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Determination of Genetic Diversity Among Antibiotic Resistant Morganella Morganii Strains Using RAPD Molecular Marks

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Determination of Genetic Diversity Among Antibiotic Resistant Morganella Morganii Strains Using RAPD Molecular Marks Flyyih et al (2020): Genetic diversity in antibiotic resistant May 2020 Vol. 23 Issue 9 Determination of genetic diversity among antibiotic resistant Morganella morganii strains using RAPD molecular marks 1* 2 3 Najat Mohammed Flyyih , Layla Saleh Abdul-Hassan ,BasimaBasim M. A. , Shrooq Ali Hussein4. 1, 3, 4- Middle technical University, Collage of Health & Medical Technology , Iraq. 2-Al-Furat Al-Awsat Technical University/College of Health and Medical Techniques/Iraq *Corresponding author: Email: [email protected] (Flyyih) ABSTRACT The study was conducted on a total of 250 clinical samples were taken from ear infection patients in Al-Sadder educational medical hospital and Al-Hakeem General hospitals, During the study period from February, 2017 to 12, 2017.This study aimed to determine Morganella morganii by using Random Amplified Polymorphic DNA ( RAPD) techniques in genetic diversity studies.The result of this study from 140(56%) samples were positive results for bacterial growth against 110(44%) samples wereconsidered negative in culture media .The biochemical test was showed that oxidase negative , catalase positive, methyl red, indole and urease positive .The percentage of antibiotic resistance was 90% , 100%, 100%,55%,100%and100% against pefloxacin , Colistin, aztreonam,ticarcillinclavulanic acid, ceftazidime and piperacillinrespectively .The result of phylogenetic tree by using RAPD analysis was similarity (0.87 ) was between (M. morganii-4 and M. morganii-12), followed by (0.80 ) between isolates (M. morganii-4and M. morganii-6 ). RAPD typing considered as a good to situations in which a large number of isolates ,cheaper and easy technique, its used in a wide range of applications, in many space of biology branch. Keywords: Ear infection, Morganella morganii, RAPD-PCR, Phylogenetic Analysis How to cite this article: Flyyih NM, Abdul-Hassan LS, et al (2020): Determination of genetic diversity among antibiotic resistant Morganella morganii strains using RAPD molecular marks, Ann Trop Med & Public Health; 23(S9): SP23920. DOI: Annals of Tropical Medicine & Public Health http://doi.org/10.36295/ASRO.2020.23920 1. Introduction Morganella morganiiis commensal, Gram-negative bacteria, bacilli in shapecolonize in intestine of humans, mammals and reptiles (Falagaset al., 2006). But this microorganism is an opportunistic pathogen present outside the intestinal tract that cause many infections and diseases including urinary tract infections (UTI), pneumonia, sepsis, musculoskeletal infections, wound infections ,central nervous system infections(CNS), pericarditis, endophthalmitis, chorioamnionitis, spontaneous bacterial peritonitis and empyema (Miller, 2012). Annals of Tropical Medicine & Public Health http://doi.org/10.36295/ASRO.2020.23920 Flyyih et al (2020): Genetic diversity in antibiotic resistant May 2020 Vol. 23 Issue 9 A few M. morganii infections are related with a high mortality rate particularly following sepsis , bacteraemia and/or hospital acquired infection have been also recorded , although most of these infections respond to adequate antibiotic drug (Lee and Liu, 2006). 2. Materials and Methodology: Isolation of Microorganisms: Microorganisms were isolated from the Ear infection from Al-Sadder educational medical hospital; Al- Hakeem general hospital.The microorganisms were isolated on MacConkey agar medium. The obtained isolates were recognized by Gram’s staining andbiochemical tests (MacFaddin, 2000). The presence of Morganella morganiiwas specified by the application of Vitec 2 system. These cultures were then used for further investigation like DNA extraction, PCR and RAPD genetic diversity. Bacterial density determination: Bacteria was cultured in 5 mL of LB medium(1% tryptone, 1% NaCl and 0.5% yeast extract) andseveral dilutions were plated on LB medium (LB with 1.5% agar) for estimation the number of colony forming units (CFU).Colony counts were made after 24 hours ofincubation at 28ºC. PCR Assay: The total genomic DNA purification kit is providedfor isolation of DNA from G -ve bacteria by extraction kit from (Promega company/USA). Gel electrophoresiswas used for DNA detection and viewed by UV-transilluminator. DNA Concentration was estimatedin spectrophotometer device by measuring its optical density(o.d.) at260 nm (Extinction coefficient of dsDNA is 50 μg/ml at260 nm) the purity of DNA solution is identified by the ratioof OD260/OD280 in a range about 1.8± 0.2 forpure DNA from contamination. RAPD analysis was performed in25μl composed of 10μl of master mix 2x, 5μl of DNA samples, 5μl of primer and 5μl of Deionized water.The thermal cycling instrument profile was as follows: 4 min.initial denaturation at 92°C, 40 cycles of 1 min. at92°C, 2 min. at 37°C, 3 min. at 72°C, followed by afinal extension at 72°C for 3 min. PCR productsin 1% concentration agarose were analyzed by gels stained with ethidium bromide stain. The primers used were OPZ04 AGGCTGTGCT 3 , OP U-17 ACCTGGGGAGand OPX-18 (5'-TGG CAA GGC A-3'), supplied byOperon Technologies (Alameda, CA, EUA). Data scoring and Dendrogram analysis: Amplicon resulted from PCR amplification were electrophoresis using 1% agarose and the fragments viewed by using UVI B and software (version 12.14) and Data were analyzed considering the presence (1) or absence (0) of bands for every isolate by Past3 program (Hammer et al.,2001) . also determent percentage polymorphic bands, polymorphic, Primer efficiency and Discrimination power were calculated for each primer using the following equations as described by Graham and Mcnicol, (1995). Results and Dissections: Source of Bacterial Isolates Through the study time from February, 2017 to 12, 2017, a total of 250 clinical samples were taken from ear infections. However, 140(56%) of samples positive results for bacterial growth against 110(44%) samples showed no colony of the bacterial when growing on MacConkey agar. Annals of Tropical Medicine & Public Health http://doi.org/10.36295/ASRO.2020.23920 Flyyih et al (2020): Genetic diversity in antibiotic resistant May 2020 Vol. 23 Issue 9 Identification of Bacterial Isolates: The isolates taken from clinical samples were identified firstlybycolonialmorphology, microscopic appearance and biochemical tests. From these isolated cultures, the identification of M.morganiiwas depended on the cultural morphology. Since the colonies of M. morganiiwere appeared on blood agar and pale in color when grown on the MacConkey agar medium, indicated that M. morganiiis cannot able to ferment lactose sugar , Microscopic examination showedthat M. morganiiwas Gram-negative bacilli with small rods in shape. The results of biochemical tests that showed in table (1) were considered as a complementary of the primary identification of M. morganii isolates. The isolates showed the general characteristics that considered oxidasenegative and catalase positive. They were motile, methyl red, urease and indolepositive. The isolates were capable to ferment glucose on Kligler iron agar (KIA), it produced (Alkaline) red color on slant and yellow-acid with gas in the bottom , but not H2S; as well as a negative result to vogues-proskauer and citrateutilization. Table (1): Biochemical tests results of Morganella morganii suspected isolates No. Test Result No. Test Result 1- Oxidase - 6- Methyl Red + 2- Catalase + 7- Vogus-Proskuar - 3- Indole + 8- Motility + 4- Citrate Utilization - 9- H2S - 5- Urease + 10- TSI K/A The last identification was carried out with the automated VITEK-2 Compact system using GN-ID cards which consists of 47 biochemical tests and one negative control well. The M. morganii clinical isolates have been estimated to be 12 (8.57%) out of the total number of growth gram-negative bacteria on MacConkey agar (n = 140). The results showed that all M. morganii isolates were considered as multidrug resistant (MDR) , The results was shown in Table ( 2) ,revealed that the resistant rate of isolates to pefloxacin and Colistin were recorded in 90% and 100% respectively. The resistance percentage rate55% isolates were resistant to ticarcillin, clavulanic acid and aztreonam appeared in 100%. The study also revealed a high rates of resistant to ceftazidime was detected in 100%. All isolates were resistant to piperacillin100%. Table (2) percentage of antibiotic sensitivity results. ANTIBIOTIC R I S 1 Colistin 100% - - 2 Pefloxacin; 90% - 10% 3 Imipenem 7% 8% 85% 4 Aztreonam 100% - - 5 Ticarcillin-clavulanic acid 55% 10% 35% 6 Ceftazidime 100% - - 7 Piperacillin 100% - - Randomly amplified polymorphic DNA (RAPD) analysis was performed using 3 unrestricted primers (OPZ-04, OPU-17 and OPX-18) and PCR technique. Annals of Tropical Medicine & Public Health http://doi.org/10.36295/ASRO.2020.23920 Flyyih et al (2020): Genetic diversity in antibiotic resistant May 2020 Vol. 23 Issue 9 As shown in Table (3), The Total bands number of 4 primers (741). OPZ-04 primer is the most competent whereas the number of bands (64), Primer efficiency (39.5%) and Discrimination power (41.26). Table (3): Information about three primers OPZ-04 OPU-17 OPX-18 Total Mean Total number of bands 64 44 54 162 54 bands 4-7 3-5 3-7 Molecular weight Range of (300-1150) (250-800) bp (250-900) bands bp bp Polymorphic bands 52 32 42 126 42 Percentage of Polymorphic 81.25 72.72 77.77 bands Primer efficiency % 39.5 27.16 33.3 Discrimination power 41.26 25.39 33.3 By using 3 primers the last results in a wide variety in genetic material (DNA) of M. morganii isolates seen as a various number of amplified bands and their molecular
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