Peripheral arterial disease (PAD) is a common complication of Diabetes Mellitus (DM) and often culminates in amputation of the affected foot. Pseudomonas aeruginosa infections associated with PAD are difficult to treat due to their multi-drug resistance. Herein we report a 38 year old male who reported with DM, chronic kidney disease (CKD) and rest pain of the right second toe in October 2011. He underwent percutaneous transluminal angioplasty (PTA) which was unsuccessful. The gangrene of the toes worsened and amputation of the right second toe was done. Bacteriological examination showed presence of P. aeruginosa which during the course of antibiotic therapy became multi-drug resistant. Gangrene and abscess of the foot worsened and amputation of the right third toe was performed. Then autologous peripheral blood mononuclear cell (PBMNC) therapy was performed but as infection control could not still be achieved, the fourth toe was amputated. A protocol of foot bath using carbonic water, local usage of antibiotics (Polymyxin-B), and basic fibroblast growth factor (b-FGF) spray was then employed after which the infection could be controlled and improvement in vascularity of the right foot could be observed in angiography. This combined approach after proper validation could be considered for similar cases.
Abscess ; Amputation ; Angiography ; Angioplasty ; Anti-Bacterial Agents ; Baths ; Carbon ; Cell- and Tissue-Based Therapy ; Diabetes Mellitus ; Drug Resistance, Multiple ; Fibroblast Growth Factor 2 ; Fibroblast Growth Factors* ; Foot ; Gangrene* ; Humans ; Infection Control* ; Kidney Failure, Chronic* ; Limb Salvage* ; Male ; Peripheral Arterial Disease ; Pseudomonas aeruginosa ; Renal Insufficiency, Chronic ; Toes* ; Water

OBJECTIVETo investigate the resistant effect of houttuyfonate sodium (SH) combined with imipenem (IMP) against Pseudomonas aeruginosa (Pa) biofilms.

METHODThe two-fold dilution method was used to examine the minimum inhibitory concentration (MIC) of the tested drug. The crystal violet staining was applied to detect the effect of the combination of 1/2MIC, 1MIC, 2MIC of SH, single IMP, 1/2MIC of SH and IMP of various concentrations on the clearance rate of adherent bacteria, growth of biofilms and alginate production. Fluorescein diacetate (FDA)-propidium iodide (PI) doubling staining assay was employed to observe the bacterial viability and morphological changes after membrane dispersion of each drug group.

RESULTSodium houttuyfonate could enhance the effect of IMP against pseudomonas aeruginosa biofilms. Particularly, the combination group with the concentration of 2MIC showed the highest effect, with P < 0.001 compared with the negative control group. The above results were proved by the bacterial viability and biofilm morphology under fluorescence microscope.

CONCLUSIONAfter being combined with imipenem, sodium houttuyfonate shows a higher effect against biofilms. It is expected that the combination of the two drugs could improve the clinical efficacy of associated infections.

Alkanes ; pharmacology ; Biofilms ; drug effects ; growth & development ; Drug Synergism ; Imipenem ; pharmacology ; Microbial Sensitivity Tests ; Microbial Viability ; drug effects ; Pseudomonas aeruginosa ; drug effects ; physiology ; Sulfites ; pharmacology

BACKGROUND: Delayed entry of blood culture bottles is inevitable when microbiological laboratories do not operate for 24 hr. There are few studies reported for prestorage of these bottles. The growth dynamics of Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa were investigated with respect to various preincubation conditions. METHODS: Fifteen or 150 colony-forming units (CFU) of bacteria were inoculated into standard aerobic or anaerobic blood culture bottles. Bottles were preincubated at 25degrees C or 37degrees C for 0, 2, 4, 8, 12, 24, or 48 hr. The time to detection (TTD) then was monitored using the BacT/Alert 3D system (bioMerieux Inc., USA). RESULTS: Significant difference in TTD was observed following preincubation for 8 hr at 25degrees C vs. 4 hr at 37degrees C for S. aureus, 4 hr at 25degrees C vs. 4 hr at 37degrees C for E. coli, 12 hr at 25degrees C vs. 4 hr at 37degrees C for P. aeruginosa, compared to no preincubation (P<0.005). TTD values did not vary significantly with bacterial CFU or with aerobic or anaerobic bottle type. The BacT/Alert 3D system returned false negatives following preincubation of P. aeruginosa for 48 hr at 25degrees C or 24 hr at 37degrees C. CONCLUSIONS: TTD was mainly affected by preincubation temperature and duration rather than by input CFU quantity or bottle type for the 3 experimental bacteria.
Bacteriological Techniques/instrumentation/*methods ; Culture Media ; Escherichia coli/growth & development/*isolation & purification ; Pseudomonas aeruginosa/growth & development/*isolation & purification ; Staphylococcus aureus/growth & development/*isolation & purification ; Temperature ; Time Factors

Biofilms are communities of surface-associated bacteria or fungi embedded in a self-produced extracellular polymeric matrix that are notoriously difficult to be eradicated and are sources of many recalcitrant infections. Treatment for biofilm infection with any individual drug is always less effective, while the combinations of different types of drugs are superior to monotherapy concerning the removing of biofilms. This paper focus on research progress in recent years for synergistic effect of drugs in combination against biofilms formed by Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli and Candida albicans.
Anti-Bacterial Agents ; pharmacology ; Antifungal Agents ; pharmacology ; Bacteria ; drug effects ; Biofilms ; drug effects ; growth & development ; Candida albicans ; drug effects ; Escherichia coli ; drug effects ; Pseudomonas aeruginosa ; drug effects ; Staphylococcus aureus ; drug effects ; Staphylococcus epidermidis ; drug effects

OBJECTIVE: To observe the effect of biyuanshu oral liquid on the formation of pseudomonas aeruginosa biofilms in vitro. METHOD: Pseudomonas aeruginosa biofilm was established by plate culture and detected by Scanning electron microscopy and AgNO3 staining. After treated with different dosages of biyuanshu oral liquid and erythromycin, the pseudomonas aeruginosa biofilms were observed by AgNO3 staining and the number of viable bacteria were measured by serial dilution. RESULT: The pseudomonas aeruginosa biofilms could be detected by SEM at the seventh culture day and it was consistent with the detection of AgNO3 staining. The biyuanshu oral liquid and erythromycin have the effect on inhibiting the formation of pseudomonas aeruginosa biofilms. But with the already formed pseudomonas aeruginosa biofilms the inhibition was not significant. The serial dilution method showed that the viable counts of bacteria of biyuanshu oral liquid and erythromycin treated groups were significantly lower than those untreated groups (P < 0.05). CONCLUSION The biyuanshu oral liquid and erythromycin can inhibit the formation of pseudomonas aeruginosa biofilms in vitro.
Anti-Bacterial Agents ; pharmacology ; Biofilms ; drug effects ; Drugs, Chinese Herbal ; pharmacology ; Erythromycin ; pharmacology ; Pseudomonas aeruginosa ; drug effects ; growth & development

OBJECTIVE: To analyze the factors and biological characteristics of infection after cochlear implantation so as to control the risk factors and improve the treatment of postoperative infection. METHOD: A retrospective study was conducted to analyze the clinical data of 316 patients receiving cochlear implantation from July 2001 to October 2011. RESULT: Postoperative infection was found in five of the 316 cases and one transferred case. The six cases recovered after clinical therapy without explantation. One case underwent explantation due to recurrent meningitis after implantation of 8 years later. CONCLUSION The pathogens of infection after cochlear implantation are staphylococcus aureus, pseudomonas aeruginosa, etc. The key infectious factor is the formation of bacterial biofilm, which can be removal by chemical agents to control the postoperative infection, especially the flap infection. It is not necessary to remove the artificial cochlea when the postoperative infection occurs. Positive perioperative interventions and postoperative infection control can improve the outcome of cochlear implantation.
Adolescent ; Aged ; Biofilms ; growth & development ; Cochlea ; Cochlear Implantation ; adverse effects ; Humans ; Postoperative Complications ; microbiology ; Pseudomonas Infections ; etiology ; Pseudomonas aeruginosa ; physiology ; Retrospective Studies ; Risk Factors ; Staphylococcal Infections ; etiology ; Staphylococcus aureus ; physiology

OBJECTIVE: To establish the rat model of chronic suppurative otitis media and observe the formation of bacterial biofilm in middle ear mucosa of animal models and discuss the role of bacterial biofilm in the pathogenesis of chronic suppurative otitis media. METHOD: Twenty-eight rats were divided into six experimental groups and a control group evenly. All rats in experimental groups were infected with pseudomonas aeruginosa solution in 1 X 10(6) cfu/ml concentration through the tympanic membrane puncture approach to bilateral middle ear cavity. On the first, sixth, tenth, fifteenth and twenty-first st day after inoculation respectively, four rats in one experimental group were narcotized, then two-sided tympanic membrane of each rat were observed by using electric otoscope. We rated on the severity of the inflammation from the general pathology level (0 for normal, four for the most serious). After the execution, the two-sided otocysts were obtained. The left was made to SEM specimen and the shape of bacterial biofilm in middle ear mucous was observed. The right was observed by CLSM. Control group were executed at the beginning of the experiment. RESULT: (1) Bacterial biofilm in line with their respective criteria were found at the six days after the inoculation, and were more typical in shape after ten days. Then the states maintained stably within three weeks. (2) By observing tympanic membrane under electric otoscope, it can be seen that the inflammation severity of otitis media aggravated gradually in the first ten days and achieved the peak, then the state continued to the third week. The differences of tympanic membrane rating between one day group and six day group, six day group and ten day group were statistically significant (P < 0.05, P < 0.01). CONCLUSION (1) In this experiment, the process of bacterial biofilm development in rats model is: from beginning to the five days, the bacteria adhere and accumulates. After six days, the 3D structure of bacterial biofilm preliminary formatted. After ten days, the bacterial biofilm achieves the mature and steady state. (2) With the growth and maturity of bacterial biofilm in middle ear mucosa, the inflammation of otitis media is gradually increasing, which suggests that the inflammation severity of otitis media and the maturation of bacterial biofilm in middle ear mucosa are closely related.
Animals ; Biofilms ; growth & development ; Disease Models, Animal ; Mucous Membrane ; microbiology ; Otitis Media, Suppurative ; microbiology ; Pseudomonas aeruginosa ; Rats ; Rats, Wistar

OBJECTIVETo explore the different expression of TGF-beta1 and collagen during the healing process of wound infected by Pseudomonas aeruginosa (PAO1).

METHODS24 female Wistar rats were randomly divided into pure wound group (group A) and wound + PAO1 group (group B). The re-epithelial rate, shrinkage rate and neutrophils number on the wounds were observed on the 1st, 3rd, 7th and 10th day after operation. The expression of TGF-beta1 and collage I, Ill was also detected.

RESULTSOn the 7th day, the re-epithelial rate in group A was higher than that in group B, while the shrinkage rate in group A was lower than that in group B. The neutrophils number increased to peak on the 1st day in group B, but on the 3rd day in group A. The TGF-beta1 expression increased after operation in both groups, but it decreased in group B on the 3rd day and re-increased after that. The TGF-beta1 expression was significantly different between the two groups on the 7th day (P < 0.05). The expression of collagen I and III decreased during healing. The expression of collagen III in group A was higher on the 3rd day and was lower on the 7th and 10th day than that in group B, showing a significant difference (P < 0.05).

CONCLUSIONSPAO1 infection could delay the expression of TGF-beta1 and collagen I, III on wound, which may interfere the healing process of wound.

Animals ; Collagen Type I ; metabolism ; Collagen Type III ; metabolism ; Female ; Pseudomonas Infections ; pathology ; Pseudomonas aeruginosa ; Rats ; Rats, Wistar ; Skin Transplantation ; Surgical Wound Infection ; metabolism ; microbiology ; Transforming Growth Factor beta1 ; metabolism ; Wound Healing

BACKGROUNDAcute lung infection due to Pseudomonas aeruginosa (P. aeruginosa) is a serious problem, especially in patients with structural lung conditions or immune compromised hosts, leading to an overwhelming threat with a high risk of morbidity and mortality. As an outcome of infection, fibrosis can be linked with chronic lung diseases. But some fibrotic manifestations, such as an irreversible decrease of lung function and fibrous bands seen on chest imaging, have been found after an acute infection with P. aeruginosa. Fibrogenesis/remodeling resulting from acute lung infection by P. aeruginosa is rarely reported. This study was designed to explore the relation between fibrogenesis/remodeling and acute infection by P. aeruginosa in vitro. We used flagellin protein from P. aeruginosa, a key initiator of acute P. aeruginosa lung infection, to elucidate mechanisms by which acute lung infection with P. aeruginosa can cause fibrogenesis/remodeling.

METHODSWe studied the effect of flagellin from P. aeruginosa (flagellin for short) on the transforming growth factor beta 1 (TGF-β1) and interleukin-8 (IL-8) expression, and the possible involvement of the signaling pathway, tumor necrosis factor receptor-associated factor 6 (TRAF6)/mitogen activated protein kinase (MAPK) pathway. Flagellin was purified from the P. aeruginosa standard strain, PAO1. Normal bronchial epithelial cells BEAS-2B were challenged with different concentrations of flagellin, and cell viability assessment was performed by cell counting kit-8. BEAS-2B cells were incubated with flagellin with the specific MAPK inhibitors or TRAF6 siRNA. Cell lysates and the cultured supernatant were collected. The level of TGF-β1 and IL-8 were detected by enzyme-linked immunosorbant assay (ELISA). Western blotting was used to detect the protein levels of MAPK signal proteins p38, c-Jun NH(2)-terminal kinase (JNK) and extracellular regulated kinase (ERK).

RESULTSExpression of TGF-β1 in BEAS-2B cells was elevated by flagellin vs. control groups ((104.3 ± 20.8) vs. (44.6 ± 4.4) pg/ml (P < 0.01)) and was ablated by either p38 or JNK inhibitors compared with flagellin treatment ((45.1 ± 18.8) vs. (104.3 ± 20.8) pg/ml and (48.1 ± 20.8) vs. (104.3 ± 20.8) pg/ml, respectively (P < 0.05)). Flagellin also elevated the expression of IL-8 in BEAS-2B cells vs. the control groups ((554.9 ± 57.7) vs. (51.4 ± 22.9) pg/ml (P < 0.01)), and p38 MAPK inhibitors weaken the expression by flagellin ((301.1 ± 155.1) vs. (554.9 ± 57.7) pg/ml (P < 0.05)). Western blotting revealed that all three MAPK proteins, p38, JNK and ERK were activated by flagellin challenge in an early phase, respectively in 15 minutes (P < 0.01), 30 minutes (P < 0.01) and 15 minutes (P < 0.01). TRAF6 siRNA which decreased expression of TRAF6, altered the activation of JNK, p38, and ERK following flagellin treatment, but its influence on the expression of TGF-β1 and IL-8 has no statistical significance.

CONCLUSIONSFlagellin from P.aeruginosa PAO1 induces TGF-β1 expression in normal bronchial epithelial cells, BEAS-2B, through the MAPK signal cascade in vitro. It suggests that the fibrogenesis/remodeling process may be initiated from an early stage of acute lung infection due to P. aeruginosa.

Blotting, Western ; Bronchi ; cytology ; Cell Line ; Cell Survival ; drug effects ; genetics ; Enzyme-Linked Immunosorbent Assay ; Epithelial Cells ; drug effects ; metabolism ; Flagellin ; pharmacology ; Humans ; Interleukin-8 ; metabolism ; Mitogen-Activated Protein Kinases ; metabolism ; Pseudomonas aeruginosa ; metabolism ; Signal Transduction ; drug effects ; genetics ; Transforming Growth Factor beta1 ; genetics ; metabolism

Bacteria survive in nature by forming biofilms on surfaces and probably most, if not all, bacteria (and fungi) are capable of forming biofilms. A biofilm is a structured consortium of bacteria embedded in a self-produced polymer matrix consisting of polysaccharide, protein and extracellular DNA. Bacterial biofilms are resistant to antibiotics, disinfectant chemicals and to phagocytosis and other components of the innate and adaptive inflammatory defense system of the body. It is known, for example, that persistence of staphylococcal infections related to foreign bodies is due to biofilm formation. Likewise, chronic Pseudomonas aeruginosa lung infections in cystic fibrosis patients are caused by biofilm growing mucoid strains. Gradients of nutrients and oxygen exist from the top to the bottom of biofilms and the bacterial cells located in nutrient poor areas have decreased metabolic activity and increased doubling times. These more or less dormant cells are therefore responsible for some of the tolerance to antibiotics. Biofilm growth is associated with an increased level of mutations. Bacteria in biofilms communicate by means of molecules, which activates certain genes responsible for production of virulence factors and, to some extent, biofilm structure. This phenomenon is called quorum sensing and depends upon the concentration of the quorum sensing molecules in a certain niche, which depends on the number of the bacteria. Biofilms can be prevented by antibiotic prophylaxis or early aggressive antibiotic therapy and they can be treated by chronic suppressive antibiotic therapy. Promising strategies may include the use of compounds which can dissolve the biofilm matrix and quorum sensing inhibitors, which increases biofilm susceptibility to antibiotics and phagocytosis.
Animals ; Antibiotic Prophylaxis ; Biofilms ; drug effects ; growth & development ; Chronic Disease ; Cystic Fibrosis ; microbiology ; Drug Resistance, Microbial ; physiology ; Foreign Bodies ; microbiology ; Humans ; Microbial Consortia ; drug effects ; genetics ; immunology ; Phagocytosis ; Pseudomonas Infections ; microbiology ; Pseudomonas aeruginosa ; drug effects ; genetics ; physiology ; Quorum Sensing ; drug effects ; genetics