Bacteria of the genusStaphylococcus are a prominent cause of acute and chronic infections. The ability of the staphylococci to establish biofilms has been linked to the persistence of chronic infections, which has drawn considerable interest from researchers over the past decade. Biofilms can be defined as sessile communities of surface-attached cells encased in an extracellular matrix, and treatment of bacteria in this mode of growth is challenging due to the resistance of biofilm structures to both antimicrobials and host defenses. In this review of the literature, we introduce Staphylococcus aureus and Staphylococcus epidermidis biofilms and summarize current antibiotic treatment approaches for staphylococcal biofilm infections. We also review recent studies on alternative strategies for preventingbiofilmformationanddispersingestablishedbiofilms,includingmatrix-degradingenzymes,small-molecule approaches, and manipulation of natural staphylococcal disassembly mechanisms. While research on staphylococcal biofilm development is still in its early stages, new discoveries in the field hold promise for improved therapies that target staphylococcal biofilm infections.

New approaches for treating staphylococcal biofilm infections.

Objectives: Isolates of Pseudomonas aeruginosa from cystic fibrosis (CF) patients are frequently hypermutable due to selection of mutants with defects in DNA repair genes such as mutS. Since P. aeruginosa grows as a biofilm within the infected CF lung, it is possible that this mode of growth enhances the mutability of the organism thereby increasing the opportunity to derive permanent hypermutators through mutation in DNA repair genes. We have now conducted experiments to examine this possibility. Methods: Using established procedures, we examined the mutability of P. aeruginosa PA01 in planktonic cultures and in biofilm cultures generated by growth in a Sorbarod system. Transcriptional profiling by DNA microarray was used to compare gene expression in planktonic and biofilm cells. Results: Mutation frequency determinations for resistance to rifampicin and ciprofloxacin demonstrated that biofilm cultures of P. aeruginosa displayed up to a 105-fold increase in mutability compared with planktonic cultures. Several genes (ahpC, katA, sodB and PA3529, a probable peroxidase) that encode enzymes conferring protection against oxidative DNA damage were down-regulated in biofilm cells. In particular, katA, which encodes the major pseudomonal antioxidant catalase, was down-regulated 7.7-fold. Conclusions: Down-regulation of antioxidant enzymes in P. aeruginosa biofilms may enhance the rate of mutagenic events due to the accumulation of DNA damage. Since P. aeruginosa forms biofilms in the CF lung, this mode of growth may enhance the direct selection of antibiotic-resistant organisms in CF patients and also increase the opportunity to derive permanent hypermutators thereby providing a further source of antibiotic-resistant mutants in the CF lung.

Increased mutability of Pseudomonas aeruginosa in biofilms

Central venous catheter (CVC)-related infection (CVC-RI) is a common complication of CVC use. The most common etiological agents of CVC-RI are gram-positive organisms, in particular, staphylococci. An in vitro model for the formation of biofilms by Staphylococcus epidermidis ATCC 35984 on polyurethane coupons in a modified Robbins device was established. Biofilm formation was confirmed by electron microscopy and was quantified by determination of viable counts. Mueller-Hinton broth was replaced with sterile physiological saline (control) or a solution of vancomycin (10 mg/ml), gentamicin (10 mg/ml), linezolid (2 mg/ml), or eperezolid (4 mg/ml). Viable counts were performed with the coupons after exposure to antimicrobials for periods of 24, 72, 168, and 240 h. The mean viable count per coupon following establishment of the biofilm was 4.6 x 10(8) CFU/coupon, and that after 14 days of exposure to physiological saline was 2.5 x 10(7) CFU/coupon. On exposure to vancomycin (10 mg/ml), the mean counts were 2.5 x 10(7) CFU/coupon at 24 h, 4.3 x 10(6) CFU/coupon at 72 h, 1.4 x 10(5) CFU/coupon at 168 h, and undetectable at 240 h. With gentamicin (10 mg/ml) the mean counts were 2.7 x 10(7) CFU/coupon at 24 h, 3.7 x 10(6) CFU/coupon at 72 h, 8.4 x 10(6) CFU/coupon at 168 h, and 6.5 x 10(6) CFU/coupon at 240 h. With linezolid at 2 mg/ml the mean counts were 7.1 x 10(5) CFU/coupon at 24 h and not detectable at 72, 168, and 240 h. With eperezolid (4 mg/ml) no viable cells were recovered after 168 h. These data suggest that linezolid (2 mg/ml) and eperezolid (4 mg/ml) achieve eradication of S. epidermidis biofilms more rapidly than vancomycin (10 mg/ml) and gentamicin (10 mg/ml).

Linezolid Compared with Eperezolid, Vancomycin, and Gentamicin in an In Vitro Model of Antimicrobial Lock Therapy for Staphylococcus epidermidis Central Venous Catheter-Related Biofilm Infections

Background. Catheter lock solutions are used for prevention and management of catheter-related bloodstream infections. We investigated the activity of daptomycin and vancomycin lock solutions against Staphylococcus aureus and Staphylococcus epidermidis in an in vitro central venous catheter (CVC) model. Methods. Biofilm-producing reference strains of S. aureus and S. epidermidis were evaluated. After 24 h of bacterial growth in a CVC model, daptomycin and vancomycin bactericidal activity (þ/� preservative-containing heparin sodium) were separately evaluated as a lock solution using 0.5, 1 and 5 mg/ml. Calcium carbonate (50 mg/l) was added to all lock solutions containing daptomycin. Each CVC was drained, flushed and sonicated at 72 h to assess CFU/ml. Results. After 72 h of exposure in the catheter lock solutions, daptomycin and vancomycin at 0.5, 1 and 5 mg/ml demonstrated bactericidal activity (>3.0 log10 CFU/ml) against S. aureus and S. epidermidis (P � 0.001). Heparin lock solution alone produced a non-significant reduction in S. aureus and S. epidermidis (1.92 � 0.07 and 1.65 � 0.03 log10 CFU/ml, respectively). Daptomycin 5 mg/ml lock solution þ/� heparin eradicated (limit of detection 2.0 log10 CFU/ml) S. epidermidis at 72 h as did the vancomycin 5 mg/ml plus heparin. S. aureus was only eradicated from the daptomycin 5 mg/ml catheter lock-solution. Conclusions. Our CVC model demonstrated that 72 h of exposure to 5 mg/ml lock solutions of daptomycin (plus calcium), þ/� heparin or 5 mg/ml of vancomycin plus heparin demonstrate promise in treating catheter infections with biofilm-producing S. epidermidis. Similarly, 5 mg/ml of daptomycin (plus calcium) as a

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In vitro activity of daptomycin and vancomycin lock solutions on staphylococcal biofilms in a central venous catheter model

Bacterial biofilm formation is a major complication of implantable medical devices that results in therapeutically challenging chronic infections, especially in cases involving antibiotic-resistant bacteria. As an approach to prevent these infections, an electrospun composite coating comprised of poly(lactic-coglycolic acid) (PLGA) nanofibers embedded in a poly(e-caprolactone) (PCL) film was developed to locally codeliver combinatorial antibiotics from the implant surface. The release of each antibiotic could be adjusted by loading each drug into the different polymers or by varying PLGA:PCL polymer ratios. In a mouse model of biofilm-associated orthopedic-implant infection, three different combinations of antibiotic-loaded coatings were highly effective in preventing infection of the bone/joint tissue and implant biofilm formation and were biocompatible with enhanced osseointegration. This nanofiber composite-coating technology could be used to tailor the delivery of combinatorial antimicrobial agents from various metallic implantable devices or prostheses to effectively decrease biofilm-associated infections in patients.

https://www.pnas.org/content/pnas/113/45/E6919.full.pdf

Polymeric nanofiber coating with tunable combinatorial antibiotic delivery prevents biofilm-associated infection in vivo

Biofilms of methicillin-susceptible and -resistant Staphylococcus aureus, a strain of coagulase-negative staphylococcus and glycopeptide-intermediate strains of S. aureus (GISA) were exposed to the oxazolidinone linezolid, and four comparator antibiotics (quinupristin/ dalfopristin, vancomycin, teicoplanin and ciprofloxacin) using a Sorbarod model. The effects of these antibiotics were assessed by monitoring the reduction in the number of cells eluted from the biofilms. The biofilms were exposed to the antibiotics by two methods. The first was an exponentially decreasing drug concentration method, where the rate of dilution was matched to the half-lives of the antibiotics and the initial concentration matched peak serum levels. The second was a constant drug concentration method, in which biofilms were exposed to antibiotics for 2 h, with the concentration of the antibiotic equalling the total amount of drug used in the exponentially decreasing method. The results indicate that linezolid produces a greater reduction in the number of cells eluted with the exponentially decreasing method compared with the constant concentration exposure against all strains tested except for one of the GISA strains, Mu 50. Overall, ciprofloxacin produced the greatest effects in the exponentially decreasing concentration experiments, but only against non-resistant strains. In the constant concentration exposure no one drug was responsible for the largest reductions in cell numbers observed. Linezolid and quinupristin/dalfopristin produced a reduction in the number of cells eluted from the biofilms of all of the strains tested in both methods of exposure and should be considered for further clinical studies of the treatment of staphylococcal biofilm-associated infections.

Katy Hayward

Papers

An investigation of the antimicrobial effects of linezolid on bacterial biofilms utilizing an in vitro pharmacokinetic model