Conceived and designed the experiments: SM LJ. Performed the experiments: NA. Analyzed the data: SM RE NA LJ. Wrote the paper: NA SM RE. Bioinformatics and array design: PS.
The authors have read the journal's policy and have the following conflicts: R. Ehricht and P. Slickers are employees of Alere technologies. S. Monecke became an employee of Alere technologies after the experimental work for this study was finished. There are no patents, products in development or marketed products to declare. This does not alter the authors' adherence to all the PLoS ONE policies on sharing data and materials, as detailed online in the guide for authors.
Worldwide, methicillin-resistant
Shortly after the introduction of methicillin, first resistant isolates were observed in England
As a result of these developments, changes within the MRSA population structure of hospitals, regions and whole countries were observed. A well documented example is Ireland. From 1971 to 2002, CC8/ST250-MRSA-I got replaced by ST239-MRSA-III which in turn was replaced by CC5-MRSA-II, ST8-MRSA-IIA/B/C/D/E, ST8-MRSA-(IV+
The aim of the present study was to monitor changes of clonal affiliations of MRSA in a German university hospital over a period of eleven years. For that purpose, a large number of isolates (30–50% of all isolates of a given year) were characterised and assigned to epidemic strains.
From 2000 to 2010, 778 isolates were genotyped,
Year | Isolates tested | Total number of patients with MRSA in that year | Coverage in % | Originated from OPD/ER | Originated from standard wards | Originated from ICU | Gender ratio, male∶female | Age > = 15 | Age 16–30 | Age 31–45 | Age 46–60 | Age >61 |
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112 | 45.5% | |||||||||
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155 | 32.3% | |||||||||
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149 | 36.2% | |||||||||
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170 | 34.1% | |||||||||
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150 | 39.3% | |||||||||
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136 | 43.4% | |||||||||
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114 | 52.6% | |||||||||
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203 | 53.7% | |||||||||
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201 | 52.7% | |||||||||
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197 | 43.1% | |||||||||
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205 | 42.4% |
If numbers for age/gender/ward affiliation do not add up to 100%, data were not available (anonymised samples from infection control unit; one case in 2002 and 2003, two cases in 2010). OPD/ER, outpatient departments and emergency rooms; ICU, intensive care units.
Strain | Total | 2000 | 2001 | 2002 | 2003 | 2004 | 2005 | 2006 | 2007 | 2008 | 2009 | 2010 |
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The most common strain, to which 270 isolates (34.7%) belonged, was a PVL-negative CC22-MRSA-IV, known as Barnim EMRSA or UK-EMRSA-15. 193 isolates originated from male patients (72.0%, two anonymous cases excluded). The mean age of patients was 65 years and their median age was 69 years. 23.4% of isolates came from intensive care units (ICUs), 51.3% from wards and 11.9% from outpatient departments or emergency rooms (OPD/ER).
Beside SCC
CC22-MRSA-IV belonged to
Approximately one out of four tested isolates (202 isolates, 26.0%) belonged to CC5-MRSA-II, also known as Rhine-Hesse EMRSA, New York-Japan Clone or UK-EMRSA-3. 134 isolates,
The macrolide/clindamycin resistance gene
Isolates of this strain belonged to
Approximately one fourth (174 isolates or 22.4%) of the isolates were identified as CC45-MRSA-IV, or Berlin EMRSA. Two-thirds of these isolates (n = 116) came from male patients. The mean age of patients was 61 years, and their median age was 63 years. 17.2% of isolates originated from ICUs, while 53.4% came from wards and 29.3% from OPD/ER.
The carriage of resistance genes in this strain was rather diverse. Neomycin/kanamycin and streptothricin resistance genes
Berlin EMRSA isolates belonged to
The fourth frequent strain was CC5/ST228-MRSA-I or South German EMRSA to which 57 isolates (7.3%) were assigned. Forty-six isolates, or 80.7%, originated from male patients. The mean age of patients was 58 and their median age 60 years. 50.9% of isolates came from ICUs, 35.1% from wards and 14.0% from OPD/ER.
Thirty-two isolates harboured a normal SCC
CC5/ST228-MRSA-I isolates belonged to
Beside the four common strains, another seventeen rare PVL-negative strains were identified.
A PVL-negative CC1-MRSA-IV strain was sporadically identified which was identical to West Australian (WA) MRSA-1/57
Sporadic CC5 strains included CC5-MRSA-IV (Paediatric clone). One isolate of a CC5/ST73-MRSA, associated with a journey to the Canary Islands, Spain, apparently had a composite SCC
Several strains belonged to CC8. The North German/Iberian clone CC8/ST247-MRSA-I was identified in a single case of a neurosurgical patient. A pandemic strain known as CC8/ST239-MRSA-III or Vienna/Hungarian/Brazilian clone was found infrequently, being involved in one outbreak and one isolated case (see below). Several PVL-negative CC8-MRSA-IV strains were discerned based on enterotoxin gene carriage. This included UK-EMRSA-14/WA-MRSA-5 (without enterotoxin genes), Lyon Clone/UK-EMRSA-2 (
Two isolates of a PVL-negative CC22-MRSA-V strain were found. These carried the
The recently emerging CC398-MRSA-V was found in six patients. This strain is mainly known from animals, especially from pigs
Very rare strains included CC7-MRSA-IV, CC88-MRSA-IV and CC30/ST36-MRSA-II which were found once each; CC97-MRSA-IV was detected in two cases.
Seven different PVL-positive strains were identified from a total of 21 patients (2.7%). Exactly two-thirds were male. Patients were clearly younger than other MRSA patients (mean, 32 years; median, 28 years). Eighteen cases were skin and soft tissue infections,
Two isolates of the PVL-positive strain CC1/ST772-MRSA-V (Bengal Bay Clone
The PVL-positive CC8-MRSA-IV strain USA300, was identified four times only, and no evidence for a spread or expansion of that clone was noted. Isolates carried the ACME locus and, in 75.0%,
The most common PVL-positive strain, the CC80-MRSA-IV (European caMRSA Clone) was found in eight cases. It is characterised by carriage of virulence-associated genes
CC152-MRSA-V was identified once. The isolate lacked enterotoxin genes and it was positive for
Temporal changes in the MRSA population are presented in
In 2000, about half of the tested isolates were assigned to CC5/ST228-MRSA-I. The vast majority belonged to the variant with a truncated SCC
In 2001, the most abundant strain was CC45-MRSA-IV, to which nearly two-thirds of typed isolates belonged. CC5-MRSA-II became more common (10.0%). The number of cases of CC5/ST228-MRSA-I significantly decreased to just one isolate and the variant with a truncated SCC
In 2002, the majority of isolates belonged to CC45-MRSA-IV. The Barnim EMRSA became more common, reaching about 15%. Three isolates of CC5/ST228-MRSA-I where found which carried un-truncated SCC
In 2003, less than half (41.4%) of MRSA isolates belonged to CC45-MRSA-IV. Most of the remaining isolates were assigned to CC5-MRSA-II (29.3%) and CC22-MRSA-IV (17.2%). CC5/ST228-MRSA-I was found only four times, including a last detection of the variant with the truncated SCC
In 2004, the majority of isolates were assigned to CC5-MRSA-II and CC22-MRSA-IV (33.9% each). In this year, the latter strain began to occur in wards and outpatient departments caring for patients with diabetic foot ulcers, where it was to become abundant in the following years. Further 22.0% of isolates belonged to CC45-MRSA-IV, including one ACME-positive isolate. Another isolate was identified as PVL-negative CC5/ST73-MRSA-IV/VI. This was acquired during a visit to the Canary Islands, Spain. Four isolates (6.8%) were identified as PVL-positive, three belonging to CC80-MRSA-IV and one to CC152-MRSA-V. Two isolates of the former strain where epidemiologically linked to each other, and an association to journeys either to Italy or to Kosovo was assumed
CC5-MRSA-II dominated in 2005 (33.9%). Other common strains were CC45-MRSA-IV (20.3%), PVL-negative CC22-MRSA-IV (27.1%) and CC5/ST228-MRSA-I (6.8%). Sporadic strains included CC5-MRSA-IV as well as CC8-MRSA-IV (USA500), the later probably being imported from Ethiopia. The livestock-associated CC398-MRSA-V and the PVL-positive CC8-MRSA-IV (USA300) were both identified for the first time with one isolate each.
In 2006, PVL-negative CC22-MRSA-IV was the most common strain (40.0%) followed by CC5-MRSA-II (28.3%) and CC45-MRSA-IV (20.0%). CC5/ST228-MRSA-I has almost disappeared, being found only twice. One of these isolates was traced back to an outbreak in another hospital nearby. Two PVL-positive MRSA strains were identified as CC80-MRSA-IV and ST30-MRSA-IV, respectively.
In 2007, approximately half of isolates (47.7%) belonged to the PVL-negative CC22-MRSA-IV. A number of these isolates originated from wards and outpatient departments specialised in care of diabetic foot ulcers, where a chronic outbreak situation emerged lasting until 2010. Nearly one quarter (22.9%) of isolates belonged to CC5-MRSA-II. CC5/ST228-MRSA-I and CC45-MRSA-IV were less common strains (11.0% and, respectively, 9.2%). CC8-MRSA-IV (Lyon clone) was found in four patients (3.7%), one of whom already carried that strain in 2006. Three PVL-positive isolates were assigned to CC80-MRSA-IV, CC30-MRSA-IV and CC59-MRSA-VT.
PVL-negative CC22-MRSA-IV (41.5%) and CC5-MRSA-II (36.8%) were almost equally abundant strains in 2008. CC45-MRSA-IV accounted for 12.3% of the tested isolates. A decrease of CC5/ST228-MRSA-I to three isolates was noted. Single isolates of six further PVL-negative strains were identified including CC8/ST239-MRSA-III (associated with a hospitalisation during travel to Turkey) and CC398-MRSA-V. One isolate of PVL-positive CC8-MRSA-IV (USA300) was found.
In 2009, Barnim EMRSA accounted for nearly the half of MRSA cases (49.4%) showing predominance in medical/diabetological wards. Another 34.1% of isolates belonged to CC5-MRSA-II. CC45-MRSA-II nearly disappeared (three isolates, 3.5%). Another three isolates belonged to CC398-MRSA-V. Three PVL-positive isolates were found (3.5%). One belonged to CC1/ST772-MRSA-V and two to CC8-MRSA-IV (USA300).
In 2010, the PVL-negative CC22-MRSA-IV was again the most prevalent strain to which more than the half (58.6%) of all isolates belonged. The prevalence of CC5-MRSA-II decreased to 26.4%. As few as 3.4% of the isolates were identified as CC45-MRSA-IV, while 2.3% belonged to CC5/ST228-MRSA-I. One case of an infection with the livestock-associated CC398-MRSA-V was diagnosed. PVL-positive strains included CC1/ST772-MRSA-V, from a patient who travelled to India in 2009. The PVL-positive CC80-MRSA-IV clone was identified three times.
Four major epidemic strains show clear and distinct trends over the study time (
PVL-negative CC22-MRSA-IV (Barnim EMRSA) predominated in recent years. This is a successful, pandemic strain which is common to abundant in many European countries
CC5-MRSA-II was very rare in the beginning of the study period and showed a marked increase. In very recent years it seems to be decreasing. It is especially common in intensive care and surgical units. Thus it might be that CC5-MRSA-II locally displaced CC5/ST228-MRSA-I which formerly was common in this type of wards. Future studies might show whether this strain is in competition with Barnim EMRSA or if they might co-exist in slightly different ecological niches.
CC5/ST228-MRSA-I decreased from approximately 50% to a much lower rate fluctuating between 4% and 11% (2007). The majority of the earlier isolates belonged to a distinct variant with a truncated SCC
CC45-MRSA-IV was found in Saxony as early as 1997–1999
The remaining PVL-negative MRSA strains occurred only sporadically and were so rare that no clear trends could be observed. For some of these strains, trends have been noted in other studies and it could be assumed that our observations mirror these developments although numbers of isolates were small. Examples are CC8/ST247-MRSA-I and CC8/ST254-MRSA. The former strain was detected only once within this study. It has been observed in the region prior to the start of the study period, from 1995–1997
PVL-positive MRSA shows strong fluctuations which, because of the small absolute number of cases, can be considered as random. PVL-MRSA in the Dresden region did not show the massive increase during the study period which was observed,
The general reasons, why MRSA strains vanish or prevail are largely unknown. One factor could be the impact of SCC
Possible other reasons for a replacement of MRSA strains by others might theoretically include specific host preferences, differences in the resistance to antibiotics, in infectivity as well as the ability to cope with environmental stress, the potential for spread, or in virulence.
Gender preference, or a lack of it, is not likely to play a role in determining the “success” of an epidemic strain. It has previously been noted that a majority of MRSA patients was male
Antibiotic resistance might be a factor in haMRSA, but many of the recently emerging caMRSA are less resistant. It will be interesting to watch in the future whether the exceptionally multi-resistant caMRSA strain CC1/ST772-MRSA-V might have an advantage compared to less resistant caMRSA strains. Changes in treatment as well as in hygiene regimes or implementation of a “search and destroy” policy should affect mainly hospital-acquired strains without having much impact on community- and livestock-associated strains.
With regard to transmissibility/infectivity, it was suggested that the presence of ACME contributed to the success of the USA300 strain
The presence of virulence factors (PVL) might have played a role in the spread of the USA300 strain. Contrarily, two other successful strains, CC22-MRSA-IV and CC398-MRSA-V, appear not to be particularly virulent, being negative for PVL, exfoliative toxin genes,
It can be assumed that the abundance of MRSA is also influenced by factors such as the presence of a susceptible host population and the absence of specific bacteriophages. A newly emerging strain might thus propagate unchecked until some kind of herd immunity evolved or until specific bacteriophages spread. Then it could be assumed that the rise and fall of MRSA strains just mirrored temporal changes in the whole
The study was performed at a tertiary care hospital in Dresden, Saxony,
MRSA isolates passed through standard clinical routine diagnostics. Specimens included screening samples and diagnostic samples from various infections. After primary culture and subculturing of single colonies, screening for clumping factor utilising the Pastorex StaphPlus kit was performed. Susceptibility tests were performed by a standard agar dilution procedure or by VITEK 1 or VITEK 2 systems (BioMerieux, Nuertingen, Germany). Methicillin resistance was confirmed by detection of PBP2a using the Innogenetics MRSA-screen agglutination assay (Innogenetics, Ghent, Belgium). Isolates were stored frozen using cryobank tubes (Microbank, Pro-Lab Diagnostics, Richmond Hill, Canada) at −80°C. For this study, one-third of the preserved isolates per year were chosen randomly and genotyped retrospectively. In addition to that, isolates were included which have been, largely prospectively, genotyped due to clinical or infection control relevance. This included all MRSA isolates from ICUs and diabetology wards, from suspected outbreak situations and from clinical conditions suggesting involvement of PVL. Only one isolate per patient and year was considered. Thus, one-third to one-half of all MRSA isolates of a given year were genotyped. Isolate numbers per year as well as patient demographics are shown in
Most isolates were characterised using the Alere StaphyType DNA microarray which covers 334 target sequences (approximately 170 distinct genes and their allelic variants) including species markers, SCC
MRSA were grown on Columbia blood agar, harvested and enzymatically lysed. DNA was prepared using commercially available systems (spin columns or EZ1, Qiagen, Hilden, Germany). DNA samples were used as templates in a linear primer elongation with one primer per target. During amplification, biotin-16-dUTP was incorporated into the amplicons. These were hybridised to the microarray followed by the addition of horseradish-peroxidase-streptavidin. Finally, hybridisations were visualised by dye precipitation. An image of the microarray was taken and analysed using a designated reader and software (Alere Technologies GmbH, Jena, Germany) using a algorithm as described previously
Multilocus sequence typing (MLST) and
(PDF)
The authors thank A. Ruppelt, H. Kanig, S. Kolewa, S. Schubert and K. Micknaß (Dresden), E. Müller, I. Engelmann and J. Sachtschal (Jena) for excellent technical assistance as well as A. Ziegler, R. Schuster and T. Uhlig for developing software used in microarray analysis. We acknowledge Prof. E. Jacobs and E. Ermantraut for their support.