Molecular characterization of antibiotic resistance and virulence genes on plasmids of Proteus mirabilis isolated from urine samples of Hospitals in Mosul City, Iraq
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Abstract
Antibiotic resistance genes when found on plasmids can be passed on to other strains causing spread of antibiotic resistance across bacteria. The present work aimed to identify virulence and resistance genes on Proteus mirabilis plasmids. A total of 37 P. mirabilis strains were isolated from 420 urine samples from patients attending different hospitals in Mosul City, Iraq, from December 2022 to April 2023 and identified using biochemical and molecular methods. Their resistance towards 18 antibiotics was tested and their plasmid DNA analysis showed that 21 of 37 Proteus strains contained plasmids. Four groups of primers were used for PCR experiments. The first group included primers used to identify six genetic regions, namely (CITM, DHAM, ACCM, EBCM, FOXM, MOXM1). Results showed that 85.71% of isolates carried FOXM on their plasmids and 14.28% carried MOXM and 4.76% carried CITM. However, DHAH, ACCM, and EBCM were not detected on plasmids. The second group included zapA, ireA (siderophore receptor), hpmA (hemolysin) and mrpA, (fimbriae) genes. zapA was detected in 80.95% of P. mirabilis plasmids, followed by ireA at a rate of 76.19%, hpmA at 14.28%, and mrpA at 4.76%. The third group included CTXG1, CTXG2, CTXG9, CTXG8 and CTXG25. The results showed that CTX9 was the highest gene detected, 76.19%, followed by CTXG1 71.42%, while CTXG2, CTXG8, and CTXG25 were only detected on the chromosome. Finally, the pathogenic genes PmIJ1 and qnrD were found on P. mirabilis plasmids at 52.38% and 47.61%, respectively. The present results showed that plasmids are increasingly spread among clinical local isolates of P. mirabilis, and serious precautions are required.
Article Details
Article Details
Antibiotic resistance genes, Proteus mirabilis, Plasmids, Virulence genes
Abdulrahman, Z. F. A. & Omar, L. A. (2012). Molecular Study of Proteus mirabilis Isolated From Urinary Tract Infections in Erbil City. MSc. thesis,
Abdulrazzaq, R. & Faisal, R. (2022). Efficiency of hichrome enterococcus faecium agar in the isolation of Enterococcus spp. and other associated bacterial genera from water. Journal of Life and Bio Sciences Research, 3(01), 01-06. https://doi.org/10.38094/jlbsr30151
Abossedgh, R., Ghane, M. & Babaeekhou, L. (2020). High Frequency of qnr genes in urinary isolates of extended-spectrum β-lactamase (ESBL)-producing Klebsiella pneumoniae in Tehran, Iran. Shiraz E-Medical Journal, 21(3). https://doi.org/10.5812/semj.92032
Aldred, K. J., Kerns, R. J. & Osheroff, N. (2014). Mechanism of quinolone action and resistance. Biochemistry, 53(10), 1565-1574. https://doi.org/10.1021/bi5000564
Al-Taie, K., Abdel-Kadhim, N. & Hassan F. (2013). The effect of containing beta-laccanum antibiotics with amino glycosides on multiple antibiotic-resistant protozoans. Journal of the University of Babylon/Pure and Applied Sciences. (12..-12.3 (:)21(4).
Al-Tamimi, A. H. & Jabbar, A. L. (2021). Isolation and identification of commonly occurrence of enterobactericeae from poultry’s meat. Biochemical & Cellular Archives, 21.
Aryal, S. C., Upreti, M. K., Sah, A. K., Ansari, M., Nepal, K., Dhungel, B. & Rijal, K. R. (2020). Plasmid-mediated AmpC β-lactamase CITM and DHAM genes among gram-negative clinical isolates. Infection and Drug Resistance, 4249-4261. DOI: 10.2147/IDR.S284751
Bedenić, B. & Meštrović, T. (2021). Mechanisms of resistance in gram-negative urinary pathogens: From country-specific molecular insights to global clinical relevance. Diagnostics, 11(5), 800. https://doi.org/10.3390/diagnostics11050800
Bie, L., Fang, M., Li, Z., Wang, M. & Xu, H. (2018). Identification and characterization of new resistance-conferring SGI1s (Salmonella genomic island 1) in Proteus mirabilis. Frontiers in Microbiology, 9, 3172. https://doi.org/10.3389/fmicb.2018.03172
Biondo, C. (2023). Bacterial Antibiotic Resistance: The Most Critical Pathogens. Pathogens, 12, 116. https://doi.org/10.3390/pathogens12010116
Bonnet, R. (2004). Growing group of extended-spectrum β-lactamases: the CTX-M enzymes. Antimicrobial agents and chemotherapy, 48(1), 1-14.
Butler, M. T., Wang, Q. & Harshey, R. M. (2010). Cell density and mobility protect swarming bacteria against antibiotics. Proceedings of the National Academy of Sciences, 107(8), 3776-3781. https://doi.org/10.1073/pnas.0910934107
Carattoli, A. (2013). Plasmids and the spread of resistance. International Journal of Medical Microbiology, 303(6–7), 298–304. https://doi.org/10.1016/j.ijmm.2013.02.001
Castanheira, M., Sader, H. S. & Jones, R. N. (2010). Antimicrobial susceptibility patterns of KPC-producing or CTX-M-producing Enterobacteriaceae. Microbial Drug Resistance, 16(1), 61–65. https://doi.org/10.1089/mdr.2009.0031
Cavaco, L. M., Hasman, H., Xia, S. & Aarestrup, F. M. (2009). qnrD, a novel gene conferring transferable quinolone resistance in Salmonella enterica serovar Kentucky and Bovismorbificans strains of human origin. Antimicrobial agents and chemotherapy, 53(2), 603-608. https://doi.org/10.1128/aac.00997-08
Cestari, S. E., Ludovico, M. S., Martins, F. H., da Rocha, S. P. D., Elias, W. P. & Pelayo, J. S. (2013). Molecular detection of HpmA and HlyA hemolysin of uropathogenic Proteus mirabilis. Current microbiology, 67, 703-707.
Clinical and Laboratory Standards Institute (CLSI) (2022). Performance Standards for Antimicrobial Susceptibility Testing; 29thed. supplement M100. Wayne, PA: Clinical and Laboratory Standards Institute.
Eisner, A., Fagan, E. J., Feierl, G., Kessler, H. H., Marth, E., Livermore, D. M. & Woodford, N. (2006). Emergence of Enterobacteriaceae isolates producing CTX-M extended-spectrum β-lactamase in Austria. Antimicrobial agents and chemotherapy, 50(2), 785-787. https://doi.org/10.1128/aac.50.2.785-787.2006
Filipiak, A., Chrapek, M., Literacka, E., Wawszczak, M., Głuszek, S., Majchrzak, M., Wróbel, G., Łysek-Gładysińska, M., Gniadkowski, M. & Adamus-Białek, W. (2020). Pathogenic factors correlate with antimicrobial resistance among clinical Proteus mirabilis strains. Frontiers in Microbiology, 11, 579389. https://doi.org/10.3389/fmicb.2020.579389
Girlich, D., Bonnin, R. A., Dortet, L. & Naas, T. (2020). Genetics of acquired antibiotic resistance genes in Proteus spp. Frontiers in Microbiology, 11, 256. https://doi.org/10.3389/fmicb.2020.00256
Grossman, T. H. (2016). Tetracycline antibiotics and resistance. Cold Spring Harbor perspectives in medicine, 6(4), a025387. http://perspectivesinmedicine.cshlp.org/
Gupta, V., Yadav, A. & Joshi, R. M. (2002). Antibiotic resistance pattern in uropathogens. Indian journal of medical microbiology, 20(2), 96-98.
Hayat, F., Khan, M., Umair, M., Akbar, S., Javed, R. & Shah, S. H. (2023). Phenotypic and Genotypic Detection of Virulence Factors Affecting Proteus mirabilis Clinical Isolates. Current Trends in OMICS, 3(1), 73-85.
Hays, J. P., Safain, K. S., Almogbel, M. S., Habib, I. & Khan, M. A. (2022). Extended spectrum-and carbapenemase-based β-lactam resistance in the arabian peninsula—a descriptive review of recent years. Antibiotics, 11(10), 1354. https://doi.org/10.3390/antibiotics11101354
He, D., Zhu, Y., Li, R., Pan, Y., Liu, J., Yuan, L. & Hu, G. (2019). Emergence of a hybrid plasmid derived from IncN1-F33: A−: B− and mcr-1-bearing plasmids mediated by IS 26. Journal of Antimicrobial Chemotherapy, 74(11), 3184–3189. https://doi.org/10.1093/jac/dkz327
Hua, X., Zhang, L., Moran, R. A., Xu, Q., Sun, L., Van Schaik, W. & Yu, Y. (2020a). Cointegration as a mechanism for the evolution of a KPC- producing multidrug resistance plasmid in Proteus mirabilis. Emerging Microbes & Infections, 9(1), 1206–1218. https://doi.org/10.1080/22221751.2020.1773322
Hutinel, M., Larsson, D. J. & Flach, C. F. (2022). Antibiotic resistance genes of emerging concern in municipal and hospital wastewater from a major Swedish city. Science of The Total Environment, 812, 151433. https://doi.org/10.1016/j.scitotenv.2021.151433
John, M. S., Nagoth, J. A., Ramasamy, K. P., Mancini, A., Giuli, G., Miceli, C. & Pucciarelli, S. (2022). Synthesis of bioactive silver nanoparticles using new bacterial strains from an antarctic consortium. Marine Drugs, 20(9), 558. https://doi.org/10.3390/md20090558
Jun K. , Myoung-Hwan Y., Hyoung-Joon K., Sang-Guen K., Chul, P. & Se-Chang, P. (2022). Antimicrobial Resistance and Virulence Factors of Proteus mirabilis Isolated from Dog with Chronic Otitis Externa. Pathogens 11, 1215. https://doi.org/10.3390/pathogens11101215
Khaleel, A. M., Faisal, R. M. & Altaii, H. A. (2023a). The efficiency of molecular methods compared to traditional methods in identifying bacteria from blood and cerebrospinal fluid samples. Malaysian Journal of Microbiology, 19(2). DOI10.21161/mjm.220105
Khaleel, A. M., Faisal, R. M. & Altaii, H. A. (2023b). Using recombinant DNA technology in bacterial identificationfrom vaginal swabs. Revis Bionatura, 8(3), 113.
Lewis, I. I. & James, S. (2022). Performance standards for antimicrobial susceptibility testing.
Li, B., Yi, Y., Wang, Q., Woo, P. C., Tan, L., Jing, H. & Liu, C. H. (2012). Analysis of drug resistance determinants in Klebsiella pneumoniae isolates from a tertiary-care hospital in Beijing, China. https://doi.org/10.1371/journal.pone.0042280
Magiorakos, A. P., Srinivasan, A., Carey, R. B., Carmeli, Y., Falagas, M. E., Giske, C. G. & Monnet, D. L. (2012). Multidrug-resistant, extensively drug-resistant and pandrug-resistant bacteria: an international expert proposal for interim standard definitions for acquired resistance. Clinical microbiology and infection, 18(3), 268-281. https://doi.org/10.1111/j.1469-0691.2011.03570.x
Mancuso, G., Midiri, A., Zummo, S., Gerace, E., Scappatura, G. & Biondo, C. (2021). Extended-spectrum β-lactamase & carbapenemase-producing fermentative Gram-negative bacilli in clinical isolates from a University Hospital in Southern Italy. New Microbiol, 44, 227-233. https://doi.org/10.3390/pathogens12040623
Mirzaei, A., Habibi, M., Bouzari, S. & Asadi Karam, M. R. (2019). Characterization of antibiotic-susceptibility patterns, virulence factor profiles and clonal relatedness in Proteus mirabilis isolates from patients with urinary tract infection in Iran. Infection and drug resistance, 3967-3979. https://doi.org/10.1111/j.1469-0691.2011.03570.x
Mo, L., Wang, J., Qian, J. & Peng, M. (2022). Antibiotic Sensitivity of Proteus mirabilis Urinary Tract Infection in Patients with Urinary Calculi. International Journal of Clinical Practice, 2022. https://doi.org/10.1155/2022/7273627
Munday, C. J., Whitehead, G. M., Todd, N. J., Campbell, M. & Hawkey, P. M. (2004). Predominance and genetic diversity of community-and hospital-acquired CTX-M extended-spectrum β-lactamases in York, UK. Journal of Antimicrobial Chemotherapy, 54(3), 628-633. https://doi.org/10.1093/jac/dkh397
Musa, H. A., Osman, M. A. M., Abdelaziz, Y. H., Mohamed, S. & Ibrahim- Saeed, M. (2019). Distribución de genes de resistencia de betalactamasas de espectro extendido TEM y CTX-M entre especies de Proteus aisladas en Sudán. Vaccimonitor, 28(2), 80–84.
Mushtaq, A., Chasan, R., Nowak, M. D., Rana, M., Ilyas, S., Paniz-Mondolfi, A. E. & Gitman, M. R. (2022). Correlation between identification of β-lactamase resistance genes and antimicrobial susceptibility profiles in gram-negative bacteria: a laboratory data analysis. Microbiology Spectrum, 10(2), e01485-21. https://doi.org/10.1128/spectrum.01485-21
Ojdana, D., Sacha, P., Wieczorek, P., Czaban, S., Michalska, A., Jaworowska, J. & Tryniszewska, E. (2014). The occurrence of blaCTX-M, blaSHV, and blaTEM genes in extended-spectrum β-lactamase-positive strains of Klebsiella pneumoniae, Escherichia coli, and Proteus mirabilis in Poland. International Journal of Antibiotics, 2014. https://doi.org/10.1155/2014/935842
Oliver, G.; Holgado, A. P. & Salim, R. (1999). Dimorphism in Candida albicans effect of cyclohximide and a cridine orange on germ tube formation. Mycopath. 79: 43-47. Ihttps://doi.org/10.1007/BF00636181
Pérez-Pérez, F. J. & Hanson, N. D. (2002). Detection of plasmid-mediated AmpC β-lactamase genes in clinical isolates by using multiplex PCR. Journal of clinical microbiology, 40(6), 2153-2162. https://doi.org/10.1128/jcm.40.6.2153-2162.2002
Qin, S., Qi, H., Zhang, Q., Zhao, D., Liu, Z.-Z. & Tian, H. (2015). Emergence of extensively drug-resistant Proteus mirabilis harboring a conjugative NDM-1 plasmid and a novel Salmonella genomic island 1 variant, SGI1-Z. Antimicrob. Agents Chemother. 59, 6601–6604. https://doi.org/10.1128/aac.00292-15
Ramsay, J. P. & Firth, N. (2017). Diverse mobilization strategies facilitate transfer of non-conjugative mobile genetic elements. Current Opinion in Microbiology, 38, 1–9. https://doi.org/10.1016/j.mib.2017.03.003
Sanches, M. S., Baptista, A. A. S., de Souza, M., Menck-Costa, M. F., Koga, V. L., Kobayashi, R. K. T. & Rocha, S. P. D. (2019). Genotypic and phenotypic profiles of virulence factors and antimicrobial resistance of Proteus mirabilis isolated from chicken carcasses: potential zoonotic risk. Brazilian Journal of Microbiology, 50, 685-694. https://doi.org/10.1007/s42770-019-00086-2
Shabeeb, B. T., Alghanimi, Y. K. & Ahmed, M. M. (2018) Molecular and Bacteriologic Study of â-lactam Resistance Proteus mirabilis associated with Urinary Tract Infection in Holy Karbala province, Iraq, Journal of Pharmaceutical Sciences and Research, 10(3), pp. 549–555.
Shanmugasundarasamy, T., Govindarajan, D. K. & Kandaswamy, K. (2022). A review on pilus assembly mechanisms in Gram-positive and Gram-negative bacteria. The Cell Surface, 8, 100077. https://doi.org/10.1016/j.tcsw.2022.100077
Shelenkov, A., Petrova, L., Fomina, V., Zamyatin, M., Mikhaylova, Y. & Akimkin, V. (2020). Multidrug-resistant Proteus mirabilis strain with cointegrate plasmid. Microorganisms, 8(11), 1775. https://doi.org/10.3390/microorganisms8111775
Swihart, K. G. & Welch, R. A. (1990). The HpmA hemolysin is more common than HlyA among Proteus isolates. Infection and immunity, 58(6), 1853-1860. https://doi.org/10.1128/iai.58.6.1853-1860.1990
Tahreer, H. S., Saba, T. H., Salma, N. M. & Bahaa, A. L.(2019). Down-Regulation of fliL Gene Expression by Ag Nanoparticles and TiO 2 Nanoparticles in Pragmatic Clinical Isolates of Proteus mirabilis and Proteus vulgaris from Urinary Tract Infection. Nano Biomed. Eng, 11(4): 321-332. doi: 10.5101/nbe.v11i4.p321-332.
Talebi, A., Momtaz, H. & Tajbakhsh, E. (2023). Frequency distribution of virulence factors and antibiotic resistance genes in uropathogenic Proteus species isolated from clinical samples. Letters in Applied Microbiology, 76(2), ovac043. https://doi.org/10.1093/lambio/ovac043
Vandepitte, J. (2003). Basic laboratory procedures in clinical bacteriology. World Health Organization.
Vasconcelos, N. G., Croda, J. & Simionatto, S. (2018). Antibacterial mechanisms of cinnamon and its constituents: A review. Microbial pathogenesis, 120, 198-203. https://doi.org/10.1016/j.micpath.2018.04.036
Wang, J. T., Chen, P. C., Chang, S. C., Shiau, Y. R., Wang, H. Y., Lai, J. F. & Lauderdale, T. L. (2014). Antimicrobial susceptibilities of Proteus mirabilis: a longitudinal nationwide study from the Taiwan surveillance of antimicrobial resistance (TSAR) program. BMC infectious diseases, 14(1), 1-10. https://doi.org/10.1186/1471-2334-14-486
Wang, M. H., Xu, X., Wu, S., Zhu, D. & Wang, M. (2008). A new plasmidmediated gene for quinolone resistance, qnrC, abstr. O207. Abstr. 18th Eur. Cong. Clin. Microbiol. Infect. Dis., Barcelona, Spain.
Wang, Z., Wu, Y., Chen, S., Hou, H. & Wang, Y. (2023). Infection of Diabetes Foot Caused by Carbapenem-Resistant Proteus penneri Mediated by a Novel Plasmid Containing blaNDM. Infection and Drug Resistance, 1099-1106. https://www.tandfonline.com/doi/epdf/10.2147/IDR.S398914
Woodford, N., Fagan, E. J. & Ellington, M. J. (2006). Multiplex PCR for rapid detection of genes encoding CTX-M extended-spectrum β-lactamases. Journal of antimicrobial chemotherapy, 57(1), 154-155. https://doi.org/10.1093/jac/dki412
Woodford, N., Ward, M. E., Kaufmann, M. E., Turton,
J., Fagan, E. J., James, D. & Livermore, D. M. (2004).
Community and hospital spread of Escherichia coli
producing CTX-M extended-spectrum β-lactamases in
the UK. Journal of Antimicrobial Chemotherapy,
54(4), 735-743. https://doi.org/10.1093/jac/dkh424
Wu, J., Ding, J. & Wang, L. (2023). Livestock and poultry infectious diseases: pathogenesis and immune mechanisms. Frontiers in Cellular and Infection Microbiology, 13, 1249034. https://doi.org/10.3389%2Ffcimb.202 3.1249034
Yasmeen, N., Aslam, B., Fang, L. X., Baloch, Z. & Liu, Y. (2023). Occurrence of extended-spectrum β-lactamase harboring K. pneumoniae in various sources: a one health perspective. Frontiers in Cellular and Infection Microbiology, 13, 1103319. https://doi.org/10.3389/fcimb.2023.1103319
Zaman, S. B., Hussain, M. A., Nye, R., Mehta, V., Mamun, K. T. & Hossain, N. (2017). A Review on Antibiotic Resistance: Alarm Bells are Ringing. Cureus, 9(6):1–9. DOI: 10.7759/cureus.1403
Zheng, M. & Lupoli, T. J. (2023). Counteracting antibiotic resistance enzymes and efflux pumps. Current Opinion in Microbiology, 75, 102334. https://doi.org/10.1016/j.mib.2023.102334
Zixuan, L., Chong, P., Gerui, Z., Yuanyu, S., Yuxuan, Z., Cong, L., Mengda, L. & Fangkun, W. (2022). Prevalence and characteristics of multidrug-resistant Proteus mirabilis from broiler farms in Shandong Province, China. Poultry Science, 101:101710 . https://doi.org/10.1016/j.psj.20 22.101710

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