Objective: The increase of multidrug resistance in bacteria has increased the efforts in search of alternative methods. The aim of the present study was to isolate and characterize the lytic phages and assess their lytic activity against a number of gram-negative bacteria. Materials and Methods: The phages and their respective hosts were isolated from wastewater collected from the municipal sewer system of Trabzon, Turkey. The lytic activities of phage were determined using the agar spot test. The identification and antibiotic susceptibility of host bacteria were determined using matrix-assisted laser desorption ionization time-of-flight mass spectrometry and Phoenix 100, respectively. The phages were characterized morphologically using transmission electron microscopy. One of the phages, Enteroc21, which has a broad-host-range, was further characterized by genome restriction endonuclease analysis and burst size. Results: Two phages infected strains of four different species, nine phages were able to infect 2-4 strains belonging to one or two species, and three phages showed lytic activity against only the hosts from which they were isolated. All phages belonged to the Siphoviridae, Myoviridae, and Podoviridae family based on trans- mission electron microscopy morphology. The Enteroc21 had more than 100 kb genome size and a burst size of 180 per infected cell. Most of the host strains were resistant to ampicillin, amoxicillin–clavulanic acid, and in particular, Achromobacter xylosoxidans TRAX 13 was multidrug-resistant showing resistance to cefepime, aztreonam, gentamicin, netilmicin, and ciprofloxacin. Conclusion: This study showed that the isolated phages have the potential to be used in phage therapy against various bacterial infections, including multidrug-resistant bacteria. Cite this article as: Khorshidtalab M, Durukan İ, Tufekci EF, Nas SS, Abdurrahman MA, Kiliç AO. Isolation and characterization of lytic bacteriophages from wastewater with phage therapy potentials against gram-negative bacteria. Eurasian J Med. 2022;54(2):157-164.

The extended-spectrum β-lactamase (ESBL)-producing bacteria have been isolated at increasing frequency worldwide. Expression of ESBL is often associated with multidrug resistance and dissemination by resistance plasmids. During a two-month period in 2000, 133 clinical isolates of enterobacterial strains were randomly collected from outpatients and inpatients at a university hospital in Turkey. The ESBL producing strains were determined by double-disk synergy (DDS) testing. Twenty ESBL producing strains (15%) including Escherichia coli (n = 9), Klebsiella pneumoniae (n = 7), Klebsiella oxytoca (n = 2) and Enterobacter aerogenes (n = 2) were detected and further analyzed for their resistance transfer features, plasmid profile and nature of the resistance genes. Plasmid transfer assays were performed using broth mating techniques. TEM- and SHV- genes were analyzed by polymerase chain reaction (PCR) and hybridization using specific probes. EcoRI restriction enzyme analyses of R plasmids were used in the detection of epidemic plasmids. Fourteen plasmid profiles (A, B1, B2, C1, and C2 to L) were obtained with EcoRI restriction enzyme analysis. Most of these plasmids were detected to carry both TEM- and SHV-derived genes by PCR, and confirmed by localizing each gene by hybridization assay. Epidemiological evidence indicated that there was an apparent horizontal dissemination of conjugative R plasmids among multidrug-resistant enterobacterial genera and species in this hospital.

A rapid micropropagation protocol was designed to produce Calamintha sylvatica plantlets by using nodal segments as explants for the shoot formation. 6-BA favored the highest shoot formation and biomass yield, whilst kinetin was found superior for the highest shoot length (38.97 ± 2.85 mm) and node numbers (2.89 ± 0.63). Rosmarinic acid was detected as major phenolic acid, ranging from 7.59 mg/100 g to 81.44 mg/100 g. Hexane extracts from natural and in vitro propagated plantlets showed activity only against Staphylococcus aureus ATCC 25923 with MIC values at 6.25 and 3.33 m/mL, respectively while in the latter case, extracts from natural plantlets exerted higher cytotoxic activity than those of micropropagated ones (IC50 values were 83 µg/mL and 98 µg/mL on HeLa cells, respectively). C. sylvatica showed high micropropagation performance and produced remarkable amount of rosmarinic acid in vitro as well as antimicrobial and cytotoxic effect. ********* In press - Online First. Article has been peer reviewed, accepted for publication and published online without pagination. It will receive pagination when the issue will be ready for publishing as a complete number (Volume 47, Issue 4, 2019). The article is searchable and citable by Digital Object Identifier (DOI). DOI link will become active after the article will be included in the complete issue. *********

The rapid rise of resistance causes existing antibiotics to become dysfunctional. Therefore, search for new antimicrobial active ingredients has increased in recent years. In this study, flower extracts of Castanea sativa were examined for antimicrobial and anti-quorum sensing aspects. The antimicrobial properties of methanol, ethyl acetate, ethanol and hexane extracts of C. sativa against some gram-positive and gram-negative bacterial species, as well as yeasts (Candida albicans and Candida parapsilosis) were investigated by the agar well diffusion method. The minimum inhibition concentration (MIC) and minimum bactericidal concentrations (MBC) of C. sativa extracts were also determined. Chromobacterium violaceum ATCC 12472, C. violaceum 35352, C. violaceum VIR07 and C. violaceum CV026 indicator strains were used for determination of the quorum sensing inhibitions, and the Pseudomonas aeruginosa PAO1 strain was used for the swarming tests. Additionally, biofilm inhibition was detected by the spectrophotometric method using the P. aeruginosa PAO1 strain. Methanol, ethanol and ethyl acetate extracts of C. sativa was found to have high antibacterial and antifungal effects, while the methanol extract also had anti-quorum sensing, anti-swarming and biofilm inhibition effects, but no activity was found in the n-hexane extract. To the best of our knowledge this is the first report revealed that methanol extract obtained from C. sativa flowers induced anti-quorum sensing activities mainly inhibited the violacein production, swarming and biofilm formation. The present investigation provided evidence that the C. sativa flower extract maybe a potential source of antimicrobial agents. Therefore, much attention should be paid to C. sativa flower content, which could be used with high efficacy against microorganisms.