Prevalence of blaCTX-M, blaTEM, and blaSHV Genes among Extended-spectrum β-lactamases-producing Clinical Isolates of Enterobacteriaceae in Different Regions of Sudan
Background: This study aimed to characterize blaCTX-M, blaTEM, and blaSHV genes among extended-spectrum beta-lactamases (ESBLs)-producing Enterobacteriaceae species in different regions of Sudan.
Methods: In this cross-sectional study, different clinical samples (n = 985) were collected randomly from symptomatic patients from four geographical regions of Sudan and cultured on chromogenic media. Following bacterial identification, phenotypic screening of ESBLs was done according to CLSI guidelines using cefotaxime (30 μg), ceftazidime (30 μg), and cefepime (30 μg) discs with and without clavulanic acid. The DNA was extracted by guanidine hydrochloride protocol, and then conventional PCR was used to detect blaCTX-M, blaTEM, and blaSHV genes. The presence of genes’ subtypes was characterized by DNA Sanger sequencing for selected samples.
Results: Enterobacteriaceae represented 31% (305/985) of all isolates, 42 (128/305) of which were ESBLs producer, confirmed by phenotypic confirmatory test (75% [96/128] of them were positive for blaCTX-M genes, 61% [78/128] for blaTEM genes, and 38% [48/128] for blaSHV genes). Fourteen isolates (11%) were negative for all genes. Forty-eight percent (63/75) of Escherichia coli isolates were positive for blaCTX-M, while in Klebsiella pneumoniae, the dominant gene was blaTEM (82%) and had a low amount of blaSHV (59%). There was a significant association (P-value = 0.001 for all except for chloramphenicol, P = 0.014, and amikacin, P = 0.017) between resistance to third-generation cephalosporins and ciprofloxacin, nalidixic acid, meropenem, chloramphenicol, and amikacin. Forty-two percent (40/96) of CTX-M-positive isolates were in Gizera State, 33% (32.96) in Sinnar, 24% (23/96) in Khartoum, and 1% (1/96) in White Nile.
Conclusion: We conclude that blaCTX-M genes are the most dominant genes in ESBLs-producing isolates and are more prevalent in big cities than in rural areas.
Keywords: phenotypic, blaCTX-M, blaTEM, and blaSHV ESBLs genes, Enterobacteriaceae, Sudan
 Kapoor, D., Kalia, S., and Kalia, A. (2019). Antibiotic sensitivity pattern of extended spectrum beta-lactamase. The Pharma Innovation Journal, vol. 8, no 2, pp. 703-708.
 Rodrigo‐Troyano, A. and Sibila, O. (2017). The respiratory threat posed by multidrug-resistant Gram‐negative bacteria. Respirology, vol. 22, no. 7, pp. 1288–1299.
 Mohajeri, P., Kavosi, S., Esmailzadeh, T., et al. (2018). Molecular characteristics of extended-spectrum-beta-lactamase-producing Klebsiella pneumoniae isolates in the West of Iran. Advances in Human Biology, vol. 8, no. 3, p. 175.
 Chong, Y., Shimoda, S., and Shimono, N. (2018). Current epidemiology, genetic evolution and clinical impact of extended-spectrum β-lactamase-producing Escherichia coli and Klebsiella pneumoniae. Infection, Genetics and Evolution, vol. 61, pp. 185–188.
 Munday, C., Whitehead, G., Todd, N., et al. (2004). Predominance and genetic diversity of community-and hospital-acquired CTX-M extended-spectrum β-lactamases in York, UK. Journal of Antimicrobial Chemotherapy, vol. 54, no. 3, pp. 628–633.
 Awad, A., Eltayeb, I., Matowe, L., et al. (2005). Self-medication with antibiotics and antimalarials in the community of Khartoum State, Sudan. Journal of Pharmacy & Pharmaceutical Sciences, vol. 8, no. 2, pp. 326–331.
 Sherif, F. M. (2008). An evaluation of the prescribing patterns of rugs in Libya. JMJ, vol. 8, no. 3, pp. 203–206.
 Sirdar, M. M., Picard, J., Bisschop, S., et al. (2012). A survey of antimicrobial residues in table eggs in Khartoum State, Sudan, 2007-2008. Onderstepoort Journal of Veterinary Research, vol. 79, no. 1, pp. 01–09.
 Altayb, H. N., Siddig, M. A., El Amin, N. M., et al. (2018). Molecular characterization of CTX-M ESBLs among pathogenic Enterobacteriaceae isolated from different regions in Sudan. Global Advanced Research Journal of Microbiology, vol. 7, no. 2, pp. 040–047.
 Ahmed, A. M., Mohammed, H., Mohammed, S. A. S., et al. (2019). CTX-M β-lactamase-producing Escherichia coli in SUDAN tertiary hospitals: detection genotypes variants and bioinformatics analysis. International Journal of Medical and Biomedical Studies, vol. 3, no. 3.
 Musa, H. A., Osman, M. A., Abdelaziz, Y. H., et al. (2019). Distribution of extended-spectrum beta-lactamase TEM and CTX-M resistance genes among Proteus species isolated in Sudan. VacciMonitor, vol. 28, no. 2, pp. 80–84.
 Kassim, A., Omuse, G., Premji, Z., et al. (2016). Comparison of Clinical Laboratory Standards Institute and European Committee on Antimicrobial Susceptibility Testing guidelines for the interpretation of antibiotic susceptibility at a University Teaching Hospital in Nairobi, Kenya: a cross-sectional study. Annals of Clinical Microbiology and Antimicrobials, vol. 15, no. 1, p. 21.
 Clinical Lab Standards Institute (CLSI). (2016). Performance Standards For Antimicrobial Susceptibility Testing. CLSI.
 Sabeel, S., Salih, M. A., Ali, M., et al. (2017). Phenotypic and genotypic analysis of multidrug-resistant Mycobacterium tuberculosis isolates from Sudanese patients. Tuberculosis Research and Treatment 2017, vol. 2017, p. 8340746.
 Cao, V., Lambert, T., and Courvalin, P. (2002). ColE1-like plasmid pIP843 of Klebsiella pneumoniae encoding extended-spectrum β-lactamase CTX-M-17. Antimicrobial Agents and Chemotherapy, vol. 46, no. 5, pp. 1212–1217.
 Siddaramappa, S., Pullela, K., Thimmappa, B., et al. (2018). Characterization of blaCTX-M sequences of Indian origin and thirteen uropathogenic Escherichia coli isolates resistant to multiple antibiotics. BMC Research Notes, vol. 11, no. 1, p. 630.
 Canton, R. and Coque, T. M. (2006). The CTX-M beta-lactamase pandemic. Current Opinion in Microbiology, vol. 9, no. 5, pp. 466–475.
 Copur Cicek, A., Saral, A., Ozad Duzgun, A., et al. (2013). Nationwide study of Escherichia coli producing extended-spectrum beta-lactamases TEM, SHV and CTX-M in Turkey. The Journal of Antibiotics, vol. 66, no. 11, pp. 647–650.
 Mekki, A. H., Hassan, A. N., and Elsayed, D. E. M. (2010). Extended spectrum beta lactamases among multi drug resistant Escherichia coli and Klebsiella species causing urinary tract infections in Khartoum. African Journal of Bacteriology Research, vol. 2, no. 3, pp. 18–21.
 Ahmed, O. B., Omar, A. O., Asghar, A. H., et al. (2013). Prevalence of TEM, SHV and CTX-M genes in Escherichia coli and Klebsiella spp urinary isolates from Sudan with confirmed ESBL phenotype. Life Science Journal, vol. 10, no, 2, pp. 191–195.
 Hamedelnil, F. and Eltayeb, H. (2012). Molecular detection of extended spectrum β-lactamases (ESBLs) genes in E. coli isolated from urine specimens. International Journal of Advanced Scientific Research. amedelnil, F. and Eltayeb, H. (2012). Molecular detection of extended spectrum β-lactamases (ESBLs) genes in E. coli isolated from urine specimens. International Journal of Advanced Scientific Research .vol. 5, no. 5, pp. 407-417.
 Poirel, L., Naas, T., Le Thomas, I., et al. (2001). CTX-M-type extended-spectrum β-lactamase that hydrolyzes ceftazidime through a single amino acid substitution in the omega loop. Antimicrobial Agents and Chemotherapy, vol. 45, no. 12, pp. 3355–3361.
 Bae, I. K., Lee, B. H., Hwang, H. Y., et al. (2006). A novel ceftazidime-hydrolysing extended-spectrum β-lactamase, CTX-M-54, with a single amino acid substitution at position 167 in the omega loop. Journal of Antimicrobial Chemotherapy, vol. 58, no. 2, pp. 315–319.
 Lautenbach, E., Patel, J. B., Bilker, W. B., et al. (2001). Extended-spectrum β-lactamase-producing Escherichia coli and Klebsiella pneumoniae: risk factors for infection and impact of resistance on outcomes. Clinical Infectious Diseases, vol. 32, no. 8, pp. 1162–1171.
 Feizabadi, M. M., Mahamadi-Yeganeh, S., Mirsalehian, A., et al. (2010). Genetic characterization of ESBL producing strains of Klebsiella pneumoniae from Tehran hospitals. The Journal of Infection in Developing Countries, vol. 4, no. 10, pp. 609–615.
 Kiratisin, P., Apisarnthanarak, A., Laesripa, C., et al. (2008). Molecular characterization and epidemiology of extended-spectrum-β-lactamase-producing Escherichia coli and Klebsiella pneumoniae isolates causing health care-associated infection in Thailand, where the CTX-M family is endemic. Antimicrobial Agents and Chemotherapy, vol. 52, no. 8, pp. 2818–2824.
 Bauernfeind, A., Stemplinger, I., Jungwirth, R., et al. (1996). Characterization of beta-lactamase gene blaPER-2, which encodes an extended-spectrum class A beta-lactamase. Antimicrobial Agents and Chemotherapy, vol. 40, no. 3, pp. 616–620.