healthmedicinet

Methicillin-resistant Staphylococcus aureus (MRSA) is involved in increasing number of serious infectious with high risk for
morbidity and mortality and it is an important cause of healthcare-associated infections
1]. Although the active surveillance and the impact of MRSA colonization on the occurrence
of S. aureus infections are unclear, some studies described that asymptomatic colonization with
MRSA is a risk factor for in a subsequent MRSA infection patients 2], 3].

In Brazil, a considerable number of hospital infections have been caused by an unique
multi-resistant MRSA clone designated as the Brazilian epidemic clone (BEC) 4]. SCCmec typing revealed that Brazilian clone isolates harbor SCCmec III5].

During a study on hand hygiene compliance, samples were obtained by nasal swab every
four days from 446 patients admitted patients at two step-down units (SDUs) of a private
tertiary care hospital in São Paulo, Brazil. Blood Culture samples were also obtained
at an intensive care unit (ICU) of the same hospital, as part of an antimicrobial
resistance surveillance project (unpublished data). The study period was from March
20, 2007 to September 20, 2007 6]. The open model ICU is a 38-bed medical-surgical unit; where approximately 2,200
patients are admitted annually and the SDUs are medical-surgical 20-bed units each.
Nasal samples were plated onto chromagar (CHROMagar, MRSA, CHROMagar, Paris, France)
and selective staphylococcal media (Mannitol Salt Agar). Blood cultures were performed
at BACTEC 9240 instrument (BACTEC 9240, BD Diagnostics, Sparks, MD) and the positive
samples were plated onto chocolate and sheep blood agar.

Twenty-one MRSA strains were isolated from nasal carriers (in the SDUs) and two MRSA
strains obtained from patients with bloodstream infections (in the ICU). Staphylococcus species were confirmed by an automated (Vitek 2 System,, Hazelwood, MO)) and manual
biochemical tests. For the susceptibility profile determination oxacillin agar screening
was used with automated system according with Clinical Laboratory Standards Institute
(CLSI) recommendations 7].

SCCmec typing was performed using a polymerase chain reaction (PCR) method 8]. All MRSA isolates from nasal cultures were typed by pulsed-field gel electrophoresis
(PFGE). Bacterial cells grown overnight were embedded in agarose, lysed and deproteinated
to isolate near intact genomic DNA. The DNA was digested with restriction endonuclease
SmaI (3 ?l/sample) (new England Biolabs, Ipswich, MA) and the restriction fragments were
separated using CHEF DR III (BioRad, Hercules, CA) under the following conditions:
1 % agarose, 0.5 × TBE, 200 v and a switch interval ranging from 5-40 sec over 21 h
period. The gel was stained in a 1.5 ?l/ml ethidium bromide solution 8].

PFGE patterns were compared to MRSA Brazilian Epidemic, Pediatric and New York-Japan
clones. Tenover’s criteria 9] were used to PFGE analyses, patterns were designated by a capital letter (e.g. A,
B, C) when all bands were shown to match. When a difference in one to six bands occurred,
the strains were assigned as a subtype or variant of the major type, and were designated
with the same capital letter followed by an Arabic number (Ex.: A1, A2, A3). Cluster
analysis was provided by BioNumeric software (Applied Maths, Kortrijk, Belgium).

Three clone profiles with different subtypes were found. Clone A defined as the Brazilian
Epidemic Clone (BEC) was presented in 13 samples, and included the following subtypes:
A1, A2 (two cases each), A3, A4, A5 (two cases), A6, A7, A8, A9, A10 and A11. Clone
B was identified in 7 cases: B1, B2 (two cases each), B3, B4 (two cases) and B5. One
case exhibited clone C. The dendrogram showed that the clone B is related to New York/Japan
clone. Blood culture isolates showed MRSA harbouring SCCmec type II with a PFGE profile similar to the New-York Japan clone, also observed in bloodstream
isolates from other hospitals (fig 1).

Fig. 1. Dendrogram with clone profiles

MRSA remains as an important pathogen in Brazilian hospitals, involved in serious
infections. While a study was performed in Brazil with strains isolated from 1995
to 1997 in several private and public hospitals showed all isolates with the same
PFGE pattern (BEC), in our sample these represent only 61.9 % 10].

Recent studies published in Brazil show Type III and even Type IVSCCmec MRSA in hospital isolates 11], 12] indicating that the strains obtained from our blood cultures do not represent a typical
pattern and are similar to New York-Japan clone.

In-patient colonization and infection with isolates of MRSA not belonging to the Brazilian
Epidemic Clone (BEC) have been observed, and include strains carrying SCCmec type II, not previously described in the city of São Paulo, showing a trend toward MRSA genetic
diversity.