RESUMEN
Accurate and timely diagnosis of appendicitis in children can be challenging, which leads to delayed admittance or misdiagnosis that may cause perforation. Surgical management involves the elimination of the focus (appendectomy) and the reduction of the contamination with peritoneal irrigation to prevent sepsis. However, the validity of conventional irrigation methods is being debated, and novel methods are needed. In the present study, the use of cold plasma treated saline solution as an intraperitoneal irrigation solution for the management of acute peritonitis was investigated. Chemical and in vitro microbiological assessments of the plasma-treated solution were performed to determine the appropriate plasma treatment time to be used in in-vivo experiments. To induce acute peritonitis in rats, the cecal ligation and perforation (CLP) model was used. Sixty rats were divided into six groups, namely, sham operation, plasma irrigation, CLP, dry cleaning after CLP, saline irrigation after CLP, and plasma-treated saline irrigation after CLP group. The total antioxidant and oxidant status, oxidative stress index, microbiological, and pathological evaluations were performed. Findings indicated that plasma-treated saline contains reactive species, and irrigation with plasma-treated saline can effectively inactivate intraperitoneal contamination and prevent sepsis with no short-term local and/or systemic toxicity.
Asunto(s)
Peritonitis , Gases em Plasma , Sepsis , Animales , Modelos Animales de Enfermedad , Cavidad Peritoneal/microbiología , Lavado Peritoneal/métodos , Peritonitis/etiología , Gases em Plasma/farmacología , Gases em Plasma/uso terapéutico , Ratas , Solución Salina , Sepsis/complicacionesRESUMEN
Peritoneal immune cells reside unanchored within the peritoneal fluid in homeostasis. Here, we examined the mechanisms that control bacterial infection in the peritoneum using a mouse model of abdominal sepsis following intraperitoneal Escherichia coli infection. Whole-mount immunofluorescence and confocal microscopy of the peritoneal wall and omentum revealed that large peritoneal macrophages (LPMs) rapidly cleared bacteria and adhered to the mesothelium, forming multilayered cellular aggregates composed by sequentially recruited LPMs, B1 cells, neutrophils, and monocyte-derived cells (moCs). The formation of resident macrophage aggregates (resMφ-aggregates) required LPMs and thrombin-dependent fibrin polymerization. E. coli infection triggered LPM pyroptosis and release of inflammatory mediators. Resolution of these potentially inflammatory aggregates required LPM-mediated recruitment of moCs, which were essential for fibrinolysis-mediated resMφ-aggregate disaggregation and the prevention of peritoneal overt inflammation. Thus, resMφ-aggregates provide a physical scaffold that enables the efficient control of peritoneal infection, with implications for antimicrobial immunity in other body cavities, such as the pleural cavity or brain ventricles.
Asunto(s)
Infecciones Bacterianas/etiología , Infecciones Bacterianas/metabolismo , Interacciones Huésped-Patógeno/inmunología , Macrófagos Peritoneales/inmunología , Macrófagos Peritoneales/metabolismo , Cavidad Peritoneal/microbiología , Animales , Biomarcadores , Microambiente Celular/inmunología , Modelos Animales de Enfermedad , Susceptibilidad a Enfermedades/inmunología , Mediadores de Inflamación/metabolismo , Ratones , Peritonitis/etiología , Peritonitis/metabolismo , Peritonitis/patologíaRESUMEN
Gut microbiota contribute to shaping the immune repertoire of the host, whereas probiotics may exert beneficial effects by modulating immune responses. Having in mind the differences in both the composition of gut microbiota and the immune response between rats of Albino Oxford (AO) and Dark Agouti (DA) rat strains, we investigated if intraperitoneal (i.p.) injection of live Lactobacillus rhamnosus (LB) may influence peritoneal cavity cell response to in vitro treatments with selected microbiota in the rat strain-dependent manner. Peritoneal cavity cells from AO and DA rats were lavaged two (d2) and seven days (d7) following i.p. injection with LB and tested for NO, urea, and H2O2 release basally, or upon in vitro stimulation with autologous E.coli and Enterococcus spp. Whereas the single i.p. injection of LB nearly depleted resident macrophages and increased the proportion of small inflammatory macrophages and monocytes on d2 in both rat strains, greater proportion of MHCIIhiCD163- and CCR7+ cells and increased NO/diminished H2O2 release in DA compared with AO rats suggest a more intense inflammatory priming by LB in this rat strain. Even though E.coli- and/or Enterococcus spp.-induced rise in H2O2 release in vitro was abrogated by LB in cells from both rat strains, LB prevented microbiota-induced increase in NO/urea ratio only in cells from AO and augmented it in cells from DA rats. Thus, the immunomodulatory properties may not be constant for particular probiotic bacteria, but shaped by innate immunity of the host.
Asunto(s)
Microbioma Gastrointestinal/inmunología , Tracto Gastrointestinal/microbiología , Inmunidad Innata , Lacticaseibacillus rhamnosus/inmunología , Macrófagos Peritoneales/microbiología , Cavidad Peritoneal/microbiología , Probióticos , Animales , Citocinas/metabolismo , Enterococcus/inmunología , Escherichia coli/inmunología , Femenino , Interacciones Huésped-Patógeno , Peróxido de Hidrógeno/metabolismo , Macrófagos Peritoneales/inmunología , Macrófagos Peritoneales/metabolismo , Óxido Nítrico/metabolismo , Fenotipo , Ratas , Especificidad de la Especie , Urea/metabolismoRESUMEN
MicroRNAs play a variety of roles in the progress of inflammation. Herein, we investigated the roles of miR-223 in governing macrophage polarization balance in the progress of sepsis. We firstly observed that miR-223 was down-regulated at the early phase and up-regulated at the late phase of sepsis in macrophages; the levels of miR-223 were positively correlated to the ratio of M2 macrophages during sepsis. In miR-223 knockout mice, we observed that miR-223 was dispensable for efficient pro-inflammatory responses, but was required for efficient M2-associated phenotype and function. miR-223 deletion increased clinical scores of sepsis, leading to increased mortality in septic mice. Furthermore, we found that miR-223 expression in M2-type macrophages was controlled by interleukin (IL)-4, but not IL-10; IL-4 antibodies were able to downregulate the levels of miR-223, increased the expression of targeted genes Nfat5 and Rasa1, reduced the ratio of M2 macrophages, resulting in a decreased survival rate in septic mice. Meanwhile, miR-223 deficient macrophages appeared a markedly decreased M2-type polarization when induced by IL-4, but did not affect macrophages skew to M2 phenotype induced by IL-10. Taken together, our results demonstrate that miR-223 acts as an important regulator to modulate IL-4-meditated M2-type polarization of macrophages via targeting to Nfat5 and Rasa1 to control the progress of sepsis.
Asunto(s)
Interleucina-4/inmunología , Macrófagos/inmunología , MicroARNs/inmunología , Sepsis/genética , Sepsis/inmunología , Animales , Carga Bacteriana , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Cavidad Peritoneal/microbiología , Fagocitosis , Fenotipo , Sepsis/microbiologíaRESUMEN
AIMS: To demonstrate a new laparoscopic sign "Sharma's Parachute sign" in abdominopelvic tuberculosis in women with infertility. METHODS: A total of 104 women who were diagnosed to have abdominopelvic tuberculosis, on endometrial sampling or on laparoscopy were enrolled in this ongoing study on tuberculosis in infertility. A new laparoscopic "Sharma's parachute sign" was looked for in these cases on laparoscopy. RESULTS: The mean age, pairty and duration of infertility was 27.6 years, 0.58 and 4.1 years respectively. Menstrual dysfuction were common especially hypomenorrhoea (34.61%), oligomenorrhoea (36.53%) along with constitutional symptoms and abdomino pelvic pain or lump. Diagnosis of abdominopelvic tuberculosis was made by identification of acid fast bacilli (AFB) on microscopy or culture of endometrial aspirate or peritoneal biopsy or positive gene Xpert or positive polymerase chain reaction (PCR) or histopathological demonstration of epithelioid granuloma on endometrial or peritoneal biopsy, various laparoscopic findings on pelvic and abdominal organs were tubercles and shaggy areas (white deposits, caseous nodules encysted ascites, abdominal and pelvic adhesions, tubal findings (hydrosalpinx, pyosalpinx, beaded or calcified tubes). A new "Sharma's parachute sign"in which ascending colon was totally adherent to anterior abdominal wall with its mesocolon looking like an open parachute with small caseous nodule was seen in 11 (10.5%) cases. CONCLUSION: Diagnostic laparoscopy is an important investigation for abdominopelvic tuberculosis showing various adhesions including new parachute sign.
Asunto(s)
Biopsia/métodos , Endometrio , Laparoscopía/métodos , Mycobacterium tuberculosis/aislamiento & purificación , Adherencias Tisulares/diagnóstico por imagen , Tuberculosis de los Genitales Femeninos , Cavidad Abdominal/microbiología , Dolor Abdominal/diagnóstico , Dolor Abdominal/etiología , Adulto , Diagnóstico Diferencial , Endometrio/microbiología , Endometrio/patología , Femenino , Humanos , Infertilidad Femenina/diagnóstico , Infertilidad Femenina/etiología , Oligomenorrea/diagnóstico , Oligomenorrea/etiología , Dolor Pélvico/diagnóstico , Dolor Pélvico/etiología , Cavidad Peritoneal/microbiología , Tuberculosis de los Genitales Femeninos/complicaciones , Tuberculosis de los Genitales Femeninos/diagnóstico por imagen , Tuberculosis de los Genitales Femeninos/fisiopatologíaRESUMEN
Mouse models of bacterial sepsis are widely used in research to investigate the underlying molecular mechanisms of sepsis and to develop clinically useful therapeutic regimens. Three commonly used mouse sepsis models include (a) injection of bacterial endotoxin, (b) infusion of cultured bacteria, and (c) cecal ligation and puncture. Here we describe the induction of bacterial sepsis in mice by intraperitoneal injection of cultured live Escherichia coli cells. The severity of the sepsis can be regulated by the number of E. coli cells injected into the peritoneal cavity of mice.
Asunto(s)
Escherichia coli/crecimiento & desarrollo , Sepsis/microbiología , Animales , Ciego/microbiología , Modelos Animales de Enfermedad , Endotoxinas/administración & dosificación , Inyecciones Intraperitoneales/métodos , Ligadura/métodos , Ratones , Ratones Endogámicos C57BL , Cavidad Peritoneal/microbiología , Punciones/métodosRESUMEN
Sepsis is a life-threatening organ dysfunction condition caused by a dysregulated host response to an infection. Here we report that the circulating levels of growth and differentiation factor-15 (GDF15) are strongly increased in septic shock patients and correlate with mortality. In mice, we find that peptidoglycan is a potent ligand that signals through the TLR2-Myd88 axis for the secretion of GDF15, and that Gdf15-deficient mice are protected against abdominal sepsis due to increased chemokine CXC ligand 5 (CXCL5)-mediated recruitment of neutrophils into the peritoneum, leading to better local bacterial control. Our results identify GDF15 as a potential target to improve sepsis treatment. Its inhibition should increase neutrophil recruitment to the site of infection and consequently lead to better pathogen control and clearance.
Asunto(s)
Bacteriemia/inmunología , Quimiocina CXCL5/inmunología , Factor 15 de Diferenciación de Crecimiento/inmunología , Neutrófilos/inmunología , Animales , Bacteriemia/genética , Bacteriemia/microbiología , Bacteriemia/prevención & control , Quimiocina CXCL5/genética , Femenino , Factor 15 de Diferenciación de Crecimiento/genética , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Infiltración Neutrófila , Cavidad Peritoneal/microbiologíaRESUMEN
Formyl peptide receptors (FPRs, mouse Fprs) belong to the G protein-coupled receptor superfamily and mediate phagocyte migration in response to bacteria- and host-derived chemoattractants; however, knowledge about their in vivo roles in bacterial pathogenesis is limited. In this study, we investigated the role of Fpr1 and Fpr2 in host defense against Escherichia coli infection. In vitro, we found that supernatants from E. coli cultures induced chemotaxis of wild-type (WT) mouse bone marrow-derived neutrophils and that the activity was significantly reduced in cells genetically deficient in either Fpr1 or Fpr2 and was almost absent in cells lacking both receptors. Consistent with this, E. coli supernatants induced chemotaxis and MAPK phosphorylation in HEK293 cells expressing either recombinant Fpr1 or Fpr2 but not untransfected parental cells. WT bone marrow -derived neutrophils could actively phagocytose and kill E. coli, whereas both activities were diminished in cells lacking Fpr1 or Fpr2; again, an additive effect was observed in cells lacking both receptors. In vivo, Fpr1 and Fpr2 deficiency resulted in reduced recruitment of neutrophils in the liver and peritoneal cavity of mice infected with inactivated E. coli Moreover, Fpr1-/- and Fpr2-/- mice had significantly increased mortality compared with WT mice after i.p. challenge with a virulent E. coli clinical isolate. These results indicate a critical role of Fprs in host defense against E. coli infection.
Asunto(s)
Infecciones por Escherichia coli/inmunología , Infecciones por Escherichia coli/metabolismo , Escherichia coli/inmunología , Receptores de Formil Péptido/inmunología , Receptores de Formil Péptido/metabolismo , Animales , Médula Ósea/inmunología , Médula Ósea/metabolismo , Médula Ósea/microbiología , Células Cultivadas , Quimiotaxis/inmunología , Células HEK293 , Humanos , Hígado/inmunología , Hígado/metabolismo , Hígado/microbiología , Ratones , Ratones Endogámicos C57BL , Proteínas Quinasas Activadas por Mitógenos/inmunología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Neutrófilos/inmunología , Neutrófilos/metabolismo , Neutrófilos/microbiología , Cavidad Peritoneal/microbiología , Fagocitosis/inmunología , Fosforilación/inmunologíaRESUMEN
BACKGROUND & AIMS: Mucosal-associated invariant T (MAIT) cells are depleted from blood in patients with advanced liver disease and show features of immune dysfunction. Because circulating MAIT cells differ from organ-resident MAIT cells, we aimed to investigate the frequency, phenotype, and function of peritoneal MAIT cells from patients with cirrhosis and spontaneous bacterial peritonitis (SBP). METHODS: MAIT cells in blood and ascitic fluid from patients with cirrhosis were characterized using flow cytometry. Healthy individuals and noncirrhotic patients undergoing peritoneal dialysis served as controls. MAIT cell migration was studied in transwell assays. Cytokine release in response to infected ascitic fluid and bacterial products was assessed in vitro. RESULTS: Peritoneal CD3+ CD161hi Vα7.2+ T cells had an inflammatory, tissue retention phenotype, expressing the alpha E integrin, the chemokine receptors CCR5 and CXCR3, and the activation marker CD69 at higher levels than their circulating equivalents. Seventy-seven percent bound to MR1 tetramers loaded with the pyrimidine intermediate 5-(2-oxopropylideneamino)-6-d-ribitylaminouracil. The ratio of peritoneal to blood MAIT cell frequency increased from 1.3 in the absence of SBP to 2.6 at diagnosis and decreased by day 3. MAIT cells migrated toward infected ascitic fluid containing CCL5 and CCL20 and released cytokines in an MR1-restricted fashion. Whereas the depleted circulating MAIT cell pool displayed features of immune exhaustion, peritoneal MAIT cells remained competent producers of inflammatory cytokines in response to bacterial products. Peritoneal MAIT activation correlated with systemic inflammation, suggesting a possible link between peritoneal and systemic immunity. CONCLUSIONS: Peritoneal MAIT cells phenotypically and functionally differ from circulating MAIT cells in decompensated cirrhosis and redistribute to the peritoneum during SBP.
Asunto(s)
Líquido Ascítico/citología , Infecciones Bacterianas/inmunología , Enfermedad Hepática en Estado Terminal/complicaciones , Cirrosis Hepática/complicaciones , Células T Invariantes Asociadas a Mucosa/inmunología , Peritonitis/inmunología , Adulto , Anciano , Anciano de 80 o más Años , Líquido Ascítico/inmunología , Infecciones Bacterianas/sangre , Infecciones Bacterianas/microbiología , Infecciones Bacterianas/patología , Estudios de Casos y Controles , Enfermedad Hepática en Estado Terminal/sangre , Enfermedad Hepática en Estado Terminal/diagnóstico , Enfermedad Hepática en Estado Terminal/inmunología , Femenino , Estudios de Seguimiento , Voluntarios Sanos , Humanos , Hígado/patología , Cirrosis Hepática/sangre , Cirrosis Hepática/diagnóstico , Cirrosis Hepática/inmunología , Masculino , Persona de Mediana Edad , Cavidad Peritoneal/microbiología , Cavidad Peritoneal/patología , Peritonitis/sangre , Peritonitis/microbiología , Peritonitis/patología , Índice de Severidad de la EnfermedadRESUMEN
OBJECTIVES: To determine whether synthetic phosphorylated hexa-acyl disaccharides provide antimicrobial protection in clinically relevant models of bacterial infection. DESIGN: Laboratory study. SETTING: University laboratory. SUBJECTS: BALB/c, C57BL/10J, and C57BL/10ScNJ mice. INTERVENTIONS: Mice were treated with lactated Ringer's (vehicle) solution, monophosphoryl lipid A, or phosphorylated hexa-acyl disaccharides at 48 and 24 hours prior to intraperitoneal Pseudomonas aeruginosa or IV Staphylococcus aureus infection. Leukocyte recruitment, cytokine production, and bacterial clearance were measured 6 hours after P. aeruginosa infection. In the systemic S. aureus infection model, one group of mice was monitored for 14-day survival and another for S. aureus tissue burden at 3 days postinfection. Duration of action for 3-deacyl 6-Acyl phosphorylated hexa-acyl disaccharide was determined at 3, 10, and 14 days using a model of intraperitoneal P. aeruginosa infection. Effect of 3-deacyl 6-Acyl phosphorylated hexa-acyl disaccharide on in vivo leukocyte phagocytosis and respiratory burst was examined. Leukocyte recruitment, cytokine production, and bacterial clearance were measured after P. aeruginosa infection in wild-type and toll-like receptor 4 knockout mice treated with 3-deacyl 6-Acyl phosphorylated hexa-acyl disaccharide or vehicle to assess receptor specificity. MEASUREMENTS AND MAIN RESULTS: During intraperitoneal P. aeruginosa infection, phosphorylated hexa-acyl disaccharides significantly attenuated infection-induced hypothermia, augmented leukocyte recruitment and bacterial clearance, and decreased cytokine production. At 3 days post S. aureus infection, bacterial burden in lungs, spleen, and kidneys was significantly decreased in mice treated with monophosphoryl lipid A or phosphorylated hexa-acyl disaccharides, which was associated with improved survival. Leukocyte phagocytosis and respiratory burst functions were enhanced after treatment with monophosphoryl lipid A or phosphorylated hexa-acyl disaccharides. A time course study showed that monophosphoryl lipid A- and 3-deacyl 6-Acyl phosphorylated hexa-acyl disaccharide-mediated protection against P. aeruginosa lasts for up to 10 days. Partial loss of augmented innate antimicrobial responses was observed in toll-like receptor 4 knockout mice treated with 3-deacyl 6-Acyl phosphorylated hexa-acyl disaccharide. CONCLUSIONS: Phosphorylated hexa-acyl disaccharides significantly augment resistance against clinically relevant Gram-negative and Gram-positive infections via enhanced leukocyte recruitment, phagocytosis, and respiratory burst functions of innate leukocytes. Improved antimicrobial protection persists for up to 10 days and is partially mediated through toll-like receptor 4.
Asunto(s)
Infección Hospitalaria/prevención & control , Citocinas/metabolismo , Disacáridos/farmacología , Hexosaminidasa A/farmacología , Cavidad Peritoneal/fisiopatología , Infecciones Estafilocócicas/fisiopatología , Análisis de Varianza , Animales , Western Blotting/métodos , Modelos Animales de Enfermedad , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Cavidad Peritoneal/microbiología , Distribución Aleatoria , Infecciones Estafilocócicas/mortalidad , Estadísticas no Paramétricas , Tasa de SupervivenciaRESUMEN
OBJECTIVE: To establish the composition of bacteria in mice following cecum ligation and puncture (CLP) through metagenomic analysis and investigate the role of TLRs on the composition of bacteria. METHODS: Total DNA extraction was done from the ascites, blood, and fecal samples from C57BL/6 mice sacrificed at 0, 4, 8, and 16 h, as well as from Tlr2-/-, Tlr4-/-, Tlr5-/-, and NF-κB-/-mice sacrificed at 16 h following CLP. Amplification of the V3-V4 regions of the bacterial 16S rRNA genes by PCR and the Illumina MiSeq sequencer was used for deep sequencing. Hierarchical clustering of the isolates was performed with Ward's method using Euclidean distances. The relative abundance according to operational taxonomic unit (OTU) number or taxa was used to compare the richness among subgroups in the experiments. RESULTS: There were 18 taxa that had significantly different abundances among the different samples of the C57BL/6 mice at 16 h following CLP. Various dynamic changes in the infectious bacteria inside the peritoneal cavity after CLP were found. While knockout of Tlr5 and NF-κB impaired the ability of bacterial clearance inside the peritoneal cavity for some kinds of bacteria found in the C57BL/6 mice, the knockout of Tlr4 enhanced clearance for other kinds of bacteria, and they presented excessive abundance in the peritoneal cavity despite their scarce abundance in the stool. CONCLUSION: NF-κB and TLRs are involved in bacterial clearance and in the expression pattern of the bacterial abundance inside the peritoneal cavity during polymicrobial infection.
Asunto(s)
Bacterias/clasificación , Metagenómica/métodos , Cavidad Peritoneal/microbiología , Receptores Toll-Like/genética , Animales , Ascitis/microbiología , Bacterias/genética , Sangre/microbiología , ADN Bacteriano , ADN Ribosómico/genética , Heces/microbiología , Técnicas de Silenciamiento del Gen , Secuenciación de Nucleótidos de Alto Rendimiento , Ratones , Ratones Endogámicos C57BL , FN-kappa B/genética , Filogenia , ARN Ribosómico 16S/genéticaRESUMEN
Macrophages resident in different organs express distinct genes, but understanding how this diversity fits into tissue-specific features is limited. Here, we show that selective expression of coagulation factor V (FV) by resident peritoneal macrophages in mice promotes bacterial clearance in the peritoneal cavity and serves to facilitate the well-known but poorly understood "macrophage disappearance reaction." Intravital imaging revealed that resident macrophages were nonadherent in peritoneal fluid during homeostasis. Bacterial entry into the peritoneum acutely induced macrophage adherence and associated bacterial phagocytosis. However, optimal control of bacterial expansion in the peritoneum also required expression of FV by the macrophages to form local clots that effectively brought macrophages and bacteria in proximity and out of the fluid phase. Thus, acute cellular adhesion and resident macrophage-induced coagulation operate independently and cooperatively to meet the challenges of a unique, open tissue environment. These events collectively account for the macrophage disappearance reaction in the peritoneal cavity.
Asunto(s)
Factor V/metabolismo , Macrófagos/metabolismo , Cavidad Peritoneal/microbiología , Cavidad Peritoneal/patología , Animales , Coagulación Sanguínea , Adhesión Celular , Tamaño de la Célula , Escherichia coli/fisiología , Macrófagos/patología , Ratones Endogámicos C57BL , Bazo/microbiologíaRESUMEN
Acinetobacter baumannii infection has become a major cause of healthcare-associated infection and a critical pathogen in the WHO antimicrobial resistance research and development priority list. Catheter-related septicemia is one of the major clinical manifestations of A. baumannii infection associated with high morbidity and mortality. In this study, we used a clinical A. baumannii strain (LAC-4) that is hypervirulent to immunocompetent C57BL/6 and BALB/c mice and established a mouse model of intraperitoneal (i.p.) A. baumannii infection. Our study showed that i.p. LAC-4 infection of C57BL/6 and BALB/c mice induces a lethal or sublethal infection with high bacterial burdens in peritoneal cavity, blood and tissues and the infected mice either succumbed to the infection within 24 hours or completely recovered from the infection. The infection induces acute peritoneal recruitment of neutrophils and other innate immune cells, and the local and systemic production of proinflammatory cytokines and chemokines (IL-1ß, IL-5, IL-6, TNF-α, RANTES, MIP-1ß, MCP-1, KC and IL-10). Mechanistic studies suggest an important role of macrophages in the host innate defense in this model in that in vitro stimulation of peritoneal macrophages with killed LAC-4 induced a similar pattern of cytokine/chemokine responses to those in the infected mice, and depletion of peritoneal macrophages rendered the mice significantly more susceptible to the infection. Thus, this mouse infection model will provide an alternative and useful tool for future pathogenesis studies of A. baumannii-associated septicemia and identification and characterization of important virulence factors, as well as serve as a surrogate model for rapid evaluation of novel therapeutics and vaccines for this emerging infectious agent.
Asunto(s)
Infecciones por Acinetobacter/microbiología , Acinetobacter baumannii/aislamiento & purificación , Acinetobacter baumannii/patogenicidad , Cavidad Peritoneal/microbiología , Infecciones por Acinetobacter/patología , Enfermedad Aguda , Animales , Carga Bacteriana , Quimiocinas/metabolismo , Interacciones Huésped-Patógeno , Inflamación/patología , Mediadores de Inflamación/metabolismo , Cinética , Activación de Macrófagos , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Viabilidad Microbiana , Cavidad Peritoneal/patología , VirulenciaRESUMEN
BACKGROUND: Choice of antibiotics for complicated appendicitis should address local antibiotic resistance patterns. As our local data showed a less than 15% resistance of Escherichia coli to co-amoxicillin (amoxicillin + clavulanic acid), we opted for this antibiotic in 2013. Subsequently, the increasing prevalence of Pseudomonas aeruginosa challenged this choice. AIM OF THE STUDY: The aim of this study was to describe the bacteriology of peritoneal swabs from cases of complicated appendicitis in our paediatric patients, and to determine the risk of infectious complications (wound and/or intra-abdominal abscesses). METHODS: We designed a retrospective cohort study including all children (<18 years old) who had surgery for complicated appendicitis between 1 January 2010 and 31 December 2016 and had a peritoneal swab culture. Microbiological results are presented descriptively. Univariate analyses were performed for potential determinants of infectious complications. All variables with a p-value <0.05 were then included in a multivariable logistic regression model, for which adjusted odds ratios (OR) and 95% confidence intervals (CI) were calculated. RESULTS: One hundred and thirty-three patients were treated for complicated appendicitis and had cultures of peritoneal fluid. Median age was 9.5 years old (IQR 5.7–12.4), and there were 53 girls (40%). E. coli was isolated in 94 patients (71%) and was resistant to co-amoxicillin in 14% of cases. P. aeruginosa was isolated in 31 patients (23%). The rate of infectious complications was 38% (8/21 patients) when the empiric antibiotic did not cover P. aeruginosa and 0% (0/10 patients) when P. aeruginosa was covered adequately (p = 0.03). In a multivariable analysis, only co-amoxicillin-resistant E. coli significantly predicted infectious complications (OR 4.7; 95% CI 1.4–16.6; p = 0.015). CONCLUSION: Results of the multivariable analysis of this small, retrospective study revealed a statistically significant increase in the risk of postoperative complications in the presence of co-amoxicillin-resistant E. coli. The choice of antibiotic should be adapted accordingly. More data are needed to justify the systematic coverage of P. aeruginosa in children with complicated appendicitis.  .
Asunto(s)
Amoxicilina/farmacología , Antibacterianos/farmacología , Apendicectomía/efectos adversos , Apendicitis/microbiología , Escherichia coli/aislamiento & purificación , Complicaciones Posoperatorias/epidemiología , Pseudomonas aeruginosa/aislamiento & purificación , Apendicitis/tratamiento farmacológico , Apendicitis/cirugía , Niño , Preescolar , Farmacorresistencia Bacteriana , Escherichia coli/efectos de los fármacos , Femenino , Humanos , Masculino , Pruebas de Sensibilidad Microbiana , Cavidad Peritoneal/microbiología , Peritonitis/epidemiología , Peritonitis/microbiología , Complicaciones Posoperatorias/microbiología , Pseudomonas aeruginosa/efectos de los fármacos , Estudios Retrospectivos , Medición de Riesgo , Factores de RiesgoRESUMEN
Bacterial spillage into a sterile environment following intestinal hollow-organ perforation leads to peritonitis and fulminant sepsis. Outcome of sepsis critically depends on macrophage activation by extracellular ATP-release and associated autocrine signalling via purinergic receptors. ATP-release mechanisms, however, are poorly understood. Here, we show that TLR-2 and -4 agonists trigger ATP-release via Connexin-43 hemichannels in macrophages leading to poor sepsis survival. In humans, Connexin-43 was upregulated on macrophages isolated from the peritoneal cavity in patients with peritonitis but not in healthy controls. Using a murine peritonitis/sepsis model, we identified increased Connexin-43 expression in peritoneal and hepatic macrophages. Conditional Lyz2cre/creGja1flox/flox mice were developed to specifically assess Connexin-43 impact in macrophages. Both macrophage-specific Connexin-43 deletion and pharmacological Connexin-43 blockade were associated with reduced cytokine secretion by macrophages in response to LPS and CLP, ultimately resulting in increased survival. In conclusion, inhibition of autocrine Connexin-43-dependent ATP signalling on macrophages improves sepsis outcome.
Asunto(s)
Conexina 43/genética , Macrófagos/metabolismo , Sepsis/genética , Adenosina Trifosfato/genética , Animales , Comunicación Autocrina/genética , Conexina 43/antagonistas & inhibidores , Modelos Animales de Enfermedad , Regulación de la Expresión Génica/genética , Células HEK293 , Humanos , Lipopolisacáridos/toxicidad , Hígado/metabolismo , Hígado/microbiología , Hígado/patología , Activación de Macrófagos/efectos de los fármacos , Activación de Macrófagos/genética , Macrófagos/efectos de los fármacos , Macrófagos/microbiología , Macrófagos/patología , Ratones , Cavidad Peritoneal/microbiología , Cavidad Peritoneal/patología , Peritonitis/tratamiento farmacológico , Peritonitis/genética , Peritonitis/microbiología , Peritonitis/patología , Probenecid/farmacología , Sepsis/inducido químicamente , Sepsis/microbiología , Sepsis/patología , Receptor Toll-Like 2/genética , Receptor Toll-Like 4/genéticaRESUMEN
Neutrophils, classified as professional phagocytes, are crucial in killing bacteria and preventing inflammation. When studying the roles of neutrophils in the development of the septic peritonitis induced by E. coli, we noticed some of the larger cells existed among peritoneal lavage fluid cells (PLCs). Besides the large size, their nuclei are segmented and flat, and squeezed to the marginal zone of the inner membrane. The cells, therefore, were designated as E. coli induced larger neutrophils (e-Neus). Further studies showed that, the e-Neus were ly6G positive, indicating the e-Neus were a type of neutrophils. The enlarged cell size and marginal nucleus of the e-Neus were caused by engulfing abundant of E. coli, marking the active participation of the e-Neus in clearance of E. coli. Functionally, the e-Neus generated reactive oxygen species (ROS) and IL-10. Furthermore, the occurrence and accumulation of the e-Neus were closely correlated with the severity of septic peritonitis and mortality of the mice. Overall, the e-Neus presented here may enrich the understandings on neutrophil transitions in response to various insults, and could be used to evaluate the severity of septic peritonitis induced by E. coli.
Asunto(s)
Infecciones por Escherichia coli/inmunología , Escherichia coli/inmunología , Neutrófilos/inmunología , Peritonitis/inmunología , Animales , Tamaño de la Célula , Infecciones por Escherichia coli/patología , Femenino , Interleucina-10/inmunología , Ratones , Ratones Endogámicos ICR , Neutrófilos/patología , Cavidad Peritoneal/microbiología , Peritonitis/microbiología , Peritonitis/patología , Especies Reactivas de Oxígeno/inmunologíaRESUMEN
Currently echinocandins are recommended in Candida peritonitis and pleuritis. We determined micafungin killing rates (k values) at therapeutic concentrations (0.25-2 mg/L) in RPMI-1640 with and without 10 and 30% serum mimicking in vivo conditions against six Candida species isolated from peritoneal and pleural fluid. In RPMI-1640, micafungin was fungicidal against C. glabrata, C. krusei and C. kefyr within 2.27 ± 10.68, 2.69 ± 10.29 and 3.10 ± 4.41 h, respectively, while was fungistatic against C. albicans, C. tropicalis and C. parapsilosis. In 10% serum, ≥ 0.25, ≥ 0.5, ≥ 0.5 and ≥ 1 mg/L micafungin produced positive k values (killing) for all C. albicans, C. glabrata, C. kefyr and C. krusei, respectively. In 30% serum, 2 mg/L micafungin produced killing against all C. albicans, C. glabrata and C. kefyr isolates, but was ineffective against C. krusei, C. parapsilosis and 2 of 3 C. tropicalis. Micafungin exposure should be increased against non-albicans species to eradicate fungi from peritoneal and pleural cavities.
Asunto(s)
Antifúngicos/farmacología , Candida/efectos de los fármacos , Candidiasis Invasiva/microbiología , Micafungina/farmacología , Pruebas de Sensibilidad Microbiana/métodos , Cavidad Peritoneal/microbiología , Cavidad Pleural/microbiología , Candida/aislamiento & purificación , Humanos , Viabilidad Microbiana/efectos de los fármacosRESUMEN
The article presents an analysis of the dynamics of enteroperitoneal translocation of bacteria on the model of acute intestinal obstruction (AIO) in rats by performing an experimental study on laboratory animals. Using the proposed model of AIO we have tried to determine the level of enteroperitoneal translocation as a function of the time of the impassable obstruction. The results which presented in the article clearly demonstrate that when AIO is developing in experimental animals the greatest level of translocation was revealed on the 3rd and 5th days. Statistically significant growth of the microflora in the lumen of the intestine above the level of obturation was observed on the 1st day and the whole period of observation was maintained, and it was also revealed that the level of CFU depends on the duration of the AIO and in the abdominal cavity it increases dramatically by 7 days, compared to 1 and 3 days. However, there is no significant correlation between enteroperitoneal translocation and the level of CFU in the lumen of the intestine and abdominal cavity.
Asunto(s)
Traslocación Bacteriana , Obstrucción Intestinal/microbiología , Enfermedad Aguda , Animales , Bacterias/aislamiento & purificación , Intestino Delgado/microbiología , Masculino , Cavidad Peritoneal/microbiología , RatasRESUMEN
The alternative sigma factor RpoS plays a key role modulating gene expression in Borrelia burgdorferi, the Lyme disease spirochete, by transcribing mammalian host-phase genes and repressing σ70-dependent genes required within the arthropod vector. To identify cis regulatory elements involved in RpoS-dependent repression, we analyzed green fluorescent protein (GFP) transcriptional reporters containing portions of the upstream regions of the prototypical tick-phase genes ospAB, the glp operon, and bba74 As RpoS-mediated repression occurs only following mammalian host adaptation, strains containing the reporters were grown in dialysis membrane chambers (DMCs) implanted into the peritoneal cavities of rats. Wild-type spirochetes harboring ospAB- and glp-gfp constructs containing only the minimal (-35/-10) σ70 promoter elements had significantly lower expression in DMCs relative to growth in vitro at 37°C; no reduction in expression occurred in a DMC-cultivated RpoS mutant harboring these constructs. In contrast, RpoS-mediated repression of bba74 required a stretch of DNA located between -165 and -82 relative to its transcriptional start site. Electrophoretic mobility shift assays employing extracts of DMC-cultivated B. burgdorferi produced a gel shift, whereas extracts from RpoS mutant spirochetes did not. Collectively, these data demonstrate that RpoS-mediated repression of tick-phase borrelial genes occurs by at least two distinct mechanisms. One (e.g., ospAB and the glp operon) involves primarily sequence elements near the core promoter, while the other (e.g., bba74) involves an RpoS-induced transacting repressor. Our results provide a genetic framework for further dissection of the essential "gatekeeper" role of RpoS throughout the B. burgdorferi enzootic cycle.IMPORTANCEBorrelia burgdorferi, the Lyme disease spirochete, modulates gene expression to adapt to the distinctive environments of its mammalian host and arthropod vector during its enzootic cycle. The alternative sigma factor RpoS has been referred to as a "gatekeeper" due to its central role in regulating the reciprocal expression of mammalian host- and tick-phase genes. While RpoS-dependent transcription has been studied extensively, little is known regarding the mechanism(s) of RpoS-mediated repression. We employed a combination of green fluorescent protein transcriptional reporters along with an in vivo model to define cis regulatory sequences responsible for RpoS-mediated repression of prototypical tick-phase genes. Repression of ospAB and the glp operon requires only sequences near their core promoters, whereas modulation of bba74 expression involves a putative RpoS-dependent repressor that binds upstream of the core promoter. Thus, Lyme disease spirochetes employ at least two different RpoS-dependent mechanisms to repress tick-phase genes within the mammal.
Asunto(s)
Adaptación Fisiológica , Proteínas Bacterianas/metabolismo , Borrelia burgdorferi/genética , Regulación Bacteriana de la Expresión Génica , Factor sigma/metabolismo , Garrapatas/microbiología , Animales , Antígenos Bacterianos/genética , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas Bacterianas/genética , Borrelia burgdorferi/fisiología , Proteínas Fluorescentes Verdes/genética , Interacciones Huésped-Patógeno , Mutación , Operón , Cavidad Peritoneal/microbiología , Regiones Promotoras Genéticas , Ratas , Factor sigma/genética , Transcripción GenéticaRESUMEN
LL37 is the only known member of the cathelicidin family of antimicrobial peptides in humans. In addition to its broad spectrum of antimicrobial activities, LL37 may modulate various inflammatory reactions. The authors previously revealed that LL37 improves the survival of a murine cecal ligation and puncture (CLP) sepsis model. In the present study, the mechanism for the protective action of LL37 was elucidated using the CLP model, focusing on the effect of LL37 on the release of neutrophil extracellular traps (NETs). The results indicated that the intravenous administration of LL37 suppressed the increase of damage-associated molecular patterns (DAMPs), including histoneDNA complex and highmobility group protein 1, in addition to interleukin1ß, tumor necrosisα and soluble triggering receptor expressed on myeloid cells (TREM)1 in plasma and peritoneal fluids. Notably, LL37 significantly suppressed the decrease of mononuclear cell number in blood, and the increase of polymorphonuclear cell (neutrophil) number in the peritoneal cavity during sepsis. Furthermore, LL37 reduced the bacterial burden in blood and peritoneal fluids. Notably, LL37 increased the level of NETs (myeloperoxidaseDNA complex) in plasma and peritoneal fluids. In addition, it was verified that LL37 induces the release of NETs from neutrophils, and NETs possess the bactericidal activity. Overall, these observations suggest that LL37 improves the survival of CLP septic mice by possibly suppressing the inflammatory responses as evidenced by the inhibition of the increase of cytokines, soluble TREM1 and DAMPs (host cell death) and the alteration of inflammatory cell numbers, and bacterial growth via the release of NETs with bactericidal activity.