Occurrence of COVID-19 in cystic fibrosis patients: a review.

Cystic fibrosis (CF) is a genetic ailment caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. This autosomal recessive disorder is characterized by diverse pathobiological abnormalities, such as the disorder of CFTR channels in mucosal surfaces, caused by inadequate clearance of mucus and sputum, in addition to the malfunctioning of mucous organs. However, the primary motive of mortality in CF patients is pulmonary failure, which is attributed to the colonization of opportunistic microorganisms, formation of resistant biofilms, and a subsequent decline in lung characteristics. In December 2019, the World Health Organization (WHO) declared the outbreak of the radical coronavirus disease 2019 (COVID-19) as a worldwide public health crisis, which unexpectedly spread not only within China but also globally. Given that the respiration system is the primary target of the COVID-19 virus, it is crucial to investigate the impact of COVID-19 on the pathogenesis and mortality of CF patients, mainly in the context of acute respiratory distress syndrome (ARDS). Therefore, the goal of this review is to comprehensively review the present literature on the relationship between cystic fibrosis, COVID-19 contamination, and development of ARDS. Several investigations performed during the early stages of the virus outbreak have discovered unexpected findings regarding the occurrence and effectiveness of COVID-19 in individuals with CF. Contrary to initial expectancies, the rate of infection and the effectiveness of the virus in CF patients are lower than those in the overall population. This finding may be attributed to different factors, including the presence of thick mucus, social avoidance, using remedies that include azithromycin, the fairly younger age of CF patients, decreased presence of ACE-2 receptors, and the effect of CFTR channel disorder on the replication cycle and infectivity of the virus. However, it is important to notice that certain situations, which include undergoing a transplant, can also doubtlessly boost the susceptibility of CF patients to COVID-19. Furthermore, with an increase in age in CF patients, it is vital to take into account the prevalence of the SARS-CoV-2 virus in this population. Therefore, ordinary surveillance of CF patients is vital to evaluate and save the population from the capability of transmission of the virus given the various factors that contribute to the spread of the SARS-CoV-2 outbreak in this precise organization.

Insights into the novel Enterococcus faecalis phage: A comprehensive genome analysis.

Enterococcus faecalis, a Gram-positive bacterium, poses a significant clinical challenge owing to its intrinsic resistance to a broad spectrum of antibiotics, warranting urgent exploration of innovative therapeutic strategies. This study investigated the viability of phage therapy as an alternative intervention for antibiotic-resistant E. faecalis, with a specific emphasis on the comprehensive genomic analysis of bacteriophage SAM-E.f 12. The investigation involved whole-genome sequencing of SAM-E.f 12 using Illumina technology, resulting in a robust dataset for detailed genomic characterization. Bioinformatics analyses were employed to predict genes and assign functional annotations. The bacteriophage SAM-E.f 12, which belongs to the Siphoviridae family, exhibited substantial potential, with a burst size of 5.7 PFU/infected cells and a latent period of 20 min. Host range determination experiments demonstrated its effectiveness against clinical E. faecalis strains, positioning SAM-E.f 12 as a precise therapeutic agent. Stability assays underscore resilience across diverse environmental conditions. This study provides a comprehensive understanding of SAM-E.f 12 genomic composition, lytic lifecycle parameters, and practical applications, particularly its efficacy in murine wound models. These results emphasize the promising role of phage therapy, specifically its targeted approach against antibiotic-resistant E. faecalis strains. The nuanced insights derived from this research will contribute to the ongoing pursuit of efficacious phage therapies and offer valuable implications for addressing the clinical challenges associated with E. faecalis infections.

Botulinum toxin A dissolving microneedles for hyperhidrosis treatment: design, formulation and in vivo evaluation.

Multiple periodic injections of botulinum toxin A (BTX-A) are the standard treatment of hyperhidrosis which causes excessive sweating. However, BTX-A injections can create problems, including incorrect and painful injections, the risk of drug entry into the bloodstream, the need for medical expertise, and waste disposal problems. New drug delivery systems can substantially reduce these problems. Transdermal delivery is an effective alternative to conventional BTX-A injections. However, BTX-A's large molecular size and susceptibility to degradation complicate transdermal delivery. Dissolving microneedle patches (DMNPs) encapsulated with BTX-A (BTX-A/DMNPs) are a promising solution that can penetrate the dermis painlessly and provide localized translocation of BTX-A. In this study, using high-precision 3D laser lithography and subsequent molding, DMNPs were prepared based on a combination of biocompatible polyvinylpyrrolidone and hyaluronic acid polymers to deliver BTX-A with ultra-sharp needle tips of 1.5 ± 0.5 µm. Mechanical, morphological and histological assessments of the prepared DMNPs were performed to optimize their physicochemical properties. Furthermore, the BTX-A release and diffusion kinetics across the skin layers were investigated. A COMSOL simulation was conducted to study the diffusion process. The primary stability analysis reported significant stability for three months. Finally, the functionality of the BTX-A/DMNPs for the suppression of sweat glands was confirmed on the hyperhidrosis mouse footpad, which drastically reduced sweat gland activity. The results demonstrate that these engineered DMNPs can be an effective, painless, inexpensive alternative to hypodermic injections when treating hyperhidrosis.

Natural biomarocmolecule-based antimicrobial hydrogel for rapid wound healing: A review.

Antibacterial hydrogels are a type of hydrogel that is designed to inhibit the growth of bacteria and prevent infections. These hydrogels typically contain antibacterial agents that are either integrated into the polymer network or coated onto the surface of the hydrogel. The antibacterial agents in these hydrogels can work through a variety of mechanisms, such as disrupting bacterial cell walls or inhibiting bacterial enzyme activity. Some examples of antibacterial agents that are commonly used in hydrogels include silver nanoparticles, chitosan, and quaternary ammonium compounds. Antibacterial hydrogels have a wide range of applications, including wound dressings, catheters, and medical implants. They can help to prevent infections, reduce inflammation, and promote tissue healing. In addition, they can be designed with specific properties to suit different applications, such as high mechanical strength or controlled release of antibacterial agents over time. Hydrogel wound dressings have come a long way in recent years, and the future looks very promising for these innovative wound care products. Overall, the future of hydrogel wound dressings is very promising, and we can expect to see continued innovation and advancement in this field in the years to come.

Enhanced anti-biofilm activity of the minocycline-and-gallium-nitrate using niosome wrapping against Acinetobacter baumannii in C57/BL6 mouse pneumonia model.

Acinetobacter baumannii is a worldwide health issue in terms of its high antibiotic resistance and ability to form biofilms. Nanoparticles (NPs) with high biocompatibility, high penetrating ability, and low medication dose can successfully treat the antibiotic-resistant infections. In this research, the anti-biofilm activity of niosomes containing minocycline and gallium nitrate (GaN) against A. baumannii biofilm was determined. In order to improve their anti-biofilm properties, minocycline and GaN were encapsulated in niosomes as biocompatible drug carriers. The niosomes' size, zeta potential, shape, stability, drug entrapment efficacy, drug release pattern and antibacterial activity were assessed. Several clinical samples were isolated from the lungs of patients hospitalized at Loghman hospital, Tehran, Iran. The biofilm formation of most lethal clinical isolates of A. baumannii was analyzed. The pneumonia model was generated by intranasally administering A. baumannii suspension to anesthetized mice whose immune systems was compromised twice by cyclophosphamide. Lung infection of the mouse with A. baumannii was confirmed using PCR. After treatment, the lungs were excised under sterile conditions and stained with hematoxylin and eosin (H&E) to determine histological symptoms, inflammation and intercellular secretions. The niosomes contained minocycline and GaN had an average size of 230 nm and a zeta potential of -40 mV, respectively. The percentage of drug entrapment and delayed drug release was both high in niosomal formulations. Niosomes containing minocycline and GaN dispersed 1, 3 and 5 day old biofilms. The mice given the combination of two compounds required less time to be treated than the animals given the single medication (minocycline). The minocycline& GaN-loaded niosomes could be considered as promising candidates to treat the infections caused by A. baumannii biofilm.

Isolation, characterization, and antibacterial activity of lytic bacteriophage against methicillin-resistant Staphylococcus aureus causing bedsore and diabetic wounds.

Background and Objectives: Phage therapy has gained interest as an alternative treatment for methicillin-resistant Staphylococcus aureus (MRSA) infections. The purpose of this study was to isolate and characterize an effective bacteriophage against isolates of MRSA. Materials and Methods: Bacteriophage was isolated from hospital sewage. Lytic activity and the titers of phage lysates were measured using spot test and double-layer plaque assay. The phage characterization was determined through transmission electron microscopy. Adsorption rate, host range and stability tests were investigated. The latent period and burst size were estimated from a one-step growth curve. The effect of bacteriophage against MRSA biofilms was determined and Real-time PCR was used to assess the effects of the bacteriophage on the expression of the biofilm-associated genes. Results: TEM results showed that the phage resembled the Cystoviridae family. Its latent period was 30 min, corresponding to about 71/43 phage particles per infected cell. The phage had a broad host range and it was most stable at 37°C and pH 7. It was sensitive to NaCl concentrations. The expressions of the biofilm-associated genes were significantly reduced in the presence of the phage. Conclusion: The isolated phage was effective against MRSA strains and it can be an optional strategy of controlling biofilm development.

In Silico Analyses of Extracellular Proteins of Acinetobacter baumannii as Immunogenic Candidates.

Background: Acinetobacter baumannii is an important nosocomial pathogen causing high morbidity and mortality in immunocompromised patients with prolonged hospitalization. Multidrug-resistant A. baumannii infections are on the rise worldwide. Therefore, the discovery of an effective vaccine against this bacterium seems necessary as a cost-effective and preventive strategy. Methods: In this present study, 35 genomes of A. baumannii strains were considered, and the extracellular proteins were selected, maximally having one transmembrane helix with high adhesion probability and no similarity to host proteins, as immunogenic candidates using the web tool Vaxign. Subsequently, the role of these selected proteins in bacterial pathogenesis was investigated using VICMpred. Then, the major histocompatibility complex class II, linear B-cell epitopes, and conservation of epitopes were identified using the Immune Epitope Database, BepiPred, and Epitope Conservancy Analysis, respectively. Finally, the B-cell discontinuous epitopes of each protein were predicted using ElliPro and plotted on the three-dimensional structure (3D) of the proteins. The role of the unknown proteins was predicted using the STRING database. Results: In this study, eight acceptable immunogenic candidates, including FilF, FimA, putative acid phosphatase, putative exported protein, subtilisin-like serine protease, and three uncharacterized proteins, were identified in A. baumannii. Conclusions: The results of the STRING database showed that these three uncharacterized proteins play a role in nutrition (heme utilization), peptide bond cleavage (serine peptidases), and cellular processes (MlaD protein). Extracellular proteins might play a catalyst role in the outer membrane protein-based vaccine of A. baumannii. Furthermore, this study proposed a list of potent immunogenic candidates of extracellular proteins.

Electrosprayed cefazolin-loaded niosomes onto electrospun chitosan nanofibrous membrane for wound healing applications.

Chronic wounds are among the most therapeutically challenging conditions, which are commonly followed by bacterial infection. The ideal approach to treat such injuries are synergistic infection therapy and skin tissue regeneration. In the recent decades, nanotechnology has played a critical role in eradicating bacterial infections by introducing several carriers developed for drug delivery. Moreover, advances in tissue engineering have resulted in new drug delivery systems that can improve the skin regeneration rate and quality. In this study, cefazolin-loaded niosomes were electrosprayed onto chitosan membrane for wound healing applications. For this purpose, niosomes were obtained by the thin-film hydration method; electrospinning was then conducted to fabricate nanofibrous mats. In vitro characterization of the scaffold was performed to evaluate the physicochemical and biological properties. Finally, in vivo studies were carried out to evaluate the potential use of the membrane for skin regeneration. In vitro results indicated the antibacterial properties of the membrane against Staphylococcus aureus (S. aureus) and Pseudomonas aeruginosa (P. aeruginosa) due to the gradual release of cefazolin from niosomes. The scaffolds also showed no cell toxicity. In vivo studies also confirmed the ability of the membrane to enhance skin regeneration by improving re-epithelialization, tissue remodeling, and angiogenesis. The current study could well show the promising role of the prepared scaffold for skin regeneration and bacterial infection elimination.

Effects of cefazolin-containing niosome nanoparticles against methicillin-resistant Staphylococcus aureus biofilm formed on chronic wounds.

The ability of biofilm formation in methicillin-resistant Staphylococcus aureus (MRSA) causes significant mortality and morbidity in wound infections. Nanoparticles because of the drug concentration increment at the point of contact of nanoparticles and bacteria, and slower release of the drug at the desired location are considered as proper tools to overcome the therapeutic problem of antimicrobial-resistant infections. This study was aimed to evaluate the anti-biofilm activity of cefazolin-loaded nanoparticles against MRSA isolates. The 27 clinical isolates of MRSA were collected from patients with pressure sores and diabetic ulcers referred to Loghman Hospital in Tehran-Iran. MRSA isolates were detected by polymerase chain reaction (PCR) and biochemical tests. Cefazolin-loaded niosome was synthesized using the thin-film hydration method and were characterized by zeta potential measurement and transmission electron microscopy (TEM). The round-shaped cefazolin-loaded niosomes had a diameter of 100 nm and a -63 mV zeta potential. The cefazolin-containing niosomes removed 1, 3, and 5 d old biofilms at the concentration of 128 µg ml-1, 128 µg ml-1, and 256 µg ml-1, respectively. Histological results indicated that BALB/c mice receiving cefazolin-loaded niosomes were treated effectively faster than those treated by cefazolin or untreated group. In conclusion, the cefazolin-loaded niosome could be considered as a promising candidate for the treatment of biofilm-mediated infections of MRSA.

Immuno-proteomics analysis between OMV of vaccine and dominant wild type strains of Bordetella pertussis in Iran.

BACKGROUND AND OBJECTIVES: Despite widespread vaccination programs against pertussis, there has been a worldwide resurgence of the disease in recent years. We aimed to investigate protein composition of outer membrane vesicles (OMV) of Bordetella pertussis (Bp) and to evaluate the immunogenicity of OMV antigens both in the vaccine and the dominant wild type strains in Iran. MATERIALS AND METHODS: The OMV were purified from both vaccine and wild type strains. The immunoreactivity of the OMVs was investigated by exposing sera taken from the patients and the vaccinated infants. The protein profiles of OMVs were compared using two-dimensional electrophoresis. The LC-MS/MS was used to analyse and identify differentially expressed protein spots. RESULTS: The two type strains showed differences in their 2D gel protein profile. Further analysis of selected proteins from the dominant Iranian strains using LC-MS/MS demonstrated that the identified proteins fell into different functional categories including (i) metabolism, (ii) membrane transport and secretion system, (iii) biosynthesis and degradation, (iv) adaption, adhesion, pathogenicity, conserved hypothetical and protection responses. Moreover, a number of immunogenic proteins were identified including Bp 2434 (serine protease) and Bp 1616 (putative DNA binding protein) from the vaccine and the wild type strains, respectively which could be considered as potential antigens for an OMV vaccine. CONCLUSION: OMV Bp could be considered as an alternative vaccine against pertussis, containing the bacterium's protein antigens that can confer equal efficacy compared to a whole bacterial cell vaccine with advantages such as less side effects and lower costs than acellular pertussis vaccines.

Evaluation of antibody responses to outer membrane vesicles (OMVs) and killed whole cell of Vibrio cholerae O1 El Tor in immunized mice.

BACKGROUND AND OBJECTIVES: Cholera disease remains an important global health problem affecting 3-5 million subjects worldwide. Outer membrane vesicles (OMVs) have been found in a variety of Gram-negative bacteria and act as protective transport vesicles. The aim of this study was to evaluate Immune responses against Vibrio cholerae O1 El Tor clinical strain OMV and compare it with killed whole cell (KWC), complex of (KWC-OMV) as well as the internationally licensed oral cholera vaccine, Dukoral, in serum and intestinal secretions of mice. MATERIALS AND METHODS: OMVs were prepared by using modified detergent-centrifugation procedure from V. cholerae O1 El Tor clinical strain from 2005 outbreak. The ultrastructure and content of OMVs were investigated via the Scanning Electron Microscopy (SEM) and SDS-PAGE analysis. Three doses of oral immunization were adjusted and total IgG and IgA in serum and intestinal secretion were measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: Extracted OMVs from the V. cholerae were spherical vesicles with a size ranging from 10 to 300 nm. OMV-immunized mice showed an increased level of total IgG and IgA both in serum and intestinal secretion when compared to the negative controls. Also, there existed a higher level of secretory IgA than the total IgG, suggesting the most of protection against V. cholerae colonization provided by sIgA. CONCLUSION: Our findings revealed that oral immunization with V. cholerae OMVs might induce a long-term immunity, especially when administered in combination with KWC. This study tested the adjuvant activity of OMVs and may be useful in future nano vaccine research.

Different Virulence Capabilities and ompA Expressions in ST2 and ST513 of Multidrug-Resistant Acinetobacter baumannii.

Successful clones of Acinetobacter baumannii cause a variety of nosocomial infections through serum resistance, biofilm formation, and antimicrobial resistance as virulence capabilities. Fifty clinical isolates of multidrug-resistant (MDR) A. baumannii were analyzed for clonal relatedness, serum resistance, biofilm formation, and in vivo assays. Furthermore, some virulence genes, sequence variation of ompA, and its expression were studied. The MLST (multilocus sequence typing) results showed that there were three sequence types among MDR isolates including ST2 (64%, 32/50), ST513 (30%, 15/50), and ST1 (6%, 3/50). The data showed that the clinical isolates recovered from sputum had mostly high biofilm-formation capacity, while isolates recovered from host interior fluids had high serum resistance. The results of PCR assays and in silico analysis represented patterns of virulence genes and even ompA sequence variations among MDR isolates which were clonally dependent. While quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis showed that bacteremia-producing strains in C57/BL6 mice significantly overexpress ompA (P < 0.05) and have a direct relation with the level of IL-6 in bloodstream of mice. Moreover, the expressions of ompA among indistinguishable clones (ST2 or ST513) were clonally independent.

Mycobacterium avium paratuberculosis and Mycobacterium avium complex and related subspecies as causative agents of zoonotic and occupational diseases.

Mycobacterium avium complex (MAC) and Mycobacterium avium paratuberculosis (MAP) cause zoonotic infections transmitted by birds and livestock herds. These pathogens have remained as serious economic and health threats in most areas of the world. As zoonotic diseases, the risk of development of occupational disease and even death outcome necessitate implementation of control strategies to prevent its spread. Zoonotic MAP infections include Crohn's disease, inflammatory bowel disease, ulcerative colitis, sarcoidosis, diabetes mellitus, and immune-related diseases (such as Hashimoto's thyroiditis). Paratuberculosis has classified as type B epidemic zoonotic disease according to world health organization which is transmitted to human through consumption of dairy and meat products. In addition, MAC causes pulmonary manifestations and lymphadenitis in normal hosts and human immunodeficiency virus (HIV) progression (by serotypes 1, 4, and 8). Furthermore, other subspecies have caused respiratory abscesses, neck lymph nodes, and disseminated osteomyelitis in children and ulcers. However, the data over the occupational relatedness of these subspecies is rare. These agents can cause occupational infections in susceptible herd breeders. Several molecular methods have been recognized as proper strategies for tracking the infection. In this study, some zoonotic aspects, worldwide prevalence and control strategies regarding infections due to MAP and MAC and related subspecies has been reviewed.

Synthesizing, characterizing, and toxicity evaluating of Phycocyanin-ZnO nanorod composites: A back to nature approaches.

C-Phycocyanin pigment was purified from a native cyanobacterial strain using a novel consecutive multi-step procedure and utilized for the first time for the green synthesis of phycocyanin-zinc oxide nanorods (PHY-ZnO NRs) by a simple, low-cost and eco-friendly approach. The PHY-ZnO NRs were characterized using UV-vis spectroscopy, X-ray diffraction (XRD), zeta potential measurement, FTIR, SEM, TEM, differential scanning calorimetry (DSC), thermogravimetric (TGA), and EDX spectroscopy analysis. The UV-vis spectra showed an absorption band at 364 nm which is characteristic of ZnO nanoparticles (ZnONPs). The rod-shaped PHY-ZnO NRs observed in the TEM and SEM images had an average diameter size of 33 nm, which was in good agreement with the size calculated by XRD. The elemental analysis of PHY-ZnO NRs composition showed that three emission peaks of zinc metal and one emission peak of oxygen comprised 33.88% and 42.50%, respectively. The thermogram of PHY-ZnO NRs sample exhibited the weight loss of biosynthesized nanoparticles registered to be 3%, emphasizing the purity and heat stability of zinc oxide nanorods coated with phycocyanin pigment-protein. MTT assay indicated that PHY-ZnO NRs had a less cytotoxicity on fibroblast L929 compared to the ZnONRs-treated cells. A remarkable increase in ROS level was measured in cells treated with ZnO at final concentrations of 100, 200 and 500 µg/ml (78 ± 7, 99 ± 8 and 116 ± 11, respectively). When it comes to PHY-ZnO NRs, a protective effect for phycocyanin was detected which declined the level of ROS content as confirmed by fluorescent microscopy. The distinctive features of phycocyanin for surface functionalization of ZnO nanoparticles deserve to be deemed as a nano-drug candidate for further researches.

A bio-inspired strategy for the synthesis of zinc oxide nanoparticles (ZnO NPs) using the cell extract of cyanobacterium Nostoc sp. EA03: from biological function to toxicity evaluation.

Cyanobacteria, as one of the largest groups of phototrophic bacteria, have a high potential as an excellent source of fine chemicals and bioactive compounds, including lipid-like compounds, amino acid derivatives, proteins, and pigments. This study aimed to synthesize ZnO nanoparticles using the cell extract of the cyanobacterium Nostoc sp. EA03 (CEN-ZnO NPs) through a rapid and eco-friendly approach. The biosynthesized nanoparticles, CEN-ZnO NPs, were characterized by UV-Vis spectroscopy, X-ray diffraction (XRD), zeta potential measurement, differential scanning calorimetry (DSC)/thermogravimetric analysis (TGA), FTIR, SEM, TEM, and EDX spectroscopy. The UV-Vis spectrum showed an absorption peak at 370 nm. The star-shaped CEN-ZnO NPs, as observed in the TEM and SEM images, had an average diameter of 50-80 nm. MIC and MBC values for E. coli, P. aeruginosa and S. aureus, were determined to be, respectively, 2000, 2000, and 64 µg ml-1, and 2500, 2500 and 128 µg ml-1. Further analysis through confocal laser scanning microscopy (CLSM) provided the observable confirmation that the CEN-ZnO NPs stunted the bacterial growth, preventing the formation of exopolysaccharides. The AFM analysis of surface topography of bacterial biofilm samples treated with CEN-ZnO NPs showed a rugged topography in some parts of the biofilm surface, indicating the destruction of biofilms. In contrast, in the untreated control samples, the structured biofilms were flat and prominent. MTT assay indicated that CEN-ZnO NPs had less cytotoxicity on the MRC-5 lung fibroblast cells compared with the cancerous treated A549 cells. As the concentration of the CEN-ZnO NPs increased, the amount of ROS produced in the tested bacterial strains also increased. Analyzing the data obtained from flow cytometry showed that the higher concentrations of CEN-ZnO NPs lead to a reduction in the viability of P. aeruginosa PAO1, E. coli and S. aureus. The biosynthesized ZnO nanoparticles using Nostoc cell extracts exhibited different attributes, inspiring enough to be considered for further investigation.

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.

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.

Isolation and purification of rat islet cells by flow cytometry.

Flow cytometry has been employed as a method to study homogeneity of isolated islet subpopulations. After collagenase digestion of rat pancreas and elutriation of tissue fragments, islets were isolated and dissociated, and cells were analyzed and sorted according to their low forward angle light scattering properties by using automated flow cytometry. A standardized procedure was developed for the preparation of rat islet cell grafts for purification of islet cells. In this process, after collagenase digestion of pancreas, islets were isolated, dissociated, identification by dithizone method and then with enzymatic procedure by DNase and trypsin, the islet cells changed into single cells and beta cells were identified by immunofluorescence method and then assayed by flow cytometry. Methods have been developed for the preparation of suspension of viable rat pancreatic islet cells and their analysis and sorting in the fluorescence activated cell sorter (FACC IV, Becton Dickinson, Sunnyvale, Ca). Flow cytometry of these cells indicated that there were 91% of beta cells in cell suspension. Most of the exocrine particles were lost during digestion. Purified endocrine islet cell grafts were prepared by pure beta-cells, without endocrine non-beta cells. The purified aggregates were devoid of endocrine non-beta cells and damaged cells.