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The Predominant Variant of the Brazilian Epidemic Clonal Complex of Methicillin-Resistant Staphylococcus aureus Has an Enhanced Ability to Produce Biofilm and to Adhere to and Invade Airway Epithelial Cells

  1. Maribel M. Amaral1,a,
  2. Leonardo R. Coelho1,a,
  3. Rosania P. Flores1,
  4. Raquel R. Souza1,
  5. Maria C. Silva-Carvalho1,
  6. Lenise A. Teixeira2,
  7. Bernadete T. Ferreira-Carvalho1 and
  8. Agnes M. S. Figueiredo1
  1. 1Institute of Microbiology Professor Paulo de Góes, Federal University of Rio de Janeiro, and
  2. 2Faculty of Pharmacy, Fluminense Federal University, Rio de Janeiro, Brazil
  1. Reprints or correspondence: Dr. Agnes M. S. Figueiredo, Instituto de Microbiologia Prof. Paulo de Góes–UFRJ, Laboratório de Biologia Molecular de Bactérias, CCS, Bloco I, 2° andar, Cidade Universitária, Rio de Janeiro, RJ, 21941-590, Brazil (agnes{at}micro.ufrj.br)
 
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Abstract

Methicillin-resistant Staphylococcus aureus (MRSA) has emerged as a therapeutic problem. In the present study, the molecular characterization by pulsed-field gel electrophoresis of MRSA isolates collected from a university hospital revealed that the predominant variant of the Brazilian epidemic clonal complex (BECC) was responsible for the increase in the incidence of MRSA strains, which reached 28% in 1998. It was verified that this predominant variant of the BECC displayed an enhanced ability to produce biofilm on inert polystyrene surfaces and to adhere to and invade epithelial airway cells. These results indicate that MRSA strains belonging to the BECC have evolved advantageous properties that might play a role in their predominance as international nosocomial pathogens

Methicillin-resistant Staphylococcus aureus (MRSA) is the leading cause of hospital infections worldwide [1 –3]. At the end of the 1980s and the beginning of the 1990s, the medical community was overwhelmed by a dramatic increase in infections caused by multidrug-resistant S. aureus in nosocomial settings. This increase was associated with an international dissemination of well-defined clonal lineages (epidemic MRSA) [1, 3]. Five major lineages (the so-called Iberian, Brazilian, Hungarian, New York/Japan, and pediatric) of pandemic MRSA clones have been defined [4, 5]. In Brazil, the Brazilian epidemic clonal complex (BECC) of MRSA has disseminated from the north to the south [6 –8]. BECC isolates have also been detected in other countries, including Argentina, Uruguay, Paraguay, Chile, Portugal, Italy, and the Czech Republic [9 –13]. The mechanisms involved in the selection of pandemic MRSA clones have not been clarified thus far. Nevertheless, the ability of S. aureus to establish infections seems to be dependent on the expression of virulence factors that enable the bacterial colonization of mucosal or skin surfaces

In the present study, we demonstrate that the predominant isolate of the BECC displays an enhanced in vitro ability to produce biofilm and to adhere to and invade the epithelial bronchial cell line 16HBE14o−. This finding strongly suggests that this isolate displays selective properties that might have contributed to the spread and predominance of the BECC of MRSA as a global nosocomial pathogen

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Materials and Methods

Bacterial isolates A total of 134 MRSA isolates were obtained from different clinical specimens from infected patients and wards in the João Barros Barreto University Hospital (Belém, Brazil) during 1995–1998. To study bacterial adherence to inert surfaces, a total of 200 S. aureus isolates were analyzed, including 150 MRSA and 50 methicillin-susceptible S. aureus (MSSA) isolates obtained during 1996–2001. Of the 150 MRSA isolates, 38 were from João Barros Barreto University Hospital, and the others were from hospitals located in different regions of Brazil or Argentina (100) and in the United States (12). The MSSA isolates studied were all associated with infections in patients in Brazilian hospitals. Seventeen MSSA isolates, 5 MRSA clones rarely detected in hospitals (sporadic MRSA clones), and 10 MRSA isolates belonging to the predominant subclone of the BECC were chosen at random for use in the adhesion and invasion experiments

Susceptibility test The disk diffusion test was performed as described elsewhere [14]. S. aureus strain ATTC 25923 was used as a control

Molecular characterization Pulsed-field gel electrophoresis (PFGE) of SmaI-digested DNA was performed on a CHEF-DR III (Bio-Rad) as described elsewhere [15]. The criteria used for interpreting the PFGE results have been described elsewhere [5]. The analyses of the ClaI/mecA and ClaI/Tn554 polymorphisms were performed as described elsewhere [16]

Biofilm Biofilm assays were performed using trypticase soy broth (TSB) (Difco Laboratories) supplemented with 1% (wt/vol) glucose. Overnight cultures (diluted 1:100) were dispensed into sterile 96-well flat-bottom tissue culture plates (Nunclon; Nunc A/S). The other procedures were performed as described elsewhere [17], except that the optical density of the culture (ODg) and of the stained biofilm (ODb) were measured at 570 nm [18]. S. epidermidis strain 70D and S. pyogenes strain 75194 were used as positive and negative controls, respectively. Biofilm production was classified on the basis of the results obtained for the negative control. The biofilm unit (BU) was calculated using the following formula: ODb/ODg. The calculated BU for S. pyogenes was 0.115. We defined S. aureus isolates with BU ⩽0.230 as nonproducers, those with BU >0.230 and ⩽0.460 as weak producers, those with BU >0.460 and ⩽0.920 as moderate producers, and those with BU >0.920 as strong producers. Because the production of biofilm is subject to phase variation, the test was performed in quadruplicate. When an isolate was negative for biofilm production, the test was repeated for up to 3 additional independent experiments. The highest BU value obtained for each isolate tested was used for the statistical calculations

Adherence The bronchial epithelial cell line 16HBE14o− [19] was provided by Dr. Maria Cristina Plotkowski (Universidade do Estado do Rio de Janeiro, Brazil). Cell monolayers were prepared on a glass coverslip (Clay Adams; Becton Dickinson) and placed in a 24-well flat-bottom plate (Gibco BRL) with M-199 medium containing 30% (wt/vol) HEPES (Gibco BRL), 2.5 mg/L fungizone, 50 mg/L gentamicin, and 10% (vol/vol) fetal calf serum. Cells were incubated for 2–3 days at 37°C in 5% CO2. Bacteria were grown up to OD610 0.4 (log phase) in TSB containing 10 μCi of [methyl-3H]thymidine (Amersham Pharmacia Biotech). Aliquots of 200 μL of the culture were laid onto the monolayer. After 1 h of incubation at 37°C, the coverslip was washed twice and was transferred to the counting scintillant (Amersham Pharmacia Biotech), and the radioactivity was measured. Three determinations were made for each isolate tested

Invasion assay Bacterial invasion was investigated using the 16HBE14o−cell line. The monolayer was prepared in 24-well flat-bottom plates. A volume of 500 μL of bacterial inoculum at log phase (OD610, 0.4) or stationary phase (OD610, 1.8) was added to the monolayers. After 3.5 h of incubation, monolayers were washed twice in M-199 medium and were incubated with 10 μg/mL lysostaphin (specific activity, 500 U/mg; Sigma) for 20 min at 37°C to lyse noninvasive bacteria. Monolayers were washed again, incubated for 5 min with 0.25% (wt/vol) trypsin (specific activity, 11,000 U/mg; Sigma), and lysed during a 5-min incubation with 0.025% (vol/vol) Triton X-100 (Sigma). The number of colony-forming units per milliliter was determined in trypticase soy agar, and the percentage of bacterial invasion was calculated as described elsewhere [20]

The isolates studied were grouped on the basis of the percentage of bacteria that invaded cells as being highly invasive (⩾10% of the bacteria invaded cells), invasive (1% to <10% of the bacteria invaded cells), poorly invasive (>0.001% to <1% of the bacteria invaded cells), or noninvasive (⩽0.001% of the bacteria invaded cells). Noninvasive isolates were defined on the basis of the amount of lysis promoted by lysostaphin

Electron microscopy After 1 h of contact with bacteria, epithelial cells were washed in 0.01 mol/L PBS (pH 7.2) and were fixed for 1 h in a 2.5% glutaraldehyde (vol/vol) and 0.1 mol/L cacodilate buffer (pH 7.2). Epithelial cells were then washed twice in PBS. The cells were postfixed in 1% (wt/vol) OsO4 dehydrated in 30%–100% (vol/vol) acetone gradients, and embedded in Epon 812 (EMS). The samples were cut, stained with uranyl acetate for 1 h, stained with lead citrate for 5 min, and examined with a Zeiss TEM900 electron transmission microscope

Effects of soluble fibronectin (Fn) and RGD peptide on bacterial invasion The bacterial Fn-binding motifs were blocked by adding 0.1 μg/mL human plasma Fn (Gibco) to the cultures of the BECC isolates GV69 and GV91. When the OD610 reached 0.4, bacterial cells were washed twice with M-199 medium and added to the epithelial cell monolayer. Bacterial invasion was measured as described above

To verify the involvement of RGD-dependent integrins on invasion by BECC isolates, the epithelial cells were treated with a 1 μg/mL synthetic RGD peptide (Gly-Arg-Gly-Asp-Thr-Pro; Gibco). The monolayer was washed twice with M-199 medium, and bacterial invasion was measured as described above. The concentrations of Fn and RGD peptide used were chosen on the basis of the results of previous studies [21, 22]

Effect of environmental conditions To determine whether bacterial entry into airway cells could be modulated by environmental conditions, 0.9% (wt/vol) dextrose, 15 μg/mL MgSO4, or 7.5% (wt/vol) NaCl were added to the bacterial culture of the BECC isolates GV69 and GV91. To study the effect of pH, bacteria were grown in liquid medium containing 0.02 mol/L sodium acetate buffer (pH 5.0). The effect of temperature variation on bacterial invasion was also evaluated after bacteria were grown at 30°C and 40°C. Bacterial invasion was measured as described above

Adherence to solid-phase Fn To study the bacterial adherence to Fn, sterile glass coverslips were covered with 50 μg/mL human plasma Fn. The coverslips were incubated for 1 h at 37°C, left for 18 h at 4°C, washed 4 times with PBS, and covered with a solution of 0.1% (vol/vol) bovine serum albumin [23]. After 1 h of incubation at 37°C, the coverslips were washed twice with PBS. For inoculum preparation, a culture of the BECC isolate GV91 was incubated with 10 μCi of [methyl-3H]thymidine and incubated at 37°C until it reached an OD610 of 0.4 (log phase). In another experiment, [methyl-3H]thymidine thymidine was added when the bacterial culture achieved an OD610 of 1.0 (stationary phase), and the incubation lasted for 3 h. The cultures were laid on the Fn-coated coverslips. After 1 h of incubation at 37°C, the coverslips were washed twice with PBS and transferred to the counting scintillant, and the radioactivity was measured. The agr − strain RN6911 (that has an increased expression of Fn-binding proteins) [24] and the isogenic agr + strain RN6390B (gift from Dr. Richard Novick, New York University, New York) were also used in these experiments

Statistical calculations The test of hypotheses on 2 population means (unpaired data) was used to compare the mean BU. In the invasion and adhesion experiments, analysis of variance was applied for comparison of the different groups. Student’s t test was used for the invasion experiments on bacterial or cellular inhibitors and for the analyses of Fn-bound activity. P<.05 was considered to be statistically significant [25]

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Results

The 134 MRSA hospital isolates were all susceptible to vancomycin. However, high-level resistance was verified for the other drugs tested (table 1). Of the 134 MRSA hospital isolates analyzed, 76 (57%) had PFGE patterns (A1) that were indistinguishable from that displayed by strain HU25 [6], whose pattern predominates among the isolates belonging to the BECC (figure 1A–B ). Forty (30%) isolates differed from pattern A1 by only 1–3 PFGE bands (patterns A2 to A21). Additionally, 15 (11%) isolates differed by 4–6 bands. Although they are possibly related to the BECC, these MRSA isolates were grouped into different types. Only 3 (2%) of the MRSA hospital isolates differed by ⩾7 bands, and they were classified as unrelated clones G and H (table 1)

Figure 1
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Figure 1

Genetic diversity as determined by pulsed-field gel electrophoresis (PFGE). A Distribution of the clonality of the methicillin-resistant Staphylococcus aureus (MRSA) population isolated from 1995 to 1998 in the João Barros Barreto University Hospital, Belém, Brazil. B PFGE patterns of the isolates analyzed: λ-ladder molecular size marker (lanes 1 and 14), MRSA clones rarely detected in hospitals (sporadic clones; lanes 2, 3, and 4), isolates belonging to the Brazilian epidemic clonal complex (BECC; lanes 5 and 6), and isolates displaying the A1 PFGE pattern that is predominant among BECC isolates (BECC A1; lanes 7–13)

All isolates classified as belonging to the BECC displayed the ClaI/mecA and ClaI/Tn554 types III and B polymorphisms, respectively (data not shown), as was reported elsewhere for the BECC [6]. It is remarkable that the percentage of isolates displaying the A1 PFGE pattern (BECC predominant variant) in this hospital increased from 38% (1995) to 53% (1996) to 65% (1997) to 79% (1998), whereas the percentage of non-A1 BECC subclones and non-BECC isolates decreased (figure 1A ). In 1994, when MRSA outbreaks began, the incidence of methicillin resistance among the S. aureus isolates in João Barros Barreto University Hospital was 7.2%. In 1995, when the present study was initiated, the incidence had increased to 15%. In subsequent years (1996–1998), the incidence varied from 26% to 28%. Data on the occurrence of MRSA in previous years were not recorded

The mean BU for BECC A1 isolates was 2.09, which was significantly higher than that for MSSA isolates (BU, 0.50; P<.001), sporadic MRSA clones (BU, 0.72; P<.001), and other BECC non-A1 subclones (BU, 0.59; P<.001). Therefore, we have great confidence in affirming that the BU for the population of BECC A1 is ∼3–4 times higher than that observed for the other 3 groups analyzed (figure 2A ). Only 2% of the BECC A1 isolates were classified as nonadherent, whereas adherences of 24%, 20%, and 14% were verified for MSSA isolates, sporadic MRSA clones, and BECC non-A1 subclones, respectively. The major contributor to the increase in the BU for the BECC A1 population was that most (74%) isolates were strong biofilm producers (figure 2B )

Figure 2
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Figure 2

Biofilm production by Staphylococcus aureus isolates by biofilm units (A) and percentage (B). The criteria used to characterize the isolates are described in Materials and Methods. BECC, isolates belonging to the Brazilian epidemic clonal complex; BECC A1, isolates displaying the A1 PFGE pattern that is predominant among BECC isolates; MSSA, methicillin-susceptible S. aureus isolates; sporadic clones, methicillin-resistant S. aureus clones rarely detected in hospitals

The percentage of adhesion of BECC A1 isolates in airway cells (6.4%) was 7–10 times higher (P<.001) than that of sporadic MRSA clones (0.87%) or MSSA isolates (0.67%) (figure 3A ). Also, there was an extraordinary difference (P<.001) in the ability of BECC A1 isolates to invade these airway cells, compared with that of the other populations (figure 3B ). Only 8 of 32 S. aureus isolates analyzed were defined as highly invasive (percentage of invasion, ⩾10%), and all of them displayed the A1 PFGE pattern. In addition, the other 2 BECC A1 isolates tested were defined as invasive (percentage of invasion, 6.1% and 4.1%). In contrast, the majority of sporadic MRSA clones and MSSA isolates (20 of 22) were classified as poorly invasive (9) or noninvasive (11). Only 2 sporadic MRSA clones were invasive (percentage of invasion, 5.7% and 2.0%). The high level of invasion by BECC A1 isolates was verified only during the log phase (figure 3C )

Figure 3
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Figure 3

Adherence and invasion of human bronchial cell line 16HBE14o− by methicillin-resistant Staphylococcus aureus (MRSA) isolates. A Percentage of adherence. Percentage of adherence was determined after 1 h of contact between the [methyl-3H]thymidine–labeled bacteria and the epithelial cell monolayer. B Percentage of invasion. Percentage of invasion was determined after 3 h of contact between the bacteria and the monolayer. The internalized bacteria were counted (in colony-forming units per milliliter) after lysis of the epithelial cells. C Percentage of invasion and optical density in different growth phases of isolate GV91. BECC, isolates belonging to the Brazilian epidemic clonal complex; BECC A1, isolates displaying the A1 pulsed-field gel electrophoresis pattern that is predominant among BECC isolates; MSSA, methicillin-susceptible S. aureus; sporadic clones, MRSA clones rarely detected in hospitals

Electron microscopy studies, performed with the BECC A1 isolate GV91, showed that the bacterial adherence to airway cells was unique, in the sense that the adhesion occurred in a delicate contact between the bacterial hairlike structures with the apical surface of 2 or 3 microvilli (figure 4B ). Indeed, the enhanced invasion involved a massive collapse of microvilli that was characterized by a shortening and loss of these structures (figure 4A and 4C ). After 1 h of contact with epithelial cells, a large number of bacteria were internalized, and most of them remained within membrane-bound vesicles, where many dividing cells (with septal rings) were visualized (figure 4D )

Figure 4
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Figure 4

Electron micrographs of the in vitro invasion of Staphylococcus aureus isolate GV91 (displaying the A1 pulsed-field gel electrophoresis pattern that is predominant among the Brazilian epidemic clonal complex) in the human bronchial cell line 16HBE14o− after 1 h of contact with the monolayer. A Intact microvilli of a noninfected airway cell. B Adhesion of bacterial hairlike structures to the apical surface of the microvilli. C Collapse of microvilli characterized by a shortening and loss of these structures. D Invasion by isolate GV91. Note that most dividing cells remain inside membrane vacuoles. Scale bar, 0.5 μm

Treatment of the bacteria with soluble Fn decreased the percentage of invasion from 31% to 1.2% for isolate GV69 and from 43.7% to 1.8% for isolate GV91 (P<.001) (figure 5A ). The entry of these 2 BECC A1 isolates was also impaired by treatment of monolayers with RGD peptide (P<.001). In addition, when protease was used, the release of proteinaceous structures from the bacterial surface abolished the bacterial entry (P<.001) (figure 5A )

Figure 5
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Figure 5

Invasion by Staphylococcus aureus isolates GV69 and GV91 (displaying the A1 pulsed-field gel electrophoresis pattern that is predominant among the Brazilian epidemic clonal complex) in epithelial airway cells under different conditions. A Bacterial cultures were treated with either nothing (control), 0.5 mg/mL protease, or 0.1 mg/mL fibronectin (Fn) before contact with epithelial cell monolayers, or epithelial cell monolayers were formed in supplemented M-199 medium (Materials and Methods) containing 1 mg/mL RGD peptide (Gly-Arg-Gly-Asp-Thr-Pro) before culture. B Effect of environmental conditions on invasion. For this experiment, bacteria were grown in the conditions indicated before contact with the epithelial cell monolayers

The effect of the environmental conditions on bacterial growth also reduced the high level of invasion by BECC A1 isolates (P<.001) (figure 5B ). Invasion by BECC A1 isolates decreased to 0.62% (isolate GV69) and 0.13% (isolate GV91) when the bacteria were grown at pH 5.0. Similarly, the addition of dextrose to the culture medium resulted in a reduction of bacterial invasion to 3.4% and 16.3% (isolates GV69 and GV91, respectively; P<.001). Ionic changes in the composition of the medium, such as increased magnesium or sodium concentrations, also blocked invasion (P<.001). When these 2 isolates were grown at 30°C or 40°C, a similar strong inhibition of bacterial invasion was verified (figure 5B )

Finally, the percentage of adhesion of isolate GV91 to solid-phase Fn (24.2%) was increased, compared with that of strain RN6390B (15.3%), for the experiments performed at log phase (OD610, 0.4; P=.048). This increase in adhesion of isolate GV91 was even greater (14% for GV91 and 2% for RN6390B) when the tests were performed at stationary phase (OD610, up to 1.8; P<.001) (figure 6). As expected, the adherence of the agr − strain RN6911 to solid-phase Fn was constant in the log (13.5%) and stationary (11.4%) phases

Figure 6
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Figure 6

Fibronectin (Fn)–binding activity of Staphylococcus aureus strain RN6390B (agr+), strain RN6911 (an isogenic agr − mutant), and isolate GV91 (a predominant isolate of the Brazilian epidemic clonal complex). Binding activity was determined at log phase (OD610, 0.4) and stationary phase (OD610, 1.8) using solid-phase Fn

Table 1
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Table 1

Resistance profiles and pulsed-field gel electrophoresis (PFGE) patterns of the methicillin-resistant Staphylococcus aureus isolates from João Barros Barreto University Hospital

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Discussion

We molecularly characterized MRSA isolates from the João Barros Barreto University Hospital. In 1995, the incidence of BECC isolates displaying the dominant A1 PFGE pattern was low, but the frequency of BECC A1 isolates increased until these isolates supplanted much of the non-A1 MRSA population. Once all non-A1 MRSA isolates collected in this hospital had similar antimicrobial susceptibility profiles, the selective pressure caused by the intensive use of drugs could not be incriminated as the only factor responsible for the prevalence of BECC A1 isolates. In addition, isolates displaying the A1 PFGE pattern also predominated among BECC isolates reported in studies published elsewhere and corresponded to 70%–80% of the MRSA population [6, 8 –10, 13]

We designed in vitro experiments to study the ability of the predominant isolate of the BECC to produce biofilm and to adhere to and invade human airway cells. Our results demonstrated that BECC A1 isolates had a significantly increased ability to adhere to the inert surface of polystyrene (3–4 times higher), compared with that in the populations formed by the BECC non-A1 subclones, sporadic MRSA clones, or MSSA isolates. The mucoid matrix of biofilm may increase bacterial persistence in the host and the pathogenic potential of bacteria [26]. Indeed, polymer-associated diseases are frequent sources of severe nosocomial infections, including catheter-associated bacteremia and infections related to the implantation of cardiac devices or orthopedic prostheses [26, 27]. Recently, Beenken et al. [27] studied the global gene expression of biofilms by S. aureus and showed that the results obtained by in vitro models to assess biofilm production, either under static or flow conditions, were confirmed by in vivo experiments using a murine model of catheter-associated biofilm formation [26]

It was determined that the adherence to the airway epithelial cells was 5-fold greater for BECC A1 isolates, compared with that of other S. aureus clones. The enhanced ability of the BECC A1 isolates to invade the epithelial cell monolayer was confirmed by electron microscopy. It is noteworthy that S. aureus was recently demonstrated to escape from the vacuoles of MAC-T cells after 1 h of infection. In contrast, the agr − mutant remained inside of vacuoles even after 3 h of infection [28]

Results of studies of S. aureus entry into epithelial cells suggested that S. aureus cannot be regarded as a classic intracellular pathogen because the S. aureus isolates tested had a low percentage of invasion [21, 29]. Nevertheless, it was suggested that the ability of S. aureus to enter mammalian cells, even at relatively low amounts, might explain its capacity to colonize and persist in host tissues [29, 30]. It is common sense that bacterial persistence in the host and the development of disseminated infections might require that bacteria have the ability to invade epithelial cells and, therefore, evade host defense mechanisms [29, 31]. In fact, in the present study, an enhanced cellular invasion was demonstrated for BECC A1 isolates. Although it is difficult to translate our in vitro studies to what exactly occurs during an in vivo pathogenic process, it follows logically that the enhanced biofilm production, adhesion, and invasion of epithelial cells displayed by BECC A1 isolates have implications for their persistence, prevalence in nosocomial infections, and widespread appearance over large geographic areas

Studies investigating the early events in invasion of epithelial cells by S. aureus showed that the entry of the bacterium involved Fn-binding proteins [30, 32] and that the interaction of S. aureus with Fn led to induction of signal transduction, tyrosine kinase activity, and cytoskeletal rearrangement [32]. Moreover, it was shown that both Fn and β1 integrins were also required for maximum uptake [32]. In the present study, we verified that the high level of invasion displayed by BECC A1 isolates was totally dependent on the presence of accessible Fn-binding domains on the bacterial surface. Furthermore, the treatment of bronchial cells with RGD peptide significantly impaired the enhanced invasion by BECC A1 isolates, which suggests that integrins served as host cell receptors for the successful bacterial entry into these cells. It was also verified that BECC A1 isolates bound more efficiently to solid-phase Fn than an agr + strain (RN6390B). In addition, we observed that, although the Fn-binding activity of BECC A1 isolates decreased, it was still higher in the stationary phase than that of an agr + strain; this is unusual among S. aureus clinical isolates, because the expression of Fn-binding proteins is down-regulated by agr in this growth phase [24]. All the evidence suggests that the mechanism associated with the enhanced invasion verified for BECC A1 isolates was similar to that of the poorly invasive process shown by most S. aureus isolates in the sense that it involves both Fn-binding activity and RGD-dependent integrins

It was reported elsewhere that an agr − mutant (RN6911) displayed enhanced adhesion and invasive properties [33]. Indeed, agr attenuation was verified in an epidemic MRSA clone (non-BECC C-MRSA-3) from Canada that also displayed increased Fn-binding activity [34]. It is remarkable that a relationship between agr dysfunction and persistent bacteremia due to MRSA infections was recently demonstrated in a study published elsewhere [35]. We are now conducting experiments to assess the involvement of global regulators, including agr RNAIII and SarA protein, in the enhanced colonization ability of BECC A1 isolates. The preliminary results of Northern blotting experiments (with a RNAIII-specific probe) of isolate GV91 revealed an agr attenuation for this BECC A1 subclone. Time course experiments showed RNAIII expression was >2-fold diminished for isolate GV91, compared with that of the agr + strain RN6390B, and both had agr polymorphism type I. This result was paralleled by an increase in Fn-binding activity (A.M.S.F, B.T.F.-C., and L.A.T., unpublished data)

Another interesting finding in the present study was that, when BECC A1 isolates were grown under different environmental conditions, the effect of these variables on high-level invasion was dramatic. That these variables had an effect indicates that the high level of invasion by these predominant MRSA isolates is tightly coordinated via a bacterial sensory system, as was verified for other important virulence traits [24, 36]

In conclusion, our results clearly show that the predominant variant of the BECC displays differential in vitro properties (adhesion and invasion) that are commonly associated with the bacterial ability to promote infections. This finding supports the hypothesis that the dominance of specific multidrug-resistant S. aureus clones, in a particular epidemiological scenario, seems to result from both the antimicrobial selective pressure and the dynamic association of different factors involved in the bacteria-host coevolution that culminate in the selection of well-adapted pathogens [37]

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Acknowledgment

We thank Wanderley de Souza (Biophysics Institute, Federal University Rio de Janeiro, Brazil) for providing the conditions for the experiments with the electron transmission microscope

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Footnotes

  • ↵Presented in part: 39th Interscience Conference of Antimicrobial Agents and Chemotherapy (ICAAC), San Francisco, 26 September 1999 (abstract B1539); 44th ICAAC, Washington, DC, 30 October 2004 (abstract K1473)

    Potential conflicts of interest: none reported

    Financial support: Conselho Nacional de Desenvolvimento Científico e Tecnológico (grants 473028/03 and 550652/2002-70); Fundação de Apoio à Pesquisa do Estado do Rio de Janeiro (grant APQ1 to A.M.S.F.); Programa de Apoio a Núcleos de Excelência (grant to A.M.S.F.); Primeiros Projetos (grants to L.A.T. and B.T.F.-C)

  • ↵M.M.A. and L.R.C. contributed equally to this work

  • Received November 30, 2004.
  • Accepted April 1, 2005.
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  1. J Infect Dis. (2005) 192 (5): 801-810. doi: 10.1086/432515
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