Genotyping and molecular investigation of plasmid-mediated carbapenem resistant clinical <i>Klebsiella pneumoniae</i> isolates in Egypt

Kholoud Baraka; Rania Abozahra; Marwa Mohammed Haggag; Sarah M Abdelhamid; Kholoud Baraka; Rania Abozahra; Marwa Mohammed Haggag; Sarah M Abdelhamid

doi:10.3934/microbiol.2023014

AIMS Microbiology

2023, Volume 9, Issue 2: 228-244. doi: 10.3934/microbiol.2023014

Previous Article Next Article

Research article

Genotyping and molecular investigation of plasmid-mediated carbapenem resistant clinical Klebsiella pneumoniae isolates in Egypt

1.
Microbiology and Immunology Department, Faculty of Pharmacy, Damanhour University, El Behira, Egypt
2.
Microbiology and Immunology Department, Faculty of Pharmacy, Sinai University, Arish Campus, Sinai, Egypt

Received: 02 January 2023 Revised: 14 March 2023 Accepted: 17 March 2023 Published: 23 March 2023

Klebsiella pneumoniae is a multidrug-resistant nosocomial pathogen. Carbapenem resistance is mediated mainly by enzymes carried on transmissible plasmids causing their dissemination among other members of Enterobacteriaceae. This study aimed to molecularly detect carbapenem resistance genes in K. pneumoniae clinical isolates, genotype them using ERIC-PCR, and investigate plasmid transformation of resistant genes by using ERIC-PCR and sequencing.

Methods
Antimicrobial resistance of sixty carbapenem-resistant K. pneumoniae strains was evaluated by using the disc diffusion method. Five carbapenemases' genes were amplified by conventional PCR. Genotyping was performed using ERIC-PCR. Gene transformation was performed for the five genes to sensitive isolates. Wild and transformed isolates were genetically investigated using ERIC-PCR and sequencing.

Results
Carbapenem resistance in our isolates was associated with high resistance to all tested antibiotics. The 60 K. pneumoniae isolates were divided into 6 resistor types. The prevalence of KPC, IMP, VIM, NDM, and OXA-48 genes were 17%, 63%, 93%, 85% and 100%, respectively. Dendrogram analysis showed 57 distinct patterns, arranged in three clusters. The five genes were transformed successfully into sensitive isolates. ERIC profiles of wild and transformed isolates showed cluster A contained all the wild isolates, and cluster B contained all transformed isolates. Genetic sequences of the 5 genes reflected high genetic similarity with the GenBank reference genes before plasmid transformation; however, a distinguishable decrease of genetic similarity was observed after transformation.

Conclusion
Plasmid-mediated carbapenem resistance in K. pneumoniae and its dissemination among different strains is a real threat to public health.
- Klebsiella,
- Egypt,
- carbapenem,
- genotyping,
- plasmid
Citation: Kholoud Baraka, Rania Abozahra, Marwa Mohammed Haggag, Sarah M Abdelhamid. Genotyping and molecular investigation of plasmid-mediated carbapenem resistant clinical Klebsiella pneumoniae isolates in Egypt[J]. AIMS Microbiology, 2023, 9(2): 228-244. doi: 10.3934/microbiol.2023014

Related Papers:

Abstract

Klebsiella pneumoniae is a multidrug-resistant nosocomial pathogen. Carbapenem resistance is mediated mainly by enzymes carried on transmissible plasmids causing their dissemination among other members of Enterobacteriaceae. This study aimed to molecularly detect carbapenem resistance genes in K. pneumoniae clinical isolates, genotype them using ERIC-PCR, and investigate plasmid transformation of resistant genes by using ERIC-PCR and sequencing.

Methods

Antimicrobial resistance of sixty carbapenem-resistant K. pneumoniae strains was evaluated by using the disc diffusion method. Five carbapenemases' genes were amplified by conventional PCR. Genotyping was performed using ERIC-PCR. Gene transformation was performed for the five genes to sensitive isolates. Wild and transformed isolates were genetically investigated using ERIC-PCR and sequencing.

Results

Carbapenem resistance in our isolates was associated with high resistance to all tested antibiotics. The 60 K. pneumoniae isolates were divided into 6 resistor types. The prevalence of KPC, IMP, VIM, NDM, and OXA-48 genes were 17%, 63%, 93%, 85% and 100%, respectively. Dendrogram analysis showed 57 distinct patterns, arranged in three clusters. The five genes were transformed successfully into sensitive isolates. ERIC profiles of wild and transformed isolates showed cluster A contained all the wild isolates, and cluster B contained all transformed isolates. Genetic sequences of the 5 genes reflected high genetic similarity with the GenBank reference genes before plasmid transformation; however, a distinguishable decrease of genetic similarity was observed after transformation.

Conclusion

Plasmid-mediated carbapenem resistance in K. pneumoniae and its dissemination among different strains is a real threat to public health.

Conflict of interest

The authors declare no conflict of interest.

References

[1]	Abbott S (2007) Klebsiella Enterobacter Citrobacter, Serratia, Plesiomonas, and Other Enterobacteriaceae. Manual of clinical microbiology. Washington: ASM Press 698-711.
[2]	Vila A, Cassata A, Pagella H, et al. (2011) Appearance of Klebsiella Pneumoniae liver abscess syndrome in Argentina: case report and review of molecular mechanisms of pathogenesis. Open Microbiol J 5: 107-113. https://doi.org/10.2174/1874285801105010107
[3]	Minarini LA, Gales AC, Palazzo IC, et al. (2007) Prevalence of community-occurring extended spectrum β-lactamase-producing Enterobacteriaceae in Brazil. Curr Microbiol 54: 335-341. https://doi.org/10.1007/s00284-006-0307-z
[4]	Daef EA, Elsherbiny NM (2012) Clinical and microbiological profile of nosocomial infections in adult intensive care units at Assiut University hospitals, Egypt. J Am Sci 8: 1239-1250.
[5]	Abdelwahab R, Alhammadi MM, Hassan EA, et al. (2021) Antimicrobial resistance and comparative genome analysis of Klebsiella pneumoniae strains isolated in Egypt. Microorganisms 9: 1880. https://doi.org/10.3390/microorganisms9091880
[6]	El Nekidy WS, Mooty MY, Attallah N, et al. (2017) Successful treatment of multidrug resistant Klebsiella pneumoniae using dual carbapenem regimen in immunocompromised patient. IDCases 9: 53-55. https://doi.org/10.1016/j.idcr.2017.06.005
[7]	McKenna M (2013) Antibiotic resistance: the last resort. Nature 499: 394-396. https://doi.org/10.1038/499394a
[8]	Paterson DL, Bonomo RA (2005) Extended-spectrum beta-lactamases: a clinical update. Clin Microbiol Rev 18: 657-686. https://doi.org/10.1128/CMR.18.4.657-686.2005
[9]	Centers for Disease Control and Prevention (U.S.)Antibiotic Resistance Threats in the United States (2019). Available from: http://dx.doi.org/10.15620/cdc:82532
[10]	Yang TY, Hsieh YJ, Kao LT, et al. (2020) In vitro and in vivo evaluations of β-lactam/β-lactamase Mono- and combined therapies against carbapenem-nonsusceptible Enterobacteriaceae in Taiwan. Microorganisms 8: 1981. https://doi.org/10.3390/microorganisms8121981
[11]	Elshamy AA, Aboshanab KM (2020) A review on bacterial resistance to carbapenems: epidemiology, detection and treatment options. Futur Sci 6. https://doi.org/10.2144/fsoa-2019-0098
[12]	Aurilio C, Sansone P, Barbarisi M, et al. (2022) Mechanisms of action of carbapenem resistance. Antibiotics 11: 421. https://doi.org/10.3390/antibiotics11030421
[13]	Yan J, Pu S, Jia X, et al. (2017) Multidrug resistance mechanisms of carbapenem resistant Klebsiella pneumoniae strains isolated in Chongqing, China. Ann Lab Med 37: 398-407. https://doi.org/10.3343/alm.2017.37.5.398
[14]	Al-baz AA, Maarouf A, Marei A, et al. (2022) Prevalence and antibiotic resistance profiles of carbapenem-resistant Klebsiella Pneumoniae isolated from tertiary care hospital, Egypt. Egypt J Hosp Med 88: 2883-2890. https://doi.org/10.21608/ejhm.2022.242765
[15]	Fanaki NH, Omar HM, Khalil AM, et al. (2018) Prevalence of carbapenem resistant Klebsiella and proteus clinical isolates: a real threat to the Egyptian Health Care System. Int J Curr Microbiol App Sci 7: 2041-2057. https://doi.org/10.20546/IJCMAS.2018.704.235
[16]	Abdelhamid SM, Abd-Elaal HM, Matareed MO, et al. (2020) Genotyping and virulence analysis of drug resistant clinical Klebsiella pneumoniae isolates in Egypt. J Pure Appl Microbiol 14. https://doi.org/10.22207/JPAM.14.3.36
[17]	Barus T, Hanjaya I, Sadeli J, et al. (2013) Genetic diversity of Klebsiella spp. isolated from tempe based on enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR). Hayati J Biosciences 20: 171-176. https://doi.org/10.4308/hjb.20.4.171
[18]	Wasfi R, Elkhatib WF, Ashour HM (2016) Molecular typing and virulence analysis of multidrug resistant Klebsiella pneumoniae clinical isolates recovered from Egyptian hospitals. Sci Rep 6: 38929. https://doi.org/10.1038/srep38929
[19]	Bailey WR, Scott EG (1986) Bailey and Scott's Diagnostic Microbiology.Mosby.
[20]	Bauer AW, Kirby WM, Sherris JC (1966) Antibiotic susceptibility testing by a standardized single disk method. Am J Clin Pathol 45: 493-496. https://doi.org/10.1093/ajcp/45.4_ts.493
[21]	Clinical and Laboratory Standards InstitutePerformance Standards For Antimicrobial Susceptibility Testing (2020). Available from: https://webstore.ansi.org/standards/clsi/clsim100ed30
[22]	Poirel L, Walsh TR, Cuvillier V, et al. (2011) Multiplex PCR for detection of acquired carbapenemase genes. Diagn Microbiol Infect Dis 70: 119-123. http://dx.doi.org/10.1016/j.diagmicrobio.2010.12.002
[23]	Mehr P, Shokoohizadeh L, Mzirzaee M, et al. (2017) Molecular typing of Klebsiella pneumoniae isolates by enterobacterial repetitive intergenic consensus (ERIC)–PCR. Infect Epidemiol Microbiol 3: 112-116. https://doi.org/10.18869/modares.iem.3.4.112
[24]	Chang AY, Chau VW, Landas JA, et al. (2017) Preparation of calcium competent Escherichia coli and heat-shock transformation. J Exp Microbiol Immunol 1: 22-25.
[25]	Patel G, Huprikar S, Factor SH, et al. (2008) Outcomes of carbapenem-resistant Klebsiella pneumoniae infection and the impact of antimicrobial and adjunctive therapies. Infect Control Hosp Epidemiol 29: 1099-1106. https://doi.org/10.1086/592412
[26]	El-sweify MA, Gomaa NI, El-maraghy NN (2015) Phenotypic detection of carbapenem resistance among Klebsiella pneumoniae in Suez Canal University Hospitals Ismailiya, Egypt. Int J Curr Microbiol Appl Sci 4: 10-18.
[27]	Gandor NHM, Amr GE, Eldin Algammal SMS, et al. (2022) Characterization of carbapenem-resistant K. pneumoniae isolated from Intensive Care Units of Zagazig University Hospitals. Antibiotics (Basel) 11: 1108. https://doi.org/10.3390/antibiotics11081108
[28]	Ragheb SM, Tawfick MM, El-Kholy AA, et al. (2020) Phenotypic and genotypic features of Klebsiella pneumoniae harboring carbapenemases in Egypt: OXA-48-Like carbapenemases as an investigated model. Antibiotics 9: 852. https://doi.org/10.3390/antibiotics9120852
[29]	Indrajith S, Kumar A, Chowdhury G, et al. (2021) Molecular insights of carbapenem resistance Klebsiella pneumoniae isolates with focus on multidrug resistance from clinical samples. J Infect Public Health 14: 131-138. https://doi.org/10.1016/j.jiph.2020.09.018
[30]	Li Y, Shen H, Zhu C, et al. (2019) Carbapenem-resistant Klebsiella pneumoniae infections among ICU admission patients in Central China: prevalence and prediction model. Biomed Res Int 27. https://doi.org/10.1155/2019/9767313
[31]	Yu X, Zhang W, Zhao Z, et al. (2019) Molecular characterization of carbapenem-resistant Klebsiella pneumoniae isolates with focus on antimicrobial resistance. BMC Genomics 20: 822. https://doi.org/10.1186/s12864-019-6225-9
[32]	Spanu T, Sanguinetti M, Tumbarello M, et al. (2006) Evaluation of the new VITEK 2 extended-spectrum beta-lactamase (ESBL) test for rapid detection of ESBL production in Enterobacteriaceae isolates. J Clin Microbiol 44: 3257-3262. https://doi.org/10.1128/JCM.00433-06
[33]	Carrër A, Poirel L, Eraksoy H, et al. (2008) Spread of OXA-48-positive carbapenem-resistant Klebsiella pneumoniae isolates in Istanbul, Turkey. Antimicrob Agents Chemother 52: 2950-2954. https://doi.org/10.1128/AAC.01672-07
[34]	Al-Agamy MH, Aljallal A, Radwan HH, et al. (2017) Characterization of carbapenemases, ESBLs, and plasmid-mediated quinolone determinants in carbapenem-insensitive Escherichia coli and Klebsiella pneumoniae in Riyadh hospitals. J Infect Public Health 11: 64-68. http://dx.doi.org/10.1016/j.jiph.2017.03.010
[35]	Stuart JC, Leverstein-Van Hall MA (2010) Guideline for phenotypic screening and confirmation of carbapenemases in Enterobacteriaceae. Int J Antimicrob Agents 36: 205-210. https://doi.org/10.1016/j.ijantimicag.2010.05.014
[36]	Del Bianco F, Morotti M, Zannoli S, et al. (2019) Comparison of four commercial screening assays for the detection of blaKPC, blaNDM, blaIMP, blaVIM, and blaOXA48 in rectal secretion collected by swabs. Microorganisms 7: 704. https://doi.org/10.3390/microorganisms7120704
[37]	Elshamy AA, Saleh SE, Alshahrani MY, et al. (2021) OXA-48 carbapenemase-encoding transferable plasmids of Klebsiella pneumoniae recovered from Egyptian patients suffering from complicated urinary tract infections. Biology (Basel) 10: 889. https://doi.org/10.3390/biology10090889
[38]	Raheel A, Azab H, Hessam W, et al. (2020) Detection of carbapenemase enzymes and genes among carbapenem-resistant Enterobacteriaceae isolates in Suez Canal University Hospitals in Ismailia, Egypt. Microbes Infect Dis 1: 24-33. https://doi.org/10.21608/mid.2020.25702.1007
[39]	Osama D, El-Mahallawy H, Mansour MT, et al. (2021) Molecular characterization of carbapenemase-producing Klebsiella pneumoniae isolated from Egyptian pediatric cancer patients including a strain with a rare gene-combination of β-Lactamases. Infect Drug Resist 14: 335-348. https://doi.org/10.2147/IDR.S284455
[40]	Amer WH, Khalil HS, Abd EL Wahab MA (2016) Risk factors, phenotypic and genotypic characterization of carpabenem resistant Enterobacteriaceae in Tanta University Hospitals, Egypt. Int J Infect Control 12: 1-11. https://doi.org/10.3396/IJIC.v12i2.012.16
[41]	Abdulall AK, Tawfick MM, El Manakhly AR, et al. (2018) Carbapenem-resistant gram-negative bacteria associated with catheter-related bloodstream infections in three intensive care units in Egypt. Eur J Clin Microbiol Infect Dis 37: 1647-1652. https://doi.org/10.1007/s10096-018-3294-7
[42]	Davarcı İ, Şenbayrak S, Aksaray S, et al. (2019) Molecular epidemiology of carbapenem-resistant Klebsiella pneumoniae isolates. Anadolu Kliniği Tıp Bilimleri Dergisi 24: 1-7. https://doi.org/10.21673/anadoluklin.423081
[43]	Tsilipounidaki K, Athanasakopoulou Z, Müller E, et al. (2022) Plethora of resistance genes in carbapenem-resistant gram-negative bacteria in Greece: no end to a continuous genetic evolution. Microorganisms 10: 159. https://doi.org/10.3390/microorganisms10010159
[44]	Alizadeh N, Ahangarzadeh Rezaee M, Samadi Kafil H, et al. (2020) Evaluation of resistance mechanisms in carbapenem-resistant Enterobacteriaceae. Infect Drug Resist 13: 1377-1385. https://doi.org/10.2147/IDR.S244357
[45]	Podschun R, Ullmann U (1998) Klebsiella spp. as nosocomial pathogens: epidemiology, taxonomy, typing methods, and pathogenicity factors. Clin Microbiol Rev 11: 589-603. https://doi.org/10.1128/CMR.11.4.589
[46]	Mohamed ER, Aly SA, Halby HM, et al. (2017) Epidemiological typing of multidrug-resistant klebsiella pneumoniae, which causes paediatric ventilator-associated pneumonia in Egypt. J Med Microbiol 66: 628-634. https://doi.org/10.1099/jmm.0.000473
[47]	Sedighi P, Zarei O, Karimi K, et al. (2020) Molecular typing of Klebsiella pneumoniae clinical isolates by enterobacterial repetitive intergenic consensus polymerase chain reaction. Int J Microbiol . https://doi.org/10.1155/2020/8894727
[48]	Oliveira ÉM, Beltrão EMB, Scavuzzi AML, et al. (2020) High plasmid variability, and the presence of IncFIB, IncQ, IncA/C, IncHI1B, and IncL/M in clinical isolates of Klebsiella pneumoniae with bla KPC and bla NDM from patients at a public hospital in Brazil. Rev Soc Bras Med Trop 53: e20200397. https://doi.org/10.1590/0037-8682-0397-2020
[49]	Sun Y, Li M, Chen L, et al. (2019) Prevalence and molecular characterization of carbapenemase-producing gram-negative bacteria from a university hospital in China. Infect Dis 48: 138-146. https://doi.org/10.3109/23744235.2015.1094822

Reader Comments

Your name:*

Email:*
© 2023 the Author(s), licensee AIMS Press. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0)