Suzannah M. Schmidt-Malan

Comparative activities of vancomycin, tigecycline and rifampin in a rat model of methicillin-resistant Staphylococcus aureus osteomyelitis

OBJECTIVES Implant-associated methicillin-resistant Staphylococcus aureus (MRSA) infections are challenging to treat. We compared antimicrobial activities in a rat model of chronic osteomyelitis in the context of retention of the foreign body without débridement. METHODS MRSA was inoculated into the proximal tibia and a wire implanted. Four weeks after infection, treatment with vancomycin 50 mg/kg every 12 h, tigecycline 14 mg/kg every 12 h, rifampin 25 mg/kg every 12 h, or the combination of vancomycin or tigecycline plus rifampin was administered intraperitoneally for 21 days. RESULTS MRSA was cultured from all tibias in the control group (median, 6.06 log10 CFU/g bone). Median bacterial counts (log10 CFU/g) at 48 h post-treatment were 6.16 for vancomycin (p = 0.753), 2.29 for vancomycin plus rifampin (p < 0.001), 5.90 for tigecycline (p = 0.270), 0.10 for tigecycline plus rifampin (p < 0.001), and 0.91 for rifampin (p = 0.044) treatment. Three deaths were observed in the tigecycline plus rifampin group. Median bacterial counts (log10 CFU/g) at two weeks post-treatment were 5.65 for vancomycin (p = 0.6), 4.05 for vancomycin plus rifampin (p = 0.105), 5.68 for tigecycline (p = 0.401), 4.05 for tigecycline plus rifampin (p = 0.028), and 5.98 for rifampin (p = 0.297) treatment. CONCLUSIONS Tigecycline plus rifampin resulted in a significant bacterial count decrease, an effect more prominent at 48 h than two weeks after treatment completion. Tigecycline was not well tolerated at the dose studied.

Antibiofilm Activity of Low-Amperage Continuous and Intermittent Direct Electrical Current

ABSTRACT Bacterial biofilms are difficult to treat using available antimicrobial agents, so new antibiofilm strategies are needed. We previously showed that 20, 200, and 2,000 μA of electrical current reduced bacterial biofilms of Staphylococcus aureus, Staphylococcus epidermidis, and Pseudomonas aeruginosa. Here, we tested continuous direct current at lower amperages, intermittent direct current, and combinations of surface materials (Teflon or titanium) and electrode compositions (stainless steel, graphite, titanium, or platinum) against S. aureus, S. epidermidis, and P. aeruginosa biofilms. In addition, we tested 200 or 2,000 μA for 1 and 4 days against biofilms of 33 strains representing 13 species of microorganisms. The logarithmic reduction factor was used to measure treatment effects. Using continuous current delivery, the lowest active amperage was 2 μA for 1, 4, or 7 days against P. aeruginosa and 5 μA for 7 days against S. epidermidis and S. aureus biofilms. Delivery of 200 μA for 4 h a day over 4 days reduced P. aeruginosa, S. aureus, and S. epidermidis biofilms on Teflon or titanium discs. A reduction of P. aeruginosa, S. aureus, and S. epidermidis biofilms was measured for 23 of 24 combinations of surface materials and electrode compositions tested. Four days of direct current delivery reduced biofilms of 25 of 33 strains studied. In conclusion, low-amperage current or 4 h a day of intermittent current delivered using a variety of electrode compositions reduced P. aeruginosa, S. aureus, and S. epidermidis biofilms on a variety of surface materials. The electricidal effect was observed against a majority of bacterial species studied.

In vitro activity of tedizolid against staphylococci isolated from prosthetic joint infections.

We determined the MIC and minimum biofilm bactericidal concentration (MBBC) of tedizolid and vancomycin against 97 isolates of Staphylococcus aureus and 74 isolates of Staphylococcus epidermidis associated with prosthetic joint infection. All isolates were vancomycin susceptible in the planktonic state; all staphylococci studied had a tedizolid MIC ≤0.5 μg/mL. The MBBC90 was >32 and >128 μg/mL for tedizolid and vancomycin, respectively.

In vitro activity of ceftolozane/tazobactam against clinical isolates of Pseudomonas aeruginosa in the planktonic and biofilm states.

Pseudomonas aeruginosa causes a variety of life-threatening infections, some of which are associated with planktonic and others with biofilm states. Herein, we tested the combination of the novel cephalosporin, ceftolozane, with the β-lactamase inhibitor, tazobactam, against planktonic and biofilm forms of 54 clinical isolates of P. aeruginosa, using cefepime as a comparator. MIC values were determined following Clinical and Laboratory Standards Institute (CLSI) guidelines. Minimum biofilm inhibitory concentration (MBIC) values were determined using biofilm-laden pegged lids incubated in antimicrobial challenge plates containing varying concentrations of ceftolozane/tazobactam. Pegged lids were then incubated in growth recovery plates containing cation-adjusted Mueller-Hinton broth to determine the minimum biofilm bactericidal concentration (MBBC). Ceftolozane/tazobactam was highly active against planktonic P. aeruginosa, with all 54 isolates studied testing susceptible (MIC ≤4/4μg/mL). On the other hand, 51/54 biofilm P. aeruginosa had MBICs ≥16/4μg/mL, and all 54 isolates had MBBCs >32μg/mL. Of the 54 isolates, 45 (83.3%) tested susceptible to cefepime, with the MIC50/MIC90 being 4/16μg/mL, respectively, and the MBIC90 and MBBC90 both being >256μg/mL. Although ceftolozane/tazobactam is a promising antimicrobial agent for the treatment of P. aeruginosa infections, it is not highly active against P. aeruginosa biofilms.

Treatment of Methicillin-resistant Staphylococcus aureus experimental Osteomyelitis with bone-targeted Vancomycin

IntroductionMethicillin-resistant S. aureus (MRSA) is a common cause of bone and joint infection. BT2-peg2-vancomycin is an investigational bone-targeted formulation of vancomycin which we hypothesized would have increased antimicrobial activity compared to conventional vancomycin in a chronic experimental MRSA osteomyelitis model.MethodsWe tested bone affinity using an hydroxyapatite (HA) binding assay, assessed the in vitro antimicrobial susceptibility of 30 MRSA isolates, and compared vancomycin and BT2-peg2-vancomycin in a rat experimental osteomyelitis model.ResultsVancomycin did not bind to hydroxyapatite (HA binding index = 0), whereas BT2-peg2-vancomycin showed appreciable binding (HA binding index = 57). The MIC50 was 1 μg/ml and the MIC90 was 2 μg/ml for both vancomycin and BT2-peg2-vancomycin. The MBC90 was 16 and 4 μg/ml for vancomycin and BT2-peg2-vancomycin, respectively. Treatment with 50 mg/kg of vancomycin every 12 hours (median, 4.73 log10 cfu/g), 63.85 mg/kg (equivalent to 50 mg/kg vancomycin) of BT2-peg2-vancomycin every 12 hours (median, 3.93 log10 cfu/g) or 63.85 mg/kg of BT2-peg2-vancomycin once per week (median, 5.00 log10 cfu/g) was more active than no treatment (median, 5.22 log10 cfu/g) (P =0.0481). Treatment with 63.85 mg/kg of BT2-peg2-vancomycin every 12 hours was more active than all other treatment regimens evaluated (P≤0.0150), but was associated with high plasma BT2-peg2-vancomycin levels, decreased animal weight, increased kidney size, creatinine and BUN, and leukocytosis with tubulointerstitial nephritis.ConclusionWith optimization of pharmacokinetic parameters to prevent toxicity, BT2-peg2-vancomycin may be useful in the treatment of MRSA osteomyelitis.

Antibiofilm Activity of Electrical Current in a Catheter Model

ABSTRACT Catheter-associated infections are difficult to treat with available antimicrobial agents because of their biofilm etiology. We examined the effect of low-amperage direct electrical current (DC) exposure on established bacterial and fungal biofilms in a novel experimental in vitro catheter model. Staphylococcus epidermidis, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and Candida parapsilosis biofilms were grown on the inside surfaces of polyvinyl chloride (PVC) catheters, after which 0, 100, 200, or 500 μA of DC was delivered via intraluminally placed platinum electrodes. Catheter biofilms and intraluminal fluid were quantitatively cultured after 24 h and 4 days of DC exposure. Time- and dose-dependent biofilm killing was observed with all amperages and durations of DC administration. Twenty-four hours of 500 μA of DC sterilized the intraluminal fluid for all bacterial species studied; no viable bacteria were detected after treatment of S. epidermidis and S. aureus biofilms with 500 μA of DC for 4 days.

Direct Electrical Current Reduces Bacterial and Yeast Biofilm Formation

New strategies are needed for prevention of biofilm formation. We have previously shown that 24 hr of 2,000 µA of direct current (DC) reduces Staphylococcus epidermidis biofilm formation in vitro. Herein, we examined the effect of a lower amount of DC exposure on S. epidermidis, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Propionibacterium acnes, and Candida albicans biofilm formation. 12 hr of 500 µA DC decreased S. epidermidis, S. aureus, E. coli, and P. aeruginosa biofilm formation on Teflon discs by 2, 1, 1, and 2 log10 cfu/cm2, respectively (p < 0.05). Reductions in S. epidermidis, S. aureus, and E. coli biofilm formation were observed with as few as 12 hr of 200 µA DC (2, 2 and 0.4 log10 cfu/cm2, resp.); a 1 log10 cfu/cm2 reduction in P. aeruginosa biofilm formation was observed at 36 hr. 24 hr of 500 µA DC decreased C. albicans biofilm formation on Teflon discs by 2 log10 cfu/cm2. No reduction in P. acnes biofilm formation was observed. 1 and 2 log10 cfu/cm2 reductions in E. coli and S. epidermidis biofilm formation on titanium discs, respectively, were observed with 12 hr of exposure to 500 µA. Electrical current is a potential strategy to reduce biofilm formation on medical biomaterials.

Dalbavancin is active in vitro against biofilms formed by dalbavancin-susceptible enterococci

We tested the in vitro activity of dalbavancin, vancomycin and daptomycin against 83 enterococcal isolates in planktonic and biofilm states. The MIC90 for vancomycin-susceptible Enterococcus faecalis was 0.125 and 4μg/mL for dalbavancin and daptomycin, respectively. For vancomycin-resistant Enterococcus faecium, the MIC90 was >16 and 2μg/mL for dalbavancin and daptomycin, respectively. Dalbavancin minimum biofilm inhibitory concentrations (MBICs) for vancomycin-susceptible and -resistant isolates were ≤0.25 and >16μg/mL, respectively. The daptomycin MBIC90 for all isolates was 4μg/mL. For E. faecalis and E. faecium, dalbavancin minimum biofilm bactericidal concentrations (MBBCs) for vancomycin-susceptible and -resistant isolates were ≤4 and >16μg/mL, respectively, whereas vancomycin MBBCs were >128μg/mL for all isolates, and daptomycin MBBC90 values for both species were 128μg/mL. In summary, dalbavancin exhibited in vitro activity against all tested isolates of vancomycin-susceptible, but not against vancomycin-resistant enterococci; activity was observed in both the planktonic and biofilm states.

Causes and Implications of the Disappearance of Rifampin Resistance in a Rat Model of Methicillin-Resistant Staphylococcus aureus Foreign Body Osteomyelitis

ABSTRACT Orthopedic foreign body-associated infections are often treated with rifampin-based combination antimicrobial therapy. We previously observed that rifampin-resistant and methicillin-resistant Staphylococcus aureus (MRSA) isolates were present 2 days after cessation of rifampin therapy in experimental foreign body osteomyelitis. Unexpectedly, only rifampin-susceptible isolates were detected 14 days after the completion of treatment. We studied two rifampin-resistant isolates recovered 2 days after treatment and one rifampin-susceptible isolate recovered 14 days after treatment. Growing these isolates alone in vitro or in vivo demonstrated no fitness defects; however, in mixed culture, rifampin-susceptible bacteria outcompeted rifampin-resistant bacteria. In vivo, two courses of rifampin treatment (25 mg/kg of body weight every 12 h for 21 days) yielded a greater decrease in bacterial quantity in the bones of treated animals 14 days following treatment than that in animals receiving a single course of treatment (P = 0.0398). In infections established with equal numbers of rifampin-resistant and rifampin-susceptible bacteria, one course of rifampin treatment did not affect bacterial quantities. Rifampin-resistant and rifampin-susceptible isolates were recovered both 2 days and 14 days following treatment completion; however, the proportion of animals with rifampin-resistant isolates was lower at 14 days than that at 2 days following treatment completion (P = 0.024). In untreated animals infected with equal numbers of rifampin-resistant and rifampin-susceptible bacteria for 4 weeks, rifampin-susceptible isolates were exclusively recovered, indicating the outcompetition of rifampin-resistant by rifampin-susceptible isolates. The data presented imply that although there is no apparent fitness defect in rifampin-resistant bacteria when grown alone, they are outcompeted by rifampin-susceptible bacteria when the two are present together. The findings also suggest that selected rifampin resistance may not persist in initially rifampin-susceptible infections following the discontinuation of rifampin.

Exposure of bacterial biofilms to electrical current leads to cell death mediated in part by reactive oxygen species

Bacterial biofilms may form on indwelling medical devices such as prosthetic joints, heart valves and catheters, causing challenging-to-treat infections. We have previously described the ‘electricidal effect’, in which bacterial biofilms are decreased following exposure to direct electrical current. Herein, we sought to determine if the decreased bacterial quantities are due to detachment of biofilms or cell death and to investigate the role that reactive oxygen species (ROS) play in the observed effect. Using confocal and electron microscopy and flow cytometry, we found that direct current (DC) leads to cell death and changes in the architecture of biofilms formed by Gram-positive and Gram-negative bacteria. Reactive oxygen species (ROS) appear to play a role in DC-associated cell death, as there was an increase in ROS-production by Staphylococcus aureus and Staphylococcus epidermidis biofilms following exposure to DC. An increase in the production of ROS response enzymes catalase and superoxide dismutase (SOD) was observed for S. aureus, S. epidermidis and Pseudomonas aeruginosa biofilms following exposure to DC. Additionally, biofilms were protected from cell death when supplemented with antioxidants and oxidant scavengers, including catalase, mannitol and Tempol. Knocking out SOD (sodAB) in P. aeruginosa led to an enhanced DC effect. Microarray analysis of P. aeruginosa PAO1 showed transcriptional changes in genes related to the stress response and cell death. In conclusion, the electricidal effect results in death of bacteria in biofilms, mediated, at least in part, by production of ROS.