Carbapenem Resistance in Gram-Negative Bacteria: A Hospital-Based Study in Egypt
Abstract
:1. Introduction
2. Material and Methods
2.1. Study Design
2.2. Inclusion and Exclusion Criteria
2.3. Sampling
2.4. Antimicrobial Susceptibility Testing
2.5. Polymerase Chain Reaction (PCR) for Detection of Carbapenemase Genes
2.6. DNA Extraction
2.7. Amplification of Carbapenemase Genes
2.8. PCR-Based Replicon Typing (PBRT) of the Plasmid
2.9. Statistical Analysis
3. Results
4. Discussion
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
- Szmolka, A.; Nagy, B. Multidrug resistant commensal Escherichia coli in animals and its impact for public health. Front. Microbiol. 2013, 4, 258. [Google Scholar] [CrossRef] [PubMed]
- Ting, S.-W.; Lee, C.-H.; Liu, J.-W. Risk factors and outcomes for the acquisition of carbapenem-resistant Gram-negative bacillus bacteremia: A retrospective propensity-matched case control study. J. Microbiol. Immunol. Infect. 2018, 51, 621–628. [Google Scholar] [CrossRef] [PubMed]
- Crespo Erchiga, V.; Ojeda Martos, A.; Vera Casano, A.; Crespo Erchiga, A.; Sanchez Fajardo, F. Malassezia globosa as the causative agent of pityriasis versicolor. Br. J. Dermatol. 2000, 143, 799–803. [Google Scholar] [CrossRef]
- Sheu, C.-C.; Chang, Y.-T.; Lin, S.-Y.; Chen, Y.-H.; Hsueh, P.-R. Infections caused by carbapenem-resistant Enterobacteriaceae: An update on therapeutic options. Front. Microbiol. 2019, 10, 80. [Google Scholar] [CrossRef]
- Walsh, T.R. Emerging carbapenemases: A global perspective. Int. J. Antimicrob. Agents 2010, 36, S8–S14. [Google Scholar] [CrossRef] [PubMed]
- Hoseini, N.; Sedighi, I.; Mozaffari Nejad, A.S.; Alikhani, M.Y. Phenotypic and Genotypic Detection of AmpC Enzymes in Clinical Isolates of Escherichia coli and Klebsiella pneumoniae. J. Krishna Inst. Med. Sci. JKIMSU 2017, 6, 10–18. [Google Scholar]
- Lutgring, J.D.; Limbago, B.M. The problem of carbapenemase-producing-carbapenem-resistant-Enterobacteriaceae detection. J. Clin. Microbiol. 2016, 54, 529–534. [Google Scholar] [CrossRef] [PubMed]
- Hammoudi Halat, D.; Ayoub Moubareck, C. The current burden of carbapenemases: Review of significant properties and dissemination among gram-negative bacteria. Antibiotics 2020, 9, 186. [Google Scholar] [CrossRef]
- Poirel, L.; Potron, A.; Nordmann, P. OXA-48-like carbapenemases: The phantom menace. J. Antimicrob. Chemother. 2012, 67, 1597–1606. [Google Scholar] [CrossRef]
- Majlesi, A.; Kakhki, R.K.; Nejad, A.S.M.; Mashouf, R.Y.; Roointan, A.; Abazari, M.; Alikhani, M.Y. Detection of plasmid-mediated quinolone resistance in clinical isolates of Enterobacteriaceae strains in Hamadan, West of Iran. Saudi J. Biol. Sci. 2018, 25, 426–430. [Google Scholar] [CrossRef]
- Wang, M.; Tran, J.H.; Jacoby, G.A.; Zhang, Y.; Wang, F.; Hooper, D.C. Plasmid-mediated quinolone resistance in clinical isolates of Escherichia coli from Shanghai, China. Antimicrob. Agents Chemother. 2003, 47, 2242–2248. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Schweizer, C.; Bischoff, P.; Bender, J.; Kola, A.; Gastmeier, P.; Hummel, M.; Klefisch, F.-R.; Schoenrath, F.; Frühauf, A.; Pfeifer, Y. Plasmid-mediated transmission of KPC-2 carbapenemase in Enterobacteriaceae in critically ill patients. Front. Microbiol. 2019, 10, 276. [Google Scholar] [CrossRef] [PubMed]
- Poirel, L.; Kieffer, N.; Liassine, N.; Thanh, D.; Nordmann, P. Plasmid-mediated carbapenem and colistin resistance in a clinical isolate of Escherichia coli. Lancet Infect. Dis. 2016, 16, 281. [Google Scholar] [CrossRef] [PubMed]
- Khattab, S.; Sweify, A.; Ali, M.; Metwally, L.; Elazab, S.; Hashem, A. Detection of plasmid-mediated colistin resistance in carbapenem-resistant Escherichia coli and Klebsiella pneumoniae isolates in Suez Canal University Hospitals. Microbes Infect. Dis. 2021, 47, 497–507. [Google Scholar] [CrossRef]
- Abouelfetouh, A.; Mattock, J.; Turner, D.; Li, E.; Evans, B.A. Diversity of carbapenem-resistant Acinetobacter baumannii and bacteriophage-mediated spread of the Oxa23 carbapenemase. Microb. Genom. 2022, 8, 000752. [Google Scholar] [CrossRef]
- Weinstein, M.P.; Limbago, B.; Patel, J.; Mathers, A.; Campeau, S.; Mazzulli, T.; Eliopoulos, G.; Patel, R.; Galas, M.; Richter, S. M100 Performance Standards for Antimicrobial Susceptibility Testing; Clinical and Laboratory Standards Institute: Wayne, PA, USA, 2018. [Google Scholar]
- Jeon, B.-C.; Jeong, S.H.; Bae, I.K.; Kwon, S.B.; Lee, K.; Young, D.; Lee, J.H.; Song, J.S.; Lee, S.H. Investigation of a nosocomial outbreak of imipenem-resistant Acinetobacter baumannii producing the OXA-23 β-lactamase in Korea. J. Clin. Microbiol. 2005, 43, 2241–2245. [Google Scholar] [CrossRef]
- Venkatachalam, I.; Teo, J.; Balm, M.N.; Fisher, D.A.; Jureen, R.; Lin, R.T. Klebsiella pneumoniae carbapenemase-producing enterobacteria in hospital, Singapore. Emerg. Infect. Dis. 2012, 18, 1381. [Google Scholar] [CrossRef]
- Nordmann, P.; Naas, T.; Poirel, L. Global spread of carbapenemase-producing Enterobacteriaceae. Emerg. Infect. Dis. 2011, 17, 1791. [Google Scholar] [CrossRef]
- Carloni, E.; Andreoni, F.; Omiccioli, E.; Villa, L.; Magnani, M.; Carattoli, A. Comparative analysis of the standard PCR-Based Replicon Typing (PBRT) with the commercial PBRT-KIT. Plasmid 2017, 90, 10–14. [Google Scholar] [CrossRef]
- Chen, H.-Y.; Jean, S.-S.; Lee, Y.-L.; Lu, M.-C.; Ko, W.-C.; Liu, P.-Y.; Hsueh, P.-R. Carbapenem-resistant enterobacterales in long-term care facilities: A global and narrative review. Front. Cell. Infect. Microbiol. 2021, 11, 601968. [Google Scholar] [CrossRef]
- Wang, C.-H.; Ma, L.; Huang, L.-Y.; Yeh, K.-M.; Lin, J.-C.; Siu, L.K.; Chang, F.-Y. Molecular epidemiology and resistance patterns of blaOXA-48 Klebsiella pneumoniae and Escherichia coli: A nationwide multicenter study in Taiwan. J. Microbiol. Immunol. Infect. 2021, 54, 665–672. [Google Scholar] [CrossRef] [PubMed]
- Jean, S.-S.; Harnod, D.; Hsueh, P.-R. Global Threat of Carbapenem-Resistant Gram-Negative Bacteria. Front. Cell. Infect. Microbiol. 2022, 236, 823684. [Google Scholar] [CrossRef]
- Raheel, A.; Azab, H.; Hessam, W.; Abbadi, S.; Ezzat, A. Detection of carbapenemase enzymes and genes among carbapenem-resistant Enterobacteriaceae isolates in Suez Canal University Hospitals in Ismailia, Egypt. Microbes Infect. Dis. 2020, 1, 24–33. [Google Scholar] [CrossRef]
- Kazmierczak, K.M.; Rabine, S.; Hackel, M.; McLaughlin, R.E.; Biedenbach, D.J.; Bouchillon, S.K.; Sahm, D.F.; Bradford, P.A. Multiyear, multinational survey of the incidence and global distribution of metallo-β-lactamase-producing Enterobacteriaceae and Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 2016, 60, 1067–1078. [Google Scholar] [CrossRef] [PubMed]
- Awoke, T.; Teka, B.; Aseffa, A.; Sebre, S.; Seman, A.; Yeshitela, B.; Abebe, T.; Mihret, A. Detection of bla KPC and bla NDM carbapenemase genes among Klebsiella pneumoniae isolates in Addis Ababa, Ethiopia: Dominance of bla NDM. PLoS ONE 2022, 17, e0267657. [Google Scholar] [CrossRef]
- El-Kholy, A.A.; Elanany, M.G.; Sherif, M.M.; Gad, M.A. High Prevalence of VIM, KPC, and NDM Expression among Surgical Site Infection Pathogens in Patients Having Emergency Surgery. Surg. Infect. 2018, 19, 629–633. [Google Scholar] [CrossRef]
- Fathy, F.E.Z.Y.; Anwar, M.A. Detection of blaKPC gene among Carbapenem Resistant Enterobacteriacae Isolates from Ain Shams University Hospital, Egypt. Egypt. J. Med. Microbiol. 2022, 31, 151–156. [Google Scholar] [CrossRef]
- Hu, L.; Zhong, Q.; Shang, Y.; Wang, H.; Ning, C.; Li, Y.; Hang, Y.; Xiong, J.; Wang, X.; Xu, Y. The prevalence of carbapenemase genes and plasmid-mediated quinolone resistance determinants in carbapenem-resistant Enterobacteriaceae from five teaching hospitals in central China. Epidemiol. Infect. 2014, 142, 1972–1977. [Google Scholar] [CrossRef]
- Khan, F.A.; Söderquist, B.; Jass, J. Prevalence and diversity of antibiotic resistance genes in Swedish aquatic environments impacted by household and hospital wastewater. Front. Microbiol. 2019, 10, 688. [Google Scholar] [CrossRef] [PubMed]
- Miller, W.R.; Munita, J.M.; Arias, C.A. Mechanisms of antibiotic resistance in enterococci. Expert Rev. Anti-Infect. Ther. 2014, 12, 1221–1236. [Google Scholar] [CrossRef] [PubMed]
- Zharikova, N.V.; Iasakov, T.R.; Bumazhkin, B.K.; Patutina, E.O.; Zhurenko, E.I.; Korobov, V.V.; Sagitova, A.I.; Kuznetsov, B.B.; Markusheva, T.V. Isolation and sequence analysis of pCS36-4CPA, a small plasmid from Citrobacter sp. 36-4CPA. Saudi J. Biol. Sci. 2018, 25, 660–671. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Carattoli, A. Resistance plasmid families in Enterobacteriaceae. Antimicrob. Agents Chemother. 2009, 53, 2227–2238. [Google Scholar] [CrossRef] [PubMed]
- Castanheira, M.; Deshpande, L.M.; Mills, J.C.; Jones, R.N.; Soave, R.; Jenkins, S.G.; Schuetz, A.N. Klebsiella pneumoniae isolate from a New York City hospital belonging to sequence type 258 and carrying blaKPC-2 and blaVIM-4. Antimicrob. Agents Chemother. 2016, 60, 1924–1927. [Google Scholar] [CrossRef] [PubMed]
- Ho, P.-L.; Li, Z.; Lo, W.-U.; Cheung, Y.-Y.; Lin, C.-H.; Sham, P.-C.; Chi-Chung Cheng, V.; Ng, T.-K.; Que, T.-L.; Chow, K.-H. Identification and characterization of a novel incompatibility group X3 plasmid carrying bla NDM-1 in Enterobacteriaceae isolates with epidemiological links to multiple geographical areas in China. Emerg. Microbes Infect. 2012, 1, 1–6. [Google Scholar] [CrossRef]
- Habeeb, M.A.; Haque, A.; Nematzadeh, S.; Iversen, A.; Giske, C.G. High prevalence of 16S rRNA methylase RmtB among CTX-M extended-spectrum β-lactamase-producing Klebsiella pneumoniae from Islamabad, Pakistan. Int. J. Antimicrob. Agents 2013, 41, 524–526. [Google Scholar] [CrossRef]
- Rozwandowicz, M.; Brouwer, M.; Fischer, J.; Wagenaar, J.; Gonzalez-Zorn, B.; Guerra, B.; Mevius, D.; Hordijk, J. Plasmids carrying antimicrobial resistance genes in Enterobacteriaceae. J. Antimicrob. Chemother. 2018, 73, 1121–1137. [Google Scholar] [CrossRef]
- Johnson, T.J.; Wannemuehler, Y.M.; Johnson, S.J.; Logue, C.M.; White, D.G.; Doetkott, C.; Nolan, L.K. Plasmid replicon typing of commensal and pathogenic Escherichia coli isolates. Appl. Environ. Microbiol. 2007, 73, 1976–1983. [Google Scholar] [CrossRef]
- Wang, L.; Gu, H.; Lu, X. A rapid low-cost real-time PCR for the detection of Klebsiella pneumonia carbapenemase genes. Ann. Clin. Microbiol. Antimicrob. 2012, 11, 1–6. [Google Scholar] [CrossRef] [PubMed]
- Pál, T.; Ghazawi, A.; Darwish, D.; Villa, L.; Carattoli, A.; Hashmey, R.; Aldeesi, Z.; Jamal, W.; Rotimi, V.; Al-Jardani, A. Characterization of NDM-7 carbapenemase-producing Escherichia coli isolates in the Arabian Peninsula. Microb. Drug Resist. 2017, 23, 871–878. [Google Scholar] [CrossRef]
Target Gene | Amplified Product (bp) | Target Amplicon | |
---|---|---|---|
blaKPC [17,18] F R | 5′-CGTTGACGCCCAATCC-3′ 5′-ACCGCTGGCAGCTGG-3′ | 390 | Class A serine enzymes |
blaIMP [17,18] F R | 5′-CATGGTTTGGTGGTTCTTG -3′ 5′-ATAATTTGGCGGACTTTGGC-3′ | 488 | Class B metallo-b-lactamases (MBLs) |
blaVIM [17,18] F R | 5′-ATTGGTCTATTTGACCGCGTC-3′ 5′-TGCTACTCAACGACTGAGCG-3′ | 780 | |
blaNDM-1 [19] F R | 5′-GGTTTGGCGATCTGGTTTTC-3′ 5′-CGGAATGGCTCATCACGATC-3′ | 621 | New Delhi metallo-b- lactamase-1 |
PCR Mix | M1 | M2 | M3 | M4 | M5 | M6 | M7 | M8 |
---|---|---|---|---|---|---|---|---|
Target site (Amplicon length, bp) | HI 1 (534) HI2 (298–308) I1α (159) | M (741) N (514) I2 (316) BO (159) | FIB (683) FIA (462) W (242) | L (854) P (534) X3 (284) I1γ (161) | T (750) A/C (418) FIIS (259–260) | U (843) X1 (370) R (251) FIIK (142–148) | Y (765) X2 (376) FIC (262) K (160) | HIB-M (570) FIB-M (440) FII (258–262) |
Sample | Organism | χ2 | p | |||||
---|---|---|---|---|---|---|---|---|
K. pneumoniae | E. coli | Ent. cloacae | P. aeruginosa | C. freundii | Total | |||
Cutaneous Wound aspirate | 6 (60%) | 4 (50%) | 2 (40%) | 2 (50%) | 0 (0%) | 14 | 26.524 | 0.622 |
Blood culture | 1 (10%) | 0 (0%) | 1 (20%) | 1 (25%) | 1 (33.3%) | 4 | ||
Catheter tip | 1 (10%) | 1 (12.5%) | 0 (0%) | 0 (0%) | 1 (33.3%) | 3 | ||
Sputum | 1 (10%) | 0 (0%) | 1 (20%) | 0 (0%) | 1 (33.3%) | 3 | ||
Peritoneal fluid | 0 (0%) | 1 (12.5%) | 0 (0%) | 1 (25%) | 0 (0%) | 2 | ||
Urine | 0 (0%) | 2 (25%) | 0 (0%) | 0 (0%) | 0 (0%) | 2 | ||
Drainage fluid | 0 (0%) | 0 (0%) | 1 (20%) | 0 (0%) | 0 (0%) | 1 | ||
Endotracheal tube | 1 (10%) | 0 (0%) | 0 (0%) | 0 (0%) | 0 (0%) | 1 | ||
Total | 10 (33.3%) | 8 (26.7%) | 5 (16.7%) | 4 (13.3%) | 3 (10%) | 30 |
Resistance Genes | K. pneumoniae | C. freundii | E. coli | E. cloacae | P. aeruginosa | Total | χ2 | p |
---|---|---|---|---|---|---|---|---|
blaIMP | 1/10(10%) | 1/3 (33.3%) | 2/8(25%) | 3/5 (60%) | 1/4(25%) | 8/30(26.7%) | 4.347 | 0.412 |
blaVIM | 1/10 (10%) | 1/3 (33.3%) | 4/8(50%) | 3/5(60%) | 0/4 (0%) | 9/30(30%) | 7.302 | 0.128 |
blaNDM-1 | 6/10 (60%) | 2/3(66.7%) | 5/8(62.5%) | 1/5(20%) | 1/4(25%) | 15/30(50%) | 4.033 | 0.457 |
blaKPC | 2/10 (20%) | 0/3(0%) | 3/8 (37.5) | 2/5 (40%) | 0/4(0%) | 7/30(23.3%) | 3.866 | 0.470 |
Replicon | Total Frequency | Frequency per Organism | χ2 | p-Value * | ||||
---|---|---|---|---|---|---|---|---|
K. pneumoniae | E. Coli | E. cloacae | P. aeruginosa | C. freundii | ||||
A/C | 6 | 3 (50%) a | 3 (37.5%) a | 0 (0%) a | 0 (0%) a | 0(0%) a | 3.207 | 0.388 |
FIA | 7 | 2 (20%) a | 2 (25%) a | 2 (40%) a | 1 (50%) a | 0(0%) a | 1.190 | 0.932 |
FIB | 8 | 3 (30%) a | 2 (25%) a | 1 (20%) a | 2 (100%) a | 0(0%) a | 4.779 | 0.218 |
FII | 6 | 1 (10%) a | 2 (25%) a | 2 (40%) a | 1 (50%) a | 0(0%) a | 2.522 | 0.541 |
FIIK | 8 | 4 (40%) a | 3 (37.5%) a | 1 (20%) a | 0 (0%) a | 0(0%) a | 1.677 | 0.657 |
FIIS | 2 | 0 (0%) a | 0 (0%) a | 1 (20%) a | 1 (50%) a | 0(0%) a | 7.337 | 0.071 |
HI2 | 1 | 1 (10%) a | 0 (0%) a | 0 (0%) a | 0 (0%) a | 0(0%) a | 1.563 | 1.000 |
I1α | 8 | 5 (50%) a | 3 (37.5%) a | 0 (0%) a | 0 (0%) a | 0(0%) a | 4.894 | 0.188 |
L | 5 | 1 (10%) a | 3 (37.5%) a | 0 (0%) a | 1 (50%) a | 0(0%) a | 4.531 | 0.209 |
FIB-M | 3 | 1 (10%) a | 2 (25%) a | 0 (0%) a | 0 (0%) a | 0(0%) a | 2.273 | 0.673 |
P | 5 | 3 (30%) a | 1 (12.5%) a | 0 (0%) a | 1 (50%) a | 0(0%) a | 3.281 | 0.387 |
R | 7 | 3 (30%) a | 3 (37.5%) a | 1 (20%) a | 0 (0%) a | 0(0%) a | 1.314 | 0.804 |
X1 | 5 | 1 (10%) a | 2 (25%) a | 1 (20%) a | 1 (50%) a | 0(0%) a | 1.875 | 0.727 |
X2 | 1 | 0 (0%) a | 0 (0%) a | 1 (20%) a | 0 (0%) a | 0(0%) a | 4.167 | 0.285 |
X3 | 1 | 1 (10%) a | 0 (0%) a | 0 (0%) a | 0 (0%) a | 0(0%) a | 1.563 | 1.000 |
Y | 2 | 0 (0%) a | 1 (12.5%) a | 1 (20%) a | 0 (0%) a | 0(0%) a | 2.242 | 0.567 |
Total replicon detected | 25 | 10 (100%) a | 8 (100%) a | 5 (100%) a | 2 (50%) a,b | 0 (0%) b | 22.8 | <0.0005 |
Organism | Genes Detected | Plasmid Replicon Detected |
---|---|---|
Klebsiella pneumoniae | _ | I1α FIIK |
Klebsiella pneumoniae | blaNDM | FIB |
Klebsiella pneumoniae | blaNDM | FIIK |
Klebsiella pneumoniae | blaVIM blaNDM | I1α X1 |
Klebsiella pneumoniae | _ | FIA FIB |
Klebsiella pneumoniae | blaIMP blaNDM | A/C X3 |
Klebsiella pneumoniae | blaNDM | FIA FII FIIK I1α FIB-M R |
Klebsiella pneumoniae | blaKPC | A/C HI2 I1α P R |
Klebsiella pneumoniae | _ | A/C FIB L P |
Klebsiella pneumoniae | blaNDM blaKPC | FIIK I1α P R |
Escherichia coli | blaVIM blaKPC | L, FIB-M, R X1 |
Escherichia coli | blaNDM | A/C FIIK I1α R X1 |
Escherichia coli | _ | A/C FIIK L |
Escherichia coli | blaNDM | FIA FIB FII L R |
Escherichia coli | blaIMP bla VIM blaNDM blaKPC | A/C FIA FIB FII I1α FIB-M P Y |
Escherichia coli | blaIMP blaVIM blaNDM | I1α |
Escherichia coli | blaVIM | L |
Escherichia coli | blaNDM blaKPC | FIIK |
Enterobacter cloacae | _ | Y |
Enterobacter cloacae | blaIMP blaVIM blaKPC | FIA FII X2 |
Enterobacter cloacae | _ | X1 |
Enterobacter cloacae | blaIMP blaVIM blaNDM blaKPC | FIA FIB FII FIIS R |
Enterobacter cloacae | blaIMP blaVIM | FIIK |
Pseudomonas aeruginosa | blaNDM | FIA FIB FII FIIS |
Pseudomonas aeruginosa | blaIMP | FIB L P X1 |
Pseudomonas aeruginosa | - | - |
Pseudomonas aeruginosa | - | - |
Citrobacter freundii | - | - |
Citrobacter freundii | blaNDM | - |
Citrobacter freundii | blaIMP blaVIM blaNDM | - |
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Elrahem, A.A.; El-Mashad, N.; Elshaer, M.; Ramadan, H.; Damiani, G.; Bahgat, M.; Mercuri, S.R.; Elemshaty, W. Carbapenem Resistance in Gram-Negative Bacteria: A Hospital-Based Study in Egypt. Medicina 2023, 59, 285. https://doi.org/10.3390/medicina59020285
Elrahem AA, El-Mashad N, Elshaer M, Ramadan H, Damiani G, Bahgat M, Mercuri SR, Elemshaty W. Carbapenem Resistance in Gram-Negative Bacteria: A Hospital-Based Study in Egypt. Medicina. 2023; 59(2):285. https://doi.org/10.3390/medicina59020285
Chicago/Turabian StyleElrahem, Amira Abd, Noha El-Mashad, Mohammed Elshaer, Hazem Ramadan, Giovanni Damiani, Monir Bahgat, Santo Raffaele Mercuri, and Wafaa Elemshaty. 2023. "Carbapenem Resistance in Gram-Negative Bacteria: A Hospital-Based Study in Egypt" Medicina 59, no. 2: 285. https://doi.org/10.3390/medicina59020285