Outbreak of methicillin-resistant Staphylococcus aureus in a Teaching Hospital — epidemiological and microbiological surveillance

Zbl. Bakt. 280, 550-559 (1994) © Gustav Fischer Verlag, Stuttgart· Jena . New York

Outbreak of Methicillin-resistant Staphylococcus aureus in a Teaching Hospital - Epidemiological and Microbiological Surveillance F. SCHUMACHER-PERDREAU, B. JANSEN, H. SEIFERT, G. PETERS, and G. PULVERER Institute of Medical Microbiology and Hygiene, University of Cologne, 50935 Cologne, Germany With 5 Figures· Received April 28, 1993 . Accepted June 4, 1993

Summary An outbreak of methicillin-resistant S. aureus (MRSA) in a large university teaching hospital occurred between December 1991 and May 1992, involving 7 different wards and more than 30 patients. Epidemiological typing was performed to control the epidemic and to identify the MRSA carriers. By a combination of various classical methods (antimicrobial susceptibility, phage typing) and molecular typing procedures (SDS-PAGE of extracellular proteins, plasmid DNA profile, restriction enzyme fragment pattern of chromosomal DNA), three different clones of MRSA could be discriminated. The epidemic clone A was recovered from 30 patients and from 3 staff members. By strict microbiological monitoring together with hygienic measures, the epidemic could be successfully controlled. It is concluded that a combination of phenotypic markers and DNA-based epidemiological markers is extremely useful in the microbiological surveillance of MRSA outbreaks.

Introduction Methicillin-resistant Staphylococcus aureus (MRSA)-strains have become important pathogens of nosocomial infections (3, 4). Although the incidence of MRSA usually does not exceed 5% in Germany, in other countries, especially in the USA, much higher frequencies have been reported (7). MRSA-strains may play an important role in nosocomial infections in university or large community hospitals, especially if outbreaks occur (11). Once established in a hospital, MRSA are difficult to eradicate and may even spread to other hospitals, e.g., by transmission of patients. As MRSA-strains can principally cause the same spectrum of (often serious) infections as methicillinsusceptible (MSSA)-strains, they pose a serious threat for the patient. Therapy of MRSA-infections usually requires the use of glycopeptides (e.g. vancomycin or teicoplanin), and thus contributes to an increase in therapy costs. Therefore, efforts should

Outbreak of Methicillin-resistant Staphylococcus aureus

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be made to control an outbreak of MRSA by optimal surveillance and eradication measures (1, 20). MRSA were first observed in 1961, shortly after the introduction of the penicillinase-stable antibiotic, methicillin (8, 9). These strains were usually resistant only to ~­ lactam antibiotics, but the MRSA strains found today are multiresistant in their majority. They are often resistant also to aminoglycosides, clindamycin, macrolides, and other antibiotics. In practice, methicillin-resistant strains should be reported as being also resistant to cephalosporines and other ~-lactam antibiotics including imipenem and ~-lactamase inhibitor combinations, even if the in vitro result demonstrates susceptibility (5, 17). There are three important resistance mechanisms found in Staphylococcus aureus (16), viz. 1. Resistance due to the production of beta-lactamase; 2. Borderline resistance, caused by hyperproduction of beta-lactamase or altered penicillin-binding proteins other than PBP2'; and 3. Methicillin resistance. MRSA produce an altered, low-affinity penicillin-binding protein (PBP2' or PBP2a), which is regarded as the main factor in determining resistance to beta-Iactam antibiotics. The genetic information for methicillin resistance in S. aureus is lecated on the mecA gene in the bacterial genome. MRSA are most often also beta-Iactamase producers, but this does not contribute significantly to the resistance against beta-Iactamasestable antibiotics. Methicillin resistance in a S. aureus population is normally expressed in a heterogenous way. This means that only a small fraction of cells appear phenotypically resistant in the normal agar disc diffusion test. As this phenotypic expression is strongly influenced by pH value, salt content, temperature and atmosphere, modifications of the susceptibility tests have been suggested in order to improve detection of MRSA strains. Currently it is recommended to perform the agar disc diffusion test at 30°C for 24 h, using 2% NaCI-supplemented Mueller-Hinton-agar (3, 17). It is generally accepted that single strains are responsible for most MRSA outbreaks and that certain epidemic MRSA are more capable of colonizing and infecting patients than others. Due to the possible impact of serious MRSA infections for the patient, it is necessary to recognize an outbreak as early as possible and to perform adequate control measures. We report here about an outbreak involving MRSA which occurred during December 1991 and May 1992 in a large university teaching hospital. Seven different wards including intensive care units were involved, and MRSA strains were detected in more than 30 patients. In the past, antibiotic susceptibility pattern and phage typing have been used to determine identity among epidemiologically related strains (2). However, these methods are often not discriminatory for MRSA strains, and in recent years, molecular typing methods have been introduced (6, 10, 12, 13, 15, 23,24). In the following, we describe microbiological methods by which a reasonable surveillance of such an outbreak can be managed. Materials and Methods

Strains. The S. aureus strains used consisted of 80 isolates recovered from 35 patients of 7 units (including 4 intensive care units) between December 1991 and May 1992 in a large university teaching hospital. The strains had been isolated from blood cultures, intravenous catheters, sputum, pus, skin of patients, and from the anterior nare of medical staff.

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Species identification. Staphylococcus aureus was identified by clumping factor and tube coagulase reaction according to the Subcommittee for Staphylococcal Taxonomy. Further, commercial test kits such as Staphaurex® (Wellcome Diagnostics), Staphyslide®, (Pastorex Pasteur), and SlideX® (API Biomerieux) were used. Hemolytic profile. The ability of isolates to produce hemolysis was tested by inoculation on blood agar base (Oxoid) supplemented with 5% sheep blood. Plates were examined for lysis after incubation for 20 h at 37°C. Antibiotic sensitivity testing. Susceptibility was tested by the agar diffusion assay, using a standard set of antibiotics. Furthermore, rifampin, fusidic acid, netilmicin and fosfomycin were occasionally tested. Phage typing (2). Bacteriophage susceptibility was determined with the international set of S. aureus typing phages by the reference laboratory (Prof. K. P. Schaal, Institute of Medical Microbiology, University of Bonn, Germany). Capsular polysaccharide typing (14). Antisera against capsular types 5 and 8 and teichoic acid were kindly provided by Dr. Karakawa (Dept. of Biochemistry, Microbiology, Molecular and Cellular Biology, Pennsylvania State University, University Park, PA, USA). Synthesis of capsular polysaccharides was enhanced by cultivating the strains in media with a low phosphate content (17). SDS-PAGE of extracellular proteins. A suspension of the strains (2 ml, 0.5% NaCl, adjusted to MacFarland 1) was prepared from an overnight blood agar base culture (Oxoid) and inoculated on CDM (chemically defined medium)-agar (25) over a washed autoclaved dialysis membrane (cut-off 14.000 D). Stock solutions (4 x) were kept at 4°C (up to 6 months); they were diluted and mixed with agar (6 gil) and glucose (3.5 gil) before use. The plates were incubated for 16-18 h at 35°C. The cultures were harvested from the membrane with 0.3-0.5 ml 0.5% NaCl and sedimented at 12500 g for 10 min. 200 J-tl supernatant was solubilized with 40 J-tl sample buffer (0.25 M Tris HCl, pH 6.8, conaining sodium dodecyl sulphate (SDS) 12.5%; glycerol 50%; dithiothreitol 7.7%; bromophenol blue sodium salt, 0.005% (Serva)) and boiled for 5 min before application to the gels. The preparation was separated on SDS-PAGE (12% separation gel, 4.5% stacking gel) as described by Laemmli (16). Gels were stained with Coomassie Brilliant Blue R 250 (0.1 %, Serva) in ethanol (50%), acetic acid (10%), and destained in ethanol (10%) and acetic acid (7%). Preparation of plasmid DNA. The extraction of plasmid DNA was performed using a modification of the method of Naidoo and Noble (21). Lysostaphin (Serva; 150 J-tglml) was added to a loopful of a blood base culture (Oxoid), suspended in 1 ml NaCI/EDTA (2.5 M NaCl, 0.05 M EDTA; pH 7.5) and incubated for 1 hat 37"C, followed by treatment with 4 mglmllysozyme (Merck, 15000 E/mg) for 1 h at 37°C. The suspension was then added to 1.5 ml of a lysis mixture (1 % Brij-58, 0.4% sodium deoxycholate and 0.6% EDTA in TRIS, 0.05 M, pH 8.0). The crude lysate was centrifuged for 1 hat 4°C at 15000 g to separate the cell debris and the chromosomal DNA from the plasmid DNA. Ribonuclease (Sigma; 100 J-tglml) and Proteinase K (Sigma; 100 J-tglml) digestion was performed for 45 min at 37°C. Isopropanol (-20°C) was used to precipitate the DNA. The pellets were dissolved in TES buffer (TRIS 0.03 M, NaCl 0.05 M, EDTA 0.005 M, pH 8.0). Chromosomal DNA extraction was performed according to Jordens and Hall (13). Approximately 1 J-tg of DNA was digested with the restriction enzymes Hind III and Cia I (Boehringer, Mannheim). Electrophoresis of plasmid DNA was performed on 0.8% agarose, chromosomal DNA on 1% agarose according to Meyers et al. (19). DNA profiles were visualized by UV light after ethidium bromide staining.

Results Between December 1991 and May 1992, 80 isolates of methicillin-resistant S. aureus involving 35 patients were cultured from clinical material from patients in a city

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hospital. The outbreak started in the neurosurgical intensive care unit (NSICU) and spread over 6 further wards including three other intenstive care units (surgery, burn patients, internal medicine units) (Fig. 1). In Fig. 2, the various isolation sites of MRSA strains from two ICU patients for the time period between January and May 1992 are illustrated. Epidemiological typing was initiated to control the epidemic, identify the carriers and limit the endemic spread. Phenotypic analysis of the strains included phage typing, antimicrobial susceptibility, slide agglutination with commercial tests to detect fibrinogen affinity, capsular typing, hemolytic activity and SDS-PAGE of extracellular proteins. Genotypic analyses included assessment of plasmid profile and restriction enzyme fragment pattern analysis of chromosomal DNA. Three different clones of methicillin-resistant S. aureus could be identified. Their antibiotic susceptibilities and molecular characteristics are listed in Tables 1 and 2. The epidemic strain (clone A) was recovered from 30 patients. It proved to be negative in the "clumping factor" test but produced coagulase in the tube test. The commercial slide agglutination tests for identification of S. aureus diverged in their ability to detect

Table 1. Molecular characteristics of three different S. aureus clones Clumping Capsular factor type (slide agglutination) Clone A Clone B Clone C

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Table 2. Antibiotic susceptibility profiles of three different S. aureus clones Antibiotic Resistance pattern

~-Lactam-Antibiotics

Quinolones Aminoglycosides Chloramphenicol Trimethoprim-Sulfamethoxazol Fusidic acid Rifampin Macrolides Clindamycin Fosfomycin Glycopeptides R = resistant; S

= susceptible

Clone A

Clone B

Clone C

R R

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these strains. Staphaurex, Slidex and Pastorex showed positive results, whereas the Staphyslide test remained negative. Clone A showed resistance to all beta-Iactam antibiotics, gentamicin, clindamycin, erythromycin, and quinolones; furthermore, it demonstrated a type 5 capsular polysaccharide. Plasmid analysis revealed one large plasmid; it showed also a specific extracellular protein profile (Fig. 3) and a unique DNA restriction pattern (Fig. 4) which helped to differentiate the strain from other MRSA clones (clone Band C) isolated during the same period. Clone A was also isolated from the anterior nare of 3 staff members. A second clone (clone B) could be differentiated by SDS-PAGE which was restricted to multiple isolates from two patients. This strain also carried a larger sized plasmid as did clone A but in contrast, it showed positivity in the clumping factor test reactivity, sensitivity to quinolones and a capsular polysaccharide type 8. A third clone C which exhibited 2 small plasmids, was positive in the clumping factor test and sensitive to quinolones was isolated from two further patients; this strain showed, in contrast to clone A and B, no hemolysis on blood agar. The detection of a slowly migrating plasmid in clones A and B was not clearly discriminative (Fig. 5). Likewise, strain identity based on phage typing was not helpful because clones A and B belonged to group III like the majority of MRS A described so far. The most conclusive data for the distinction of strains from clones A and B resulted from their unique extracellular protein profiles, differences in resistance to quinolones and affinity to fibrinogen (clumping factor), capsular polysaccharide typing and restriction endonuclease profiles.

Fig. 3. Extracellular protein profiles of methicillin-resistant Staphylococcus aureus. Isolates belonged to Clone (A) and Clone (B). The profiles (C) and (D) demonstrate strains isolated during the same period in other city hospitals.

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Fig. 4. Agarose-gel electrophoresis of Hind III and CIa I restriction endonuclase-digested total cellular DNA of various clones A-E; A = reference marker.

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Fig. 5. Agarose-gel electrophoresis of the plasmids of the three clones A, B, C. chr chromosomal DNA. Molecular weight marker is shown on the left lane.

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Discussion The epidemiological assessment of an outbreak of methicillin-resistant S. aureus in a large teaching hospital demonstrated that three clones were involved. Clone A demonstrated a far superior capacity to spread within the hospital, as it could be recovered from patients from 7 wards. It was isolated from blood cultures in 3 patients, and from colonized skin, nares and sputum from other patients. Two clones were found to be present simultaneously in two wards. Antibiotic resistance patterns were homogenous within one clone and demonstrated identical minimal inhibitory concentrations (MIC's). The determination of the antimicrobial resistance phenotype was accurate enough for identifying the epidemic S. aureus clone A from endogenous strains of the same patient. For the discrimination of the MRSA clones found in the epidemic the plasmid profile, however, was not able to clearly differentiate between clones A and B due to their closely related high molecular weight plasmids. Only the extracellular protein profile and the restriction enzyme analysis of DNA allowed a clear discrimination of the various clones and the prompt identification of single isolates. The combination of several typing methods is necessary to identify single clones. The choice of methods varies with the isolates (20). Extracellular protein profiles proved to be a reliable epidemiological tool for the typing of staphylococci (23, 24). To control an outbreak of MRSA in a hospital or hospital unit, it is necessary to undertake a strict microbiological monitoring. As it may happen that different clones of MRSA are involved in the same epidemic, phenotypic markers together with DNAbased epidemiological markers are extremely helpful in identification of MRSA in carriers and patients and to follow the transmission routes. A combination of microbiological monitoring together with hygienic measures (strict use of hand desinfection, isolation of patients and eradication of MRSA in carriers by use of Mupirocin ointment) was successful to control the MRSA outbreak in the present case.

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10. Goering, R. V. and T. D. Duesning: Rapid field invesion gel electrophoresis in combination with an rRNA gene probe in the epidemiological evaluation of staphylococci. J. Clin. Microbiol. 28 (1990) 42~29 11. Grosserode, M. H. and R. P. Wenzel: The continuing importance of staphylococci as major hospital pathogens. J. Hosp. Infect. 19, Suppl. B (1991) 3-17 12. Hartstein, A. V. H. Morthland, S. Eng, G. L. Archer, F. D. Schoenknecht, and A. L. Rashad: Restriction enzyme analysis of plasmid DNA and bacteriophage typing of paired Staphylococcus aureus blood culture isolates. J. Clin. Microbiol. 27 (1989) 1874-1879 13. Jordens, J. Z. and L. M. C. Hall: Characterization of methicillin-resistant Staphylococcus aureus isolates by restriction endonuclease digestion of chromosomal DNA. J. Med. Microbiol. 27 (1988) 117-123 14. Karakawa, W. W., J. M. Fournier, W. F. Vann, R. Arbeit, R. S. Schneerson, and J. B. Robbins: Methods for the serological typing of the capsular polysaccharides of Staphylococcus aureus. J. Clin. Microbiol. 22 (1985) 445~47 15. Kaufhold, A., C. Livdahl, and P. Ferrieri: Characterization of methicillin-susceptible and methicillin-resistant Staphylococcus aureus isolates by molecular typing methods. Zbl. Bakt. 277 (1992) 309-319 16. Laemmli, U. K.: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227 (1970) 680-685 17. Lyon, B. R. and R. Skurray: Antimicrobial resistance of Staphylococcus aureus: Genetic Basis. Microbiol. Rev. 51 (1987) 88-134 18. McDougal, L. K. and C. Thornsberry: New recommendations for disk diffusion antimicrobial susceptibility tests for methicillin-resistant (heteroresistant) staphylococci. J. Clin. Microbiol. 19 (1984) 482~88 19. Meyers, J. A., D. Sanchez, L. P. Ellwell, and S. Falkow: Simple agarose gel electrophoresis method for the identification and characterisation of plasmid desoxyribonucleic acid. J. Bact. 127 (1976) 1529-1537 20. Mulligan, M. E. and R. D. Arbeit: Epidemiologic and clinical utility of typing systems for differentiating among strains of methicillin-resistant Staphylococcus aureus. Infect. Control. Hosp. Epidem. 12 (1991) 20-28 21. Naidoo, J. and W. C. Noble: Skin as a source of transferable antibiotic resistance in coagulase-negative staphylococci.ln: G. Pulverer, P. G. Quie, G. Peters, eds.), Pathogenicity and clinical relevance of coagulase-negative staphylococci, pp. 225-232. G. Fischer Verlag, Stuttgart (1987) 22. Sompolinsky, D., Z. Samra, W. W. Karakawa, W. F. Vann, R. Schneerson, and Z. Malik: Encapsulation and capsular types in isolates of Staphylococcus aureus from different sources and relationship to phage types. J. Clin. Microbiol. 22 (1985) 828-834 23. Schumacher-Perdreau, F., B. Jansen, G. Peters, and G. Pulverer: Typing of coagulasenegative staphylococci isolated from foreign body infections. Eur. J. Clin. Microbiol. 7 (1988) 270-273 24. Schumacher-Perdreau, F., W. Karakawa, W. F. Vann, R. Krause, G. Peters, and G. Pulverer: Typing of multiple-resistant Staphylococcus aureus from two outbreaks in a burned patient intensive care unit. Zbl. Bakt. Suppl. 21 (1991) 446~48 25. Van der Ryn, I. and R. E. Kessler: Growth characteristics of group A streptococci in a new chemically defined medium. Infect. Immun. 27 (1985) 444~48

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Dr. Francoise Schumacher-Perdreau, Institut fiir Med. Mikrobiologie und Hygiene der Universitiit, Goldenfelsstr. 19-21, D-50935 Koln, Germany