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Basic science. Investigate the ability of local applicaiton of vancomycin, either in powder form or suspended within poly(lactic-co-glycolic acid) microspheres (MS), to treat infection using a rat spinal model. Surgical site infections (SSIs) are a serious complication after spine surgery and are associated with high morbidity and mortality and often caused my coagulase negative staphylococci. A comprehensive approach to reduce SSIs has been recommended including the use of topical vancomycin. Animal and human studies have shown improved control of infection with local compared to systemic antibiotics. K-wires seeded with methicillin-resistant Staphylococcus epidermidis RP62A (MRSE) were treated with vancomycin powder, carboxymethylcellulose sodium salt (CMC) (microsphere carrier), vancomycin powder, blank MS or vancomycin-loaded MS for 24 or 48 h in vitro after which bacteria were enumerated. In addition, a spinal instrumentation model was developed in rats with a bacterial seeded K-wire implanted into the right side of L4 and L5. Rats underwent no treatment or were treated locally with either vancomycin powder, blank MS or vancomycin-loaded MS. After 8 weeks, the K-wire, bone, soft tissue and wire fastener were cultured and results analyzed. Vancomycin powder and vancomycin-loaded MS resulted in significantly fewer bacteria remaining in vitro than did CMC. Vancomycin powder- treated animals’ cultures were significantly lower than all other groups (P

Omadacycline, vancomycin, and rifampin, as well as rifampin combination therapies, were evaluated in an experimental rat model of methicillin-resistant Staphylococcus aureus (MRSA) osteomyelitis. All treatment groups had less MRSA recovered than saline-treated animals. The emergence of rifampin resistance was observed in 3 of 16 animals with rifampin monotherapy and none with rifampin combination therapy. After treatment, the median tibial bacterial loads were 6.04, 0.1, 4.81, and 5.24 log10 CFU/g for saline-, rifampin-, vancomycin-, and omadacycline-treated animals, respectively. Omadacycline or vancomycin administered with rifampin yielded no detectable MRSA. Omadacycline administered with rifampin deserves evaluation in humans as a potential treatment for osteomyelitis.

Drug-resistant Gram-positive bacterial infections are still a substantial burden on the public health system, with two bacteria ( Staphylococcus aureus and Streptococcus pneumoniae ) accounting for over 1.5 million drug-resistant infections in the United States alone in 2017. In 2019, 250,000 deaths were attributed to these pathogens globally. We have developed a preclinical glycopeptide antibiotic, MCC5145, that has excellent potency (MIC 90 ≤ 0.06 μg/ml) against hundreds of isolates of methicillin-resistant S. aureus (MRSA) and other Gram-positive bacteria, with a greater than 1000-fold margin over mammalian cell cytotoxicity values. The antibiotic has therapeutic in vivo efficacy when dosed subcutaneously in multiple murine models of established bacterial infections, including thigh infection with MRSA and blood septicemia with S. pneumoniae , as well as when dosed orally in an antibiotic-induced Clostridioides difficile infection model. MCC5145 exhibited reduced nephrotoxicity at microbiologically active doses in mice compared to vancomycin. MCC5145 also showed improved activity against biofilms compared to vancomycin, both in vitro and in vivo, and a low propensity to select for drug resistance. Characterization of drug action using a transposon library bioinformatic platform showed a mechanistic distinction from other glycopeptide antibiotics.

Microbial communities provide protection to their hosts by resisting pathogenic invasion. Microbial residents of a host often exclude subsequent colonizers, but this protection is not well understood. The Enterococcus faecalis plasmid pCF10, whose conjugative transfer functions are induced by a peptide pheromone, efficiently transfers in the intestinal tract of mice. Here we show that an invading donor strain established in the gastrointestinal tract of mice harboring resident recipients, resulting in a stable, mixed population comprised of approximately 10% donors and 90% recipients. We also show that the plasmid-encoded surface protein PrgB (Aggregation Substance), enhanced donor invasion of resident recipients, and resistance of resident donors to invasion by recipients. Imaging of the gastrointestinal mucosa of mice infected with differentially labeled recipients and donors revealed pheromone induction within microcolonies harboring both strains in close proximity, suggesting that adherent microcolonies on the mucosal surface of the intestine comprise an important niche for cell-cell signaling and plasmid transfer.

After staphylococci, streptococci and enterococci are the most frequent causes of periprosthetic joint infection (PJI). MICs and minimum biofilm bactericidal concentrations of rifampin, rifabutin, and rifapentine were determined for 67 enterococcal and 59 streptococcal PJI isolates. Eighty-eight isolates had rifampin MICs of ≤1 μg/ml, among which rifabutin and rifapentine MICs were ≤ 8 and ≤4 μg/ml, respectively. There was low rifamycin in vitro antibiofilm activity except for a subset of Streptococcus mitis group isolates. IMPORTANCE Rifampin is an antibiotic with antistaphylococcal biofilm activity used in the management of staphylococcal periprosthetic joint infection with irrigation and debridement with component retention; some patients are unable to receive rifampin due to drug interactions or intolerance. We recently showed rifabutin and rifapentine to have in vitro activity against planktonic and biofilm states of rifampin-susceptible periprosthetic joint infection-associated staphylococci. After staphylococci, streptococci and enterococci combined are the most common causes of periprosthetic joint infection. Here, we investigated the in vitro antibiofilm activity of rifampin, rifabutin, and rifapentine against 126 Streptococcus and Enterococcus periprosthetic joint infection isolates. In contrast to our prior findings with staphylococcal biofilms, there was low antibiofilm activity of rifampin, rifabutin, and rifapentine against PJI-associated streptococci and enterococci, apart from some Streptococcus mitis group isolates.

The most common organism-type causing orthopedic foreign body infection is the staphylococci, of which Staphylococcus aureus and Staphylococcus epidermidis are especially common. These organisms form biofilms on orthopedic foreign body surfaces, rendering such infections challenging and time consuming to treat. Our group evaluates novel therapeutics for orthopedic foreign body infection in animal models. A current limitation of most animal models is that that they only allow for the removal of one sample per animal, at the time of sacrifice. Herein, we describe a novel rat model of foreign body osteomyelitis that allows removal of foreign bodies at different time points, from the same infected animal. We demonstrate that this model can be used for both S. aureus and S. epidermidis orthopedic foreign body infection, with 3.56, 3.60 and 5.51 log10 cfu/cm2 S. aureus recovered at four, five and six weeks, respectively, after infection, and 2.08, 2.17 and 2.62 log10 cfu/cm2 S. epidermidis recovered at four, five and six weeks, respectively, after infection. We evaluated the model with S. aureus infection treated with rifampin 25 mg/kg twice daily for 21 days. Using quantitative cultures, we were no longer able to detect bacteria as of the 14th day of treatment with bacteria becoming detectable again 7 days following the discontinuation of rifampin a period. This novel model allows monitoring of evolution of infection at the infection site in the same animal.

MRSA periprosthetic 1 joint infection (PJI) can be challenging to treat due to biofilm formation, alongside sometimes limited vancomycin activity (1-3).

Introduction: Antibiotic envelopes are being developed for cardiac implantable electronic device (CIED) wrapping to reduce the risk of infections. Methods: Fifteen CIED infection-associated bacterial isolates of Staphylococcus aureus, Staphylococcus epidermidis and Cutibacterium acnes were used to assess in vitro biofilm formation on Hylomate® compared to titanium, silicone and polyurethane coupons pre-treated with vancomycin (400 µg/ml), bacitracin (1000 U/ml) or a combination of rifampin (80 µg/ml) plus minocycline (50 µg/ml). Scanning electron microscopy (SEM) was performed to visualize bacteria on Hylomate®. Results: There was significantly less (p

Cefiderocol (CFDC) is a siderophore cephalosporin with activity against Gram-negative bacterial species that are resistant to carbapenems and other drugs. The MICs of CFDC were determined for 610 Gram-negative bacilli, including 302 multinational Enterobacterales isolates with characterized mechanisms of beta-lactam resistance, 180 clinical isolates from the Mayo Clinic and Mayo Clinic Laboratories not characterized for specific resistance mechanisms, and 128 isolates with CFDC MICs of ≥8 μg/ml obtained from International Health Management Associates, Inc. (IHMA, Schaumburg, IL). Broth microdilution using standard cation-adjusted Mueller-Hinton broth (BMD) and iron-depleted cation-adjusted Mueller-Hinton broth (ID-BMD), and agar dilution (AD) using standard Mueller-Hinton agar were performed according to Clinical and Laboratory Standards Institute (CLSI) guidelines. MICs were interpreted according to the investigational CLSI, FDA, and EUCAST breakpoints, and results were compared. MICs inhibiting 50 and 90% of organisms (MIC50 and MIC90, respectively), essential agreement (EA), categorical agreement (CA), and error of different types were determined. Results showed considerable discordance between AD and ID-BMD. CFDC showed low EA and CA rates and high error rates for AD in comparison to ID-BMD. Overall, this study does not support use of standard AD for determining CFDC MICs.

Ureaplasma species, including Ureaplasma parvum and Ureaplasma urealyticum, are challenging to culture and maintain. Here, we describe a novel bioreactor for growing high-titer liquid Ureaplasma cultures in a stable manner.

Cefiderocol (CFDC) is a siderophore cephalosporin with activity against Gram-negative bacterial species that are resistant to carbapenems and other drugs. The MICs of CFDC were determined for 610 Gram-negative bacilli, including 302 multinational Enterobacterales isolates with characterized mechanisms of beta-lactam resistance, 180 clinical isolates from the Mayo Clinic and Mayo Clinic Laboratories not characterized for specific resistance mechanisms, and 128 isolates with CFDC MICs of ≥8 μg/ml obtained from International Health Management Associates, Inc., Cefiderocol (CFDC) is a siderophore cephalosporin with activity against Gram-negative bacterial species that are resistant to carbapenems and other drugs. The MICs of CFDC were determined for 610 Gram-negative bacilli, including 302 multinational Enterobacterales isolates with characterized mechanisms of beta-lactam resistance, 180 clinical isolates from the Mayo Clinic and Mayo Clinic Laboratories not characterized for specific resistance mechanisms, and 128 isolates with CFDC MICs of ≥8 μg/ml obtained from International Health Management Associates, Inc. (IHMA, Schaumburg, IL). Broth microdilution using standard cation-adjusted Mueller-Hinton broth (BMD) and iron-depleted cation-adjusted Mueller-Hinton broth (ID-BMD), and agar dilution (AD) using standard Mueller-Hinton agar were performed according to Clinical and Laboratory Standards Institute (CLSI) guidelines. MICs were interpreted according to the investigational CLSI, FDA, and EUCAST breakpoints, and results were compared. MICs inhibiting 50 and 90% of organisms (MIC50 and MIC90, respectively), essential agreement (EA), categorical agreement (CA), and error of different types were determined. Results showed considerable discordance between AD and ID-BMD. CFDC showed low EA and CA rates and high error rates for AD in comparison to ID-BMD. Overall, this study does not support use of standard AD for determining CFDC MICs.

Background Owing to patient intolerance or drug interactions, alternative agents to rifampin are needed for management of staphylococcal periprosthetic joint infection. In the current study, we evaluated rifabutin, rifapentine and rifampin, with and without vancomycin, in a rat model of foreign body osteomyelitis. Methods Proximal tibiae were inoculated with methicillin-resistant Staphylococcus aureus (MRSA) and a Kirschner wire (K-wire) implanted in each. After 4 weeks of infection, rifampin, rifabutin, or rifapentine were administered, alone or with vancomycin. Tibiae and K-wires were cultured, and medians were reported as log10 colony-forming units (CFUs) per gram of bone or log10 CFUs per K-wire, respectively. Results Rifampin, rifabutin or rifapentine administered with vancomycin yielded less MRSA from bones (0.10, 3.02, and 0.10 log10 CFUs/g, respectively) than did no treatment (4.36 log10 CFUs/g) or vancomycin alone (4.64 log10 CFUs/g) (both P ≤ .02). The K-wires of animals receiving no treatment or vancomycin monotherapy recovered medians of 1.76 and 2.91 log10 CFUs/g per K-wire, respectively. In contrast, rifampin, rifabutin and rifapentine administered with vancomycin yielded medians of 0.1 log10 CFUs per K-wire, respectively. Rifampin resistance was detected in a single animal in the rifampin monotherapy group. Conclusions Rifabutin or rifapentine with vancomycin were as active as rifampin with vancomycin against MRSA in rat foreign body osteomyelitis, suggesting that rifabutin and/or rifapentine may be alternatives to rifampin in the clinical management of staphylococcal periprosthetic joint infections.

This study evaluated the in vitro activity of oritavancin in combination with rifampin or gentamicin against methicillin-resistant Staphylococcus epidermidis (MRSE) biofilms. Oritavancin, rifampin, and gentamicin were tested against 20 MRSE isolates recovered from prosthetic joint infection (PJI). Time-kill studies were used to evaluate the activities of the three antimicrobial agents individually vs. combinations of oritavancin with rifampin or gentamicin against MRSE biofilms formed on Teflon coupons. At 24 h, the combination of oritavancin and rifampin resulted in a significant (P≤0.001) reduction in biofilm density compared with either antimicrobial alone for 85% (17/20) of isolates. Oritavancin combined with gentamicin showed a significant (P≤0.001) reduction in biofilm density compared with either antimicrobial alone against 55% (11/20) of isolates at 24 h. Synergy (defined as a ≥2 log10 cfu/cm2 decrease at 24 h for the antimicrobial combination compared with the most active single antimicrobial) was observed against 65% (13/20) of the isolates for oritavancin in combination with rifampin and 35% (7/20) of the isolates for oritavancin in combination with gentamicin. Oritavancin in combination with rifampin or gentamicin demonstrated bactericidal activity (defined as a ≥3 log10 cfu/cm2 reduction at 24 h from the starting biofilm bacterial density) for 85% (17/20) and 80% (16/20) isolates, respectively. Our study suggests that oritavancin and rifampin combination therapy may be an option for antimicrobial management of PJIs caused by MRSE.

Clay is a substance historically utilized by indigenous cultures for the treatment of superficial wound infections. This study evaluated the effects of a recently identified clay – OMT Blue Clay – against staphylococci, streptococci, Enterobacteriaceae and non-fermenting Gram-negative bacilli. The clay and its aqueous leachate were evaluated against the bacteria in biofilm and planktonic states. Time-kill studies were used to assess planktonic activity. Biofilms on medical-grade Teflon discs were treated with a hydrated clay suspension or leachate. For the planktonic studies, clay and leachate exhibited bactericidal activity against all strains tested, with the exception of leachate against Staphylococcus aureus IDRL-6169 and USA300. All strains treated with clay suspension and leachate resulted in statistically significant biofilm population reductions compared with controls, except S. aureus IDRL-6169 and USA300 (P ≤ 0.05). OMT Blue Clay and its aqueous leachate exhibited bactericidal activity against a range of human pathogens in the planktonic and biofilm states.

Background Disclosing agents are dyes used in dentistry to colorize plaque (biofilm) and may offer a means for intraoperative detection of biofilms on orthopedic implants. Methylene blue (MB) stains biofilm and is safely used in orthopedic applications. Injection of MB into acutely infected prosthetic knees before debridement may enable visualization of biofilm, which could influence treatment decisions. The aims of this study were to determine if MB could be used to visualize biofilm on total knee arthroplasty (TKA) implants and to determine if MB staining has an antimicrobial effect that might interfere with subsequent culture. Methods Staphylococcus epidermidis biofilms were formed on TKA polyethylene liners and polymethylmethacrylate (PMMA) and Teflon discs. After staining biofilms on these implants, the bacterial densities were determined through sonication and quantitative culture. The antimicrobial activity of MB staining was determined by measuring the bacterial density of S. epidermidis biofilms on PMMA discs incubated in 0.05% MB for 24 hours vs 30 seconds and comparing it with controls unexposed to MB. Results MB stained S. epidermidis biofilms grown on TKA implants and Teflon and PMMA discs in vitro. Sonication and quantitative culture of the stained implants showed that bacterial densities were at supraphysiological levels. Staining did not affect the ability to culture the organism. Conclusion MB is a possible cost-effective and novel method to expeditiously identify intraoperative biofilm. To further evaluate MB staining and its potential clinical usefulness, future studies are needed to assess the ability of MB to stain physiological levels of biofilm.

Periprosthetic infections are devastating for patients and more efficacious preventive strategies are needed. Surface-modified implants using antibacterial coatings represent an option to cope with this problem; however, manufacturing limitations and cytotoxicity have curbed clinical translation. Among metals with antibacterial properties, copper has shown superior in vitro antibacterial performance while maintaining an acceptable cytotoxicity profile. A thin film containing copper could prevent early biofilm formation to limit periprosthetic infections. This pilot study presents the in vitro antibacterial effect, cytotoxicity, and copper ion elution pattern of a thin film of titanium-copper oxide (TiCuO). (1) Do titanium alloy (Ti6Al4V) discs coated with a thin film of TiCuO reduce Staphylococcus epidermidis biofilm and planktonic cell density compared with uncoated discs? (2) Do Ti6Al4V discs coated with a thin film of TiCuO affect normal human osteoblast viability compared with untreated cells? (3) Is copper ion concentration generated by coated discs lower than previously published copper ion concentrations that cause 50% toxicity in similar human cell lines in vitro (TC50)? Ninety Ti6Al4V discs (12.5 mm diameter; 1.25 mm thick) were used in this study. Seventy-two Ti6Al4V discs were coated with a thin film of either titanium oxide (TiO) or TiCuO containing 20%, 40%, or 80% copper using high-power impulse magnetron sputtering (HiPIMS). Eighteen Ti6Al4V discs remained uncoated for control purposes. We tested antibacterial properties of S epidermidis grown on discs in wells containing growth medium. After 24 hours, planktonic bacteria as well as biofilms removed by sonication were quantitatively cultured. Annexin/Pi staining was used to quantify in vitro normal human osteoblast cell viability at 24 hours and Day 7, respectively. Copper elution was measured at Days 1, 2, 3, 7, 14, and 28 using an inductively coupled plasma mass spectrometer to analyze aliquots of culture medium. Copper ion concentration achieved at 24 hours was compared with previously published TC50 for gingival fibroblast, a phenotypically similar cell line with available data regarding copper ion exposure. Discs coated with TiCuO 80% copper showed greater biofilm and planktonic cell density reduction when compared with other tested compositions (analysis of variance [ANOVA]; p < 0.001). Discs coated with TiCuO 80% copper showed mean biofilm and planktonic cell density of 4.0 log10 (SD = 0.4) and 5.7 log10 (SD = 0.2). Discs coated with TiCuO 80% showed a mean difference in biofilm and planktonic cell density of 2.5 log10 (95% confidence interval [CI], 1.9–3.1 log10; p < 0.001) and 1.2 (95% CI, 0.6–1.8; p < 0.001), respectively, when compared with uncoated discs. Normal human osteoblast viability did not differ among all groups at 24 hours (ANOVA; p = 0.2) and Day 7 (ANOVA; p = 0.7). Discs coated with TiCuO 80% copper showed a mean difference (95% CI) in relative cell viability (%) at 24 hours and Day 7 of 31.1 (95% CI, −19.4 to 81.7; p = 0.4) and −5.0 (95% CI, −7.8 to 17.9; p = 0.9), respectively, when compared with untreated cells. For all TiCuO-coated discs, copper ion elution peaked at 24 hours and slowly decreased in a curvilinear fashion to nearly undetectable levels by Day 28. Discs coated with TiCuO 80% copper showed mean copper ion concentration at 24 hours of 269.4 µmol/L (SD = 25.2 µmol/L) and this concentration was lower than previously published TC50 for similar human cell lines at 24 hours (344 µmol/L, SEM = 44 µmol/L). This pilot study demonstrates a proof of concept that a thin-film implant coating with TiCuO can provide a potent local antibacterial environment while remaining relatively nontoxic to a human osteoblast cell line. Further research in an animal model will be necessary to establish efficacy and safety of this technique and whether it might be useful in the design of implants. A thin film coating with TiCuO demonstrates high antibacterial activity and low cellular cytotoxicity to human osteoblasts in vitro. Taken together, these properties represent a potential strategy for preventing periprosthetic infection if further work in animal models can confirm these results in vivo.

We tested the in vitro activity of oritavancin against 185 staphylococci associated with prosthetic joint infection, including 37 methicillin-resistant S. aureus, 67 methicillin-susceptible S. aureus, 59 methicillin-resistant S. epidermidis (MRSE), and 22 methicillin-susceptible S. epidermidis (MSSE) isolates. The oritavancin MIC

We tested the in vitro activity of oritavancin against 60 vancomycin-susceptible enterococci (VSE) and 27 vancomycin-resistant enterococci (VRE). The oritavancin MIC ranged from ≤0.002 to 0.5μg/mL; the minimum biofilm bactericidal concentration ranged from ≤0.002 to 2μg/mL. Oritavancin has promising in vitro activity against VSE and VRE in both planktonic and biofilm states.

The in vitro activities of rifampin, rifabutin, rifapentine, and rifaximin were tested against 200 periprosthetic joint infection (PJI)-associated staphylococci. Seven rifampin-resistant isolates had MICs of ≥4 μg/ml. Three isolates had rifampin MICs of 0.25 to 1 μg/ml and harbored an Asp471Gly RpoB variant, suggesting that the CLSI rifampin-susceptible staphylococcal breakpoint of ≤1 μg/ml may be too high. The remaining isolates had rifampin MICs of ≤0.016 μg/ml, and the rifampin, rifabutin, rifapentine, and rifaximin minimum biofilm bactericidal concentrations (MBBC) for ≥50% of isolates were 8, 1, 2, and 4 μg/ml (for S. aureus) and 2, 0.06, 0.25, and 0.5 μg/ml (for S. epidermidis), respectively, for rifampin-susceptible isolates. Nonrifampin rifamycins have promising staphylococcal activity, including antibiofilm activity.

Bacteriophage-derived lysins are being developed as anti-infective agents. In an acute osteomyelitis methicillin-resistant Staphylococcus aureus (MRSA) model, rats receiving no treatment or treatment with daptomycin, exebacase (CF-301), or daptomycin plus exebacase had means of 5.13, 4.09, 4.65, and 3.57 log(10) CFU/gram of bone, respectively. All treated animals had fewer bacteria than did untreated animals (P ≤ 0.0001), with daptomycin plus exebacase being more active than daptomycin (P = 0.0042) or exebacase (P < 0.001) alone.

We are developing electrical approaches to treat biofilm‐associated orthopedic foreign‐body infection. Although we have previously shown that such approaches have antibiofilm activity, the effects on bone have not been assessed. Herein, low‐amperage 200 μA fixed direct current (DC) was compared with no current, in a rat femoral foreign‐body infection model. In the infected group, a platinum implant seeded with S. epidermidis biofilm (105 CFU/cm2), plus 50 μL of a 109 CFU suspension of bacteria, were placed in the femoral medullary cavity of 71 rats. One week later, rats were assigned to one of four groups: infected with no current or DC, or uninfected with no current or DC. After 2 weeks, bones were removed and subjected to histopathology, micro‐computed tomography (μCT), and strength testing. Histopathology showed no inflammation or bony changes/remodeling in the uninfected no current group, and some osteoid formation in the DC group; bones from the infected no current group had evidence of inflammation without bony changes/remodeling; along with inflammation, there was moderate osteoid present in the DC group. μCT showed more cortical bone volume and density, trabecular thickness, and cancellous bone volume in the DC group compared with the no current group, for both uninfected and infected bones (p 0.05). © 2018 The Authors. JBMR Plus Published by Wiley Periodicals, Inc. on behalf of the American Society for Bone and Mineral Research.

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.

Background The vancapticins are modified vancomycin derivatives developed by adding membrane targeting motifs to the C-terminus of vancomycin. We determined the in vitro activity of a lead vancapticin candidate against periprosthetic joint infection-associated methicillin-resistant Staphylococcus aureus (MRSA) in the planktonic and biofilm states, and the effect of adding 0.002% polysorbate 80 (P-80; Sigma-Aldrich) on vancapticin susceptibility testing. Methods Thirty-seven clinical isolates of MRSA collected at Mayo Clinic (Rochester, Minnesota) were studied. Vancapticin minimum inhibitory concentrations (MICs) were determined using Clinical and Laboratory Standards Institutes guidelines. Minimum biofilm bactericidal concentrations (MBBCs) were determined using a pegged lid microtiter plate assay. Vancapticin MIC and MBBC values were assessed with and without P-80. Vancapticin, vancomycin, and dalbavancin biofilm time-kill assays were performed using biofilms formed by 10 MRSA isolates on Teflon coupons. Results Vancapticin MICs with and without P-80 ranged from 0.015 to 0.12 μg/mL and 0.25 to 1 μg/mL, respectively. Vancapticin MBBCs with and without P-80 ranged from 0.25 to 4 μg/mL and 1 to 8 μg/mL, respectively. Reductions of biofilm bacterial densities on Teflon coupons after 8 and 24 hours of incubation with vancapticin, vancapticin with P-80, vancomycin, or dalbavancin with P-80 were less than 3-log10 cfu/cm2 for all isolates tested. Conclusion Vancapticin has promising in vitro activity against planktonic MRSA and MRSA in a pegged lid biofilm assay, but was not bactericidal against biofilms on Teflon coupons. P-80 decreased vancapticin MICs and MBBCs. Disclosures Mark A. Blaskovich, PhD, MAB Consulting (Consultant)The University of Queensland (Employee, Grant/Research Support, Other Financial or Material Support, Inventor on patent) Robin Patel, MD, Accelerate Diagnostics (Grant/Research Support)CD Diagnostics (Grant/Research Support)Contrafect (Grant/Research Support)Curetis (Consultant)GenMark Diagnostics (Consultant)Heraeus Medical (Consultant)Hutchison Biofilm Medical Solutions (Grant/Research Support)Merck (Grant/Research Support)Next Gen Diagnostics (Consultant)PathoQuest (Consultant)Qvella (Consultant)Samsung (Other Financial or Material Support, Dr. Patel has a patent on Bordetella pertussis/parapertussis PCR issued, a patent on a device/method for sonication with royalties paid by Samsung to Mayo Clinic, and a patent on an anti-biofilm substance issued.)Selux Dx (Consultant)Shionogi (Grant/Research Support)Specific Technologies (Consultant)

Diagnosis of persistent infection at the time of reimplantation for staged revision of infected arthroplasties is challenging. Implant sonication culture for the diagnosis of prosthetic joint infection (PJI) has improved sensitivity compared to standard periprosthetic tissue culture. We report our experience with periprosthetic tissue culture and sonication culture of antimicrobial agent-containing cement spacers (ACSs) collected during second stages of staged revisions for arthroplasty infection. We studied 87 ACSs from 66 patients undergoing two-stage revision arthroplasty for PJI submitted for sonication culture, along with conventional periprosthetic tissue cultures. Two or more positive periprosthetic tissue cultures with the same organism were considered a positive tissue culture. For sonication culture, ≥20 CFU of bacteria per 10 ml of sonicate fluid was considered positive. The sensitivity and specificity of periprosthetic tissue and ACS sonication culture in detecting persistent infection, as well as their association with outcome, were assessed. Persistent infection occurred in 26% of cases. Periprosthetic tissue and sonicate fluid culture had specificities of 96.3 and 100% (P = 0.50), respectively, and sensitivities of 31.6 and 26.3% (P = 1.00), respectively, for the diagnosis of persistent infection. Thirteen subjects deemed not to have persistent infection at time of reimplantation and who had negative periprosthetic tissue and sonicate fluid cultures subsequently developed overt infection. Sonication culture of cement spacers identifies a similar proportion of patients with persistent infection during staged revisions, as detected by periprosthetic tissue cultures; both have low sensitivities to detect persistent infection.

We have previously demonstrated that culturing periprosthetic tissue in blood culture bottles (BCBs) improves sensitivity compared to conventional agar and broth culture methods for diagnosis of prosthetic joint infection (PJI). We have also shown that prosthesis sonication culture improves sensitivity compared to periprosthetic tissue culture using conventional agar and broth methods. The purpose of this study was to compare the diagnostic accuracy of tissue culture in BCBs (subsequently referred to as tissue culture) to prosthesis sonication culture (subsequently referred to as sonicate fluid culture). We studied 229 subjects who underwent arthroplasty revision or resection surgery between March 2016 and October 2017 at Mayo Clinic in Rochester, Minnesota. Using the Infectious Diseases Society of America (IDSA) PJI diagnostic criteria (omitting culture criteria) as the gold standard, the sensitivity of tissue culture was similar to that of the sonicate fluid culture (66.4% versus 73.1%, P = 0.07) but was significantly lower than that of the two tests combined (66.4% versus 76.9%, P < 0.001). Using Bayesian latent class modeling, which assumes no gold standard for PJI diagnosis, the sensitivity of tissue culture was slightly lower than that of sonicate fluid culture (86.3% versus 88.7%) and much lower than that of the two tests combined (86.3% versus 99.1%). In conclusion, tissue culture in BCBs reached sensitivity similar to that of prosthesis sonicate fluid culture for diagnosis of PJI, but the two tests combined had the highest sensitivity without compromising specificity. The combination of tissue culture in BCBs and sonicate fluid culture is recommended to achieve the highest level of microbiological diagnosis of PJI.

We are developing electrical approaches to treat biofilm‐associated orthopedic foreign‐body infection. Although we have previously shown that such approaches have antibiofilm activity, the effects on bone have not been assessed. Herein, low‐amperage 200 μA fixed direct current (DC) was compared with no current, in a rat femoral foreign‐body infection model. In the infected group, a platinum implant seeded with S. epidermidis biofilm (105 CFU/cm2), plus 50 μL of a 109 CFU suspension of bacteria, were placed in the femoral medullary cavity of 71 rats. One week later, rats were assigned to one of four groups: infected with no current or DC, or uninfected with no current or DC. After 2 weeks, bones were removed and subjected to histopathology, micro‐computed tomography (μCT), and strength testing. Histopathology showed no inflammation or bony changes/remodeling in the uninfected no current group, and some osteoid formation in the DC group; bones from the infected no current group had evidence of inflammation without bony changes/remodeling; along with inflammation, there was moderate osteoid present in the DC group. μCT showed more cortical bone volume and density, trabecular thickness, and cancellous bone volume in the DC group compared with the no current group, for both uninfected and infected bones (p 0.05). © 2018 The Authors. JBMR Plus Published by Wiley Periodicals, Inc. on behalf of the American Society for Bone and Mineral Research.

The antibiofilm activity of oritavancin in combination with rifampin, gentamicin, or linezolid was evaluated against 10 prosthetic joint infection (PJI)-related methicillin-resistant Staphylococcus aureus (MRSA) isolates by time-kill assays. Oritavancin combined with rifampin demonstrated statistically significant bacterial reductions compared with those of either antimicrobial alone for all 10 isolates ( P ≤ 0.001), with synergy being observed for 80% of the isolates.

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.

Staphylococcus aureus is a common cause of prosthetic joint infection (PJI). The prevalence of superantigens (SAgs) among PJI-associated S. aureus is unknown. Eighty-four S. aureus isolates associated with PJI isolated between 1999 and 2006 were studied. SAg genes, sea , seb , sec , sed , see , seg , seh , sei , and tst , were assayed by PCR. Seventy-eight (92.9%) isolates carried at least 1 SAg gene studied, with 61 (72.6%) harboring more than 1. seg was most commonly (70.2%), and seh was least frequently (4.8%) detected. tst -positive isolates were associated with early infection and increased erythrocyte sedimentation rate at diagnosis ( P =0.006 and P =0.021, respectively). seg and sei were associated with methicillin resistance ( P =0.008 and P =0.002, respectively). A majority of PJI-associated isolates studied produced biologically active SAgs in both planktonic and biofilm growth modes. SAg genes are prevalent in S. aureus causing PJI.

The frequency of superantigen production among Staphylococcus aureus isolates associated with endocarditis is not well defined. We tested 154 S. aureus isolates from definite infective endocarditis cases for the presence of staphylococcal enterotoxins A-E, H, and TSST-1 by PCR, enzyme-linked immunosorbent assay, and using an HLA-DR3 transgenic mouse splenocyte proliferation assay. Sixty-three isolates (50.8%) tested positive for at least 1 superantigen gene, with 21 (16.9%) testing positive for more than 2. tst (28.6%) was most common, followed by seb (27%), sea (22.2%), sed (20.6%), see (17.5%), and sec (11.1%). Of 41 methicillin-resistant S. aureus, 21 had superantigen genes, with sed being more frequently detected in this group compared to methicillin-susceptible S. aureus (P < 0.05). Superantigen genes were not associated with mortality (P = 0.81). 75% of PCR-positive isolates induced robust splenocyte proliferation. Overall, more than half of S. aureus isolates causing endocarditis carry superantigen genes, of which most are functional.

Background Orthopedic foreign body-associated infection can be difficult to treat due to the formation of biofilms protecting microorganisms from both antimicrobials and the immune system. Exebacase (EXE) is a phage-derived lysin which acts as a direct lytic agent by hydrolyzing the peptidoglycan cell wall of Staphylococcus aureus. In this study, the activity of EXE was evaluated in comparison to daptomycin against MRSA biofilms on orthopedic Kirschner wires (K-wires). Methods MRSA strain IDRL-6169 was studied; it has a MIC of 0.5 µg/mL for both daptomycin (DAP) and EXE. Biofilms were formed in 1 mL of 106 cfu/mL tryptic soy broth on 0.5x0.1 mm threaded stainless steel K-wires for 10 hours, after which the wires were removed from the media and placed into 0.04 mL of either DAP or EXE at 0 (vehicle only), 0.098, 0.98, or 9.8 mg/mL. DAP+EXE was also tested, each at 0.098 mg/mL. Bacteria were quantified after 0, 2, 4, 8, and 12 hours of incubation at 37ºC. Testing was performed in triplicate. Results were reported as log10 cfu/K-wire reduction relative to vehicle alone. A 3-log10 cfu/K-wire reduction was considered bactericidal. P-values were calculated using Kruskal–Wallis. Results The bacterial burden of vehicle alone ranged from 5.49- to 6.33-log10 cfu/K-wire at all time points. Bacterial reductions for each treatment compared with carrier solution are shown in the table. DAP showed no bactericidal activity. EXE showed bactericidal activity at all concentrations at all time points studied except 0.098 mg/mL at 8 hours. There was no significant difference between EXE at 0.098 and 0.98 mg/mL at any time point but EXE at 9.8 mg/mL did show superiority over the lower concentrations. DAP+EXE 0.098 mg/mL was bactericidal at all time points. Conclusion EXE showed a rapid effect against MRSA biofilms on orthopedic K-wires apparent within the first 2 hours of exposure and was more active than daptomycin alone at the same concentrations. Disclosures All authors: No reported disclosures.

We previously showed that culture of samples obtained by prosthesis vortexing and sonication was more sensitive than tissue culture for prosthetic joint infection (PJI) diagnosis. Despite improved sensitivity, culture-negative cases remained; furthermore, culture has a long turnaround time. We designed a genus-/group-specific rapid PCR assay panel targeting PJI bacteria and applied it to samples obtained by vortexing and sonicating explanted hip and knee prostheses, and we compared the results to those with sonicate fluid and periprosthetic tissue culture obtained at revision or resection arthroplasty. We studied 434 subjects with knee ( n = 272) or hip ( n = 162) prostheses; using a standardized definition, 144 had PJI. Sensitivities of tissue culture, of sonicate fluid culture, and of PCR were 70.1, 72.9, and 77.1%, respectively. Specificities were 97.9, 98.3, and 97.9%, respectively. Sonicate fluid PCR was more sensitive than tissue culture ( P = 0.04). PCR of prosthesis sonication samples is more sensitive than tissue culture for the microbiologic diagnosis of prosthetic hip and knee infection and provides same-day PJI diagnosis with definition of microbiology. The high assay specificity suggests that typical PJI bacteria may not cause aseptic implant failure.

Drugs such as linezolid that inhibit bacterial protein synthesis may be beneficial in treating infections caused by toxigenic Staphylococcus aureus. As protein synthesis inhibitors have no effect on preformed toxins, neutralization of pathogenic exotoxins with anti-toxin antibodies may be beneficial in conjunction with antibacterial therapy. Herein, we evaluated the efficacy of human-mouse chimeric high-affinity neutralizing anti-staphylococcal enterotoxin B (SEB) antibodies in the treatment of experimental pneumonia caused by SEB-producing S. aureus. Since HLA class II transgenic mice mount a stronger systemic immune response following challenge with SEB and are more susceptible to SEB-induced lethal toxic shock than conventional mice strains, HLA-DR3 transgenic mice were used. Lethal pneumonia caused by SEB-producing S. aureus in HLA-DR3 transgenic mice was characterized by robust T cell activation and elevated systemic levels of several pro-inflammatory cytokines and chemokines. Prophylactic administration of a single dose of linezolid 30 min prior to the onset of infection attenuated the systemic inflammatory response and protected from mortality whereas linezolid administered 60 min after the onset of infection failed to confer significant protection. Human-mouse chimeric high-affinity neutralizing anti-SEB antibodies alone, but not polyclonal human IgG, mitigated this response and protected from death when administered immediately after initiation of infection. Further, anti-SEB antibodies as well as intact polyclonal human IgG, but not its Fab or Fc fragments, protected from lethal pneumonia when followed with linezolid therapy 60 min later. In conclusion, neutralization of superantigens with high-affinity antibodies may have beneficial effects in pneumonia.

A relationship between hyperammonemia and Ureaplasma infection has been shown in lung transplant recipients. We have demonstrated that Ureaplasma urealyticum causes hyperammonemia in a novel immunocompromised murine model. Herein, we determined whether Ureaplasma parvum can do the same. Male C3H mice were given mycophenolate mofetil, tacrolimus, and prednisone for 7 days, and then challenged with U. parvum intratracheally (IT) and/or intraperitoneally (IP), while continuing immunosuppression over 6 days. Plasma ammonia concentrations were determined and compared using Wilcoxon rank-sum tests. Plasma ammonia concentrations of immunosuppressed mice challenged IT/IP with spent broth (median, 188 μmol/L; range, 102–340 μmol/L) were similar to those of normal (median, 226 μmol/L; range, 154–284 μmol/L, p > 0.05), uninfected immunosuppressed (median, 231 μmol/L; range, 122–340 μmol/L, p > 0.05), and U. parvum IT/IP challenged immunocompetent (median, 226 μmol/L; range, 130–330 μmol/L, p > 0.05) mice. Immunosuppressed mice challenged with U. parvum IT/IP (median 343 μmol/L; range 136–1,000 μmol/L) or IP (median 307 μmol/L; range 132–692 μmol/L) had higher plasma ammonia concentrations than those challenged IT/IP with spent broth (p

Foreign-body-associated infections are often difficult to treat, given that the associated microorganisms are in a biofilm state. Previously, we showed that a low-amperage direct electrical current (DC) reduces Propionibacterium acnes biofilms formed on implant-associated materials in vitro . In this study, low-amperage DC was compared to ceftriaxone treatment or no treatment in a novel rat femur model of foreign-body osteomyelitis. A platinum implant seeded with a P. acnes biofilm (10 7 CFU/cm 2 ) and 10 9 CFU of planktonic P. acnes was placed in the femoral medullary cavity. One week later, rats were assigned to one of three treatment groups: no treatment, ceftriaxone treatment, or 200-μA-DC treatment. After 2 weeks of treatment, there were fewer bacteria in the bones of the ceftriaxone group (3.06 log 10 CFU/g of bone [ P = 0.0209]) and the 200-μA-DC group (0.5 log 10 CFU/g [ P = 0.0015]) than in those of the control group (6.58 log 10 CFU/g). The DC-exposed animals exhibited fewer bacteria than the ceftriaxone-treated animals ( P = 0.0330). There were fewer bacteria on the implanted wires in the groups treated with ceftriaxone (0.1 log 10 CFU/cm 2 ) or a 200-μA DC (0.1 log 10 CFU/cm 2 ) than in the control group (2.53 log 10 CFU/cm 2 [ P , 0.0003 for both comparisons]). Low-amperage DC may be useful for treating, or aiding in the treatment of, foreign-body infections caused by P. acnes .

Summary The human gut is colonized by a large number of microorganisms (∼10 13 bacteria) that support various physiologic functions. A perturbation in the healthy gut microbiome might lead to the development of inflammatory diseases, such as multiple sclerosis (MS). Therefore, gut commensals might provide promising therapeutic options for treating MS and other diseases. We report the identification of human gut-derived commensal bacteria, Prevotella histicola , which can suppress experimental autoimmune encephalomyelitis (EAE) in a human leukocyte antigen (HLA) class II transgenic mouse model. P. histicola suppresses disease through the modulation of systemic immune responses. P. histicola challenge led to a decrease in pro-inflammatory Th1 and Th17 cells and an increase in the frequencies of CD4 + FoxP3 + regulatory T cells, tolerogenic dendritic cells, and suppressive macrophages. Our study provides evidence that the administration of gut commensals may regulate a systemic immune response and may, therefore, have a possible role in treatment strategies for MS.

New strategies are needed for prevention of biofilm formation. We have previously shown that 24 hr of 2,000 µA of direct current (DC) reducesStaphylococcus epidermidisbiofilm formationin vitro. Herein, we examined the effect of a lower amount of DC exposure onS. epidermidis,Staphylococcus aureus,Escherichia coli,Pseudomonas aeruginosa,Propionibacterium acnes, andCandida albicansbiofilm formation. 12 hr of 500 µA DC decreasedS. epidermidis,S. aureus,E. coli, andP. aeruginosabiofilm formation on Teflon discs by 2, 1, 1, and 2 log10cfu/cm2, respectively (p<0.05). Reductions inS. epidermidis,S. aureus, andE. colibiofilm 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/cm2reduction inP. aeruginosabiofilm formation was observed at 36 hr. 24 hr of 500 µA DC decreasedC. albicansbiofilm formation on Teflon discs by 2 log10 cfu/cm2. No reduction inP. acnesbiofilm formation was observed. 1 and 2 log10 cfu/cm2reductions inE. coliandS. epidermidisbiofilm 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.

Beards in the operating room are controversial because of their potential to retain and transmit pathogenic organisms. Many bearded orthopedic surgeons choose to wear nonsterile hoods in addition to surgical masks to decrease contamination of the operative field. The goal of this study was to determine whether nonsterile surgical hoods reduce the risk of bacterial shed ding posed by beards. Bearded (n=10) and clean-shaven (n=10) subjects completed 3 sets of standardized fa cial motions, each lasting 90 seconds and performed over blood agar plates, while unmasked, masked, and masked and hooded. The plates were cultured for 48 hours under aerobic and anaerobic conditions. Colony-forming units (CFUs) were quantified, expanded, and identified. Overall, the addition of surgical hoods did not decrease the total number of anaerobic and aerobic CFUs isolated per subject, with a mean of 1.1 CFUs while hooded compared with 1.4 CFUs with the mask alone ( P =.5). Unmasked subjects shed a mean of 6.5 CFUs, which was significantly higher than the number of CFUs shed while masked ( P =.02) or hooded ( P =.01). The bearded group did not shed more than the clean-shaven group while unmasked (9.5 vs 3.3 CFUs, P =.1), masked (1.6 vs 1.2 CFUs, P =.9), or hooded (0.9 vs 1.3 CFUs, P =.6). Bearded surgeons did not appear to have an increased likelihood of bacterial shedding compared with their nonbearded counter parts while wearing surgical masks, and the addition of nonsterile surgical hoods did not decrease the amount of bacterial shedding observed. [ Orthopedics. 2016; 39(2):e290–e294.]

Hyperammonemia syndrome is an often fatal complication of lung transplantation which has been recently associated with Ureaplasma infection. It has not been definitely established that Ureaplasma species can cause hyperammonemia. We established a novel immunocompromised murine model of Ureaplasma urealyticum infection and used it to confirm that U. urealyticum can cause hyperammonemia. Male C3H mice were pharmacologically immunosuppressed with mycophenolate mofetil, tacrolimus and oral prednisone for seven days, and then challenged intratracheally (IT) and/or intraperitoneally (IP) with 107 CFU U. urealyticum over six days, while continuing immunosuppression. Spent U. urealyticum-free U9 broth was used as a negative control, with uninfected immunocompetent mice, uninfected immunosuppressed mice, and infected immunocompetent mice serving as additional controls. Plasma ammonia concentrations were compared using Wilcoxon ranks sum tests. Plasma ammonia concentrations of immunosuppressed mice challenged IT/IP with spent U9 broth (n = 14) (range 155-330 μmol/L) were similar to those of normal mice (n = 5), uninfected immunosuppressed mice (n = 5), and U. urealyticum IT/IP challenged immunocompetent mice (n = 5) [range 99-340 μmol/L, p = 0.60]. However, immunosuppressed mice challenged with U. urealyticum IT/IP (n = 20) or IP (n = 15) had higher plasma ammonia concentrations (range 225-945 μmol/L and 276-687 μmol/L, respectively) than those challenged IT/IP with spent U9 broth (p

Periprosthetic tissue and/or synovial fluid PCR has been previously studied for prosthetic joint infection (PJI) diagnosis; however, few studies have assessed the utility of PCR on biofilms dislodged from the surface of explanted arthroplasties using vortexing and sonication (i.e., sonicate fluid PCR). We compared sonicate fluid 16S rRNA gene real-time PCR and sequencing to culture of synovial fluid, tissue, and sonicate fluid for the microbiologic diagnosis of PJI. PCR sequences generating mixed chromatograms were decatenated using RipSeq Mixed. We studied sonicate fluids from 135 and 231 subjects with PJI and aseptic failure, respectively. Synovial fluid, tissue, and sonicate fluid culture and sonicate fluid PCR had similar sensitivities (64.7, 70.4, 72.6, and 70.4%, respectively; P > 0.05) and specificities (96.9, 98.7, 98.3, and 97.8%, respectively; P > 0.05). Combining sonicate fluid culture and PCR, the sensitivity was higher (78.5%, P < 0.05) than those of individual tests, with similar specificity (97.0%). Thirteen subjects had positive sonicate fluid culture but negative PCR, and 11 had negative sonicate fluid culture but positive PCR (among which 7 had prior use of antimicrobials). Broad-range PCR and culture of sonicate fluid have equivalent performance for PJI diagnosis.