Immunization with lytic polysaccharide monooxygenase CbpD induces protective immunity against Pseudomonas aeruginosa pneumonia.

Pseudomonas aeruginosa (PA) CbpD belongs to the lytic polysaccharide monooxygenases (LPMOs), a family of enzymes that cleave chitin or related polysaccharides. Here, we demonstrate a virulence role of CbpD in PA pneumonia linked to impairment of host complement function and opsonophagocytic clearance. Following intratracheal challenge, a PA ΔCbpD mutant was more easily cleared and produced less mortality than the wild-type parent strain. The x-ray crystal structure of the CbpD LPMO domain was solved to subatomic resolution (0.75Å) and its two additional domains modeled by small-angle X-ray scattering and Alphafold2 machine-learning algorithms, allowing structure-based immune epitope mapping. Immunization of naive mice with recombinant CbpD generated high IgG antibody titers that promoted human neutrophil opsonophagocytic killing, neutralized enzymatic activity, and protected against lethal PA pneumonia and sepsis. IgG antibodies generated against full-length CbpD or its noncatalytic M2+CBM73 domains were opsonic and protective, even in previously PA-exposed mice, while antibodies targeting the AA10 domain were not. Preexisting antibodies in PA-colonized cystic fibrosis patients primarily target the CbpD AA10 catalytic domain. Further exploration of LPMO family proteins, present across many clinically important and antibiotic-resistant human pathogens, may yield novel and effective vaccine antigens.

Bacterial Membrane-Derived Vesicles Attenuate Vancomycin Activity against Methicillin-Resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) has evolved numerous antimicrobial resistance mechanisms and is identified as a serious public health threat by the World Health Organization and U.S. Centers for Disease Control and Prevention. The glycopeptide vancomycin (VAN) remains a cornerstone of therapy for severe MRSA infections despite increasing reports of therapeutic failure in hospitalized patients with bacteremia or pneumonia. Recently, the role of released bacterial-derived membrane vesicles (MVs) in antibiotic resistance has garnered attention. Here we examined the effect of exogenous MRSA-derived MVs on VAN activity against MRSA in vitro, using minimum inhibitory concentration and checkerboard assays, and ex vivo, incorporating components of host innate immunity such as neutrophils and serum complement present in blood. Additionally, the proteome of MVs from VAN-exposed MRSA was characterized to determine if protein expression was altered. The presence of MVs increased the VAN MIC against MRSA to values where clinical failure is commonly observed. Furthermore, the presence of MVs increased survival of MRSA pre-treated with sub-MIC concentrations of VAN in whole blood and upon exposure to human neutrophils but not human serum. Unbiased proteomic analysis also showed an elevated expression of MV proteins associated with antibiotic resistance (e.g., marR) or proteins that are functionally linked to cell membrane/wall metabolism. Together, our findings indicate MRSA-derived MVs are capable of lowering susceptibility of the pathogen to VAN, whole-blood- and neutrophil-mediated killing, a new pharmacodynamic consideration for a drug increasingly linked to clinical treatment failures.

Azithromycin Exerts Bactericidal Activity and Enhances Innate Immune Mediated Killing of MDR Achromobacter xylosoxidans.

Azithromycin (AZM), the most commonly prescribed antibiotic in the United States, is thought to have no activity against multidrug-resistant Gram-negative pathogens such as Achromobacter xylosoxidans (AX) per standard minimum inhibitory concentration testing in cation-adjusted Mueller Hinton Broth. Here we provide the first report of AZM bactericidal activity against carbapenem-resistant isolates of AX, with a multifold decrease in minimum inhibitory concentration across 12 clinical isolates when examined under physiologic testing conditions that better recapitulate the in vivo human environment. This pharmaceutical activity, evident in eukaryotic tissue culture media, is associated with enhanced AZM intracellular penetration and synergistic killing with human whole blood, serum, and neutrophils. Additionally, AZM monotherapy inhibited preformed AX biofilm growth in a dose-dependent manner together with a reduction in viable bacteria. In an illustrative case, AZM in combination with piperacillin-tazobactam exerted clear therapeutic effects in a patient with carbapenem-resistant AX mediastinitis, sternal osteomyelitis, and aortic graft infection. Our study reinforces how current antimicrobial testing practices fail to recapitulate the host environment or host-pathogen interactions and may misleadingly declare complete resistance to useful agents, adversely affecting patient outcomes. We conclude that AZM merits further exploration in the treatment of drug-resistant AX infections. Novel approaches to antimicrobial susceptibility testing that better recapitulate the host environment should be considered, especially as infections caused by multidrug-resistant Gram-negative bacterial pathogens are expanding globally with high morbidity and mortality.

Staphylococcus aureus Membrane-Derived Vesicles Promote Bacterial Virulence and Confer Protective Immunity in Murine Infection Models.

Staphylococcus aureus produces membrane-derived vesicles (MVs), which share functional properties to outer membrane vesicles. Atomic force microscopy revealed that S. aureus-derived MVs are associated with the bacterial surface or released into the surrounding environment depending on bacterial growth conditions. By using a comparative proteomic approach, a total of 131 and 617 proteins were identified in MVs isolated from S. aureus grown in Luria-Bertani and brain-heart infusion broth, respectively. Purified S. aureus MVs derived from the bacteria grown in either media induced comparable levels of cytotoxicity and neutrophil-activation. Administration of exogenous MVs increased the resistance of S. aureus to killing by whole blood or purified human neutrophils ex vivo and increased S. aureus survival in vivo. Finally, immunization of mice with S. aureus-derived MVs induced production of IgM, total IgG, IgG1, IgG2a, and IgG2b resulting in protection against subcutaneous and systemic S. aureus infection. Collectively, our results suggest S. aureus MVs can influence bacterial-host interactions during systemic infections and provide protective immunity in murine models of infection.

Interaction of Antibiotics with Innate Host Defense Factors against Salmonella enterica Serotype Newport.

This study examines the pharmacodynamics of antimicrobials that are used to treat Salmonella with each other and with key components of the innate immune system. Antimicrobial synergy was assessed using time-kill and checkerboard assays. Antimicrobial interactions with innate immunity were studied by employing cathelicidin LL-37, whole-blood, and neutrophil killing assays. Ceftriaxone and ciprofloxacin were found to be synergistic in vitro against Salmonella enterica serotype Newport. Ceftriaxone, ciprofloxacin, and azithromycin each demonstrated synergy with the human cathelicidin defense peptide LL-37 in killing Salmonella. Exposure of Salmonella to sub-MICs of ceftriaxone resulted in enhanced susceptibility to LL-37, whole blood, and neutrophil killing. The activity of antibiotics in vivo against Salmonella may be underestimated in bacteriologic media lacking components of innate immunity. The pharmacodynamic interactions of antibiotics used to treat Salmonella with each other and with components of innate immunity warrant further study in light of recent findings showing in vivo selection of antimicrobial resistance by single agents in this pathogen. IMPORTANCE It is becoming increasingly understood that the current paradigms of in vitro antimicrobial susceptibility testing may have significant shortcomings in predicting activity in vivo. This study evaluated the activity of several antibiotics alone and in combination against clinical isolates of Salmonella enterica serotype Newport (meningitis case) utilizing both conventional and physiological media. In addition, the interactions of these antibiotics with components of the innate immune system were evaluated. Azithromycin, which has performed quite well clinically despite high MICs in conventional media, was shown to be more active in physiological media and to enhance innate immune system killing. Alternatively, chloramphenicol did not show enhanced immune system killing, paralleling its inferior clinical performance to other antibiotics that have been used to treat Salmonella meningitis. These findings are important additions to the building understanding of current in vitro antimicrobial assay limitations that hopefully will amount to future improvements in these assays to better predict clinical efficacy and activity in vivo.