Artigo High prevalence of methicillin-resistant Staphylococcus aureus with SCC mec type III in cystic fibrosis patients in southern , Brazil Alta prevalência de Staphylococcus aureus resistente à meticilina com SCC mec tipo III em pacientes com fibrose cística no Sul do Brasil

1. Medical Sciences Post-graduate Program, Medicine School, Universidade Federal do Rio grande do Sul, Porto Alegre, RS, Brazil. 2. Microbiology and Molecular Biology Unit, Hospital de Clínicas de Porto Alegre, Porto Alegre, RS, Brazil. 3. Pharmacy School, Universidade Federal do Rio grande do Sul, Porto Alegre, RS, Brazil. Address to: Dra. Keli Cristine Reiter. Laboratório de Pós-graduação em Patologia/UFSCPA. Rua Sarmento Leite 245, 90050-170 Porto Alegre, RS, Brasil. Fax: 55 51 2101-8607 e-mail: kelir@ufcspa.edu.br Received in 23/04/2010 Accepted in 14/05/2010 ABSTRACT Introduction: Bacterial colonization of the lungs is the main cause of morbidity in cystic fibrosis (CF). Pathogens such as Staphylococcus aureus are very well adapted to the pulmonary environment and may persist for years in the same patient. genetic determinants of these bacteria, such as the presence of SCCmec have recently emerged as a problem in this population of patients. Methods: Staphylococcus aureus isolates obtained from different clinical materials coming from CF and non-CF patients attended at a cystic fibrosis reference hospital were compared according to SCCmec type and antibiotic susceptibility profile. Results: Three hundred and sixty-four single-patient Staphylococcus aureus isolates were collected, of which 164 (45%) were from CF patients. Among the latter, 57/164 (44.5%) were MRSA, and among the non-CF patients, 89/200 (35%) were MRSA. Associated pathogens were found in 38 CF patients. All 57 MRSA from CF patients harbored the multiresistant cassette type III. In contrast, 31/89 MRSA from non-CF patients harbored SCCmec type I (35%) and 44/89 harbored type III (49%). The antibiotic susceptibility pattern was similar between CF and non-CF patients. Conclusions: The high prevalence of multiresistant SCCmec type III among CF patients compared with non-CF patients in our institution may make it difficult to control disease progression through antibiotic therapy for promoting the survival of this kind of patient. Key-words: Cystic fibrosis. SCCmec. MRSA. Multiresistance

Cystic fibrosis pathogens are well adapted to the CF pulmonary environment and are difficult to eradicate, and Staphylococcus aureus may persist for years in the same patient 6 .In addition to resistance to methicillin, Staphylococcus aureus may also present other genetic determinants that contribute to pathogenicity and virulence, such as hypermutator populations and biofilm production, which make it more difficult to eradicate these pathogens 7,8 .These and other features, such as presence of the pvl gene and SCCmec distribution, have not been well studied in CF patients but have recently been detected as an emerging problem in this population 9 .
The aim of this study was to determine the presence of SCCmec types and the antibiotic susceptibility profile in a MRSA population recovered from CF patients at our institution.

Setting
Hospital de Clínicas de Porto Alegre (HCPA) is a tertiary care medical center in Porto Alegre, Brazil, with 785 inpatient beds.HCPA is a reference center for cystic fibrosis treatment in Brazil and had cared for 210 patients (130 children or adolescents and 80 adults) at the time of the study.

RESULTS
Cystic fibrosis patients are attended at any time when an acute exacerbation occurs, or routinely every three months.Patients may elect to be admitted to hospital annually, but hospitalization occurs mainly when there is an exacerbation.

Bacterial isolates
Staphylococcus aureus isolates from CF and non-CF patients in all clinical settings (hospitalized patients, emergency departments and outpatient care) were compared.All the isolates from CF patients were obtained from the respiratory tract (sputum and oropharyngeal swab).The isolates from non-CF patients were obtained from a variety of clinical specimens, but most of them were also obtained from sputum.The antibiotic susceptibility profile of each isolate was determined using the following antibiotics: gentamicin, doxycycline, levofloxacin, erythromycin, cefoxitin, rifampicin, sulfamethoxazoletrimethoprim, clindamycin and chloramphenicol.All the assays were performed in accordance with the CLSI 10 .Isolates with intermediate test results were considered to be resistant.

Molecular typing of MRSA isolates
The presence of the mecA gene was assessed for all Staphylococcus aureus isolates, and the SCCmec type of all positive mecA strains was determined from the molecular architecture of the ccr and mec complexes using PCR, as previously described 11 .

Patient information
For each MRSA patient-isolate, the medical records were reviewed to determine the demographic information, the date of specimen procurement, the anatomical site of the culture, the clinical indication for the culture and the antibiotic susceptibility.

Definitions of CA-MRSA and HA-MRSA
Patient-isolates were designated as community-acquired (CA) or hospital-acquired (HA) MRSA, based on the SCCmec type.SCCmec type IV isolates were considered to be CA-MRSA, and SCCmec types I, II or III were considered to be HA-MRSA 12 .

Study population
A total of 364 single-patient Staphylococcus aureus isolates were selected for the present study.One hundred and four (45%) isolates were from CF patients, and 57 (44.5%) of them were MRSA.Among the isolates from non-CF patients, 89/200 (35%) were MRSA.The majority of the CF patients went to hospital for routine care after an exacerbation had occurred, and 172/200 non-CF patients had an infection during hospitalization for other reasons.

Molecular typing of MRSA isolates
All of the 57 MRSA isolates from CF patients harbored SCCmec type III, but one isolate harbored SCCmec type I simultaneously with type III.In contrast, 31/89 MRSA isolates from non-CF patients harbored SCCmec type I (35%) and 44/89 harbored type III (49%) (Figure 1).Also, only one isolate harbored type I and III simultaneously.Thirteen (15%) MRSA isolates harbored an untypable SCCmec element, which could not be identified by means of the PCR method used.All of these were obtained from non-CF patients.
It was possible to compare the results regarding SCCmec cassettes according to the clinical specimens from CF versus non-CF patients (i.e.sputum, which was the most prevalent type of specimen).We observed that that the distributions of SCCmec elements I and III were similar, regardless of the clinical specimen (Table 1).

Antimicrobial susceptibility
All the MRSA isolates underwent antimicrobial susceptibility testing (Table 2).Also, the antibiotic susceptibility of MRSA isolates was compared between those harboring SCCmec type I and those harboring type III (Table 3).

FINANCIAL SUPPORT CONFLICT OF INTEREST
The authors declare that there is no conflict of interest.

REFERENCES DISCUSSION
MRSA isolates are increasingly populating the respiratory tract of CF patients.Hypermutational phenotypes, the ability to form biofilms and high antimicrobial resistance rates have been demonstrated in CF pathogens, and these may be the result from an adaptive process under their special habitat conditions 5 .Millar et al demonstrated that colonization with Pseudomonas aeruginosa or Staphylococcus aureus between 1985 and 2005 remained stable (77 to 82%), but that MRSA colonization increased (1 to 6%) 13 .Data from the Cystic Fibrosis Foundation Patient Registry in the United States also showed that the prevalence of MRSA increased, from 0.1% in 1995 to 17.2% in 2005 14 .
This study only characterized Staphylococcus aureus isolates, but it also found the presence of a second or third pathogen in the same patient, in many cases.For 38 CF patients, other pathogens were also identified: Pseudomonas aeruginosa was the most frequent, recovered from 26 CF patients, while Burkholderia cepacea was only recovered in four patients.This low recovery of Burkholderia cepacea may be a reflection of increasing use of single rooms for inpatients who had previously been colonized by this pathogen, in accordance with clinical segregation policies.These procedures have controlled the spread of Burkholderia cepacea among all CF patients attending our hospital.
In our study, there were pathogens that could not be identified, despite recent improvements in microbiological identification techniques; these were classified as non-fermenting bacilli (n = 3) 13 .In addition, we found significantly low prevalence of Haemophilus influenzae (n=1), which is a very common respiratory pathogen.This was perhaps a result of widespread use of antibiotics such as imipenem and meropenem, which are used to treat Pseudomonas aeruginosa but have concurrent activity against Haemophilus influenzae.
We found remarkable differences between our CF patients and patients with diseases other than CF.All the MRSA isolates from CF patients that we typed were the so-called HA-MRSA strains.These isolates harbored SCCmec type III, which was much more prevalent than in isolates from non-CF patients (100% vs 49%, respectively).Conversely, other studies have shown predominance of SCCmec type I (97%) or type II (89%) in CF patients, and also the presence of lower rates of type IV 5,15 .In order to minimize study bias, we also compared the distribution of SCCmec considering only respiratory specimens.From this, we found that the distribution of SCCmec types was similar, regardless of the origin of the Staphylococcus aureus.This result confirms the high prevalence of SCCmec type III among CF patients in our institution.
The antibiotic susceptibility profile was similar between CF and non-CF patients, as shown in Table 2.All the isolates harboring SCCmec type III demonstrated high rate of resistance to the antibiotics used in this study, except chloramphenicol.Comparing the MRSA isolates from all the patients, it was found that isolates harboring type III were more resistant to non-β-lactam antibiotics then were isolates harboring type I.This difference was statistically significant for three antibiotics: trimethoprim/sulfamethoxazole, doxycycline and rifampicin (p < 0.001) (Table 3).In fact, molecular studies have shown that the presence of resistance genes in type III is higher than in type I, although both types of SCCmec are found mostly in hospital-acquired isolates 11,12,16 .
Patients with MRSA have been found to present larger numbers of respiratory exacerbations than do patients with MSSA 17 .Whereas some pathogens detected in CF respiratory cultures are usually transient events, a previous study found that MRSA infection tended to be persistent and confirmed the hypothesis that MRSA is a marker of more severe lung disease 2 .The high prevalence of multiresistant SCCmec type III among the CF patients in our institution, compared with other institutions, may suggest that serious risks exist in relation to treatment of lung colonization and control over disease progression.Until now, antibiotic use has been one of many therapies that have contributed towards improved lung function and ultimately the survival of CF patients.Further studies are necessary to evaluate the possible genetic relatedness between these MRSA isolates.
The present study was supported by Fundação de Incentivo à Pesquisa

FIgURE 1 -
FIgURE 1 -Distribution of SCCmec types between CF and non-CF patients.
c p value obtained by c 2 test.

TABLE 1 -Clinical specimens and the distribution of SCCmec types between CF and non-CF patients. Specimen Total mecA + SCCmec I SCCmec III SCCmec I-III untypeable Non-CF-patients
BAL: bronchoalveolar lavage, NA: not applicable.
b c