Molecular Analysis of Gram-Negative Bacterial Strains in Patients from Koya City

Implications for Diagnostic Approaches

  • Mohammed A. Hama–Ali Department of Medical Microbiology, Faculty of Science and Health, Koya University, Koya KOY45, Kurdistan Region - F.R. Iraq https://orcid.org/0000-0002-1210-0290
  • Ayad H. Hasan Department of Medical Microbiology, Faculty of Science and Health, Koya University, Koya KOY45, Kurdistan Region - F.R. Iraq, Department of Biomedical Sciences, College of Applied Sciences, Cihan University-Erbil, Kurdistan Region, Iraq https://orcid.org/0000-0001-7280-2254
  • Mohammed I. Jameel Department of Medical Microbiology, Faculty of Science and Health, Koya University, Koya KOY45, Kurdistan Region - F.R. Iraq, Department of Biomedical Sciences, College of Applied Sciences, Cihan University-Erbil, Kurdistan Region, Iraq https://orcid.org/0000-0002-2964-2520
  • Hewa A. Muhammed Department of Medical Microbiology, Faculty of Science and Health, Koya University, Koya KOY45, Kurdistan Region - F.R. Iraq https://orcid.org/0009-0009-2376-7494
  • Sada N. khazaye Department of Medical Microbiology, Faculty of Science and Health, Koya University, Koya KOY45, Kurdistan Region - F.R. Iraq https://orcid.org/0009-0009-8735-2044
  • Marwa B. Muhammed Amin Department of Medical Microbiology, Faculty of Science and Health, Koya University, Koya KOY45, Kurdistan Region - F.R. Iraq https://orcid.org/0009-0008-1186-1347
  • Khoshy A. Muhammedsaid Department of Medical Microbiology, Faculty of Science and Health, Koya University, Koya KOY45, Kurdistan Region - F.R. Iraq https://orcid.org/0009-0004-4210-6281
DOI: https://doi.org/10.24086/cuesj.v8n2y2024.pp110-115
Keywords: Gram-negative bacteria, 16SrRNA gene, Sequencing, molecular identification

Abstract

This study addresses the critical issue of Gram-negative bacteria (GNB) in patients from Koya City, acknowledging their significant resistance to antibiotics and the consequent threat to public health. A total of 14 samples were collected from diverse patient demographics and sources, including urine, wounds, vaginal swabs, seminal fluid, and throat swabs. The samples were subjected to analysis through a multifaceted approach involving culturing and molecular identification. Following the isolation of bacterial samples, the 16SrRNA gene was targeted using polymerase chain reaction (PCR). Subsequently, the obtained PCR products underwent Sanger sequencing, providing a detailed analysis of the nucleotide sequences. The resulting sequences were then compared against existing prokaryotic DNA sequences in the BLAST database to determine the identity of the isolated bacteria. Out of the 14 samples, 13 yielded sequences of good quality, while one exhibited a noise sequence. The sequences were further aligned, and 11 samples demonstrated a 100% identity with previously identified bacteria such as Escherichia coli (100 %), Klebsiella pneumoniae (100 %), and Shigella flexneri (100 %) Notably, two samples displayed a >99% identity with Escherichia coli (99.5%), highlighting the diversity within the bacterial strains. This study sheds light on the prevalence and identity of Gram-negative bacteria in Koya City patients. The findings underscore the necessity of molecular techniques, such as PCR and sequencing, in clinical diagnostics. The diverse bacterial strains identified highlight the complexity of bacterial infections, emphasizing the importance of targeted approaches in addressing antibiotic resistance.

Downloads

Download data is not yet available.

Author Biographies

Mohammed A. Hama–Ali, Department of Medical Microbiology, Faculty of Science and Health, Koya University, Koya KOY45, Kurdistan Region - F.R. Iraq

Mohammed A. Aziz Hama-Ali is Assistant Lecturer at the Department of Medical Microbiology, Faculty of Science and Health, Koya University. He got the B.Sc. degree in Medical Microbiology and the M.Sc. degree in Molecular Microbiology. His research interests are in Microbiome, Molecular Microbiology, and antimicrobial agents. 

Ayad H. Hasan, Department of Medical Microbiology, Faculty of Science and Health, Koya University, Koya KOY45, Kurdistan Region - F.R. Iraq, Department of Biomedical Sciences, College of Applied Sciences, Cihan University-Erbil, Kurdistan Region, Iraq

Ayad H. Hassan is  is a professor of Molecular Microbiology. He obtained his BSc degree in Biology and MSc degree in Biotechnology from Mosul University, and later obtained his PhD in Molecular Microbiology from Leeds University in the UK. His research interests focus on secondary metabolism in Streptomyces, Metagenomics, Bioinformatics, Natural Products, Drug Discovery, Gut Microbiota, and Plastic Degradation.

Mohammed I. Jameel, Department of Medical Microbiology, Faculty of Science and Health, Koya University, Koya KOY45, Kurdistan Region - F.R. Iraq, Department of Biomedical Sciences, College of Applied Sciences, Cihan University-Erbil, Kurdistan Region, Iraq

Mohammed I. Jameel is Lecturer at the Department of Medical Microbiology, Faculty of Science and Health, Koya University. He got the B.Sc. degree in Biotechnology and the M.Sc. degree in Bioinformatics. His research interests are in Microarray, Whole genome sequencing data analysis, Metagenomics and R programming.

Hewa A. Muhammed, Department of Medical Microbiology, Faculty of Science and Health, Koya University, Koya KOY45, Kurdistan Region - F.R. Iraq

Hewa A. Mohammed got the B.Sc. degree in Medical Microbiology at Department of Medical Microbiology, Faculty of Science and Health, Koya University. currently working as a laboratory assistant at Koya University and studying M.Sc in the same field. His research interests are in, Microbiology, Molecular Microbiology, and Anti-Fungal agents. 

Sada N. khazaye, Department of Medical Microbiology, Faculty of Science and Health, Koya University, Koya KOY45, Kurdistan Region - F.R. Iraq

Sada N. khazye got the B.Sc. degree in Medical Microbiology at Department of Medical Microbiology, Faculty of Science and Health, Koya University. Currently working in Shaheed Dr. Khaleed Hospital at Koya City. Her research interests are in, Microbiology and Molecular Microbiology. 

Marwa B. Muhammed Amin, Department of Medical Microbiology, Faculty of Science and Health, Koya University, Koya KOY45, Kurdistan Region - F.R. Iraq

Marwa B. MuhammedAmin got the B.Sc. degree in Medical Microbiology at Department of Medical Microbiology, Faculty of Science and Health, Koya University. Worked in Shaheed Dr. Khaled Hospital at koya city and also at Koya Central Laboratory. Her research interests are in, Molecular Microbiology, and Antimicrobial agents. 

Khoshy A. Muhammedsaid, Department of Medical Microbiology, Faculty of Science and Health, Koya University, Koya KOY45, Kurdistan Region - F.R. Iraq

Khoshy A. Muhammed Said got the B.Sc. degree in Medical Microbiology at Department of Medical Microbiology, Faculty of Science and Health, Koya University. Worked in Shaheed Dr. Khaled Hospital at koya city and also at Koya Central Laboratory. Her research interests are in, Microbiology, Molecular Microbiology, and antimicrobial agents. 

References

D. M. Livermore. As the era of untreatable infections arrived? Journal of Antimicrobial Chemotherapy, vol. 64, no. Supplement 1, pp. i29-i36, 2009.

J. Oliveira and W.C. Reygaert. Gram-negative bacteria. In: StatPearls. StatPearls Publishing, Treasure Island, FL, 2023.

M. Tumbarello, T. Spanu, M. Sanguinetti, R. Citton, E. Montuori, F. Leone, G. Fadda and R. Cauda. Bloodstream infections caused by extended-spectrum-β-lactamase-producing Klebsiella pneumoniae: Risk factors, molecular epidemiology, and clinical outcome. Antimicrobial Agents and Chemotherapy, vol. 50, no. 2, pp. 498-504, 2006.

E. Wenzler, D. R. Fraidenburg, T. Scardina and L. H. Danziger. Inhaled antibiotics for gram-negative respiratory infections. Clinical Microbiology Reviews, vol. 29, no. 3, pp. 581-632, 2016.

R. M. AL-Hilali and H. D. K. Al-Mozan. Isolation and identification of gram negative bacteria that cause diarrhea. University of Thi-Qar Journal of Science, vol. 10, no. 1, pp. 175-180, 2023.

J. Zhuo, B. Liang, H. Zhang, Y. Chi and Y. Cai. An overview of gram-negative bacteria with difficult-to-treat resistance: Definition, prevalence, and treatment options. Expert Review of Anti-infective Therapy, vol. 21, no. 11, pp. 1203-1212, 2023.

J. Brosius, M. L. Palmer, P. J. Kennedy and H. F. Noller. Complete nucleotide sequence of a 16S ribosomal RNA gene from Escherichia coli. Proceedings of the National Academy of Sciences, vol. 75, no. 10, pp. 4801-4805, 1978.

S. H. Yoon, S. M. Ha, S. Kwon, J. Lim, Y. Kim, H. Seo and J. Chun. Introducing EzBioCloud: A taxonomically united database of 16S rRNA gene sequences and whole-genome assemblies. International Journal of Systematic and Evolutionary Microbiology, vol. 67, no. 5, pp. 1613-1617, 2017.

P. Y. Lee, J. Costumbrado, C. Y. Hsu and Y. H. Kim. Agarose gel electrophoresis for the separation of DNA fragments. Journal of Visualized Experiments, vol. 3923, no. 62, p. 3923, 2012.

M. A. Hama-Ali and A. H. Hasan. Profiling of bacterial species from Covid-19 faecal samples in Kurdistan Region-Iraq. Science Journal of University of Zakho, vol. 11, no. 1, pp. 11-15, 2023.

P. Ioannou, E. Tsagkaraki, A. Athanasaki, C. Tsioutis and A. Gikas. Gram-negative bacteria as emerging pathogens affecting mortality in skin and soft tissue infections. Hippokratia, vol. 22, no. 1, pp. 23-28, 2018.

J. M. Sahuquillo-Arce, R. Menéndez, R. Méndez, I. Amara‐ Elori, R. Zalacain, A. Capelastegui, J. Aspa, L. Borderías, J. J. Martín‐Villasclaras, S. Bello, I. Alfageme, F. R. de Castro, J. Rello, L. Molinos, J. Ruiz-Manzano and A. Torres. Age-related risk factors for bacterial aetiology in community-acquired pneumonia. Respirology, vol. 21, no. 8, pp. 1472- 479, 2016.

S. Morris and E. Cerceo. Trends, epidemiology, and management of multi-drug resistant gram-negative bacterial infections in the hospitalized setting. Antibiotics (Basel), vol. 9, no. 4, p. 196, 2020.

E. K. Costello, C. L. Lauber, M. Hamady, N. Fierer, J. I. Gordon and R. Knight. Bacterial community variation in human body habitats across space and time. Science, vol. 326, no. 5960, pp. 1694-1697, 2009.

L. M. Abayasekara, J. Perera, V. Chandrasekharan, V. S. Gnanam, N. A. Udunuwara, D. S. Liyanage, N. E. Bulathsinhala, S. Adikary, J. V. S. Aluthmuhandiram, C. S. Thanaseelan, D. P. Tharmakulasingam, T. Karunakaran and J. Ilango. Detection of bacterial pathogens from clinical specimens using conventional microbial culture and 16S metagenomics: A comparative study. BMC Infectious Diseases, vol. 17, no. 1, p. 631, 2017.

J. G. Cappuccino and C. Welsh. Microbiology: A Laboratory Manual. 12th ed. Pearson, New York, pp. 151-217, 2019.

E. A. Makky, R. D. Thanoon and Y. Kumar. Antibiotic susceptibility pattern of Enterococcus spp. Isolated from poultry feces. Cihan University-Erbil Scientific Journal, vol. 3, no. 1, pp. 50-55, 2019.

G. Kaur and N. Das. Molecular cloning, expression and in silico analyses of calcium-dependent protein kinase 2 (CDPK2) in potato (Solanum tuberosum L.). South African Journal of Botany, vol. 148, pp. 634-642, 2022.

M. D. Younus, O. F. Bahjat and S. A. Rashid. Antibiotic susceptibility pattern, molecular characterization of virulence genes among Pseudomonas Aeruginosa isolated from burn patients. Cihan University-Erbil Scientific Journal, vol. 5, no. 1, pp. 36-41, 2021.

G. Mishra, R. K. Meena, R. Kant, S. Pandey, H. S. Ginwal and M. S. Bhandari. Improved Shorea robusta genomic DNA extraction protocol with high PCR fidelity. Biology Methods & Protocols, vol. 8, no. 1, p. bpad039, 2023.

J. Singh, N. Birbian, S. Sinha and A. Goswami. A critical review on PCR, its types and applications. International Journal of Advanced Research in Biological Sciences, vol. 1 no. 7, pp. 65-80, 2014.

B. M. Crossley, J. Bai, A. Glaser, R. Maes, E. Porter, M. L. Killian, T. Clement and K. Toohey-Kurt. Guidelines for sanger sequencing and molecular assay monitoring. Journal of Veterinary Diagnostic Investigation, vol. 32, no. 6, pp. 767-775, 2020.

Published
2024-11-10
How to Cite
1.
Hama–Ali M, Hasan A, Jameel M, Muhammed H, khazaye S, Muhammed Amin M, Muhammedsaid K. Molecular Analysis of Gram-Negative Bacterial Strains in Patients from Koya City. cuesj [Internet]. 10Nov.2024 [cited 26Jan.2025];8(2):110-5. Available from: https://journals.cihanuniversity.edu.iq/index.php/cuesj/article/view/1237
Issue
Vol 8 No 2 (2024)
Section
Research Article

Copyright (c) 2024 Mohammed A. Hama–Ali, Ayad H. Hasan, Mohammed I. Jameel, Hewa A. Muhammed, Sada N. khazaye, Marwa B. Muhammed Amin, Khoshy A. Muhammedsaid

Creative Commons License

This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.

Authors who publish with this journal agree to the following terms:
1. Authors retain copyright and grant the journal right of first publication with the work simultaneously licensed under a Creative Commons Attribution License [CC BY-NC-ND 4.0] that allows others to share the work with an acknowledgment of the work's authorship and initial publication in this journal.
2. Authors are able to enter into separate, additional contractual arrangements for the non-exclusive distribution of the journal's published version of the work (e.g., post it to an institutional repository or publish it in a book), with an acknowledgment of its initial publication in this journal.
3. Authors are permitted and encouraged to post their work online (e.g., in institutional repositories or on their website) prior to and during the submission process, as it can lead to productive exchanges, as well as earlier and greater citation of published work (See The Effect of Open Access).