Evaluating the antibacterial effect of meropenem-loaded chitosan/sodium tripolyphosphate (TPP) nanoparticles on Acinetobacter baumannii isolated from hospitalized patients.

BACKGROUND: Acinetobacter baumannii is a health threat due to its antibiotic resistance. Herein, antibiotic susceptibility and its association with the Toxin-antitoxin (TA) system genes in A. baumannii clinical isolates from Iran were investigated. Next, we prepared meropenem-loaded chitosan nanoparticles (MP-CS) and investigated their antibacterial effects against meropenem-susceptible bacterial isolates. METHODS: Out of 240 clinical specimens, 60 A. baumannii isolates were assessed. Antibiotic resistance of the isolates against conventional antibiotics was determined alongside investigating the presence of three TA system genes (mazEF, relBE, and higBA). Chitosan nanoparticles were characterized in terms of size, zeta potential, encapsulation efficiency, and meropenem release activity. Their antibacterial effects were assessed using the well diffusion method, minimum inhibitory concentration (MIC), and colony-forming unit (CFU) counting. Their cytotoxic effects and biocompatibility index were determined via the MTT, LDH, and ROS formation assays. RESULTS: Ampicillin, ceftazidime, and colistin were the least effective, and amikacin and tobramycin were the most effective antibiotics. Out of the 60 isolates, 10 (16.7%), 5 (8.3%), and 45 (75%) were multidrug-resistant (MDR), extensively drug-resistant (XDR), and pandrug-resistant (PDR), respectively. TA system genes had no significant effect on antibiotic resistance. MP-CS nanoparticles demonstrated an average size of 191.5 and zeta potential of 27.3 mV alongside a maximum encapsulation efficiency of 88.32% and release rate of 69.57%. MP-CS nanoparticles mediated similar antibacterial effects, as compared with free meropenem, against the A. baumannii isolates with significantly lower levels of meropenem. MP-CS nanoparticles remarkably prevented A549 and NCI-H292 cell infection by the A. baumannii isolates alongside demonstrating a favorable biocompatibility index. CONCLUSION: Antibiotic-loaded nanoparticles should be further designed and investigated to increase their antibacterial effect against A. baumannii and assess their safety and applicability in vivo settings.

Possible Molecular Targeting of Biofilm-Associated Genes by Nano-Ag in Candida albicans.

The treatment of candidiasis infections is hindered by the presence of biofilms. Here, we report the biofilm-associated genes as potential molecular targets by silver nanoparticles (nano-Ag) in Candida albicans. Nano-Ag was biosynthesized using Bacillus licheniformis, Bacillus cereus, and Fusarium oxysporum. The physicochemical properties of the microbial-synthesized of nano-Ag are widely characterized by visual observation, ultraviolet-visible spectroscopy, scanning electron microscopy, X-ray diffraction spectroscopy, and Fourier transform infrared spectroscopy. Characterization results revealed the formation of nano-Ag. Antiplanktonic cells and antibiofilm activities of nano-Ag were also demonstrated by minimum inhibition concentrations (MIC), minimum fungicidal concentration (MFC), MFC/MIC ratio, crystal violet staining, 2,3-bis (2-methoxy-4-nitro-5 sulfophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide (XTT), and microscopic image analysis. We have analyzed the expressions of biofilm-associated genes in C. albicans treated with different concentrations of nano-Ag based on MIC. The expression profile of BCR1, ALS1, ALS3, HWP1, and ECE1 showed downregulated genes involved in these pathways by the treatment with nanoparticles. Negative regulators, TUP1, NRG1, and TOR1, were upregulated by the treatment of nano-Ag. Our study suggests that nano-Ag affects gene expression and may subsequently decrease the pathogenesis of C. albicans by inhibiting biofilm formation. Molecular targeting of biofilm-associated genes involved in biofilm formation by nano-Ag may be an effective treatment strategy for candidiasis infections.

Antibacterial and Anti-Biofilm Activities of Microbial Synthesized Silver and Magnetic Iron Oxide Nanoparticles Against Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a human bacterial pathogen causing devastating diseases and equipped with various virulence factors like biofilm formation. Common antibiotic treatment has limited efficacy for the P. aeruginosa present in biofilms because of the increased resistance. In this study, we focused our attention on the antibacterial and anti-biofilm activities of various microbial synthesized silver (nano-Ag) and magnetic iron oxide (nano-Fe3O4) nanoparticles against clinical isolates of P. aeruginosa that displayed ceftazidime resistance. The nano-Ag and nano-Fe3O4 represented great antibacterial properties. Nano-Ag and nano-Fe3O4 exhibited a reduction in the biofilm formation by P. aeruginosa reference strain as determined by crystal violet and XTT assays and light microscopy method. Among all, nano-Ag-2 and 7 owing to inherent attributes and mechanisms of resistance in the bacterial biofilm, exhibited anti-biofilm efficacy against ceftazidime resistance clinical isolate of P. aeruginosa. Moreover, nano-Ag and nano-Fe3O4 changed the relative expression of biofilm-associated genes, PELA and PSLA in a concentration dependent manner by P. aeruginosa reference strain. As revealed by qRT-PCR, the expression levels of biofilm-associated genes were downregulated in P. aeruginosa biofilms treated with nano-Ag, while selected biofilm-associated genes were low expressed under treated with nano-Fe3O4. Results of the study demonstrate the potential of microbial synthesized nano-Ag-2 and 7 to act as anti-biofilm agents against ceftazidime resistance clinical isolate of P. aeruginosa. Molecular targeting of biofilm-associated genes by nano-Ag and nano-Fe3O4 may be candidate for new therapeutics against P. aeruginosa diseases.

Biofilm-associated genes as potential molecular targets of nano-Fe3O4 in Candida albicans.

BACKGROUND: There are few effective treatments for Candida biofilm-associated infections. The present study demonstrated changes in the expression of biofilm-associated genes in Candida albicans treated with magnetic iron oxide nanoparticles (denoted as nano-Fe3O4). METHODS: Nano-Fe3O4 was biologically synthesized using Bacillus licheniformis, Bacillus cereus, and Fusarium oxysporum. Additionally, the biologically synthesized nano-Fe3O4 was characterized by visual observation; ultraviolet-visible spectroscopy, scanning electron microscopy, X-ray diffraction spectroscopy, and Fourier transform infrared spectroscopy. The biologically synthesized nano-Fe3O4 was tested for growth and biofilm formation in C. albicans. Furthermore, quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR) was used to study the inhibition of biofilm-associated genes in C. albicans treated with nano-Fe3O4. RESULTS: The production of biologically synthesized nano-Fe3O4 was confirmed using extensive characterization methods. The nano-Fe3O4 inhibited growth and biofilm formation. Nano-Fe3O4 exhibited growth inhibition with minimum inhibition concentrations (MICs) of 50 to 200 µg mL-1. The anti-biofilm effects of nano-Fe3O4 were shown by 2,3-bis (2-methoxy-4-nitro-5 sulfophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide (XTT) reduction assay, crystal violet staining, and light field microscopy. The gene expression results showed that the downregulation of BCR1, ALS1, ALS3, HWP1, and ECE1 genes inhibited the biofilm formation in C. albicans. ALS1 reduction was greater than others, with downregulation of 1375.83-, 1178.71-, and 768.47-fold at 2 × MIC, 1 × MIC, and ½ × MIC of nano-Fe3O4, respectively. CONCLUSION: Biofilm-associated genes as potential molecular targets of nano-Fe3O4 in C. albicans may be an effective novel treatment strategy for biofilm-associated infections.

An "olivomycin A" derivative from a sponge-associated Streptomyces sp. strain SP 85.

We isolated an actinobacterium, Streptomyces sp. strain SP 85 from the marine sponge Dysidea avara. Polyphasic identification of the microorganism showed that the strain SP 85 had high 16S rRNA gene similarity (99%) with Streptomyces olivaceus strain NBRC 12805, while some physiological and biochemical differences were observed. A cytotoxic compound, SP 85 was isolated from the active culture extract of the strain SP 85 by bioassay-guided purification over silica gel column chromatography, preparative TLC, and HPLC. The structure elucidation based on the spectroscopic analysis, including UV, ESI-MS, and 13C NMR data revealed that SP 85 compound is an analog of anti-tumor drug, "olivomycin A". The SP 85 compound showed high cytotoxic activity against three human cancer cell lines, including SW480, HepG2, and MCF7 with IC50 values of 16, 93, and 78 nM, respectively. SP 85 exhibited significantly (2-10 times) higher cytotoxicity against the tumor cell lines in comparison with HUVECs as the normal cell line, which also induced apoptosis in the tested cancerous cell line. This is the first report on the production of an "olivomycin A" derivative by a sponge-associated Streptomyces, showing the great potential of sponge-associated actinobacteria in producing cytotoxic natural products.

Molecular detection of virulence factors and biofilm formation in Pseudomonas aeruginosa obtained from different clinical specimens in Bandar Abbas.

BACKGROUND AND OBJECTIVES: Pseudomonas aeruginosa is a ubiquitous opportunistic pathogen. The presence of several virulence factors such as exotoxin and exoenzyme genes and biofilm may contribute to its pathogenicity. The purpose of this study was to investigate the presence of toxA, exoU and exoS, the determination of biofilm production and antimicrobial susceptibility patterns among clinical isolates of P. aeruginosa. MATERIALS AND METHODS: In this study, 75 isolates of P. aeruginosa were recovered from various clinical specimens. Antimicrobial susceptibility pattern of isolates were identified. Virulence genes toxA, exoU and exoS were determined using PCR. The ability of biofilm production was assessed. RESULTS: Antimicrobial susceptibility test showed that 12 strains were resistant to more than 8 antibiotics (17.14%). The most effective antibiotic was colistin as 98.6% of isolates were sensitive. The frequencies of exoU and exoS genes were detected as 36.6% and 55.7%, respectively. In addition, 98.6% of the isolates were biofilm producers. Exotoxin A was detected in sixty-eight isolates (95.7%). CONCLUSION: The findings of this study showed that, the presence of P. aeruginosa exotoxin and exoenzyme genes, particularly, the exoU gene is the most common virulence factors in the bacterial isolates from urine samples. Biofilm is a serious challenge in the treatment of P. aeruginosa infection.

Anti-cancer Parasporin Toxins of New Bacillus thuringiensis Against Human Colon (HCT-116) and Blood (CCRF-CEM) Cancer Cell Lines.

Bacillus thuringiensis is one of the most important microorganisms used against cancer cell lines in latest studies all over the world. This study aims to perform the isolation, molecular identification, and to identify novel B. thuringiensis strains that specifically targeting human cancer cell-killing activities in Iran. A total of 88 B. thuringiensis isolates were recovered from Iran. Upon the treatment of the non-hemolytic crystal proteins by proteinase K, five isolates belonging to three biotypes, thuringiensis, kurstaki and sotto of B. thuringiensis are found to have different cytotoxicity toward HCT-116 and CCRF-CEM cell lines. Digested inclusions of the isolates consisted of one major poly peptide of 34-kDa, as estimated by sodium dodecyl-sulfate polyacrylamide gel electrophoresis. The structure, molecular identification, and functionality of five isolates inclusion proteins have shown to be closely like to parasporin-2 but their size of activated protein is not similar to this parasporin. It is unclear that discovered damaging proteins are parasporin-2. This 34-kD protein exhibited varying degrees of cytocidal activity toward human colon and blood cancer cells and caused cell swelling and the formation of blebs in the surface of the cells or alteration in cytoskeleton. The soil in the humid and temperate climates of Iran is a good reservoir for parasporin producing B. thuringiensis. The isolated B. thuringiensis strains exhibit specific and different cytocidal activities against human colon and blood cancer cells. Parasporin is a novel cytotoxic protein to human cancer cells produced by B. thuringiensis and these toxins appeared to attack an identical target on human cancer cells.

Antibiotic Resistance Patterns and Virulence Determinants of Different SCCmec and Pulsotypes of Staphylococcus Aureus Isolated from a Major Hospital in Ilam, Iran.

AIMS & OBJECTIVES: The aim of this study is to evaluate genetic relatedness, antibiotic resistance pattern, and virulence characteristics of different types of S. aureus isolated from air, surfaces, staff, and patients in a Public hospital in Ilam. METHODS & MATERIALS: A total of 88 of 140 staphylococci identified as S. aureus by conventional and molecular methods were used in this study. Isolate samples were obtained from surfaces, staff, patients, and hospital indoor air. The sampling from staff and surfaces was done through using swab and air by standard pump. Antimicrobial susceptibility testing and presence different resistant and virulence determinants was assessed. Isolates were then typed by pulsed-field gel electrophoresis (PFGE) and SCCmec typing methods. RESULTS: Out of 88isolates, 36 of them (40.9%) were MRSA. Among MRSA isolates, the range of resistance to antibiotic was 0% in vancomycin to 83.3% in gentamycin. The most prevalent resistant genes among gentamicin resistant S. aureus were acc (6')/aph (2")Ia and aph(3")IIIa. The most common erythromycin resistant gene was ermC. Surprisingly, SCCmec types I (30.5%), II (25%)were highly distributed. PFGE analysis showed 33 different pulsotypes. CONCLUSION: This study confirms that different isolates of MSSA and MRSA circulate in Ilam which differ in antimicrobial susceptibility, content of resistance, and virulence determinants.

Analysis of DNA gyrA Gene Mutation in Clinical and Environmental Ciprofloxacin-Resistant Isolates of Non-Tuberculous Mycobacteria Using Molecular Methods.

BACKGROUND: During the past several years, nontuberculous mycobacteria (NTM) have been reported as some of the most important agents of infection in immunocompromised patients. OBJECTIVES: The aim of this study was to evaluate the ciprofloxacin susceptibility of clinical and environmental NTM species isolated from Isfahan province, Iran, using the agar dilution method, and to perform an analysis of gyrA gene-related ciprofloxacin resistance. MATERIALS AND METHODS: A total of 41 clinical and environmental isolates of NTM were identified by conventional and multiplex PCR techniques. The isolates were separated out of water, blood, abscess, and bronchial samples. The susceptibility of the isolates to 1 µg/mL, 2 µg/mL and 4 µg/mL of ciprofloxacin concentrations was determined by the agar dilution method according to CLSI guidelines. A 120-bp area of the gyrA gene was amplified, and PCR-SSCP templates were defined using polyacrylamide gel electrophoresis. The 120-bp of gyrA amplicons with different PCR-SSCP patterns were sequenced. RESULTS: The frequency of the identified isolates was as follows: Mycobacterium fortuitum, 27 cases; M. gordonae, 10 cases; M. smegmatis, one case; M. conceptionense, one case; and M. abscessus, two cases. All isolates except for M. abscessus were sensitive to all three concentrations of ciprofloxacin. The PCR-SSCP pattern of the gyrA gene of resistant M. abscessus isolates showed four different bands. The gyrA sequencing of resistant M. abscessus isolates showed 12 alterations in nucleotides compared to the M. abscessus ATCC 19977 resistant strain; however, the amino acid sequences were similar. CONCLUSIONS: This study demonstrated the specificity and sensitivity of the PCR-SSCP method for finding mutations in the gyrA gene. Due to the sensitivity of most isolates to ciprofloxacin, this antibiotic should be considered an appropriate drug for the treatment of related diseases.

First time isolation of Photobacterium damselae subsp. damselae from Caranx sexfasciatus in Persian Gulf, Iran.

BACKGROUND AND OBJECTIVE: Photobacterium damselae subsp. damselae is a marine pathogenic bacterium which causes disease in marine animals and human. This bacterium mostly found in coastal shallow seawater. So, the aim of this study was isolation and characterization of Photobacterium damselae subsp. damselae from edible fish of Persian Gulf, Bandar Abbas. MATERIAL AND METHODS: Totally 100 fish from different species were evaluated and out of that 5 different types of fish with external symptoms including: Caranx sexfasciatus, Lethrinus olivaceus, Scomberoid tol, Auxis thazard and Liza macrolepis, were collected from Bandar Abbas local fish market in September 2013. The samples were cultured on Marin Agar 2216 and Thiosulfate Citrate Bile salts Sucrose Agar media and incubated at 25°C for 48 hrs. Then the isolates were characterized using biochemical (API 20 NE system) and molecular techniques. In addition, antibiotic susceptibility, presence of poly ß hydroxy butyrate and hemolysis activity of isolates were evaluated. RESULTS AND CONCLUSION: Entirely, 30 Gram negative bacterial colonies were isolated from the selected fish. Among the isolates, two suspected colonies were identified as Photobacterium damselae from Caranx sexfasciatus with API 20NE biochemical test. This results confirmed by 16s rRNA sequencing method. Both isolates showed α hemolytic with existence of ß hydroxyl butyrate. Furthermore, the isolates were susceptible to ciprofloxacin, chloramphenicol and nalidixic acid. CONCLUSION: Overall, the study indicated first time isolation of this bacterium from one type of fish caught from Persian Gulf, which warns us to pay more attention to fishery in this geographical area.

Plasmid mediated antibiotic resistance in isolated bacteria from burned patients.

BACKGROUND: Nowadays, the treatment of burned patients is difficult because of the high frequency of infection with antibiotic resistance bacteria. OBJECTIVES: This study was conducted to evaluate the level of antibiotic resistance in Gram-negative bacteria and its relation with the existence of plasmid. MATERIALS AND METHODS: The samples were collected from two hundred twenty hospitalized burned patients in Isfahan burn hospital during a three-month period (March 2012 to June 2012). The samples were isolated and the Gram-negative bacteria were identified using phenotypic method and API 20E System. Antibiotic susceptibility and plasmid profile were determined by standard Agar disc diffusion and plasmid spin column extraction methods. RESULTS: Totally 117 Gram-negative bacteria were isolated, the most common were Pseudomonas aerugionsa (37.6%), P. fluorescens (25.6%), Acinetobacter baumanii (20/5%) and Klebsiella pneumoniae (7.6%), respectively. The isolates showed high frequency of antibiotic resistance against ceftazidime and co-amoxiclave (100%) and low frequency of antibiotic resistance against amikacin with (70%).The results indicated that 60% of the isolates harboured plasmid. On the other hand, the patients infected with A. baumanii and P. aeruginosa were cured (with 60% frequency) whereas, those infected with P. fluorescens were not cured. Hence, probably antibiotic resistance markers of A. baumanii and P. aeruginosa are plasmid mediated; however, P. fluorescens is chromosomally mediated. CONCLUSIONS: Based on our findings, P. aerugionsa is a major causative agent of wound infections and amikacin could be considered as a more effective antibiotic for treatment of the burned patients.

The effect of temperature on antibacterial activity of biosynthesized silver nanoparticles.

The purpose of this study was the evaluation of two different temperatures on antibacterial activity of the biosynthesized silver nanoparticles. 38 silver nanoparticles-producing bacteria were isolated from soil and identified. Biosynthesis of silver nanoparticles by these bacteria was verified through visible light spectrophotometry. Two strains were relatively active for production of silver nanoparticles. These strains were subjected for molecular identification and recognized as Bacillus sp. and Acinetobacter schindleri. In the present study, the effect of temperatures was evaluated on structure and antimicrobial properties of the silver nanoparrticles by transmission electron microscopy (TEM), X-ray diffraction (XRD) analysis and antimicrobial Agar well diffusion methods. The silver nanoparticles showed antibacterial activity against all the pathogenic bacteria; however, this property was lost after treatment of the silver nanoparticles by high temperatures (100 and 300 °C). TEM images showed that the average sizes of heated silver nanoparticles were >100 nm. However, these were <100 nm for non-heated silver nanoparticles. Although, XRD patterns showed the crystalline structure of heated silver nanoparticles, their antibacterial activities were less. This was possible because of the sizes and accordingly less penetration of the particles into the bacterial cells. In addition, elimination of the capping agents by heat might be considered another reason.

Chemotactic behavior of Campylobacter spp. in function of different temperatures (37 °C and 42 °C).

The chemotactic behaviour of Campylobacter strains was determined in the presence of different amino acids at two temperatures (37 °C and 42 °C). Two strains of catalase positive (Campylobacter jejuni) and negative (Campylobacter sputurum) Campylobacter were isolated from river water in Tonekabon, Iran and identified by phenotyping and 16srRNA Gene sequencing methods. Chemotactic responses of the isolates were assessed toward a variety of amino acids viz., L-cystine, L-asparagine, L-histidine, L-aspartic acid, L-serine, L-phenylalanine, L-leucine and L-tryptophan by disc and capillary methods at two temperatures: 37 °C and 42 °C. C. jejuni showed positive chemotactic response towards L-cystine,L-tryptophan, L-phenylalanine, - L-leucine, L-asparagine and L-Serine at both, 37 °C and 42 °C however, it was greater at 37 °C. C. sputurum showed negative or weak response towards all of the amino acids. In addition, C. jejuni illustrated strong chemotactic response to L-asparagine follow by L-serine and weak chemotaxis response to L-phenylalanine and L-cysteine at 37 °C. Overall, C. jejuni showed relatively strong chemotactic response to some amino acids, likewise it was greater at 37 °C. Hence, the human body temperature (37 °C) in compared to avian body temperature (42 °C) probably promotes chemotactic response of C. jejuni, which it might be a reason for causing disease in human being compared to avian.