Greenwood‐Quaintance, Kerryl E.

ABSTRACTWe 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). There was no difference in torsional strength or stiffness between the no current versus DC groups, for both infected and uninfected 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.

Metagenomic shotgun sequencing has the potential to transform how serious infections are diagnosed by offering universal, culture-free pathogen detection. This may be especially advantageous for microbial diagnosis of prosthetic joint infection (PJI) by synovial fluid analysis since synovial fluid cultures are not universally positive and since synovial fluid is easily obtained preoperatively.

ABSTRACT 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.

ABSTRACT Cell-cell communication mediated by peptide pheromones (cCF10 [CF]) is essential for high-frequency plasmid transfer in vitro in Enterococcus faecalis . To examine the role of pheromone signaling in vivo , we established either a CF-producing (CF+) recipient or a recipient producing a biologically inactive variant of CF (CF− recipient) in a germfree mouse model 3 days before donor inoculation and determined transfer frequencies of the pheromone-inducible plasmid pCF10. Plasmid transfer was detected in the upper and middle sections of the intestinal tract 5 h after donor inoculation and was highly efficient in the absence of antibiotic selection. The transconjugant/donor ratio reached a maximum level approaching 1 on day 4 in the upper intestinal tract. Plasmid transfer was significantly lower with the CF− recipient. While rescue of the CF− mating defect by coculture with CF+ recipients is easily accomplished in vitro , no extracellular complementation occurred in vivo . This suggests that most pheromone signaling in the gut occurs between recipient and donor cells in very close proximity. Plasmid-bearing cells (donors plus transconjugants) steadily increased in the population from 0.1% after donor inoculation to about 10% at the conclusion of the experiments. This suggests a selective advantage of pCF10 carriage distinct from antibiotic resistance or bacteriocin production. Our results demonstrate that pheromone signaling is required for efficient pCF10 transfer in vivo . In the absence of CF+ recipients, a low level of transfer to CF− recipients occurred in the gut. This may result from low-level host-mediated induction of the donors in the gastrointestinal (GI) tract, similar to that previously observed in serum. IMPORTANCE Horizontal gene transfer is a major factor in the biology of Enterococcus faecalis , an important nosocomial pathogen. Previous studies showing efficient conjugative plasmid transfer in the gastrointestinal (GI) tracts of experimental animals did not examine how the enterococcal sex pheromone response impacts the efficiency of transfer. Our study demonstrates for the first time pheromone-enhanced, high-frequency plasmid transfer of E. faecalis plasmid pCF10 in a mouse model in the absence of antibiotic or bacteriocin selection. Pheromone production by recipients dramatically increased plasmid transfer in germfree mice colonized initially with recipients, followed by donors. The presence of a coresident community of common gut microbes did not significantly reduce in vivo plasmid transfer between enterococcal donors and recipients. In mice colonized with enterococcal recipients, we detected plasmid transfer in the intestinal tract within 5 h of addition of donors, before transconjugants could be cultured from feces. Surprisingly, pCF10 carriage provided a competitive fitness advantage unrelated to antibiotic resistance or bacteriocin production.

ABSTRACT Although carbapenems are effective for treating serious multidrug-resistant Pseudomonas aeruginosa infections, carbapenem-resistant P. aeruginosa (CRPA) is now being reported worldwide. Ceftolozane-tazobactam (C/T) demonstrates activity against many multidrug-resistant isolates. We evaluated the activity of C/T and compared its activity to that of ceftazidime-avibactam (C/A) using a well-characterized collection of non-carbapenemase-producing CRPA isolates. Forty-two non-carbapenemase-producing CRPA isolates from a previous study (J. Y. Lee and K. S. Ko, Int J Antimicrob Agents 40:168–172, 2012, https://doi.org/10.1016/j.ijantimicag.2012.04.004 ) were included. All had been previously shown to be negative for bla IMP , bla VIM , bla SPM , bla GIM , bla SIM , and bla KPC by PCR. In the prior study, expression of oprD , ampC , and several efflux pump genes had been defined by quantitative reverse transcription-PCR. Here, antimicrobial susceptibility was determined by broth microdilution according to Clinical and Laboratory Standards Institute (CLSI) guidelines. Time-kill curve assays were performed using three C/T- and C/A-susceptible CRPA isolates. Among 42 non-carbapenemase-producing CRPA isolates, overall susceptibility to C/T was 95.2%, compared to 71.4%, 42.9%, 23.8%, 21.4%, and 2.4% for C/A, ceftazidime, piperacillin-tazobactam, cefepime, and meropenem, respectively. The C/T resistance rate was significantly lower than that of C/A among isolates showing decreased oprD and increased mexB expression (5.1% versus 25.6%, P = 0.025, and 4.3% versus 34.8%, P = 0.022, respectively). In time-kill curve studies, C/T was less bactericidal than C/A against an isolate with decreased oprD and increased ampC expression. C/T was active against 95.2% of non-carbapenemase-producing CRPA clinical isolates. No apparent correlation of C/T MIC values with specific mutation-driven resistance mechanisms was noted.

Abstract Background Detection and identification of microorganism(s) involved in periprosthetic joint infection (PJI) can inform surgical management and directed antibiotic therapy. Metagenomic shotgun sequencing is a powerful tool with the potential to change how many PJIs are diagnosed as it allows direct detection and identification of pathogens in clinical specimens. In the largest series to date, we utilized a metagenomics-based approach applied to SF to define potential microbial etiologies of failed total knee arthroplasties (TKAs). Methods Synovial fluid was collected from 112 failed TKAs [74 PJI and 38 aseptic implant failure (AF)] via preoperative arthrocentesis. Cell count and differential, standardized culture and DNA-based metagenomic shotgun sequencing were performed. Human DNA was depleted using the MolYsis basic kit prior to DNA extraction, whole genome amplification, and sequencing. Taxonomic assignment of reads and pathogen identification was achieved using a pipeline incorporating k-mer- and marker gene-based classification software. A scheme for analysis and filtration of false-positives was created and applied, incorporating cut-offs for the number of reads, quality scores, and coverage across a reference genome. Patients were classified as having PJI using the IDSA criteria and expert review. Analyses were recorded as percent agreement, with 95% confidence intervals (CI), of metagenomics to SF culture. Results Metagenomic analysis identified the known pathogen in 54 (90%) (CI, 79.5%–96.2%) of the 60 culture-positive PJIs analyzed and one (2%) (CI, 0.0%–8.9%) potential polymicrobial infection not detected by culture. For the 14 culture-negative PJIs tested, metagenomics showed 79% (CI, 49.2%–95.3%) agreement for negative findings; potential pathogens were identified in three (21%) (CI, 4.7%–50.8%) culture-negative PJI cases, with one being polymicrobial. Of the 37 culture-negative AF cases, metagenomics showed 97% (CI, 85.8%–99.9%) agreement with negative culture and identified one (3%) (CI, 0.0%–14.2%) potential pathogen. For the one culture-positive AF case, metagenomic results were negative, suggesting possible culture contamination. Conclusion Metagenomic shotgun sequencing performed on SF can be used to diagnose PJI and may be particularly useful for culture-negative PJI. Disclosures R. Patel, ASM: Board Member, None; CD Diagnostics, BioFire, Curetis, Merck, Hutchison Biofilm Medical Solutions, Accelerate Diagnostics, Allergan, and The Medicines Company: Grant Investigator, Grant recipient; Curetis: Consultant, Monies paid to my employer; 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: Patents, Patents, any money is paid to my employer; Actelion: DSMB, Money paid to my employer; ASM and IDSA: Editor’s stipends, Editor’s stipends; NBME, Up-to-Date and the Infectious Diseases Board Review Course: NBME, Up-to-Date and the Infectious Diseases Board Review Course, Honoraria; Roche, ASM, and IDSA: Travel reimbursement, Travel reimbursement

ABSTRACT Whole-genome amplification (WGA) is a useful tool for amplification of very small quantities of DNA for many uses, including metagenomic shotgun sequencing for infection diagnosis. Depending on the application, background DNA from WGA kits can be problematic. Three WGA kits were tested for their utility in a metagenomics approach to identify the pathogens in sonicate fluid comprised of biofilms and other materials dislodged from the surfaces of explanted prosthetic joints using sonication. The Illustra V2 Genomiphi, Illustra single cell Genomiphi, and Qiagen REPLI-g single cell kits were used to test identical sonicate fluid samples. Variations in the number of background reads, the genera identified in the background, and the number of reads from known pathogens known to be present in the samples were observed between kits. These results were then compared to those obtained with a library preparation without prior WGA using an NEBNext Ultra II paired-end kit, which requires a very small amount of input DNA. This approach also resulted in the presence of contaminant bacterial DNA and yielded fewer reads from the known pathogens. These findings highlight the impact that WGA kit selection can have on metagenomic analysis of low-biomass samples and the importance of the careful selection and consideration of the implications of using these tools.