The effect of different antibiotic combinations in calcium sulfate cement on the growth of Cutibacterium acnes and Staphylococcus periprosthetic shoulder infection isolates.

BACKGROUND: Periprosthetic joint infections (PJI) of the shoulder are a devastating complication of shoulder arthroplasty and are commonly caused by Staphylococcus and Cutibacterium acnes. Absorbable calcium sulfate (CS) beads are sometimes used for delivering antibiotics in PJI. This study evaluates the in vitro effect of different combinations of gentamicin, vancomycin, and ertapenem in beads made from CS cement on the growth of C acnes and coagulase-negative Staphylococcus (CNS) strains. METHODS: Three strains of C acnes and 5 strains of CNS from clinically proven shoulder PJI were cultured and plated with CS beads containing combinations of vancomycin, gentamicin, and ertapenem. Plates with C acnes were incubated anaerobically while plates with Staphylococcus were incubated aerobically at 37 °C. Zones of inhibition were measured at intervals of 3 and 7 days using a modified Kirby Bauer technique, and beads were moved to plates containing freshly streaked bacteria every seventh day. This process was run in triplicate over the course of 56 days. Statistical analysis was conducted using SPSS v. 28 with repeated measures analysis of variance (ANOVA) and pairwise comparisons with Tukey correction. RESULTS: In experiments with C acnes, beads containing ertapenem + vancomycin and vancomycin alone formed the largest zones of inhibition over time (P < .001). In experiments with Staphylococcus, beads containing vancomycin alone formed the largest zones of inhibition over time for all 5 strains (P < .001). Zones of inhibition were 1.4x larger for C acnes than for Staphylococcus with beads containing vancomycin alone. For both C acnes and Staphylococcus, beads containing ertapenem had the strongest initial effect, preventing all bacterial growth in C acnes and almost all growth for Staphylococcus during the first week but dropping substantially by the second week. Beads containing gentamicin alone consistently created smaller zones of inhibition than beads containing vancomycin alone, with vancomycin producing zones 5.3x larger than gentamicin in C acnes and 1.3x larger in Staphylococcus (P < .001). DISCUSSION: These data suggest that for both C acnes and Staphylococcal species, CS beads impregnated with vancomycin were most effective at producing a robust antibiotic effect. Additionally, ertapenem may be a viable supplement in order to create a more potent initial antibiotic effect but is not as effective as vancomycin when used alone. Gentamicin alone was not effective in maintaining consistent and long-term antibiotic effects. These results indicate that amongst the antibiotics currently commercially available to be used with CS, vancomycin is consistently superior to gentamicin in the setting of C. acnes and CNS.

Role of the SOS Response in the Generation of Antibiotic Resistance In Vivo.

The SOS response to DNA damage is a conserved stress response in Gram-negative and Gram-positive bacteria. Although this pathway has been studied for years, its relevance is still not familiar to many working in the fields of clinical antibiotic resistance and stewardship. Under some conditions, the SOS response favors DNA repair and preserves the genetic integrity of the organism. On the other hand, the SOS response also includes induction of error-prone DNA polymerases, which can increase the rate of mutation, called the mutator phenotype or "hypermutation." As a result, mutations can occur in genes conferring antibiotic resistance, increasing the acquisition of resistance to antibiotics. Almost all of the work on the SOS response has been on bacteria exposed to stressors in vitro. In this study, we sought to quantitate the effects of SOS-inducing drugs in vivo, in comparison with the same drugs in vitro. We used a rabbit model of intestinal infection with enteropathogenic Escherichia coli strain E22. SOS-inducing drugs triggered the mutator phenotype response in vivo as well as in vitro. Exposure of E. coli strain E22 to ciprofloxacin or zidovudine, both of which induce the SOS response in vitro, resulted in increased antibiotic resistance to 3 antibiotics: rifampin, minocycline, and fosfomycin. Zinc was able to inhibit the SOS-induced emergence of antibiotic resistance in vivo, as previously observed in vitro. Our findings may have relevance in reducing the emergence of resistance to new antimicrobial drugs.

Spatio-temporal focal spot characterization and modeling of the NIF ARC kilojoule picosecond laser.

The advanced radiographic capability (ARC) laser system, part of the National Ignition Facility (NIF) at Lawrence Livermore National Laboratory, is a short-pulse laser capability integrated into the NIF. The ARC is designed to provide adjustable pulse lengths of ∼1-38ps in four independent beamlets, each with energies up to 1 kJ (depending on pulse duration). A detailed model of the ARC lasers has been developed that predicts the time- and space-resolved focal spots on target for each shot. Measurements made to characterize static and dynamic wavefront characteristics of the ARC are important inputs to the code. Modeling has been validated with measurements of the time-integrated focal spot at the target chamber center (TCC) at low power, and the space-integrated pulse duration at high power, using currently available diagnostics. These simulations indicate that each of the four ARC beamlets achieves a peak intensity on target of up to a few 1018W/cm2.

Metal Nanoparticles in Infection and Immunity.

Background Enthusiasm for the use of metal nanoparticles in human and veterinary medicine is high. Many articles describe the effects of metal nanoparticles on microbes in vitro, and a smaller number of articles describe effects on the immune system, which is the focus of this review. Methods Articles were retrieved by performing literature searches in Medline, of the National Institute of Medicine, as well as via Google Scholar. Results In vitro studies show that metal nanoparticles have antimicrobial effects. Some metal nanoparticles augment innate host immune defenses, such as endogenous antimicrobial peptides, and nitric oxide. Metal nanoparticles may also function as vaccine adjuvants. Metal nanoparticles can migrate to locations distant from the site of administration, however, requiring careful monitoring for toxicity. Conclusions Metal nanoparticles show a great deal of potential as immunomodulators, as well as direct antimicrobial effects. Before metal particles can be adopted as therapies; however, more studies are needed to determine how nanoparticles migrate though the body and on possible adverse effects.

Injection laser system for Advanced Radiographic Capability using chirped pulse amplification on the National Ignition Facility.

We report on the design, performance, and qualification of the injection laser system designed to deliver joule-level chirped pulse beamlets arranged in dual rectangular beam formats into two main laser amplifier beamlines of the National Ignition Facility. The system is designed to meet the requirements of the Advanced Radiographic Capability upgrade with features that deliver performance, adjustability, and long-term reliability.

Biological Activities of Uric Acid in Infection Due to Enteropathogenic and Shiga-Toxigenic Escherichia coli.

In previous work, we identified xanthine oxidase (XO) as an important enzyme in the interaction between the host and enteropathogenic Escherichia coli(EPEC) and Shiga-toxigenic E. coli(STEC). Many of the biological effects of XO were due to the hydrogen peroxide produced by the enzyme. We wondered, however, if uric acid generated by XO also had biological effects in the gastrointestinal tract. Uric acid triggered inflammatory responses in the gut, including increased submucosal edema and release of extracellular DNA from host cells. While uric acid alone was unable to trigger a chloride secretory response in intestinal monolayers, it did potentiate the secretory response to cyclic AMP agonists. Uric acid crystals were formed in vivo in the lumen of the gut in response to EPEC and STEC infections. While trying to visualize uric acid crystals formed during EPEC and STEC infections, we noticed that uric acid crystals became enmeshed in the neutrophilic extracellular traps (NETs) produced from host cells in response to bacteria in cultured cell systems and in the intestine in vivo Uric acid levels in the gut lumen increased in response to exogenous DNA, and these increases were enhanced by the actions of DNase I. Interestingly, addition of DNase I reduced the numbers of EPEC bacteria recovered after a 20-h infection and protected against EPEC-induced histologic damage.

Immunotherapy for Infectious Diseases: Past, Present, and Future.

Passive immunotherapy for established infections, as opposed to active immunization to prevent disease, remains a tiny niche in the world of antimicrobial therapies. Many of the passive immunotherapies currently available are directed against bacterial toxins, such as botulism, or are intended for agents of bioterrorism such as anthrax, which fortunately has remained rare. The emergence of Ebola virus and multi-drug resistant pathogens, however, may breathe new life into the immunotherapy field as researchers seek non-antibiotic interventions for infectious diseases.

Zinc protects against Shiga-toxigenic Escherichia coli by acting on host tissues as well as on bacteria.

BACKGROUND: Zinc supplements can treat or prevent enteric infections and diarrheal disease. Many articles on zinc in bacteria, however, highlight the essential nature of this metal for bacterial growth and virulence, suggesting that zinc should make infections worse, not better. To address this paradox, we tested whether zinc might have protective effects on intestinal epithelium as well as on the pathogen. RESULTS: Using polarized monolayers of T84 cells we found that zinc protected against damage induced by hydrogen peroxide, as measured by trans-epithelial electrical resistance. Zinc also reduced peroxide-induced translocation of Shiga toxin (Stx) across T84 monolayers from the apical to basolateral side. Zinc was superior to other divalent metals to (iron, manganese, and nickel) in protecting against peroxide-induced epithelial damage, while copper also showed a protective effect.The SOS bacterial stress response pathway is a powerful regulator of Stx production in STEC. We examined whether zinc's known inhibitory effects on Stx might be mediated by blocking the SOS response. Zinc reduced expression of recA, a reliable marker of the SOS. Zinc was more potent and more efficacious than other metals tested in inhibiting recA expression induced by hydrogen peroxide, xanthine oxidase, or the antibiotic ciprofloxacin. The close correlation between zinc's effects on recA/SOS and on Stx suggested that inhibition of the SOS response is one mechanism by which zinc protects against STEC infection. CONCLUSIONS: Zinc's ability to protect against enteric bacterial pathogens may be the result of its combined effects on host tissues as well as inhibition of virulence in some pathogens. Research focused solely on the effects of zinc on pathogenic microbes may give an incomplete picture by failing to account for protective effects of zinc on host epithelia.

Pro-inflammatory effects of uric acid in the gastrointestinal tract.

Uric acid can be generated in the gastrointestinal (GI) tract from the breakdown of nucleotides ingested in the diet or from purines released from host cells as a result of pathogen-induced cell damage. Xanthine oxidase (XO) is the enzyme that converts hypoxanthine or xanthine into uric acid, a reaction that also generates hydrogen peroxide. It has been assumed that the product of XO responsible for the pro-inflammatory effects of this enzyme is hydrogen peroxide. Recent literature on uric acid, however, has indicated that uric acid itself may have biological effects. We tested whether uric acid itself has detectable pro-inflammatory effects using an in vivo model using ligated rabbit intestinal segments ("loops") as well as in vitro assays using cultured cells. Addition of exogenous uric acid increased the influx of heterophils into rabbit intestinal loops, as measured by myeloperoxidase activity. In addition, white blood cells adhered avidly to uric acid crystals, forming large aggregates of cells. Uric acid acts as a leukocyte chemoattractant in the GI tract. The role of uric acid in enteric infections and in non-infectious disorders of the GI tract deserves more attention.

SOS-Inducing Drugs Trigger Nucleic Acid Release and Biofilm Formation in Gram-Negative Bacteria.

Our laboratory recently reported that induction of the SOS response, triggered by SOS-inducing drugs, was accompanied by a large release of DNA from enteric bacteria. The SOS response release had not previously been reported to include release of extracellular DNA from bacterial cells. We followed up on those observations in this current study and found that not just double-stranded DNA was being released, but also single-stranded DNA, RNA, and protein. SOS-inducing drugs also triggered formation of biofilm at the air-fluid interface on glass, and the biofilms contained DNA. We extended our study to test whether inhibitors of the SOS response would block DNA release and found that SOS inhibitors, including zinc salts, nitric oxide donors, and dequalinium, inhibited SOS-induced DNA release. The understanding that SOS-induced DNA release is associated with formation of biofilms increases our appreciation of the role of the SOS response in pathogenesis, as well as in emergence of new antibiotic resistance. Our findings with SOS inhibitors also suggest that regimens might be devised that could block the deleterious effects of the SOS response, at least temporarily, when this is desired.

Role of host xanthine oxidase in infection due to enteropathogenic and Shiga-toxigenic Escherichia coli.

Xanthine oxidase (XO), also known as xanthine oxidoreductase, has long been considered an important host defense molecule in the intestine and in breastfed infants. Here, we present evidence that XO is released from and active in intestinal tissues and fluids in response to infection with enteropathogenic Escherichia coli (EPEC) and Shiga-toxigenic E. coli (STEC), also known as enterohemorrhagic E. coli (EHEC). XO is released into intestinal fluids in EPEC and STEC infection in a rabbit animal model. XO activity results in the generation of surprisingly high concentrations of uric acid in both cultured cell and animal models of infection. Hydrogen peroxide (H(2)O(2)) generated by XO activity triggered a chloride secretory response in intestinal cell monolayers within minutes but decreased transepithelial electrical resistance at 6 to 22 h. H(2)O(2) generated by XO activity was effective at killing laboratory strains of E. coli, commensal microbiotas, and anaerobes, but wild-type EPEC and STEC strains were 100 to 1,000 times more resistant to killing or growth inhibition by this pathway. Instead of killing pathogenic bacteria, physiologic concentrations of XO increased virulence by inducing the production of Shiga toxins from STEC strains. In vivo, exogenous XO plus the substrate hypoxanthine did not protect and instead worsened the outcome of STEC infection in the rabbit ligated intestinal loop model of infection. XO released during EPEC and STEC infection may serve as a virulence-inducing signal to the pathogen and not solely as a protective host defense.

Antimicrobial susceptibility of Propionibacterium acnes isolates from shoulder surgery.

Orthopedic surgeons at our institution have noticed an increase in the number of infections due to Propionibacterium acnes, especially following operations on the shoulder. We collected P. acnes isolates from our hospital microbiology laboratory for 1 year and performed antimicrobial susceptibility testing on 28 strains from the shoulder. Antibiotics with the lowest MIC values against P. acnes (MIC50 and MIC90) included penicillin G (0.006, 0.125), cephalothin (0.047 and 0.094), and ceftriaxone (0.016, 0.045), while others also showed activity. Strains resistant to clindamycin were noted.

Role of Extracellular DNA in Bacterial Response to SOS-Inducing Drugs.

The SOS response is a conserved stress response pathway that is triggered by DNA damage in the bacterial cell. Activation of this pathway can, in turn, cause the rapid appearance of new mutations, sometimes called hypermutation. We compared the ability of various SOS-inducing drugs to trigger the expression of RecA, cause hypermutation, and produce elongation of bacteria. During this study, we discovered that these SOS phenotypes were accompanied by the release of large amounts of DNA into the extracellular medium. The release of DNA was accompanied by a form of bacterial aggregation in which the bacteria became tightly enmeshed in DNA. We hypothesize that DNA release triggered by SOS-inducing drugs could promote the horizontal transfer of antibiotic resistance genes by transformation or by conjugation.

Virulence inhibition by zinc in shiga-toxigenic Escherichia coli.

Previously, our laboratories reported that zinc inhibited expression of several important virulence factors in enteropathogenic Escherichia coli (EPEC) and reduced EPEC-induced intestinal damage in vivo. Since EPEC is genetically related to Shiga-toxigenic E. coli (STEC), we wondered whether the beneficial effects of zinc extended to STEC as well. Treatment options for STEC infection are very limited, since antibiotics tend to exacerbate disease via enhanced toxin production, so a safe intervention for this infection would be welcome. In this study, we report that in STEC strains zinc inhibits adherence to cultured cells as well as expression of EHEC secreted protein A (EspA). In addition, zinc inhibits the expression of Shiga toxin (Stx) at both the protein and the RNA level. Zinc inhibits basal and antibiotic-induced Stx production and inhibits both Stx1 and Stx2 by ≥90% at a concentration of 0.4 mM zinc. Rabbit EPEC strains were selected for acquisition of Stx-encoding bacteriophages, and these rabbit STEC strains (designated RDEC-H19A and E22-stx2) were used to test the effects of zinc in vivo in ligated rabbit intestinal loops. In vivo, zinc reduced fluid secretion into loops, inhibited mucosal adherence, reduced the amount of toxin in the loops, and reduced STEC-induced histological damage (villus blunting). Zinc has beneficial inhibitory effects against STEC strains that parallel those observed in EPEC. In addition, zinc strongly inhibits Stx expression; since Stx is responsible for the extraintestinal effects of STEC infection, such as hemolytic-uremic syndrome (HUS), zinc might be capable of preventing severe sequelae of STEC infection.

Psychoactive Drugs Induce the SOS Response and Shiga Toxin Production in Escherichia coli.

Several classes of non-antibiotic drugs, including psychoactive drugs, proton-pump inhibitors (PPIs), non-steroidal anti-inflammatory drugs (NSAIDs), and others, appear to have strong antimicrobial properties. We considered whether psychoactive drugs induce the SOS response in E. coli bacteria and, consequently, induce Shiga toxins in Shiga-toxigenic E. coli (STEC). We measured the induction of an SOS response using a recA-lacZ E. coli reporter strain, as RecA is an early, reliable, and quantifiable marker for activation of the SOS stress response pathway. We also measured the production and release of Shiga toxin 2 (Stx2) from a classic E. coli O157:H7 strain, derived from a food-borne outbreak due to spinach. Some, but not all, serotonin selective reuptake inhibitors (SSRIs) and antipsychotic drugs induced an SOS response. The use of SSRIs is widespread and increasing; thus, the use of these antidepressants could account for some cases of hemolytic-uremic syndrome due to STEC and is not attributable to antibiotic administration. SSRIs could have detrimental effects on the normal intestinal microbiome in humans. In addition, as SSRIs are resistant to environmental breakdown, they could have effects on microbial communities, including aquatic ecosystems, long after they have left the human body.

Inhibition of SOS Response by Nitric Oxide Donors in Escherichia coli Blocks Toxin Production and Hypermutation.

Background: Previous reports have differed as to whether nitric oxide inhibits or stimulates the SOS response, a bacterial stress response that is often triggered by DNA damage. The SOS response is an important regulator of production of Shiga toxins (Stx) in Shiga-toxigenic E. coli (STEC). In addition, the SOS response is accompanied by hypermutation, which can lead to de novo emergence of antibiotic resistance. We studied these effects in vitro as well as in vivo. Results: Nitric oxide donors inhibited induction of the SOS response by classical inducers such as mitomycin C, ciprofloxacin, and zidovudine, as measured by assays for E. coli RecA. Nitric oxide donors also inhibited Stx toxin protein production as well as stx2 RNA in vitro and in vivo. In vivo experiments were performed with ligated ileal segments in the rabbit using a 20 h infection. The NO donor S-nitroso-acetylpenicillamine (SNAP) reduced hypermutation in vitro and in vivo, as measured by emergence of rifampin resistance. SNAP blocked the ability of the RecA protein to bind to single-stranded DNA in an electrophoretic mobility shift assay (EMSA) in vitro, an early event in the SOS response. The inhibitory effects of SNAP were additive with those of zinc acetate. Conclusions: Nitric oxide donors blocked the initiation step of the SOS response. Downstream effects of this blockade included inhibition of Stx production and of hypermutation. Infection of rabbit loops with STEC resulted in a downregulation, rather than stimulation, of nitric oxide host defenses at 20 h of infection.

Cross-Talk between Probiotic Nissle 1917 and Human Colonic Epithelium Affects the Metabolite Composition and Demonstrates Host Antibacterial Effect.

Colonic epithelium-commensal interactions play a very important role in human health and disease development. Colonic mucus serves as an ecologic niche for a myriad of commensals and provides a physical barrier between the epithelium and luminal content, suggesting that communication between the host and microbes occurs mainly by soluble factors. However, the composition of epithelia-derived metabolites and how the commensal flora influences them is less characterized. Here, we used mucus-producing human adult stem cell-derived colonoid monolayers exposed apically to probiotic E. coli strain Nissle 1917 to characterize the host-microbial communication via small molecules. We measured the metabolites in the media from host and bacterial monocultures and from bacteria-colonoid co-cultures. We found that colonoids secrete amino acids, organic acids, nucleosides, and polyamines, apically and basolaterally. The metabolites from host-bacteria co-cultures markedly differ from those of host cells grown alone or bacteria grown alone. Nissle 1917 affects the composition of apical and basolateral metabolites. Importantly, spermine, secreted apically by colonoids, shows antibacterial properties, and inhibits the growth of several bacterial strains. Our data demonstrate the existence of a cross-talk between luminal bacteria and human intestinal epithelium via metabolites, which might affect the numbers of physiologic processes including the composition of commensal flora via bactericidal effects.

Effect of Blue Light and Photosensitizers on Cutibacterium acnes on Shoulder Periprosthetic Joint Infection Isolates.

Introduction: Cutibacterium acnes is gaining recognition as a leading pathogen after orthopaedic shoulder procedures. Photodynamic therapy, a combination of light and a photosensitizer, has demonstrated antimicrobial activity against C. acnes in the treatment of acne vulgaris. We sought to evaluate the effect of photodynamic therapy using blue light and photosensitizers on C. acnes isolates from shoulder prosthetic joint infections. Methods: C. acnes strains isolated from 19 patients with shoulder PJI were exposed to blue light alone (415 nm) or in combination with photosensitizers (fluorescein, riboflavin and demeclocycline). C. acnes strains were divided into 4 categories: Highly Sensitive (HS), Sensitive (S), Weakly Sensitive (WS), Resistant to blue light. Results: 13 of 19 C. acnes strains (68%) were S or HS to blue light alone. Of these 19 strains tested, 11 were tested with blue light and fluorescein or blue light plus riboflavin. Fluorescein (1 µg/mL) enhanced the effect of blue light in 6 of 11 strains (55%). Blue light plus riboflavin (10 µg/mL) resulted enhanced killing in 3 of 11 strains (27%), but produced a paradoxical photoprotective effect in 4 of 11 strains (36%), resulting in a net decrease compared to blue light alone. Demeclocycline, however, enhanced the effect of blue light in 16 of 17 strains (94 %). Conclusions: Blue light with the addition of photosensitizers killed C. acnes from periprosthetic shoulder infections in vitro, with demeclocycline having the most pronounced effect.

Pathogenic genetic variations of C. acnes are associated with clinically relevant orthopedic shoulder infections.

Many surgeons continue to face the clinical dilemma of interpreting a positive aspiration or unexpected positive Cutibacterium acnes (C. acnes) culture. There are factors that complicate the interpretation of positive cultures including variations in both frequency of false positive cultures and virulence properties. As indices of virulence, hemolytic strains, from previously confirmed clinically infected shoulders, were compared with non-hemolytic isolates determined to be contaminants, by RNA-sequencing (RNA-Seq). Six C. acnes isolates from patients who underwent revision total shoulder arthroplasty (TSA) were identified based on previously described infection criteria. Three C. acnes isolates from each group underwent RNA-Seq. Differential gene expression analysis, principal component analysis (PCA), and heatmap analysis were used to determine the gene variation and patterning between the definite infection and probable contaminant isolates. Differential gene expression analysis identified genes that were differentially expressed between the isolates classified as definite infection and isolates classified as probable contaminants. PCA using a 500 gene subset of identified genes was able to find combinations of these genes that separated out the definite infection and probable contaminants isolates. The heatmap demonstrated similar gene expression in the three Definite Infections isolates, and significantly different expression when compared with the probable contaminant isolates. Clinical significance: C. acnes revision TSA isolates classified as definite infection and probable contaminant demonstrated a similar gene expression pattern to each respective group and different gene expression pattern when compared between groups. These findings indicate distinct differences in C. acnes strains associated with clinically relevant orthopedic TSA infections.