Mortality Risk Profiling of Staphylococcus aureus Bacteremia by Multi-omic Serum Analysis Reveals Early Predictive and Pathogenic Signatures.

Staphylococcus aureus bacteremia (SaB) causes significant disease in humans, carrying mortality rates of ∼25%. The ability to rapidly predict SaB patient responses and guide personalized treatment regimens could reduce mortality. Here, we present a resource of SaB prognostic biomarkers. Integrating proteomic and metabolomic techniques enabled the identification of >10,000 features from >200 serum samples collected upon clinical presentation. We interrogated the complexity of serum using multiple computational strategies, which provided a comprehensive view of the early host response to infection. Our biomarkers exceed the predictive capabilities of those previously reported, particularly when used in combination. Last, we validated the biological contribution of mortality-associated pathways using a murine model of SaB. Our findings represent a starting point for the development of a prognostic test for identifying high-risk patients at a time early enough to trigger intensive monitoring and interventions.

TLR4 signaling and macrophage inflammatory responses are dampened by GIV/Girdin.

Sensing of pathogens by Toll-like receptor 4 (TLR4) induces an inflammatory response; controlled responses confer immunity but uncontrolled responses cause harm. Here we define how a multimodular scaffold, GIV (a.k.a. Girdin), titrates such inflammatory response in macrophages. Upon challenge with either live microbes or microbe-derived lipopolysaccharides (a ligand for TLR4), macrophages with GIV mount a more tolerant (hypo-reactive) transcriptional response and suppress proinflammatory cytokines and signaling pathways (i.e., NFkB and CREB) downstream of TLR4 compared to their GIV-depleted counterparts. Myeloid-specific gene-depletion studies confirmed that the presence of GIV ameliorates dextran sodium sulfate-induced colitis and sepsis-induced death. The antiinflammatory actions of GIV are mediated via its C-terminally located TIR-like BB-loop (TILL) motif which binds the cytoplasmic TIR modules of TLR4 in a manner that precludes receptor dimerization; such dimerization is a prerequisite for proinflammatory signaling. Binding of GIV's TILL motif to TIR modules inhibits proinflammatory signaling via other TLRs, suggesting a convergent paradigm for fine-tuning macrophage inflammatory responses.

Paired Siglec receptors generate opposite inflammatory responses to a human-specific pathogen.

Paired immune receptors display near-identical extracellular ligand-binding regions but have intracellular sequences with opposing signaling functions. While inhibitory receptors dampen cellular activation by recognizing self-associated molecules, the functions of activating counterparts are less clear. Here, we studied the inhibitory receptor Siglec-11 that shows uniquely human expression in brain microglia and engages endogenous polysialic acid to suppress inflammation. We demonstrated that the human-specific pathogen Escherichia coli K1 uses its polysialic acid capsule as a molecular mimic to engage Siglec-11 and escape killing. In contrast, engagement of the activating counterpart Siglec-16 increases elimination of bacteria. Since mice do not have paired Siglec receptors, we generated a model by replacing the inhibitory domain of mouse Siglec-E with the activating module of Siglec-16. Siglec-E16 enhanced proinflammatory cytokine expression and bacterial killing in macrophages and boosted protection against intravenous bacterial challenge. These data elucidate uniquely human interactions of a pathogen with Siglecs and support the long-standing hypothesis that activating counterparts of paired immune receptors evolved as a response to pathogen molecular mimicry of host ligands for inhibitory receptors.

Hepatic Mucinous Cystic Neoplasm Versus Simple Biliary Cyst: Assessment of Distinguishing Imaging Features Using CT and MRI.

OBJECTIVE. The purpose of our study was to identify the imaging features that differentiate a hepatic mucinous cystic neoplasm (MCN) from a simple biliary cyst. MATERIALS AND METHODS. Surgically resected hepatic MCNs and simple biliary cysts over a 20-year period (October 29, 1997-January 23, 2018) with preoperative CT, MRI, or both were retrospectively identified. Included cases underwent histopathologic confirmation of diagnosis based on the 2010 World Health Organization criteria and blinded imaging review. Various imaging features, including cyst shape and septal enhancement, were assessed for performance. For septate cysts, the relationship of the septation to the cyst wall-that is, arising from the wall without an indentation versus arising from an external macrolobulation-was recorded. Statistical analysis was performed for the imaging features with the chi-square test. RESULTS. The study group comprised 22 hepatic MCNs and 56 simple biliary cysts. A unilocular hepatic cystic lesion was highly predictive of a simple biliary cyst (positive predictive value = 95.2%). The imaging feature of septations arising only from macro-lobulations was 100% specific for a simple biliary cyst on CT (p = 0.001). The presence of septations arising from the cyst wall without indentation was 100% sensitive for hepatic MCN but was only 56.3% specific on CT. Septal enhancement reached 100% sensitivity for hepatic MCN on MRI (p = 0.018). CONCLUSION. The presence of septations, relationship of the septations to the cyst wall, and septal enhancement were sensitive imaging features in the detection of hepatic MCN. The imaging feature of septations arising only from macrolobulations in the cyst wall was specific for simple biliary cysts on CT and helped differentiate simple biliary cysts from hepatic MCNs.

Chordoma: 18F-FDG PET/CT and MRI imaging features.

OBJECTIVE: Examine the 18F-FDG PET/CT and MRI imaging characteristics of chordoma. MATERIALS AND METHODS: Biopsy-proven chordoma with a pre-therapy 18F-FDG PET/CT from 2001 through 2019 in patients > 18 years old were retrospectively reviewed. Multiple PET/CT and MRI imaging parameters were assessed. RESULTS: A total of 23 chordoma patients were included (16 M, 7 F; average age of 60.1 ± 13.0 years) with comparative MRI available in 22 cases. This included 13 sacrococcygeal, 9 mobile spine, and one clival lesions. On 18F-FDG PET/CT, chordomas demonstrated an average SUVmax of 5.8 ± 3.7, average metabolic tumor volume (MTV) of 160.2 ± 263.8 cm3, and average total lesion glycolysis (TLG) of 542.6 ± 1210 g. All demonstrated heterogeneous FDG activity. On MRI, chordomas were predominantly T2 hyperintense (22/22) and T1 isointense (18/22), contained small foci of T1 hyperintensity (17/22), and demonstrated heterogeneous enhancement (14/20). There were no statistically significant associations found between 18F-FDG PET/CT and MRI imaging features. There was no relationship of SUVmax (p = 0.53), MTV (p = 0.47), TLG (p = 0.48), maximal dimension (p = 0.92), or volume (p = 0.45) to the development of recurrent or metastatic disease which occurred in 6/22 patients over a mean follow-up duration of 4.1 ± 2.0 years. CONCLUSION: On 18F-FDG PET/CT imaging, chordomas demonstrate moderate, heterogeneous FDG uptake. Predominant T2 hyperintensity and small foci of internal increased T1 signal are common on MRI. The inherent FDG avidity of chordomas suggests that 18F-FDG PET/CT may be a useful modality for staging, evaluating treatment response, and assessing for recurrent or metastatic disease.

Real-time jitter correction in a photonic analog-to-digital converter.

A real-time jitter meter is used to measure and digitally sample the pulse-to-pulse timing error in a laser pulse train. The jitter meter is self-referenced using a single-pulse delay line interferometer and measures timing jitter using optical heterodyne detection between two frequency channels of the pulse train. Jitter sensitivity down to 3×10-10fs2/Hz at 500 MHz has been demonstrated with a pulse-to-pulse noise floor of 1.6 fs. As a proof of principle, the digital correction of the output of a high-frequency photonic analog-to-digital converter (PADC) is demonstrated with an emulated jitter signal. Up to 23 dB of jitter correction, down to the noise floor of the PADC, is accomplished with radio-frequency modulation up to 40 GHz.

Macrophage-like nanoparticles concurrently absorbing endotoxins and proinflammatory cytokines for sepsis management.

Sepsis, resulting from uncontrolled inflammatory responses to bacterial infections, continues to cause high morbidity and mortality worldwide. Currently, effective sepsis treatments are lacking in the clinic, and care remains primarily supportive. Here we report the development of macrophage biomimetic nanoparticles for the management of sepsis. The nanoparticles, made by wrapping polymeric cores with cell membrane derived from macrophages, possess an antigenic exterior the same as the source cells. By acting as macrophage decoys, these nanoparticles bind and neutralize endotoxins that would otherwise trigger immune activation. In addition, these macrophage-like nanoparticles sequester proinflammatory cytokines and inhibit their ability to potentiate the sepsis cascade. In a mouse Escherichia coli bacteremia model, treatment with macrophage mimicking nanoparticles, termed MΦ-NPs, reduced proinflammatory cytokine levels, inhibited bacterial dissemination, and ultimately conferred a significant survival advantage to infected mice. Employing MΦ-NPs as a biomimetic detoxification strategy shows promise for improving patient outcomes, potentially shifting the current paradigm of sepsis management.

Collective Resistance in Microbial Communities by Intracellular Antibiotic Deactivation.

The structure and composition of bacterial communities can compromise antibiotic efficacy. For example, the secretion of ß-lactamase by individual bacteria provides passive resistance for all residents within a polymicrobial environment. Here, we uncover that collective resistance can also develop via intracellular antibiotic deactivation. Real-time luminescence measurements and single-cell analysis demonstrate that the opportunistic human pathogen Streptococcus pneumoniae grows in medium supplemented with chloramphenicol (Cm) when resistant bacteria expressing Cm acetyltransferase (CAT) are present. We show that CAT processes Cm intracellularly but not extracellularly. In a mouse pneumonia model, more susceptible pneumococci survive Cm treatment when coinfected with a CAT-expressing strain. Mathematical modeling predicts that stable coexistence is only possible when antibiotic resistance comes at a fitness cost. Strikingly, CAT-expressing pneumococci in mouse lungs were outcompeted by susceptible cells even during Cm treatment. Our results highlight the importance of the microbial context during infectious disease as a potential complicating factor to antibiotic therapy.

Tamm-Horsfall Protein Protects the Urinary Tract against Candida albicans.

Urinary tract infections (UTIs) caused by the human fungal pathogen Candida albicans and related species are prevalent in hospitalized patients, especially those on antibiotic therapy, with indwelling catheters, or with predisposing conditions such as diabetes or immunodeficiency. Understanding of key host defenses against Candida UTI is critical for developing effective treatment strategies. Tamm-Horsfall glycoprotein (THP) is the most abundant urine protein, with multiple roles in renal physiology and bladder protection. THP protects against bacterial UTI by blocking bacterial adherence to the bladder epithelium, but its role in defense against fungal pathogens is not yet described. Here we demonstrate that THP restricts colonization of the urinary tract by C. albicans THP binds to C. albicans hyphae, but not the yeast form, in a manner dependent on fungal expression of the Als3 adhesion glycoprotein. THP directly blocks C. albicans adherence to bladder epithelial cells in vitro, and THP-deficient mice display increased fungal burden in a C. albicans UTI model. This work outlines a previously unknown role for THP as an essential component for host immune defense against fungal urinary tract infection.

Marine Natural Product Honaucin A Attenuates Inflammation by Activating the Nrf2-ARE Pathway.

The cyanobacterial marine natural product honaucin A inhibits mammalian innate inflammation in vitro and in vivo. To decipher its mechanism of action, RNA sequencing was used to evaluate differences in gene expression of cultured macrophages following honaucin A treatment. This analysis led to the hypothesis that honaucin A exerts its anti-inflammatory activity through activation of the cytoprotective nuclear erythroid 2-related factor 2 (Nrf2)-antioxidant response element/electrophile response element (ARE/EpRE) signaling pathway. Activation of this pathway by honaucin A in cultured human MCF7 cells was confirmed using an Nrf2 luciferase reporter assay. In vitro alkylation experiments with the natural product and N-acetyl-l-cysteine suggest that honaucin A activates this pathway through covalent interaction with the sulfhydryl residues of the cytosolic repressor protein Keap1. Honaucin A presents a potential therapeutic lead for diseases with an inflammatory component modulated by Nrf2-ARE.

Natural Product Anacardic Acid from Cashew Nut Shells Stimulates Neutrophil Extracellular Trap Production and Bactericidal Activity.

Emerging antibiotic resistance among pathogenic bacteria is an issue of great clinical importance, and new approaches to therapy are urgently needed. Anacardic acid, the primary active component of cashew nut shell extract, is a natural product used in the treatment of a variety of medical conditions, including infectious abscesses. Here, we investigate the effects of this natural product on the function of human neutrophils. We find that anacardic acid stimulates the production of reactive oxygen species and neutrophil extracellular traps, two mechanisms utilized by neutrophils to kill invading bacteria. Molecular modeling and pharmacological inhibitor studies suggest anacardic acid stimulation of neutrophils occurs in a PI3K-dependent manner through activation of surface-expressed G protein-coupled sphingosine-1-phosphate receptors. Neutrophil extracellular traps produced in response to anacardic acid are bactericidal and complement select direct antimicrobial activities of the compound.

Classical ß-Lactamase Inhibitors Potentiate the Activity of Daptomycin against Methicillin-Resistant Staphylococcus aureus and Colistin against Acinetobacter baumannii.

We asked whether beta-lactamase inhibitors (BLIs) increased the activity of daptomycin (DAP) against methicillin-resistant Staphylococcus aureus (MRSA), the peptide antibiotic colistin (COL) against the emerging Gram-negative nosocomial pathogen Acinetobacter baumannii, and the human host defense peptide cathelicidin LL37 against either pathogen. DAP and LL37 kill curves were performed with or without BLIs against MRSA, vancomycin-intermediate S. aureus (VISA), and heterogeneous VISA (hVISA). COL and LL37 kill curves were performed against A. baumannii Boron-dipyrromethene (BODIPY)-labeled DAP binding to MRSA grown with the BLI tazobactam (TAZ) was assessed microscopically. The combination of COL plus TAZ was studied in a murine model of A. baumannii pneumonia. TAZ alone lacked in vitro activity against MRSA or A. baumannii The addition of TAZ to DAP resulted in a 2- to 5-log10 reduction in recoverable MRSA CFU at 24 h compared to the recoverable CFU with DAP alone. TAZ plus COL showed synergy by kill curves for 4 of 5 strains of A. baumannii tested. Growth with 20 mg/liter TAZ resulted in 2- to 2.5-fold increases in the intensity of BODIPY-DAP binding to MRSA and hVISA strains. TAZ significantly increased the killing of MRSA and A. baumannii by LL37 in vitro TAZ increased the activity of COL in a murine model of A. baumannii pneumonia. Classical BLIs demonstrate synergy with peptide antibiotics. Since BLIs have scant antimicrobial activity on their own and are thus not expected to increase selective pressure toward antibiotic resistance, their use in combination with peptide antibiotics warrants further study.

Tamm-Horsfall glycoprotein engages human Siglec-9 to modulate neutrophil activation in the urinary tract.

Urinary tract infections are a major problem in human medicine for which better understanding of native immune defenses may reveal new pathways for therapeutic intervention. Tamm-Horsfall glycoprotein (THP), the most abundant urinary protein, interacts with bacteria including uropathogenic Escherichia coli (UPEC) as well host immune cells. In addition to its well-studied functions to antagonize bacterial colonization, we hypothesize that THP serves a critical host defense function through innate immune modulation. Using isolated human neutrophils, we found that THP binds neutrophils and that this interaction reduces reactive oxygen species generation, chemotaxis and killing of UPEC. We discovered that THP engages the inhibitory neutrophil receptor sialic acid-binding Ig-like lectin-9 (Siglec-9), and mouse functional ortholog Siglec-E, in a manner dependent on sialic acid on its N-glycan moieties. THP-null mice have significantly more neutrophils present in the urine compared with wild-type mice, both with and without the presence of inflammatory stimuli. These data support THP as an important negative regulator of neutrophil activation in the urinary tract, with dual functions to counteract bacterial colonization and suppress excessive inflammation within the urinary tract.

Role of Hypoxia Inducible Factor-1α (HIF-1α) in Innate Defense against Uropathogenic Escherichia coli Infection.

Uropathogenic E. coli (UPEC) is the primary cause of urinary tract infections (UTI) affecting approximately 150 million people worldwide. Here, we revealed the importance of transcriptional regulator hypoxia-inducible factor-1 α subunit (HIF-1α) in innate defense against UPEC-mediated UTI. The effects of AKB-4924, a HIF-1α stabilizing agent, were studied using human uroepithelial cells (5637) and a murine UTI model. UPEC adherence and invasion were significantly reduced in 5637 cells when HIF-1α protein was allowed to accumulate. Uroepithelial cells treated with AKB-4924 also experienced reduced cell death and exfoliation upon UPEC challenge. In vivo, fewer UPEC were recovered from the urine, bladders and kidneys of mice treated transurethrally with AKB-4924, whereas increased bacteria were recovered from bladders of mice with a HIF-1α deletion. Bladders and kidneys of AKB-4924 treated mice developed less inflammation as evidenced by decreased pro-inflammatory cytokine release and neutrophil activity. AKB-4924 impairs infection in uroepithelial cells and bladders, and could be correlated with enhanced production of nitric oxide and antimicrobial peptides cathelicidin and ß-defensin-2. We conclude that HIF-1α transcriptional regulation plays a key role in defense of the urinary tract against UPEC infection, and that pharmacological HIF-1α boosting could be explored further as an adjunctive therapy strategy for serious or recurrent UTI.

Nipple Sparing Mastectomy in Patients With Prior Breast Scars: Is It Safe?

BACKGROUND: Nipple-sparing mastectomy (NSM) preserves the native skin envelope, including the nipple-areolar skin, and has significant benefits including improved aesthetic outcome and psychosocial well-being. Patients with prior breast scars undergoing NSM are thought to be at increased risk for postoperative complications, such as skin and/or nipple necrosis. This study describes our experience performing NSM in patients who have had prior breast surgery and aims to identify potential risk factors in this subset of patients. METHODS: A retrospective review of all patients undergoing nipple sparing mastectomy at The University of Utah from 2005 to 2011 was performed. Fifty-two patients had prior breast scars, for a total of 65 breasts. Scars were categorized into 4 groups depending on scar location: inframammary fold, outer quadrant, periareolar, and circumareolar. Information regarding patient demographics, social and medical history, treatment intent, and postoperative complications were collected and analyzed. RESULTS: Eight of the 65 breasts (12%) developed a postoperative infection requiring antibiotic treatment. Tobacco use was associated with an increased risk of infection in patients with prior breast scars (odds ratio [OR], 7.95; 95% confidence interval [CI], 1.37-46.00; P = 0.0206). There was a 13.8% rate of combined nipple and skin flap necrosis and receipt of chemotherapy (OR, 5.00; CI, 1.11-22.46; P = 0.0357) and prior BCT (OR, 12.5; CI, 2.2-71.0; P = 0.004) were found to be associated with skin flap or NAC necrosis. CONCLUSIONS: Nipple-sparing mastectomy is a safe and viable option for patients with a prior breast scar. Our results are comparable to the published data in patients without a prior scar. Caution should be exercised with patients who have a history of tobacco use or those requiring chemotherapy because these patients are at increased risk for infection and NAC/skin flap necrosis, respectively, when undergoing NSM in the setting of a prior breast scar.

Synergy between Ursolic and Oleanolic Acids from Vitellaria paradoxa Leaf Extract and ß-Lactams against Methicillin-Resistant Staphylococcus aureus: In Vitro and In Vivo Activity and Underlying Mechanisms.

Combining antibiotics with resistance reversing agents is a key strategy to overcome bacterial resistance. Upon screening antimicrobial activities of plants used in traditional medicine, we found that a leaf dichloromethane extract from the shea butter tree (Vitellaria paradoxa) had antimicrobial activity against methicillin-resistant Staphylococcus aureus (MRSA) with further evidence of synergy when combined with ß-lactams. Using HPLC-MS, we identified ursolic (UA) and oleanolic acids (OA) in leaves and twigs of this species, and quantified them by HPLC-UV as the major constituents in leaf extracts (21% and 6% respectively). Both pure triterpenic acids showed antimicrobial activity against reference and clinical strains of MRSA, with MICs ranging from 8-16 mg/L for UA to 32-128 mg/L for OA. They were highly synergistic with ß-lactams (ampicillin and oxacillin) at subMIC concentrations. Reversion of MRSA phenotype was attributed to their capacity to delocalize PBP2 from the septal division site, as observed by fluorescence microscopy, and to disturb thereby peptidoglycan synthesis. Moreover, both compounds also inhibited ß-lactamases activity of living bacteria (as assessed by inhibition of nitrocefin hydrolysis), but not in bacterial lysates, suggesting an indirect mechanism for this inhibition. In a murine model of subcutaneous MRSA infection, local administration of UA was synergistic with nafcillin to reduce lesion size and inflammatory cytokine (IL-1ß) production. Thus, these data highlight the potential interest of triterpenic acids as resistance reversing agents in combination with ß-lactams against MRSA.

Penicillin Binding Protein 1 Is Important in the Compensatory Response of Staphylococcus aureus to Daptomycin-Induced Membrane Damage and Is a Potential Target for ß-Lactam-Daptomycin Synergy.

The activity of daptomycin (DAP) against methicillin-resistant Staphylococcus aureus (MRSA) is enhanced in the presence of ß-lactam antibiotics. This effect is more pronounced with ß-lactam antibiotics that exhibit avid binding to penicillin binding protein 1 (PBP1). Here, we present evidence that PBP1 has a significant role in responding to DAP-induced stress on the cell. Expression of the pbpA transcript, encoding PBP1, was specifically induced by DAP exposure whereas expression of pbpB, pbpC, and pbpD, encoding PBP2, PBP3, and PBP4, respectively, remained unchanged. Using a MRSA COL strain with pbpA under an inducible promoter, increased pbpA transcription was accompanied by reduced susceptibility to, and killing by, DAP in vitro. Exposure to ß-lactams that preferentially inactivate PBP1 was not associated with increased DAP binding, suggesting that synergy in the setting of anti-PBP1 pharmacotherapy results from increased DAP potency on a per-molecule basis. Combination exposure in an in vitro pharmacokinetic/pharmacodynamic model system with ß-lactams that preferentially inactivate PBP1 (DAP-meropenem [MEM] or DAP-imipenem [IPM]) resulted in more-rapid killing than did combination exposure with DAP-nafcillin (NAF) (nonselective), DAP-ceftriaxone (CRO) or DAP-cefotaxime (CTX) (PBP2 selective), DAP-cefaclor (CEC) (PBP3 selective), or DAP-cefoxitin (FOX) (PBP4 selective). Compared to ß-lactams with poor PBP1 binding specificity, exposure of S. aureus to DAP plus PBP1-selective ß-lactams resulted in an increased frequency of septation and cell wall abnormalities. These data suggest that PBP1 activity may contribute to survival during DAP-induced metabolic stress. Therefore, targeted inactivation of PBP1 may enhance the antimicrobial efficiency of DAP, supporting the use of DAP-ß-lactam combination therapy for serious MRSA infections, particularly when the ß-lactam undermines the PBP1-mediated compensatory response.

Cefazolin and Ertapenem, a Synergistic Combination Used To Clear Persistent Staphylococcus aureus Bacteremia.

Ertapenem and cefazolin were used in combination to successfully clear refractory methicillin-susceptible Staphylococcus aureus (MSSA) bacteremia. In addition, recent work has demonstrated activity of combination therapy with beta-lactams from different classes against methicillin-resistant S. aureus (MRSA). The ertapenem-plus-cefazolin combination was evaluated for synergy in vitro and in vivo in a murine skin infection model using an index MSSA bloodstream isolate from a patient in whom persistent bacteremia was cleared with this combination and against a cadre of well-described research strains and clinical strains of MSSA and MRSA. Against the index MSSA bloodstream isolate, ertapenem and cefazolin showed synergy using both checkerboard (fractional inhibitory concentration [FIC] index = 0.375) and time-kill assays. Using a disk diffusion ertapenem potentiation assay, the MSSA isolate showed a cefazolin disk zone increased from 34 to 40 mm. In vitro pharmacokinetic/pharmacodynamic modeling at clinically relevant drug concentrations demonstrated bactericidal activity (>3 log10-CFU/ml reduction) of the combination but bacteriostatic activity of ether drug alone at 48 h. A disk diffusion potentiation assay showed that ertapenem increased the cefazolin zone of inhibition by >3 mm for 34/35 (97%) MSSA and 10/15 (67%) MRSA strains. A murine skin infection model of MSSA showed enhanced activity of cefazolin plus ertapenem compared to monotherapy with these agents. After successful use in clearance of MSSA bacteremia, the combination of ertapenem and cefazolin showed synergy against MSSA in vitro and in vivo This combination may warrant consideration for future clinical study in MSSA bacteremia.

Standard susceptibility testing overlooks potent azithromycin activity and cationic peptide synergy against MDR Stenotrophomonas maltophilia.

OBJECTIVES: The Gram-negative bacillus Stenotrophomonas maltophilia (SM) is an emerging MDR opportunistic pathogen. Recent studies identify a potentially relevant activity of azithromycin against Gram-negative bacteria overlooked in standard bacteriological testing. We investigated azithromycin activity against SM in testing conditions incorporating mammalian tissue culture medium and host defence factors. METHODS: MIC testing, chequerboard assays, time-kill assays and fluorescence microscopy were performed for azithromycin, the cationic peptide antibiotic colistin and the human defence peptide cathelicidin LL-37 alone or in combination in cation-adjusted Mueller-Hinton broth or mammalian tissue culture media. Azithromycin sensitization of SM to host immune clearance was tested in a human neutrophil killing assay and a murine pneumonia model. RESULTS: We observed potent bactericidal activity of azithromycin against SM in mammalian tissue culture medium absent in bacteriological medium. Colistin and LL-37 strongly potentiated azithromycin killing of SM by increasing drug entry. Additionally, azithromycin sensitized SM to neutrophil killing and increased SM clearance in the murine pneumonia model. CONCLUSIONS: Despite lack of activity in standard MIC testing, azithromycin synergizes with cationic peptide antibiotics to kill SM in medium mimicking tissue fluid conditions. Azithromycin, alone or in combination with colistin, merits further exploration in therapy of drug-resistant SM infections.