Cytokine patterns in cystic fibrosis patients with different microbial infections in oropharyngeal samples.

BACKGROUND: Cytokines play a crucial role in the immune system's regulation by mediating protective responses to infections. anti-inflammatory and pro-inflammatory cytokines are in equilibrium. Therefore, any alteration in cytokine production or cytokine receptor expression might result in pathological illnesses and health issues. Cystic fibrosis (CF) is a genetic disease caused by mutations in the CF transmembrane regulator (CFTR) gene. Lung infection in these patients is related to chronic bacterial airway infection and inflammation, which is triggered by some inflammatory cytokines. Our goal was to compare the cytokine patterns in CF patient's serum and PBMCs caused by microbial pathogens that colonized their airways to controls. METHODS: ELISA and Real-time PCR were used to determine the levels of IL-10, IFN-γ, IL-4, TGF-ß, IL-8, and IL-17 in serum and PBMC cells. Blood parameters in both patients and healthy people were studied. RESULTS: An increase in IL-10, IFN-γ, IL-4 (p-v = 0.03, 0.024 and 0.003) levels and a decrease in IL-17 (p-v = 0.004) was found in Pseudomonas aeruginosa positive patients. There were no different in TGF-ß and IL-8 (p-value = 0.778 and 0.903) in this patients. IL-10, IFN-γ, and IL-4 (p-value = 0.023, 0.001 and 0.002) levels were high in Staphylococcus aureus positive patients and TGF-ß, IL-17, and IL-8 (p-value = 0.085, 0.167 and 0.362) were not significantly different in the patient and control groups. IFN-γ and IL-4 levels were higher in patients without infection who had normal microbiota (p-v = 0.002 and 0.024). In patients with P. aeruginosa, WBC and platelets increased, and MCH and MCV decreased. Patients with normal microbiota had less MCV. CONCLUSION: According to our research, patients with P. aeruginosa, S. aureus, and normal microbiota are exposed to cytokine alterations and changes in blood factors. The link between the CF patient's airway microbiota and the kind of generated cytokines might lead to the modulation of inflammatory cytokines alone or in combination with antibiotics, reducing disease-causing effects while avoiding drug resistance.

One step green synthesis of Cu nanoparticles by the aqueous extract of Juglans regia green husk: assessing its physicochemical, environmental and biological activities.

Juglans regia (J. regia) green husk is an abundant agricultural waste. In this study, an economical, rapid and green synthetic route was introduced for the biosynthesis of copper nanoparticles (CuNPs) by applying the aqueous extract of J. regia green husk at the ambient conditions. Ultra Violet-Visible (UV-Visible) analysis revealed that the Surface Plasmon Resonance (SPR) of the CuNP was 212 nm. The average hydrodynamic and metallic core diameters of the CuNPs were about 53-28 nm, respectively. X-ray Diffraction (XRD) analysis presented that the CuNPs were amorphous. The CuNPs exhibited the highest free radical 1,1-diphenyl-2-picryl-hydrazyl (DPPH) scavenging efficiency. These nanoparticles (NPs) showed antibacterial, antifungal and antibiofilm properties. They presented photocatalytic activity against Methyl Orange (MO). Besides, the potential of these NPs for the fast and precise colorimetric detection of Hg2+ was remarkable. The biosynthesized CuNPs are introduced as a multifunctional nanomaterial with various applications in medicine and environmental cases. The CuNPs were produced through an environmentally green process by the aqueous extract of dried J. regia green husk at the ambient condition. The CuNPs confirmed that this type of nanomaterial is a multifunctional agent with significant antibacterial, antifungal, antibiofilm, antioxidant, photocatalytic activities. Besides, it is a promising colorimetric sensor for the detection of Hg2+ in an aqueous complex media.

Involvement of Pseudomonas aeruginosa in the occurrence of community and hospital acquired diarrhea, and its virulence diversity among the stool and the environmental samples.

Transmission of Pseudomonas aeruginosa along the food chain could cause gastrointestinal infections. To show this involvement, the prevalence, putative virulence genotype, and antibiotic resistance phenotype of P. aeruginosa isolates from stool of 1482 patients with community and hospital acquired diarrhea were compared with 87 isolates from the environmental samples. The results showed infection with P. aeruginosa in 3.4% of the cases, while 57.4% of vegetable samples were contaminated. Significantly higher frequency of lasB (98%), aprA (98%), exoY (98%), and exoS (90%), but lower rate of exoT (39.2%), was detected among the stool isolates. Multi-drug resistance (MDR) phenotype was detected in 25.5% and 4% of the stool and vegetable isolates, respectively. A higher rate of studied virulence genes was detected among the MDR strains vs non-MDR strains. These results indicate P. aeruginosa as a causative agent of diarrhea either among the hospitalized patients and those with community-acquired diarrhea.

Ecological role of Acinetobacter calcoaceticus GSN3 in natural biofilm formation and its advantages in bioremediation.

This study assessed the role of a new Acinetobacter calcoaceticus strain, GSN3, with biofilm-forming and phenol-degrading abilities. Three biofilm reactors were spiked with activated sludge (R1), green fluorescent plasmid (GFP) tagged GSN3 (R2), and their combination (R3). More than 99% phenol removal was achieved during four weeks in R3 while this efficiency was reached after two and four further operational weeks in R2 and R1, respectively. Confocal scanning electron microscopy revealed that GSN3-gfp strains appeared mostly in the deeper layers of the biofilm in R3. After four weeks, almost 7.07 × 107 more attached sludge cells were counted per carrier in R3 in comparison to R1. Additionally, the higher numbers of GSN3-gfp in R2 were unable to increase the efficiency as much as measured in R3. The presence of GSN3-gfp in R3 conveyed advantages, including enhancement of cell immobilization, population diversity, metabolic cooperation and ultimately treatment efficiency.

Highly efficient phenol degradation in a batch moving bed biofilm reactor: benefiting from biofilm-enhancing bacteria.

In this study, the efficiency improvement of three moving bed biofilm reactors (MBBRs) was investigated by inoculation of activated sludge cells (R1), mixed culture of eight strong phenol-degrading bacteria consisted of Pseudomonas spp. and Acinetobacter spp. (R2) and the combination of both (R3). Biofilm formation ability of eight bacteria was assessed initially using different methods and media. Maximum degradation of phenol, COD, biomass growth and also changes in organic loading shock were used as parameters to measure the performance of reactors. According to the results, all eight strains were determined as enhanced biofilm forming bacteria (EBFB). Under optimum operating conditions, more than 90% of initial COD load of 2795 mg L-1 was reduced at 24 HRT in R3 while this reduction efficiency was observed in concentrations of 1290 mg L-1 and 1935 mg L-1, in R1 and R2, respectively. When encountering phenol loading shock-twice greater than optimum amount-R1, R2 and R3 managed to return to the steady-state condition within 32, 24 and 18 days, respectively. SEM microscopy and biomass growth measurements confirmed the contribution of more cells to biofilm formation in R3 followed by R2. Additionally, established biofilm in R3 was more resistant to phenol loading shock which can be attributed to the enhancer role of EBFB strains in this reactor. It has been demonstrated that the bacteria with both biofilm-forming and contaminant-degrading abilities are not only able to promote the immobilization of other favorable activated sludge cells in biofilm structure, but also cooperate in contaminant degradation which all consequently lead to improvement of treatment efficiency.

Comparison of culture and PCR-DGGE methods to evaluate the airways of cystic fibrosis patients and determination of their antibiotic resistance profile.

Background and Objectives: Respiratory infections are the most serious condition in cystic fibrosis (CF) patients; therefore, a thorough comprehension of the diversity and dominant microbial species in CF airways has a crucial role in treatment. Our objective was to determine the antibiotic resistance profile of CF airways microbiota and compare culture methods and PCR-DGGE to evaluate bacterial diversity. Materials and Methods: Pharyngeal swabs from 121 CF patients were collected. The samples were then cultured, identified and antibiotic resistance testing was performed. Thirty samples were subjected to further molecular surveys. DNA contents of these samples were extracted and amplified using nested-PCR technique and their bacterial diversity was assessed by DGGE. The DGGE patterns were visualized and certain bands were excised and purified. Next, the DNA was amplified by another round of PCR and sent out for sequencing. Results: Staphylococcus aureus, Pseudomonas aeruginosa, and Klebsiella pneumoniae were the most prevalent species isolated using culture methods. S. aureus was the most common bacteria among 6 years and younger patients; while, P. aeruginosa had more prevalence among older ones. The PCR-DGGE results showed more diversity than culture methods, particularly in younger patients who exhibited more bacterial diversity than the older groups. Sequencing results unveiled the presence of certain bacterial species including Haemophilus parainfluenzae and Stenotrophomonas maltophilia which were completely missed in culture. Conclusion: Even though culture-dependent methods are cost-effective, PCR-DGGE appeared to be more efficient to determine bacterial diversity. PCR-DGGE detects less abundant species, though their viability could not be determined using this method.

Bacterial variation in the oral microbiota in multiple sclerosis patients.

BACKGROUND: Microorganisms in oral cavity are called oral microbiota, while microbiome consists of total genome content of microorganisms in a host. Interaction between host and microorganisms is important in nervous system development and nervous diseases such as Autism, Alzheimer, Parkinson and Multiple Sclerosis (MS). Bacterial infections, as an environmental factor in MS pathogenesis play role in T helper 17(Th17) increase and it enhancing the production of pro-inflammatory cytokines such as Interlukin-21(IL-21), IL-17 and IL -22. Oral microbiota consists diverse populations of cultivable and uncultivable bacterial species. Denaturing gradient gel electrophoresis (DGGE) is an acceptable method for identification of uncultivable bacteria. In this study, we compared the bacterial population diversity in the oral cavity between MS and healthy people. METHODS: From October to March 2019, samples were taken at Kermanshah University of Medical Sciences' MS patients center. A total of 30 samples were taken from MS patients and another 30 samples were taken from healthy people. Phenotypic tests were used to identify bacteria after pure cultures were obtained. DNA was extracted from 1 mL of saliva, and PCR products produced with primers were electrophoresed on polyacrylamide gels. RESULTS: The genera Staphylococcus, Actinomyces, Fusobacterium, Bacteroides, Porphyromonas, Prevotella, Veillonella, Propionibacterium and uncultivable bacteria with accession number MW880919-25, JQ477416.1, KF074888.1 and several other un-culturable strains were significantly more abundant in the MS group while Lactobacillus and Peptostreptococcus were more prevalent in the normal healthy group according to logistic regression method. CONCLUSION: Oral micro-organisms may alleviate or exacerbate inflammatory condition which impact MS disease pathogenesis. It may be assumed that controlling oral infections may result in reduction of MS disease progression.

Biochemical and computational study of an alginate lyase produced by Pseudomonas aeruginosa strain S21.

OBJECTIVES: Alginates play a key role in mucoid Pseudomonas aeruginosa colonization, biofilm formation, and driving out of cationic antibiotics. P. aeruginosa alginate lyase (AlgL) is a periplasmic enzyme that is necessary for alginate synthesis and secretion. It also has a role in depolymerization of alginates. Using AlgLs in cystic fibrosis patients along with antibiotics enhances bacterial killing and host healing. In this study, we investigated the different biochemical properties of a newly isolated AlgL from P. aeruginosa S21 to complete the databank of AlgLs. MATERIALS AND METHODS: The enzyme was extracted from the periplasmic space of the bacteria by the heat shock method. Using the TBA method, the enzyme activity and biochemical properties were assessed. The mutability of P. aeruginosa S21 AlgL to increase its thermal stability was investigated. The most favorable mutations were studied computationally. The molecular dynamics simulation (MDS) package GROMACS was used for determining the effect of S34R mutation on enzyme's thermal stability. RESULTS: Data showed that this enzyme has the best activity at 37 °C and pH 7.5 and it can degrade mannuronate blocks, guluronate blocks, and sodium alginate. After 7 hr at 80 °C, 45% of the enzyme activity was retained. This enzyme needed 15 min to completely degrade accessible sodium alginate. Tris buffer, pH 8.5 and Britton-Robinson buffer, pH 7.0 were the preferable buffers for the enzyme activity. MDS of native and mutated enzymes showed desirable results. CONCLUSION: P. aeruginosa S21 AlgL can be used in medical and industrial applications to degrade alginates.

Immunoinformatics design of multivalent chimeric vaccine for modulation of the immune system in Pseudomonas aeruginosa infection.

Increasing in drug-resistant Pseudomonas aeruginosa and high mortality and morbidity rate have become a health challenge worldwide; therefore, developing the novel therapeutic strategies such as immunogenic vaccine candidate are required. Despite a substantial research effort, the future of immunization against P. aeruginosa due to failure in covering two separate stages of infection, and furthermore, inducing ineffective type of immune response, still remains controversial. In this study, immunoinformatics approach was utilized to design multivalent chimeric vaccine from both stages of infection containing Lectin, HIV TAT peptide, N-terminal fragment of exotoxin A and Epi8 of outer membrane protein F (OprF) with hydrophobic linkers which have a high density of B-cell, T Lymphocytes (HTL), T Lymphocytes (CTL), and IFN-γ epitopes. The physicochemical properties, antigenicity, and allergenicity for designed vaccine were analyzed. 3D model generation and refinement further validation of the final vaccine were followed by computational docking with molecular dynamics analyses that demonstrated high- affinity interaction between vaccine and TLR-4. Finally, designed vaccine was in silico cloned in pET22b. We have expected that the designed vaccine able to elucidate innate, humoral and cellular innate immune responses and control the interaction of P. aeruginosa with host and maybe overcome to P. aeruginosa vaccines drawback.

Biosynthesised AgCl NPs using Bacillus sp. 1/11 and evaluation of their cytotoxic activity and antibacterial and antibiofilm effects on multi-drug resistant bacteria.

Silver nanoparticles (AgNPs) have attracted the attention of researchers due to their properties. Biological synthesis of AgNPs is eco-friendly and cost-effective preferred to physical and chemical methods, which utilize environmentally harmful agents and large amounts of energy. Microorganisms have been explored as potential biofactories to synthesize AgNPs. Bacterial NP synthesis is affected by Ag salt concentration, pH, temperature and bacterial species. In this study, Bacillus spp., isolated from soil, were screened for AgNP synthesis at pH 12 with 5 mM Ag nitrate (AgNO3) final concentration at room temperature. The isolate with fastest color change and the best ultraviolet-visible spectrum in width and height were chosen as premier one. AgNO3 and citrate salts were compared in terms of their influence on NP synthesis. Spherical Ag chloride (AgCl) NPs with a size range of 35-40 nm were synthesized in 1.5 mM Ag citrate solution. Fourier transform infrared analysis demonstrated that protein and carbohydrates were capping agents for NPs. In this study, antimicrobial and antitumor properties of the AgNP were investigated. The resulting AgCl NPs had bacteriostatic activity against four standard spp. And multi-drug resistant strain of Pseudomonas aeruginosa. These NPs are also cytotoxic to cancer cell lines MCF-7, U87MG and T293.

Screening of Alginate Lyase-Producing Bacteria and Optimization of Media Compositions for Extracellular Alginate Lyase Production.

BACKGROUND: Alginate is a linear polysaccharide consisting of guluronate (polyG) and mannuronate (polyM) subunits. METHODS: In the initial screening of alginate-degrading bacteria from soil, 10 isolates were able to grow on minimal medium containing alginate. The optimization of cell growth and alginate lyase (algL) production was carried out by the addition of 0.8% alginate and 0.2-0.3 M NaCl to the culture medium. Of 10 isolates, one was selected based on its fast growth rate on minimal 9 medium containing 0.4% sodium alginate. The selected bacterium, identified based on morphological and biochemical characteristics as well as 16S rDNA sequence data, was confirmed to be an isolate belonging to the genus Bacillus and designated as Bacillus sp. TAG8. Resuls: The results showed the ability of Bacillus sp. TAG8 to utilize alginate as a sole carbon source. Bacillus sp. TAG8 growth and algL production were augmented with an increase in sodium alginate concentration and also by the addition of 0.2-0.3 M NaCl. Molecular analysis of TAG8 algL gene showed 99% sequence identity with algL of Pseudomonas aeruginosa PAO1. algL produced by Bacillus sp. TAG8 cleaved both polyM and polyG blocks in alginate molecule as well as acetylated alginate residues, confirming the bifunctionality of the isolated lyase. CONCLUSION: The identification of novel algL genes from microbial communities constitutes a new approach for exploring lyases with specific activity against bacterial alginates and may thus contribute to the eradication of persistent biofilms from clinical samples.

Improved effect of amikacin-loaded poly(D,L-lactide-co-glycolide) nanoparticles against planktonic and biofilm cells of Pseudomonas aeruginosa.

PURPOSE: Amikacin is one of the most effective antibiotics against Pseudomonas aeruginosa infections, but because of its high toxicity, the use of this antibiotic has been clinically limited. In the present study, amikacin was successfully loaded into a new formulation of nanoparticles (NPs) based on poly(d,l-lactide-co-glycolide) 50 : 50 in order to enhance the treatment efficacy. The synthetized amikacin-loaded PLGA nanoparticles with high drug loading and stability were used to eliminate P. aeruginosa cells in planktonic and biofilm states. METHODOLOGY: P. aeruginosa PAO1 biofilm susceptibility studies were done using the minimum biofilm eradication concentration assay. The association of fluorescently labeled amikacin-loaded nanoparticles (A-NPs) with mouse monocyte macrophage cells (RAW 264.7), and the nanoparticles ability to interact and eradicate the bacterial cells even in the form of biofilms, was investigated using Flow cytometric studies and confocal laser scanning microscopy. RESULTS: Flow cytometric studies showed that these NPs were able to interact with planktonic and biofilm bacterial cells. Moreover, following 1 h of incubation of A-NPs with 1-day-old biofilm, it was found that particles penetrate through the entire biofilm thickness. Live/dead fluorescent staining followed by CLSM analysis showed that the A-NPs were more effective than free drug in biofilm eradication. CONCLUSION: The good antibacterial and antibiofilm activities of A-NPs, in addition to their ability to enter macrophages without any cytotoxicity for these cells, make them a potential candidate to treat P. aeruginosa infections.

Analysis of Pseudomonas aeruginosa PAO1 Biofilm Protein Profile After Exposure to n-Butanolic Cyclamen coum Extract Alone and in Combination with Ciprofloxacin.

Pseudomonas aeruginosa biofilm-related infections are the major cause of premature death in cystic fibrosis patients. Strategies to induce biofilm dispersal are of interest, because of their potential in preventing biofilm-related infections. Our previous work demonstrated that n-butanolic Cyclamen coum extract with ciprofloxacin could eliminate 1- and 3-day-old P. aeruginosa PAO1 biofilms. To gain new insights into the role of C. coum extract and its synergistic effect with ciprofloxacin in eliminating P. aeruginosa PAO1 biofilms, two-dimensional gel electrophoresis (2-DE) in combination with mass spectrometry-based protein identification were used. Changes in the bacterial protein expression were analyzed when 3-day-old biofilm cells were exposed to the C. coum extract alone and in combination with ciprofloxacin. Proteins involved in alginate biosynthesis, quorum sensing, adaptation/protection, carbohydrate and amino acid metabolism showed a weaker expression in the C. coum extract-ciprofloxacin-treated biofilm cells compared to those in the untreated cells. Interestingly, the proteome of C. coum extract-ciprofloxacin-treated biofilm revealed more resemblance to the planktonic phenotype than to the biofilm phenotype. It appears that saponin extract in combination with ciprofloxacin causes biofilm disruption due to several mechanisms such as motility induction, cell envelope integrity perturbation, stress protein expression reduction, and more importantly, signal transduction perturbation. In conclusion, exposure to a combination of biofilm dispersal such as saponin extract and antimicrobial agents may offer a novel strategy to control preestablished, persistent P. aeruginosa biofilms and biofilm-related infections.

A comparison of antibiotic disks from different sources on Quicolor and Mueller-Hinton agar media in evaluation of antibacterial susceptibility testing.

BACKGROUND AND OBJECTIVES: Antibacterial susceptibility testing of clinical bacterial isolates through disk diffusion method plays a major role in antibacterial treatment. One of the main factors affecting the result of these tests is the type, structure and quality of the disks. The main objective of this study was to compare the agreement of antibiotic disks originated from three companies on Quicolor and Mueller-Hinton agar. MATERIALS AND METHODS: Quicolor and Mueller-Hinton agar media were used in disk diffusion method. Seventy clinical isolates from Enterobacteriaceae family (21 Klebsiella spp., 36 Escherichia coli, 1 Enterobacter spp. and 12 Shigella spp.) were investigated in the study. After obtaining data, the results were interpreted as resistant, sensitive or intermediate. Kappa coefficient measured the agreement of two media. Coefficient of variation (CV) was also calculated for antibiotic disks. RESULTS: The kappa agreement values for three types of antibiotic disks on Quicolor and Mueller-Hinton agar plates were good or excellent for all the examined antibiotics. CV values were also very satisfactory in the majority of cases. CONCLUSION: Antibiotic disks from three manufacturers can successfully be used on both Quicolor and Mueller-Hinton agar plates.

Amikacin loaded PLGA nanoparticles against Pseudomonas aeruginosa.

Amikacin is a very effective aminoglycoside antibiotic but according to its high toxicity, the use of this antibiotic has been limited. The aim of this study was to formulate and characterize amikacin loaded PLGA nanoparticles. Nanoparticles were synthetized using a solid-in-oil-in-water emulsion technique with different ratio of PLGA 50:50 (Resomer 502H) to drug (100:3.5, 80:3.5 and 60:3.5), two different concentrations of stabilizer (pluronic F68) (0.5% or 1%) and varied g forces to recover the final products. The most efficient formulation based on drug loading (26.0±1.3µg/mg nanoparticle) and encapsulation efficiency (76.8±3.8%) was the one obtained with 100:3.5 PLGA:drug and 0.5% luronic F68, recovered by 20,000×g for 20min. Drug release kinetic study indicated that about 50% of the encapsulated drug was released during the first hour of incubation in phospahte buffer, pH7.4, 37°C, 120rpm. Using different cell viability/cytotoxicity assays, the optimized formulation showed no toxicity against RAW macrophages after 2 and 24h of exposure. Furthermore, released drug was active and maintained its bactericidal activity against Pseudomonas aeruginosa in vitro. These results support the effective utilization of the PLGA nanoparticle formulation for amikacin in further in vivo studies.

Optimization of tetrazolium salt assay for Pseudomonas aeruginosa biofilm using microtiter plate method.

Pseudomonas aeruginosa is one of the most important pathogenic bacteria related to biofilm infections. Due to the biofilm multi-drug resistance, methods of biofilm formation enumeration are of interest for assessment of efficient drug regimen development for biofilm inhibition or eradication. There are many different assay methods to determine the biofilm formation, using vital or non-vital dyes. The primary aim of the current study was to develop an assay using a member of tetrazolium salts family, 2,3,5-triphenyl-tetrazolium chloride (TTC), for detection of P. aeruginosa biofilm formation in 96-well microtiter plates and also a method of Minimum Biofilm Inhibitory Concentration (MBIC) determination of antibiotics against P. aeruginosa PAO1. Furthermore, the assay was optimized for TTC concentration, wavelength and period of incubation for 4 different antibiotics. The optimized condition was then compared with two other prevalent methods: the crystal violet (CV) assay and the 2,3-bis (2-methoxy-4-nitro-5-sulfophenly)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide (XTT) assay. In general, the optimized TTC assay (0.5% TTC, 6h of incubation and absorbance measurement at 405nm for biofilm assay and 1% TTC, 5h of incubation and absorbance measurement at 490nm for MBIC determination) distinguished between biofilms formed by different concentrations of bacteria and also was able to detect lower amounts of biofilm formed in contrast to the other two assay methods suggesting that TTC assay is more sensitive and also less expensive than other vital staining methods.

Assessment of Biofilm Formation and Resistance to Imipenem and Ciprofloxacin among Clinical Isolates of Acinetobacter baumannii in Tehran.

BACKGROUND: Biofilms are communities of bacteria attached to the surfaces in an extracellular polymeric matrix which are associated with many chronic infections in humans. Acinetobacter spp. are emerging as a major cause of nosocomial infections and Acinetobacter baumannii is the predominant species associated with this kind of infections. OBJECTIVES: In the present study, the potential of biofilm formation of clinical isolates, A. baumannii, was assessed by using crystal violet method. Furthermore, susceptibility pattern of these strains to ciprofloxacin and imipenem was determined. METHODS AND MATERIALS: Biofilm formation by 75 A. baumannii isolates was evaluated by using microtiter plate and tube methods and crystal violet staining. Tube method was carried out under static and shaking conditions. Then, the susceptibility of isolates to ciprofloxacin and imipenem was determined. RESULTS: Results showed that in tube method under shaking, 22% of clinical isolates were strong biofilm producers while 23% of them were not able to form biofilms. In this experiment, 18% and 42% of isolates were considered as moderate and weak biofilm-forming strains, respectively. In microtiter plate tests, 18% of strains were strong-biofilm producers and 25% of them were notable biofilm producers. In this assessment, 10% and 47% were considered as moderate and weak biofilm-forming isolates, respectively. The susceptibility tests, using microdilution method, confirmed that 92% of these isolates were resistant and 6.6% were susceptible to ciprofloxacin, although these results for imipenem were 68% and 24%, respectively. CONCLUSIONS: It can be concluded that most of A. baumannii isolates can form biofilm in microtiter plate and tube. The results also verified that most of these isolates were resistant to ciprofloxacin and imipenem.

Synergistic Effects of Bismuth Thiols and Various Antibiotics Against Pseudomonas aeruginosa Biofilm.

BACKGROUND: Pseudomonas aeruginosa is an opportunistic pathogen that takes advantages of some weaknesses in the immune system to initiate an infection. Biofilms of P. aeruginosa can cause chronic opportunistic infections in immunocompromised and elderly patients. This bacterium is considered as a model organism to study antibiotic resistance as well as biofilm formation. In the biofilm structures, bacteria are protected from many harmful environmental factors such as fluctuations in the level of oxygen and nutrients, and the alterations of pH as well as sensitivity to antibiotics. Decreased permeability of biofilms is one of the important reasons of antimicrobial resistance in bacteria. OBJECTIVES: In this study the anti-biofilm activity of bismuth thiols in combination with ciprofloxacin, imipenem and ceftazidime against the P. aeruginosa biofilm was investigated. MATERIALS AND METHODS: Checkerboard method was used to test the susceptibility of biofilms against various antimicrobial combinations. The biofilm formation was measured by 2,3-bis (2-methoxy-4-nitro-5-sulfo-phenyl)-2H-tetrazolium-5-carboxanilide (XTT) colorimetric assay. The fractional bio-film inhibitory concentration was reported for each agent. RESULTS: The combination of bismuth ethanedithiol with ciprofloxacin showed synergistic inhibitory effect on the P. aeruginosa biofilm formation. The combination of bismuth ethanedithiol ciprofloxacin, ceftazidime and imipenem showed synergistic inhibitory effects on the biofilm formation. Furthermore, the combination of bismuth ethanedithiol, imipenem and ceftazidime did not show any synergistic inhibitory effect on biofilm formation. CONCLUSIONS: Our studies show that using appropriate concentrations of bismuth thiols in combination with various antibiotics can act synergistically against P. aeruginosa biofilm formation.

Eradication of Pseudomonas aeruginosa Biofilms Using the Combination of n-butanolic Cyclamen coum Extract and Ciprofloxacin.

BACKGROUND: Biofilm formation is a major pathogenic factor in different bacteria such as Pseudomonas aeruginosa. A number of studies have reported that bacterial biofilms show different levels of antibiotic resistance. In order to re-sensitize the bacterial biofilms to antibiotics, biofilms should be dispersed. OBJECTIVES: In this study, the effect of n-butanolic Cyclamen coum extract in combination with ciprofloxacin was examined on one, three and five day old P. aeruginosa biofilms. The synergistic effect of n-butanolic C. coum extract and ciprofloxacin towards dispersing pre-established P. aeruginosa biofilms was also studied. MATERIALS AND METHODS: The ability of biofilm formation by six different P. aeruginosa strains was confirmed by microtiter plate method and PCR assay for the cupA gene. The extraction of C. coum tubers was achieved by fractionation method using different solvents. The minimum inhibitory concentration (MIC) of n-butanolic C. coum extract and ciprofloxacin against planktonic cells was evaluated using agar well diffusion and microdilution methods. The microdilution chequerboard method was used to determine the fractional biofilm eradication concentration index (FBCI), when the combination of n-butanolic C. coum extract and ciprofloxacin were used against P. aeruginosa biofilms. RESULTS: The ability of biofilm formation by P. aeruginosa strains was quantitatively confirmed. The PCR method confirmed the existence of cup A gene (172 bp) in all studied strains. Saponin content of the n-butanolic C. coum extract was 156 µg/mL. The extract revealed antibacterial activity against planktonic cells of P. aeruginosa strains. The results showed that one and three day old biofilms are affected by either ciprofloxacin or n-butanolic C. coum extract. However, n-butanolic C. coum extract in combination with ciprofloxacin was significantly more effective against P. aeruginosa biofilms. CONCLUSIONS: Using n-butanolic C. coum extract in combination with ciprofloxacin offers a novel strategy to control biofilm-based infections caused by P. aeruginosa.