Dual antimicrobial-loaded biodegradable nanoemulsions for synergistic treatment of wound biofilms.

Wound biofilm infections caused by multidrug-resistant (MDR) bacteria constitute a major threat to public health; acquired resistance combined with resistance associated with the biofilm phenotype makes combatting these infections challenging. Biodegradable polymeric nanoemulsions that encapsulate two hydrophobic antimicrobial agents (eugenol and triclosan) (TE-BNEs) as a strategy to combat chronic wound infections are reported here. The cationic nanoemulsions efficiently penetrate and accumulate in biofilms, synergistically eradicating MDR bacterial biofilms, including persister cells. Notably, the nanoemulsion platform displays excellent biocompatibility and delays emergence of resistance to triclosan. The TE-BNEs are active in an in vivo murine model of mature MDR wound biofilm infections, with 99% bacterial elimination. The efficacy of this system coupled with prevention of emergence of bacterial resistance highlight the potential of this combination platform to treat MDR wound biofilm infections.

Nanotherapeutics using all-natural materials. Effective treatment of wound biofilm infections using crosslinked nanoemulsions.

Bacterial wound infections are a threat to public health. Although antibiotics currently provide front-line treatments for bacterial infections, the development of drug resistance coupled with the defenses provided through biofilm formation render these infections difficult, if not impossible, to cure. Antimicrobials from natural resources provide unique antimicrobial mechanisms and are generally recognized as safe and sustainable. Herein, an all-natural antimicrobial platform is reported. It is active against bacterial biofilms and accelerates healing of wound biofilm infections in vivo. This antimicrobial platform uses gelatin stabilized by photocrosslinking using riboflavin (vitamin B2) as a photocatalyst, and carvacrol (the primary constituent of oregano oil) as the active antimicrobial. The engineered nanoemulsions demonstrate broad-spectrum antimicrobial activity towards drug-resistant bacterial biofilms and significantly expedite wound healing in an in vivo murine wound biofilm model. The antimicrobial activity, wound healing promotion, and biosafety of these nanoemulsions provide a readily translatable and sustainable strategy for managing wound infections.

Desirability of Outcome Ranking for the Management of Antimicrobial Therapy (DOOR MAT) Reveals Improvements in the Treatment of Bloodstream Infections Caused by Escherichia coli and Klebsiella pneumoniae in Patients from the Veterans Health Administration.

BACKGROUND: Reductions in the use of broad-spectrum antibiotics is a cornerstone of antimicrobial stewardship. We aim to demonstrate use of the Desirability of Outcome Ranking Approach for the Management of Antimicrobial Therapy (DOOR MAT) to evaluate the treatment of Escherichia coli and Klebsiella pneumoniae bloodstream infections in patients from the Veterans Health Administration (VHA) across a decade. METHODS: Using electronic records, we determined empiric and definitive antibiotic treatments, clinical characteristics, and 30-day mortality of patients with monomicrobial E. coli and K. pneumoniae bloodstream infections hospitalized in VHA medical centers from 2009 to 2018. Focusing on patients treated with parenteral ß-lactams and with available antibiotic susceptibility testing results, we applied a range of DOOR MAT scores that reflect the desirability of antibiotic choices according to spectrum and activity against individual isolates. We report trends in resistance and desirability of empiric and definitive antibiotic treatments. RESULTS: During the 10-year period analyzed, resistance to expanded-spectrum cephalosporins and fluoroquinolones increased in E. coli but not in K. pneumoniae, while resistance to carbapenems and piperacillin-tazobactam remained unchanged. In 6451 cases analyzed, we observed improvements in DOOR MAT scores consistent with deescalation. Improvement in desirability of definitive treatment compared with empiric treatment occurred in 26% of cases, increasing from 16% in 2009 to 34% in 2018. Reductions in overtreatment were sustained and without negative impact on survival. CONCLUSIONS: DOOR MAT provides a framework to assess antibiotic treatment of E. coli and K. pneumoniae bloodstream infections and can be a useful metric in antimicrobial stewardship.

In Vitro Activity of Rifampin, Rifabutin, Rifapentine, and Rifaximin against Planktonic and Biofilm States of Staphylococci Isolated from Periprosthetic Joint Infection.

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.

Microbiology of polymicrobial prosthetic joint infection.

Prosthetic joint infection (PJI) is a rare but challenging complication of arthroplasty. Herein, we describe the epidemiology and microbiology of PJI, with a focus on analyzing differences between the microbiology of polymicrobial versus monomicrobial infection of hip, knee, and shoulder prostheses. In addition, we report the most frequent co-pathogens in polymicrobial infections, as detected by culture. A total of 373 patients diagnosed with PJI at Mayo Clinic were studied. For hip and knee arthroplasties, a higher proportion of fractures (P = 0.02) and a shorter time between the implantation and symptom onset (P < 0.0001) were noted in polymicrobial versus monomicrobial PJI. The most common microorganism detected, Staphylococcus epidermidis, was more frequently detected in polymicrobial (60%) versus monomicrobial (35%) PJI (P = 0.0067). Among polymicrobial infections, no co-pathogens were more frequently found than others, except S. epidermidis and Enterococcus faecalis which were found together in 5 cases. In addition to coagulase-negative staphylococci and enterococci, Corynebacterium species and Finegoldia magna were common in polymicrobial infections. Conversely, there was no difference between the prevalence of Staphylococcus aureus, Gram-negative bacilli, or Cutibacterium acnes between the polymicrobial and monomicrobial groups. The microbiology of polymicrobial PJI is different from that of monomicrobial PJI.

Rapid Molecular Diagnostics to Inform Empiric Use of Ceftazidime/Avibactam and Ceftolozane/Tazobactam Against Pseudomonas aeruginosa: PRIMERS IV.

BACKGROUND: Overcoming ß-lactam resistance in pathogens such as Pseudomonas aeruginosa is a major clinical challenge. Rapid molecular diagnostics (RMDs) have the potential to inform selection of empiric therapy in patients infected by P. aeruginosa. METHODS: In this study, we used a heterogeneous collection of 197 P. aeruginosa that included multidrug-resistant isolates to determine whether 2 representative RMDs (Acuitas Resistome test and VERIGENE gram-negative blood culture test) could identify susceptibility to 2 newer ß-lactam/ß-lactamase inhibitor (BL-BLI) combinations, ceftazidime/avibactam (CZA) and ceftolozane/tazobactam (TOL/TAZO). RESULTS: We found that the studied RMD platforms were able to correctly identify BL-BLI susceptibility (susceptibility sensitivity, 100%; 95% confidence interval [CI], 97%, 100%) for both BLs-BLIs. However, their ability to detect resistance to these BLs-BLIs was lower (resistance sensitivity, 66%; 95% CI, 52%, 78% for TOL/TAZO and 33%; 95% CI, 20%, 49% for CZA). CONCLUSIONS: The diagnostic platforms studied showed the most potential in scenarios where a resistance gene was detected or in scenarios where a resistance gene was not detected and the prevalence of resistance to TOL/TAZO or CZA is known to be low. Clinicians need to be mindful of the benefits and risks that result from empiric treatment decisions that are based on resistance gene detection in P. aeruginosa, acknowledging that such decisions are impacted by the prevalence of resistance, which varies temporally and geographically.

Activity of fixed direct electrical current in experimental Staphylococcus aureus foreign-body osteomyelitis.

Fixed DC was compared to ceftriaxone, ceftriaxone with 200 µA fixed DC, or no treatment in a rat model of methicillin-susceptible Staphylococcus aureus foreign-body osteomyelitis. After 3 weeks, fewer bacteria were present in bones of the ceftriaxone group (5.71 log10cfu/g [P = 0.0004]) and the ceftriaxone/DC group (3.53 log10cfu/g [P = 0.0002]) than untreated controls (6.70 log10cfu/g). Fewer bacteria were present in the ceftriaxone/DC group than in the ceftriaxone-alone and DC-alone groups (P = 0.0012 and 0.0008, respectively). There were also fewer bacteria on the implanted wires in the groups treated with ceftriaxone (5.47 log10cfu/cm2) or ceftriaxone/DC (2.82 log10cfu/cm2) than in the untreated controls (6.44 log10cfu/cm2 [P = 0.0003 and 0.0002, respectively]). There were fewer bacteria in the ceftriaxone/DC rats than in the ceftriaxone-alone- and fixed DC-alone-treated rats (P = 0.0017 and 0.0016, respectively). Fixed DC with an antibiotic may be useful for treating foreign-body infections caused by S. aureus.

Informing Antibiotic Treatment Decisions: Evaluating Rapid Molecular Diagnostics To Identify Susceptibility and Resistance to Carbapenems against Acinetobacter spp. in PRIMERS III.

The widespread dissemination of carbapenem-resistant Acinetobacter spp. has created significant therapeutic challenges. At present, rapid molecular diagnostics (RMDs) that can identify this phenotype are not commercially available. Two RMD platforms, PCR combined with electrospray ionization mass spectrometry (PCR/ESI-MS) and molecular beacons (MB), for detecting genes conferring resistance/susceptibility to carbapenems in Acinetobacter spp. were evaluated. An archived collection of 200 clinical Acinetobacter sp. isolates was tested. Predictive values for susceptibility and resistance were estimated as a function of susceptibility prevalence and were based on the absence or presence of beta-lactamase (bla) NDM, VIM, IMP, KPC, and OXA carbapenemase genes (e.g., blaOXA-23, blaOXA-24/40, and blaOXA-58 found in this study) against the reference standard of MIC determinations. According to the interpretation of MICs, 49% (n = 98) of the isolates were carbapenem resistant (as defined by either resistance or intermediate resistance to imipenem). The susceptibility sensitivities (95% confidence interval [CI]) for imipenem were 82% (74%, 89%) and 92% (85%, 97%) for PCR/ESI-MS and MB, respectively. Resistance sensitivities (95% CI) for imipenem were 95% (88%, 98%) and 88% (80%, 94%) for PCR/ESI-MS and MB, respectively. PRIMERS III establishes that RMDs can discriminate between carbapenem resistance and susceptibility in Acinetobacter spp. In the context of a known prevalence of resistance, SPVs and RPVs can inform clinicians regarding the best choice for empiric antimicrobial therapy against this multidrug-resistant pathogen.

Rifampin-Based Combination Therapy Is Active in Foreign-Body Osteomyelitis after Prior Rifampin Monotherapy.

Staphylococcal prosthetic joint infections (PJIs) are associated with biofilm formation, making them difficult to treat; if managed with debridement and implant retention, rifampin-based therapy is usually employed. Rifampin resistance potentially challenges PJI treatment. In investigating the effects of rifampin monotherapy on methicillin-resistant Staphylococcus aureus (MRSA) foreign-body osteomyelitis in rats, we previously demonstrated that rifampin resistance was selected but that it disappeared 14 days following rifampin monotherapy (1) and that rifampin resistance occurred less frequently following two rounds than following one round of rifampin monotherapy (2). Here, we compared rifampin monotherapy followed by rifampin-vancomycin combination therapy to rifampin-vancomycin combination therapy alone in experimental MRSA foreign-body osteomyelitis. Animals treated with rifampin monotherapy followed by rifampin-vancomycin combination therapy had decreased quantities of bacteria 14 days following treatment completion (P = 0.034) compared to those in animals treated with combination therapy alone. Additionally, some isolates recovered from animals treated with combination therapy alone, although still susceptible to rifampin, had higher MIC, minimum biofilm-inhibitory concentration (MBIC), and minimum biofilm-bactericidal concentration (MBBC) values than those of the inoculating strain. This suggests that rifampin may remain a feasible treatment option in foreign-body-associated orthopedic infections following the selection of rifampin resistance.

Activity of Electrical Current in Experimental Propionibacterium acnes Foreign-Body Osteomyelitis.

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 (107 CFU/cm2) and 109 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 log10 CFU/g of bone [P = 0.0209]) and the 200-µA-DC group (0.5 log10 CFU/g [P = 0.0015]) than in those of the control group (6.58 log10 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 log10 CFU/cm2) or a 200-µA DC (0.1 log10 CFU/cm2) than in the control group (2.53 log10 CFU/cm2 [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.

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.

Antimicrobial Susceptibility and Clonality of Clinical Ureaplasma Isolates in the United States.

Ureaplasma urealyticum and Ureaplasma parvum are pathogens involved in urogenital tract and intrauterine infections and also in systemic diseases in newborns and immunosuppressed patients. There is limited information on the antimicrobial susceptibility and clonality of these species. In this study, we report the susceptibility of 250 contemporary isolates of Ureaplasma (202 U. parvum and 48 U. urealyticum isolates) recovered at Mayo Clinic, Rochester, MN. MICs of doxycycline, azithromycin, ciprofloxacin, tetracycline, erythromycin, and levofloxacin were determined by broth microdilution, with MICS of the last three interpreted according to CLSI guidelines. Levofloxacin resistance was found in 6.4% and 5.2% of U. parvum and U. urealyticum isolates, respectively, while 27.2% and 68.8% of isolates, respectively, showed ciprofloxacin MICs of ≥4 µg/ml. The resistance mechanism of levofloxacin-resistant isolates was due to mutations in parC, with the Ser83Leu substitution being most frequent, followed by Glu87Lys. No macrolide resistance was found among the 250 isolates studied; a single U. parvum isolate was tetracycline resistant. tet(M) was found in 10 U. parvum isolates, including the single tetracycline-resistant isolate, as well as in 9 isolates which had low tetracycline and doxycycline MICs. Multilocus sequence typing (MLST) performed on a selection of 46 isolates showed high diversity within the clinical Ureaplasma isolates studied, regardless of antimicrobial susceptibility. The present work extends previous knowledge regarding susceptibility to antimicrobial agents, resistance mechanisms, and clonality of Ureaplasma species in the United States.

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

The in vitro activity of dalbavancin was tested against biofilms of 171 staphylococci associated with prosthetic joint infection. Dalbavancin minimum biofilm bactericidal concentration (MBBC) values were: MBBC50 for Staphylococcus aureus and Staphylococcus epidermidis, 1µg/mL; MBBC90 for S. aureus, 2µg/mL; MBBC90 for S. epidermidis, 4µg/mL.

Impact of Cefepime Susceptible-Dose-Dependent MIC for Enterobacteriaceae on Reporting and Prescribing.

The CLSI cefepime breakpoints were revised to include a susceptible-dose-dependent (SDD) category for Enterobacteriaceae, with the intention that higher cefepime doses be used for these isolates. In this study, 1.6% of Enterobacteriaceae isolates were reported as SDD, with Escherichia coli the most common SDD organism. Cefepime was prescribed in three cases (4.8%); the majority of SDD isolates were treated with a carbapenem. Enterobacteriaceae with cefepime SDD MICs were not commonly treated with cefepime at our institution.

Improved Diagnosis of Prosthetic Joint Infection by Culturing Periprosthetic Tissue Specimens in Blood Culture Bottles.

UNLABELLED: Despite known low sensitivity, culture of periprosthetic tissue specimens on agars and in broths is routine. Culture of periprosthetic tissue samples in blood culture bottles (BCBs) is potentially more convenient, but it has been evaluated in a limited way and has not been widely adopted. The aim of this study was to compare the sensitivity and specificity of inoculation of periprosthetic tissue specimens into blood culture bottles with standard agar and thioglycolate broth culture, applying Bayesian latent class modeling (LCM) in addition to applying the Infectious Diseases Society of America (IDSA) criteria for prosthetic joint infection. This prospective cohort study was conducted over a 9-month period (August 2013 to April 2014) at the Mayo Clinic, Rochester, MN, and included all consecutive patients undergoing revision arthroplasty. Overall, 369 subjects were studied; 117 (32%) met IDSA criteria for prosthetic joint infection, and 82% had late chronic infection. Applying LCM, inoculation of tissues into BCBs was associated with a 47% improvement in sensitivity compared to the sensitivity of conventional agar and broth cultures (92.1 versus 62.6%, respectively); this magnitude of change was similar when IDSA criteria were applied (60.7 versus 44.4%, respectively; P = 0.003). The time to microorganism detection was shorter with BCBs than with standard media (P < 0.0001), with aerobic and anaerobic BCBs yielding positive results within a median of 21 and 23 h, respectively. Results of our study demonstrate that the semiautomated method of periprosthetic tissue culture in blood culture bottles is more sensitive than and as specific as agar and thioglycolate broth cultures and yields results faster. IMPORTANCE: Prosthetic joint infections are a devastating complication of arthroplasty surgery. Despite this, current microbiological techniques to detect and diagnose infections are imperfect. This study examined a new approach to diagnosing infections, through the inoculation of tissue samples from around the prosthetic joint into blood culture bottles. This study demonstrated that, compared to current laboratory practices, this new technique increased the detection of infection. These findings are important for patient care to allow timely and accurate diagnosis of infection.

In vitro activity of ceftaroline against staphylococci from prosthetic joint infection.

We tested the in vitro activity of ceftaroline by Etest against staphylococci recovered from patients with prosthetic joint infection, including 97 Staphylococcus aureus isolates (36%, oxacillin resistant) and 74 Staphylococcus epidermidis isolates (74%, oxacillin resistant). Ceftaroline inhibited all staphylococci at ≤0.5 µg/mL. The ceftaroline MIC(90/50) values for methicillin-susceptible S. aureus, methicillin-susceptible S. epidermidis, methicillin-resistant S. aureus, and methicillin-resistant S. epidermidis were 0.19/0.125, 0.094/0.047, 0.5/0.38, and 0.38/0.19 µg/mL, respectively. Based on these in vitro findings, ceftaroline should be further evaluated as a potential therapeutic option for the treatment of prosthetic joint infection caused by methicillin-susceptible and methicillin-resistant S. aureus and S. epidermidis.

Antibiofilm Activity of Electrical Current in a Catheter Model.

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.

Elution of High Dose Amphotericin B Deoxycholate From Polymethylmethacrylate.

Fungal periprosthetic joint infections are rare, devastating complications of arthroplasty. There is conflicting evidence as to the efficacy of amphotericin B elution from cement spacers. The purpose of this study was to determine whether concentrations of amphotericin B released from bone cement over time would be efficacious in treating a periprosthetic infection. A continuous flow chamber was used to evaluate the in vitro release of amphotericin from cement beads containing 7.5% amphotericin. Following polymerization, 3.3% of the initially loaded amphotericin B was detected. The peak mean concentration eluted from the bone cement was 0.33 µg/mL at 8 hours. The AUC0-24 was 2.79 µg/mL/h; 0.20% of the amphotericin B was released. In conclusion, amphotericin B is released from bone cement at a clinically useful concentration.

Superantigens in Staphylococcus aureus isolated from prosthetic joint infection.

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.

Streptococcus suis-related prosthetic joint infection and streptococcal toxic shock-like syndrome in a pig farmer in the United States.

Streptococcus suis is an emerging swine-associated zoonotic agent that can cause meningitis and septicemia in humans. We present, to our knowledge, the first case of S. suis arthroplasty infection and streptococcal toxic shock-like syndrome due to an nonencapsulated serotype 5 strain in North America.