Available online on 15.12.2023 at ijmspr.com

 International Journal of Medical Sciences and Pharma Research 

Open Access to Medical Science and Pharma Research

Copyright  © 2023 The  Author(s): This is an open-access article distributed under the terms of the CC BY-NC 4.0 which permits unrestricted use, distribution, and reproduction in any medium for non-commercial use provided the original author and source are credited

Open Access                                                                                                                                                                                                                                         Research Article

 

Characterization of VIM, VEB and CTX-M Beta-lactamase Gene in Escherichia coli and Pseudomonas aeruginosa Isolated from Urine Samples of Patients Visiting a Tertiary Hospital in Abakaliki

Donatus Chukwuma Ilang ¹, Ikemesit Udeme Peter ² *, Ifeanyichukwu Romanus Iroha ³ 

¹ Department of Microbiology, Alex Ekwueme Federal University, Ndufu-Alike Ikwo, Ebonyi State Nigeria

² Department of Public Health, Federal College of Dental Technology and Therapy, Trans-Ekulu, Enugu, Nigeria

³ Department of Applied Microbiology, Ebonyi State University, Abakaliki, Ebonyi State Nigeria

 

 

INTRODUCTION

Escherichia coli and Pseudomonas aeruginosa are Gram-negative rods associated with health care, and are significant causes for infection with antibiotic resistance burden ^{1, 2, 3, 4, 5}. (Yusof et al., 2022; Ogba et al., 2022; Nomeh et al., 2023; Egwu et al., 2023; Mustafai et al., 2023). The treatment of human infection associated with this pathogen are commonly with beta-lactam antimicrobials agent. The World Health Organization has classified beta-lactam antimicrobials such as extended-spectrum cephalosporins and carbapenems as 'last resort' and 'critically important antimicrobials' because antimicrobial alternatives for treating last resort antimicrobial resistant bacteria are limited ⁶. (WHO, 2019).

Most Escherichia coli and Pseudomonas aeruginosa resistance to beta-lactams is mainly due to the production of beta-lactamases enzymes, which are often encoded either on the chromosome or plasmid ^{2, 3, 4,} (Ogba et al., 2022; Nomeh et al., 2023; Egwu et al., 2023). Beta-lactamases, such as carbapenemase beta-lactamases and extended-spectrum beta-lactamases (ESBLs), are increasingly being identified in food and companion animals, wildlife, humans, and the environment around the world ^{3, 4, 7, 8}. (Egwu et al., 2023; Nomeh et al., 2023; Awosile et al., 2022; Joseph et al., 2023). The spread and convergence of multiple beta-lactamase genes across distinct resistant bacterial populations from various hosts and settings demonstrates that antimicrobial resistance (AMR) is a One Health issue ⁹. (Rousham et al., 2018). Beta-lactamase production in Escherichia coli and Pseudomonas aeruginosa is a public health concern due to the possibility of therapeutic failure, serious consequences for infection control and increased risk of morbidity and mortality in animals and humans ^{7, 10.} (Awosile et al., 2022; EFSA, 2011). The predominant ESBL genes encountered are blaCTX-M, blaTEM, and blaSHV while for carbapenemases, blaOXA-48 and blaNDM-1 have been reported globally ^7. (Awosile et al., 2022).  Although beta-lactamase genes are globally disseminated, they are not equally prevalent among human bacteria. Also, the occurrence and prevalence of these resistance genes varies across different geographic regions. For instance while blaCTX-M is widely disseminated and has been reported in almost every region of the world, blaVIM and VEB has been mostly encountered in Asian and Arabic pennisula in both animal and human hosts ^{11, 12, 13, 14, 15}. (Hashemizadeh et al., 2020; Tian et al., 2018; Haghighi and  Goli, 2022; Haghighifar et al., 2021; Zafer et al., 2014). For this reason, ongoing monitoring of beta-lactamase resistance genes is necessary in order to gain a deeper understanding of the regional distribution of these genes.

MATERIAL AND METHODS

Study area and Duration

The study was conducted at Alex Ekwueme Federal University Teaching Hospital, Abakaliki (AEFUTHA). The tertiary hospital is located at latitude 6.32629167 and longitude 8.11168167 in Abakaliki, Ebonyi State, South eastern Nigeria ¹⁶. (Adibe-Nwafor et al., 2023). The duration of the study was between January-August. 2023.

Ethical Approval

The ethical approval for the study was granted in 2022 by the AE-FUTHA Ethics and Research Committee with reference number: SMOH/ERC/043/22. Every fundamental study was done in accordance with the ARRIVE guidelines.

Sample collection and bacteriological examination

A total of three hundred (300) urine samples were collected from patients visiting AE-FUTHA and the collected samples were subjected to bacteriological examination using culture, Gram staining and biochemical techniques for routine microbiological identification. A loopful of the urine sample were enriched in Brain-heart infusion broth (Merck Co., Germany) and incubated at 35◦C for 18–24 h. After overnight incubation, the turbid broth culture were aseptically streak on solidified plate of CM1046 brilliance™ E. coli Agar (Thermo-fisher Scientific, U. S.A) and Pseudomonas Isolation Agar (PIA) (Merck Co., Germany) and incubated at 35◦C for 18–24 h. After incubation, E. coli and Pseudomonas aeruginosa was identified by standard microbiology techniques such as colonial morphology, Gram staining, motility, and biochemical tests as previously described and further confirmed using the VITEK-2 Automated System (Biomerieux, France)

Antimicrobial susceptibility Testing

This was done by disc diffusion technique on MHA according to the guidelines of the Clinical and Laboratory Standards Institute ¹⁷. (CLSI, 2019). The following antibiotics were tested against isolated bacteria: ceftriaxone (30 µg), aztreonam (30 µg), cefotaxime (30 µg), ceftazidime (30 µg), cefoxitin (30 µg), cefepime (10 µg), aztreonam (30 µg), amoxycillinclavulanic acid (20/10 µg), Piperacillin/tazobactam (30 µg), (Oxoid, UK). Zones of inhibition diameters were measured, recorded, and interpreted as resistant or susceptible according to established criteria ^{18, 19}. (Oke et al., 2020; Uzoije et al., 2021).

Molecular analysis and Amplification of beta-lactamase genes 

The bacteria DNA were extracted according to the manufacturer’s instructions using advanced BioRobot EZ1 XL instrument (QIAGEN, Germany). The Master Mix QuantiTect Probe PCR Kit (QIAGEN, Hilden, Germany) and PCR specific primers were used for amplification of extended spectrum β-lactamase genes (bla_VIM, bla_VEB, and bla_CTX-M) according to previous studies ^{2, 3, 4, 13}. (Ogba et al., 2022; Nomeh et al., 2023; Egwu et al., 2023; Haghighi and  Goli, 2022).   The Amplified gene were detected by electrophoresis on agarose gels containing SYBR-safe (Invitrogen, USA), along with a DNA molecular weight marker (BenchTop pGEM^®DNA Marker, Promega, Madison, WI, USA). Visualization of gels was displayed using The BenchTop pGEM®DNA Marker (Promega, Madison, WI, USA) under ultraviolet illumination.

RESULTS

Of the 300 urine samples samples, the prevalence rate of 187 (62.3%) and 91 (30.3 %) E. coli and P. aeruginosa were recorded through standard microbiology techniques as presented in figure 1. The proportion of 2.5 %, 27.3 % and 45.6 % of E. coli were susceptible to Azetronam, ceftriaxone, cefepime while resistance rate of 100 % to amoxycillin/clavulanic acid and cefoxitin, Piperacillin/tazobactam 89.7 % as shown in figure 2. 

P. aeruginosa exhibit resistance to aztreonam 100 %, cefoxitin 100 %, cefotaxime 91.5 %, amoxycillin/clavulanic acid 89.6 %, ceftriaxone 78.4 % and Piperacillin/tazobactam 50 % but displayed low susceptibility to cefotaxime 8.5 % and amoxycillin/clavulanic acid 10.4 % as presented in figure 3.

The proportion of beta-lactamase genes; blaVEB 143 (76.5 %), blaCTX-M 175(93.5 %), blaVIM 77 (41.2 %) were identified in E. coli while P. aeruginosa harbored blaVEB 91 (100 %), blaCTX-M 63 (69.2%) and blaVIM 48 (52.7 %) as shown in figure 4.

 

 

Figure 1: Percentage prevalence of E. coli and P. aeruginosa

 

Figure 2: Percentage susceptibility profile of E. coli from urine samples

Key: CRO: ceftriaxone (30 µg), ATM: Aztreonam (30 µg), CTX: cefotaxime (30 µg), CAZ: ceftazidime (30 µg), FOX: cefoxitin (30 µg), FEP: cefepime (10 µg), AMC: amoxycillinclavulanic acid (20/10 µg), TZP: Piperacillin/tazobactam (30 µg),

Figure 3: Percentage susceptibility profile of P. aeruginosa from urine samples

Key: CRO: ceftriaxone (30 µg), ATM: Aztreonam (30 µg), CTX: cefotaxime (30 µg), CAZ: ceftazidime (30 µg), FOX: cefoxitin (30 µg), FEP: cefepime (10 µg), AMC: amoxycillinclavulanic acid (20/10 µg), TZP: Piperacillin/tazobactam (30 µg),

Figure 4: Percentage distribution of beta-lactamase gene.

 

DISCUSSION

In this study, bla_CTX-M gene was the most predominant gene in E. coli and the second most predominant gene in P. aeruginosa but such observed prevalence does not undermine it global spread and predominant in different continent of the world. In practice, earlier study, in Kano, Nigeria, bla_CTX-M 73.3% were the most predominant resistance genes in the ESBLs positive E. coli ²⁰. (Saka et al., 2020) while in another study, among the 27 patients with previous hospitalizations, bla_CTX-M were found in 18 (67%) of patients ²¹. (Rodríguez-Baño et al., 2004). In yet another study detection of β-lactamase genes in 59 ESBL-producing E. coli isolates showed high prevalence of bla_CTX-M 69.5%, over other genes, respectively, using specific PCR primers ²². (Mirkalantari et al., 2020) while the frequency of bla_CTX-M in P. aeruginosa has been reported in Jordan 68.9% ²³. (Al-dawodeyahin., 2018), India 7.0% ²⁴. (Murugan et al., 2017), Saudi Arabia 11%, Brazil 2.8% ²⁵. (de Almeida Silva et al., 2017). CTX-M gene are known to contained a plasmid mediated cefotaxime hydrolyzing bla_CTX-M enzyme encoded on a 42 kb plasmid which may be associated with transferable resistance to ceftriaxone, ceftazidime and other advanced generation cephalosporins observed in this study and have been reported as a vital mechanism of resistance ^{26, 27}. (Ugbo et al., 2020; Hussain et al., 2011). 

Our study reports carbapenemase bla_VIM 41.2 % and 52.7 % in E. coli and P. aeruginosa respectively, earlier in the same country this gene was first reported in Kano ²⁸ (Aminu, et al., 2022) and Abakaliki, Nigeria ³.(Nomeh et al., 2023).  Elsewhere isolate positive for the bla_VIM gene has been found by other researchers ^{11, 12, 29}. (Hashemizadeh et al., 2020; Tian et al., 2018; Murugan et al., 2019). The dissemination of bla_VIM among Gram-negative rod should not be underrate and in recent time, bla_{VIM, IMP, NDM} have become the two most prevalent class B carbapenemases in worldwide P. aeruginosa isolates ^30.  (Yoon and Jeong, 2021).

The rate of bla_VEB gene was 76.5 % and 100 % in E. coli and P. aeruginosa respectively. The bla_VEB was first detected in Escherichia coli isolates in Vietnam in 1996 and then detected in many members of Enterobacteriaceae and Pseudomonas ^14. (Haghighifar et al., 2021). In an Egyptian study conducted by ¹⁵. Zafer et al. in 2014, 7.4% of the P. aeruginosa isolates were ESBL positive, while 10.4% of them carried the bla_VEB-1 gene, and 60.6%, 45.1%, and 25.4% of their isolates were resistant to ceftazidime, aztreonam, and piperacillin-tazobactam, respectively ¹⁵. (Zafer et al., 2014) while 93.02% P. aeruginosa carried bla_VEB-1 gene in Iran ¹³. (Haghighi and  Goli, 2022). This genotype are highly prevalent and significant in clinical isolates of P. aeruginosa ¹³. (Haghighi and  Goli, 2022). The bla_VEB enzyme also promotes resistance to ceftazidime, aztreonam, and cefepime. In the present study, 50.0%-100% of the isolates resistant to aztreonam, ceftazidime, ceftriaxone, cefuroxime, indicating the important role of the bla_VEB enzyme in resistance to these antibiotics. The genes encoding these enzymes are usually carried by transposons and can move between Gram-negative bacteria ³¹ (John-Onwe et al., 2023) and may cause the creation of MDR strains that make it difficult to choose the appropriate treatment for any GNB disease condition.

CONCLUSION

Our study provides an in-depth characterization of bla_VIM, bla_VEB and bla_CTX-M beta-lactamase gene and the proportion of the amplified gene reported in our study may apparently revealed the endemic convergence of this gene in our study area. Thus, it is of paramount importance for each hospital to have an antibiotic guidance or stewardship program for all pharmacists and the physicians based on the most accurate microbiological data. In conjunction with this guidance, a continuous effort in hospital surveillance, infection control, and clinical audits must be conducted to fight against the rapid development of antibiotic-resistant pathogens.

Acknowledgment

We sincerely appreciate Professor Ifeanyichukwu Romanus Iroha and Staff of Alex Ekwueme Federal University Teaching Hospital, Abakaliki (AEFUTHA) for their unflinching support.

Author’s Contribution

All authors investigated the study, did literature searches and did data Validation and Visualization. All the authors reviewed and approved the final draft, and are responsible for all aspects of the work

Funding Source: None 

Conflict of Interest: None 

Ethical consideration: Ethical approval with reference No: AEFUTHA/ERC/179/R22 was obtained from the Research and Ethics Committee of AEFUTHA.

REFERENCES 

1. Yusof, N.Y., Norazzman, N.I.I., Hakim, S.N. W. A., Azlan, M.M., Anthony, A.A., Mustafa, F.H., Ahmed, N., Rabaan, A.A., Almuthree, S.A., Alawfi, A. Prevalence of Mutated Colistin-Resistant Klebsiella pneumoniae: A Systematic Review and Meta Analysis. Tropical Medicine and Infectious Disease, 7:414-419. https://doi.org/10.3390/tropicalmed7120414 PMid:36548669 PMCid:PMC9782491

2. Ogba, R. C., Nomeh, O. L, Edemekong, C. I., Nwuzo, A. C., Akpu, P. O., Peter, I. U and Iroha, I. R. Molecular Characterization of Carbapenemase Encoding Genes in Pseudomonas aeruginosa from Tertiary Healthcare in South Eastern Nigeria. Asian Journal of Biology, Genetic and Molecular Biology, 2022;12(4):161-168. https://doi.org/10.9734/ajbgmb/2022/v12i4281

3. Nomeh, L. O., Federica, O. I., Joseph, O. V., Moneth, E. C., Ogba, R. C., Nkechi, O. A., Peter, I. U., Akpu, P. O., Edemekong, C. I and Iroha, I. R. Detection of Carbapenemase-Producing Escherichia coli and Klebsiella pneumoniae Implicated in Urinary Tract Infection. Asian Journal of Research in Infectious Diseases, 2023;2 (1):15-23. https://doi.org/10.9734/ajrid/2023/v12i1234

4. Mustafai, M. M., Hafeez, M., Munawar, S., Basha, S., Rabaan, A. A., Halwani, M. A., Alawfi, A., Alshengeti, A., Najim, M. A and Alwarthan, S. Prevalence of Carbapenemase and Extended-Spectrum β-Lactamase Producing Enterobacteriaceae: A Cross-Sectional Study. Antibiotics, 2023;12:148-149 https://doi.org/10.3390/antibiotics12010148 PMid:36671350 PMCid:PMC9854900

5. Egwu, E., Ibiam, F. A, Moses, I. B., Iroha, C. S Orji, I., Okafor-Alu, F. N., Eze, C. O and Iroha, I. R. Antimicrobial Susceptibility and Molecular Characteristics of Beta-lactam- and Fluoroquinolone-resistant E. coli from Human Clinical Samples in Nigeria. Scientific African 2023;21:18-63 https://doi.org/10.1016/j.sciaf.2023.e01863

6. World Health Organization. Critically important antimicrobials for human medicine: Ranking of antimicrobial agents for risk management of antimicrobial resistance due to non-human use [homepage on the Internet]. Vol. 5th rev. Geneva; 2017. Available from: http://apps.who.int/iris/bitstre am/10665/255027/1/9789241512220-eng.pdf 

7. Awosile BB, Agbaje M, Adebowale O, Kehinde O, Omoshaba E. Beta-lactamase resistance genes in Enterobacteriaceae from Nigeria. Afr J Lab Med. 2022;11(1):a1371 https://doi.org/10.4102/ajlm.v11i1.1371 PMid:35282396 PMCid:PMC8905388

8. Joseph I S, Okolo I O, Udenweze E C, Nwankwo C E, Peter I U, Ogbonna I P, Iroha I R, Comparison of Antibiotic-Resistant Pattern of Extended Spectrum Beta-Lactamase and Carbapenem- Resistant Escherichia coli Isolates from Clinical and Non-Clinical Sources, Journal of Drug Delivery and Therapeutics, 2023; 13(7):107-118 https://doi.org/10.22270/jddt.v13i7.5918

9. Rousham EK, Unicomb L, Islam MA. Human, animal and environmental contributors to antibiotic resistance in low-resource settings: Integrating behavioural, epidemiological and one health approaches. Proc Roy Soc B: Biol Sci. 2018;285(1876):20180332. https://doi.org/10.1098/rspb.2018.0332 PMid:29643217 PMCid:PMC5904322

10. EFSA Panel on Biological Hazards (BIOHAZ). Scientific opinion on the public health risks of bacterial strains producing extended-spectrum β-lactamases and/or AmpC β-lactamases in food and food-producing animals: ESBL/AmpC in foodproducing animals and foods. EFSA J. 2011;9(8):2322. https://doi.org/10.2903/j.efsa.2011.2322

11. Hashemizadeh, Z., Hosseinzadeh, Z., Azimzadeh, N and Motamedifar, M. Dissemination Pattern of Multidrug Resistant Carbapenemase Producing Klebsiellapneumoniae Isolates Using Pulsed-Field Gel Electrophoresis in Southwestern Iran. Infection and Drug Resistance, 2020;13:921-929. https://doi.org/10.2147/IDR.S227955 PMid:32280248 PMCid:PMC7125322

12. Tian, X., Wang, Q., Perlaza-Jiménez, L., Zheng, X., Zhao, Y., Dhanasekaran, V., Fang, R., Li, J., Wang,C., Liu, H., Lithgow, T., Cao, J and Zhou, T. First Description of Antimicrobial Resistance in Carbapenem-susceptible Klebsiella pneumoniae after Imipenem Treatment, driven by Outer Membrane Remodeling. Biomedical Complement of Microbiology, 2020;20:218-219 https://doi.org/10.1186/s12866-020-01898-1 PMid:32689945 PMCid:PMC7372807

13. Haghighi, S and Goli, H R. High prevalence of blaVEB, blaGES and blaPER genes in beta-lactam resistant clinical isolates of Pseudomonas aeruginosa. AIMS Microbiology, 2022;8(2):153-166 https://doi.org/10.3934/microbiol.2022013 PMid:35974990 PMCid:PMC9329875

14. Haghighifar, E., Dolatabadi, R. K and Norouzi, F. Prevalence of blaVEB and blaTEM genes, antimicrobial resistance pattern and biofilm formation in clinical isolates of Pseudomonas aeruginosa from burn patients in Isfahan, Iran. Gene Report, 2021;23:10-45 https://doi.org/10.1016/j.genrep.2021.101157

15. Zafer, M. M., Al-Agamy, M. H., El-Mahallawy, H. A., Amin, M. A and Ashour, M. S. Antimicrobial Resistance Pattern and their Beta-lactamase Encoding Genes among Pseudomonas aeruginosa Strains isolated from cancer patients. Biomedical Research International, 2014;101635(10):23-34. https://doi.org/10.1155/2014/101635 PMid:24707471 PMCid:PMC3953503

16. Adibe-Nwafor, J. O., Uduku, N. D., Iroha, C. S., Ibiam, F. A., Onuora, A. L., Nwafor, K. A., Peter, I. U., Iroha, I. R. Distribution and Antibiotic Resistance Profile of Extended Spectrum Beta-Lactamase Producing Escherichia coli from Fish Farms within Abakaliki Metropolis. Advance in Research, 2023;24(5):175-184. https://doi.org/10.9734/air/2023/v24i5968

17. Clinical and Laboratory Standards Institute (CLSI). Performance standards for antimicrobial susceptibility testing; twenty-eighth edition (M100). Wayne, PA: Clinical and Laboratory Standards Institute; 2019

18. Oke B, Iroha I R, Moses I B, Egwu I H, Elom E, Uzoh C V, et al. Killing Rate Kinetics of Commercially Available Brands of Ciprofloxacin and Cefotaxime on Clinical Bacterial Isolates Subjected to in vitro Antibiotic Treatments. Int. J. Pharm. Sci. Rev. Res. 2020;64(2):87-97 https://doi.org/10.47583/ijpsrr.2020.v64i02.015

19. Uzoije U N, Moses I B, Nwakaeze E A, Uzoeto H O, Otu J O, Egbuna N R, et al. Prevalence of Multidrug-resistant Bacteria Isolates in Waste Water from Different Hospital Environment in Umuahia, Nigeria Int. J. Pharm. Sci. Rev. Res. 2021;69(2):25-32. https://doi.org/10.47583/ijpsrr.2021.v69i02.003

20. Saka, H. K., García-Soto, S., Dabo, N. T., Lopez-Chavarrias, V., Muhammad, B., Ugarte-Ruiz M aand Alvarez, J. Molecular detection of Extended Spectrum β-lactamase Genes in Escherichia coli Clinical Isolates from Diarrhoeic Children in Kano, Nigeria. Public Library of Science One, 2020;15(12):24-3130 https://doi.org/10.1371/journal.pone.0243130 PMid:33270734 PMCid:PMC7714196

21. Rodríguez-Baño, J., Navarro, M. D., Romero, L., Martínez-Martínez, L., Muniain, M.A., Perea, E. J., Pérez-Cano, R and Pascual, A. Epidemiology and Clinical Features of Infections Caused by Extended-Spectrum Beta-Lactamase-Producing Escherichia coli in Non-hospitalized Patients. Journal of clinical Microbiology, 2004; 42(3):1089-1094 https://doi.org/10.1128/JCM.42.3.1089-1094.2004 PMid:15004058 PMCid:PMC356843

22. Mirkalantari, S., Masjedian, F., Irajian, G., Siddig, E E and Fattahi, A. Determination of the frequency of β-lactamase genes (bla SHV, bla TEM, bla CTX-M) and Phylogenetic Groups among ESBL-producing Uropathogenic Escherichia coli isolated from outpatients. Journal of Laboratory Medicine, 2020; 23:24-67 https://doi.org/10.1515/labmed-2018-0136

23. Al-dawodeyahin, H. Y., Obeidat, N., Abu-Qatouseh, L. F and Shehabi, A. A. Antimicrobial Resistance and Putative Virulence Genes of Pseudomonas aeruginosa Isolates from Patients with Respiratory Tract Infection. Germs, 2018;8:31-40. https://doi.org/10.18683/germs.2018.1130 PMid:29564246 PMCid:PMC5845973

24. Murugan, N., Malathi, J., Therese, K. L and Madhavan, H. N. R. Application of six multiplex PCR's among 200 clinical isolates of Pseudomonas aeruginosa for the Detection of 20 Drug Resistance Encoding Genes. Kaohsiung Journal of Medical Science, 2017;1:9-10. https://doi.org/10.1016/j.kjms.2017.09.010 PMid:29413231

25. de Almeida Silva K de C. F., Calomino, M. A., Deutsch, G., de Castilho, S. R., de Paula, G. R and Esper, L. M. R. Molecular Characterization of Multidrug-resistant (MDR) Pseudomonas aeruginosa Isolated in a Burn Center. Burns, 2017;43:137-43. https://doi.org/10.1016/j.burns.2016.07.002 PMid:27595453

26. Ugbo EN, Anyamene CO, Moses IB, Iroha IR, Babalola OO, Ukpai EG, Chukwunwejim CR, Egbule CU, Emioye AA, Okata-Nwali OD, Igwe OF, Ugadu, IO, Prevalence of blaTEM, blaSHV, and blaCTX-M genes among extended spectrum beta-lactamase-producing Escherichia coli and Klebsiella pneumoniae of clinical origin, Gene Report, 2020; 21:34-45 https://doi.org/10.1016/j.genrep.2020.100909

27. Hussain, M., Hasan, F., Shah, A. A., Hameed, A., Jung, M and Rayamajhi, N. Prevalence of Class A and AmpC β-lactamases in Clinical Escherichia coli Isolates from Pakistan Institute of Medical Science, Islamabad, Pakistan. Japanese Journal of Infectious Disease, 2011;64(3):249-252. https://doi.org/10.7883/yoken.64.249

28. Aminu, A., Daneji, I. M., Yusuf, M. A., Jalo, R. I., Tsiga-Ahmed, F. I and Yahaya, M. Carbapenem resistant Enterobacteriaceae Infections among Patients Admitted to Intensive Care Units in Kano, Nigeria. Sahel Medical Journal, 2021;24:1-9.

29. Murugan, M. S., Sinha, D. K., Vinodh Kumar, O. R., Yadav, A. K., Pruthvishree, B. S., Vadhana, P., Nirupama, K. R., Bhardwaj, M and Singh, B. R (2019). Epidemiology of Carbapenem Resistant Escherichia coli and First Report of blaVIM Carbapenemases Gene in Calves from India. Epidemiology and Infection, 147:159, 1-5. https://doi.org/10.1017/S0950268819000463 PMid:31063112 PMCid:PMC6518490

30. Yoon, E. J and Jeong, S. H. Mobile Carbapenemase Genes in Pseudomonas aeruginosa. Frontier in Microbiology, 2021;12:614-058. https://doi.org/10.3389/fmicb.2021.614058 PMid:33679638 PMCid:PMC7930500

31. John-Onwe BN, Ibiam FA, Udenweze EC, Iroha CS, Edemekong CI, Peter IU, Iroha IR, Co-expression of Extensively drug resistant (XDR) clinical isolates of Pseudomonas aeruginosa harboring FOX and MOX ampicillinase Gene, International Journal of Medical Sciences & Pharma Research, 2023;9(3):14-19 https://doi.org/10.22270/ijmspr.v9i3.76