Tissue Bacterial Burden Research Articles

The multifaceted role of TolA protein in promoting survival, biofilm formation and virulence of avian pathogenic Escherichia coli

Avian pathogenic Escherichia coli (APEC) can spread beyond the intestines and cause systemic infections, leading to various clinical manifestations, including airsacculitis, pericarditis, perihepatitis and colisepticemia. The mechanisms facilitating this extraintestinal infections are not fully understood. In this study, we investigate how the tolA gene affects APEC virulence by encoding a protein involved in maintaining outer membrane integrity. We constructed a tolA deletion mutant of APEC strain E058 and evaluated its growth and survival in various environments, including in vitro cultures and in vivo infection models in chickens. We found that the motility-defective ΔtolA mutant exhibits reduced biofilm formation ability and weakened resistance to the environmental stresses, suggesting an important role for TolA in APEC's survival. The lack of tolA gene affects the bacterial ability to resist the host's immune system, such as complement-mediated serum killing or phagocytosis, as shown by the serum killing and macrophage phagocytosis assays. Additionally, in vivo infection studies using chickens demonstrated that the ΔtolA mutant displayed attenuated virulence, evidenced by reduced mortality and lower tissue bacterial burden. Reverse transcription quantitative real-time PCR (RT-qPCR) analysis revealed that inactivation of tolA led to downregulation of virulence genes associated with serum resistance (traT) and flagellar biosynthesis (fliR). Taken together, our findings demonstrate the multifaceted role of TolA protein in promoting the survival, immune evasion, biofilm formation, and virulence of APEC E058. This suggests that targeting TolA could potentially offer new strategies for combating APEC infections.

Development of rabbit models of ventilator-associated bacterial pneumonia produced by carbapenem-resistant Pseudomonas aeruginosa.

Ventilator-associated bacterial pneumonia (VABP) is among the most intractable of carbapenem-resistant Gram-negative bacterial infections. New antimicrobial agents are critically needed for the treatment of VABP. However, current conventionally used animal model systems are inadequate to meet this challenge. We, therefore, developed rabbit models of VABP caused by carbapenem-resistant Pseudomonas aeruginosa. Persistently neutropenic New Zealand White rabbits were used throughout the study. The early-phase intubated model (0-24 h) received mechanical ventilation, while the late-phase intubated model (72-96 h) was ambulatory. The following outcome parameters were studied: survival, residual tissue bacterial burden (CFU/g), residual BAL bacterial burden (CFU/mL), lung weights, pulmonary lesion score, histology, O2 saturation, radiographic imaging, and histology. Each anesthetized rabbit received a predetermined endotracheal bacterial inoculum, and ventilators were set to FiO2 = 40% and PEEP = 8 mmHg. Within the first 12 h post-inoculation, mean bacterial burdens in lung tissue and BAL fluid, respectively, were established at approximately 107 CFU/g and 106 CFU/mL, persisted through 24 h in the early-phase model and increased in the late-phase model to approximately 108 CFU/g and 107 CFU/mL. Mean max SpO2 was ≥98 mmHg, and mean nadir SpO2 was ≥68 mmHg. Serial thoracic radiographs demonstrated progressive multilobar pneumonic infiltrates. Lung histology revealed progressive focal bronchopneumonia, coagulative necrosis, intra-alveolar hemorrhage, alveolar epithelial cell necrosis, and bacterial microcolonies. The new rabbit model of VABP produced by carbapenem-resistant Pseudomonas aeruginosa recapitulates the pathophysiological, microbiological, diagnostic imaging, and histological patterns of human disease by which to assess critically needed new antimicrobial agents against this lethal infection.

Differential cellular immune responses against Orientia tsutsugamushi Karp and Gilliam strains following acute infection in mice.

Scrub typhus is a leading cause of febrile illness in endemic countries due to infection with Orientia tsutsugamushi (Ot), a seriously understudied intracellular bacterium. Pulmonary involvement associated with vascular parasitism in patients is common and can develop into life threatening interstitial pneumonia. The diverse antigenicity of Ot genotypes and inter-strain differences in genome content are connected to varied virulence and clinical outcomes; however, detailed studies of strain-related pulmonary immune responses in human patients or small animal models of infection are lacking. In this study, we have used two clinically prevalent bacterial strains (Karp and Gilliam) to reveal cellular immune responses in inflamed lungs and potential biomarkers of disease severity. The results demonstrate that outbred CD-1 mice are highly susceptible to both Karp and Gilliam strains; however, C57BL/6 (B6) mice were susceptible to Karp, but resistant to Gilliam (with self-limiting infection), corresponding to their tissue bacterial burdens and lung pathological changes. Multicolor flow cytometric analyses of perfused B6 mouse lungs revealed robust and sustained influx and activation of innate immune cells (macrophages, neutrophils, and NK cells), followed by CD4+ and CD8+ T cells, during Karp infection, but such responses were greatly attenuated during Gilliam infection. The robust cellular responses in Karp-infected B6 mice positively correlated with significantly early and high levels of serum cytokine/chemokine protein levels (CXCL1, CCL2/3/5, and G-CSF), as well as pulmonary gene expression (Cxcl1/2, Ccl2/3/4, and Ifng). In vitro infection of B6 mouse-derived primary macrophages also revealed bacterial strain-dependent immune gene expression profiles. This study provided the lines of evidence that highlighted differential tissue cellular responses against Karp vs. Gilliam infection, offering a framework for future investigation of Ot strain-related mechanisms of disease pathogenesis vs. infection control.

2557. Development of Rabbit Models of Ventilator-Associated Carbapenem-Resistant Acinetobacter baumannii Pneumonia

Abstract Background Carbapenem-resistant (CR) Gram-negative bacteria are emerging worldwide as global public health threats. Mortality rates from these pathogens are especially high in immunocompromised patients. Ventilator-associated bacterial pneumonia (VABP) is among the most intractable of CR Gram-negative bacterial infections. We therefore aimed to develop rabbit animal models of VABP caused by CR strains of Acinetobacter baumannii. Methods Persistently neutropenic NZW rabbits were used throughout the study. A novel endotracheal tracheostomy system was developed for intubation. The early-phase intubated model (0-24h) received mechanical ventilation, while the late-phase intubated model (72-96h) was ambulatory. Ventilators were set to FiO2=40% and PEEP=8mmHg. The following outcome variables were assessed: survival, residual tissue bacterial burden (log CFU/g), residual BAL bacterial burden (log CFU/mL), lung weights, pulmonary lesion score, histology, pulse O2 saturation, radiographic imaging, pro-inflammatory cytokines, quantitative tissue and BAL PCR, and histology. Results Within the first 12h post-inoculation, mean bacterial burdens in lung tissue and BAL fluid, respectively, which were established at approximately 106 CFU/g and 105 CFU/mL, persisted through 24h in the early-phase model and were increased in the late-phase model (72-96h) to approximately 107 CFU/g, and 106 CFU/mL. Mean Max SpO2 was ≥99 mmHg and mean nadir SpO2 was ≥74 mmHg. Serial thoracic radiographs over the course of 12-96h demonstrated progressive multilobar pneumonic infiltrates, while lung histology revealed progressive development of focal bronchopneumonia, intra-alveolar hemorrhage, alveolar epithelial cell necrosis, macrophage activation, monocytic infiltration, alveolar septal disruption, and microcolonies of bacteria. Conclusion The new rabbit model of VABP produced by CR A. baumannii recapitulates the pathophysiological, microbiological, diagnostic imaging, and histological patterns of human disease by which to assess critically needed new antimicrobial agents against this lethal infection. Disclosures All Authors: No reported disclosures

MP34-12 BURDEN OF BLADDER-RESIDENT BACTERIA IN TRIGONE BIOPSIES CORRELATES WITH INCREASED RISK OF RUTI RELAPSE IN POSTMENOPAUSAL WOMEN FOLLOWING ELECTROFULGURATION

You have accessJournal of UrologyCME1 Apr 2023MP34-12 BURDEN OF BLADDER-RESIDENT BACTERIA IN TRIGONE BIOPSIES CORRELATES WITH INCREASED RISK OF RUTI RELAPSE IN POSTMENOPAUSAL WOMEN FOLLOWING ELECTROFULGURATION Jashkaran G. Gadhvi, Parker R. M. Kenee, Fatima Khan, Philippe E. Zimmern, and Nicole J. De Nisco Jashkaran G. GadhviJashkaran G. Gadhvi More articles by this author , Parker R. M. KeneeParker R. M. Kenee More articles by this author , Fatima KhanFatima Khan More articles by this author , Philippe E. ZimmernPhilippe E. Zimmern More articles by this author , and Nicole J. De NiscoNicole J. De Nisco More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003268.12AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Antibiotic-recalcitrant recurrent urinary tract infection (rUTI) is common in postmenopausal women. Therefore, many elect to undergo electrofulguration (EF) of areas of chronic cystitis when standard antibiotic therapies have failed. Previously, tissue-resident bacteria have been observed in the bladder walls of postmenopausal women with rUTI by fluorescence in situ hybridization (FISH). We hypothesized that increased urothelial bacterial burden decreases the likelihood of complete rUTI resolution following EF. METHODS: Following IRB approval, bladder biopsies were obtained from 23 consenting postmenopausal women who elected EF for the advanced management of rUTI. Biopsies were processed and 16S rRNA fluorescence in situ hybridization (FISH) was performed as described previously1 using the following probe sets: 1. Scramble-AlexaFluor488/647 (control), 2. Universal 16s rRNA-AlexaFluor647 (all bacteria), and 3. Escherichia 16s rRNA-AlexaFluor488. Images analysis was performed blinded by two separate evaluators to quantify tissue bacterial burden. Time to rUTI relapse after EF was recorded for all patients during a 1-year follow-up period. The group was dichotomized about the median of quantified bladder bacterial burden and Kaplan-Meir analysis was performed to determine if bladder bacterial burden was associated with risk of rUTI relapse. RESULTS: Women with bladder bacterial burden above the median value had a significantly (p=0.0191) higher risk of rUTI relapse within 6 months of EF (HR=5.276, 95% CI: 1.389-20.02) (Figure 1). Interestingly, bladder-resident E. coli, as detected by genus-specific FISH, was not significantly associated with a higher risk of rUTI relapse. CONCLUSIONS: In this pilot study, we observed that total bladder bacterial burden as quantified by FISH (universal probe) was associated with an ∼5.3x increased risk of rUTI relapse within 6-months in postmenopausal women following EF. A continued analysis of this relationship between bladder bacterial burden and rUTI outcomes may prove useful in the management of these challenging patients. Reference: De Nisco NJ et al. J Mol Biol. 2019 doi: 10.1016/j.jmb.2019.04.008 Source of Funding: N/A © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e463 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Jashkaran G. Gadhvi More articles by this author Parker R. M. Kenee More articles by this author Fatima Khan More articles by this author Philippe E. Zimmern More articles by this author Nicole J. De Nisco More articles by this author Expand All Advertisement PDF downloadLoading ...

Vi polysaccharide and conjugated vaccines afford similar early, IgM or IgG-independent control of infection but boosting with conjugated Vi vaccines sustains the efficacy of immune responses.

Vaccination with Vi capsular polysaccharide (Vi-PS) or protein-Vi typhoid conjugate vaccine (TCV) can protect adults against Salmonella Typhi infections. TCVs offer better protection than Vi-PS in infants and may offer better protection in adults. Potential reasons for why TCV may be superior in adults are not fully understood. Here, we immunized wild-type (WT) mice and mice deficient in IgG or IgM with Vi-PS or TCVs (Vi conjugated to tetanus toxoid or CRM197) for up to seven months, with and without subsequent challenge with Vi-expressing Salmonella Typhimurium. Unexpectedly, IgM or IgG alone were similarly able to reduce bacterial burdens in tissues, and this was observed in response to conjugated or unconjugated Vi vaccines and was independent of antibody being of high affinity. Only in the longer-term after immunization (>5 months) were differences observed in tissue bacterial burdens of mice immunized with Vi-PS or TCV. These differences related to the maintenance of antibody responses at higher levels in mice boosted with TCV, with the rate of fall in IgG titres induced to Vi-PS being greater than for TCV. Therefore, Vi-specific IgM or IgG are independently capable of protecting from infection and any superior protection from vaccination with TCV in adults may relate to responses being able to persist better rather than from differences in the antibody isotypes induced. These findings suggest that enhancing our understanding of how responses to vaccines are maintained may inform on how to maximize protection afforded by conjugate vaccines against encapsulated pathogens such as S. Typhi.

Single-cell profiling identifies ACE+ granuloma macrophages as a nonpermissive niche for intracellular bacteria during persistent Salmonella infection.

Macrophages mediate key antimicrobial responses against intracellular bacterial pathogens, such as Salmonella enterica. Yet, they can also act as a permissive niche for these pathogens to persist in infected tissues within granulomas, which are immunological structures composed of macrophages and other immune cells. We apply single-cell transcriptomics to investigate macrophage functional diversity during persistent S. enterica serovar Typhimurium (STm) infection in mice. We identify determinants of macrophage heterogeneity in infected spleens and describe populations of distinct phenotypes, functional programming, and spatial localization. Using an STm mutant with impaired ability to polarize macrophage phenotypes, we find that angiotensin-converting enzyme (ACE) defines a granuloma macrophage population that is nonpermissive for intracellular bacteria, and their abundance anticorrelates with tissue bacterial burden. Disruption of pathogen control by neutralizing TNF is linked to preferential depletion of ACE+ macrophages in infected tissues. Thus, ACE+ macrophages have limited capacity to serve as cellular niche for intracellular bacteria to establish persistent infection.

Efficacy of phage therapy in preclinical models of bacterial infection: a systematic review and meta-analysis

Antimicrobial resistance of bacterial pathogens is an increasing clinical problem and alternative approaches to antibiotic chemotherapy are needed. One of these approaches is the use of lytic bacterial viruses known as phage therapy. We aimed to assess the efficacy of phage therapy in preclinical animal models of bacterial infection. In this systematic review and meta-analysis, MEDLINE/Ovid, Embase/Ovid, CINAHL/EbscoHOST, Web of Science/Wiley, Cochrane Central Register of Controlled Trials, Cochrane Database of Systematic Reviews, and Google Scholar were searched from inception to Sept 30, 2021. Studies assessing phage efficacy in animal models were included. Only studies that assessed the efficacy of phage therapy in treating established bacterial infections in terms of survival and bacterial abundance or density were included. Studies reporting only in-vitro or ex-vivo results and those with incomplete information were excluded. Risk-of-bias assessment was performed using the Systematic Review Centre for Laboratory Animal Experimentation tool. The main endpoints were animal survival and tissue bacterial burden, which were reported using pooled odds ratios (ORs) and mean differences with random-effects models. The I2 measure and its 95% CI were also calculated. This study is registered with PROSPERO, CRD42022311309. Of the 5084 references screened, 124 studies fulfilled the selection criteria. Risk of bias was high for 70 (56%) of the 124 included studies; therefore, only studies classified as having a low-to-moderate risk of bias were considered for quantitative data synthesis (n=32). Phage therapy was associated with significantly improved survival at 24 h in systemic infection models (OR 0·08 [95% CI 0·03 to 0·20]; I2=55% [95% CI 8 to 77]), skin infection (OR 0·08 [0·04 to 0·19]; I2 = 0% [0 to 79]), and pneumonia models (OR 0·13 [0·06 to 0·31]; I2=0% [0 to 68]) when compared with placebo. Animals with skin infections (mean difference -2·66 [95% CI -3·17 to -2·16]; I2 = 95% [90 to 96]) and those with pneumonia (mean difference -3·35 [-6·00 to -0·69]; I2 = 99% [98 to 99]) treated with phage therapy had significantly lower tissue bacterial loads at 5 ± 2 days of follow-up compared with placebo. Phage therapy significantly improved animal survival and reduced organ bacterial loads compared with placebo in preclinical animal models. However, high heterogeneity was observed in some comparisons. More evidence is needed to identify the factors influencing phage therapy performance to improve future clinical application. Swiss National Foundation and Swiss Heart Foundation.

Predominance of Escherichia-Shigella in Gut Microbiome and Its Potential Correlation with Elevated Level of Plasma Tumor Necrosis Factor Alpha in Patients with Tuberculous Meningitis.

Tuberculous meningitis (TBM), the most lethal and disabling form of tuberculosis (TB), may be related to gut microbiota composition, warranting further study. Here we systematically compared gut microbiota compositions and blood cytokine profiles of TBM patients, pulmonary TB patients, and healthy controls. Notably, the significant gut microbiota dysbiosis observed in TBM patients was associated with markedly high proportions of Escherichia-Shigella species as well as increased blood levels of tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6). Next, we obtained a fecal bacterial isolate from a TBM patient and administered it via oral gavage to mice in order to develop a murine gut microbiota dysbiosis model for use in exploring mechanisms underlying the observed relationship between gut microbial dysbiosis and TBM. Thereafter, cells of commensal Escherichia coli (E. coli) were isolated and administered to model mice by gavage and then mice were inoculated with Mycobacterium tuberculosis (M. tuberculosis). Subsequently, these mice exhibited increased blood TNF-α levels accompanied by downregulated expression of tight junction protein claudin-5, increased brain tissue bacterial burden, and elevated central nervous system inflammation relative to corresponding indicators in controls administered PBS by gavage. Thus, our results demonstrated that a signature dysbiotic gut microbiome profile containing a high proportion of E. coli was potentially associated with an increased circulating TNF-α level in TBM patients. Collectively, these results suggest that modulation of dysbiotic gut microbiota holds promise as a new strategy for preventing or alleviating TBM. IMPORTANCE As the most severe form of tuberculosis, the pathogenesis of tuberculous meningitis (TBM) is still unclear. Gut microbiota dysbiosis plays an important role in a variety of central nervous system diseases. However, the relationship between gut microbiota and TBM has not been identified. In our study, significant dysbiosis in gut microbiota composition with a high proportion of E. coli and increased levels of TNF-α in plasma was noted in TBM patients. A commensal E. coli was isolated and shown to increase the plasma level of TNF-α and downregulate brain tight junction protein claudin-5 in the murine model. Gavage administration of E. coli aggravated the bacterial burden and increased the inflammatory responses in the central nervous system after M. tuberculosis infection. Dysbiosis of gut microbiota may be a promising therapeutic target and biomarker for TBM prevention or treatment.

Duration of peripheral blood cellular immune responses induced in dogs in response to a modified live attenuated vaccine for Ehrlichia chaffeensis

Abstract Ehrlichia chaffeensis (ECH) is an obligate intracellular gram-negative bacterium and a frequent cause of severe and fatal tick-borne infection in people in North America. The canine is a common incidental host and a useful model for vaccine development and assessing immune responses to ECH infection. We recently established mutagenesis methods for ECH and demonstrated that immunization of dogs with an ECH mutant with a functional disruption in the ECH_0660 gene protected the vertebrate host from both tick-transmitted and intravenous wildtype ECH challenge when given 4 weeks after immunization. In the current study, we evaluated the duration of immunity offered by the ECH_0660 gene mutant live attenuated vaccine (MLAV) against wildtype infection challenge. Groups of 12 dogs were immunized with the ECH MLAV and then challenged 2, 4, 8 and 12 months later via tick-transmitted or blood-transmitted infection with wildtype ECH. Animals were monitored for 4 weeks after challenge and then sacrificed for evaluation of tissue pathology and bacterial burden. Immunization with the MLAV induced ECH-specific T cell responses, detectable by ELISPOT for IFNγ; and differentiation of IFNγ- and/or TNFα-producing ECH-specific T effector memory CD4 T cells (CD45RAneg CD45RO+ CD62Lneg). Antigen-specific recall responses were detectable in the peripheral blood of most (27/32) vaccinated dogs for 4 months after immunization, but only in 5/24 of the dogs after 8 months. This result suggests that a single MLAV vaccination induces cellular immune responses and differentiation of memory helper T cells. Future work will investigate the correlations between cellular immunity, humoral responses and bacterial load. Supported by grants from NIH (R01 AI152418)

Distinct Role of TNFR1 and TNFR2 in Protective Immunity Against Orientia tsutsugamushi Infection in Mice.

Infection with Orientia tsutsugamushi, an obligate intracellular bacterium, can cause mild or severe scrub typhus. Some patients develop acute lung injury, multi-organ failure, and fatal infection; however, little is known regarding key immune mediators that mediate infection control or disease pathogenesis. Using murine models of scrub typhus, we demonstrated in this study the requirement of TNF-TNFR signaling in protective immunity against this infection. Mice lacking both TNF receptors (TNFR1 and TNFR2) were highly susceptible to O. tsutsugamushi infection, displaying significantly increased tissue bacterial burdens and succumbing to infection by day 9, while most wild-type mice survived through day 20. This increased susceptibility correlated with poor activation of cellular immunity in inflamed tissues. Flow cytometry of lung- and spleen-derived cells revealed profound deficiencies in total numbers and activation status of NK cells, neutrophils, and macrophages, as well as CD4 and CD8 T cells. To define the role of individual receptors in O. tsutsugamushi infection, we used mice lacking either TNFR1 or TNFR2. While deficiency in either receptor alone was sufficient to increase host susceptibility to the infection, TNFR1 and TNFR2 played a distinct role in cellular responses. TNF signaling through TNFR1 promoted inflammatory responses and effector T cell expansion, while TNFR2 signaling was associated with anti-inflammatory action and tissue homeostasis. Moreover, TNFRs played an intrinsic role in CD8+ T cell activation, revealing an indispensable role of TNF in protective immunity against O. tsutsugamushi infection.

CXCL20a, a Teleost-Specific Chemokine That Orchestrates Direct Bactericidal, Chemotactic, and Phagocytosis-Killing–Promoting Functions, Contributes to Clearance of Bacterial Infections

The major role of chemokines is to act as a chemoattractant to guide the migration of immune cells to the infectious sites. In the current study, we found that CiCXCL20a, a teleost-specific chemokine from grass carp (Ctenopharyngodon idella), demonstrates broad-spectrum, potent, direct bactericidal activity and immunomodulatory functions to bacterial infections, apart from the chemotaxis. CiCXCL20a kills bacteria by binding, mainly targeting acid lipids, perforating bacterial membrane, resulting in bacterial cytoplasm leakage and death. CiCXCL20a aggregates and neutralizes LPS, agglutinates Gram-negative bacteria, and binds to peptidoglycan and Gram-positive bacteria, but not agglutinate them. All the complexes may be phagocytized and cleared away. CiCXCL20a chemoattracts leukocytes, facilitates phagocytosis of myeloid leukocytes, not lymphoid leukocytes, and enhances the bacteria-killing ability in leukocytes. We further identified its receptor CiCXCR3.1b1. Furthermore, we investigated the physiological roles of CiCXCL20a against Aeromonas hydrophila infection in vivo. The recombinant CiCXCL20a increases the survival rate and decreases the tissue bacterial loads, edema, and lesions. Then, we verified this function by purified CiCXCL20a Ab blockade, and the survival rate decreases, and the tissue bacterial burdens increase. In addition, zebrafish (Danio rerio) DrCXCL20, an ortholog of CiCXCL20a, was employed to verify the bactericidal function and mechanism. The results indicated that DrCXCL20 also possesses wide-spectrum, direct bactericidal activity through membrane rupture mechanism. The present study, to our knowledge, provides the first evidence that early vertebrate chemokine prevents from bacterial infections by direct bactericidal and phagocytosis-killing-promoting manners. The results also demonstrate the close functional relationship between chemokines and antimicrobial peptides.

A Multi-Modal Approach to Closing Exploratory Laparotomies Including High-Risk Wounds.

BackgroundLaparotomy incisions with contamination have a high incidence of surgical site infection (SSI). One strategy to reduce SSI has been to allow these wounds to heal by secondary intention; however, this results in an ongoing need for wound care after discharge.MethodsA prospectively maintained Acute and Critical Care Surgery database was queried for patients who underwent exploratory laparotomy during 2008-2018. Patients were stratified into two groups: 2008-2015 (no protocol [NP]) and 2016-2018 (closure protocol [CP]). CP patients were operated on by a single surgeon utilizing a multi-modal high-risk incisional closure protocol, which included dilute chlorhexidine lavage, closed suction drains for incisions deeper than 3 centimeters, and incisional negative-pressure wound therapy (iNPWT). The CDC (Centers for Disease Control and Prevention) guidelines were used to determine wound classification and SSI based on chart review. Groups were compared using univariate and multivariate analysis.ResultsA total of 139 patients met the study criteria. The overall SSI rate, including superficial and deep space infections, was no different in NP versus CP (21.6 vs. 24.1%; p=0.74). The rate of superficial SSI was similar between NP and CP (11.8 vs. 8.4%; p=0.53). Rates of wound closure at discharge were higher in the CP group than the NP group across wound classes, with the greatest difference among dirty wounds (50.0% NP vs. 94.9% CP; p<0.01). CP significantly increased the likelihood of wound closure (OR=179.2; p<0.001) even after controlling for body mass index, wound classification, ASA (American Society of Anesthesiologists) status, and initially open abdomen.ConclusionsBy addressing both tissue factors and bacterial burden through the use of a multi-modal high-risk incisional closure protocol involving iNPWT, all wounds can be considered for closure without increasing the risk of SSI.

Ischemic stroke alters immune cell niche and chemokine profile in mice independent of spontaneous bacterial infection.

IntroductionStroke‐associated pneumonia (SAP) is a major cause of mortality in patients who have suffered from severe ischemic stroke. Although multifactorial in nature, stroke‐induced immunosuppression plays a key role in the development of SAP. Previous studies using a murine model of transient middle cerebral artery occlusion (tMCAO) have shown that focal ischemic stroke induction results in functional defects of lymphocytes in the spleen, thymus, and peripheral blood, leading to spontaneous bacterial infection in the lungs without inoculation. However, how ischemic stroke alters immune cell niche and the expression of cytokines and chemokines in the lungs has not been fully characterized.MethodsIschemic stroke was induced in mice by tMCAO. Immune cell profiles in the brain and the lungs at 24‐ and 72‐hour time points were compared by flow cytometric analysis. Cytokine and chemokine expression in the lungs were determined by multiplex bead arrays. Tissue damage and bacterial burden in the lungs following tMCAO were evaluated.ResultsIschemic stroke increases the percentage of alveolar macrophages, neutrophils, and CD11b+ dendritic cells, but reduces the percentage of CD4+ T cells, CD8+ T cells, B cells, natural killer cells, and eosinophils in the lungs. The alteration of immune cell niche in the lungs coincides with a significant reduction in the levels of multiple chemokines in the lungs, including CCL3, CCL4, CCL5, CCL17, CCL20, CCL22, CXCL5, CXCL9, and CXCL10. Spontaneous bacterial infection and tissue damage following tMCAO, however, were not observed.ConclusionThis is the first report to demonstrate a significant reduction of lymphocytes and multiple proinflammatory chemokines in the lungs following ischemic stroke in mice. These findings suggest that ischemic stroke directly impacts pulmonary immunity.

Modulation of Th17 and regulatory T-cell responses during murine pregnancy contributes to increased maternal susceptibility to Salmonella Typhimurium infection.

Salmonella Typhimurium (S. Tm) infection in pregnant mice results in massive placental infection, fetal loss, and exacerbated systemic infection. The Th17 host response can aid control of S. Tm infection, whereas successful pregnancy correlates to a dampened inflammatory and enhanced regulatory T-cell (Treg) response. Mice were infected systemically with S. Tm and tissue bacterial burden, splenic Th17 and Treg cell numbers, and serum cytokines were analyzed. Splenic and/or placental mRNA expression of IL-17A, RORγ-t, IL-10, and TNF was determined. The effects of in vivo CD25+ cell depletion and TLR4 blockade on the course of S. Tm infection and Th17 response were determined. Enhanced S. Tm burden in pregnant mice was associated with time-dependent increased serum inflammatory cytokines. In vivo, TLR4 blockade reduced splenic S. Tm burden, suggesting detrimental TLR4-mediated inflammation. However, the splenic and placental Th17 response was reduced in S. Tm-infected pregnant mice relative to non-pregnant controls. Alternatively, there was an increase in splenic Treg frequency in pregnant mice and depletion of this subset reduced bacterial burden and increased the Th17 response. Downregulation of Th17 cell responses by Tregs during pregnancy potentially contributes to exacerbation of S. Tm infection in pregnant mice.

Modulation of Th17 and Regulatory T cell responses during murine pregnancy contributes to increased maternal susceptibility to Salmonella Typhimurium infection

PROBLEM Salmonella Typhimurium (S. Tm) infection in pregnant mice results in massive placental infection, fetal loss, and exacerbated systemic infection. The Th17 host response can aid control of S. Tm infection, whereas successful pregnancy correlates to a dampened inflammatory and enhanced regulatory T-cell (Treg) response. METHOD OF STUDY Mice were infected systemically with S. Tm and tissue bacterial burden, splenic Th17 and Treg cell numbers, and serum cytokines were analyzed. Splenic and/or placental mRNA expression of IL-17A, RORγ-t, IL-10, and TNF was determined. The effects of in vivo CD25+ cell depletion and TLR4 blockade on the course of S. Tm infection and Th17 response were determined. RESULTS Enhanced S. Tm burden in pregnant mice was associated with time-dependent increased serum inflammatory cytokines. In vivo, TLR4 blockade reduced splenic S. Tm burden, suggesting detrimental TLR4-mediated inflammation. However, the splenic and placental Th17 response was reduced in S. Tm-infected pregnant mice relative to non-pregnant controls. Alternatively, there was an increase in splenic Treg frequency in pregnant mice and depletion of this subset reduced bacterial burden and increased the Th17 response. CONCLUSION Downregulation of Th17 cell responses by Tregs during pregnancy potentially contributes to exacerbation of S. Tm infection in pregnant mice.

Hepcidin Protects Against Extracellular Infections By Eliminating Non-Transferrin-Bound Iron

The production of the iron-regulatory hormone hepcidin is strongly induced by infection and inflammation, causing hypoferremia. This is proposed to be a host defense response to limit microbial replication. Patients with hereditary hemochromatosis or β-thalassemia, who are hepcidin-deficient and iron-overloaded, are susceptible to infections with gram-negative bacteria. The specific mechanism by which hepcidin and hypoferremia mediate resistance to infection, and the spectrum of microbes affected, are poorly understood.We tested the role of hepcidin in infections with several groups of bacteria: 1) siderophilic gram-negative Yersinia enterocolitica and Vibrio vulnificus, known to have increased virulence in patients with iron overload; 2) clinically common gram-negative Klebsiella pneumoniae and gram-positive Staphylococcus aureus; and 3) primarily intracellular Mycobacterium tuberculosis.In vivo role of hepcidin and iron was assessed by comparing survival and tissue bacterial burden between wild type (WT) and hepcidin knockout (KO) mice. Hepcidin KO mice were tested both in iron-loaded and iron-depleted condition to assess for any iron-independent roles of hepcidin in infection.Hepcidin played the most prominent host defense role in infections with siderophilic pathogens Y. enterocolitica and V. vulnificus. At inocula where WT mice were resistant and had 0% mortality, iron-loaded hepcidin KO mice were highly susceptible to these infections and displayed 100% mortality (Figure). Compared to WT mice, iron-loaded hepcidin KO were also more susceptible to infection with K. pneumoniae, and displayed more rapid and higher mortality (Figure). In all of these models, treatment of infected iron-loaded hepcidin KO mice with hepcidin analogs prevented or decreased mortality. Short-term administration of hepcidin analogs altered serum iron concentrations but not iron stores, indicating that only extracellular iron availability plays a role in infection with these extracellular, gram-negative pathogens. Additional experiments indicated that the protective effect of hepcidin and minihepcidin was not caused by any direct antimicrobial activity but by their effect on extracellular iron.In contrast, in mouse models of infection with gram-positive S. aureus or with M. tuberculosis, there was no difference in susceptibility between hepcidin KO and WT mice. We further tested the effect of parenteral iron administration in WT mice infected with S. aureus and M. tuberculosisbut iron loading of these mice had no effect on bacterial burden. These results indicated that neither hepcidin nor iron availability affect the virulence of these bacteria.We next examined the mechanism by which hepcidin deficiency promotes the growth of gram- negative pathogens. Hepcidin causes a decrease in iron-transferrin concentrations, however iron-transferrin is accessible only to a few highly host-adapted pathogens and most bacteria cannot utilize this form of iron. On the other hand, non-transferrin-bound iron (NTBI), a form of iron that appears in the circulation when the iron-binding capacity of transferrin is exceeded, may be readily utilizable by microbes. Indeed, mice that had increased susceptibility to infection in our models also had detectable NTBI in plasma. We developed an in vitro model to test the dependence of bacterial growth on iron-transferrin or NTBI concentrations. Human plasma was supplemented with graded amounts of ferric ammonium citrate and agar plates generated with a range of increasing transferrin saturation and eventually increasing NTBI concentrations. V. vulnificus, Y. enterocolitica and K. pneumoniaeonly grew when sufficient iron was added to saturate transferrin and generate NTBI. Similarly, bacterial CFU counts in vivo correlated with the presence of NTBI in mouse models but not with transferrin saturation when NTBI was absent.Using mouse models of different bacterial infections, we demonstrated that hepcidin is essential for resistance to infection with certain gram-negative pathogens, but plays no role in the susceptibility to intracellular M. tuberculosis or gram-positive S. aureus. We further provide evidence that the primary host defense function of hepcidin is the elimination of NTBI, and this can be potentiated by the use of synthetic hepcidin analogues to prevent progression of infection and associated mortality. [Display omitted] DisclosuresGanz:Intrinsic LifeSciences: Equity Ownership; Merganser biotech: Consultancy, Equity Ownership; Silarus Therapeutics: Equity Ownership. Nemeth:Merganser Biotech: Consultancy, Equity Ownership; Intrinsic LifeSciences: Equity Ownership; Silarus Therapeutics: Equity Ownership, Research Funding.

Vaccination sequence effects on immunological response and tissue bacterial burden in paratuberculosis infection in a rabbit model.

Paratuberculosis (PTB), a chronic granulomatous enteritis produced by Mycobacterium avium subspecies paratuberculosis (MAP), is considered as one of the diseases with the highest economic impact in the ruminant industry. Vaccination against MAP is recommended during the first months after birth on the basis that protection would be conferred before the first contact with mycobacteria. However, little is known about the therapeutic effect of MAP vaccination in controlled experimental conditions. The current study was designed to evaluate the efficacy of vaccination before and after challenge with MAP in a rabbit infection model. The rabbits were divided into four groups: non-infected control (NIC, n = 4), infected control challenged with MAP (IC, n = 5), vaccinated and challenged 1 month after with MAP (VSI, n = 5) and challenged with MAP and vaccinated 2 months later (IVS, n = 5). The results from this study show a quick increase in IFN-γ release upon stimulation with bovine, avian and johnin PPD in animals vaccinated before MAP challenge. All vaccinated animals show an increased humoral response as seen by western blot and ELISA. The final bacteriology index (considering tissue culture and qPCR) shows that the IC group was the most affected. Vaccination after infection (IVS) produced the lowest bacteriology index showing significant differences with the IC group (p = 0.034). In conclusion, vaccination against MAP shows positive effects in a rabbit model. However, vaccination after infection shows a slightly stronger protective effect compared to vaccination before infection, suggesting a therapeutic effect. This feature could be applied to previously infected adult animals under field conditions.

Murine Models of Bacteremia and Surgical Wound Infection for the Evaluation of Staphylococcus aureus Vaccine Candidates.

There is an unmet need for an effective vaccine to prevent infections caused by Staphylococcus aureus. Murine models of staphylococcal infections are useful tools for evaluation of experimental vaccines and adjuvants in preclinical studies. Mice can be actively immunized with vaccines or passively immunized with antibodies prior to bacterial challenge. We described two infection models, bacteremia and surgical wound infection, that are relevant to human disease. To achieve a persistent bacteremia, mice are challenged with a sublethal inoculum of S. aureus by the intraperitoneal route. Bacteremia is assessed 2 h after challenge, and weight loss and renal infection are quantified after 4 days. Surgical wound infection can be achieved by inoculation of S. aureus directly into the sutured incision of a thigh muscle. After 3 days the tissue bacterial burden and weight loss are evaluated in this localized infection. Protective efficacy of experimental vaccines is analyzed by comparison with mice immunized with appropriate control vaccines.

DosS Is required for the complete virulence of mycobacterium tuberculosis in mice with classical granulomatous lesions.

Mycobacterium tuberculosis (Mtb) must counter hypoxia within granulomas to persist. DosR, in concert with sensor kinases DosS and DosT, regulates the response to hypoxia. Yet Mtb lacking functional DosR colonize the lungs of C57Bl/6 mice, presumably owing to the lack of organized lesions with sufficient hypoxia in that model. We compared the phenotype of the Δ-dosR, Δ-dosS, and Δ-dosT mutants to Mtb using C3HeB/FeJ mice, an alternate mouse model where lesions develop hypoxia. C3HeB/FeJ mice were infected via aerosol. The progression of infection was analyzed by tissue bacterial burden and histopathology. A measure of the comparative global immune responses was also analyzed. Although Δ-dosR and Δ-dosT grew comparably to wild-type Mtb, Δ-dosS exhibited a significant defect in bacterial burden and pathology in vivo, accompanied by ablated proinflammatory response. Δ-dosS retained the ability to induce DosR. The Δ-dosS mutant was also attenuated in murine macrophages ex vivo, with evidence of reduced expression of the proinflammatory signature. Our results show that DosS, but not DosR and DosT, is required by Mtb to survive in C3HeB/FeJ mice. The attenuation of Δ-dosS is not due to its inability to induce the DosR regulon, nor is it a result of the accumulation of hypoxia. That the in vivo growth restriction of Δ-dosS could be mimicked ex vivo suggested sensitivity to macrophage oxidative burst. Anoxic caseous centers within tuberculosis lesions eventually progress to cavities. Our results provide greater insight into the molecular mechanisms of Mtb persistence within host lungs.