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Keywords:

  • Austria;
  • MRSA;
  • PVL;
  • typing;
  • USA300

Abstract

  1. Top of page
  2. Abstract
  3. Acknowledgements
  4. Transparency Declaration
  5. References

Clin Microbiol Infect 2011; 17: 920–923

Abstract

Between 2006 and 2009, all MRSA isolates recovered from human patients in Upper Austria were subjected to molecular biological analysis. Whereas the isolate number decreased from year to year, the proportion of the most common sequence types (ST5, ST8 and ST22) as well as the frequency of associated PFGE subtypes and spa-types remained similar. The rate of PVL-positive MRSA increased, whereupon the most common sequence types were ST152, ST8 including clone USA300, ST5, ST777 and ST88. The frequency of ST398 was high (25%) in relation to the PVL-positive clones. Thus, we consider a special focus on community-associated MRSA to be necessary.

Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most important human pathogens. About one-third of all human individuals is colonized with S. aureus and infections can affect all tissues and anatomical sites [1]. Furthermore, MRSA can be acquired not only in, but also, with increasing frequency, outside of health care facilities.

The Austrian federal province Upper Austria has an MRSA surveillance project that is called ‘MRSA-Registry Upper Austria’. This project has existed since 2006 and aims to systematically compile and analyse the data on all human MRSA isolates that have been collected there. All nine Upper Austrian laboratories and all 22 Upper Austrian hospitals take part in this project. In this study, we investigated, for the first time, the trend in the occurrence of MRSA types and subtypes in Upper Austria within a time period of 4 years.

Between January 2006 and December 2009, we received all collected primary clinical MRSA isolates (one isolate per person per hospitalization; one isolate per outpatient per 6 months) from the participating hospitals and laboratories. Each hospital had its own protocol for taking samples for molecular biological diagnostics. Thus, the isolates were comprised of samples from patients with infection (48.7%) as well as from patients with colonization (47.6%); some isolates could not be categorized (3.8%).

All isolates were cultured and analysed as described [2,3]. Briefly, DNA was isolated and PCR was used to detect the presence of mecA and femA as well as the PVL genes lukS-lukF. SmaI macrorestriction digestion was performed with subsequent PFGE. Additionally, isolates were analysed by spa-typing and, if necessary, multi-locus sequence typing (MLST) as described [4]. USA300 isolates were confirmed by multiplex PCR-based agr grouping [5] and detection of the ACME gene cluster by arcA-PCR [6].

We received 1492 human primary MRSA isolates. The isolate number decreased over time (2006, n = 461; 2007, n = 374; 2008, n = 385; 2009, n = 272). This is in accordance with other data for Austria (MRSA bacteraemia decreased from 15.3% in 2003 to 7.5% in 2008 [7]), as well as with trends in other European countries [8]. As shown in Fig. 1, 30 different MRSA types were detected using PFGE as well as MLST. Interestingly, the frequency of ST5, ST8 and ST22 remained fairly constant over time. This indicates that these sequence types have probably some selective advantages, and are therefore not replaced by others.

image

Figure 1.  Proportion of the five most common MRSA sequence types and their corresponding main spa-types in Upper Austria from 2006 to 2009. These five types represented 85.1% of all isolates. The table lists the mean percentage of each sequence type with regard to all MRSA isolates, and for each sequence type the percentage of its most common spa-type. ST8 Austrian clone is described in Krziwanek et al. [2] (the main difference to other ST8 clones is the PFGE-pattern and spa-type). The following sequence types were also detected: ST1, ST30, ST34, ST36, ST39, ST45, ST72, ST80, ST88, ST97, ST152, ST217, ST225, ST239, ST247, ST254, ST398, ST449, ST617, ST772, ST777, ST778, ST1430 and ST1473.

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In more detail, we detected predominant PFGE subtypes within these main MRSA sequence types that also remained similarly frequent: ST5 – subtype 2 (48.3% mean), ST8 – subtype 1 (78.8% mean), ST8 Austrian clone – subtype 0 (51.7% mean), and ST22 – subtype 0 (54.0% mean). These data suggest that it is not the sequence type as a whole, but only a single strain that has selection advantages. The differences between these predominant and other strains should be further investigated.

Within the group of predominant sequence types, ST228 was exceptional in that its proportion within the group of all MRSA isolates decreased, it was multifarious, and showed no predominant PFGE-subtypes (in contrast to spa-type, see below).

Comparable to the PFGE-subtypes, also the main spa-types of the predominant sequence types (including ST228) remained the predominant types, although their proportions varied (Fig. 1).

The proportion of PVL-positive isolates began to increase in 2008 (2006, 8.0%; 2007, 5.6%; 2008, 6.2%; 2009, 11.8%), which corresponds to tendencies in countries with low MRSA rates [9,10]. However, compared with, for example, Germany (1.74% from 2003 to 2006) [9], the Upper Austrian PVL-positive MRSA rates were rather high. One reason for this might be the decrease of the total MRSA rate in Upper Austria. Even stable amounts of PVL-positive MRSA would imply an increase in the proportion of PVL-positive MRSA relative to total MRSA.

As shown in Fig. 2, most of the PVL-positive isolates belonged to ST152, which is still a relatively rare sequence type in Europe and for which HA-MRSA isolates have not been reported so far. It is distributed across the middle of Europe, from the Balkans to Saxony, and has also been reported in Mali [11]. The second and third most common PVL-positive strains belonged to sequence types ST8 and ST5. PVL-positive ST8, especially the clone USA300 (defined as CC8/ST8/t008, PVL-positive and ACME-positive, characteristic PFGE-pattern; 76.2% of all PVL-positive ST8), was the main cause of the increase of the PVL-positive MRSA. Although relative to all MRSA isolates the proportion of USA300 isolates was low, it increased steadily from 0.4% in 2006 to 2.2% in 2009. This increase and the fact that USA300 was also reported from the Austrian provinces Salzburg, Lower Austria and Vienna [12] should be a focus of future attention. Although the 100% PVL-positive sequence type CC59/ST777 is rarely reported in Europe, the corresponding spa-type t437 is detected in many European countries, New Zealand, Taiwan and China (http://www.ridom.de/spaserver). Due to the same spa-type and similar PFGE-patterns compared with Coombs et al. [13], our ST777 strains seem to be related to Taiwan Clone ST59-V. Interestingly, the widely disseminated European clone ST80 occurred only to a low extent in Upper Austria.

image

Figure 2.  Proportion of the six most common CA-MRSA sequence types and their corresponding predominant spa-types in Upper Austria from 2006 to 2009. These six sequence types represented 81.6% of all CA-MRSA isolates. The table lists the absolute number of each isolate and its percentage with regard to the total number of CA-MRSA (PVL-positive and ST398 MRSA) as well as the percentage of the main spa-type for each strain. The main spa-type among the ST398 isolates was t011, which occurred at a relatively constant frequency between 2006 (71.4%) and 2008 (85.7%). In 2009, its proportion decreased to 40.0%, whereas spa-type t034 increased from 0% (2006) to 40.0% (2009). The following PVL-positive sequence types were also detected: ST1, ST8 Austrian Clone, ST22, ST30, ST72, ST80 and ST772.

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Acting on the assumption that PVL can be used as a marker for CA-MRSA [3,9], the biggest part of all of the Upper Austrian CA-MRSA was represented by the animal-associated clone ST398, as shown in Fig. 2. This is in contrast to observations of Witte from Germany, who describes ST398 as rare relative to PVL-positive CA-MRSA [9]. The proportion of ST398 among our MRSA isolates varied between 1.5% and 3.7%, showing no upward- or downward tendency.

In conclusion, the MRSA population in Upper Austria is not as dynamic as might be expected. No matter which typing method was used, the predominant MRSA clonal lineages, PFGE-subtypes and spa-types identified in 2006 remained the most frequent ones until 2009 and were not replaced by minor clones. The increase in the proportion of PVL-positive isolates since 2007 is partly a consequence of the occurrence of USA300 in Austria and partly due to decreasing HA-MRSA rates. Our data suggest that MRSA surveillance, with an additional focus on community-associated MRSA, should be continued.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Acknowledgements
  4. Transparency Declaration
  5. References

We thank our colleagues in the hospitals of Upper Austria, especially the infection control and laboratory teams, who participated in the ‘MRSA-Registry Upper Austria’ project and thereby enabled this study. A part of the results was presented on a poster at the 20th European Congress of Clinical Microbiology and Infectious Diseases in Vienna, Austria, on 11 April 2010.

Transparency Declaration

  1. Top of page
  2. Abstract
  3. Acknowledgements
  4. Transparency Declaration
  5. References

This work has been partly supported by a grant from the provincial government of Upper Austria (Reference Number: San-121654/16-2006-Hi/Shn). No data were generated as part of routine activities. We affirm the absence of dual or conflicting interests.

References

  1. Top of page
  2. Abstract
  3. Acknowledgements
  4. Transparency Declaration
  5. References