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Staphylococcal Chromosomal Cassette mec Type I, spa Type, and Expression of Pls Are Determinants of Reduced Cellular Invasiveness of Methicillin-Resistant Staphylococcus aureus Isolates

  1. Cornelia Werbick1,
  2. Karsten Becker1,
  3. Alexander Mellmann2,
  4. Katri M. Juuti3,
  5. Christof von Eiff1,
  6. Georg Peters1,
  7. Pentti I. Kuusela4,5,
  8. Alexander W. Friedrich2 and
  9. Bhanu Sinha1,a
  1. 1 Institute of Medical Microbiology, Germany
  2. 2 Institute for Hygiene, University Hospital of Münster, Germany
  3. 3 Department of Biological and Environmental Sciences, General Microbiology, Helsinki, Finland
  4. 4 Department of Bacteriology and Immunology, Haartman Institute, University of Helsinki, Helsinki, Finland
  5. 5 5Division of Clinical Microbiology, Helsinki University Central Hospital (HUCH) Laboratory Diagnostics, HUCH, Helsinki, Finland
  1. Reprints or correspondence: Dr. Bhanu Sinha, Institute of Hygiene and Microbiology, University of Würzburg, Josef-Schneider-Str. 2, Bldg. E1, 97080 Würzburg, Germany (Bhanu.Sinha{at}gmx.de).

  1. Presented in part: 10th International Symposium on Staphylococci and Staphylococcal Diseases, Charleston, South Carolina, 23–27 October 2004 (poster PA-34); 56th Annual Meeting of the German Society for Hygiene and Microbiology, Münster, Germany, 26–29 September 2004 (poster MPP005).

  • ↵a Present affiliation: Institute of Hygiene and Microbiology, University of Würzburg, Würzburg, Germany.

 
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Abstract

We have shown previously that pls, which codes for the surface protein Pls of methicillin-resistant Staphylococcus aureus (MRSA), reduces adhesion to immobilized fibronectin, fibrinogen, laminin, and immunoglobulin G as well as invasion of host cells. Here, we tested a collection of 66 clinical MRSA isolates—48 negative for pls/Pls (pls-/Pls-), 15 positive for pls/Pls (pls+/Pls+), and 3 harboring the pls gene but not expressing Pls (pls+/Pls-)—for cellular invasiveness. Invasion of 293 cells by pls+/Pls+ strains was lower than that by the pls-/Pls- strains (median [range], 36% [22%–70%] vs. 93% [25%–162%]). The 3 pls+/Pls- strains (median [range], 95% [63%–103%]) were as invasive as the pls-/Pls- strains. In addition, we identified a pls+/Pls+ staphylococcal chromosomal cassette mec (SCCmec) IV strain. In conclusion, 3 properties—pls/Pls, SCCmec type, and spa type—strongly predicted the cellular invasiveness of MRSA strains, as indicated by good clustering. In contrast, the spa type–deduced multilocus sequence typing clonal complex (MLST-CC) was not able to predict the invasiveness of MRSA strains equally well. The underlying mechanism remains to be elucidated.

Staphylococcus aureus is a major cause of severe nosocomial and community-acquired infections [1]. Treatment of infections is complicated by the emergence of methicillin-resistant S. aureus (MRSA), some of which— especially those belonging to staphylococcal chromosomal cassette mec (SCCmec) types I–III—have also acquired multidrug resistance.

SCCmec is a potentially mobile genetic element [2]. To date, 5 different SCCmec types have been identified, but new types may be generated continuously [3]. Recently, SCCmec type VI was identified as a structural variant of type IV [4].

The pathogenic potential of S. aureus is in part refflected by a large number of surface proteins with adhesive and invasive functions. One of these MRSA surface proteins is Pls (plasmin-sensitive surface protein). The pls gene is located in the SCCmec. SCCmec usually also contains the mecA gene, which codes for a modified penicillin-binding protein 2 (PBP2a or PBP2′) that causes resistance to methicillin. So far, pls has been found only in type I SCCmec element [5, 6].

Another S. aureus surface protein is protein A, which is encoded by the spa gene. Because protein A is known to carry polymorphic regions [7], DNA sequencing of the repeat region (X region) of spa has been proven to be a useful alternative to existing techniques, such as pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST), for molecular typing of MRSA [8, 9, 10].

We and others have elucidated the molecular mechanism for cellular invasion by S. aureus [11, 12, 13, 14, 15] (reviewed in [16, 17]). Invasion is dependent on fibronectin (Fn)–binding proteins (FnBPs) and host cell α5β1 integrins, which are linked by Fn as a bridging molecule.

We have previously shown that presence of pls/Pls decreases the adherence of MRSA strains on host proteins such as immobilized Fn, fibrinogen (Fg), IgG, and laminin as well as on soluble Fn and Fg [6, 18]. Furthermore, we have demonstrated that pls/Pls also decreases the invasiveness of MRSA strains for 293 cells (human embryonic kidney cells). Disruption of the pls gene of strain 1061 increased adherence [6], and complementation restored the low level of invasiveness [18]. The aim of the present study was (1) to test a large and well-characterized strain collection for their invasive properties and for the presence of pls/Pls and (2) to search for predictors of cellular invasiveness other than pls, such as the SCCmec and spa type.

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Materials and Methods

Reagents, enzymes, and antibodies. Recombinant lysostaphin (Ambicin L) was supplied by Applied Microbiology, and human serum albumin was supplied by Behring. SmaI was purchased from Roche, and the lambda ladder (no. N0340S) was purchased from New England Biolabs. Monoclonal antibodies against Pls purified from strain 1061 were produced as described elsewhere [19], with the modification that Dulbecco's modified Eagle medium (DMEM) was substituted by RPMI 1640.

Bacterial strains and cultures. S. aureus strain Cowan 1 and S. carnosus strain TM300 were used as reference isolates. The strains used in this study were provided by W. Witte (National Reference Center for Staphylococci, Wernigerode, Germany), K. Hiramatsu (Juntendo University, Department of Bacteriology, Tokyo, Japan), and J. É tienne (Centre National de Référence Staphylocoques, Lyon, France) and were collected at the University Hospital of Münster (Germany) and at Finnish hospitals (table 1). Species identification and determination of methicillin resistance were confirmed by use of VITEK 2 (bio-Mérieux). In addition, all strains were phenotypicallycompared (colony size, pigmentation, and hemolysis) on sheep blood agar. Strains with ambiguous results in phenotypic tests were analyzed by 16s rRNA gene sequencing, as described elsewhere [26]. Methicillin resistance was confirmed by mecA-specific polymerase chain reaction (PCR) [27].

Table 1.
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Table 1.

Sources of the bacterial strains used in the present study

PFGE and Southern blotting for pls. PFGE analysis and Southern blotting for pls were performed as described elsewhere [18].

Cell culture. All media components were fromGibco-BRL. 293 cells (adenovirus type 5 DNA–transformed primary human embryonic kidney cells) were obtained from either the American Type Culture Collection (no. CRL-1573) or the German Collection of Microorganisms and Cell Cultures (no. ACC 305); maintained in DMEM/F-12 nutrient mixture (containing Glu-taMAX-I, a stable glutamine dipeptide; Invitrogen) supplemented with 10% fetal calf serum, 50 IU/mL penicillin, and 50 µg/mL streptomycin; and split 1:4 twice weekly by trypsinization. Cells had been passaged for a maximum of 35 times after freezing before use in the experiments.

Flow cytometric invasion assay. MRSA strains were assayed for cellular invasion in a blinded fashion by use of a fflow cytometric invasion assay described elsewhere [11, 12], with minor modifications [18, 28]. A fresh bacterial culture was used for each experiment. Results were expressed as the mean ± SE values from at least 3 independent experiments performed in duplicate.

spa typing. For molecular subtyping of the strains, the sequence of the repeat region (X region) of the protein A gene (spa) was determined [8, 10]. Therefore, the primers spa-1113f (5′-TAAAGACGATCCTTCGGTGAGC-3′) and spa-1514r (5′-CAGCAGTAGTGCCGTTTGCT-3′) were used for spa amplification, and Taq cycle sequencing DNA sequences were obtained with an ABI Prism 3100 Avant Genetic Analyzer (Applied Biosystems) and were analyzed with StaphType software (version 1.0; Ridom) [9]. The spa type nomenclature used was in accordance with that used at http://spaserver.ridom.de, which has been developed by Ridom and is curated by the SeqNet.org initiative (http://www.seqnet.org) [29].

Clonal complex (CC) typing. MLST-CCs were deduced from the data on the MLST-spa mapping of the spaserver (http://spa.ridom.de/mlst.shtml). The corresponding MLST for each spa type was used, and the associated MLST-CC was determined in concordance with the MLST database (http://saureus.mlst.net) (table 2).

Table 2.
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Table 2.

Presence of pls/Pls, staphylococcal chromosomal cassette mec (SCCmec) types, spa types, and spa type–deduced clonal complexes (CCs) of the strains used in the present study.

SCCmec classification. Staphylococci were collected from 0.5 mL of an overnight brain-heart infusion broth culture. Lysis of staphylococci and isolation of DNA was performed as described elsewhere [30]. To determine SCCmec types I–IV, 2 different multiplex PCR typing strategies—as described by Oliveira and de Lencastre [31] and by Hiramatsu's group [3, 5, 23]—were applied. SCCmec type V was determined as published by Ito et al. [3]. Uniplex combinations of the corresponding primer pairs were also performed in cases of nonassignable or missing bands, to avoid putative amplification biases due to the multiplex approach [32]. For further confirmation, the typing strategy described by Branger et al. [33] was also used. The amplification was performed in an iCycler (Bio-Rad). After thermal cycling, 10 µL of the amplified product was run on a 2% (wt/vol) agarose gel, stained with ethidium bromide, and visualized under UV light.

In ∼21% of the strains, the 3 methods did not yield consistent results for SCCmec classification, demonstrating a lack of unambiguity. Except for SCCmec type V, strains were assigned to an SCCmec type if at least the results of 2 methods were consistent.

Statistical analysis. Student's 2-tailed t test was used for the comparison of pls+/Pls+ versus pls-/Pls- strains and of pls+/Pls+ versus pls+/Pls- strains. If variances between the groups were not equal, the t test probabilities were calculated using the Cochran and Cox approximation. P < .05 and P < .001 were considered to be statistically significant and highly statistically significant, respectively.

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Results

PFGE analysis, spa typing, and characterization of strains. As opposed to methicillin-susceptible S. aureus (MSSA) isolates, MRSA isolates with SCCmec types I, II, or III [34]—but not with type IV [24] and potentially with type V—tend to have a higher probability of being clonally related. To exclude clonal identity between the strains used in the present study, genomic PFGE analysis and spa typing were performed. Only isolates originating from different patients and showing either a difference of at least 1 band in PFGE pattern or a different spa type were included, as described elsewhere [18]. Some strains appeared to be closely related to each other, showing either a difference of only 1–3 bands or an identical PFGE pattern but different spa types. The majority, however, had larger differences, suggesting more-distant or no relatedness. In general, the prevalence of SCCmec in naturally occurring MRSA appears to be limited to relatively few genetic backgrounds of related S. aureus [35]. Although these criteria allow only the exclusion of identical isolates, not related ones, the presented results are based on a selection of strains with rather diverse genetic backgrounds.

The SCCmec type most frequently found was I (24%), followed by type III and nontypeable (21% each), type IV (17%), type II (11%), and type V (6%). SCCmec type V was not very common in our strain collection, refflecting the notion that it is a new type [3] and was not very widespread until recently, especially given that our most recent strains were collected in 2002 (table 2).

Prediction of cellular invasiveness of MRSA isolates on the basis of expression of Pls and SCCmec type. Fifteen (23%) of 66 MRSA strains were pls+/Pls+, as demonstrated by Southern blotting with a pls region A probe and by Western blotting of cell wall extracts with monoclonal antibodies against Pls, respectively. Three strains (4%) were pls+ but did not express Pls (pls+/Pls-). All other 48 MRSA strains (73%) were negative for pls and Pls (pls-/Pls-) (table 2). Surprisingly, we identified 1 pls+/Pls+ strain that carried the SCCmec type IV, rather than the usual SCCmec type I. Of note, this strain was Panton-Valentine leukocidin negative, as determined by PCR. Additionally, 1 strain that possessed the pls gene and expressed Pls was nontypeable with respect to the SCCmec (table 2).

To avoid selection bias and clearly determine the function of pls/Pls, we tested 66 strains for invasiveness in a blinded fashion. Subsequently, the presence of pls and Pls by Southern and Western blotting was determined for each strain. In agreement with our previous results [18], we found that, on average, pls-/Pls- strains were as invasive as Cowan 1 (median, 93%; range, 25%–162%), whereas pls+/Pls+ strains were significantly less invasive (median, 36%; range, 22%–70%) ( P < .0001 ) (figure 1). Interestingly, the 3 strains that possessed the pls gene but did not express Pls (figure 2) were as invasive as pls-/Pls- strains (median, 95%; range, 63%–103%) (for the difference between pls+/Pls+ and pls+/Pls- strains, P < .05). Thus, we conclude that Pls has to be expressed to affect the invasiveness of MRSA strains.

Figure 1.
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Figure 1.

Reduction of the cellular invasiveness of methicillin-resistant Staphylococcus aureus (MRSA) isolates by the expression of Pls. The invasiveness of 66 MRSA isolates is shown. Results are given as mean ± SE values from 3 independent experiments run in duplicate and are expressed as the relative invasiveness compared with strain Cowan 1. Solid lines indicate the median invasiveness of each group, and dotted lines indicate the mean invasiveness of each group. The pls gene and its expression were detected as described in Materials and Methods. Thirteen strains that have been presented previously [18] are indicated as white symbols. pls+/Pls+, strains positive for pls and Pls; pls-/Pls-, strains negative for pls and Pls; pls+/Pls-, strains that carry the pls gene but do not express Pls.

Figure 2.
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Figure 2.

Southern and Western blot analysis of discordant (pls+/Pls-) isolates. A, Presence of the pls gene, determined by Southern blotting; B, Expression of the Pls protein, determined by Western blotting of cell wall extracts with anti-Pls monoclonal antibodies (as described in Materials and Methods [18]). The arrows show the pls/Pls bands. The 2 bands in panel B correspond to full-length and cleaved protein, respectively.

MRSA strains belonging to SCCmec types other than type I appear to be at least as invasive as MSSA and penicillin-susceptible S. aureusx strains. Sixteen MRSA strains belonging to SCCmec type I were less invasive (median, 45%; range, 22%– 103%) than 50 MRSA strains belonging to other SCCmec types (median, 93%; range, 36%–162%) (figure 3). However, SCCmec type I was not an independent predictor of reduced invasiveness, because all SCCmec type I isolates were pls+. Inversely, there were 2 exceptions. Interestingly, we identified 1 strain that possessed the pls gene and expressed the Pls protein but that belonged to SCCmec type IV (strain 1106/98; mean ± SE invasiveness, 36% ± 2%).

Figure 3.
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Figure 3.

Reduced invasiveness of methicillin-resistant Staphylococcus aureus (MRSA) of staphylococcal chromosomal cassette mec (SCCmec) type I, compared with MRSA of other SCCmec types. Shown is the median invasiveness of the 66 methicillin-resistant S. aureus isolates tested in this study by SCCmec classification. Results of the invasion assay are given as mean ± SE values. SCCmec classification was performed by multiplex polymerase chain reaction. Strains were included in an SCCmec type if the results of at least 2 methods were consistent (for details on SCCmec classification, see Materials and Methods). Solid lines indicate the median invasiveness of each group, and dotted lines indicate the mean invasiveness of each group. NT, nontypeable.

Ability of the spa type (related groups) to predict the invasiveness of a given strain better than the spa type–deduced CC. The 66 MRSA strains fell into 31 different spa types, and the spa type strongly predicted invasiveness (figure 4A). It was remarkable that the SCCmec and spa types seemed to correlate very closely: spa type t001 contained only strains belonging to SCCmec type I, and spa type t002 contained only strains belonging to SCCmec type II. This correlation could be recognized in almost every spa type with just a few exceptions (spa type t008 contained strains belonging to SCCmec types I and IV; spa type t037 contained 1 strain that was nontypeable, with all other strains belonging to SCCmec type III; and spa type t051 contained 1 strain that could not be clearly assigned to either SCCmec type I or IV, with all other strains belonging to SCCmec type I).

By contrast, the spa type–deduced CC only moderately predicted the invasiveness of MRSA strains, compared with SCCmec or spa type (figure 4B). This is not surprising, given that the major CCs—5, 8, 22, 30, and 45—contained MRSA strains with different SCCmec types (table 2). This finding is in accordance with previous observations that led to the conclusion that MRSA clones have emerged on >1 occasion in strains of the same genetic background [36]. An exception was CC239, a descendant of CC8, which appeared to contain strains belonging to SCCmec type III only (along with 1 nontypeable isolate) [37].

Figure 4.
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Figure 4.

Reduced invasiveness of methicillin-resistant Staphylococcus aureus (MRSA) of spa types t001 and t051 relative to MRSA of other spa types, and better prediction of invasiveness by spa types than by spa type–deduced clonal complexes (CCs). Shown are results for the most common spa types (t001, t002, t004, t008, t009, t030, t037, and t051; A) and for the major spa type–deduced CCs (1, 5, 8, 22, 30, 45, and 239; B), in correlation with the median invasiveness of the corresponding MRSA isolates. Results of the invasion assay are given as mean ± SE values. The spa gene was detected by DNA sequencing. spa types that contained only 1 or 2 MRSA isolates are not shown. Solid lines indicate the median invasiveness of each group, and dotted lines indicate the mean invasiveness of each group. For CC22, the mean and median are identical (n = 2). On the basis of current data, a CC has not yet been assigned to strains W5260 (sequence type [ST] 398), 01093 (ST72), 16219a (ST1), 4/16–6N (ST1), and C20100101 (ST1). For strains SAP411 and 91 2619, no ST or CC could be determined.

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Discussion

We have previously shown that the presence of pls/Pls reduces cellular invasiveness and adhesion to host proteins in MRSA [6, 18]. Here, we corroborated these results in a greater collection of clinical and laboratory MRSA strains: 13 strains that expressed Pls were markedly less invasive than 48 strains that did not harbour pls and, therefore, could not express Pls (median invasiveness, 36% vs. 93%). Most importantly, we found that both SCCmec and spa types correlated with the invasive properties of MRSA strains. Furthermore, we showed that expression of the Pls protein (or the pls gene) appears to be required for reduced cellular invasiveness of MRSA isolates. This could suggest that steric hindrance mediates the effect of Pls on cellular invasiveness.

SCCmec type clearly predicted the invasiveness of MRSA strains in our study. Strains belonging to SCCmec type II or IV and strains that were nontypeable were very similar in invasiveness, whereas strains belonging to SCCmec type III were even more invasive. Strains belonging to SCCmec type I were the least invasive, coinciding with the fact that all SCCmec type I strains possessed the pls gene. On the other hand, some strains that were poorly invasive did not possess the pls gene. This might be explained by regulatory differences (e.g., in global regulators [38, 39]) that would lead to a low expression of FnBPs. However, these isolates appeared to be heterogeneous, because they were phenotypically diverse (e.g., with regard to pigmentation and hemolysis).

Our present and previously observed [18] data stand in apparent contrast to data obtained for desquamated nasal epithelial cells, for which Pls appeared to increase staphylococcal adherence on expression in S. aureus 8325-4. In this case, Pls would act as an adhesin, potentially recognizing an unknown epithelial cell receptor [40]. Thus, it is conceivable that Pls may possess different functional domains or that Pls is expressed at different levels during the process of host infection. However, this question could potentially be answered by an in vivo study. The epidemiological relevance of pls is still unknown. Reduced invasiveness (potentially associated with reduced virulence) might increase or decrease biological fitness. In addition, Pls seems to act as a staphylococcal virulence factor in septic arthritis [41].

To our knowledge, this is the first report of the presence of the pls gene in an SCCmec type other than type I: 1 MRSA strain that possessed the pls gene and expressed Pls belonged to SCCmec type IV (strain 1106/98; mean ± SE invasiveness, 36% ± 2%). The low invasiveness of this strain argues for the plausibility of this finding, given that the median invasiveness of the other pls+/Pls+ strains was ∼36% (range, 22%–70%). This finding might be the result of the close relation between SCCmec types I and IV in size and structure [42]. Further proof for this close relation was the fact that strains belonging to SCCmec type I or IV were found in the same spa type (t008; table 2). Additionally, 1 strain that possessed the pls gene and expressed Pls was nontypeable with respect to SCCmec (table 2). It seemed to belong to SCCmec type I by the Hiramatsu method, to SCCmec type IV by the Oliveira method, and to SCCmec type II by the Branger method. Because the Branger method cannot discriminate properly between SCCmec types II and IV, it is likely that the strains belongs to either type I or IV, which would fit with the known occurrence of pls.

That 14 strains were nontypeable in the SCCmec classification may underline the proposal of Ito et al. [3] that the SCCmec element is undergoing rearrangement processes in which new SCCmec elements are generated. The recent publication of a community-acquired MRSA genome might help to elucidate a better knowledge of the genetic events [43].

It appears to be worthwhile to investigate the correlation between the invasiveness of MRSA strains and their clinical behavior. Potentially, low invasiveness may correlate with less-severe infection; thus, the combination of detection of pls and spa typing might be a suitable method for predicting the outcome of severe MRSA infections. Additionally, the knowledge of the expected severity of an infection could guide therapeutic decisions concerning the duration and aggressiveness of antibiotic treatment.

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Acknowledgments

We thank Katrin Strangfeld, Martina Schulte, Ursula Keckevoet, and Sirpa Kuisma, for excellent experimental assistance. We are grateful to Wolfgang Witte (Robert Koch Institute, National Reference Center for Staphylococci, Wernigerode, Germany) and Keiichi Hiramatsu (Department of Bacteriology, Juntendo University, Tokyo, Japan) for kindly providing some of the S. aureus clinical and reference isolates used in this study.

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Footnotes

  • Potential conflicts of interest: none reported.

  • Financial support: This work was supported by the Deutsche Forschungsgemeinschaft (Collaborative Research Center 492, project B9), in part by grant Si2/039/06 from the Interdisciplinary Center for Clinical Research (IZKF Münster), by a grant from the Academy of Finland (project 1206356), and by a grant from Bundesministerium für Bildung und Forschung, Germany (Pathogenomic Plus Network PTJ-BIO/0313801B).

  • Received August 21, 2006.
  • Accepted December 29, 2006.
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  1. J Infect Dis. (2007) 195 (11): 1678-1685. doi: 10.1086/517517
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