An animal model of limitation of gut colonization by carbapenemase-producing Klebsiella pneumoniae using rifaximin.

Current knowledge suggests that infection by carbapenem-resistant enterobacteria is preceded by gut colonization. It is hypothesized that colonization is eradicated by non-absorbable antibiotics like rifaximin. We investigated the effect of rifaximin against carbapenem-resistant Klebsiella pneumoniae (CRKP) in vitro and in a mouse model. We studied the in vitro efficacy of rifaximin against 257 CRKP clinical isolates, 188 KPC producers and 69 OXA-48 producers, by minimum inhibitory concentration and time-kill assays. We then developed a model of gut colonization by feeding 30 C57Bl6 mice with 108 cfu of one KPC-KP isolate for 7 days; mice were pre-treated orally with saline, omeprazole or ampicillin. Then, another 60 mice with established KPC-2 gut colonization received orally for 7 consecutive days rifaximin 180 mg/kg dissolved in ethanol and 4% bile or vehicle. On days 0, 3 and 7 stool samples were collected; mice were sacrificed for determination of tissue outgrowth. At a concentration of 1000 µg/ml rifaximin inhibited 84.8% of CRKP isolates. Α 3 × log10 decrease of the starting inoculum was achieved by 100, 250 and 500 µg/ml of rifaximin after 24 h against 25, 55 and 55% of isolates. Pre-treatment with ampicillin was necessary for gut colonization by KPC-KP. Treatment with rifaximin succeeded in reducing KPC-KP load in stool and in the intestine. Rifaximin inhibits at clinically meaningful gut concentrations the majority of CRKP isolates and is efficient against gut colonization by KPC-KP.

Efficacy of tigecycline alone or in combination for experimental infections by KPC carbapenemase-producing Klebsiella pneumoniae.

Although in vitro data suggest that tigecycline is active against Klebsiella pneumoniae carbapenemase-producing K. pneumoniae (KPC-Kp), experimental and clinical data are limited. We studied the effect of tigecycline alone or in combination for experimental infections by KPC-Kp. A total of 540 male C57BL/6 mice were infected with three genetically diverse KPC-Kp isolates susceptible to tigecycline with meropenem minimum inhibitory concentrations (MICs) of 4, 16 and 256 µg/mL, respectively. Mice were randomly treated with water for injection, tigecycline, meropenem and colistin alone, and double or triple combinations of tigecycline, colistin and meropenem. Mouse survival was recorded for 14 days. In separate experiments, mice were sacrificed 6 h and 24 h after bacterial challenge for quantitative culture of tissues and serological analysis. Time-kill curves were performed. Tigecycline, colistin and meropenem concentrations were measured in tissues and serum by high-performance liquid chromatography (HPLC). Survival was significantly prolonged when mice were treated with tigecycline alone and tigecycline-containing regimens compared with control mice and mice treated with tigecycline-sparing regimens. Tigecycline-sparing regimens were active only against the isolate with a meropenem MIC of 4 µg/mL. Mortality was associated with progression to multiple organ failure. Tigecycline and tigecycline-containing regimens achieved a rapid decrease of bacterial loads both in tissues and in vitro. Tigecycline concentrations in tissues were negatively correlated with tissue bacterial load. Tigecycline alone or in combination with meropenem and/or colistin achieves effective treatment of experimental KPC-Kp infections irrespective of the meropenem MIC.

Host cystathionine-γ lyase derived hydrogen sulfide protects against Pseudomonas aeruginosa sepsis.

Hydrogen sulfide (H2S) has recently been recognized as a novel gaseous transmitter with several anti-inflammatory properties. The role of host- derived H2S in infections by Pseudomonas aeruginosa was investigated in clinical and mouse models. H2S concentrations and survival was assessed in septic patients with lung infection. Animal experiments using a model of severe systemic multidrug-resistant P. aeruginosa infection were performed using mice with a constitutive knock-out of cystathionine-γ lyase (Cse) gene (Cse-/-) and wild-type mice with a physiological expression (Cse+/+). Experiments were repeated in mice after a) treatment with cyclophosphamide; b) bone marrow transplantation (BMT) from a Cse+/+ donor; c) treatment with H2S synthesis inhibitor aminooxyacetic acid (ΑΟΑΑ) or propargylglycine (PAG) and d) H2S donor sodium thiosulfate (STS) or GYY3147. Bacterial loads and myeloperoxidase activity were measured in tissue samples. The expression of quorum sensing genes (QS) was determined in vivo and in vitro. Cytokine concentration was measured in serum and incubated splenocytes. Patients survivors at day 28 had significantly higher serum H2S compared to non-survivors. A cut- off point of 5.3 µΜ discriminated survivors with sensitivity 92.3%. Mortality after 28 days was 30.9% and 93.7% in patients with H2S higher and less than 5.3 µΜ (p = 7 x 10-6). In mice expression of Cse and application of STS afforded protection against infection with multidrug-resistant P. aeruginosa. Cyclophosphamide pretreatment eliminated the survival benefit of Cse+/+ mice, whereas BMT increased the survival of Cse-/- mice. Cse-/- mice had increased pathogen loads compared to Cse+/+ mice. Phagocytic activity of leukocytes from Cse-/- mice was reduced but was restored after H2S supplementation. An H2S dependent down- regulation of quorum sensing genes of P.aeruginosa could be demonstrated in vivo and in vitro. Endogenous H2S is a potential independent parameter correlating with the outcome of P. aeruginosa. H2S provides resistance to infection by MDR bacterial pathogens.

Association of modulation of pro-inflammatory responses by dectin-2 with preterm delivery: An experimental model.

PROBLEM: Pro-inflammatory responses of pathogen recognition receptors (PRR) are implicated in preterm delivery (PTD). Dectin-2 is one PRR recognizing unselective carbohydrate structures; its participation in PTD has never been studied before. METHOD OF STUDY: In an experimental model, PTD was induced in female pregnant wild-type (WT) mice and mice with homologous deficiency for dectin-2 by the intraperitoneal injection of bacterial lipopolysaccharide (LPS) on day 14 of pregnancy. Time to delivery and fetal mortality were recorded. Challenged mice were killed for tissue collection and splenocyte isolation 6 hours later. Concentrations of tumour necrosis factor-alpha (TNFα), interleukin (IL)-1α, and IL-1ß were measured. RESULTS: Delivery was induced significantly earlier in WT than dectin-2-/- mice; however, fetal mortality was higher among dectin-2-/- mice. Candida albicans challenge could not lead to these changes. Sacrifice experiments showed that LPS challenge led to significant increase of TNFα, IL-1α, and IL-1ß in maternal tissues of WT; this was further enhanced for TNFα and IL-1ß in dectin-2-/- mice. Pre-treatment with the prostaglandin inhibitor diclofenac delayed time to delivery of WT mice, but not of dectin2-/- mice. TNFα stimulation of splenocytes of dectin2-/- mice was enhanced with the addition of anti-TLR4 and decreased in the presence of lipid A. CONCLUSIONS: Dectin-2 delays LPS-induced PTD by enhancing the production of pro-inflammatory cytokines.

Early increase of VEGF-A is associated with resolution of ventilator-associated pneumonia: Clinical and experimental evidence.

BACKGROUND AND OBJECTIVE: The role of vascular endothelial growth factor (VEGF)-A in the resolution of ventilator-associated pneumonia (VAP) was investigated in clinical and mouse pneumonia models. METHODS: VEGF-A was measured for seven consecutive days by an immunosorbent assay in sera of 82 patients with VAP and changes from baseline were correlated with the resolution of VAP. Experimental animals were challenged intratracheally with Pseudomonas aeruginosa. Mouse bronchoalveolar lavage (BAL) samples and segments of lung tissue were obtained at 24, 48 and 124 h after bacterial challenge. Levels of VEGF-A, tumour Necrosis Factor alpha (TNF-α), interleukin (IL)-1ß, interferon-gamma (IFNγ) and myeloperoxidase (MPO) activity were measured in these samples. RESULTS: VAP resolved in 36.1% of patients with a less than 45% increase of VEGF-A on day 5 compared to 65.2% of patients with a more than 45% increase (P = 0.014). This was also accompanied by an earlier resolution of VAP (log-rank: 7.99; P = 0.005) and it was not pathogen-specific. The increase of VEGF-A was an independent variable associated with VAP resolution in forward logistic regression analysis where Acute Physiology and Chronic Health Evaluation (APACHE) II and Sequential Organ Failure Assessment (SOFA) scores were included as independent variables. VEGF-A in mouse BAL and lung tissue increased significantly at 124 h but not with the other mediators. In mice pre-treated with bevacizumab, VEGF-A concentrations decreased while TNF-α and MPO significantly increased. CONCLUSION: In patients, an association between increased levels of circulating VEGF-A and VAP resolution was observed. The mouse study suggests that elevated VEGF-A levels may be associated with lung inflammation resolution. CLINICAL TRIAL REGISTRATION: NCT00297674 at www.clinicaltrials.gov.

Immunomodulatory intervention with interferon-γ in Escherichia coli pyelonephritis.

PURPOSE: We investigated the efficacy of recombinant human interferon-γ in experimental pyelonephritis due to Escherichia coli. MATERIALS AND METHODS: Pyelonephritis was induced by intrapelvic inoculation of bacteria after ureteral ligation in 38 rabbits assigned to 1 of 3 groups, including group 1-16 controls, group 2-14 rabbits treated with intravenous recombinant human interferon-γ and group 3-8 rabbits treated with intravenous recombinant human interferon-γ plus amikacin. Bacterial counts, cytokines and malondialdehyde were measured in blood. Peripheral blood mononuclear cells were isolated to measure TNFα transcripts, cytokine stimulation and apoptosis. Survival was recorded, and the tissue bacterial load and myeloperoxidase activity were measured after sacrifice. RESULTS: The mortality rate in groups 1, 2 and 3 was 66.7%, 25% and 12.5%, respectively. The circulating bacterial count and tissue bacterial load were less in group 2 than in group 1. Circulating malondialdehyde negatively correlated with the bacterial load of the spleen. Although the number of TNFα transcripts in circulating peripheral blood mononuclear cells did not differ, peripheral blood mononuclear cells isolated from group 2 at 48 hours produced much greater concentrations of tumor necrosis factor-α after stimulation with Pam3Cys. In parallel, the apoptosis rate of circulating monocytes was increased in group 2 at 48 hours. Lung myeloperoxidase activity at 24 hours, serving as indirect evidence of neutrophil infiltration, was decreased in group 2. CONCLUSIONS: Recombinant human interferon-γ administration prolonged survival in rabbits with experimental E. coli urosepsis. Its action was probably related to increased bacterial phagocytosis after modulation of oxidant status and reversal of monocyte immunoparalysis.

The in vitro effective antiviral action of povidone-iodine (PVP-I) may also have therapeutic potential by its intravenous administration diluted with Ringer's solution.

The use of povidone-iodine (PVP-I) is well known in clinical medical practice. In vitro studies of cell cultures infected by HIV and H5N1 virus have shown that PVP-I has an antiviral action, while the cell hosts were not affected and survived. It is therefore worth investigating whether PVP-I, diluted with Ringer's solution, may have a therapeutic effect by parenteral administration. Specifically, the question is whether small concentrations of intravenous PVP-I could be well tolerated by the human organism, and in addition, if it would be possible to detect a beneficial activity. Its intravenous use may have a potential value against infections (by microbes, viruses, fungi and parasites), as well as an anti-inflammatory activity, especially in cases where antibiotics are ineffective. It could be used as a blood disinfectant, for treating burns, for the prevention of cancer, for the therapy of H5N1 influenza after its mutation, and other potential applications.

Oleuropein: a novel immunomodulator conferring prolonged survival in experimental sepsis by Pseudomonas aeruginosa.

Oleuropein, a novel immunomodulator derived from olive tree, was assessed in vitro and in experimental sepsis by Pseudomonas aeruginosa. After addition in monocyte and neutrophil cultures, malondialdehyde, TNF-alpha, IL-6, and bacterial counts were estimated in supernatants. Acute pyelonephritis was induced in 70 rabbits after inoculation of pathogen in the renal pelvis. Intravenous therapy was administered in four groups postchallenge by one multidrug-resistant isolate (A, controls; B, oleuropein; C, amikacin; D, both agents) and in three groups postchallenge by one susceptible isolate (E, controls; F, oleuropein; G, amikacin). Survival was recorded; bacterial growth in blood and organs was counted; endotoxins (LPS), malondialdehyde, total antioxidant status, and TNF-alpha in serum were estimated. TNF-alpha and IL-6 of cell supernatants were not increased compared with controls when triggered by LPS and P. aeruginosa. Counts of multidrug-resistant P. aeruginosa were decreased in monocyte supernatants. Median survival of groups A, B, C, D, E, F, and G were 3.00, 6.00, 2.00, 10.00, 1.00, 5.00, and 1.00 days, respectively. Bacteria in blood were lower at 48 h in groups B and D compared with A and in groups F and G compared with E. Total antioxidant status decreased steadily over time in groups A, C, D, and G, but not in groups B and F. TNF-alpha of groups B, C, and D was lower than A at 48 h. Tissue bacteria decreased in group F compared with E. Oleuropein prolonged survival in experimental sepsis probably by promoting phagocytosis or inhibiting biosynthesis of proinflammatory cytokines.

Clarithromycin is an effective immunomodulator in experimental pyelonephritis caused by pan-resistant Klebsiella pneumoniae.

OBJECTIVES: To apply clarithromycin as an immunomodulatory treatment in experimental infection caused by pan-resistant Klebsiella pneumoniae. METHODS: Acute pyelonephritis was induced in 80 rabbits after inoculation of the test isolate in the renal pelvis. Rabbits were divided into eight groups, with 10 animals in each group. In groups A-D, therapy was administered simultaneously with bacterial challenge as follows: A, controls; B, intravenous clarithromycin; C, amikacin; and D, both agents. In groups E-H, therapy was administered 24 h after bacterial challenge as follows: E, controls; F, intravenous clarithromycin; G, amikacin; and H, both agents. Blood was sampled for estimation of tumour necrosis factor-alpha (TNF-alpha) and malondialdehyde (MDA); monocytes were isolated for determination of intracellular activity of caspase-3 and ex vivo TNF-alpha secretion. Four days after bacterial challenge, animals were sacrificed for quantitative cultures and biopsies of organs. RESULTS: Serum TNF-alpha at 48 h was lower in groups B, C and D compared with group A. Activity of caspase-3 of monocytes was lower at 48 h in group D compared with group A. Bacterial loads of liver and spleen were decreased in group D compared with those of group A. The numbers of inflammatory cells of spleen of group B were lower compared with those of group A; those of kidney and mesenteric lymph nodes of group D were lower than those of group A. Serum MDA of group H was lower than that of group E and serum TNF-alpha of group F was lower compared with that of group E. TNF-alpha of monocyte supernatants and activity of caspase-3 of monocytes of group F were lower than those of group E. Bacterial tissue loads did not differ among groups E, F, G and H. The numbers of inflammatory cells of liver of groups F and H were lower compared with those of group E; those of kidney of groups F, G and H were lower compared with those of group E. CONCLUSIONS: Clarithromycin administered intravenously in experimental infection caused by pan-resistant K. pneumoniae attenuated systemic inflammatory response and local tissue damage. This effect is probably attributed to immunomodulatory intervention on blood monocytes.