While disagreement remains about the efficacy of the biofilm matrix as a diffusion barrier to antibiotics, altered microenvironments within the biofilm clearly play a role in antibiotic protection. Oxygen limitation in particular has been extensively investigated, and numerous studies have revealed the presence of hypoxic zones deep within biofilms. A recent microarray study of E. coli biofilms found an upregulation of the cydAB and b2997-hybABC gene clusters, which are known to be transcribed in oxygen-limiting conditions (Schembri et al. 2003). Similarly, nutrient diffusion through biofilms is restricted. Oxygen and nutrient deprivation consequently result in a decrease in bacterial metabolic activity and cessation of bacterial growth (Donlan and Costerton 2002; Dunne 2002). Indeed, experimental measurements have revealed a severe reduction in bacterial growth rates within biofilms compared to planktonic cultures (Anderl et al. 2003; Borriello et al. 2004). Even in planktonic cultures of P. aeruginosa and K. pneumoniae, deprivation of oxygen or nutrients, respectively, has resulted in slow growth and antibiotic resistance
(Anderl et al. 2003; Field et al. 2005). Because antibiotics typically act upon rapidly growing bacteria, slow or nongrowing microorganisms would be protected from killing (Fig. 1) (Brown et al. 1988).
2.2.1 Oxygen Limitation, Metabolism, and Antibiotic Killing
Several studies have shown a correlation between oxygen limitation, metabolic activity, and protection from antibiotic killing in biofilms. Alkaline phosphatase activity and expression of green fluorescent protein (GFP), as measures of general bacterial protein production, have been used to show restriction of bacterial metabolism to the medium-exposed edge of P. aeruginosa biofilms (Borriello et al. 2004; Walters et al. 2003; Xu et al. 1998). In these same studies, oxygen microelectrodes were utilized to analyze the dissolved oxygen at various depths within the biofilm. Intriguingly, oxygen penetration was also restricted to the medium-exposed edge, suggesting that decreased oxygen tension throughout the rest of the biofilm inhibited metabolic activity and, consequently, increased antibiotic resistance (Walters et al. 2003; Xu et al. 1998). Similarly, diffusion of glucose and oxygen was inhibited through intact K. pneumoniae biofilms, which corresponded to a decrease in bacterial growth and resistance to ampicillin (Anderl et al. 2003). In both of these cases, antibiotics completely permeated the biofilm, yet the drugs only affected the biofilm edge (Anderl et al. 2003; Walters et al. 2003). Thus, limited metabolic activity within these biofilms, created by oxygen and nutrient gradients, protects the constituent bacteria from antibiotic killing.
Discussion of the metabolic pathways used during anaerobic growth can shed some light on the genetic mechanisms governing the reduced killing of slow-growing bacteria. P. aeruginosa, for instance, can utilize NO3_ and NO2_ for anaerobic respiration (Hassett et al. 2002). These processes are carried out by the sequential actions of the nar, nir, nor, and nos genetic loci, which reduce the nitrogenous substances to N2. P. aeruginosa tightly regulates these genes in order to prevent buildup of toxic intermediates in the pathway (such as the production of nitric oxide). In fact, altered regulation of these loci in mutants of the quorum sensing gene rhlR under anaerobic conditions leads to rapid cell death (Hassett et al. 2002). Consequently, drugs targeting quorum sensing or nitrogen utilization pathways may be efficacious in destroying tenacious biofilms. Intriguingly, treating mature P. aeruginosa biofilms under anaerobic conditions with a combination of NO3_ and either cipro-floxacin or tobramycin significantly enhanced killing of the microorganisms compared to antibiotic treatment alone (Borriello et al. 2006). However, these effects were not apparent in younger biofilms (Borriello et al. 2004). Obviously, the age and metabolic state of the biofilm plays a major role in determining its susceptibility to antibiotic treatment.
2.2.3 Stationary Phase and Stress Response Similarities
The slow growth and altered metabolic activity apparent in biofilms have led some researchers to suggest that the biofilm bacteria are in a stationary-phase state (Anderl et al. 2003). One of the hallmark features of stationary-phase bacteria is the activity of rpoS, the stationary-phase sigma factor instrumental in regulating expression of stress response factors. Microarray analysis of E. coli biofilms revealed the upregulation of nearly 50% of all rpoS-regulated genes (Schembri et al. 2003). In the same study, an rpoS mutant failed to form a biofilm. On the other hand, a P. aeruginosa rpoS mutant formed a much larger and more antibiotic resistant biofilm than wild type (Whiteley et al. 2001).
Additional studies have further implicated stress response factors as integral components of bacterial biofilms. For instance, microarray analysis of tobramy-cin-treated wild type P. aeruginosa biofilms showed upregulation of the stress response chaperones groES and dnaK (Whiteley et al. 2001). Studies with K. pneumo-niae demonstrated expression of catalase in stationary-phase planktonic cells and in biofilms, but not in exponentially growing planktonic bacteria (Anderl et al. 2003). Catalase breaks down hydrogen peroxide and consequently protects expressing microorganisms from destruction. In P. aeruginosa, the constitutive catalase gene katA and the hydrogen peroxide inducible catalase gene katB were found to be important in resistance and adaptation to hydrogen peroxide stress in biofilms (Elkins et al. 1999). Thus, stress responses activated within bacterial biofilms may impact bacterial resistance to biocides and potentially to other antimicrobial agents.
In summary, it is clear that altered metabolism within biofilms promotes the creation of a bacterial subpopulation with altered sensitivity to antibiotics (Fig. 1). By decreasing the growth rate and activating vigorous stress responses, biofilms increase their chances of surviving antimicrobial treatment. In this sense, these metabolic changes represent a vital innate biofilm antibiotic resistance mechanism.
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