ANNUAL REPORT ON STAPHYLOCOCCUS AUREUS BACTERAEMIA CASES IN DENMARK 2008 (part I)

ANNUAL REPORT ON STAPHYLOCOCCUS AUREUS BACTERAEMIA CASES IN DENMARK 2008 (part I) STAPHYLOCOCCUS LABORATORY, STATENS SERUM INSTITUT 1

Staphylococcus aureus bacteraemia annual report, part I The format of the annual reports changed in 2007 and is now published in two parts. Part I includes bacteriological characteristics of Staphylococcus aures isolates (typing and antimicrobial susceptibility testing), and Part II additionally describes patient characteristics (age, gender, site of acquisition, co-morbidities and secondary manifestations). Since 1957 clinical and epidemiological information for the majority of patients with S. aureus bacteraemia (SAB) in Denmark have been registered at the Staphylococcus Laboratory at Statens Serum Institut. Members of the Danish Staphylococcus aureus bacteraemia group Thomas Benfield, MD DMSc Frank Espersen, MD DMSc Niels Frimodt-Møller, MD DMSc. Allan Garlik Jensen, MD DMSc. Lars V. Pallesen, M. Sc. (Chem. Eng), PhD Andreas Petersen, MSc, PhD Henrik C. Schønheyder, Prof. MD DMSc. Peter Skinhøj, Prof. MD DMSc Robert Skov, MD Henrik Westh, MD DMSc. Acknowledgement All Danish Departments of Clinical Microbiology have collected and sent the SAB isolates reported in this annual report: Esbjerg Herlev Herning Hillerød Hvidovre Næstved/Nykøbing Falster Odense Rigshospitalet Slagelse Statens Serum Institut Sønderborg Vejle Viborg Aarhus Aalborg STAPHYLOCOCCUS LABORATORY, STATENS SERUM INSTITUT 2

Summary This annual report is based on data from the 1,340 bacteraemia cases notified voluntarily to the Staphylococcus Laboratory by all of the 15 Danish Departments of Clinical Microbiology in 2008. Typing of SAB isolates Beginning in 2007, typing of the staphylococcal protein A gene variation (spa typing) has been used for all SAB isolates, replacing phage typing. spa typing offers a higher level of discrimination of isolates. Furthermore, spa typing is widely used internationally, which will facilitate a comparison of Danish isolates with those of other countries. A total of 434 different spa types were demonstrated. spa types that could be assigned to clonal complex CC45 constituted 21% of all isolates. Antimicrobial susceptibility The present annual report includes susceptibility testing to the following antimicrobial agents: Penicillin, cefoxitin, erythromycin, clindamycin, tetracycline, streptomycin, kanamycin, rifampin, fusidic acid, norfloxacin, linezolid and mupirocin. Resistance frequencies for 2008 are shown with 2007 and 2006 for comparison in Table 3. The proportion of methicillin resistant S. aureus (MRSA) was 1.3% which is a doubling compared to 2007 (0.6%) but at comparable levels to 2005 and 2006 (1.5% and 1.4%, respectively). For the other antimicrobials frequencies were at levels comparable with previous years (Table 3 and Figure 2). Resistance varied between isolates belonging to different MLST clonal complexes (CC; Table 4). Most notably were 69% of isolates from CC1 resistant to fusidic acid (all isolates 9%) and 15% of isolates belonging to CC22 were resistant to norfloxacin (all isolates 2%). STAPHYLOCOCCUS LABORATORY, STATENS SERUM INSTITUT 3

Materials and methods Staphylococcus aureus bacteraemia (SAB) isolates During 2008, SAB isolates (repeats excluded) were referred from the regional Departments of Clinical Microbiology to the Staphylococcus Reference Laboratory, SSI. Isolates from the same patients were regarded as belonging to separate episodes if the intervening period was more than one month. Results of spa typing and antimicrobial susceptibility testing of all SAB isolates were performed at SSI and comprise the material presented in this report. spa typing The method and primers described by Harmsen et al. (2003) were used. The isolates were analysed and spa types were annotated using Bionumerics 5.10 (Applied Maths, Sint-Martens- Latem, Belgium) and Ridom StaphType 1.4 (Ridom GmbH, Würzburg, Germany). spa types were assigned to multi locus sequence typing (MLST) clonal complexes (CC). Antimicrobial susceptibility testing Susceptibility testing was performed by disc diffusion testing using Neosensitabs (Rosco, Taastrup, DK) on Danish blood Agar (SSI, Copenhagen, DK) containing 5% horse blood with semi confluent growth and overnight incubation at 35-36 C in atmospheric air. The following antibiotics were tested: Erythromycin, clindamycin, kanamycin, rifampicin, penicillin, cefoxitin, fusidic acid, norfloxacin, streptomycin, linezolid, tetracycline and mupirocin (interpreration was done based on the observed distribution of zone diameters for all isolates with the breakpoints as shown in Table 1 (epidemiological breakpoints)). Differences in resistance prevalence between specific CC groups and all isolates were examined with 2x2 contingency table and Fisher s exact test. MRSA were screened for glycopeptide resistance using spot test on agar plates containing teicoplanin (5 mg/l) (Fitzgibbon et al. 2007). Isolates demonstrating 10 or more colony forming units in the spot were subjected to the Etest macro method. Brain Heart Infusion agar plates (BD) were inoculated with a McFarland 2 suspension, incubated at 35-36 C in atmospheric air. Etest values were read after 24 and 48 hours. Isolates with MIC- values 8 mg/l for both van- STAPHYLOCOCCUS LABORATORY, STATENS SERUM INSTITUT 4

comycin and teicoplanin or an MIC 12 mg/l for teicoplanin were tested with the PAP-AUC method (Walsh et al. 2001) Table 1. The breakpoints used for interpretation of antimicrobial resistance. Resistant < mm Susceptible mm Erythromycin 27 29 Clindamycin* 29 30 Kanamycin 23 25 Rifampicin 34 34 Penicillin 28 28 Cefoxitin 29 29 Fusidic acid 23 24 Norfloxacin 12 18 Streptomycin 24 26 Linezolid 21 24 Mupirocin 19 19 Tetracycline 25 27 *Inducible resistance to clindamycin resulting in D- shaped inhibition zone is recorded as resistant regardless of the zone diameter. PCR detection of resistance and virulence genes All isolates with a cefoxitin zone diameter <29 mm were investigated for the presence of the meca gene using PCR. All isolates were routinely screened for the presence of the Panton- Valentine leukocidin genes by PCR. STAPHYLOCOCCUS LABORATORY, STATENS SERUM INSTITUT 5

Results The total number of SAB cases in the 15 Danish counties was 1,341, which is at the same level as during the last decade (Figure 1). In total 1,290 patients were diagnosed with SAB of whom 44 had two episodes of SAB and 3 had three or more episodes detected. This corresponds to an incidence rate of SAB of 23.6/100,000 inhabitants/year. Figure 1. Number of S. aureus bacteraemia (SAB) cases 1960-2008 and 5 years moving average 1600 1400 1200 1000 800 600 400 200 0 1960 1962 1964 1966 1968 1970 1972 1974 1976 1978 1980 1982 1984 1986 1988 1990 1992 1994 1996 1998 2000 2002 2004 2006 2008 No. of SAB Number of SAB moving average (5 years) Typing spa types were obtained from 1323/1,341 isolates (99%). The remaining isolates were indeterminate because either the isolates were unavailable or the sequence was of low quality. In total, 434 different spa types were identified, with ten predominant spa types comprising 36% of the isolates (Table 1). Nine of the ten most predominant types in 2008 were also among the most frequent in 2007 (Table 1). Annotation to multi locus sequence type clonal complex (MLST CC) inferred from spa-repeats was possible for 1216 isolates; in 125 isolates spa types were either too short (< 5 repeats) or demonstrated spa types not yet assigned to known MLST types. STAPHYLOCOCCUS LABORATORY, STATENS SERUM INSTITUT 6

Table 1. Predominant spa types among Danish S. aureus bacteraemia isolates in 2007 and 2008 spa type MLST CC Number of isolates in 2008 (percent of total isolates) Number of isolates in 2007 (percent of total isolates) t230 CC45 93 (6.9) 88 (6.5) t084 CC15 61 (4.5) 48 (3.6) t002 CC5 58 (4.3) 41 (3.0) t012 CC30 54 (4.0) 34 (2.5) t127 CC1 44 (3.3) 41 (3.0) t015 CC45 44 (3.3) 42 (3.1) t021 CC30 44 (3.3) 44 (3.3) t091 CC7 30 (2.2) 29 (2.2) t216 CC59 25 (1.9) 17 (1.3 - not in top 10) t008 CC8 24 (1.8) 32 (2.4) Based on spa typing the isolates were grouped into 24 different MLST CC groups (Table 2). SAB isolates belonging to the CC80 was demonstrated in 2008, comprising three different spa types. This group was not demonstrated in 2007 where systematic spa typing of SAB isolates started. The remaining 23 groups were also demonstrated in 2007. CC45 was the most prevalent group covering 21% of the isolates and a total of 54 different spa types. Table 2. Frequencies of multi locus sequence type (MLST) clonal complex (CC) based on spa types. MLST CC Number of isolates 2008 (% of total) % of total in 2007 Number of different spa types Dominating spa type(s) CC45 283 (21) 24 73 t230, t015, t026 CC30 207 (15) 17 59 t012, t021 CC15 159 (12) 11 37 t084, t346 CC5 97 (7) 6 22 t002 CC1 86 (6) 5 13 t127, t189 CC8 86 (6) 7 34 t008 CC22 48 (4) 3 21 t005 CC7 40 (3) 3 9 t091 CC59 37 (3) 2 10 t216 CC12 30 (2) 3 10 t160 CC25 25 (2) 3 12 t078 others* 118 55 t056, t164, t375 unknown or 125 91 unassigned * CC97, CC101, CC121, CC509, CC20, CC9, CC398, CC182, CC72, CC80, CC88, CC151, CC395 and ST152/377 STAPHYLOCOCCUS LABORATORY, STATENS SERUM INSTITUT 7

Antimicrobial Susceptibility Testing Resistance to the tested antimicrobials in 2008 is shown in Table 3. Results from 2006 and 2007 are provided for comparison. The proportion of MRSA was 1.3% which is twice as high as in 2007 but at the same level as recorded in 2005 and 2006. All MRSA isolates were negative for glycopeptide resistance in the teicoplanin spot test. The proportion of isolates that was susceptible to all antimicrobials (16.5%) was at the same level as previous year. The resistance to fusidic acid (8.9%) was comparable to the level in 2007 (9.0%). In Figure 2 resistance percentages of Danish S. aureus bacteraemia isolates 1980-2008 is shown together with percentage of isolates sensitive to all tested antimicrobials. Table 3. Prevalence of resistance among Danish S. aureus bacteraemia isolates 2006-2008 Resistance to 2008 (%) 2007 (%) 2006 (%) Penicillin 77.4 78.2 80.4 Cefoxitin 1.3 0.6 1.4 Erythromycin 4.5 4.3 5.2 Clindamycin 3.7 3.2 4.0 Tetracycline 2.5 2.0 2.7 Streptomycin 0.2 0.4 0.8 Kanamycin 1.0 0.6 1.4 Rifampicin 0.4 0.6 0.7 Fusidic acid 8.9 9.0 9.7 Norfloxacin 2.2 1.1 2.2 Linezolid 0 0 0 Mupirocin 0.6 0.5 0 Sensitive to all antimicrobials 16.5 18.0 15.6 The resistance frequencies for each CC group are listed in Table 4. The frequency of resistance seems in part to depend on the genetic background of the isolates. Thus, resistance to penicillin was significantly higher in isolates belonging to CC30 and CC15 compared with all isolates and lower in CC5, CC59 and CC12. CC5 isolates demonstrated significantly higher prevalence of resistance to erythromycin and clindamycin compared to the prevalence for all isolates. Fusidic acid resistance was unevenly distributed (range 0% for CC22 and CC7 to 68.6% for CC1, all isolates 8.9%). A significantly higher frequency of resistance to norfloxacin was demonstrated in CC22. STAPHYLOCOCCUS LABORATORY, STATENS SERUM INSTITUT 8

Table 4 Antimicrobial resistance in major MLST CC groups Antimicrobial CC45 CC30 CC15 CC5 CC1 CC8 CC22 CC7 CC59 CC12 CC25 No. of isolates 283 207 159 97 86 86 48 40 37 30 25 Penicillin 74.9 96.1 86.8 54.6 68.6 80.2 83.3 77.5 56.8 46.7 72.0 Erythromycin 1.8 5.3 3.8 11.3 4.7 8.1 6.3 2.5 2.7 0 0 Clindamycin 1.4 4.3 2.5 9.3 3.5 5.8 6.3 2.5 2.7 0 0 Tetracycline 1.4 0.5 3.1 1.0 3.5 5.8 2.1 2.5 0 3.3 0 Fusidic acid 4.6 0.5 1.9 2.1 68.6 11.6 0 0 2.7 6.7 4.0 Norfloxacin 0.7 1.9 1.9 4.1 0 4.7 14.6 2.5 2.7 0 0 Figure 2. Resistance percentages of Danish S. aureus bacteraemia isolates (1980-2008) and proportion of isolates sensitive to all tested antimicrobials. 20 18 16 14 12 10 8 6 4 2 0 1980 1982 1984 1986 1988 1990 1992 1994 1996 1998 Percentage (%) 2000 2002 2004 2006 2008 Meth/Oxa/Fox Erythromycin Clindamycin Tetracycline Streptomycin Gentamicin Kanamycin Rifampicin Fusidic acid Flouroquinolone Sensitive to all Year Figure note: Due to the long time span susceptibility has been monitored the antimicrobials tested have changed. Thus, in 2003 kanamycin replaced gentamicin, norfloxacin replaced ciprofloxacin and cefoxitin replaced oxacillin which again had replaced methicillin in 2001. STAPHYLOCOCCUS LABORATORY, STATENS SERUM INSTITUT 9

References Fitzgibbon, M. M., Rossney, A. S. and O'Connell B. 2007. Investigation of reduced susceptibility to glycopeptides among methicillin-resistant Staphylococcus aureus isolates from patients in Ireland and evaluation of agar screening methods for detection of heterogeneously glycopeptideintermediate S. aureus. J Clin Microbiol. 45(10):3263-9. Harmsen D, Claus H., Witte W, Rothgänger J, Claus H, Turnwald D, Vogel U. 2003. Typing of methicillin-resistant Staphylococcus aureus in a university hospital setting by using novel software for spa repeat determination and database management. J Clin Microbiol. 41(12):5442-8 Walsh, T. R., A. Bolmstrom, A. Qwarnstrom, P. Ho, M. Wootton, R. A. Howe, A. P. MacGowan, and D. Diekema. 2001. Evaluation of current methods for detection of staphylococci with reduced susceptibility to glycopeptides. J Clin Microbiol. 39:2439-2444. STAPHYLOCOCCUS LABORATORY, STATENS SERUM INSTITUT 10