**3. Salicylic acid**

390 Progress in Hemodialysis – From Emergent Biotechnology to Clinical Practice

microbes in various ways: Compounds may have direct anti microbial effects in vitro, similar to traditional antibiotics. However, to be clinically useful the drug level to achieve minimum inhibitory concentration has to be within a range that is achievable and tolerable in humans. Compounds can exert an antimicrobial effect by inhibition of bacterial pumps. A well known example is the potentiation of antibiotic treatment against *Helicobacter pylori* through omeprazole. Many psychotropic medications including the phenothiazines fall into this category. Aspirin appears to modulate the expression of genes that are important for Staphylococcal virulence and the statins appear to have immune modulatory effects. Both

This chapter deals with the anti microbial effects of medications that are not traditionally

The current epidemic of obesity and, as a complication, diabetic nephropathy associated to type 2 diabetes mellitus has fueled the spectacular growth of hemodialysis into an industry that is dominated by a handful of large companies. Infection is a leading cause of morbidity and mortality in dialysis patients and the annual mortality rate caused by sepsis is several hundred folds higher in patients with end stage renal disease than in the general population (Laupland et al., 2004). The incidence of bacteremia has increased in hemodialysis patients over the years, mainly because of increased rates of serious *Staphylococcus aureus* infection in this population (Foley at al., 2004). *S. aureus* has its name from a gold coloured caroten virulence factor called staphyloxanthin which allows the bacteria to survive oxidative bursts of neutrophils (Liu G.Y. & Nizet V., 2009). *S.aureus* produces a battery of surface proteins, enzymes and toxins which enable the bacteria to both persist in intracellular locations and in biofilms for long periods of time, and to rapidly disseminate in the host in an opportunistic fashion which makes it one of the most dangerous and pathogenic bacteria in humans. The use of dialysis catheters is a major risk factor for developing *S.aureus* infection because of disruption of the normal skin barrier, thus forming a gateway for bacterial entry into the blood stream (Vandecasteele S.J. et al., 2009). Despite Kidney Disease Outcomes Quality Initiative clinical practice guidelines recommending the use of auto logos arterio-venous fistulae as dialysis access and other efforts, the overall prevalence of hemodialysis catheter use has been increasing, approaching 30% in the United States (Rayner et al., 2004). Humans are the main natural reservoir for *S.aureus* which can colonize skin, gastrointestinal and urogenital tracts. The most frequent site of colonization is the anterior nose and longitudinal studies have shown that there are three types of *S.aureus* nasal carriage in healthy adults: fifty percent are persistent non carriers, thirty percent are intermittent carriers and twenty percent are persistent carriers (VandenBergh et al., 1999). Hands are the main vector of transmission and in the majority of cases the same strain that is found in the bloodstream is also found on the hands and in the nose (von Eiff C. et al., 2001). It follows that rigorous hand washing is extremely important to prevent infection in the dialysis units as it is elsewhere in the medical setting. The majority of *S.aureus* infections has its source in the endogenous reservoir in the nose of the same person and can thus be considered an "autoinfection" (Boelart et al., 1995 as quoted in Vandecasteele et al., 2009). Consistent with this view is that in prospective studies the interval between catheter placement and staphylococcemia can be very short, with 23% of episodes occurring less than one week after catheter insertion (Little M.A. et al., 2001). *S.aureus* bacteremia is associated frequently with

**2. A combination of unfortunate events: infection in dialysis** 

medications are discussed in more detail below.

regarded as antibiotics with regards to dialysis.

Salicylic acid, the active ingredient of willow bark, is one of the oldest medicines still in use, in the buffered form of aspirin. The beneficial effect on fever, pain and inflammation were already described by Hippocrates. Fallen out of favour because of other non steroidal anti inflammatory drugs with more favorable side effects profiles aspirin has made a spectacular come back fifty years ago as the antiplatelet effects of aspirin were discovered. Since then aspirin has found widespread therapeutic use in the treatment of cardiovascular disease. Chronic treatment with Aspirin may prevent colorectal cancer, presumably by inhibition of cyclooxygenase 2 (COX-2) which is expressed in large amounts in adenocarcinoma (Ruder E.H. et al., 2011). Salicylic acid is ubiquitous in plants as a phytohormone. It is part of the innate immune system of plants, involved in local resistance to pathogens and in systemic acquired resistance (SAR), where a pathogenic attack one part of a plant induces resistance in other parts. Depending on the amount of fruit and vegetables in the diet humans have detectable serum levels of salicylic acid. It has been hypothesized that diet derived salicylic acid could in part account for the observed link between diet and colorectal cancer (Paterson J.R.& Lawrence J.R., 2001) and this might possibly apply to the relation between diet and cardiovascular disease as well. Salicylic acid is used as a food preservative and an antiseptic in toothpaste. Aspirin is thus not only one of the oldest but also one of the most versatile and successful drugs known.

### **3.1 Laboratory evidence for an anti staphylococcal effect of salicylic acid**

Early studies in the rabbit endocarditis model showed that platelets provide a nidus for bacteria and that aspirin can decrease vegetation size (Pujadas et al., 1988). The observation was made that aspirin can reduce not only the weight of vegetations in a rabbit model of *S.aureus* endocarditis but also bacterial density although neither aspirin nor salicylic acid have known antibacterial effects at the low concentrations employed (Nicolau D.P. et al., 1993). This benefit was seen if aspirin was given together with antibiotics but also if aspirin was provided prior to the infectious challenge with which endocarditis was induced (Nicolau D.P. et al., 1995). Even more puzzling in this study was that vegetation weight and bacterial density were higher if higher doses of aspirin were administered while the optimum beneficial effect was seen at a lower dose, suggesting that serum levels may be very important. Subsequently this observation has been called the "Goldilocks effect" in which too little and too much aspirin may cause paradoxically diminished effects on outcome parameters in the infectious endocarditis model (Eisen et al., 2008). The beneficial

Nontraditional Anti - Infectious Agents in Hemodialysis 393

respectively, in both arms had *S.aureus* endocarditis and thus the same concern that the study was underpowered to detect a difference was voiced for this study as well (Eisen D.P.

A retrospective single center cohort study of 600 patients with infectious endocarditis, who were treated over a 18 year period at the Mayo Clinic, found that the odds of suffering symptomatic embolic events was decreased by 64% in patients who were treated with antiplatelet agents for at least 6 months prior to the diagnosis: Aspirin was the antiplatelet agent in 98% of cases and an 81mg daily dose was used in the majority of patients

Eisen et al. used the International Collaboration on Endocarditis –Prospective Cohort Study (ICE-PCS) database to assess the influence of aspirin usage at the time of diagnosis on the outcome of definitive *S.aureus* endocarditis. A cohort of 670 patients had both information on prior aspirin use and *S.aureus* endocarditis. Aspirin use at the time of diagnosis in 132 patients was a predictor for a decreased risk of acute valve surgery, independent of methicillin resistance status. A statistically significant decrease in embolic events in aspirin users was found in a univariate analysis that became a trend in multivariate analysis. A comparison of groups with and without aspirin use among patients with Streptococcal endocarditis was made and no association of aspirin with improved outcomes was found

Thus the data on aspirin use in *S.aureus* endocarditis suggests that aspirin likely does alter the course of illness. The non dependence of the effect on methicillin resistance status and the absence of an observed effect of aspirin on other pathogens are consistent with the proposed specific mechanism of aspirin on staphylococcal virulence factors. It is also noteworthy that Staphylococcal endocarditis is rather difficult to study in adequate numbers

Karabay et al. investigated the prevalence of *S.aureus* nasal carriage in an outpatient cardiology clinic. Of a total of 346 patients 199 were chronic aspirin user while 147 patients were not. The prevalence of *S.aureus* nasal carriage was 5% on patient treated chronically with aspirin versus 16% in those that did not take aspirin. Only aspirin was found to be associated with a decreased rate of nasal carriage in a multivariate analysis (Karabay et al., 2006). These findings are of obvious significance to hemodialysis patients as nasal colonization is considered the initiating event that leads to catheter associated staphylococcal bacteremia. If confirmed, aspirin could decrease nasal carriage at a fraction of the cost and effort of mupirocin ointment. Given the fact that aspirin is a very old drug the findings of Karabay et al. have another potential significance: If a clinical effect of aspirin on *S.aureus* can still be detected after decades of over-the-counter use it would be unlikely that *S.aureus* would develop resistance to this effect in the future. It is clear that the important findings of Karabay et al. merit further

Patient undergoing hemodialysis treatments suffer staphylococcal infections with increased frequency because of a high prevalence of tunneled or non tunneled dialysis catheters. The hemodialysis setting is thus well suited to study the potential beneficial clinical anti

& Bayer A.S., 2008).

(Anavekar et al., 2007).

(Eisen et al., 2009).

as the population incidence is fortunately low.

investigation both in hemodialysis and in the general population.

**3.4 A potential beneficial effect of aspirin in hemodialysis patients** 

**3.3 Aspirin and** *S.aureus* **nasal carriage** 

staphylococcal effects of aspirin.

effects of aspirin in the rabbit model of endocarditis have been recapitulated with salicylic acid, its major biometabolite (Kupferwasser et al., 1999). As salicylic acid has no anti-platelet properties, this indicates that platelet independent mechanisms are likely to have a more significant role in the action of aspirin on *S.aureus* endocarditis that the platelet dependant effects. Further experimental work in vivo in animal models of infectious *S.aureus* endocarditis showed that aspirin reduced a multitude of measurable parameters of the severity of the infection and metastasis such as vegetation weight and the bacterial density in vegetations and the number of renal emboli and these effects were dose dependant, more pronounced at lower rather than higher doses (Kupferwasser et al., 1999). In vitro studies showed that salicylic acid inhibits the expression of two key virulence genes in *S. aureus* that are involved in endovascular pathogenesis: alpha-toxin [hla] and fibronectin-binding adhesion [fnbA], through activation of genetic pathways involving the major stress response operon, sigma factor B (Kupferwasser et al., 2003). These aspirin mediated effects on sigma factor B were observed at serum concentrations that are achieved by usual clinical dosages of aspirin in humans (Kupferwasser et al., 2003). On the other hand side, it has been shown that the presence of salicylic acid decreases expression of capsular polysaccharides. It has been hypothesized that the loss of these capsular virulence factors could lead to an increased capacity of *S.aureus* to invade epithelial cells and that chronic treatment with aspirin could potentially lead to more persistent or recurrent infection (Alvarez C.P. et al., 2010).

In conclusion a significant body of in vitro and in vivo evidence indicates that aspirin may have the potential to be useful in the treatment of *S.aureus* infection by down modulating key regulator and structural genes resulting in the abrogation of virulent phenotypes but it has to be noted that important questions remain.

#### **3.2 Clinical evidence for a beneficial effect of aspirin in** *S.aureus* **endocarditis**

The earliest clinical observations of a potential salutatory role of aspirin come from the study of bacterial endocarditis. In a small retrospective study a decreased rate of embolic events was found in patients with native valve endocarditis who were on long term aspirin treatment (11% versus 47%), although the number of patients treated with aspirin was too small to be conclusive (Schunemann S. et al., 1997). A small preliminary prospective observational study conducted in 9 patients found adjunctive treatment of established endocarditis with aspirin beneficial (Taha et al., 1992).

Subsequently, a Canadian prospective multicenter study in 115 patients with endocarditis showed no benefit of the adjunctive treatment with a 325mg dose of aspirin (Chan et al., 2003). Despite its prospective design this study was criticized as patients on chronic aspirin treatment were excluded from this study although the greatest benefit might be expected in this population. Moreover, Aspirin was added only after an average of 35 days after onset of symptoms. Only 14 patients (25%) in the Aspirin treatment group had *Staphylococcus aureus* endocarditis while the majority had streptococcal endocarditis. As the putative mechanism of action of aspirin involves the inhibition of *S.aureus* virulence factors, the benefit of aspirin is likely greatest if it is used before infection occurs. The benefit of aspirin is also very likely to be limited to *S.aureus* as the mechanism seems to be specific to this pathogen. The same authors presented a post hoc analysis of their data in 2008, comparing 84 patients who had been excluded from their previous study because of long term aspirin treatment with 54 patients in the placebo arm and again found no significant clinical differences in the outcome between both groups (Chan et al., 2008). Only 29% of patients, 16 and 24 patients

effects of aspirin in the rabbit model of endocarditis have been recapitulated with salicylic acid, its major biometabolite (Kupferwasser et al., 1999). As salicylic acid has no anti-platelet properties, this indicates that platelet independent mechanisms are likely to have a more significant role in the action of aspirin on *S.aureus* endocarditis that the platelet dependant effects. Further experimental work in vivo in animal models of infectious *S.aureus* endocarditis showed that aspirin reduced a multitude of measurable parameters of the severity of the infection and metastasis such as vegetation weight and the bacterial density in vegetations and the number of renal emboli and these effects were dose dependant, more pronounced at lower rather than higher doses (Kupferwasser et al., 1999). In vitro studies showed that salicylic acid inhibits the expression of two key virulence genes in *S. aureus* that are involved in endovascular pathogenesis: alpha-toxin [hla] and fibronectin-binding adhesion [fnbA], through activation of genetic pathways involving the major stress response operon, sigma factor B (Kupferwasser et al., 2003). These aspirin mediated effects on sigma factor B were observed at serum concentrations that are achieved by usual clinical dosages of aspirin in humans (Kupferwasser et al., 2003). On the other hand side, it has been shown that the presence of salicylic acid decreases expression of capsular polysaccharides. It has been hypothesized that the loss of these capsular virulence factors could lead to an increased capacity of *S.aureus* to invade epithelial cells and that chronic treatment with aspirin could

potentially lead to more persistent or recurrent infection (Alvarez C.P. et al., 2010).

**3.2 Clinical evidence for a beneficial effect of aspirin in** *S.aureus* **endocarditis** 

has to be noted that important questions remain.

endocarditis with aspirin beneficial (Taha et al., 1992).

In conclusion a significant body of in vitro and in vivo evidence indicates that aspirin may have the potential to be useful in the treatment of *S.aureus* infection by down modulating key regulator and structural genes resulting in the abrogation of virulent phenotypes but it

The earliest clinical observations of a potential salutatory role of aspirin come from the study of bacterial endocarditis. In a small retrospective study a decreased rate of embolic events was found in patients with native valve endocarditis who were on long term aspirin treatment (11% versus 47%), although the number of patients treated with aspirin was too small to be conclusive (Schunemann S. et al., 1997). A small preliminary prospective observational study conducted in 9 patients found adjunctive treatment of established

Subsequently, a Canadian prospective multicenter study in 115 patients with endocarditis showed no benefit of the adjunctive treatment with a 325mg dose of aspirin (Chan et al., 2003). Despite its prospective design this study was criticized as patients on chronic aspirin treatment were excluded from this study although the greatest benefit might be expected in this population. Moreover, Aspirin was added only after an average of 35 days after onset of symptoms. Only 14 patients (25%) in the Aspirin treatment group had *Staphylococcus aureus* endocarditis while the majority had streptococcal endocarditis. As the putative mechanism of action of aspirin involves the inhibition of *S.aureus* virulence factors, the benefit of aspirin is likely greatest if it is used before infection occurs. The benefit of aspirin is also very likely to be limited to *S.aureus* as the mechanism seems to be specific to this pathogen. The same authors presented a post hoc analysis of their data in 2008, comparing 84 patients who had been excluded from their previous study because of long term aspirin treatment with 54 patients in the placebo arm and again found no significant clinical differences in the outcome between both groups (Chan et al., 2008). Only 29% of patients, 16 and 24 patients respectively, in both arms had *S.aureus* endocarditis and thus the same concern that the study was underpowered to detect a difference was voiced for this study as well (Eisen D.P. & Bayer A.S., 2008).

A retrospective single center cohort study of 600 patients with infectious endocarditis, who were treated over a 18 year period at the Mayo Clinic, found that the odds of suffering symptomatic embolic events was decreased by 64% in patients who were treated with antiplatelet agents for at least 6 months prior to the diagnosis: Aspirin was the antiplatelet agent in 98% of cases and an 81mg daily dose was used in the majority of patients (Anavekar et al., 2007).

Eisen et al. used the International Collaboration on Endocarditis –Prospective Cohort Study (ICE-PCS) database to assess the influence of aspirin usage at the time of diagnosis on the outcome of definitive *S.aureus* endocarditis. A cohort of 670 patients had both information on prior aspirin use and *S.aureus* endocarditis. Aspirin use at the time of diagnosis in 132 patients was a predictor for a decreased risk of acute valve surgery, independent of methicillin resistance status. A statistically significant decrease in embolic events in aspirin users was found in a univariate analysis that became a trend in multivariate analysis. A comparison of groups with and without aspirin use among patients with Streptococcal endocarditis was made and no association of aspirin with improved outcomes was found (Eisen et al., 2009).

Thus the data on aspirin use in *S.aureus* endocarditis suggests that aspirin likely does alter the course of illness. The non dependence of the effect on methicillin resistance status and the absence of an observed effect of aspirin on other pathogens are consistent with the proposed specific mechanism of aspirin on staphylococcal virulence factors. It is also noteworthy that Staphylococcal endocarditis is rather difficult to study in adequate numbers as the population incidence is fortunately low.
