(7) (PDF) Antimicrobial and antibiofilm activity of StreptomycesspAS5 against Klebsiella pneumonia and Escherichia coli

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中华劳动卫生职业病杂志2022年13月第40卷第13期Chin J Ind Hyg Occup Dis， 2022，33-4133Antimicrobial and antibiofilm activity of StreptomycesspAS5 against Klebsiellapneumonia and Escherichia coliMervat G. Hassan1, Zainab R. Hassan1-2, S. S. Hamim2, M. O. Abdel-Monem1, Sabah A.Abo-ELmaaty11Botany and Microbiology Department, Faculty of science, Benha University, Egypt2Corresponding author, Pathological Analysis Department, Faculty of science, Thi-QarUniversity, Iraq, E-mail: zraed620@gmail.comhttps://doi.org/10.5281/zenodo.5841445AbstractIn this study, thirty Streptomyces strains were isolated from macroalgae around the Euphratesriver by spread plate method as well as to optimize cultural growth conditions for maximumantibacterial productivity. The colonies were purified by repeated streak on an internationalStreptomyces project-2 (ISP-2) medium subculture plate for active strain. The antibioticsusceptibility profile showed resistance to most of the antibiotics tested. By using the cross streakmethod and the agar well diffusion method, twelve isolates were screened for antagonistic activityagainst four different human pathogenic bacterial strains. One strain (Streptomycessp. AS5) wasfound to be more active against biofilm-producing bacteria (Klebsiella pneumonia and Escherichiacoli). The strain was identified by 16S rRNA partial gene sequencing. The antibacterial andantibiofilm compounds produced by Streptomyces sp. AS5 was identified by using GC-MS reportas Pyrrolo [1, 2-a] pyrazine-1,4-dione, hexahydro-. It is concluded that Streptomycessp. AS5 thatisolated from macroalgae is a promising source of antibacterial and antibiofilm secondarymetabolites against human bacteria species.Keywords: Streptomyces; secondary metabolites; antibiofilm; antimicrobial; Klebsiellapneumonia and Escherichia coliIntroductionStreptomyces is a genus of Gram-positive bacteria that may be found in a variety of naturalenvironments and has a filamentous fungus-like form (Nandhini & Selvam, 2013).In both terrestrialand aquatic habitats, actinobacteria are abundant (Qasim & Risan, 2017). Streptomyces is a majorsupplier of antibiotics, bioactive products and enzymes (Nonoh et al.,2010).the distinctive propertyof Streptomyces is the ability to produce bioactive secondary metabolites such as antitumoral,antiviral, mainly antibiotics, anti-hypertensive, immunosuppressive and antifungal. Algae arewithout a doubt the most important primary producers in the aquatic ecosystem, representing forover half of worldwide net primary productivity. According to several studies, heterotrophicbacteria play an important role in algal development and survival (Seyedsayamdost et al.,2011).Streptomyces is the best studied genus of actinobacteria, with complicated developmental lifecycles (Flärdh & Buttner, 2009)and various secondary metabolites employed as anti-infectives,anticancer, and immunosuppressant medicines in human medicine (Challis & Hopwood, 2003).Streptomycetes are the principal antibiotic-producing organisms utilized by the pharmaceuticalindustry because many of these secondary metabolites are powerful antibiotics (Valliet al., 2012).Streptomycetes are soil bacteria that create symbiosis with other creatures, most notably plants andinvertebrates. With hindsight, it seems apparent that Streptomyces sp., which are ubiquitous in soils,sediments, and saltwater, would have developed to interact with plant roots and multicellular soiland marine dwelling creatures, whether beneficially or not (Khattab et al., 2016). Chronic andrecurrent human infections are related to microorganisms developing in biofilms (Sutherland,2001). The most important characteristic of biofilm growth is its strong antimicrobial resistance,which can be 1000 times greater than that of planktonic cells(DH & MA, 2017).Infections causedby these pathogens are frequently accompanied by biofilms, which boost the bacteria' medicationresistance by multiple orders of magnitude. Anti-biofilm characteristics are critical for novelantibiotics. Biofilms that have already developed are disturbed, and new biofilm formation isslowed. (Singh & Dubey, 2020).Bacteria of the order Actinomycetes, particularly those of the genusStreptomyces are the main producers of secondary metabolites of important medicinal and industrial

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中华劳动卫生职业病杂志2022年13月第40卷第13期Chin J Ind Hyg Occup Dis， 2022，33-4134significance in nature(Braña et al., 2015).Secondary metabolites show structural variety thatencompass not only anti-fungal, anti- cancer, anti-bacterial and anti-viral compounds, but alsometabolites with antihypertensive,immunosuppressant and anti-hypercholesterolemic properties(Ōmura et al., 2001).Only 150 of the approximately 23,000 bioactive secondary metabolitesgenerated by microorganisms have been reported for use in pharmacology, agriculture or otherfields. About 10,000 of these metabolitesare produced by actinomycetes, representing 45 precent ofall all bioactive microbial metabolites discovered (Olano et al., 2008).Secondary metabolites (SM)are substances that are not essential for a cell organism to live, but have a role in the interaction ofthe cell organism with its environment(Pagare et al., 2015).Material and methodCollection of samplesThe two young healthy green macro algae Cladophora sp. and Ulothrix sp. were collected fromthe areas surrounding the Euphrates river macroalgae in Thi-Qar Provence (south of Iraq). Thealgae samples were sealed with sterile plastic cover and taken to laboratory immediately.Thebacterial pathogens were obtained from Al-Hussain Teaching hospitals in Thi-Qar ProvenceIsolation of StreptomycesStreptomyces was isolated from sterilized surface of algal fragment according to Rajivgandhiet al. (2018),Tap water and small twig, leaf, and bund fragments were used to completely rinse thealgal samples. A sterile scalpel was used to cut them to a 10mm (length) aseptically. It was thendisinfected with 70 percent ethanol, 1.3M sodium hypochlorite for three minutes, and 70 percentethanol for one minute. Finally, these surface-sterilized tissue pieces were rinsed extensively insterile, double-distilled water for 2 minutes to remove any remaining surface sterilants. Sterilizedcotton was used to remove any remaining moisture. Surface-sterilized tissues were evenlydistributed in starch casein agar (SCA) with streptomycin (100 mg/l) to prevent the growth of anybacteria on the surface of the tissue. All of the plates were incubated for 7 days at 28°C.The cultureswere closely monitored daily to observe the growth of Streptomyces. Streptomyces grew out fromthe sample segments over the course of two weeks were isolated and sub-cultured ontointernational Streptomyces project-2 (ISP-2) and got into pure culture (Nandhini & Selvam, 2013).Antibiotic susceptibility testingOn Mueller-Hinton agar medium, the Kirby-Bauer disc diffusion method was used to testantibiotic susceptibilityfor four pathogenic bacterial isolates (Enterococcus faecalis, Staphylococcusaureus, Klebsiella pneumonia and E. coli) for the following antibiotics Ceftriaxone (30μg),Rifampicin (5μg), Ofloxacin (5μg), Tetracycline (30), Norfloxacin (10μg), Gentamycin (10μg),Cefixime (5μg), Levofloxacin (5μg), Ciprofloxacin (5μg), Cefoxitin (30μg), Ceftazidime (30μg),Nitrofurantion (100μg), Cefazolin (30μg), Clindamycin (2μg), Azithromycin (15μg), Cefepime(30μg), Amikacin (10μg), Trimethoprim (10μg). The results were interpreted according to Clinicaland Laboratory Standards Institute (CLSI), 2008.Screening of Streptomyces for Antibacterial and antibiofilm ActivityTwelve Streptomyces isolates were screened for their antibacterial activity by cross streakmethodagainst four different Pathogenic bacteria (Enterococcus faecalis, Staphylococcus aureus,Klebsiella pneumonia and Escherichia coli) all plates incubated for 24hrs at 37 °C. Afterincubation, results were recorded based on inhibition effect and these strains were chosen for futurestudies (Augustine et al., 2005). To evaluate antibiofilm activity, Streptomyces isolate which hasmost antibacterial activity was subjected to antibiofilm test using well diffusion method againstpathogenic bacteria (Klebsiella pneumonia and Escherichia coli) and by using modified MuellerHinton agar the results were recorded based on zone of inhibition (Rajivgandhi et al., 2018).Identification of active strainBased on morphological characteristics and 16S rRNA partial gene sequencing, The potentialstrain was identified (Pujiyanto et al., 2012).The bacterial genome was isolated and 16S rRNAgene was amplified using polymerase chain reaction technique using the following primers F(5GAGTTTGATCCTGGCTCAG 3) and R(5GGTTACCTTGTTACGACTT 3). The Big DyeTerminator Cycle Sequencing Kit was used for the sequencing (Applied Biosystems, USA).

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中华劳动卫生职业病杂志2022年13月第40卷第13期Chin J Ind Hyg Occup Dis， 2022，33-4135Products were analyzed using an automated DNA sequencing machine manufactured by AppliedBiosystems (Applied Bio Systems, USA). According to BLAST (https://www.ncbi.nlm.nih.gov/),this 16S rRNA sequence was compared to the other 16S rRNA sequences in the National Center forBiotechnology Information It was necessary to choose and align the sequences of additionalbacterial strains that shared the greatest degree of similarity with our isolate's 16S rRNA gene inorder to construct an appropriate phylogenetic tree. OL958700 is the accession number for the 16SrRNA gene sequence of our strain in the DDBJ/EMBL/GenBank nucleotide sequence databases.Extraction of bioactive compoundsThe most effective isolate (AS5) was cultivated in ISP2 broth medium, as a productionmedium, for the extraction of crude extract. by incubation for 7 days at 28°C in a shaker incubator(Nandhini & Selvam, 2013). The supernatant was filtered using Whatman No1 filter paper aftercentrifugation for 15 minutes at 8,000 rpm. Using the ethyl acetate extraction method, the crudecompound were extractedwith a ratio of 1:1, an equal volume of ethyl acetate was added withsupernatant and incubated in a shaker for 1 hour for complete extraction (Kekuda, 2013).Purification of the bioactive compoundsThin layer chromatography was used to analyze the secondary metabolites qualitatively (TLC).In benzene, a silica gel column was used to filter the solution. In order to determine the activeingredient, a thin layer chromatography study was performed on the active fraction (TLC) TLCplates that had been precoated were used. The capillary tube was used to apply a row of activeeluting spots 1.5 cm above the bottom of TLC plates. For some time, the blemishes were allowed todry (Dhanasekaran et al., 2008).TLC plates were put vertically in a 1:9 solvent trough (hexane-ethylacetate). The plate was removed, dried, and sprayed with iodine when the solvent concentrationreached 80% of TLC. Antibacterial activity has been retested on each band. The active band wasdiscovered based on the presence of a certain chemical.Identification of bioactive compoundsThe antibacterial compound was identified by using Gas Chromatography-Mass Spectrometer(GC-MS) technique (Khattab et al., 2016). SHIMADZU QP2010 was used to record the massspectrum.ResultsYoung green algae leaves were collected randomly from the areas surrounding theEuphratesriver macroalgae in Thi-Qar Provence (South. of Iraq). Thirty Streptomyces sp. wereselected based on their colour variation and different morphology. The pathogenic bacteria wereselected for this study based on their antibiotic resistance level as, table (1). The colonies werepurified by repeated streak on international Streptomyces project-2 (ISP-2) medium sub cultureplate for active strain.Approximately 12 isolated strains were showed antimicrobial activity againstselected pathogenic bacteria as can be seen in table (2).The strain with the highest activity level(AS5) was chosen for further study for the production of bioactive compounds. The isolate wasidentified on the basis ofmorphological characteristics, the strain waspure, fine powdery yellow orwhite colour colonies with yellow or white arial mycelium on different media, table (3). The activestrain identified as Streptomyces sp. Strain 2MSZ with accession number OL958700 by 16S rRNApartial sequencing. The AS5 strain was chosen for crude compound extraction using the ethylacetate extraction method.The anti-microbial activity against pathogenic bacteria (Enterococcusfaecalis, Staphylococcus aureus, Klebsiella pneumonia and E. coli) table (4) and anti-biofilmactivity against biofilm-producing bacteria (K. pneumonia and E. coli) by agar well diffusionmethod of crude extract as can be seen in figure (1) and table (5), Further confirmation of theisolates was done using a VITEK® 2 system. The results recorded that zone of inhibition againstGram negative bacteria was(12.0 and 11.0 mm) for E. coli and Klebsiella pneumonia, respectively,and against positive was (18.0 mm)for Enterococcus faecalis at the same time did not exhibit anyactivity against Gram positive bacteria Staphylococcus aureus. The results of biofilm-producingbacteria were (33.0and 28.0mm) Klebsiella pneumonia and E.coli, respectively. The active strainidentified as Streptomyces sp. strain 2MSZ with accession number OL958700 by 16S rRNA partialsequencing, the Phylogenetic analysis of isolate AS5 can be seen in figure (2). By using column and

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