Methicillin-resistant Staphylococcus aureus DNA electrophoretic pattern: temporal changes in an endemic hospital environment
Patrón electroforético del ADN de Staphylococcus aureus resistente a la meticilina: cambios temporales en un medio hospitalario endémico
Gleice Cristina LeiteI; Maria Clara PadovezeII; Maria Luiza MorettiIII
IPós-Graduação em Clínica Médica, Departamento de Clínica Médica, Faculdade de Ciências Médicas, Universidade Estadual de Campinas, Sao Paulo, Brazil
IIDepartamento de Enfermagem em Saúde Coletiva, Escola de Enfermagem, Universidade de São Paulo, Sao Paulo, Brazil
IIIDepartamento de Clínica Médica, Faculdade de Ciências Médicas, Universidade Estadual de Campinas, Sao Paulo, Brazil
OBJECTIVE: To describe the analysis of geographical and temporal distribution of DNA profiles determined by pulsedfield gel electrophoresis (PFGE) of methicillinresistant Staphylococcus aureus (MRSA) strains isolated from hospitalized patients in a tertiary care university hospital in Brazil.
METHODS: Ninetynine samples of MRSA obtained from 89 patients in the period 1999 2004 were studied. MRSA strains were isolated from central venous catheters (33 isolates) and bloodstream infections (66 strains). PFGE with 20 units of SmaI restriction endonuclease was used for genomic typing.
RESULTS: Analysis of DNA PFGE of 99 strains of MRSA revealed 26 profiles and their respective related profiles. The mean time interval for detecting MRSA infection was 26 days from hospital admission. Forty-nine patients (57.6%) had a recent hospitalization. The DNA PFGE MRSA profiles were distributed in three clonal groupsI, II, and IIIaccording to the period of time when the MRSA strains were isolated. DNA PFGE MRSA profiles were spread homogeneously through all hospital wards.
CONCLUSIONS: Changes in the distribution of DNA PFGE MRSA profiles were largely temporal, with clonal groups being replaced over time, without predominance in any hospital ward or any specific area of the hospital.
Key words: Staphylococcus aureus; methicillin-resistant Staphylococcus aureus; electrophoresis, gel, pulsed-field; drug resistance, microbial; Brazil.
OBJETIVO: Analizar la distribución geográfica y temporal de los perfiles de ADN determinados mediante electroforesis en gel de campo pulsado (PFGE) de cepas de Staphylococcus aureus resistente a la meticilina (SARM) aisladas de pacientes internados en un hospital universitario de atención terciaria en el Brasil.
MÉTODOS: Se estudiaron 99 muestras de SARM obtenidas 89 de pacientes en el período 19992004. Las cepas de SARM se aislaron de infecciones de catéteres venosos centrales (33 aislados) y del torrente sanguíneo (66 cepas). Para la tipificación genómica se empleó PFGE con 20 unidades de endonucleasa de restricción SmaI.
RESULTADOS: El análisis del ADN de 99 cepas de SARM mediante PFGE reveló 26 perfiles, con sus respectivos perfiles relacionados. El intervalo medio de detección de la infección por SARM fue de 26 días desde el ingreso al hospital. En 49 pacientes (57,6%) había habido una hospitalización previa reciente. Los perfiles de ADN de SARM determinados mediante PFGE se distribuyeron en tres grupos clonales I, II y III según el período en el que se aislaron las cepas de SARM. Estos perfiles de ADN se encontraban distribuidos de manera homogénea en todos los servicios del hospital.
CONCLUSIONES: Los cambios en la distribución de los perfiles de ADN de SARM determinados mediante PFGE fueron en gran medida temporales, con reemplazo de los grupos clonales con el transcurso del tiempo, y sin predominio en ningún servicio ni área específica del hospital.
Palabras clave: Staphylococcus aureus; Staphylococcus aureus resistente a meticilina; electroforesis en gel de campo pulsado; farmacorresistencia microbiana; Brasil.
Staphylococcus aureus is a pathogen responsible for a variety of community and hospital-acquired infections; it also colonizes skin and nares of healthy individuals (1). S. aureus resistance to β-lactam antibiotics started soon after the introduction of penicillin in clinical practices in the mid-1940s; the most common mechanism is production of penicillinase induced by the bla gene, which is usually carried on a plasmid (2). The first description of methicillin-resistant S. aureus (MRSA) appeared at about the same time penicillinasestable β-lactams became available. The main mechanism of methicillin resistance is mediated by the acquired PBP2a, encoded by the mecA gene.
MRSA represents a serious threat to public health throughout the world, spreading fast and showing a great diversity of pandemic clones with relevant virulence and antimicrobial resistance (3). MRSA strains were mainly isolated in hospitals and in ambulatory health care centers (4). Recently, however, MRSA infections emerged, causing severe illness in community scenarios (5).
Molecular typing methods have been applied to help researchers map the spread and evolution of MRSA clones, including pulsedfield gel electro-phoresis (PFGE), multilocus sequencing strain, and staphylococcal cassette chromosome mec typing (6, 7). PFGE is still considered a standard reference molecular technique for analyzing dissemination of hospital and community-acquired MRSA and has proved to be one of the most discriminatory methods (8). It has been an excellent laboratory tool for emergency identification of new clones (9).
This study aims to describe the analysis of geographical and temporal distribution of DNA profiles, determined by PFGE, of MRSA strains isolated from hospitalized patients in a tertiary care university hospital.
MATERIALS AND METHODS
The study was conducted in a 400-bed tertiary care, university hospital (Hospital and Clinics HCUNICAMP) in Campinas, Sao Paulo, Brazil, which provides all major medical services and is the reference hospital for 5 million inhabitants. A descriptive study was performed in a collection of 99 MRSA isolates obtained from 89 patients who were hospitalized from January 1999 to February 2004. MRSA strains were isolated from central venous catheter (33 isolates) and bloodstream (66 isolates) infections. Eighty isolates were individually obtained from 80 patients. A second or third isolate was included in the study, if there had been a minimum interval of 10 days. Nine patients had more than one MRSA isolate.
The isolates were collected in the presence of clinical signs and symptoms of infection. The results from the growth of > 15 colony-forming units from a 5cm segment of the central venous catheter tip by semiquantitative (roll plate) cultures were considered positive. Blood samples were collected and cultivated by means of the Bactec® automated system. The clinical pathology laboratory had previously identified the MRSA strains and stored them in 10% skim milk, at 20ºC, in the molecular epidemiology and infectious diseases laboratory.
The strains were inoculated on blood agar plates and S. aureus was confirmed with the Staphy test commercial kit (Probac, Sao Paulo, Brazil). Oxacillin resistance was confirmed by means of the disc diffusion method according to the recommendations of the National Committee for Clinical Laboratory Standards using 1μg oxacillin and 30μg cefoxitin discs (10).
Genomic DNA preparations for PFGE were done as described by Goering and Duensing (11) with modifications by Branchini et al. (12) using 20 units of SmaI restriction endonuclease (Gibco Life Technologies, Grand Island, New York, United States of America). Restriction fragments were separated by means of the CHEFDR® III (BioRad, Hercules, California, United States) electrophoresis system. Pulse time ranged from 5 to 35 seconds for 18 hours at 6 V/cm. A DNA ladder was used as a molecular weight marker. The gel was stained with ethidium bromide and photographed.
The genetic relationship between two given strains was estimated after images were captured with a digital imaging system (Bio-Capt version 99, Biogene software, Vilbert Loumart, France). The PFGE pattern dendogram was generated by using the Dice coefficient of similarity (CS) (13). Isolates were considered to originate from the same clone if CS = 1. Isolates were considered related if CS < 1 and ≥ 0.90. Isolates were considered as having a different profile if CS < 0.90.
Clinical data were obtained by revising patient records using a standardized form. The following variables were analyzed: age, gender, date and duration of hospitalization, surgical procedure, HIV status, outcome, day care surgical procedure, prior hospitalization if ≤ 1 year (recent) or ≥ 1 year (late), interval (days) of hospitalization, surgical procedures, and recent and late prior hospitalization until a positive MRSA culture was reported.
Eighty-nine patients with documented MRSA infections (blood culture or cath-eter-related infection) were included in the study; 61 (68.5%) were males from 20 to 50 years old, and 48 (56.4%) had had a surgical procedure. HIV serology was performed in 85 patients and 84.7% had a negative result. The mortality rate in the studied group was 63.5%.
Patients were in different units of the hospital, including clinical and surgical emergencies, bone marrow transplant, hematology, pediatrics, adult and pediatric intensive care wards, oncology, rheumatology, neurosurgery, orthopedics, internal medicine, infectious diseases, nephrology, cardiology, pneumology, gastric surgery, and AIDS day care center. Forty-nine (57.6%) patients had a prior hospitalization; among them, 38 (44.7%) had a hospitalization < 1 year before and 11 (12.9%) had been hospitalized > 1 year before (Table 1).
Analysis of the DNA PFGE profile of 99 MRSA strains showed 26 profiles, which were identified with numbers from 1 to 26, and their respective related profiles were named with lowercase letters from a to g (Table 2). Patients with more than one sample evaluated showed a different DNA PFGE profile.
The DNA PFGE profiles were distributed in three clonal groupsI, II, and IIIaccording to the time when the MRSA strains were isolated. Clonal group I was composed mainly of MRSA strains isolated from January 1999 to April 2002 (DNA PFGE profiles 17), clonal group II consisted of isolates predominantly recovered from June 2001 to October 2003 (DNA PFGE profiles 820), and clonal group III included isolates from February 2003 to February 2004 (DNA PFGE profiles 2126). DNA PFGE profile 4 was recovered during the entire study period (Figure 1).
DNA PFGE MRSA profiles were spread homogeneously across all hospital wards (Table 3). No specific localization of DNA PFGE profile with regard to the physical location of hospital wards was observed.
MRSA has been a major concern causing colonization and infections in hospitalized patients worldwide. Hospital infections with MRSA are reported to be caused by strains belonging to a single clone or to clones related to an endemic one (14). A large diversity of MRSA DNA profiles was identified by PFGE during this 6year study. This study observed that changes in the distribution of DNA PFGE MRSA profiles were largely temporal, with clonal groups being replaced over time, without predominance in any hospital ward or in any specific area of the hospital. Of note, DNA PFGE profile 4 remained present throughout the study period, although it was not a predominant pattern.
Most patients in this study had been previously exposed to a hospital environment either by prior hospitalization or by outpatient surgery. Therefore, despite the mean time of about 3 weeks from hospital admission or inpatient surgery to MRSA detection, whether they had become colonized earlier on is not known. In that case, the DNA PFGE profile would represent the prevalent strain in periods other than the studied period.
A previous study conducted in the same hospital by Padoveze and Branchini (15) demonstrated a high similarity of DNA PFGE MRSA profiles. This study was carried out in the 1990s in two infectious disease wards and mainly included AIDS patients. A later study performed in our institution demonstrated an increasing DNA PFGE profile diversity compared with the previous study (14).
These studies may support a better understanding of the hospital epidemiology by MRSA strains and suggest a progression toward a greater diversity in DNA PFGE profile while MRSA remains endemic in our hospital. The diversity and replacement of DNA PFGE MRSA profiles observed over time in this study might be explained by microevolution of the pathogen or by strain competition to adapt in the hospital environment.
Trinidad et al. (17) reported a high variability in MRSA-related profiles of the Brazilian endemic clone (BEC), where the predominant profile was observed in 15% of the strains. Oliveira (18) reported that 70% of the strains of a study conducted in hospitals in different regions of Brazil belonged to the BEC profile. Da Silva Coimbra et al. (19) reported that 49% of MRSA isolates from three hospitals in Argentina had the profile of the BEC. In a German hospital, Ghebremedhin et al. (20) reported greater heterogeneity among MRSA strains during a 1-year study. Changing patterns of MRSA were also detected in a 16-year study in Portugal (21). Blanc et al. (22) identified MRSA clone replacement by other emerging clones, suggesting a rapid change.
This study was limited to evaluating the DNA profile defined by PFGE of samples obtained from blood and catheter-related infections. For a better understanding of the characteristics of clonal replacement, appropriate additional research is necessary, including staphylococcal cassette chromosome mec typing of all MRSA infection and colonization samples.
In conclusion, this study showed that the distribution of DNA MRSA profiles determined by PFGE was temporally rather than geographically defined in the hospital, demonstrating that temporal changes in the electrophoretic pattern can occur in an endemic environment.
Several studies using different molecular typing methods have demonstrated the diversity of MRSA genomic patterns, either in MRSA strains from the same hospital unit or among genotypes disseminated locally, nationally, and internationally. This fact supports the hypothesis that the greater the geographical spread the greater is the genomic evolution (16).
The authors received financial support for this study from the Internal Medicine Department, Faculty of Medical Sciences, State University of Campinas.
1. Tiwari HK, Das AK, Sapkota D, Sivarajan K, Pahwa VK. Methicillin resistant Staphylococcus aureus: prevalence and antibiogram in a tertiary care hospital in western Nepal. J Infect Dev Ctries. 2009;3(9):6814.
2. Ito T, Ma XX, Takeuchi F, Okuma K, Yuzawa H, Hiramatsu K. Novel type V staphylococcal cassette chromosome mec driven by a novel cassette chromosome recombinase, ccrC. Antimicrob Agents Chemother. 2004;48(7): 263751.
3. Rodríguez-Noriega E, Seas C. The changing pattern of methicillin-resistant Staphylococcus aureus clones in Latin America: implications for clinical practice in the region. Braz J Infect Dis. 2010;14(Suppl 2):S8796.
4. Naimi TS, LeDell KH, Como-Sabetti K, Brochardt SM, Boxrud DJ, Etienne J, et al. Comparison of community and health careassociated methicillin-resistant Staphylococcus aureus infection. JAMA. 2003;290(22):297684.
5. Gorwitz RJ. Understanding the success of methicillin-resistant Staphylococcus aureus strains causing epidemic disease in the community. J Infect Dis. 2008;197(2):17982.
6. Cookson BD, Robinson DA, Monk AB, MurchanS, Deplano A, de Ryck R, et al. Evaluation of molecular typing methods in characterizing a European collection of epidemic methicillin-resistant Staphylococcus aureus strains: the HARMONY collection. J Clin Microbiol. 2007;45(6):18307.
7. Donnio PY, Février F, Bifani P, Dehern M, Kervégant C, Wilhelm N, et al. Molecular and epidemiological evidence for spread of multiresistant methicillin-susceptible Staphylococcus aureus strains in hospitals. Antimicrob Agents Chemother. 2007;51(12):434250.
8. Aires de Souza M, de Lencastre H. Bridges from hospitals to the laboratory: genetic portraits of methicillin-resistant Staphylococcus aureus clones. FEMS Immunol Med Microbiol. 2004;40(2):1011.
9. Carriço JA, Pinto FR, Simas C, Nunes S, Sousa NG, de Lencastre H, et al. Assessment of band-based similarity coefficients for automatic type and subtype classification of microbial isolates analyzed by pulsedfield gel electrophoresis. J Clin Microbiol. 2005; 43(11):548390.
10. National Committee for Clinical Laboratory Standards. Performance standards for antimicrobial disk susceptibility tests. Villanova, Pennsylvania: National Committee for Clinical Laboratory Standards; 2005. M02-15;14(16).
11. Goering RV, Duensing TD. Rapid field inversion gel electrophoresis in combination with an rRNA gene probe in the epidemiological evaluation of staphylococci. J Clin Microbiol. 1990;28(5):4269.
12. Branchini MLM, Morthland VH, Tresoldi AT, von Nowakonski A, Dias MB, Pfaller MA. Application of genomic DNA subtyping by pulsed field gel electrophoresis and restriction enzyme analysis of plasmid DNA to characterize methicillin-resistant Staphylococcus aureus from two nosocomial outbreaks. Diagn Microbiol Infect Dis. 1993;17(4):27581.
13. Dice LR. Measures of the amount of ecology association between species. Ecology. 1945; 26(3):297302.
14. Beretta AL, Trabasso P, Stucchi RB, Moretti ML. Use of molecular epidemiology to monitor the nosocomial dissemination of methicillin-resistant Staphylococcus aureus in a university hospital from 1991 to 2001. Bras J Med Biol Res. 2004;37(9):134551.
15. Padoveze MC, Tresoldi AT, von Nowakonski A, Aoki FH, Branchini ML. Nasal MRSA colonization of AIDS patients cared for in a Brazilian university hospital. Infect Control Hosp Epidemiol. 2001;22(12):7835.
16. van Leeuwen W, van Belkum A, Kreiswirth B, Verbrugh H. Genetic diversification of methicillin-resistant Staphylococcus aureus as a function of prolonged geographic dissemination and as measured by binary typing and other genotyping methods. Res Microbiol. 1998;149(7):497507.
17. Trindade PA, Pacheco RL, Costa SF, Rossi F, Barone AA, Mamizuka EM, et al. Prevalence of SCCmec type IV in nosocomial bloodstream isolates of methicillin-resistant Staphylococcus aureus. J Clin Microbiol. 2005;43(7):34357.
18. Oliveira GA. Caracterização de cepas de Staphylococcus aureus isolados de diferentes regiões do Brasil baseada em métodos fenotípicos e genotípicos [dissertação]. São Paulo: Faculdade de Ciências Farmacêuticas, Universidade de Sao Paulo; 1998.
19. Da Silva Coimbra MV, Teixeira LA, Ramos RL, Predari SC, Castello L, Famiglietti A, et al. Spread of the Brazilian epidemic clone of a multiresistant MRSA in two cities in Argentina. J Med Microbiol. 2000;49(2):18792.
20. Ghebremedhin B, König W, König B. Hetero-geneity of methicillin-resistant Staphylococcus aureus strains at a German university hospital during a 1-year period. Eur J Clin Microbiol Infect Dis. 2005;24(6):38898.
21. Airesde-Sousa M, Correia B, de Lencastre H, Multilaboratory Project Collaborators. Changing patterns in frequency of recovery of five methicillin-resistant Staphylococcus aureus clones in Portuguese hospitals: surveillance over a 16-year period. J Clin Microbiol. 2008; 46(9):29127.
22. Blanc DS, Petignat C, Wenger A, Kuhn G, Vallet Y, Fracheboud D, et al. Changing molecular epidemiology of methicillin-resistant Staphylococcus aureus in a small geographic area over an eight-year period. J Clin Microbiol. 2007;45(11):372936.
Manuscript received on 9 April 2011. Revised version accepted for publication on 31 October 2011.