Isolation and Identification of Effective Probiotics on Drug-Resistant Acinetobacter baumannii Strains and Their Biofilms.

Introduction: This study aimed to identify, assess, and isolate strong lactobacilli demonstrating broad antibacterial and anti-biofilm activity against drug-resistant strains of Acinetobacter baumannii. Additionally, the mechanism of inhibition of these organisms was to be determined. Methods: Over a 6-month period (from December 2021 to June 2022), 53 clinical A. baumannii strains were collected from clinical samples. Twenty probiotic strains were isolated from local dairy products. Antibacterial activity of Lactobacillus strains' cell-free supernatant (CFS) was identified using the agar well diffusion method and the microbroth dilution test. Anti-biofilm effect was performed by the microtiter plate assay. The MTT assay was also used to look into the probiotics' cytotoxic effects on the L929 fibroblast cell line. Results: During the 6-month period, 53 clinical A. baumannii strains were obtained and identified. Out of 20 lactobacillus strains, the CFS of a lactobacillus strain (named L9) showed an inhibitory effect against all A. baumannii strains. Using the broth microdilution method, it was shown that the minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of CFS extracts of L9 strains against A. baumannii strains were both » mg/mL. The result of the anti-biofilm showed that the selected probiotic could inhibit biofilm formation. The most common organic acid produced by all Lactobacillus strains, according to the HPLC method, was lactic acid, which was followed by acetic acid. The L929 fibroblast cell line was used in the cytotoxicity assay, which revealed that 100% of the cells in the L929 fibroblast cell line survived treatment with successive doses of CFSs for a full day. Conclusion: The probiotic strain isolated from local yogurt in this study showed potential anti-biofilm and antimicrobial properties against all drug-resistant Acinetobacter isolates. Given the increasing interest in probiotic microorganisms based on their high health benefits, further studies are recommended on the mechanisms of action between probiotics and A. baumannii strains to find new solutions for biological control and treatment of these infections without the use of antibiotics.

Investigating the Antagonistic Effect of Indigenous Probiotics on Carbapenem-Resistant Pseudomonas aeruginosa Strains.

Introduction: With the increase of hospital infections due to the indiscriminate use of antibiotics, multidrug resistance has increased, decreasing the effectiveness of antibiotics against these infections. For this reason, the identification of alternative agents such as probiotics has been considered. The aim of this study was to isolate and identify effective probiotics against carbapenem-resistant Pseudomonas aeruginosa strains. Material and Methods. During a period of eight months, isolates of P. aeruginosa were collected from patients in three hospitals in Isfahan. The presence of metallo-beta-lactamase enzymes was determined by the combination disc test (CDT). The inhibitory and antimicrobial activities of 20 probiotic bacteria isolated from local dairy products against these strains were investigated by agar dilution. Two probiotic strains that showed broader inhibition results were selected, and the values of the lowest inhibitory concentration (MIC) and the lowest lethal concentration (MBC) and their antibiofilm effect were determined using the microtiter plate method. The concentration of organic acids was done by HPLC. Findings. Of the 100 samples isolated and identified, 61 samples (61%) exhibited multiple drug resistance (MDR) and were selected for further investigation. Phenotypic diagnosis of the presence of metallo-beta-lactamase enzymes revealed that 74.5% of the strains were positive. The results showed that these two probiotics killed P. aeruginosa strains after only one hour, and the inhibition mechanism was due to the presence of lactic acid and acetic acid. The antibiofilm effect of these two probiotics was at concentrations of 1/2 and 1/4. Conclusion: The two Lactobacillus isolates had potential antimicrobial and antibiofilm properties against all carbapenem-resistant P. aeruginosa strains, even at thinner dilutions. Considering the broad activity of this strain, it can potentially be used for biocontrol of these strains.

Antibacterial activity of epsilon-poly-l-lysine produced by Stenotrophomonas maltophilia HS4 and Paenibacillus polymyxa HS5, alone and in combination with bacteriophages.

Over the past decades, antibiotic resistance has become a major clinical problem, and searching for new therapeutic strategies seems to be necessary. Using novel natural compounds, antimicrobial peptides, and bacteriophages is the most promising solution. In this study, various cationic metabolite-producer bacteria were isolated from different soil samples. Two isolates were identified as Stenotrophomonas maltophilia HS4 (accession number: MW791428) and Paenibacillus polymyxa HS5 (accession number: MW791430) based on biochemical characteristics and phylogenetic analysis using 16S rRNA gene sequences. The cationic compound in the fermentation broth was precipitated and purified with sodium tetraphenylborate salt. The purified cationic peptide was confirmed to be epsilon-poly-l-lysine by structural and molecular analysis using High-Performance Liquid Chromatography, Sodium dodecyl-sulfate-polyacrylamide gel electrophoresis, and Fourier-transform infrared spectroscopy. The antibacterial activity of epsilon-poly-l-lysine was evaluated against Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922, Enterococcus faecalis ATCC 29212, Serratia marcescens ATCC 13880, and Klebsiella pneumoniae ATCC 13883 by microdilution method. Furthermore, the antibacterial effects of purified epsilon-poly-l-lysine in combination with two long non-contractile tail bacteriophages against vancomycin-resistant Enterococcus faecalis and colistin-resistant Klebsiella pneumoniae were investigated. The results indicated great antibacterial activity of epsilon-poly-l-lysine which was produced by two novel bacteria. The epsilon-poly-l-lysine as a potent cationic antimicrobial peptide is demonstrated to possess great antimicrobial activity against pathogenic and also antibiotic-resistant bacteria.

Investigation of antioxidant and anticancer activities of unsaturated oligo-galacturonic acids produced by pectinase of Streptomyces hydrogenans YAM1.

Pectin, a diverse carbohydrate polymer in plants consists of a core of α-1,4-linked D-galacturonic acid units, includes a vast portion of fruit and agricultural wastes. Using the wastes to produce beneficial compounds is a new approach to control the negative environmental impacts of the accumulated wastes. In the present study, we report a pectinase producing bacterium Streptomyces hydrogenans YAM1 and evaluate antioxidative and anticancer effects of the oligosaccharides obtained from pectin degradation. The production of oligosaccharides due to pectinase activity was detected by thin layer chromatography (TLC) and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Our results revealed that S. hydrogenans YAM1 can degrade pectin to unsaturated pectic oligo-galacturonic acids (POS) with approximately 93% radical scavenging activity in 20 mg/mL which it is more than 50% of the same concentration of pectin. Flow cytometric analysis revealed that MCF-7 cells viability decreased more than 32 and 92% following treatment with 6 and 20 mg/mL POS after 24 h, respectively. It is suggested that pectin degradation by S. hydrogenans YAM1 is not only a new approach to produce highly active compounds from fruit wastes, but also is an effective method to remove fibrous pollutants from different environments.

Molecular investigation of two novel bacteriophages of a facultative methylotroph, Raoultella ornithinolytica: first report of Raoultella phages.

Bacteria of the genus Raoultella are known to inhabit aquatic environments and can be found in medical samples. The pathogenicity of Raoultella ornithinolytica isolates in human has become increasingly important, and several cases of infections have been reported recently. However, there are no reports of isolation of bacteriophages infecting this bacterium. In this study, two novel phages (ISF3 and ISF6) of a methylotrophic Raoultella strain were isolated from sewage. To characterize the isolated phages, morphological features, protein profiles, restriction digestion patterns, and partial genome sequences were studied. Despite morphological differences, electron microscopy revealed that both phages had an icosahedral capsid connected to a contractile tail, suggesting that ISF3 and ISF6 both belong to the family Myoviridae. Partial nucleotide sequences of the ISF3 genome showed 99% to 100% identity to DNA of Klebsiella pneumonia phages KP15, KP27 and BMBT1; however, the restriction digestion profiles of ISF3 genome digested by EcoRI and EcoRV differed from those of Klebsiella phages KP15 and KP27. A partial sequence alignment showed that ISF6 can be classified as a member of a new viral genus due to its significant differences from other previously identified phages. To the best of our knowledge, this is the first report of the isolation and characterization of the specific Raoultella phages that have potential to be used as new pharmaceuticals against R. ornithinolytica.

Comparative genomic analysis of Lactococcus garvieae phage WP-2, a new member of Picovirinae subfamily of Podoviridae.

To date, a few numbers of bacteriophages that infect Lactococcus garvieae have been identified, but their complete genome sequences have not yet been investigated. For the first time, herein, the complete DNA sequence of a new phage of L. garvieae (phage WP-2) is reported and analyzed. The morphological characteristics indicated that the phage had a small isometric head along with a short and non-contractile tail, suggesting that WP-2 belongs to the family Podoviridae. Bioinformatic analysis revealed that phage WP-2 can be classified as a new member of Ahjdlikevirus in the Picovirinae subfamily because it had a small dsDNA of 18,899 bp with 24 open reading frames and a protein-primed DNA polymerase. The phage nucleotide sequence and predicted protein products have been identified to share very limited evidence of homology with complete genome and proteome of other phages. To our knowledge, this is the first Ahjdlikevirus bacteriophage which can infect a member of the Lactococcus genus.

Insights into new bacteriophages of Lactococcus garvieae belonging to the family Podoviridae.

Lactococcus garvieae is an emerging pathogen responsible for lactococcosis, a serious disease in trout aquaculture. The identification of new bacteriophages against L. garvieae strains may be an effective way to fight this disease and to study the pathogen's biology. Three L. garvieae phages, termed WP-1, WWP-2 and SP-2, were isolated from different environments, and their morphological features, genome restriction profiles and structural protein patterns were studied. Random cloning of HindIII-cut fragments was performed, and the fragments were partially sequenced for each phage. Although slight differences were observed by transmission electron microscopy, all of the phages had hexagonal heads and short non-contractile tails and were classified as members of the family Podoviridae. Restriction digestion analysis of the nucleic acids of the different phages revealed that the HindIII and AseI digests produced similar DNA fragment patterns. Additionally, SDS-PAGE analysis indicated that the isolated phages have similar structural proteins. The sequence BLAST results did not show any significant similarity with other previously identified phages. To the best of our knowledge, this study provides the first molecular characterization of L. garvieae phages.

Complete genome sequence of a novel phage, vB_MoxS-ISF9, infecting methylotrophic Microbacterium: first report of a virulent Microbacterium phage.

Here, we report the first genome sequence of a new virulent phage of Microbacterium oxydans, termed vB_MoxS-ISF9, which was isolated from sewage. Transmission electron microscopy showed that the isolated phage, which has a hexagonal head of about 80 nm in diameter and a long non-contractile tail of about 240 nm, belongs to the family Siphoviridae. The vB_MoxS-ISF9 DNA was completely sequenced and found to be 59,254 bp in length, with a G+C content of 62.76% and 120 putative open reading frames (ORFs). The predicted protein products of the ORFs were identified, and their sequences were analyzed. In a comparison with all available phage genomes, vB_MoxS-ISF9 did not show any significant similarity to other previously reported bacteriophages. To the beast of our knowledge, this is the first report of the isolation and complete genomic sequencing of a virulent phage against a member of the genus Microbacterium.

Development of an eco-friendly approach for biogenesis of silver nanoparticles using spores of Bacillus athrophaeus.

The biological synthesis methods have been emerging as a promising new approach for production of nanoparticles due to their simplicity and non-toxicity. In the present study, spores of Bacillus athrophaeus were used to achieve the objective of developing a green synthesis method of silver nanoparticles. Enzyme assay revealed that the spores and their heat inactivated forms (microcapsules) were highly active and their enzymatic contents differed from the vegetative cells. Laccase, glucose oxidase, and alkaline phosphatase activities were detected in the dormant forms, but not in the vegetative cells. Although no nanoparticle was produced by active cells of B. athrophaeus, both spores and microcapsules were efficiently capable of reducing the silver ions (Ag⁺) to elemental silver (Ag°) leading to the formation of nanoparticles from silver nitrate (AgNO3). The presence of biologically synthesized silver nanoparticles was determined by obtaining broad spectra with maximum absorbance at 400 nm in UV-visible spectroscopy. The X-ray diffraction analysis pattern revealed that the nanoscale particles have crystalline nature with various topologies, as confirmed by transmission electron microscopy (TEM). The TEM micrograph showed the nanocrystal structures with dimensions ranging from 5 to 30 nm. Accordingly, the spore mixture could be employed as a factory for detoxification of heavy metals and subsequent production of nanoparticles. This research introduces an environmental friendly and cost effective biotechnological process for the extracellular synthesis of silver nanoparticles using the bacterial spores.