Molecularly specific detection of bacterial lipoteichoic acid for diagnosis of prosthetic joint infection of the bone

Discriminating sterile inflammation from infection, especially in cases of aseptic loosening versus an actual prosthetic joint infection, is challenging and has significant treatment implications. Our goal was to evaluate a novel human monoclonal antibody (mAb) probe directed against the Gram-positive bacterial surface molecule lipoteichoic acid (LTA). Specificity and affinity were assessed in vitro. We then radiolabeled the anti-LTA mAb and evaluated its effectiveness as a diagnostic imaging tool for detecting infection via immunoPET imaging in an in vivo mouse model of prosthetic joint infection (PJI). In vitro and ex vivo binding of the anti-LTA mAb to pathogenic bacteria was measured with Octet, ELISA, and flow cytometry. The in vivo PJI mouse model was assessed using traditional imaging modalities, including positron emission tomography (PET) with [18F]FDG and [18F]NaF as well as X-ray computed tomography (CT), before being evaluated with the zirconium-89-labeled antibody specific for LTA ([89Zr]SAC55). The anti-LTA mAb exhibited specific binding in vitro to LTA-expressing bacteria. Results from imaging showed that our model could reliably simulate infection at the surgical site by bioluminescent imaging, conventional PET tracer imaging, and bone morphological changes by CT. One day following injection of both the radiolabeled anti-LTA and isotype control antibodies, the anti-LTA antibody demonstrated significantly greater (P < 0.05) uptake at S. aureus-infected prosthesis sites over either the same antibody at sterile prosthesis sites or of control non-specific antibody at infected prosthesis sites. Taken together, the radiolabeled anti-LTA mAb, [89Zr]SAC55, may serve as a valuable diagnostic molecular imaging probe to help distinguish between sterile inflammation and infection in the setting of PJI. Future studies are needed to determine whether these findings will translate to human PJI

Postoperative Staphylococcus aureus Infections in Patients With and Without Preoperative Colonization

Importance Staphylococcus aureus surgical site infections (SSIs) and bloodstream infections (BSIs) are important complications of surgical procedures for which prevention remains suboptimal. Contemporary data on the incidence of and etiologic factors for these infections are needed to support the development of improved preventive strategies.Objectives To assess the occurrence of postoperative S aureus SSIs and BSIs and quantify its association with patient-related and contextual factors.Design, Setting, and Participants This multicenter cohort study assessed surgical patients at 33 hospitals in 10 European countries who were recruited between December 16, 2016, and September 30, 2019 (follow-up through December 30, 2019). Enrolled patients were actively followed up for up to 90 days after surgery to assess the occurrence of S aureus SSIs and BSIs. Data analysis was performed between November 20, 2020, and April 21, 2022. All patients were 18 years or older and had undergone 11 different types of surgical procedures. They were screened for S aureus colonization in the nose, throat, and perineum within 30 days before surgery (source population). Both S aureus carriers and noncarriers were subsequently enrolled in a 2:1 ratio.Exposure Preoperative S aureus colonization.Main Outcomes and Measures The main outcome was cumulative incidence of S aureus SSIs and BSIs estimated for the source population, using weighted incidence calculation. The independent association of candidate variables was estimated using multivariable Cox proportional hazards regression models.Results In total, 5004 patients (median [IQR] age, 66 [56-72] years; 2510 [50.2%] female) were enrolled in the study cohort; 3369 (67.3%) were S aureus carriers. One hundred patients developed S aureus SSIs or BSIs within 90 days after surgery. The weighted cumulative incidence of S aureus SSIs or BSIs was 2.55% (95% CI, 2.05%-3.12%) for carriers and 0.52% (95% CI, 0.22%-0.91%) for noncarriers. Preoperative S aureus colonization (adjusted hazard ratio [AHR], 4.38; 95% CI, 2.19-8.76), having nonremovable implants (AHR, 2.00; 95% CI, 1.15-3.49), undergoing mastectomy (AHR, 5.13; 95% CI, 1.87-14.08) or neurosurgery (AHR, 2.47; 95% CI, 1.09-5.61) (compared with orthopedic surgery), and body mass index (AHR, 1.05; 95% CI, 1.01-1.08 per unit increase) were independently associated with S aureus SSIs and BSIs.Conclusions and Relevance In this cohort study of surgical patients, S aureus carriage was associated with an increased risk of developing S aureus SSIs and BSIs. Both modifiable and nonmodifiable etiologic factors were associated with this risk and should be addressed in those at increased S aureus SSI and BSI risk

Development and validation of a rabbit model of Pseudomonas aeruginosa non-ventilated pneumonia for preclinical drug development

BackgroundNew drugs targeting antimicrobial resistant pathogens, including Pseudomonas aeruginosa, have been challenging to evaluate in clinical trials, particularly for the non-ventilated hospital-acquired pneumonia and ventilator-associated pneumonia indications. Development of new antibacterial drugs is facilitated by preclinical animal models that could predict clinical efficacy in patients with these infections.MethodsWe report here an FDA-funded study to develop a rabbit model of non-ventilated pneumonia with Pseudomonas aeruginosa by determining the extent to which the natural history of animal disease reproduced human pathophysiology and conducting validation studies to evaluate whether humanized dosing regimens of two antibiotics, meropenem and tobramycin, can halt or reverse disease progression.ResultsIn a rabbit model of non-ventilated pneumonia, endobronchial challenge with live P. aeruginosa strain 6206, but not with UV-killed Pa6206, caused acute respiratory distress syndrome, as evidenced by acute lung inflammation, pulmonary edema, hemorrhage, severe hypoxemia, hyperlactatemia, neutropenia, thrombocytopenia, and hypoglycemia, which preceded respiratory failure and death. Pa6206 increased &gt;100-fold in the lungs and then disseminated from there to infect distal organs, including spleen and kidneys. At 5 h post-infection, 67% of Pa6206-challenged rabbits had PaO2 &lt;60 mmHg, corresponding to a clinical cut-off when oxygen therapy would be required. When administered at 5 h post-infection, humanized dosing regimens of tobramycin and meropenem reduced mortality to 17-33%, compared to 100% for saline-treated rabbits (P&lt;0.001 by log-rank tests). For meropenem which exhibits time-dependent bactericidal activity, rabbits treated with a humanized meropenem dosing regimen of 80 mg/kg q2h for 24 h achieved 100% T&gt;MIC, resulting in 75% microbiological clearance rate of Pa6206 from the lungs. For tobramycin which exhibits concentration-dependent killing, rabbits treated with a humanized tobramycin dosing regimen of 8 mg/kg q8h for 24 h achieved Cmax/MIC of 9.8 ± 1.4 at 60 min post-dose, resulting in 50% lung microbiological clearance rate. In contrast, rabbits treated with a single tobramycin dose of 2.5 mg/kg had Cmax/MIC of 7.8 ± 0.8 and 8% (1/12) microbiological clearance rate, indicating that this rabbit model can detect dose-response effects.ConclusionThe rabbit model may be used to help predict clinical efficacy of new antibacterial drugs for the treatment of non-ventilated P. aeruginosa pneumonia

Identification of Immunogenic and Serum Binding Proteins of Staphylococcus epidermidis

Staphylococcus epidermidis is a commensal of human skin and a leading cause of nosocomial bloodstream infections. Limited information is available about S. epidermidis proteins that are expressed upon transition to the bloodstream or those involved in host-pathogen interactions. A cell surface fraction from S. epidermidis 0-47 grown in rabbit serum to mimic environmental signals encountered during a bloodstream infection was separated by two-dimensional (2D) gel electrophoresis. Following 2D separation, the proteins were transferred to nitrocellulose and detected with either pooled sera generated in rabbits immunized with live S. epidermidis 0-47 or with biotin-labeled serum proteins eluted from the surface of bacteria grown in rabbit serum. Twenty-nine immunoreactive or serum binding proteins of S. epidermidis were identified by mass spectrometry. Twenty-seven of the corresponding genes were expressed in Escherichia coli, and the purified recombinant proteins were used to immunize mice. In a preliminary screen, 12 of the 27 recombinant proteins induced a response that reduced the number of bacteria recovered from the spleen or bloodstream of infected mice. In subsequent vaccination studies, 5 of the 12 proteins resulted in a statistically significant reduction in the number of bacteria. The identification of five candidate vaccine antigens from the initial screen of only 29 proteins demonstrates the utility of this approach

Alpha-Toxin Contributes to Biofilm Formation among Staphylococcus aureus Wound Isolates

Biofilms complicate treatment of Staphylococcus aureus (SA) wound infections. Previously, we determined alpha-toxin (AT)-promoted SA biofilm formation on mucosal tissue. Therefore, we evaluated SA wound isolates for AT production and biofilm formation on epithelium and assessed the role of AT in biofilm formation. Thirty-eight wound isolates were molecularly typed by pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (ST), and spa typing. We measured biofilm formation of these SA isolates in vitro and ex vivo and quantified ex vivo AT production. We also investigated the effect of an anti-AT monoclonal antibody (MEDI4893*) on ex vivo biofilm formation by methicillin-resistant SA (USA 300 LAC) and tested whether purified AT rescued the biofilm defect of hla mutant SA strains. The predominant PFGE/ST combinations were USA100/ST5 (50%) and USA300/ST8 (33%) for methicillin-resistant SA (MRSA, n = 18), and USA200/ST30 (20%) for methicillin-susceptible SA (MSSA, n = 20). Ex vivo AT production correlated significantly with ex vivo SA wound isolate biofilm formation. Anti-alpha-toxin monoclonal antibody (MEDI4893*) prevented ex vivo biofilm formation by MRSA USA300 strain LAC. Wild-type AT rescued the ex vivo biofilm defect of non-AT producing SA strains. These findings provide evidence that AT plays a role in SA biofilm formation on epithelial surfaces and suggest that neutralization of AT may be useful in preventing and treating SA infections

Candida albicans Augments Staphylococcus aureus Virulence by Engaging the Staphylococcal agr Quorum Sensing System

Relatively little is known about the complex interactions and signaling events that occur between microbes and even less so about how microbial “cross talk” shapes human health and disease. Candida albicans (a fungus) and Staphylococcus aureus (a bacterium) are formidable human nosocomial pathogens, causing severe morbidity and mortality. Moreover, they are frequently coisolated from central venous catheters and deep-seated infections, including intra-abdominal sepsis. In this work, we have shown that coinfection with C. albicans and S. aureus is highly lethal, leading to >80% mortality by day 1 postinfection, whereas monoinfection with C. albicans or S. aureus does not cause mortality. This infectious synergism is dependent on the expression of staphylococcal alpha-toxin, and secretion of this potent virulence factor is actually augmented by C. albicans via an agr-dependent mechanism. Moreover, prophylactic neutralization of alpha-toxin with a monoclonal antibody is sufficient to elicit protection during coinfection. Therefore, we have demonstrated that a pathogenic fungus can enhance virulence determinants of a bacterium in vivo with devastating consequences to the host. These results have important implications in the surveillance and treatment of polymicrobial disease and highlight the dynamic intersection of environment, pathogens, and host.Candida albicans and Staphylococcus aureus are among the most prevalent nosocomial pathogens that are responsible for severe morbidity and mortality, even with appropriate treatment. Using a murine model of polymicrobial intra-abdominal infection (IAI), we have previously shown that coinfection with these pathogens results in synergistic lethality that is partially dependent on exacerbated prostaglandin signaling, while monomicrobial infection is nonlethal. Therefore, the objective of this study was to identify staphylococcal virulence determinants that drive lethal synergism during polymicrobial IAI. Using the toxigenic S. aureus strain JE2, we observed that coinfection with C. albicans led to a striking 80 to 100% mortality rate within 20 h postinoculation (p.i.) while monomicrobial infections were nonlethal. Use of a green fluorescent protein (GFP)-P3 promoter S. aureus reporter strain revealed enhanced activation of the staphylococcal agr quorum sensing system during in vitro polymicrobial versus monomicrobial growth. Analyses by quantitative real-time PCR (qPCR), Western blot, and toxin functional assays confirmed enhanced agr-associated gene transcription and increases in secreted alpha- and delta-toxins. C. albicans-mediated elevated toxin production and hemolytic activity were determined to be agrA dependent, and genetic knockout and complementation of hla identified alpha-toxin as the key staphylococcal virulence factor driving lethal synergism. Analysis of mono- and polymicrobial infections 8 h p.i. demonstrated equivalent bacterial burdens in the peritoneal cavity but significantly elevated levels of alpha-toxin (3-fold) and the eicosanoid prostaglandin E2 (PGE2) (4-fold) during coinfection. Importantly, prophylactic passive immunization using the monoclonal anti-alpha-toxin antibody MEDI4893* led to significantly improved survival rates compared to those following treatment with isotype control antibody. Collectively, these results define alpha-toxin as an essential virulence determinant during C. albicans-S. aureus IAI and describe a novel mechanism by which a human-pathogenic fungus can augment the virulence of a highly pathogenic bacterium in vivo

Microbial burden and viral exacerbations in a longitudinal multicenter COPD cohort

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is a heterogeneous disease characterized by frequent exacerbation phenotypes independent of disease stage. Increasing evidence shows that the microbiota plays a role in disease progression and severity, but long-term and international multicenter assessment of the variations in viral and bacterial communities as drivers of exacerbations are lacking. METHODS: Two-hundred severe COPD patients from Europe and North America were followed longitudinally for 3 years. We performed nucleic acid detection for 20 respiratory viruses and 16S ribosomal RNA gene sequencing to evaluate the bacterial microbiota in 1179 sputum samples collected at stable, acute exacerbation and follow-up visits. RESULTS: Similar viral and bacterial taxa were found in patients from the USA compared to Bulgaria and Czech Republic but their microbiome diversity was significantly different (P &lt; 0.001) and did not impact exacerbation rates. Virus infection was strongly associated with exacerbation events (P &lt; 5E-20). Human rhinovirus (13.1%), coronavirus (5.1%) and influenza virus (3.6%) constitute the top viral pathogens in triggering exacerbation. Moraxella and Haemophilus were 5-fold and 1.6-fold more likely to be the dominating microbiota during an exacerbation event. Presence of Proteobacteria such as Pseudomonas or Staphylococcus amongst others, were associated with exacerbation events (OR &gt; 0.17; P &lt; 0.02) but more strongly associated with exacerbation frequency (OR &gt; 0.39; P &lt; 4E-10), as confirmed by longitudinal variations and biotyping of the bacterial microbiota, and suggesting a role of the microbiota in sensitizing the lung. CONCLUSIONS: This study highlights bacterial taxa in lung sensitization and viral triggers in COPD exacerbations. It provides a global overview of the diverse targets for drug development and explores new microbiome analysis methods to guide future patient management applications

Protective Efficacy of Monoclonal Antibodies Neutralizing Alpha-Hemolysin and Bicomponent Leukocidins in a Rabbit Model of Staphylococcus aureus Necrotizing Pneumonia.

Staphylococcus aureus is a major human pathogen that causes a wide range of infections by producing an arsenal of cytotoxins. We found that passive immunization with either a monoclonal antibody (MAb) neutralizing alpha-hemolysin or a broadly cross-reactive MAb neutralizing Panton-Valentine leukocidin, leukocidin ED, and gamma-hemolysins HlgAB and HlgCB conferred only partial protection, whereas the combination of those two MAbs conferred significant protection in a rabbit model of necrotizing pneumonia caused by the USA300 methicillin-resistant S. aureus epidemic clone