# Biology and Regulation of Staphylococcusococcal Biofilm
## 8 A Glimpse into Staphylococcal Biofilm Matrix and Physiology  
Durable catheters are most frequently colonized with staphylococci, followed by _Acinetobacter calcoacetic_s or _Pseudomonas aeruginosa_. The first microscopic studies of a staphylococcal biofilm were performed by the group of [PERSON] [125]. Scanning electron microscopy (SEM, magnification \(\times\) 5000) of _S. epidermidis_ grown for 24 h on a cellulose acetate surface showed closely packed bacterial cells embedded in a slimy matrix. An image of such an _S. epidermidis_ biofilm on a catheter is shown in Figure 4A. We now know that the slimy matrix consists of the exopolysaccharideicating PIA/dPNAG. The question that concerned the researchers was which metabolism would be predominant in such a biofilm. It was shown that an _S. aureus sarA_ (staphylococcal accessory regulator A protein) mutant was impaired in biofilm formation. A transcriptome analysis with this mutant suggested that cells grow essentially anaerobically in mature biofilms cells, and that the genes of the acid tolerance response, such as the global regulators SigB and SarA, are upregulated in response to an acidic (pH 5.5) environment [44]. A comparative transcriptome analysis between biofilm- and planktonic-grown _S. aureus_ showed that the envelope appeared to be a very active compartment in biofilm-associated cells. Indeed, genes that encode binding proteins, proteins involved in murein and PIA/dPNAG synthesis, and enzymes involved in cell envelope metabolism were significantly upregulated [81]. In addition, formate fermentation (formate dehydrogenase), urease activity, the response to oxidative stress (staphyloxanthin), acid and ammonium production, and the arginine deaminase cluster were upregulated in a biofilm. On the other hand, toxins and proteases were upregulated under planktonic growth conditions. Interesting, the _ica_ operon was highly upregulated during the first 8 h of biofilm growth compared to planktonic organisms. The expression level in the biofilm cells then decreased, but remained 3-fold higher than the expression level in a planktonic state. It has been assumed that enzymes have a long half-life [126]; therefore, the upregulation of these genes might not be needed in aged, surface-associated cells, as biofilm formation has begun and cell growth is retarded due to nutrient depletion [81]. A comparative proteome analysis essentially confirmed the transcriptome results [10]. Compared to planktonic growth, biofilm cells expressed higher levels of proteins associated with cell adhesion, peptidoglycan synthesis, fibrinogen-binding proteins, and enzymes involved in pyruvate and formate metabolism as well as SarA, which is in accordance with the positive effect of SarA on _ica_ locus expression. All data indicate that anoxic conditions prevail in the biofilm, as also indicated by the upregulation of pyruvate formate lyase (Pfl) and NAD-dependent formate dehydrogenase (Fdh) in _S. aureus_ biofilms. To investigate their physiological role, _fdh_ and _pfl_ deletion mutants were constructed (_\(\Delta\)fdh_ and _\(\Delta\)pfl_) and their impact on the biofilm was analyzed [127]. The absence of formate production was recorded in the _pfl_ mutant, and glucose consumption was delayed. Thus, as ethanol production was decreased, acetate and lactate production were unaffected. All metabolic alterations could be restored by the addition of formate or complementation of the _\(\Delta\)pfl_ mutant. All results suggest the model proposed in Figure 4B. The upregulation of _pfl_ takes place in the deeper layer of the biofilm where anoxic conditions are prevalent (Pfl is oxygen sensitive) and nutrients are limited. Pfl is necessary under these conditions, allowing for the formation of C\({}_{1}\) units (formate) for formyl-THF synthesis and for protein and purine biosynthesis. Interestingly, not only _fdh_ and _pfl_ but also the formyl-THF synthetase gene (_fhs_) were upregulated under biofilm conditions [81]. It was suggested that Fdh plays a role in the microaerobic area of the biofilm.