Treatment of Staphylococcus aureus Biofilm Infection by the Quorum-Sensing Inhibitor RIP

Abstract

The quorum-sensing inhibitor RIP inhibits staphylococcal TRAP/agr systems and both TRAP- and agr-negative strains are deficient in biofilm formation in vivo, indicating the importance of quorum sensing to biofilms in the host. RIP injected systemically into rats has been found to have strong activity in preventing methicillin-resistant Staphylococcus aureus graft infections, suggesting that RIP can be used as a therapeutic agent.

Millions of indwelling medical devices are implanted annually and are at risk of persistent infections caused by bacteria organized as a biofilm. Such biofilms are resistant to antibiotics, are difficult to treat, and are common causes of morbidity and mortality (1, 14, 17, 26, 29, 13, 34, 35). Staphylococcus aureus and S. epidermidis are common causes of device-associated infections. S. aureus (like Pseudomonas aeruginosa) regulates virulence through two quorum-sensing (QS) systems that regulate one another (4, 23, 33, 28, 41). One QS uses the 33-kDa autoinducer RNAIII-activating protein (RAP), which induces the phosphorylation of the target of RAP (TRAP) (4, 5, 8, 22, 23, 24). The other QS uses the autoinducing peptide AIP, which phosphorylates AgrC, resulting in the production of RNAIII and toxins (10, 18, 25, 27, 30, 31, 32, 37, 38).

TRAP is a 21-kDa protein that was shown to regulate the expression of the many toxins and their regulator, agr (9, 23). In the absence of TRAP expression or phosphorylation, the bacteria do not produce toxins and do not cause disease (as determined from tests with multiple strains and species of S. aureus and S. epidermidis) (9, 22, 39, 40). To directly test the biofilm formation by TRAP and agr mutants in vivo, TRAP- and agr-negative mutants and their parent strains were injected onto a graft (n = 10) by using the rat graft model. The bacteria used were S. aureus 8325-4, a wild-type (WT) laboratory strain, and its isogenic derivative with an inactivated TRAP gene (the TRAP-negative strain) (22) and WT laboratory strain S. aureus RN6390 and its agr-negative isogenic derivative, strain RN6911 (30, 31). The results (Fig. 1) show that the TRAP-negative mutants formed very little biofilm (27 ± 5 CFU/ml) compared to the amount formed by control parent strain S. aureus 8325-4 (5.0 × 10⁵ ± 1.1 × 10⁵ CFU/ml) (P < 0.0001) and that mutated agr (S. aureus RN6911) demonstrated a reduced biofilm-forming capacity (7.9 × 10³ ± 1.3 × 10³ CFU/ml) compared to that of parent strain S. aureus RN6390 (4.8 × 10⁵ ± 1.7 × 10⁵ CFU/ml) (P < 0.0001). These results indicate that TRAP regulates multiple genes important for biofim formation in vivo, in addition to those regulated by agr, and further indicates the usefulness of TRAP as a therapy target site.

FIG. 1.

Formation of a biofilm in vivo by TRAP-negative (TRAP−) or agr-negative (agr−) mutants was tested by using the rat graft model, in which 1-cm² collagen-sealed Dacron grafts (Albograft; Italy) were implanted subcutaneously into Wistar rats. Exponentially growing bacteria (2 × 10⁷) were inoculated onto the grafts, the grafts were removed 10 days later, and the bacterial loads on the grafts were determined and expressed as the numbers of CFU/ml (lower limit of detection, 10 CFU/ml). Comparisons of the results were performed by analysis of variance of the log-transformed data. Significance was defined as a P value ≤0.05. The study was approved by the animal research ethics committee of the INRCA, IRRCS, University of Ancona. Rats (n = 10) were challenged with S. aureus RN6390 (WT), RN6911 (agr-negative), 8325-4 (WT), or TRAP-negative strains.

TRAP phosphorylation can be inhibited by the QS inhibitor RNAIII-inhibiting peptide (RIP). RIP (YSPWTNF-NH₂) (21) has already been tested in multiple animal models and has been found to have strong activity in preventing staphylococcal infections, including those caused by drug-resistant strains, like methicillin-resistant S. aureus (MRSA), glycopeptide-intermediate S. aureus, and vancomycin-intermediate S. aureus strains (2, 3, 5-7, 9, 11, 12, 15, 16, 19-21, 36). No toxicity has been noted, and no resistant strains have emerged.

To test if RIP can also be used to treat preformed device-associated staphylococcal infections (biofilm), a graft rat model was used (see the legend to Fig. 1). As a model for parenteral treatment, S. aureus strain Smith diffuse (SD) (5) was injected onto the graft. Animals (n = 5) received intraperitoneal injections with various doses of RIP (0, 10, 20, and 30 mg/kg of body weight given every day for 1, 4, or 7 days); the RIP doses were administered immediately after graft implantation or starting 2 days after graft implantation. All grafts were explanted on day 10. Some of the grafts from control challenged but untreated animals were removed on day 3 to evaluate the biofilm. As shown in Fig. 2A, all control untreated animals demonstrated evidence of graft infection (6.9 × 10⁷ ± 1.8 × 10⁷ CFU/ml). In contrast, all rats included in the prophylaxis group (which received a single dose of RIP injected immediately after implantation) showed concentration-dependent reductions in the bacterial load, with counts of 5.0 × 10⁴ ± 2.1 × 10⁴ CFU/ml if they were given 10 mg/kg RIP, 9.4 × 10³ ± 3.3 × 10³ CFU/ml if they given 20 mg/kg RIP, and 2.3 × 10³ ± 0.8 × 10³ CFU/ml if they were given 30 mg/kg RIP. RIP administered as a single dose 2 days after graft implantation and bacterial challenge also demonstrated biofilm reductions (Fig. 2A); rats administered a single dose of 10 mg/kg RIP had a count of 8.4 × 10⁶ ± 3.2 × 10⁶ CFU/ml, rats administered a single dose of 20 mg/kg RIP had a count of 3.0 × 10⁶ ± 1.1 × 10⁶ CFU/ml, and rats administered a single dose of 30 mg/kg RIP had a count of 7.6 × 10⁵ ± 2.8 × 10⁵ CFU/ml. All results were significant (P < 0.0001 with respect to the results for the controls).

FIG. 2.

(A) Prevention and treatment of S. aureus infection with a single dose of RIP. By using the rat graft model, rats (n = 5) were challenged with S. aureus strain SD and injected parenterally with a single dose of RIP immediately after or 2 days after bacterial challenge. The grafts were removed on day 10, and the bacterial loads on the grafts were determined and expressed as the numbers of CFU/ml. (B) Treatment of S. aureus infection with multiple doses of RIP. By using the rat graft model, rats (n = 5) were challenged with S. aureus strain SD and injected parenterally 2 days later with multiple doses of RIP (every day for 1, 4, or 7 days). The grafts were removed on day 10, and the bacterial loads on the grafts were determined and expressed as the numbers of CFU/ml. (C) Treatment of MRSA biofilm by RIP (10 mg/kg) and/or teicoplanin (3 mg/kg). By using the rat graft model, rats (n = 15) were challenged with MRSA and treated 3 days later with seven doses of RIP and/or teicoplanin (administered every day for 7 days). The grafts were removed on day 10, and the bacterial loads on the grafts were determined and expressed as the numbers of CFU/ml. As a control (Cont), the grafts of challenged and untreated animals were removed on days 3 and 10.

RIP treatment demonstrated a notably greater suppression of 2-day-old biofilms when it was administered in multiple doses. Treatment activity correlated with both the dose and the duration of treatment. As shown in Fig. 2B, rats administered four doses of 10 mg/kg RIP had a count of 5.3 × 10⁴ ± 1.9 × 10⁴ CFU/ml, rats administered four doses of 20 mg/kg RIP had a count of 6.3 × 10³ ± 2.0 × 10³ CFU/ml, and rats administered four doses of 30 mg/kg RIP had a count of 8.5 × 10² ± 3.2 × 10² CFU/ml (P < 0.0001 with respect to the results for the controls).

As also shown in Fig. 2B, rats given seven doses of 10 mg/kg RIP had a count of 6.7 × 10³ ± 1.7 × 10³ CFU/ml, rats given seven doses of 20 mg/kg RIP had a count of 4.1 × 10² ± 1.8 × 10² CFU/ml, and rats given seven doses of 30 mg/kg RIP had a count of 2.9 × 10² ± 0.5 × 10² CFU/ml (P < 0.0001 with respect to the results for the controls).

To test the synergistic effect of RIP and antibiotics, grafts were implanted and the animals (n = 15) were challenged with MRSA ATCC 43300. Three days after graft implantation and biofilm establishment, the animals received intraperitoneal injections of RIP (10 mg/kg) and/or teicoplanin (3 mg/kg) every day for 7 days. To evaluate treatment activity, the grafts were explanted on day 10. As controls, the grafts were explanted on day 3 and day 10 to evaluate the biofilms in the untreated animals. As shown in Fig. 2C, 38 × 10⁷ ± 1.3 × 10⁷ CFU/ml was present on the graft on day 3 and 5.8 × 10⁸ ± 1.8 × 10⁸ CFU/ml was present on day 10, suggesting that a biofilm already existed on the day of treatment initiation. Similar results were obtained with the grafts explanted on day 2 of biofilm formation by strain SD (data not shown). As also shown in Fig. 2C, animals treated with seven doses of 10 mg/kg RIP had a count of 7.8 × 10⁴ ± 3.0 × 10⁴ CFU/ml, animals treated with 3 mg/kg teicoplanin had a count of 3.6 × 10⁴ ± 1.2 × 10⁴ CFU/ml, and animals treated with 10 mg/kg RIP and 3 mg/kg teicoplanin had a count of 4.4 ×10² ± 1.6 × 10² CFU/ml. These results clearly demonstrate that the reduction in the bacterial load was significant (P < 0.0001 with respect to the results for the controls) when the animals were treated jointly with RIP and antibiotics.

From these data, we demonstrate that a biofilm is well formed by the second day of graft infection, making it possible to treat the animal from day 2, excise the graft by day 10, and test whether or not treatment can enable biofilm eradication. Treatment of rats with RIP was most effective when RIP was administered in multiple doses, with suggestions of dose- and duration-dependent effects on biofilm load reduction. We also show that low-dose RIP treatment, when it is combined with antibiotics like teicoplanin, results in augmented activity relative to the activity of either agent alone. These results provide encouraging support for the further evaluation of RIP as a therapeutic agent for device-associated infections, including those caused by drug-resistant strains.