Improving the Inhibitory Effect of Phages against Pseudomonas aeruginosa Isolated from a Burn Patient Using a Combination of Phages and Antibiotics.

Antibiotic resistance causes around 700,000 deaths a year worldwide. Without immediate action, we are fast approaching a post-antibiotic era in which common infections can result in death. Pseudomonas aeruginosa is the leading cause of nosocomial infection and is also one of the three bacterial pathogens in the WHO list of priority bacteria for developing new antibiotics against. A viable alternative to antibiotics is to use phages, which are bacterial viruses. Yet, the isolation of phages that efficiently kill their target bacteria has proven difficult. Using a combination of phages and antibiotics might increase treatment efficacy and prevent the development of resistance against phages and/or antibiotics, as evidenced by previous studies. Here, in vitro populations of a Pseudomonas aeruginosa strain isolated from a burn patient were treated with a single phage, a mixture of two phages (used simultaneously and sequentially), and the combination of phages and antibiotics (at sub-minimum inhibitory concentration (MIC) and MIC levels). In addition, we tested the stability of these phages at different temperatures, pH values, and in two burn ointments. Our results show that the two-phages-one-antibiotic combination had the highest killing efficiency against the P. aeruginosa strain. The phages tested showed low stability at high temperatures, acidic pH values, and in the two ointments. This work provides additional support for the potential of using combinations of phage-antibiotic cocktails at sub-MIC levels for the treatment of multidrug-resistant P. aeruginosa infections.

Evaluation of cinnamon extract effects on clbB gene expression and biofilm formation in Escherichia coli strains isolated from colon cancer patients.

BACKGROUND: Colon cancer is one of the most common malignancies and the fourth leading cause of cancer-related mortality in the world. Colibactin, which is synthesized by the pks genomic island of E. coli interfere with the eukaryotic cell cycle. Cinnamon has an antimicrobial effect and considered as a colon cancer-preventing agent. The aim of the study was to evaluate the effects of cinnamon extract and cinnamaldehyde on clbB gene expression and biofilm formation in clinical isolates of E. coli. METHODS: Thirty E. coli carrying pks gene were isolated from the colon cancer patients, inflammatory bowel disease and healthy subjects. Antibiotic susceptibility was evaluated by disk diffusion method and the minimum inhibitory concentration of cinnamon essential oil and cinnamaldehyde by microdilution broth method. In vitro biofilm formation of E.coli isolates was monitored using a microtiter plate method. The presence of clbB, clbA and clbQ genes in E.coli isolates were evaluated by PCR. The effect of cinnamaldehyde and cinnamon essential oil on clbB gene expression was evaluated by Real-Time PCR. RESULTS: The highest antibiotic resistance was obtained with 94.4% for ticarcillin-clavulanic acid, azithromycin, amoxicillin, and amikacin. The MIC for all clinical isolates was 32 µl/ml of cinnamon essential oil and the MIC of cinnamaldehyde was between 0.00002 to 0.03 µl/ml. After exposure of isolates to cinnamon extract and cinnamaldehyde, 40 and 13.3% were weakly biofilm producers, respectively. The frequencies of clbB, clbA, and clbQ genes were 23.3, 23.3, and 26.7%, respectively. The expression of clbB gene in the presence of the Sub-MIC concentration of cinnamon essential oil and cinnamaldehyde was decreased in 8 isolates compared to untreated isolates (p-value < 0.05). CONCLUSIONS: The antibacterial activity of cinnamaldehyde and cinnamon essential oil allows the use of these herbal compounds for treatment or supplements in infections caused by E. coli and in patients with suspected colorectal cancer.

Detection of adeABC efllux pump encoding genes and antimicrobial effect of Mentha longifolia and Menthol on MICs of imipenem and ciprofloxacin in clinical isolates of Acinetobacter baumannii.

BACKGROUND: Acinetobacter baumannii is an opportunistic pathogen that causes nosocomial infections especially in patients in intensive care units (ICUs). Accordingly, the aim of our study was to detection of adeABC efllux pump encoding genes and antimicrobial effect of the essential oil of Mentha longifolia and Menthol on the minimum inhibitory concentration (MIC) of imipenem and ciprofloxacin in clinical isolates of A. baumannii. METHODS: A total of 75 clinical isolates of A. baumannii were collected. The presence of efflux pump genes was detected by polymerase chain reaction (PCR). The minimum inhibitory concentration (MIC) of the essential oil of Mentha longifolia and Menthol and their combined effect with antibiotics were measured by microbroth dilution method and fractional inhibitory concentration (FIC) index. RESULTS: The frequency of adeA, adeB, and adeC genes in clinical isolates of A. baumannii were 86.7, 90.7, and 92%, respectively. When the essential oil of Mentha longifolia was combined with ciprofloxacin and imipenem, MICs decreased 4- and 8-fold, respectively. In the combination of menthol with imipenem, the resistance to imipenem was reduced from 0- to 16-fold in 90% (63/70) of the isolates. CONCLUSION: The presence of efflux pump genes in more than 90% of A. baumannii isolates indicates its potential role in inducing imipenem- and ciprofloxacin-resistance in this bacterium. Menthol has an antimicrobial effect as an active ingredient in Mentha longifolia. In the future, the combination of medicinal plants with antibiotics can be used as a complement in treating diseases caused by drug-resistant bacteria such as A. baumannii infections.

The bactericidal effect of lysostaphin coupled with liposomal vancomycin as a dual combating system applied directly on methicillin-resistant Staphylococcus aureus infected skin wounds in mice.

BACKGROUND AND AIM: Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most common causes of surgical infection, and its resistance to numerous conventional antibiotics makes treatment difficult. Although vancomycin is often an effective agent for the initial therapy of MRSA, clinical failure sometimes occurs. Therefore, there is an urgent need to develop better therapies. Here, we prepared some vancomycin-loaded nanoliposomes coupled with anti-staphylococcal protein (lysostaphin) and evaluated their in vitro and in vivo efficacy as a topical MRSA therapy. METHODS: Vancomycin was encapsulated in liposomes, and the coupling of lysostaphin with the surface of liposomes was carried out through cyanuric functional groups. The bactericidal efficacies and a full characterization were evaluated. To define different nanoliposomal-bacterium interactions and their bactericidal effect, flow cytometry was employed. Finally, in vivo, the topical antibacterial activity of each formulation was measured against surgical wound MRSA infection in a mouse model. RESULTS: High encapsulation and conjugation efficiency were achieved for all formulations. All the formulations showed a significant reduction in bacterial counts (p<0.05). The targeted liposomes more effectively suppress bacterial infection in vitro and in vivo relative to equivalent doses of untargeted vancomycin liposome. The flow cytometry results confirmed liposome-bacterium interactions, which increased during the incubation time. The maximum binding rate and the bactericidal effect were significantly higher in targeted liposomes (p<0.05) compared with control liposomes. CONCLUSION: Our data suggest a novel nano-vehicle (lysostaphin-conjugated coupled liposomal vancomycin) which could be used as a great topical antimicrobial construct for treatment of MRSA skin infections.

The effect of antimicrobial photodynamic therapy on the expression of biofilm associated genes in Staphylococcus aureus strains isolated from wound infections in burn patients.

PURPOSE: Burn patients are particularly susceptible to microbial infection. Staphylococcus aureus causes burn wound, impetigo and cellulitis. Although sub-lethal antimicrobial photodynamic therapy (aPDT) would not result in microorganism killing, it can considerably influence microbial virulence factor. METHODS: Twelve methicillin-resistant S. aureus (MRSA) and methicillin-sensitive S. aureus (MSSA) isolated from burns patients. To determine the sub-lethal dose of aPDT, 12 clinical isolates of S. aureus photosensitized with 100 µg ml -1 toluidine blue O (TBO) and irradiated by light emitting diode (LED) with a wavelength of 630 ± 10 nm and energy densities of 52.0, 104.1, and 156.2 J/cm2, then bacterial viability was measured. The effects of sub-lethal aPDT on the expression levels of ica ABCD and ica R genes were assessed by quantitative Real-time PCR (qRT-PCR) method. RESULT: Fifty and 100 µg ml-1 of TBO significantly reduced the mean cell survival in the MRSA (2.5 - 3 log10) and MSSA (2.75-3.1 log10) isolates. The average expression levels of icaA, ica B, ica C, and ica D in the MRSA and MSSA isolates were decreased by (12, 14, 11, and 9) and (13, 14.5, 12, and 9.5) fold change, respectively (P < 0.05). However, the expression of ica R gene was decreased by 6 and 8 folds change in MRSA and MSSA, respectively. CONCLUSION: The potential of TBO-mediated aPDT could reduce the expression of ica ABCD as important genes involved in biofilm formation and ica R gene as a repressor of the ica operon. Therefore, the use of aPDT agents as a complementary therapy in wound infections of burn patients is recommended.