Abstract

Objectives

The aim of this paper is to attribute Panton-Valentine leucocidin (PVL)-positive methicillin-resistant Staphylococcus aureus (MRSA) to clonal lineages by molecular typing with special reference to isolates exhibiting spa type t008/multilocus sequence type (MLST) ST8 [widely disseminated in the USA as ‘community-associated MRSA (caMRSA) USA300’].

Methods

PVL-positive MRSA (n = 117) were detected among 4815 MRSA sent to the German National Reference Laboratory for typing. These isolates were analysed by PFGE, spa typing, multilocus sequence typing, grouping of SCCmec elements and PCR detection of arcA, msr(A), mph(B) and the ≥6 AT repeat unit in the SACOL0058 sequence.

Results

Among the 117 isolates, 80 exhibited spa type t044 (corresponding to MLST ST80) and 23 exhibited spa type t008/MLST ST8. Other spa types were sporadically represented. Further characterization of isolates exhibiting t008/ST8 by PCR [arcA, msr(A), mph(B), ≥6 AT repeat signature] indicates the arrival of caMRSA ‘USA300’ in Central Europe.

Conclusions

caMRSA ST80 still predominate; however, caMRSA ST8 exhibiting the characteristics of the ‘USA300’ clone became the second most frequent. Routine detection of this clone in clinical bacteriology can be easily performed by PCR.

Introduction

Methicillin-resistant Staphylococcus aureus (MRSA) are a common cause of nosocomial infections worldwide. Hospital-associated MRSA (haMRSA) are often resistant to multiple antibiotic classes, which limits treatment options.1 Since the end of the 1990s, however, MRSA infections in the general population without the risk factors known for acquisition of haMRSA have been described, first in the USA and later worldwide.2–4 Community-associated MRSA (caMRSA) are usually less broadly resistant to antibiotics; they often contain the genes lukS-PV lukF-PV coding for Panton-Valentine leucocidin (PVL) and SCCmec elements of types IV and V. Most of haMRSA are associated with 8 of the 10 major clonal lineages of the S. aureus population. caMRSA belong to various other clonal lineages than haMRSA.4–8 However, only a few of them are widely disseminated and therefore can be regarded as epidemic. In the USA, two lineages, namely multilocus sequence type (MLST) ST1 (‘USA400’ according to the PFGE pattern) and ST8 (‘USA300’), are the most frequent and widely disseminated caMRSA.9 In Oceania, this is ST3010 and in Europe ST80.4,6,11 caMRSA ‘USA300’ has become the predominant cause of community-onset S. aureus skin and soft tissue infections in a number of areas in the USA;12 there are also first reports of healthcare-associated infections.13,14 Therefore, timely recognition of this clone in other parts of the world is an essential prerequisite for the prevention of further spread.

caMRSA ‘USA300’ exhibits MLST ST8. Although PVL-negative methicillin-susceptible S. aureus (MSSA) of this clonal lineage are still common among S. aureus isolates from colonization and infections,15 MRSA have evolved from ST8 MSSA at several times and on several occasions by acquisition of SCCmec elements of types I, II and IV.16,17 PVL-negative MRSA ST8, SCCmec IV, are particularly epidemic in Irish hospitals.18 Reliable recognition of caMRSA of ST8 not only requires discrimination from other lineages of caMRSA, but also from nosocomial MRSA, particularly those exhibiting ST8.

Traditional molecular typing methods for the detection of caMRSA ST8 include SmaI-macrorestriction patterns, multilocus sequence typing, spa sequence typing and PCR demonstration of lukS-PV lukF-PV.

An additional arginine deiminase pathway contained by a chromosomal cassette element adjacent to SCCmec IV seems to be specific for caMRSA ‘USA300’ strains.19 Furthermore, plasmid-located macrolide resistance genes msr(A) and mph(B) coding for macrolide efflux and a macrolide phosphotransferase, respectively, have been reported for ‘USA300’.20 These resistance genes are more frequent among coagulase-negative staphylococci and so far are rare among S. aureus.21 A signature AT repeat consisting of ≥6 AT repeats in the vicinity of the SCCmec integration site has been described as a further characteristic of caMRSA ‘USA300’.22

Here, we report on types of caMRSA from Germany in 2005 and 2006 with special emphasis on caMRSA of lineage ST8.

Materials and methods

Origin of S. aureus isolates investigated

The German National Reference Center for Staphylococci at the Robert Koch Institute, Wernigerode Branch has established a network based on diagnostic microbiological laboratories that send isolates for typing, all together 145 laboratories in all of Germany. MRSA isolates thought to possibly be caMRSA had been collected and submitted to the reference centre based on the following criteria: (i) originating from infections acquired in the community in the absence of risk factors associated with haMRSA; and (ii) MRSA from hospital-acquired infections (more than 48 h after admission) when associated with deep-seated infections of skin and soft tissue and from necrotizing pneumonia.

All isolates were primarily grown on sheep blood agar and confirmed by standard procedures as S. aureus.

Antimicrobial susceptibility testing was performed by broth microdilution according to DIN 58940, Deutsches Institut für Normung.23

Molecular typing

For SmaI-macrorestriction patterns, the HARMONY protocol as described by Murchan et al.24 was followed. The resulting patterns were analysed using the Bionumerics software (Applied Maths, Ghent, Belgium) for relatedness evaluation and dendrogram generation. Multilocus sequence typing was performed according to Enright et al.,25 except for the use of an alternative forward primer for the tpi amplimer.26

spa sequence typing was performed according to the Ridom Staph Type standard protocol (www.ridom.com) and by use of the Ridom Staph Type software package for assigning spa types.27 SCCmec elements of types I–V were identified by using a combination of different PCRs, as described previously.26,28

PCR experiments

Procedures for DNA extraction and detection of antibiotic resistance genes mecA, erm(A), erm(B) and erm(C) by PCR were performed using primers and conditions as reported previously.29 PCR for the detection of lukS-PV lukF-PV was performed using primers as described by Lina et al.30 and conditions as reported previously.29

For the detection of msr(A), we used the primer pair msr(A)-f 5′-GAAGCACTTGAGCGTTCT (1687–1704 in ABO13298) and msr(A)-r 5′-CCTTGTATCGTGTGATGT (1887–1869), and for the detection of mph(B), we used primers mph(B)-f 5′-CATGGAGTAGAATGGGTT (2417–2434 in ABO13298) and mph(B)-r 5′-TGGACTTATTGTGGCTGC (2604–2587).

PCR for arcA (ACME gene cluster) was performed using primers arcA-f 5′-TCAAACTTTGAGAGATGA (169–188, locus SAUSA300-0065 in CP000255) and arcA-r (346–329), the cycling scheme according to Strommenger et al.29 and an annealing temperature of 50°C.

PCR for the detection of the SACOL0058 sequence and its AT repeats was performed by use of primers and the cycling scheme as described by Bonnstetter et al.,22 with the exception of an annealing temperature of 55°C.

Results

caMRSA of different clonal lineages and infections caused by them

The number of MRSA encoding PVL that were referred for investigation was 46 among 2497 (1.8%) in 2005 and 71 among 2318 (3.1%) in 2006.

Data on the clinical origin and results of typing are shown in Table 1. Attribution to clonal lineages was deduced from spa sequence types using the BURP algorithm. For selected isolates of spa types t008 (ST8), t437 (ST59) and t305 (ST617), MLSTs were determined and assigned. For only two patients, a nosocomial acquisition was likely. The isolates mostly originated from deep-seated infections of skin and soft tissue; there were, however, also five cases of pneumonia, two of them with a fatal outcome.

Table 1

Clinical origin and molecular typing of caMRSA from infections in 2005 and 2006 in Germany

   Clonal lineages as deduced from spa types via eBURP 
 Number of isolates t002/ST5 SCCmect008/ST8 SCCmec IV t310/ST22 SCCmec IV t021/ST30 SCCmec IV t437/ST59 SCCmec IV t044/ST80 SCCmec IV t355/ST152 SCCmec IV t305/ST617 SCCmec IV 
Clinical origin 2005 2006 2005 2006 2005 2006 2005 2006 2005 2006 2005 2006 2005 2006 2005 2006 2005 2006 
Abscess (skin, soft tissue) 30 45 – – 22 29 – – – 
Furuncle 11 – – – – – – – – 10 – – – – 
Diabetic ulcer – – – – – – – – – – – – – – – – 
Wound infection – – – – – – – – – – – – 
Folliculitis – – – – – – – – – – – – – 
Phlegmonous inflammation – – – – – – – – – – – – – – – – 
Pneumonia – – – – – – – – – – – – – 
Septicaemia – – – – – – – – – – – – – – – – 
Nasal colonization – – – – – – – – – – – – – – 
Total 46 71 10 13 – – 33 47 – – 
   Clonal lineages as deduced from spa types via eBURP 
 Number of isolates t002/ST5 SCCmect008/ST8 SCCmec IV t310/ST22 SCCmec IV t021/ST30 SCCmec IV t437/ST59 SCCmec IV t044/ST80 SCCmec IV t355/ST152 SCCmec IV t305/ST617 SCCmec IV 
Clinical origin 2005 2006 2005 2006 2005 2006 2005 2006 2005 2006 2005 2006 2005 2006 2005 2006 2005 2006 
Abscess (skin, soft tissue) 30 45 – – 22 29 – – – 
Furuncle 11 – – – – – – – – 10 – – – – 
Diabetic ulcer – – – – – – – – – – – – – – – – 
Wound infection – – – – – – – – – – – – 
Folliculitis – – – – – – – – – – – – – 
Phlegmonous inflammation – – – – – – – – – – – – – – – – 
Pneumonia – – – – – – – – – – – – – 
Septicaemia – – – – – – – – – – – – – – – – 
Nasal colonization – – – – – – – – – – – – – – 
Total 46 71 10 13 – – 33 47 – – 

Among the 117 isolates investigated, 80 (68%) were spa type t044 (suggesting CC80), 23 isolates (20%) were t008 (suggesting CC8) and 2 non-typeable isolates were found to be ST8. Other clonal lineages were sporadically represented. Besides three isolates exhibiting spa type t002 and containing SCCmec V, PCR typing indicated SCCmec IV elements for all the other isolates investigated. Isolates exhibiting spa type t044 originated from various areas of Germany (federal states). The first cases of infections with caMRSA spa type t008 were recorded in the West of Germany in 2005, with other cases occurring elsewhere in 2006. Cases associated with isolates of spa type t310 were restricted to the South of Germany in 2005–06.

Patients with infections caused by caMRSA t008, PVL-positive, SCCmec IVa and their relationship to the USA

In 2005, a patient suffering from a skin abscess had stayed in the USA for several months. In addition, there were two episodes of infections associated with a US military base. In early 2006, a lukS-PV lukF-PV-positive caMRSA isolate exhibiting ST8, t008, SCCmec IVa and positive for lukS-lukF was obtained from blood culture and tracheal secretion from a 2-month-old child with severe pneumonia. The family belonged to the staff of a US military base. Further investigations of family members revealed vaginal carriage for MRSA exhibiting the same characteristics. In November/December 2006, five staff members of the same military base were affected by skin abscesses. The isolates were ST8, lukS-PV lukF-PV-positive and SCCmec IVa and showed pulse types as seen in lane 13 of Figure 1. Three isolates were spa t008 and two were spa non-typeable.

Figure 1

SmaI-macrorestriction patterns of MRSA and MSSA of lineage ST8 in comparison with major clonal lineages of epidemic haMRSA and caMRSA.

Figure 1

SmaI-macrorestriction patterns of MRSA and MSSA of lineage ST8 in comparison with major clonal lineages of epidemic haMRSA and caMRSA.

Introduction of caMRSA t008 into hospitals

During 2005, two patients in different hospitals were admitted for surgical treatment of abscesses. MRSA exhibiting the same characteristics, as those given earlier, were later isolated from post-operative wound infections from other patients who were in the same ward at the same time, suggesting that nosocomial transmission had occurred.

Further characterization of isolates exhibiting spa type t008/MLST ST8

All of the 23 isolates were phenotypically resistant to oxacillin (MIC ≥ 4 mg/L) and to erythromycin. Three isolates were also resistant to clindamycin and harboured erm(C) in addition to msr(A) and mph(B). PCR analysis indicated the presence of an SCCmec IVa element.

As shown in Figure 1, SmaI-macrorestriction patterns of MRSA t008/ST8 form a separate cluster when compared with major clonal lineages of haMRSA and caMRSA. MRSA ST8, SCCmec VIa, PVL-positive exhibit patterns that are similar to each other, but differ from those of PVL-negative MRSA by more than three fragments.

PCR for markers characteristic for caMRSA ‘USA300’

Although PVL-positive MRSA exhibiting ST8 but lacking other characteristics of ‘USA300’ have not been reported so far, a rapid identification of this particular clone by PCR is important. Therefore, we tested isolates of other clonal lineages of caMRSA as well as representative isolates of haMRSA clonal lineages for the presence of arcA, msr(A), mph(B) and ≥6 AT repeats in the SACOL0058 sequence (Table 2). The number of ≥6 AT repeats in the SACOL0058 sequence seems to be characteristic for S. aureus of the ST8 clonal lineage. PCR for the ACME-associated arcA gene was positive in 21 of 23 PVL-positive MRSA of t008/ST8, but also for 3 from 12 PVL-negative MRSA of this clonal lineage. This marker was not detected in isolates affiliated to other clonal lineages of haMRSA and caMRSA. Macrolide resistance genes msr(A) and mph(B) have not been found in isolates from other clonal lineages with the exception of one MRSA isolate of lineage ST5.

Table 2

PCR detection of characteristics for discrimination of caMRSA ST8 ‘USA300’ from other lineages of MRSA in clonal complex 8 and from other lineages of caMRSA and major clonal lineages of haMRSA

    PCR demonstration 
 spa type Corresponding MLST Number of isolates mecA lukS-PV lukF-PV arcA msr(A) mph(B) ≥6 AT repeats in SACOL0058 
Clonal complex CC8 t008 21 21 19 21 21 21 
 − 
 t008 12 − − − − − 12 
 t008 12 − − − 12 
 t009 254 − − − − 
 t036 254 − − − − 
 t051 247 − − − − 
 t037 239 − − − − 
Other caMRSA lineages t175 − − − − 
 t002 − − − 
 t310 22 − − − − 
 t021 30 − − − − 
 t437 59 − − − − 
 t044 80 50 50 − − − − 
 t355 152 − − − − 
Major haMRSA lineages t002 12 − − − − − 
 t001 228 12 − − − − − 
 t022, t032 22 15 − − − − − 
 t003 225 15 − − − − − 
 t004 45 15 − − − − − 
    PCR demonstration 
 spa type Corresponding MLST Number of isolates mecA lukS-PV lukF-PV arcA msr(A) mph(B) ≥6 AT repeats in SACOL0058 
Clonal complex CC8 t008 21 21 19 21 21 21 
 − 
 t008 12 − − − − − 12 
 t008 12 − − − 12 
 t009 254 − − − − 
 t036 254 − − − − 
 t051 247 − − − − 
 t037 239 − − − − 
Other caMRSA lineages t175 − − − − 
 t002 − − − 
 t310 22 − − − − 
 t021 30 − − − − 
 t437 59 − − − − 
 t044 80 50 50 − − − − 
 t355 152 − − − − 
Major haMRSA lineages t002 12 − − − − − 
 t001 228 12 − − − − − 
 t022, t032 22 15 − − − − − 
 t003 225 15 − − − − − 
 t004 45 15 − − − − − 

Discussion

As reported from other European countries4,6,8,11 caMRSA ST80 still predominate among PVL-positive MRSA of community onset. caMRSA affiliated to clonal lineages ST1, ST5, ST9, ST22, ST30 and ST59 are less frequent, but were already observed in Germany in 200511 and were also reported from other European countries. The first PVL-positive caMRSA ST8, t008 were recorded in Germany in early 2005, and 22 further cases followed until the end of 2006. Interestingly, caMRSA ST8 had already been reported from the Netherlands in 2003.31

Because of the typical pattern of characteristics, there is no doubt that the 23 PVL-positive MRSA ST8 are a progeny of caMRSA ‘USA300’, and further importation from the USA is likely to occur. However, PVL-positive caMRSA ST8 have already been reported from other European countries, particularly Belgium, Denmark, the Netherlands6,8,32,33 and Ireland.34 There are also reports on PVL-positive MRSA ST8 from Japan35 and from Hong Kong.36 Although we have to assume that these isolates also represent ‘USA300’, an independent evolution of lukS-PV lukF-PV-containing MRSA of lineage ST8 cannot be excluded as suggested by a report from the UK on caMRSA of this clonal lineage containing lukS-PV lukF-PV, SCCmec I and surprisingly the superantigen determinants sec and tst.37 PVL-negative MSSA ST8 are widely disseminated among nasal carriers in the community. ST8 MRSA emerged in the early 1970s (‘ancestral MRSA’ of clonal complex CC817), and they still exist in German hospitals (26 nosocomial and 3 community isolates among 2318 MRSA sent to the authors' laboratory in 2006).

To date, ST8 caMRSA do not appear to be widely disseminated in Europe. This might be due to differences with respect to predisposing conditions among the communities in Europe and in the USA, where an association between non-white race and soft-tissue infections caused by caMRSA was established.12 However, the reasons for this association are unclear, and there might be confounding factors not recorded (e.g. HIV status).

Apart from the cluster of infections in a US military base and the two cases for which nosocomial transmission was likely, the other 14 cases were sporadic. Intrafamilial transmission as already reported from the USA38 and from the Netherlands39 was observed in one case only.

Nevertheless, reliable, early detection in the clinical bacteriological laboratory is necessary and an essential prerequisite to prevent further dissemination. This can be achieved by PCR detection of marker genes that are characteristic for caMRSA ‘USA300’ such as arcA and msr(A). Although not frequently, spa type t008 can be found in isolates from other clonal lineages of clonal complex CC8. Moreover, 2 of 23 isolates of PVL-positive MRSA ST8 described here were non-typeable by spa typing. Therefore, PCR for ≥6 AT repeats in SACOL0058 is useful to confirm the attribution of PVL-positive MRSA to lineage ST8. The observation of some arcA-negative isolates among PVL-positive MRSA ST8 and arcA-positive isolates among PVL-negative MRSA ST8 means that PCR for the presence of msr(A) is useful as an additional marker.

However, care must be exercised when using molecular epidemiological markers on transferable genetic elements, as events such as loss or acquisition of these elements by other clonal lineages, e.g. arcA in ST5-MRSA-SCCmec II from the USA,40 could mislead.

Funding

This study was supported by a grant from the German Federal Ministry of Health to support the work of the authors' laboratory as German National Reference Center for Staphylococci. There were no financial supports from other sides.

Transparency declarations

None to declare.

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