ANNUAL REPORT ON STAPHYLOCOCCUS AUREUS BACTERAEMIA CASES IN DENMARK 2009 (part I)

Transcription

1 ANNUAL REPORT ON STAPHYLOCOCCUS AUREUS BACTERAEMIA CASES IN DENMARK 2009 (part I) STAPHYLOCOCCUS LABORATORY, STATENS SERUM INSTITUT 1

2 Staphylococcus aureus Bacteraemia Annual Report, Part I The annual reports are 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. Anders Rhod Larsen, MSc, PhD Lars V. Pallesen, MSc (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

3 Summary This annual report is based on data from the 1,480 bacteraemia cases notified voluntarily to the Staphylococcus Laboratory by all of the 15 Danish Departments of Clinical Microbiology in 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 is highly discriminatory and isolates can in most cases be related to sequence type and clonal complex (CC). A total of 456 different spa types were demonstrated in SAB isolates from The four predominant spa types were t230 (6.1%), t012 (4.0%), t002 (3.9%) and t084 (3.8%), whereas CC45 was the largest CC including 22.0% 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 2009 are shown with 2008, 2007 and 2006 for comparison in Table 3. The proportion of methicillin resistant S. aureus (MRSA) was 1.6% which is comparable to previous years. Erythromycin and clindamycin demonstrated significant increase compared to 2008, from % and %, respectively. For the other antimicrobials frequencies were at levels comparable with previous years (Table 3 and Figure 2). Resistance varied between different spa types (Table 4). Most notably were 65.9% of spa type t127 resistant to fusidic acid (average for all isolates were 8.6%). Panton-Valentine Leukocidin (PVL) The genes encoding Panton Valentine Leukocidin (luks/f-pv) were detected in 24/1,480 (1.6%) of SAB isolates. STAPHYLOCOCCUS LABORATORY, STATENS SERUM INSTITUT 3

4 Materials and methods Staphylococcus aureus bacteraemia (SAB) isolates During 2009, 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 Larsen et al. (2008) 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 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). Intermediate zone diameters were interpreted as susceptible. Differences in resistance prevalence 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 C in atmospheric air. Etest values were read after 24 and 48 hours. Isolates with MIC- values 8 mg/l for both vancomycin and teicoplanin or an MIC 12 mg/l for teicoplanin were tested with the PAP-AUC method (Walsh et al. 2001) STAPHYLOCOCCUS LABORATORY, STATENS SERUM INSTITUT 4

5 Table 1. The breakpoints used for interpretation of antimicrobial resistance. Resistant < mm Susceptible mm Erythromycin Clindamycin* Kanamycin Rifampicin Penicillin Cefoxitin Fusidic acid Norfloxacin Streptomycin Linezolid Mupirocin Tetracycline *Inducible resistance to clindamycin resulting in D- shaped inhibition zone was recorded as resistant regardless of the zone diameter. PCR detection of resistance and virulence genes All isolates were investigated for the presence of the meca gene and the Panton-Valentine leukocidin genes by PCR (Larsen et al 2008). STAPHYLOCOCCUS LABORATORY, STATENS SERUM INSTITUT 5

6 Results The total number of SAB cases in the 15 Danish counties was 1,480, which is at the same level as during the last decade (Figure 1). In total 1,409 patients were diagnosed with SAB of whom 57 had two episodes of SAB and six had three or more episodes detected. This corresponds to an incidence rate of SAB of 25.5/100,000 inhabitants/year. Figure 1. Number of S. aureus bacteraemia (SAB) cases and 5 years moving average No. of SAB Number of SAB moving average (5 years) STAPHYLOCOCCUS LABORATORY, STATENS SERUM INSTITUT 6

7 Typing spa types were obtained from 1,462/1,480 isolates (99%). The remaining isolates were indeterminate because either the isolates were unavailable or were spa negative. In total, 456 different spa types were identified, with ten predominant spa types comprising 33% of the isolates (Table 1). Nine of the ten most predominant types in 2009 were also among the most frequent in 2008 (Table 1). Annotation to multi locus sequence type clonal complex (MLST CC) inferred from spa-repeats was possible for 1,354 isolates; in 108 isolates spa types were either too short (< 5 repeats) or demonstrated spa types not yet assigned to known MLST types. Table 1. Predominant spa types among Danish S. aureus bacteraemia isolates in 2008 and 2009 spa type MLST CC Number of isolates in 2009 (percent of total isolates) Number of isolates in 2008 (percent of total isolates) t230 CC45 91 (6.1) 93 (6.9) t012 CC30 59 (4.0) 54 (4.0) t002 CC5 57 (3.9) 58 (4.3) t084 CC15 56 (3.8) 61 (4.5) t021 CC30 45 (3.0) 44 (3.3) t127 CC1 44 (3.0) 44 (3.3) t015 CC45 40 (2.7) 44 (3.3) t008 CC8 34 (2.3) 24 (1.8) t065 CC45 31 (2.1) 12 (0.9 - not in top 10) t216 CC59 24 (1.6) 25 (1.9) Based on spa typing the isolates were grouped into 25 different MLST CC groups (Table 2). CC45 was as in the previous years the most prevalent group constituting 22.0% of the isolates and a total of 84 different spa types. STAPHYLOCOCCUS LABORATORY, STATENS SERUM INSTITUT 7

8 Table 2. Frequencies of multi locus sequence type (MLST) clonal complex (CC) based on spa types. MLST CC Number of isolates 2009 (% of total) % of total in 2008 Number of different spa types Dominating spa type(s) CC (22.0) t230, t015, t065 CC (16.5) t012, t021, t166 CC (10.2) t084, t254, t346 CC5 108 (7.3) t002 CC8 107 (7.2) t008, t701, t024 CC1 95 (6.4) t127, t189, t948 CC22 45 (3.0) t005, t223 CC59 36 (2.4) t216 CC97 33 (2.2) t267 CC7 29 (2.0) t091 CC12 26 (1.8) t160 CC (1.8) t375, t525 others* t056, t078 unknown or unassigned * CC101, CC25, CC121, CC20, CC182, CC72, CC398, CC88, CC151, CC9, CC80, CC395 and ST152/377 Antimicrobial Susceptibility Testing Resistance to the tested antimicrobials in 2009 is shown in Table 3. Results from are provided for comparison. The proportion of MRSA was 1.6% which is comparable to 1.3% in All MRSA isolates were negative for glycopeptide resistance in the teicoplanin spot test. Resistance to erythromycin and clindamycin demonstrated a significant increase compared to 2008 (p<0.05 and p<0.01, respectively). The proportion of isolates that was susceptible to all antimicrobials was 21.6%, which is a slight increase to previous years. The resistance to fusidic acid (8.6%) was comparable to the level in 2008 (8.9%). In Figure 2 resistance percentages of Danish S. aureus bacteraemia isolates is shown together with percentage of isolates sensitive to all tested antimicrobials. STAPHYLOCOCCUS LABORATORY, STATENS SERUM INSTITUT 8

9 Table 3. Prevalence of resistance among Danish S. aureus bacteraemia isolates Resistance to 2009 (%) 2008 (%) 2007 (%) 2006 (%) Penicillin Cefoxitin Erythromycin 6.7* Clindamycin 5.9* Tetracycline Streptomycin Kanamycin Rifampicin Fusidic acid Norfloxacin Linezolid Mupirocin Sensitive to all antimicrobials The resistance frequencies for the most numerous spa types 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 t012 compared with all isolates and lower in t127. Fusidic acid resistance was unevenly distributed (range 0% to 65.9% for t127, all isolates 8.6%), but has steadily decreased since Table 4. Antimicrobial resistance in major spa types spa type t230 t012 t002 t084 t021 t127 t015 Number of isolates Antimicrobial Penicillin Erythromycin Clindamycin Tetracycline Fusidic acid Norfloxacin STAPHYLOCOCCUS LABORATORY, STATENS SERUM INSTITUT 9

10 Figure 2. Resistance percentages of Danish S. aureus bacteraemia isolates ( ) and proportion of isolates sensitive to all tested antimicrobials Percentage (%) 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 had replaced methicillin in STAPHYLOCOCCUS LABORATORY, STATENS SERUM INSTITUT 10

11 Panton-Valentine Leukocidin (PVL) The genes encoding Panton Valentine Leukocidin (luks/f-pv) were detected in 24/1480 (1.6%) of SAB isolates. Five of the isolates were MRSA (two t044 and one of t008, t127, and t355). The remaining 19 MSSA were sporadic cases, with only t021 (CC30) found more than once (four occasions). Isolates belonging to CC30 dominated the PVL positive isolates including 7/24 (29.2%). References Fitzgibbon, M. M., Rossney, A. S. and O'Connell B 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): Larsen AR, Stegger M, Sørum M spa typing directly from a meca, spa and pvl multiplex PCR assay-a cost-effective improvement for methicillin-resistant Staphylococcus aureus surveillance. Clin Microbiol Infect. 14: Walsh, T. R., A. Bolmstrom, A. Qwarnstrom, P. Ho, M. Wootton, R. A. Howe, A. P. MacGowan, and D. Diekema Evaluation of current methods for detection of staphylococci with reduced susceptibility to glycopeptides. J Clin Microbiol. 39: STAPHYLOCOCCUS LABORATORY, STATENS SERUM INSTITUT 11

12 Staphylococcus Laboratory Artillerivej 5 Mail: staphylococcus@ssi.dk 2300 Copenhagen S Web: Denmark STAPHYLOCOCCUS LABORATORY, STATENS SERUM INSTITUT 12