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Department of Clinical Microbiology, Education and Research Centre, Beaumont Hospital, Royal College of Surgeons in Ireland, Dublin, Ireland
ABSTRACT
Synthesis of a polysaccharide adhesin by icaADBC-encoded enzymes is currently the best-understood mechanism of staphylococcal biofilm development. In four methicillin-resistant Staphylococcus aureus isolates, environmental activation of icaADBC did not always correlate with increased biofilm production. Moreover, glucose-mediated biofilm development in these isolates was icaADBC independent. Apparently, an environmentally regulated, ica-independent mechanism(s) of biofilm development exists in S. aureus clinical isolates.
TEXT
Production of a polysaccharide adhesion, termed polysaccharide intercellular adhesion or polymeric N-acetylglucosamine, by ica operon-encoded enzymes is currently the best-understood mechanism of staphylococcal biofilm development (18, 19). Although the majority of clinical Staphylococcus aureus isolates contain the ica operon (2, 7, 10, 11), the expression of the ica operon and biofilm production are tightly regulated under in vitro conditions (20). In the laboratory, CO2 levels, anaerobicity, glucose, and osmotic stress can all influence ica operon expression and/or biofilm development (1, 8, 14, 22). Cramton et al. (7) demonstrated that deletion of the ica operon in S. aureus ATCC 35556 resulted in impaired polysaccharide intercellular adhesion/polymeric N-acetylglucosamine production and a biofilm-negative phenotype. However, a recent study demonstrated that mutation of the ica locus in S. aureus strain UAMS-1 had little impact on biofilm formation in vitro or in vivo (5). In addition, Lim et al. (17) recently identified a new gene, rbf (regulator of biofilm formation), which was required for biofilm formation in S. aureus but did not regulate ica operon expression. It is not clear whether these findings apply to clinical as well as reference isolates.
To investigate the contribution of the ica locus to biofilm development in S. aureus clinical isolates, the environmental regulation of biofilm development by NaCl and glucose was investigated in four ica-positive, methicillin-resistant strains (BH1S, BH1P, BH1Y, and BH1CC) recovered from intensive care unit patients (Table 1). Biofilm assays using Nunclon tissue culture-treated (Surface) 96-well polystyrene plates (Nunc, Denmark) were performed as described previously (5, 15). Biofilm formation was increased four- to eightfold in all four of these isolates when grown in brain heart infusion (BHI) medium supplemented with glucose compared to BHI alone (Fig. 1A). In contrast, growth in BHI supplemented with NaCl failed to induce biofilm in these isolates (Fig. 1A).
To examine the contribution of ica operon expression to the biofilm phenotype of these isolates, we used reverse transcriptase PCR (RT-PCR) as described previously (4-6, 12). The methicillin-resistant S. aureus (MRSA) isolate BH1CC was grown in BHI medium and in BHI medium supplemented with NaCl, glucose, or both NaCl and glucose (Fig. 1B). The addition of NaCl to the growth medium activated ica operon expression in BH1CC even though this ica activation was not associated with any increase in biofilm-forming capacity (Fig. 1A and B). In addition, the glucose-mediated induction of biofilm formation was not associated with increased ica operon transcription in BH1CC. An examination of ica operon expression in BH1S, BH1P, and BH1Y also revealed little correlation between ica expression levels and the biofilm phenotype (Fig. 1C). Glucose-mediated induction of biofilm formation correlated with substantially increased ica operon expression only in isolate BH1S (Fig. 1C). Similar to results with BH1CC, NaCl activated ica operon expression in BH1S but was not associated with a biofilm-positive phenotype (Fig. 1A and C). These findings suggest that in contrast to recent findings with Staphylococcus epidermidis (4-6, 9, 16, 21), there appears to be little correlation between ica operon expression and biofilm formation in S. aureus clinical isolates and that the ica operon may not be required for biofilm development in the isolates examined.
In order to more carefully assess the contribution of the ica operon to biofilm development in S. aureus, an ica operon deletion mutant of MRSA isolate BH1CC was constructed. As described above, biofilm development by this strain, which was isolated from a patient with central venous catheter infection, is induced approximately fivefold when grown in BHI-glucose, even though ica operon expression was activated only in BHI-NaCl and not in BHI-glucose. An allele replacement strategy was employed to replace the ica operon on the chromosome of BH1CC with the tetracycline resistance cassette (ica::tet) as described previously (7). PCR with the primers SAdel1 (5'-TGC-AAA-TGC-CCT-TGA-TGT-AA-3') and SALR2 (5'-GGC-GGA-AAG-TCA-GGT-TAC-AA-3'), which amplify the entire icaR-icaADBC locus, was used to confirm the presence of the ica::tet allele on the chromosome of candidate mutants (data not shown). Interestingly, an analysis of BH1CC and an isogenic ica::tet mutant grown in BHI, BHI-NaCl, and BHI-glucose revealed no substantial differences in biofilm-forming capacity (Fig. 2A). These data are consistent with our earlier findings that ica operon expression and regulation do not influence biofilm development in BH1CC and further suggest that the mechanism of glucose-induced biofilm formation in this strain is entirely ica independent. Interestingly, Beenken et al. (3) recently reported that in the S. aureus clinical isolate UAMS-1, the deletion of the ica locus also had no effect on biofilm development in tryptic soy broth media supplemented with both NaCl and glucose. However, it may be significant that the ica operon expression by the MRSA isolate BH1CC examined in the present study was induced by glucose alone but inhibited by both NaCl and glucose together (Fig. 1B), perhaps suggesting that one or more ica-independent mechanism of biofilm development may exist.
Our evidence that the ica operon was not required for biofilm development in BH1CC was inconsistent with the findings of Cramton et al. (7), which revealed that the ica locus was required for biofilm development in S. aureus ATCC 35556. To further investigate the contribution of the ica locus to biofilm development in different S. aureus strains, we constructed ica::tet deletion mutants in the NCTC 8325-derived laboratory strains 8325-4, SH1000, and RN4220. In contrast to results with BH1CC, we have previously observed that biofilm formation in 8325-4, SH1000, and RN4220 was strongly induced by NaCl (15). By using allele replacement, the ica::tet deletion mutation was constructed in the laboratory strain RN4220, a chemically mutagenized derivative of 8325-4 that can readily accept foreign DNA. Phage 80 was then used to transduce the ica::tet allele from RN4220 to 8325-4 and SH1000, an rsbU-repaired (B-positive) derivative of 8325-4 (13). In contrast to results with S. aureus BH1CC, deletion of the ica locus in RN4220 (Fig. 2B) resulted in a biofilm-negative phenotype under all environmental conditions. A similar phenotype was associated with ica deletion mutations in 8325-4 and SH1000 (data not shown).
The ica::tet allele was also transduced into the MRSA isolates BH1S, BH1P, and BH1Y, which, like BH1CC, display glucose-induced biofilm formation. Interestingly, similar to our findings with S. aureus BH1CC, deletion of the ica locus in these isolates did not result in a biofilm-negative phenotype and all three of these isolates displayed a glucose-induced, ica-independent biofilm phenotype (data not shown).
It is also relevant to note that all three 8325-4 derivative strains used in this study produced black, dry, crusty colonies when grown on Congo red agar (CRA), which is indicative of a biofilm-positive phenotype in S. epidermidis (6, 12). In contrast, the four clinical isolates produced red, smooth colonies on CRA. Deletion of the ica operon in the 8325-4 derivatives resulted in a red, smooth phenotype on CRA and, predictably, had no effect on the CRA phenotype in the clinical isolates. These findings may suggest that the ica operon is required for NaCl-induced biofilm in S. aureus strains that produce black, crusty colonies on CRA but not in isolates that form red, smooth colonies on CRA. This finding may be significant given that in a survey of 31 ica-positive S. aureus clinical isolates (F. Fitzpatrick, H. Humphreys, and J. P. O'Gara, unpublished findings), all produced red smooth colonies on CRA, suggesting that the ica operon may not play an important role in biofilm development among clinical isolates with this phenotype.
These studies suggest that the regulatory pathways controlling the biofilm phenotype in reference strains may be different than those used by clinical isolates. Such differences may, in turn, be driven by genetic exchange and exposure to environmental stimuli in the complex in vivo infection milieu from which the clinical isolates are recovered. Further studies are under way to elucidate an alternative, ica-independent mechanism(s) of biofilm development in clinical isolates of methicillin-resistant S. aureus.
ACKNOWLEDGMENTS
This study was funded by a Clinical Research Training Fellowship from the Health Research Board (Ireland) to F. Fitzpatrick and grants from the Research Committee of the Royal College of Surgeons in Ireland and the Irish Research Council for Science, Engineering, and Technology to J. P. O'Gara. We are grateful to Pfizer (Ireland) for generously supporting the establishment of the RCSI Microbiology Laboratory at the RCSI Education and Research Centre.
We thank P. D. Fey, University of Nebraska Medical Center, for generously providing phage 80; S. Foster, University of Sheffield, for the gift of strain SH1000; and E. G. Smith, Beaumont Hospital, Dublin, for the provision of clinical isolates. Plasmid pSC23 was a kind gift from S. Cramton and F. Gotz, Universitt Tübingen, Tübingen, Germany. We thank Kevin Conlon, Ciara Kennedy, Sinead O'Donnell, and Tracey Dillane for experimental advice and assistance throughout the study.
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