997 and 2000 a single multidrug-susceptible methicillin-resistant Staphylococcus aureus clone, M (sequence type 30 [ST30]-staphylococcal cassette chromosome mec [SCCmec] type IV), was present in a pediatric hospital in Mexico City, Mexico. In 2001 the international multidrug-resistant New York-Japan clone (ST5-SCCmec type II) was introduced into the hospital, completely replacing clone M by 2002.

Staphylococcus aureus is one of the most important human pathogens causing skin and tissue infections, deep abscess formation, pneumonia, endocarditis, osteomyelitis, toxic shock syndrome, and bacteremia (34). The emergence of penicillin-, methicillin-, and, recently, high-level vancomycin-resistant strains (7-9) showed that S. aureus could easily adapt to antibiotic pressure and acquire resistance genes of heterologous origin. Methicillin-resistant S. aureus (MRSA), besides having established itself in the hospital setting, is now beginning to appear in the wider community as well as among people without typical associated risk factors for MRSA acquisition (18, 19). There are only a limited number of nosocomial MRSA clones spread worldwide (15, 28), and the genetic backgrounds of community-acquired MRSA strains do not correspond to that of hospital-acquired MRSA in the same geographic region (35). The success in colonization and production of disease by this bacterium is largely due to the expression of a battery of virulence factors, which promote adhesion, acquisition of nutrients, and evasion of the host immunologic response (23).

The prevalence of resistant bacteria has been increasing rapidly in Mexican hospitals during the last few years. According to different studies performed in the 1980s, 1990s, and 2000, the prevalence of MRSA in Mexican hospitals was estimated to vary from 7 to 30% (5, 6, 21, 33). However, reports from Mexico documenting the clonality of MRSA isolates are very scarce, and to the best of our knowledge there was just one publication describing the molecular characterization of isolates from Mexico (4).

The aim of the present study was to define the MRSA clonal types and their evolution over time (1997 to 2003) in a tertiary care pediatric hospital in Mexico City by different molecular typing methods, including pulsed-field gel electrophoresis (PFGE), spa typing, multilocus sequence typing (MLST), and staphylococcal cassette chromosome mec (SCCmec) typing. Additionally, the presence of several staphylococcal virulence determinants and accessory gene regulator (agr) types was also determined in the different MRSA clones.

A total of 659 S. aureus strains were isolated at the Hospital de Pediatría of Centro Médico Nacional, Siglo XXI-IMSS, in Mexico City, Mexico, between January 1997 and May 2003. During this period a total of 98 single-patient S. aureus isolates were identified as MRSA, each of which was available for molecular typing. The frequency of MRSA varied between 17 and 23% of the total of S. aureus isolates during the 5 years up to 2001, after which it dropped to 4% in 2002 and to 0% in 2003. The decrease in the frequency of MRSA strains since 2002 was most probably related to the intervention of the infection control committee of the hospital at the end of 2001.

Antimicrobial susceptibility testing was performed by the MicroScan automated method (Dade-Behring, Sacramento, Calif.) for penicillin, oxacillin, amoxicillin, cefotaxime, cephalothin, cefazolin, imipenem, trimethoprim-sulfamethoxazole, ciprofloxacin, chloramphenicol, clindamycin, erythromycin, clarithromycin, gentamicin, rifampin, tetracycline, and vancomycin. The MIC of vancomycin was determined by broth microdilution according to the National Committee for Clinical Laboratory Standards (NCCLS) guidelines (25).

PFGE of SmaI digests of chromosomal DNAs (10), hybridization of SmaI digests with a Tn554 probe (12), spa typing (32), MLST (14), and SCCmec typing (26) were performed as previously described. For spa typing and MLST, sequences of both strands were determined at Macrogen, Seoul, South Korea.

Sequences specific for the genes encoding staphylococcal enterotoxins A to E (SEA to SEE, respectively) and G to J (SEG to SEJ, respectively) and the toxic shock syndrome toxin were detected by a multiplex PCR strategy (24). Beta, gamma, and gamma variant hemolysins; exfoliatin toxins A, B, and D (ETA, ETB, and ETD); Panton-Valentine leukocidin; leukocidin LukE-LukD; and exotoxin of the epidermal cell differentiation inhibitor were detected as previously described (20, 35). The agr type was determined by PCR as described elsewhere (16).

The 98 MRSA isolates were distributed into two PFGE types, M (six subtypes) and C (three subtypes) (Fig. 1A). All 75 MRSA isolates recovered between 1997 and 2000 belonged to clone M. In 2001 clones M and C represented 39 and 61% of the isolates, respectively, and in 2002 clone M was completely replaced by clone C. The 82 isolates classified as PFGE pattern M belonged to a clear major subtype, M (n = 65), and interestingly, all isolates collected during 2001 belonged to the second most frequent subtype, M5 (n = 7). Each of the remaining subtypes of pattern M included fewer than five isolates. Clone C was represented by 16 isolates, out of which the large majority (n = 14) belonged to subtype C1.

FIG. 1.

(A) SmaI PFGE patterns of the MRSA clones found in the Hospital de Pediatría of Centro Médico Nacional, Siglo XXI-IMSS, Mexico City, Mexico, and international MRSA clones. (B) SmaI PFGE of panel A hybridized with a Tn554 probe. (C) Dendrogram of data from panel A. The relatedness among PFGE profiles was evaluated by using Bionumerics software (version 3.0; Applied Maths, Ghent, Belgium). Patterns were clustered by the unweighted pair group method using arithmetic averages, and the similarity coefficients were generated from a similarity matrix calculated with the Jaccard coefficient. NY, New York; USA, United States; UK, United Kingdom.

Clones M and C could be easily distinguished not only by PFGE but also by antibiogram and other molecular properties as well (Table 1). (i) Clone M showed a limited pattern of resistance (resistance to β-lactams and gentamicin only), while clone C was multidrug resistant (resistance to β-lactams, gentamicin, ciprofloxacin, clindamycin, and erythromycin). (ii) The hybridization with a Tn554 probe of one representative of each PFGE subtype found in the present study (subtypes M to M5 and C1 to C3) indicated that strains belonging to clone M did not have homology with transposon Tn554, while strains characterized by PFGE pattern C carried two copies of Tn554 in SmaI fragments of approximately 646 and 200 kb (Fig. 1B). (iii) Representatives of clone C (two isolates belonging to the clearly dominant subtype C1) showed spa type 2 (TJMBMDMGMK), sequence type 5 (ST5), and SCCmec type II, whereas representatives of the two major clone M subtypes (one isolate of M and one isolate of M5) were characterized by spa type 183 (WGKAKAOKMQ), ST30, and SCCmec type IV. (iv) The same isolates were tested for the presence of virulence determinants and agr type, showing that clone C was positive for hlg-v (the gamma-hemolysin variant gene) and lukE-lukD, while clone M was positive for hlb (the beta-hemolysin gene), hlg (the gamma-hemolysin gene), and tst (the toxic shock syndrome toxin gene). Clone C was of agr type 2, and clone M was of agr type 3. Figure 2 shows the agarose gel electrophoresis of PCR products of virulence determinants and agr group for representatives of clones C and M.

TABLE 1.

Phenotypic and genotypic properties of the two MRSA clones present in a pediatric hospital in Mexico City, Mexico (1997 to 2003)

Property	Result for clone:
	M	C
Antibiograma	β-Lactams, GEN	β-Lactams, GEN, CIP, CD, ERYb
PFGE pattern	M-M5	C1 to C3
spa typec	183 (WGKAKAOKMQ)	2 (TJMBMDMGMK)
ST	30	5
SCCmec type	IV	II
agr type	3	2
Virulence gened
hlg-v	−	+
hlg	+	−
lukE-lukD	−	+
tst	+	−
seg	+	+
sei	+	+
hlb	+	−

^aAntibiotic abbreviations: GEN, gentamicin; CIP, ciprofloxacin; CD, clindamycin; ERY, erythromycin.

^bTwo isolates were also resistant to trimethoprim-sulfamethoxazole.

^cspa types 2 and 183 were assigned according to the work of Shopsin et al. (32) or to the work of B. N. Kreiswirth and S. Naidich (personal communication), respectively.

^dResults for virulence genes absent in both clones (Panton-Valentine leukocidin genes, edinAC, eta, etb, etd, sea, seb, sec, sed, see, seh, sej, sek, sel, and sem) are not presented; +, presence of toxin gene; −, absence of toxin gene.

FIG. 2.

Agarose gel electrophoresis of PCR products of virulence determinants and agr group. (A) hlg-v and hlb. Lane 1, 1 Kb Plus (Gibco BRL); lanes 2 to 5 (hlg-v), RIMD 31092 (positive control), MEXC80 (clone M), MEXC79 (clone C), and MEXC78 (clone C), respectively; lanes 6 to 9 (hlb), NCTC 7428 (positive control), MEXC79, MEXC78, and MEXC80, respectively. (B) lukE-lukD and hlg. Lanes 1 to 4 (lukE-lukD), FRI-913 (positive control), MEXC80, MEXC79, and MEXC78, respectively; lane 5, 1 Kb Plus (Gibco BRL); lanes 6 to 9 (hlg), ATCC 49775 (positive control), MEXC79, MEXC78, and MEXC80, respectively. (C) tst (559 bp), seg (327 bp), and sei (465 bp). Lane 1, 1 Kb Plus (Gibco BRL); lane 2, MEXC79; lane 3, MEXC78; lane 4, MEXC80; lane 5, FRI913 (see⁺ tst⁺); lane 6, FRI472 (seg⁺ sed⁺ sei⁺). Amplification of a 228-bp band corresponding to 16S rRNA was used as an internal control. (D) agr group. Lane 1, 1 Kb Plus (Gibco BRL); lanes 2 to 4, MEXC79 (type 2), MEXC78 (type 2), and MEXC80 (type 3), respectively. Numbers at left are molecular sizes in kilobases.

Clone M, previously designated the Mexican clone (4), was also found in a hospital in Patras, Greece, and is related to the EMRSA-16 clone (1). Although representatives of the Mexican clone M and of EMRSA-16 showed very similar PFGE patterns (one-band difference [Fig. 1A]), they had distinct sequence types (ST30 and ST36, respectively) and SCCmec types (type IV and type II, respectively, which in part explains the multidrug resistance of the latter clone). Therefore, it is important that clones be identified by a combination of molecular typing methods, namely, PFGE, MLST, and SCCmec typing, as previously pointed out by others (15, 28). Curiously, although clone M represented all the MRSA isolates found between 1997 and 2000 at the pediatric hospital in Mexico City, it has not been reported in other countries of Latin America so far.

As previously shown in other studies from our laboratory (2), clone M, which showed a limited resistance pattern, was replaced by a more resistant clone, clone C. One isolate belonging to each subtype of clone C (C1 to C3) was compared to strains belonging to previously characterized MRSA clones sharing identical sequence types (ST5), 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 (Fig. 1C). Clone C showed a high degree of similarity to the pediatric (75.9%) and the New York-Japan (77.7%) clones (Fig. 1C). The multidrug-resistant New York-Japan clone (3, 30) and the multidrug-susceptible pediatric MRSA clone (31) shared very similar spa types; spa type 2 (TJMBMDMGMK) and spa type 45 (TJMBDMGMK), respectively, were both ST5 but differed in the type of SCCmec (types II and IV, respectively) (27). The isolates found in Colombia in 1996 to 1998 that belonged to the pediatric clone harbored SCCmec type IV (27). The combination of characteristics strongly suggested that clone C, found at the pediatric hospital in Mexico since 2001, was very similar to the multiresistant New York-Japan clone, which was widely spread in different states of the United States (11, 22, 29, 30), and to the clone spread in Colombia in 1996 to 1998 (17). Therefore, clone C belongs to the New York-Japan lineage and might have been transferred from the United States to Mexico. The replacement of a multidrug-susceptible clone by this multiresistant clone that has spread in a pediatric hospital could create a serious public health threat since the therapeutic options would become even more limited. In addition, seven out of the eight vancomycin-intermediate S. aureus strains isolated in the United States (22) and the first two high-level vancomycin-resistant S. aureus isolates (7, 9), which acquired in vivo Tn1546 from Enterococcus faecalis, belonged to the New York lineage (22, 36), reinforcing concerns of potential emergence of similar vancomycin-resistant S. aureus strains in Mexico.

Eady and Cove (13) suggested that MRSA isolates associated with community-acquired skin and soft tissue infections could represent the displacement of some hospital clones, namely, the EMRSA-15 clone, the pediatric clone, and the gentamicin-susceptible MRSA from French hospitals, all harboring SCCmec type IV and therefore showing a limited susceptibility pattern similar to that of community-acquired MRSA. Besides containing SCCmec type IV, community-acquired MRSA also usually belongs to agr type 3 (35), as is the case with clone M from Mexico. The overall characteristics of the Mexican clone suggest that it may be a candidate for transfer from the hospital to the community.