We evaluated the morphology of mixed species biofilms of S. epidermidis and C. albicans, in vitro. We observed enhancement of biofilms in a mixed species environment. In a mouse subcutaneous catheter model of biofilm infection, we noted increased catheter infection and systemic dissemination of S. epidermidis in a mixed species environment. To further explore the reasons for increased pathogenicity of S. epidermidis in mixed species biofilm infections with C. albicans, we evaluated the transcriptome of S. epidermidis in a mixed species environment and found that the repressors of autolysis, lrgA and lrgB were highly down regulated. Down regulation of repressors of autolysis, is associated with increased eDNA in the biofilm matrix, possibly by increased bacterial autolysis. We confirmed the significance of increased biofilm eDNA by evaluating its degradation by DNAse.
Mixed species biofilms of S. epidermidis and C. albicans were significantly thicker and voluminous compared to single species biofilms of either organism in vitro. Increased thickness of mixed species biofilms can be due to increase in the number of organisms or increase in the extracellular matrix or possibly both. In mixed species biofilm infections in vivo, at 8 days of infection, we observed increase in catheter CFU/ml of S. epidermidis associated with blood dissemination. Mixed species biofilms in vivo may further be modified by environmental milieu e.g. conditioning of the catheter implants with host proteins that may increase biofilm adhesion and aggregation.
In mixed species biofilms of other bacteria with Candida species, bacterial association with hyphae predominates association with yeast cells
[22, 23]. Hogan et al. evaluated interactions of Pseudomonas aeruginosa and Candida, and found that Pseudomonas aeruginosa had a predilection for the hyphal form without affecting the yeast form of the fungus
. In studies of mixed species infections of S. aureus and C. albicans, similar to P. aeruginosa, adherence to the Candida hyphae was nearly 30-fold more than adherence to the yeast form of Candida
. In our experiments (data not shown) we found adherence of S. epidermidis to both yeasts and hyphae of Candida which may facilitate mixed species biofilms of these two organisms and partly contribute to the increased clinical frequency of mixed species biofilm infections of C. albicans and S. epidermidis. The yeast and hyphal forms of C. albicans may act as a scaffold on which biofilms of S. epidermidis are formed
. Candida infection is associated with tissue invasion by hyphae and it been hypothesized that staphylococcal tissue infection is facilitated by its association with Candida hyphae
. Synergistic effects of C. albicans and S. epidermidis have been reported by other investigators
[16, 17]. In mixed species biofilms of C. albicans and S. epidermidis, presence of slime producing strains of S. epidermidis decreases antifungal susceptibility related to decreased penetration of the fluconazole through the ECM and conversely the fungal cells protected slime negative S. epidermidis against vancomycin
. In an in vitro study of mixed species biofilms of C. albicans and S. epidermidis, enhanced the growth of S. epidermidis was observed
We used a clinically relevant model of subcutaneous catheter biofilm infection to evaluate the clinical implications of mixed species biofilm infection
. In mixed species biofilms, catheter biofilm infection of S. epidermidis increased in the presence of C. albicans. Pre-insertion cultures revealed lower catheter infection of S. epidermidis in mixed species infection compared to single species S. epidermidis but on day 8 of insertion in vivo, we found increased catheter infection of S. epidermidis in the mixed species infection. This suggests that mixed species environment facilitates biofilm aggregation and not the initial phase of S. epidermidis adhesion to catheters. Enhanced biofilm aggregation was associated with enhanced dispersal that led to increased systemic dissemination of S. epidermidis in the mixed species infection. Increased virulence and mortality has been described in mouse models of dual infection with C. albicans and S. aureus but not with S. epidermidis[12–14]. Peters et al. using proteomic techniques found that the global transcriptional repressor of virulence was down regulated thereby increasing virulence of the dual species biofilms of S. aureus and C. albicans. Enhancement of biofilms, increased catheter infection and dissemination of S. epidermidis in mixed species biofilms in vivo may partly explain clinical therapeutic failures and contribute to increased mortality and morbidity in polymicrobial infections.
We performed microarrays to delineate changes in staphylococcal gene expression that lead to increased catheter infection and dissemination in mixed species biofilms with C. albicans. We noted that the lrg operon comprising lrgA and lrgB was highly down regulated (36 fold and 27 fold change respectively) in mixed species biofilms. Lrg operon along with the cidR operon represents the molecular elements of programmed cell death or apoptosis in Staphylococcus aureus[25–27]. The lrg operon is a repressor of murein hydrolase activity that hydrolyzes components of the cell wall, involved in autolysis. Lrg protein has also been shown to affect antibiotic tolerance, biofilm formation (by release of eDNA which is a structural component of the biofilm) and acetoin production in S. aureus[25, 26, 28, 29]. Lrg operon is regulated by the LytSR two component regulatory system in S. aureus and transcriptional regulators agr and sar that regulate virulence also influence the lrg operon
[28, 29]. Down regulation of the lrg operon (autolysis repressors) in mixed species biofilms is associated with enhanced release of eDNA possibly by autolysis
[25, 30]. Extracellular DNA plays a significant role in biofilm aggregation
[18, 19] and it is conceivable that increased eDNA enhances aggregation of mixed species biofilms of S. epidermidis and C. albicans. Most bacteria have cardiolipin synthases that convert bacterial membrane phosphatidyl glycerol to cardiolipin, during the transition from logarithmic phase to the stationary phase and may help survival during prolonged high salt stress conditions
. S. aureus and S. epidermidis have 2 ORFs cls1 and cls2 and we found cardiolipin synthetase (cls2) was significantly down regulated. Other down regulated genes included those associated with carbohydrate, amino acid and nucleotide metabolism, transporters and other proteins. Biofilm as a whole may be metabolically less active compared to actively dividing planktonic organisms and that may explain the down regulation of metabolic processes and overall more down regulated genes (6%) than upregulated genes (2.7%)
Genes upregulated in mixed species biofilms include transcriptional regulators (sarR and hrcA the heat inducible transcriptional repressor), genes associated with nucleic acid metabolism, some transporters and other proteins. sarR is known inhibitor of sarA, a transcriptional regulator that represses extracellular proteases and that may influence virulence determinants in S. aureus[34–36] but its role in S. epidermidis is not known. Therefore the net effect of sarR upregulation is to facilitate secretion of extracellular proteases that may function as virulence factors. Heat shock protein GrpE protein of the DnaK family of shock proteins is upregulated indicating an adaptive response to polymicrobial stress by S. epidermidis in mixed species biofilms. Adaptation to competition for iron in mixed species environments is facilitated by the increased transcription of transferrin receptor, which facilitates uptake of iron from human transferrin by a receptor-mediated energy dependent process
[37, 38]. Genes related to nucleic acid and glycerol metabolism (guaC, purC, purM, glpD, apt and uraA) were also upregulated.
We measured the eDNA content in the extracellular matrix of single and mixed-species biofilms and confirmed that S. epidermidis derived eDNA predominated in mixed species biofilms. Candida derived eDNA was barely detected indicating the predominant role for bacterial eDNA in the enhancement of mixed-species biofilms. Low Candida eDNA may be also partly due to decreased growth of Candida in mixed species biofilms. Indirectly, this indicates that bacterial autolysis, the most important mechanism for producing bacterial eDNA, is strongly implicated in the enhancement of mixed species biofilms.
We evaluated the effects of disrupting eDNA by DNAse on mature (24 hr) and developing single and mixed species biofilms of S. epidermidis and C. albicans. DNAse decreased biofilm metabolic activity (as measured by XTT method) by a concentration dependent manner in both single and mixed species biofilms. We also evaluated the effects of DNAse on a developing biofilms by initiating exposure to DNAse at different time points (0, 6 and 18 hrs). Exposure at earlier time-points would decrease adhesion of the microbial cells and exposure later would affect biofilm aggregation. We observed that DNAse decreased biofilm formation significantly at both adhesion and aggregation stages in biofilm development. The reduction in biofilm formation as a percentage of that of untreated biofilms was more pronounced in mixed species biofilms compared to single species biofilms, due to an increased eDNA content in the mixed species biofilms. Other investigators have found similar inhibiting effects of DNAse on biofilm adhesion and aggregation outlining the essential role of eDNA in biofilm development
We confirmed increased eDNA in mixed species biofilms by quantitation of eDNA in the biofilm extracellular matrix. Increased eDNA in the biofilm matrix is probably caused by autolysis as active secretion of eDNA has not been reported in S. epidermidis biofilms. Staphylococcal biofilm aggregation is enhanced by eDNA and increased quantity of eDNA may explain the increased thickness of mixed-species biofilms. Significant down regulation of repressors of autolysis (lrg operon) also point to increased bacterial autolysis in mixed species biofilms. The lrg operon that represses murein hydrolase activity and thereby autolysis in S. aureus has not been studied in S. epidermidis so far. In Staphylococcus aureus, cidA and lrgA genes encode homologous hydrophobic proteins that function similar to bacteriophage coded holin (causes autolysis) and antiholin (inhibits autolysis), respectively. The S. aureus cidB and lrgB genes also encode homologous hydrophobic proteins, but their functions are unknown
. In a model proposed by Bayles et al., the LytSR two-component regulatory system senses decreases in cell membrane potential due to cell membrane damage and responds by inducing lrgAB transcription. The CidR protein, a LysR-type transcription regulator, enhances cidABC in response to carbohydrate metabolism that enhance murein hydrolase activity thereby enhancing autolysis
[26, 43]. LrgAB operon in S. aureus also influences penicillin (that causes cell lysis) tolerance
. In S. epidermidis, LytSR knockout strain exhibited decreased extracellular murein hydrolase activity and mildly increased biofilm formation but did not differ in Triton X-100 mediated autolysis or in murein hydrolase zymogram patterns from the parent strain
. Mutation of SaeRS (another two component signal system) in S. epidermidis increased autolysis and biofilm forming ability
. Association of autolysis and increased biofilm formation is also confirmed by studies on autolysin atlE in S. epidermidis. Therefore, autolysis and release of eDNA has a significant role to play in Staphylococcal biofilm formation and enhancement of mixed species biofilms.
The limitations of the study include using a single clinical strain each of S. epidermidis and C. albicans. Findings of this study will have to be confirmed using multiple strains of S. epidermidis and C. albicans. The subcutaneous catheter biofilm infection in mice is an appropriate and reproducible model to evaluate foreign device biofilm infections i.e. pacemaker and shunt infections but an intravenous catheter model will be more appropriate for indwelling vascular catheter infections. Nevertheless the subcutaneous catheter model has been successfully used to study biofilm infections and to evaluate anti-biofilm strategies. In our microarray experiments, S. epidermidis probes on the microarray that might hybridize with Candida RNA were eliminated in the design of the microarray. Also, those probes that actually hybridized with Candida RNA were also eliminated from data analysis. It is possible that some transcriptome data was lost due to the elimination of Candida cross-reacting probes.