J Med Microbiol 57 (2008), 612-616; DOI: 10.1099/jmm.0.47757-0
© 2008 Society for General Microbiology
ISSN 1473-5644
Distribution of spa types among meticillin-resistant Staphylococcus aureus isolates during a 6 year period at a low-prevalence university hospital
Lukas Fenner1,
Andreas F. Widmer2,
Marc Dangel2 and
Reno Frei1
1 Microbiology Laboratory, University Hospital Basel, Petersgraben 4, CH-4031 Basel, Switzerland
2 Division of Infectious Diseases and Hospital Epidemiology, University Hospital Basel, Petersgraben 4, CH-4031 Basel, Switzerland
Correspondence
Reno Frei
rfrei{at}uhbs.ch
Received 8 November 2007
Accepted 8 February 2008
This study describes the distribution and frequencies of strain types by protein A-encoding gene (spa) typing among a total of 200 meticillin-resistant Staphylococcus aureus (MRSA) single-patient isolates collected between 2000 and 2005 at the University Hospital Basel, Switzerland. Nine frequent spa types accounted for 49.5 % of MRSA isolates, whereas spa type t041 (15 % of all isolates) belonged to a local epidemic strain that is also a common strain type in southern Germany. Successful control of the outbreak strain was documented by epidemiological data and confirmed by spa typing results. The spa type t044 (3.5 %), corresponding to a widely disseminated European community-acquired MRSA (CA-MRSA), was first observed in 2002. The well-known CA-MRSA USA300 clone was detected in four patients (2 %). Sporadic strains occurring less than four times (32 different spa types) accounted for 23 % of isolates. No predominant spa type was seen, indicating a great genetic diversity. Only 34.5 % of patient isolates were acquired nosocomially. The presence of one or more of ten common virulence genes was shown in 79 % of strains. It was demonstrated that the sequence-based spa typing method allows analysis of local MRSA epidemiology in relation to other regions and countries over time.
Abbreviations: CA-MRSA, community-acquired MRSA; MLST, multilocus sequence typing; MRSA, meticillin-resistant Staphylococcus aureus; spa-CC, spa clonal complex; UHBS, University Hospital Basel.
 |
INTRODUCTION
|
|---|
Staphylococcus aureus is a frequent cause of human infections and is one of the most important nosocomial pathogens. The emergence and spread of both healthcare- and community-associated meticillin-resistant S. aureus (MRSA) are challenging infection control interventions and limiting treatment options. An epidemiological work-up of clusters requires typing of potentially related strains. Analysis of chromosomal DNA restriction patterns by PFGE is still regarded as the gold standard for strain typing, but lacks an appropriate interlaboratory comparability (Van Belkum et al., 1998). Currently, sequence-based methods, such as protein A-encoding gene (spa) typing, offer excellent intra- and inter-laboratory reproducibility, and the opportunity to compare results internationally (Harmsen et al., 2003; Strommenger et al., 2006). spa typing has also been validated for long-term nationwide surveillance studies (Hallin et al., 2007). Recently, virulence genes such as those encoding Panton–Valentine leukocidin (pvl) or exfoliative toxin D (etd) have been associated with emerging MRSA clones and specific clinical presentation (Vandenesch et al., 2003; Yamasaki et al., 2006).
The prevalence of MRSA at University Hospital Basel (UHBS), Switzerland, is low, as shown previously (Mertz et al., 2007). Little is known about spa type distribution in low-prevalence hospital settings. In the present study, we evaluated the distribution and frequency of spa types among non-replicate isolates obtained from patients with MRSA admitted to UHBS from 2000 to 2005 and compared them with the global spa type database. Furthermore, a representative selection of strains was characterized by the presence or absence of important virulence-associated genes.
|
METHODS
|
|---|
Isolates, cultivation and DNA extraction.
At UHBS, each non-repetitive clinical MRSA isolate has been typed by PFGE since 1994. Single-patient isolates obtained from patients between 2000 and 2005 at UHBS differing by at least one band in the PFGE pattern were stored at –70 °C in our laboratory. The isolates were identified as S. aureus on the basis of a positive catalase reaction, agglutination with Slidex Staph Plus (bioMérieux), aurease detection (RAPIDEC Staph; bioMérieux) and the presence of the femA gene by PCR (Mehrotra et al., 2000). The meticillin resistance gene (mecA) was detected by PCR (Murakami et al., 1991). Isolates were thawed from frozen storage, subcultured on sheep blood agar (bioMérieux) and DNA was extracted using an LC MagnaPure system (Roche Diagnostic) according to the manufacturer's instructions. If more than one isolate per band pattern was available, only the first isolate was chosen.
PFGE and spa typing.
PFGE was carried out as described previously (Stranden et al., 2003). For spa typing, the polymorphic X region of the spa gene was amplified using primers spa-1113f and spa-1514r, and sequenced as described previously (Harmsen et al., 2003; Aires de Sousa et al., 2005). Sequences were analysed using Ridom StaphType software version 1.4 (Ridom GmbH) and synchronized using SpaServer (www.spaserver.ridom.de). To test the assumption that isolates with an identical PFGE pattern had the same spa type, ten randomly chosen isolates were subsequently typed; all ten of these isolates were of the expected spa type. Clustering analysis into spa clonal complexes (spa-CCs) was carried out using the clustering algorithm BURP (based upon repeat pattern) using default parameters (spa types were clustered if calculated costs were less than or equal to four and spa types shorter than five repeats were excluded) (Strommenger et al., 2006).
Detection of virulence-associated genes by PCR.
PCR was used to detect the following genes: pvl, toxic shock syndrome toxin-encoding gene (tsst), exfoliative toxin A, B and D encoding genes (eta, etb and etd), and staphylococcal enterotoxin A–E encoding genes (sea–see) as described elsewhere (Lina et al., 1999; Mehrotra et al., 2000; Yamasaki et al., 2006). Amplification of the 16S rRNA gene was used to confirm the absence of PCR inhibitors. PCR products were visualized on ethidium bromide-stained 3 % agarose electrophoresis gels.
Clinical data.
Data from all MRSA patients were collected prospectively, stored in a case-report form, and categorized into nosocomial and non-nosocomial cases according to guidelines issued by the Centers for Disease Control and Prevention (Garner et al., 1988).
|
RESULTS AND DISCUSSION
|
|---|
General description of patients and MRSA strains
Between 2000 and 2005, a total of 200 MRSA patients was identified, with a median age of 64 years (interquartile range 40–79 years). Among these, 69 patients (34.5 %) had acquired their MRSA strain nosocomially. The most common site of isolation was the nasopharynx (56 %), followed by wounds (12.5 %), the respiratory tract (10.0 %), urine (7.5 %), vagina/cervix (6.5 %), biopsies (4.5 %) and blood (2.5 %). Of the 200 isolates, 126 were recovered from hospitalized patients (surgery 21.5 %, geriatrics 11.5 %, gynaecology 11.5 %, internal medicine 10.5 % and intensive care unit 8.0 %) and 74 isolates from patients in the emergency department (14.0 %), outpatient clinics (14.5 %) and dermatology (8.5 %).
Among these 200 isolates, 102 strains showed a unique pattern by PFGE (at least one band difference) of which 86 were available for testing. spa typing discriminated 42 different spa types, whilst 1 strain was not typable. When PFGE bands were analysed using the Dice similarity coefficient and a dendrogram for similarity was built using the unweighted pair-group method, PFGE clustering showed good concordance with spa-CC analysis (data not shown).
Distribution and frequency of spa types
Over the 6 year period, 23 % of MRSA isolates corresponding to 32 spa types were observed less than four times (Table 1
). The ten most common spa types are listed in Table 1. spa type t002 and t041 correspond to strains present in southern Germany situated close to Basel. spa type t008 (13 % of all isolates) is represented in most European countries and comprises the Archaic/Iberian/Irish clone, as well as more recently the USA300 MRSA and other clones (Deurenberg et al., 2007). spa types t002 and t008 were constantly present throughout the observed period (Fig. 1). Four of the twenty-six isolates with spa t008 showed the typical PFGE pattern of the community-acquired MRSA (CA-MRSA) USA300 clone recently imported from the USA to Switzerland (Tietz et al., 2005). Another well-known CA-MRSA (spa type t044), widely disseminated in European countries and positive for the pvl and etd genes, was present at UHBS from 2002 (Vandenesch et al., 2003; Yamasaki et al., 2006; Deurenberg et al., 2007). Three of the fifteen globally most frequent spa types in the Ridom database (t011, t012 and t084) were not detected at UHBS. Other globally frequent spa types such as t001, t004, t015, t024, t032 and t037 were only isolated occasionally (Table 1). An outbreak of MRSA spa type t041 was experienced, affecting 20 geriatric patients in 2002 (Fig. 1). Further transmission was stopped and successful control of the MRSA clone at the hospital was documented by spa typing. A cluster of nine patients with spa type t400 was seen in 2004: five of these belonged to a defined outbreak. Looking at the distribution among the various clinical departments revealed that CA-MRSA of spa type t044 was detected only in the emergency department, dermatology, outpatient clinics and the surgical department. In the department of internal medicine, the intensive care unit and the geriatric clinic, spa type t008 was less common (0–5 % vs 18–28 %). Based upon BURP spa clustering, the most frequent spa-CCs were spa-CC002 (29.5 %) and spa-CC008 (22 %), followed by spa-CC037 (4.5 %), spa-CC1510 (3.5 %) and three other unnamed clusters (12.5 %). Nosocomially acquired MRSA corresponded mainly to spa types t041, t400 and t030 (Table 2).
View this table:
[in this window]
[in a new window]
|
Table 1. Frequency of the ten most common spa types among MRSA single-patient isolates recovered between 2000 and 2005 at UHBS compared with relative global frequency and geographical spread
|
|
View larger version (36K):
[in this window]
[in a new window]
|
Fig. 1. Distribution of the most frequent spa types among single-patient MRSA isolates between 2000 and 2005 at UHBS. Only spa types occurring at least four times are included. O, Other spa types; NA, strain not available.
|
|
View this table:
[in this window]
[in a new window]
|
Table 2. Gene profiles of virulence-associated determinants and spa types among 86 MRSA isolates representing a unique pattern in PFGE
Columns do not add up to 100 % as several gene profile configurations are possible.
|
|
By using spa typing, Harmsen et al. (2003) identified spa types t001, t003 and t023 as the predominant types at a German university hospital and demonstrated the dynamics of MRSA clone circulation over two different study periods. Typing results of non-replicate MRSA strains from another German university hospital in 154 patients and 21 staff members over 1 year revealed 34 different spa types (Mellmann et al., 2005). In a study from Austria, the most common spa types among 382 MRSA isolates found were t001 (28.8 % of all isolates), t190 (27.0 %), t008 (14.1 %), and t041 (11.3 %) (Ruppitsch et al., 2006), whereas the 42 remaining spa types accounted for less than or equal to 2.4 % each. However, the strain collection consisted of arbitrarily chosen isolates from different origins.
The method of spa typing is impressive for its speed, reproducibility, ease of interpretation and suitability for international comparison, despite its limits as a single-locus-based method (Hallin et al., 2007). Multilocus sequence typing (MLST) is another excellent typing tool based on sequence analysis of seven S. aureus housekeeping genes and is part of the current nomenclature of MRSA strains (Deurenberg et al., 2007). MLST is especially suitable for investigating the clonal evolution of MRSA, but it is laborious and less discriminatory than spa typing (Deurenberg et al., 2007).
Frequencies of virulence-associated genes
Out of 86 representative MRSA strains with a unique PFGE pattern, 68 isolates (79 %) were positive for one or more of the virulence-associated genes tested: 13 (15.1 %) were positive for pvl, 5 (5.8 %) for tsst, 3 (3.5 %) for etd and 57 (66 %) for one of the staphylococcal enterotoxin A–D encoding genes, but no eta, etb or see was detected (Table 2
). All tsst-positive strains were also positive for the sec gene. Strains that were positive for pvl were associated with 7 different spa types, tsst-positive strains with 4, sea–sed-positive strains with 30 different spa types. Only etd was exclusively associated with a single spa type, t044, consistent with the findings of Vandenesch et al. (2003). Within a group of the same spa type, strains harboured different virulence genes, suggesting parallel evolution by acquisition of additional mobile elements as observed previously (Hallin et al., 2007). Overall, toxin expression in our MRSA collection was comparable with other reports. The frequency of pvl-positive strains was higher (15 vs 6 %) than in a previous study (Ghebremedhin et al., 2005) and the proportion of strains positive for one or more staphylococcal enterotoxins was lower (66 vs 89 %). The frequencies of virulence gene-positive strains in our study could be biased by the selection of the tested strains, as only isolates differing by at least one band in the PFGE pattern were included.
Conclusion
Among a total of 200 MRSA isolates obtained from patients admitted to UHBS between 2000 and 2005, some of the frequent spa types corresponded to those frequently represented in the global spa database and corresponded to frequent types in the neighbouring country of Germany. Nine frequent spa types accounted for 49.5 % of MRSA isolates and can be considered to be endemic at UHBS, whereas spa type t041 (15 % of all isolates) belonged to a local epidemic strain. Two well-known international CA-MRSA clones (corresponding to USA300 and MLST ST80 clones, 5.5 % of the total strains) were also present at our university hospital. Another 32 spa types occurring less than four times accounted for 23 %. These were considered to be sporadic strains, possibly indicating the introduction of new MRSA clones from outside. Genetic diversity was considerable and there was no dominant ‘UHBS’ spa type throughout the study period except for outbreak strains.
We demonstrated that strain types and strain type frequencies can easily be compared with other countries using spa typing and that clusters as well as the control of outbreak strains can be clearly documented. Based on our results, we cannot draw any conclusion regarding implementation of infection control measures by spa typing as has been suggested by Mellmann et al. (2005). Thus, further studies should focus on the usefulness of spa typing with regard to efficient infection control measures. The impact of spa clustering analysis also needs to be addressed in order to be able to detect variations in endemic spa types.
|
ACKNOWLEDGEMENTS
|
|---|
We are indebted to Clarisse Straub and Elisabeth Schultheiss for excellent technical assistance.
|
REFERENCES
|
|---|
Aires de Sousa, M., Conceicao, T., Simas, C. & de Lencastre, H. (2005). Comparison of genetic backgrounds of methicillin-resistant and -susceptible Staphylococcus aureus isolates from Portuguese hospitals and the community. J Clin Microbiol 43, 5150–5157.[Abstract/Free Full Text]
Deurenberg, R. H., Vink, C., Kalenic, S., Friedrich, A. W., Bruggeman, C. A. & Stobberingh, E. E. (2007). The molecular evolution of methicillin-resistant Staphylococcus aureus. Clin Microbiol Infect 13, 222–235.[CrossRef][Medline]
Garner, J. S., Jarvis, W. R., Emori, T. G., Horan, T. C. & Hughes, J. M. (1988). CDC definitions for nosocomial infections, 1988. Am J Infect Control 16, 128–140.[CrossRef][Medline]
Ghebremedhin, B., Konig, W. & Konig, B. (2005). Heterogeneity of methicillin-resistant Staphylococcus aureus strains at a German university hospital during a 1-year period. Eur J Clin Microbiol Infect Dis 24, 388–398.[CrossRef][Medline]
Hallin, M., Deplano, A., Denis, O., De Mendonca, R., De Ryck, R. & Struelens, M. J. (2007). Validation of pulsed-field gel electrophoresis and spa typing for long-term, nationwide epidemiological surveillance studies of Staphylococcus aureus infections. J Clin Microbiol 45, 127–133.[Abstract/Free Full Text]
Harmsen, D., Claus, H., Witte, W., Rothganger, 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, 5442–5448.[Abstract/Free Full Text]
Lina, G., Piemont, Y., Godail-Gamot, F., Bes, M., Peter, M. O., Gauduchon, V., Vandenesch, F. & Etienne, J. (1999). Involvement of Panton–Valentine leukocidin-producing Staphylococcus aureus in primary skin infections and pneumonia. Clin Infect Dis 29, 1128–1132.[CrossRef][Medline]
Mehrotra, M., Wang, G. & Johnson, W. M. (2000). Multiplex PCR for detection of genes for Staphylococcus aureus enterotoxins, exfoliative toxins, toxic shock syndrome toxin 1, and methicillin resistance. J Clin Microbiol 38, 1032–1035.[Abstract/Free Full Text]
Mellmann, A., Friedrich, A. W., Kipp, F., Hinder, F., Keckevoet, U. & Harmsen, D. (2005 ). Evidence-based infection control methods using spa genotyping for MRSA spread in hospitals. Dtsch Med Wochenschr 130, 1364–1368 (in German).[CrossRef][Medline]
Mertz, D., Frei, R., Jaussi, B., Tietz, A., Stebler, C., Fluckiger, U. & Widmer, A. F. (2007). Throat swabs are necessary to reliably detect carriers of Staphylococcus aureus. Clin Infect Dis 45, 475–477.[CrossRef][Medline]
Murakami, K., Minamide, W., Wada, K., Nakamura, E., Teraoka, H. & Watanabe, S. (1991). Identification of methicillin-resistant strains of staphylococci by polymerase chain reaction. J Clin Microbiol 29, 2240–2244.[Abstract/Free Full Text]
Ruppitsch, W., Indra, A., Stoger, A., Mayer, B., Stadlbauer, S., Wewalka, G. & Allerberger, F. (2006). Classifying spa types in complexes improves interpretation of typing results for methicillin-resistant Staphylococcus aureus. J Clin Microbiol 44, 2442–2448.[Abstract/Free Full Text]
Stranden, A., Frei, R. & Widmer, A. F. (2003). Molecular typing of methicillin-resistant Staphylococcus aureus: can PCR replace pulsed-field gel electrophoresis? J Clin Microbiol 41, 3181–3186.[Abstract/Free Full Text]
Strommenger, B., Kettlitz, C., Weniger, T., Harmsen, D., Friedrich, A. W. & Witte, W. (2006). Assignment of Staphylococcus isolates to groups by spa typing, SmaI macrorestriction analysis, and multilocus sequence typing. J Clin Microbiol 44, 2533–2540.[Abstract/Free Full Text]
Tietz, A., Frei, R. & Widmer, A. F. (2005). Transatlantic spread of the USA300 clone of MRSA. N Engl J Med 353, 532–533.[Free Full Text]
Van Belkum, A., Van Leeuwen, W., Kaufmann, M. E., Cookson, B., Forey, F., Etienne, J., Goering, R., Tenover, F., Steward, C. & other authors (1998). Assessment of resolution and intercenter reproducibility of results of genotyping Staphylococcus aureus by pulsed-field gel electrophoresis of SmaI macrorestriction fragments: a multicenter study. J Clin Microbiol 36, 1653–1659.[Abstract/Free Full Text]
Vandenesch, F., Naimi, T., Enright, M. C., Lina, G., Nimmo, G. R., Heffernan, H., Liassine, N., Bes, M., Greenland, T. & other authors (2003). Community-acquired methicillin-resistant Staphylococcus aureus carrying Panton–Valentine leukocidin genes: worldwide emergence. Emerg Infect Dis 9, 978–984.[Medline]
Yamasaki, O., Tristan, A., Yamaguchi, T., Sugai, M., Lina, G., Bes, M., Vandenesch, F. & Etienne, J. (2006). Distribution of the exfoliative toxin D gene in clinical Staphylococcus aureus isolates in France. Clin Microbiol Infect 12, 585–588.[CrossRef][Medline]
TOP
INTRODUCTION
METHODS
