J Med Microbiol 53 (2004), 545-549; DOI: 10.1099/jmm.0.05406-0
© 2004 Society for General Microbiology
ISSN 0022-2615
Genetic polymorphism of the accessory gene regulator (agr) locus in Staphylococcus epidermidis and its association with pathogenicity
M. Li,
M. Guan,
X. F. Jiang,
F. Y. Yuan,
M. Xu,
W. Z. Zhang and
Y. Lu
Center of Laboratory Medicine, Huashan Hospital, Fudan University, Shanghai, China
Correspondence Y. Lu yuanlu{at}hsh.stn.sh.cn
Received July 29, 2003
Accepted December 26, 2003
Staphylococcus epidermidis has become one of the most important causes of nosocomial infections in recent years. The staphylococcal accessory gene regulator (agr) is the most important locus responsible for the regulation of virulence factors, and it has been shown to be polymorphic. The aim of this study was to investigate the agr locus and its genetic polymorphisms in different Chinese S. epidermidis isolates and the relationship between genetic polymorphisms and pathogenicity. Specific PCR was used to amplify the different agr groups. Results were confirmed by restriction enzyme digestion and sequence analysis. agr mutations were detected and three agr groups of S. epidermidis were determined. Of the isolates, 12 % were pathogenic S. epidermidis and 17 % had naturally occurring agr mutations (P > 0.05). Pathogenic S. epidermidis isolates comprised 68.2 % agr group I, 19.3 % group II and 12.5 % group III, while isolates from healthy controls comprised 39 % agr group I, 51 % group II and 10 % group III (P < 0.01). The percentages of agr locus mutants and the three agr groups in different hospitals showed no significant differences (P > 0.05). The percentage of agr group I S. epidermidis isolated from catheters and blood was higher than that isolated from the other clinical specimens. This is the first study to investigate the genetic polymorphism of agr in S. epidermidis in China. The mean percentage of agr locus mutants was 14.9 % (12 % in clinical specimens; 17.7 % in controls). Genetic polymorphism of agr in S. epidermidis was linked to its pathogenicity; group I was associated with pathogenicity, while most isolates from healthy subjects were group II. The mechanism is to be investigated.
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INTRODUCTION
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Until recently, Staphylococcus epidermidis had been regarded as a commensal inhabitant of human skin and mucus that rarely caused disease. Since the early 1980s, it has gained substantial attention because it has been recognized as one of the most important causes of nosocomial infections (Vuong & Otto, 2002; Raad et al., 1998). S. epidermidis is mainly found in nosocomial cardiovascular infections, bloodstream infections and infections of the eye, ear, nose and throat (von Eiff et al., 2002).
The staphylococcus accessory gene regulator (agr) locus is the most important locus responsible for growth-phase-dependent regulation of virulence factors (Otto et al., 1998, 1999, 2001). The gene structure and sequences of the S. epidermidis agr system are very similar to those of Staphylococcus aureus, and the locus may play a comparable role. The agr system, in both S. aureus and S. epidermidis, is approximately 3.5 kb in size with 68 % similarity, and comprises the genes agrA, agrB, agrC and agrD, which are co-transcribed (RNAII). As a gene for the effector molecule of the agr system, RNAIII also encodes delta toxin (hld). RNAIII controls expressions of target genes in an unknown manner (Vuong et al., 2000a). The agr system is activated during the transition from exponential to stationary growth phase by an auto-regulatory mechanism, which may involve a modified pheromone peptide. About a quarter of S. aureus clinical isolates are naturally occurring agr mutants, most of which show increased ability to attach to polystyrene compared with agr+ strains (Vuong & Otto, 2002). This mechanism probably also occurs in agr mutants of S. epidermidis, but the mutation rate of agr in S. epidermidis is unknown.
The agr locus in staphylococci has been shown to be polymorphic (Moore & Lindsay, 2001; Dufour et al., 2002; Jarraud et al., 2000, 2002). The agr locus in S. aureus can be divided into four distinct genetic groups. Jarraud et al. (2000, 2002) reported that S. aureus agr groups were associated with the pattern of S. aureus diseases. As far as S. epidermidis is concerned, Dufour et al. (2002) recently used sequencing to determine (based on a limited number of S. epidermidis strains) that S. epidermidis has three agr groups. Similar to S. aureus agr, sequence variation of S. epidermidis is particularly marked in agrD, the C-terminal two-thirds of agrB and the N-terminal half of agrC. The size of the hypervariable region is about 1000 bp. Genetic polymorphisms of the agr locus in S. epidermidis have not been reported in China to our knowledge, and the relationship between genetic polymorphisms of the agr locus and its pathogenicity is unknown.
The aim of this study was to investigate the agr locus and its genetic polymorphisms in different Chinese S. epidermidis isolates and the relationship between genetic polymorphisms and pathogenicity.
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METHODS
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Bacterial isolates.
Between November 2002 and May 2003, 100 S. epidermidis isolates were collected from three different hospitals: Huashan Hospital (58), Ruijin Hospital (22) and Children's Hospital (20). Isolates were taken from catheters (11), blood cultures (24), urine (20), wounds (12), cerebrospinal fluid and dialysate (16) and sputa (17). A further 62 isolates of S. epidermidis were isolated from the skin of healthy volunteers. There were 20 isolates of other staphylococci, including five isolates of S. aureus, three of Staphylococcus hominis, five of Staphylococcus haemolyticus, three of Staphylococcus lugdunensis and four of Staphylococcus xylosus. All isolates were characterized by Gram stain and by catalase and coagulase activity on rabbit plasma (bioMérieux). Staphylococcal species were identified by the API STAPH test (bioMérieux) (Petinaki et al., 2001).
Genomic DNA extraction.
Cells from an overnight culture in Luria–Bertani broth were collected by centrifugation and suspended in 1 ml buffer (0.1 M Tris/HCl, pH 7.5, 0.1 M EDTA, 0.15 M NaCl) with 0.2 mg lysostaphin (Sigma). After incubation at 37 °C for 30 min, samples were extracted with phenol/chloroform/isoamyl alcohol (25 : 24 : 1). DNA was precipitated by addition of two volumes of 100 % ethanol and was resuspended in distilled water.
DNA amplification.
Primers are given in Table 1. Sequences of the three agr groups of S. epidermidis were taken from the GenBank sequence database of the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/) (accession nos Z49220, AF346724, AF346725). Primers were designed by the Primer3 program (http://www.genome.wi.mit.edu/genome_software/other/primer3.html) based on the gene sequence information. The A primers were selected from conserved sequences of agr, which are common to agr groups I, II and III, to amplify a 1022 bp fragment. The B primers were selected from the hypervariable region, which is common to agr groups II and III but not to group I. The C primers were also selected from the hypervariable region and were specific for group II.
Primers were synthesized using a PolyPlex DNA synthesizer (Genomic Solutions). Amplification was carried out on a GeneAmp-9700 (Applied Biosystems) under the following conditions: an initial 5-min denaturation at 94 °C followed by 35 cycles of 30 s denaturation at 94 °C, 30 s annealing at 55 °C and 1 min extension at 72 °C, with a final extension at 72 °C for 7 min. In each PCR, a positive control and a negative control (distilled water) were included. Yield of amplicons was determined by electrophoresis in the presence of ethidium bromide (0.3 µg ml–1).
Restriction endonuclease digestion and sequence analysis of PCR products.
Restriction endonuclease digestions of all amplicons were performed with HindIII and AvaI (Promega) according to the manufacturer's instructions. HindIII and AvaI digestions were visualized on 2 % (w/v) agarose gel (TaKaRa), stained with ethidium bromide. Two PCR products of each agr group amplified by specific primers were randomly selected and sequenced using fluorescent-labelled primers on a LI-COR sequencer (MWG-Biotech) (Vuong et al., 2000b). Sequences were aligned with CLUSTAL X software (Aiyar, 2000).
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RESULTS AND DISCUSSION
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In recent years, S. epidermidis has emerged as one of the most important pathogens in nosocomial infections. Effective antibiotic treatment of S. epidermidis has been difficult because a biofilm-forming slime capsule that surrounds colonies of the bacterium can be barely penetrated by many antibiotics (Ji et al., 1997; Raad et al., 1998; O'Gara & Humphreys, 2001). The situation has become even more severe because of the emergence of multidrug-tolerant strains, especially vancomycin-tolerant S. epidermidis strains. Although these problems have been recognized for a number of years, identification of S. epidermidis virulence factors and investigation of their regulation have not kept pace with research performed in S. aureus (Hoyle & Costerton, 1991; Day et al., 2001; Lina et al., 2003), thus very little is known about the regulation of virulence genes in S. epidermidis.
The agr locus was first identified as a regulator of virulence factors in S. aureus (Peschel et al., 1999; Papakyriacou et al., 2000). In an agr mutant, expression of exotoxins, such as 
-, ß- and 
-toxin, serine protease, DNase, fibrinolysin, enterotoxin B and toxic shock syndrome toxin-1, was decreased, whereas expression of many surface proteins was increased (Recsei et al., 1986; Abdelnour et al., 1993). The most important virulence factor of S. epidermidis is considered to be biofilm formation (Huebner & Goldmann, 1999; Mah & O'Toole, 2001). The agr system of S. epidermidis seems to affect biofilm formation via regulation of expression of autolysin (AtlE), which is involved in primary adhesion to polymer surfaces (Heilmann et al., 1997), and it is reported that the S. epidermidis agr system inhibits expression of AtlE (Vuong et al., 2003). About a quarter of S. aureus clinical isolates are naturally occurring agr mutants (Otto, 2001); the mutation rate of the agr locus in S. epidermidis is unknown.
In this study, we used specific primers to amplify different agr groups. The A primers were selected from conserved sequences to amplify a 1022-bp fragment of agr that is common to agr groups I, II and III. The PCR result would be negative if there were mutations in this conserved region. The B primers were selected from the hypervariable region to amplify a 453-bp fragment with a sequence common to agr groups II and III. The PCR result for agr group I was negative but that of groups II and III was positive. Using the pair of C primers to amplify a 615-bp fragment specific for agr group II, PCR of agr groups I and III was negative (Fig. 1). In order to confirm that the PCR results from the three agr groups of S. epidermidis amplified by specific primers were correct, all of the PCR products were digested with HindIII and AvaI. The 615-bp amplicon of agr group II could be digested into two fragments, of 220 and 395 bp; the 453-bp amplicon of agr group III could be digested into two fragments, of 253 and 200 bp and the 1022-bp amplicon of agr group I could not be digested by these two enzymes (Fig. 2). Two PCR products of each agr group amplified by specific primers were randomly selected and sequenced. Sequences were aligned for analysis by using CLUSTAL X software. Sequence analysis revealed that amplicons of the three agr groups amplified by specific primers concurred with the GenBank sequences. To confirm the specificity of the three primer sets used in PCR for S. epidermidis, we used genomic DNA of 20 other staphylococcal strains as templates and carried out PCR with the same three primer sets under standard conditions. A PCR product of the expected size was only obtained when DNA from S. epidermidis isolates was used as the template (data not shown), thereby confirming the specificity of the PCR assay for S. epidermidis DNA. Of the 100 S. epidermidis isolates collected from clinical specimens and 62 isolates from the skin of healthy volunteers, the rates of failure to detect the agr locus were 12 and 17.7 %, respectively (P > 0.05) (Table 2). It has been reported that agr mutants of S. aureus show an increased ability to attach to polystyrene compared with agr+ strains, which is at least partially due to the lack of the detergent-like peptide -toxin (Vuong & Otto, 2002). This mechanism probably also occurs in agr mutants of S. epidermidis. In our study, we used a quantitative plate test (Ammendolia et al., 1999; Christensen et al., 1985) to determine production of slime. Results showed that, for both pathogenic S. epidermidis isolates and isolates from healthy volunteers, agr mutants showed an increased ability to attach to polystyrene (data not shown).
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Fig. 1. PCR products with primer sets A, B and C, separated on a 2 % (w/v) agarose gel. Lanes: 1 and 8, 100-bp DNA ladder (fragment sizes 100–1031 bp in 100-bp increments); 2 and 3, 615-bp fragment amplified by C primers (lane 2, negative; lane 3, positive); 4 and 5, 453-bp fragment amplified by B primers (lane 4, negative; lane 5, positive); 6 and 7, 1022-bp fragment amplified by A primers (lane 6, negative; lane 7, positive).
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Fig. 2. PCR products of three agr groups following digestion with restriction endonucleases HindIII and AvaI, separated on a 2 % (w/v) agarose gel. Lanes: 1 and 2, 1022-bp amplicon of agr group I before and after digestion (1 h); 4 and 5, 615-bp amplicon of agr group II before and after digestion (1 h; digested into two fragments, 220 and 395 bp); 7 and 8, 453-bp amplicon of agr group III before and after digestion (1 h; digested into two fragments, 253 and 200 bp); 3, 6 and 9, DNA ladder (100, 250, 500, 750, 1000 and 2000 bp).
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Table 2. Comparison of genetic polymorphism of the agr locus in different S. epidermidis isolates Values are numbers (percentages) of isolates.
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The agr locus in staphylococci has been shown to be polymorphic. S. aureus has four agr groups. Jarraud et al. (2000, 2002) reported that S. aureus agr groups were associated with the pattern of S. aureus diseases. For instance, agr group IV strains are associated with generalized exfoliate syndromes and endocarditic strains belong mainly to agr groups I and II. In our study, we found agr mutations and three agr groups of S. epidermidis. Similar to the findings of Dufour et al. (2002), most pathogenic S. epidermidis isolates were in agr group I. Pathogenic S. epidermidis isolates comprised 68.2 % group I, 19.3 % group II and 12.5 % group III, while isolates from healthy subjects with genetic polymorphisms of the agr locus comprised 39 % group I, 51 % group II and 10 % group III (P < 0.01). The percentage of S. epidermidis agr group I was higher in isolates from blood (77.3 %), and especially from catheters (100 %), than in isolates from other clinical specimens (mean 68.2 %) (Table 3). In clinics, catheter-related infections are the most serious induced by S. epidermidis (Caputo et al., 1987). It seems, therefore, that genetic polymorphisms of the agr locus in S. epidermidis are linked to its pathogenicity; group I was associated with pathogenicity while most isolates from healthy people were group II. The relationship between agr group III and pathogenicity was not very clear. There was no significant difference (P > 0.05) in the percentage of agr locus mutants or the relative percentage of the three agr groups found in different hospitals (Table 4).
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Table 3. Comparison of genetic polymorphism of the agr locus in isolates from different specimens Values are numbers (percentages) of isolates. CSF, Cerebrospinal fluid.
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Table 4. Comparison of genetic polymorphism of the agr locus in isolates from different hospitals Values are numbers (percentages) of isolates.
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The finding that agr genetic polymorphisms in S. epidermidis are linked to its pathogenicity is a significant step in our initial approach towards understanding gene regulation in this pathogen. The obvious question is: why is pathogenicity different in different agr groups? Perhaps the groups have a different genetic background (Novick & Muir, 1999), or virulence-related proteins or enzymes are involved. This is the first study to investigate genetic polymorphisms of the agr locus in S. epidermidis and the relationship between genetic polymorphisms and pathogenicity in China. This result may help to clarify the function of agr in S. epidermidis and the relationship between agr and biofilm formation. It may also be valuable in the development of new preventive and therapeutic measures.
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ACKNOWLEDGEMENTS
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This work was supported by the fund of the Shanghai Project for 100 Academic Leaders and the 211 project grant ‘Functional Genomics of Important Pathogenic Microorganisms'. We thank Professors Y. M. Wen, D. Qu and Q. Gao and Dr H. L. Li (Laboratory of Medical Molecular Virology, Shanghai Medical College, Fudan University, China) for their technical assistance and for sourcing the healthy volunteers who provided S. epidermidis isolates.
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INTRODUCTION
METHODS
