Effect of subinhibitory doses of rifaximin on in vitro Pseudomonas aeruginosa adherence and biofilm formation to biotic and abiotic surface models.

BACKGROUND: The adhesion of Pseudomonas aeruginosa to biotic and abiotic surfaces is responsible for the persistence and development of bacterial infection. OBJECTIVES: To fill the gap in the knowledge regarding the relationship between rifaximin susceptibility and biofilm formation, and to investigate the effect of subinhibitory doses of rifaximin on the adhesion and biofilm formation. MATERIAL AND METHODS: A total of 10 isolates of P. aeruginosa were obtained from 110 urine samples of urinary tract infection (UTI) patients. Biofilm formation on polystyrene microtiter plates, minimum inhibitory concentrations (MICs) of rifaximin against the 10 isolates of P. aeruginosa (Pa1-Pa10), the effect of sub-MICs of rifaximin (0.5 × MIC, 0.25 × MIC, 0.125 × MIC, and 0.06 × MIC) on biofilm formation by the Pa4 isolate to polystyrene microtiter plates, and the adhesion to human epithelial cells (HECs) in vitro were evaluated. RESULTS: The MICs of rifaximin against 10 isolates ranged from 62.5 µg/mL to 1000 µg/mL. The Pa4 isolate produced the highest level of biofilm formation, while the MIC of Pa4 was 125 µg/mL. There was no correlation between bacterial susceptibility to rifaximin and biofilm formation (r: -0.016; p > 0.05). Sub-MIC doses of rifaximin significantly reduced the biofilm formation on abiotic surfaces, while only 0.5 × MIC, 0.25 × MIC and 0.12 × MIC of rifaximin reduced the adhesion to HECs significantly (p < 0.05) in a dose-dependent manner. CONCLUSIONS: This pioneering study demonstrated the negative effect of sub-MIC doses of rifaximin on biofilm formation and adhesion to abiotic and biotic surfaces in vitro.

Extracellular product of Pseudomonas aeruginosa in growth medium is involved in the pro-inflammatory cytokine response of human oral epithelial cells in vitro.

BACKGROUND: Epithelial cells are the first barrier to any microbial invasion. Finding a safe and affordable substance to stimulate the innate immune response of epithelial cells is one of the main challenges immunologists and vaccine manufacturers are facing. OBJECTIVES: This study aimed to show the comparative effect of sterile bacterial secretion (SBS) and Pseudomonas aeruginosa bacterial cell isolates obtained from burn wound infections on the ability of human epithelial cells (HECs) to produce interleukin (IL)-1ß and tumor necrosis factor alpha (TNF-α) in vitro. MATERIAL AND METHODS: The HEC cultures were exposed to P. aeruginosa 8 (Pa 8), Pa 2 and Pa 1 bacterial cells (isolated from burn wound infections). The other 3 groups of HECs were exposed to 50 µL of sterile, endotoxin-free SBS of Pa 8, Pa 2 and Pa 1. The time course of changes in IL-1ß mRNA, TNF-α mRNA, IL-1ß, and TNF-α was examined. RESULTS: Moderate (p < 0.05) elevations of IL-1ß mRNA in HECs and IL-1ß protein in the supernatant of the HEC culture were observed following exposure to SBS of Pa 8, Pa 2 and Pa 1 at most time points. High elevation (p < 0.05) of IL-1ß was seen in the supernatant of the HEC culture that was exposed to bacterial cells (Pa 8, Pa 2 and Pa 1). Similar results were found when TNF-α mRNA was measured in HECs and TNF-α in the supernatant of the HEC cultures after exposure to bacterial cells (Pa 8, Pa 2 and Pa 1) and the SBS of Pa 8, Pa 2 and Pa 1. CONCLUSIONS: This is the first time that the capacity of SBS to generate epithelial cell pro-inflammatory cytokines in vitro has been shown. In other words, SBS enhanced a nonspecific immune response, which opens the door to the possibility of using SBS from P. aeruginosa as an adjuvant in the future.

Bacterial secretions in growth medium stimulate the mouse respiratory innate immune response.

Introduction. Finding a safe innate immune response stimulator is one of the greatest challenges facing immunologists and vaccine manufacturers.Gap statement. The role of sterile bacterial secretions (SBSs) of Pseudomonas aeruginosa in stimulating the innate immune response was not investigated previously.Aim. The comparative effect of SBSs and bacterial cells of P. aeruginosa isolates isolated from freshwater (PAE) and infected wounds (PAC) on the respiratory tract innate immune response.Methodology. Four test mice groups were instilled intranasally (i.n.) with 106 c.f.u of PAC, 106 c.f.u of PAE, SBS of PAC, and SBS of PAE. Two control groups were given i.n. either LB broth or PBS. Time-course changes in IL-1 beta mRNA, TNF-alpha mRNA, IL-1ß and TNF-α, leukocyte count, bacterial uptake, and intracellular bacterial killing by mouse alveolar macrophages (AMs) and histological changes were examined. Lung bacterial burdens were counted in first and second test groups.Results. The maximum level of IL-1ß was seen as early as 2 h (1360±180 pg ml-1) post-instillation (i.n.) with SBS of PAC and 1 h (1910±244 pgml-1) post-instillation with SBS of PAE. The maximum level of TNF-α was seen as early as 4 h (953±192 pg ml-1) post-instillation with SBS of PAC and (1197±298 pg ml-1) post-instillation with SBS of PAE. These values were almost in line with IL-1ß and TNF-α gene expression. Moderate infiltration of leukocytes in bronchoalveolar lavage (BAL) and lung sections and moderate activity of AMs (bacterial uptake and bacterial killing) were observed. The above innate immune response parameters in mice instilled i.n. with PAC and PAE were higher (P<0.05) than in the mice groups instilled i.n. with SBSs. The PAC was persistent in the lungs of mice for up to 72 h (3.5±0.22 log10 of c.f.u. g-1) and up to 48 h (2.05±0.21 log10 of c.f.u. g-1) for PAE.Conclusion. The administration of mice with SBS i.n. stimulates cellular and molecular arms of the innate immune response in the respiratory tract, opening the door to the possibility of using SBS of P. aeruginosa as an adjuvant.

Coating indwelling urinary catheters with moxifloxacin prevents biofilm formation by Burkholderia cepacia.

BACKGROUND: Burkholderia cepacia adhesion and biofilm formation onto abiotic surfaces is an important feature of clinically relevant isolates. The in vitro biofilm formation of B. cepacia onto coated indwelling urinary catheters (IDCs) with moxifloxacin has not been previously investigated. OBJECTIVES: To examine the ability of B. cepacia to form biofilms on IDCs and the effect of coating IDCs with moxifloxacin on biofilm formation by B. cepacia in vitro. MATERIAL AND METHODS: The adhesion of B. cepacia to coated and uncoated IDCs with moxifloxacin was evaluated. Pieces of IDCs were coated with moxifloxacin (adsorption method). The spectrophotometric method was used to check moxifloxacin leaching into tubes. Coated and uncoated tubes were incubated with 107 colony forming units (cfu)/mL of B. cepacia. The viable bacterial count was used to count the number of bacteria adhered to coated and uncoated IDC pieces. RESULTS: A significant adhesion of B. cepacia to uncoated IDC pieces started 15 min after the incubation in a bacterial suspension (107 cfu/mL). A maximum adhesion was observed at 48 h. The pretreatment of IDCs with 100 µg/mL of moxifloxacin produced the best adsorption of antibiotic onto the IDCs. Coating IDC pieces with moxifloxacin significantly reduced the adhesion and biofilm formation of B. cepacia (p < 0.05) at various time intervals (1 h, 4 h and 24 h). CONCLUSIONS: The present study has demonstrated for the first time that coated IDCs with moxifloxacin reduce B. cepacia adhesion and biofilm formation. This finding has opened the door to the production of the new generation IDCs that prevent bacteria from attaching and forming biofilms.

Moxifloxacin reduces Stenotrophomonas maltophilia adhesion to mouse intestinal tract in vitro.

Stenotrophomonas maltophilia is an important opportunistic pathogen that affects immunocompromised individuals. Viable bacterial count method was used to count the number of adhered bacteria. The current study showed the efficiency of S. maltophilia (Sm2) adhesion on different parts of mouse intestinal tract (IT), small intestinal tract (SIT), large intestinal tract (LIT) and rectum (P<0.05) and this ability was equal for each part of IT [ANOVA test (P > 0.05)]. Moxifloxacin (0.03 x MIC) resulted a significant decrease in adhesion of S. maltophilia to SIT (P<0.05) versus control and other sub-inhibitory moxifloxacin concentrations (0.06 x and 1.2 x MIC). It can be concluded from the current study that the S. maltophilia (Sm2) has a good ability to adhere to mouse IT and the lowest concentrations of moxifloxacin (0.03 x MIC) reduced the ability of this bacterium to infect IT by reducing the ability of this bacterium to adhere to IT.

Direct role of antibody-secreting B cells in the severity of chronic hepatitis B.

Chronic hepatitis B involves different immune cells. The direct role of antibody-secreting B cells in the severity of chronic hepatitis B unclear. In this study, the number of plaque forming cells [PFC-(IgG, IgM, anti-HBc IgG, and anti-HBc IgM)], liver function tests (LFT) [alkaline phosphatase (ALP), alanine aminotransferase (ALT), and total serum bilirubin (TSB)], the levels of IL-10 in sera and in lymphocyte cultures, the number of CD4(+) and CD8(+) cells were measured in the peripheral blood of patients and in the controls. In addition, the hepatocytotoxic effect of anti-HBc and anti-HBe in vitro was studied. The largest number of PFCs was observed in the peripheral blood of patients with chronic hepatitis B. This was concomitant with a decrease in CD4(+) /CD8(+) ratio versus this ratio in asymptomatic HBV carriers and in healthy volunteers (P < 0.05). An increase in immunoglobulin (IgG and IgM) levels, anti-HBc IgG, and anti-HBc IgM levels and LFTs in peripheral blood of patients with chronic hepatitis B was seen. Anti-HBc induced hepatocytotoxicity in vitro. The expression of mRNA and protein for IL-10 production was observed at a significant level in culture of lymphocytes isolated from patients with chronic hepatitis B. In addition, a high level of IL-10 was found only in the sera of patients with chronic hepatitis B. It is concluded that the antibody-secreting B cells and the antibodies, which are produced, play an important role in the severity of chronic hepatitis B, which was related negatively with CD4(+) /CD8(+) ratio and positively with IL-10 expression.

Involvement of (IgG and IgM)-secreting B lymphocytes in severity of autoimmune hepatitis type 1.

Autoimmune hepatitis type 1 (AIH1) is an autoimmune disease attacks the liver and characterized by periportal inflammation, elevated immunoglobulins, and autoantibodies. The central role of B lymphocyte in pathogenicity of AIH1 is unclear. Here, the effect of antibody-secreting cells activity in terms of number of plaque-forming cells (PFC) on severity of AIH1 was evaluated. The high number of PFC-(IgG and IgM) in peripheral blood of patients with AIH1 was observed and this was concomitant with increase in the number of CD4+ T lymphocyte, immunoglobulin (Ig) (IgG and IgM) concentrations, and levels of liver function tests (LFTs). However, the negative correlation (r < -0.5, P < 0.05) between the numbers of PFC-(IgG and IgM) and CD8+ T lymphocytes was reported here. The present study showed the positive relationship between the concentrations of Ig (IgG and IgM) and levels of LFTs (r > +0.5, P < 0.05). The high expression of IL-10 mRNA was found in the tissue culture of peripheral blood lymphocytes obtained from patients with AIH1 as compared with that isolated from control group (P < 0.05). The current study proved the direct role of (IgG and IgM)-secreting cells in severity of AIH1. This was associated with CD4+ cell numbers and IL-10 mRNA expression, and mediated by IgG and IgM.

Immunological and biological relationship among flagellin of Pseudomonas aeruginosa, Burkholderia cepacia and Stenotrophomonas maltophilia.

The flagellar protein (flagellin) was isolated and purified from strains of Pseudomonas aeruginosa, Burkholderia cepacia and Stenotrophomonas maltophilia. A significant difference was observed in the molecular weight of different flagellin preparations obtained from these bacterial isolates. Antiserum prepared against S. maltophilia flagellin did not react with flagellin of P. aeruginosa or/and B. cepacia on Immunoblot or in indirect ELISA. In addition the anti-flagellin did not agglutinate P. aeruginosa and B. cepacia. No inhibition of motility of P. aeruginosa and B. cepacia was observed in presence of antiserum; though the latter inhibited the motility of S. maltophilia. The results of the present study prove that no specific relationship existed among all the studied flagellar proteins obtained from closely related bacteria.

Escherichia coli flagellin stimulates pro-inflammatory immune response.

Flagellin, a principal component of bacterial flagella, is a virulence factor that is recognized by the innate immune system. Recognition of flagellin by innate immune receptors stimulates the production of cytokines necessary for the development of effective immunity. Here, we demonstrated that the intranasal (i.n.) instillation of different amount of Escherichia coli K-12 flagellin preparation (0.5, 1, 2, 4 µg) in BALB/c mice induced pro-inflammatory immune response. Instillation i.n. of 1 µg of flagellin induced the maximum expression of interleukin 1 beta (IL-1ß), tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6) mRNA and production of pro-inflammatory cytokines (IL-1ß, TNF-α and IL-6) in mice lungs. The same dose of flagellin induced neutrophil polymorphonuclear cells infiltration in peribronchial and perivascular regions. High number of neutrophil in bronchoalveolar lavage fluid was found at 24 h after i.n. instillation of flagellin (1 µg). These findings were concomitant with the maximum production of myeloperoxidase and nitric oxide in mice lungs. Present study showed that the maximum pro-inflammatory mediator levels were found when mice instilled i.n. with 1 µg E. coli flagellin. The amount of flagellin of E. coli K-12 that achieve the maximum stimulation of mucosal pro-inflammatory immune response in mice lungs was explored in this study.

Stenotrophomonas maltophilia flagellin restricts bacterial colonization in BALB/c mouse lung in vivo.

Stenotrophomonas maltophilia is an emerging drug-resistant pathogen. Here, we demonstrated that S. maltophilia flagellin could locally activate innate immunity thereby increase the resistance to respiratory tract infection (RTI) and improved bacterial clearance in the lungs of BALB/c mice. The test group consisted of BALB/c mice instilled with 5 µg of purified flagellin and challenged 4 h later with S. maltophilia. In this group, increased production of pro-inflammatory cytokines (IL-1ß and TNF-α), myeloperoxidase activity, caspase-1 activity and nitric oxide (NO) was seen in lung homogenates in significant amounts (P < 0.05) as compared to control groups. On the contrary, low level of malondialdehyde was detected in the test group. Activation of alveolar macrophages in terms of bacterial engulfment and intracellular bacterial killing at an early stage and delayed production of anti-inflammatory cytokine (IL-10) was also detected. Concomitant with elevation of pro-inflammatory mediators, high number of leukocytes infiltration was detected in bronchoalveolar lavage of treated mice. The generated mucosal immune response was found to be protective against S. maltophilia as well as Staphylococcus aureus infection. In conclusion, this study emphasizes the nonspecific protection mediated by flagellin against homologous as well as heterologous bacterium that might be exploited therapeutically to prevent the development of RTI.

Flagellin administration protects respiratory tract from Burkholderia cepacia infection.

Burkholderia cepacia is an important pathogen that often causes pneumonia in immunocompromised individuals. Here, it was demonstrated that the TLR5 agonist flagellin could locally activate innate immunity. This was characterized by rapid expressions of IL-1beta, TNF-alpha, and iNOS mRNA and a delay in the expression of IL-10 mRNA. A significant elevation in the IL-1beta, TNF-alpha, and nitric oxide levels was also noted. In the respiratory tract, flagellin induced neutrophil infiltration into the airways, which was observed by histopathological examination and confirmed by the neutrophil count and level of myeloperoxidase activity. This was concomitant with a high activity of alveolar macrophages that engulfed and killed B. cepacia in vitro. The flagellin mucosal treatment improved the B. cepacia clearance in the mouse lung. Thus, the present findings illustrate the profound stimulatory effect of flagellin on the lung mucosal innate immunity, a response that needs to be exploited therapeutically to prevent the development of respiratory tract infection by B. cepacia.

The effect of high temperature on the kinetics of lipopolysaccharide (LPS)-induced human monocytes activity in vitro.

Human peripheral blood monocytes were stimulated with lipopolysaccharide at different temperatures (34, 37 and 40 °C) and TNF-α, IL-1ß and NO production was measured. Levels of TNF-α mRNA and IL-1ß mRNA were measured by RT-PCR. Phagocytic activity of LPS-stimulated monocytes in terms of bacterial uptake and intracellular bacterial killing was checked at different conditions in vitro. Early elevation of TNF-α, IL-1ß and NO production was found in LPS-stimulated monocytes that incubated at 40 °C followed by cells that incubated at 37 °C and lowest level was detected at 34 °C. Similar results were observed in the phagocytic activity. Expression of TNF-α mRNA and IL-1ß mRNA was observed as early as 30 min post exposure to LPS in all studied temperatures and these, decreased sharply after 12 h post exposure to LPS in LPS-stimulated monocytes that incubated at 40 °C only. This report describes the striking effects of incubation temperature on activity of LPS-stimulated monocytes.

Adhesion of Stenotrophomonas maltophilia to mouse tracheal mucus is mediated through flagella.

Adhesion of Stenotrophomonas maltophilia, an opportunistic pathogen, to different surfaces has been reported in the literature. However, its ability to adhere to mucus and the involvement of different bacterial appendages in this process has not been elucidated. In this study, bacterial adhesion to mouse tracheal mucus as well as the role of flagella in the adhesion process were investigated using clinical isolates of S. maltophilia. All the clinical isolates adhered to mouse tracheal mucus to varying degrees, showing isolate-to-isolate variation. Isolate Sm2 was selected to study the kinetics of bacterial adhesion to mouse tracheal mucus. The process of bacterial adhesion started after 30 min of incubation, and significant adhesion was detected after 1 h. Bacteria pre-treated with S. maltophilia anti-flagellin antibody were used to determine the role of flagellin in bacterial adhesion. The attachment of S. maltophilia flagellin preparation to mucus was assessed by enzyme immunoassay. Pre-treatment of the bacteria with anti-flagellin antibody resulted in a significant decrease in adhesion to mucus and this decrease was antibody concentration dependent. A similar observation was made when pure flagellin was allowed to interact with mucus. Pre-treatment of mouse tracheal mucus with flagellin led to a significant decrease in bacterial adhesion at concentrations of 40 and 80 µg ml⁻¹ (P<0.05). The ability of S. maltophilia to adhere to mucus was also reduced when mechanically deflagellated bacteria were checked for this property (P<0.005). It was concluded that S. maltophilia has the ability to adhere to mouse tracheal mucus and that flagella play an important role in this process. However, further studies using genetically defined mutants lacking flagella are needed to support this observation.

Immunological and pathological aspects of respiratory tract infection with Stenotrophomonas maltophilia in BALB/c mice.

A comprehensive study on the production of inflammatory mediators in the lungs of BALB/c mice following infection with Stenotrophomonas maltophilia was conducted. The levels of pro-inflammatory cytokines, tumor necrosis factor alpha (TNF-alpha) and Interleukin-1beta (IL-1 beta) was raised in the lungs of infected mice compared to control. The production of anti-inflammatory cytokine IL-10 was slightly delayed. Its peak level was on 2nd day whereas the peak of pro-inflammatory cytokines was observed on day one after intranasal challenge. This was accompanied with a rise in Myeloperoxidase (MPO) and Malondialdehyde (MDA) on day one. The increase in MPO levels matched with histopathological observations as neutrophils infiltration was detected on the first day. Alveolar macrophages (AMs) obtained from infected animals showed higher rate of uptake and killing when exposed to bacteria in vitro compared to similar experiment conducted with AMs from normal mice (control). This suggests that AMs were more efficient in cleaning the bacteria. The nitric oxide (NO) production though started early during infection but reached its maximum on 3rd day. No mortality was observed among the infected animals and infection was resolved by 5th post infection day. No drastic changes in the lung tissue were observed on histopathological examination.

Stenotrophomonas maltophilia flagellin induces a compartmentalized innate immune response in mouse lung.

Intranasal (i.n.) instillation of different amounts of purified Stenotrophomonas maltophilia flagellin preparation (1, 5 and 15 microg) in BALB/c mice stimulated a transient innate immune response in the lungs. This was characterized by infiltration of different kinds of leukocytes (neutrophils, monocytes and lymphocytes), production of various inflammatory mediators (tumour necrosis factor alpha, interleukin 1 beta, interleukin 10, nitric oxide, myeloperoxidase and malondialdehyde) and activated alveolar macrophages (AMs). The proinflammatory cytokine production resulted in accumulation of activated neutrophils and macrophages and their products following immunostimulation with flagellin. The activation of AMs by flagellin was non-specific as AMs obtained from flagellin-treated animals, even after 4 h of exposure, were found to engulf and kill S. maltophilia and Staphylococcus aureus efficiently compared to macrophages obtained from control animals. i.n. instillation of 5 microg flagellin resulted in the generation of an effective innate immunity compared to other flagellin doses. Our data provide strong evidence that S. maltophilia flagellin stimulates innate immunity in mouse lung.