Obvious Lack of Association Between Dynamics of Epidemic Methicillin-Resistant Staphylococcus aureus in Central Europe and agr Specificity Groups
- First Online:
- Cite this article as:
- Strommenger, B., Cuny, C., Werner, G. et al. Eur J Clin Microbiol Infect Dis (2004) 23: 15. doi:10.1007/s10096-003-1046-8
- 100 Views
During the past 8 years, changes in the prevalence and spread of different epidemic methicillin-resistant Staphylococcus aureus (MRSA) have been observed in central Europe, with the emergence of new strains possessing fewer resistance characters. This has also been demonstrated at the level of particular hospitals. Since variation in agr specificity type has been proposed as a possible reason for population dynamics in Staphylococcus aureus, the agr specificity groups of different epidemic MRSA strains were investigated by PCR using agr group-specific primers. Four of the “old” as well as two “new” epidemic strains exhibited agr specificity group I. One group of epidemic MRSA strains, which has been observed since the beginning of the 1990s, exhibited the agr specificity group II. Sequencing the variable part (agrB-D-C) of the agr locus revealed only six relevant nucleotide changes within this region, with three of them modifying the Shine-Dalgarno sequence region of agrC. On the basis of the results obtained, it is proposed that the dynamics observed in the population of MRSA in Germany is not due to different agr group specificities in “old” and “new” epidemic clones.
Staphylococcus aureus is a major nosocomial pathogen that causes a variety of infections including septicemia, endocarditis, pneumonia, and osteomyelitis. During the past 15 years, the prevalence of methicillin-resistant Staphylococcus aureus (MRSA) has increased in many parts of the world. As shown by several studies, this is due mainly to the emergence and spread of epidemic strains [1, 2, 3]. In central Europe, a particular dynamic of epidemic strains has been observed: strains widely disseminated during the beginning of the 1990s have become less frequent, whereas an increasing spread of newly emerging strains has been recorded . Changes in the population of bacteria can occur for different reasons. One putative reason is the impact of selective pressure due to the use of antibiotics, which results in the acquisition of new resistance genes. Since the newly emerging strains are even less broadly resistant [4, 5, 6], acquisition of resistance genes may not play a role in the spread of new epidemic MRSA in Germany.
As already suggested and elucidated in previous studies, the staphylococcal quorum-sensing system mediated by the accessory gene regulator (agr) is probably involved in competition between strains for sites of colonization or infection [7, 8, 9]. Quorum sensing by the agr global regulatory system is mediated by a post-transcriptionally processed autoinducing peptide that is excised from the agrD gene product and that interacts with a transmembrane receptor encoded by the agrC gene. As a part of a two-component regulatory system, AgrC mediates phosphorylation of the agrA gene product, which autoactivates the transcription of the agr operon from two divergent promotors, P2 and P3. This results in downregulation of surface proteins, including putative colonization factors, and in upregulation of genes encoding extracellular proteins via the regulatory RNA III transcribed from promotor P3 . Until now, four different groups of autoinducer-receptor specificity have been described [7, 9]. The secreted autoinducing peptide can activate the agr response in strains in the same specificity group and can inhibit it in strains from other groups.
Epidemic Staphylococcus aureus strains have to compete successfully with resident staphylococcal microflora. If the agr quorum-sensing system plays a role in this competition, this should be reflected by the agrC-agrD polymorphism of epidemic strains. Therefore, the aim of this study was to compare the agr group specificity of older epidemic MRSA strains with that of the newer ones that have replaced them in recent years.
Materials and Methods
Staphylococcus aureus isolates investigated in this study originate from the strain collection of our laboratory at the National Reference Center for Staphylococci in Germany. Criteria for selection of MRSA for molecular typing have been described previously [3, 4]. Epidemic MRSA had been identified by molecular typing on the basis of SmaI macrorestriction patterns and supplementary polymerase chain reaction (PCR) typing  and cluster analysis [3, 5]. Representative isolates from epidemic MRSA in Germany had been included into the European HARMONY collection  and further subjected to MLST typing . The Staphylococcus aureus strains 8325-4 (agr group I), 705/92 (agr group II), PS29 (agr group III), PS55 (agr group IV), and 282/94 and 306/94 (for clonal group V of Staphylococcus aureus) were selected as reference strains.
Polymerase Chain Reaction
Primers used in the study
Reference or source of sequence
5′-CAC TTA TCA TCA AAG AGC C-3′
5′-CCA CTA ATT ATA GCT GG-3′
5′-GTA GAG CCG TAT TGA TTC-3′
5′-GTA TTT CAT CTC TTT AAG G-3′
5′-CAA GCT ATT ACA TTA CTA CCA-3′
AF00178 pos. no. 510–530
5′-AAT GCT TCC ACT TAC TATC-3′
AF00178 pos. no. 728-743
5′-GCT CAA TTC ATG CAA TTA-3′
AF288215 pos. no. 1207–1223
5′-ATG GTA CTG TAA ACA TTA-3′
AF288215 pos. no. 1502–1520
5′-TAT GCT CCT GCA GCA ACT AA-3′
5′-CTT GCG CAT TTC GTT GTT TGA-3′
Restriction of Polymerase Chain Reaction Products
Amplicons were purified using the Qiagen PCR Purification Kit (Qiagen, Germany) and digested with DraI (Roche Biochemicals, Germany) according to the manufacturer’s instructions. Resulting restriction fragments were analyzed on a 1.4% agarose gel.
DNA Sequencing and Analysis of Sequence Data
Sequencing reactions were carried out using the ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction Kit (Perkin Elmer Applied Biosystems Division, USA) according to the manufacturer’s protocol. Sequence alignment and analysis of sequence polymorphism among the different agr genes were performed with the DNASTAR software package (DNASTAR, USA).
Emergence and Spread of Epidemic Methicillin-Resistant Staphylococcus aureus
Dynamics of epidemic MRSA in German hospitals
Grouping according to molecular typing
Resistance phenotypesa and genotypesb
Frequency of occurrence of epidemic MRSA in different German hospitalsc
agr specificity groupd
Northern German epidemic MRSA ST247
PEN, OXA, GEN, ERY, CLI, OTE, SXT, RIF, CIP
mecA, aph2”-aac6’, ermA, tetM, dfrA
Southern German epidemic MRSA ST228
PEN, OXA, ERY, CLI, CIP, (GEN), (OTE)e
mecA, aph2”-aac6’, ermA, tetM, dfrA
Hannover area epidemic MRSA
PEN, OXA, GEN, ERY, CLI, SXT, CIP
mecA, aph2”-aac6’, ermA, tetM, dfrA
Vienna epidemic MRSA ST254
PEN, OXA, GEN, ERY, CLI, SXT, CIP, OTE, (FUS)
mecA, aph2”-aac6’, ermA, tetM
Berlin epidemic MRSA ST45
PEN, OXA, CIP, (GEN, ERY, ERY-CLI, SXT)
mecA, grlA, (aph2”-aac6’, ermC, dfrA)f
Barnim epidemic MRSA ST22
PEN, OXA, ERY, CLI, CIP, GEN
mecA, ermC, grlA, (aph2”-aac6’, rpoB)f
Rhine-Hesse epidemic MRSA ST5
PEN, OXA, ERY, CLI, CIP
mecA, ermA, grlA
PEN, OXA, (ERY, CLI)
No. of hospitals with epidemic MRSA
Dynamics of epidemic MRSA in tertiary care hospital G
No. of patients affected
Northern German epidemic MRSA
Southern German epidemic MRSA
Hannover area epidemic MRSA
Berlin epidemic MRSA
Barnim epidemic MRSA
Determination of agr Specificity Group and Resistance Genotype
The agr specificity group and acquired resistance genes were determined by PCR for four independent isolates of each epidemic strain. The clones dominating in the year 2000 are clearly less broadly resistant compared to the older strains. Except for the Southern German epidemic MRSA and the Rhine-Hesse epidemic MRSA, which belong to specificity group II, the strains all belong to agr specificity group I (Table 2).
Sequence Comparison of the agr Locus
It was of particular interest to check whether the reported nucleotide changes are specific for both groups of newly emerging MRSA or whether they are already present in supposed methicillin-susceptible ancestors of the Berlin epidemic MRSA (Staphylococcus aureus of group V) [6, 13]. The corresponding agr sequence of these strains is identical to that obtained for isolates of the Berlin epidemic MRSA (Fig. 1).
Comparison of the nucleotide sequence of the Southern German epidemic MRSA, which belong to agr specificity type II, with the corresponding sequence of the agr specificity type II reference strain Staphylococcus aureus SA 502A (GenBank accession no. AF001782) revealed no relevant nucleotide changes.
All agr sequences from Staphylococcus aureus strains belonging to different clonal groups can be classified in one of the agr groups defined by Ji et al. . Except for the Southern German epidemic MRSA and the Rhine-Hesse epidemic MRSA (agr group II), all epidemic MRSA belong to agr group I, with the new emerging ones included. Similar findings were reported in a previous study by Van Leeuwen et al. . In a worldwide collection of MRSA, the majority of the strains belonging to different clonal lineages were also attributed to agr group I, with minor nucleotide polymorphism within this group. Therefore, agr specificity does not seem to play a prominent role in the dynamics of epidemic MRSA. Otherwise, we would expect the emergence of MRSA strains differing in agr specificity from their predecessors or exhibiting at least amino acid changes in either agrD or agrC and leading to a modified agr specificity as described by Mayville et al.  for modifications of AgrD that resulted in inhibition of the agr response. Modifications of agr specificity by such kind of mutations are obviously rare, as indicated by identical sequences of the older epidemic MRSA and strain 8325, which was first described in 1945 .
In this study, we could identify only three relevant point mutations that resulted in amino acid modifications. The nucleotide changes in the Shine-Dalgarno sequence region (ribosomal binding site) between agrD and agrC are in thus far remarkable in that they are identical in the Barnim epidemic MRSA and the Berlin epidemic MRSA, both of which belong to the dominating clones in 2000 on the one hand, but can be classified into quite different clonal groups on the other [5, 6]. The same genotype has recently been described for a Staphylococcus aureus strain representing a subtype of the Canadian epidemic CMRSA-1 (7; GenBank accession no. AF210055). As Shine-Dalgarno sequences in Staphylococcus aureus include the “classical” -GAGA- part , it seems possible that a mutation resulting in -GAAA- affects the expression of the receptor protein AgrC. A change in the expression pattern of AgrC could lead finally to a reduced autoactivation of the agr locus. As a result, the “switch” from the expression of surface proteins to the expression of secreted proteins, mediated by the expression of RNA III from the promotor P3, would be diminished, which could be advantageous for colonization. Such a balance of virulence factors expression was also discussed by Papakyriacou et al. , who suggested that the protein expression pattern could be influenced by point mutations in the agr locus. It should also be kept in mind that these mutations were already present in methicillin-susceptible Staphylococcus aureus from which the Berlin epidemic MRSA had very likely originated and which became increasingly prevalent by the beginning of the 1990s .
However, to verify our hypothesis, further experiments are required to show whether the nucleotide changes concerning the agrC Shine-Dalgarno sequence region affect the expression pattern of agrC.