Sudan Journal of Medical Sciences Volume 16, Issue no. 1, DOI 10.18502/sjms.v16i1.8933 Production and Hosting by Knowledge E Research Article Prevalence of blaCTX-M, blaTEM, and blaSHV Genes among Extended-spectrum 𝛽-lactamases-producing Clinical Isolates of Enterobacteriaceae in Different Regions of Sudan Hisham N Altayb1, Mohamed A M Siddig3, Nagwa M El Amin4, and Maowia M. Mukhtar5 1Biochemistry Department, Faculty of Sciences, King Abdulaziz University, Jeddah, 21452, Saudi Arabia 3Botany department, Faculty of Science, University of Khartoum, Sudan 4Department of Microbiology, Faculty of Medicine, Qassim University, KSA 5Bioscience Research Institute, Ibn Sina University, P.O. Box 11463, Khartoum, Sudan ORCID: Mohamed A M Siddig: http://orcid.org/0000-0002-8884-5336 Abstract 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 How to cite this article: Hisham N Altayb, Mohamed A M Siddig, Nagwa M El Amin, and Maowia M. Mukhtar (2021) “Prevalence of blaCTX-M, blaTEM, and blaSHV Genes among Extended-spectrum 𝛽-lactamases-producing Clinical Isolates of Enterobacteriaceae in Different Regions of Sudan,” Sudan Journal of Medical Sciences, vol. 16, Issue no. 1, pages 5–16. DOI 10.18502/sjms.v16i1.8933 Page 5 Corresponding Author: Hisham N Altayb; Biochemistry Department, Faculty of Sciences, King Abdulaziz University, Jeddah, 21452, Saudi Arabia email: hdemmahom@kau.edu.sa Received 7 February 2021 Accepted 20 March 2021 Published 31 March 2021 Production and Hosting by Knowledge E Hisham N Altayb et al. This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited. Editor-in-Chief: Prof. Mohammad A. M. Ibnouf http://www.knowledgee.com mailto:hdemmahom@kau.edu.sa https://creativecommons.org/licenses/by/4.0/ https://creativecommons.org/licenses/by/4.0/ https://creativecommons.org/licenses/by/4.0/ Sudan Journal of Medical Sciences Hisham N Altayb et al 1. Introduction Extended-spectrum beta-lactamases (ESBLs) are the types of enzymes that cause resis- tance to most beta-lactam ring containing antibiotics [1]. Enterobacteriaceae sp. can resist a wide range of antibiotics, including cephalosporin and carbapenems, used as last-line antibiotics [2]. Infections caused by ESBLs-producing Enterobacteriaceae are increasingly being reported worldwide, causing high mortality rates, prolonged hospital stay, and rising medical costs [3]. Escherichia coli and Klebsiella pneumoniae species are considered among species associated with the high spread of ESBLs genes globally, especially the blaCTX-M genes, which have become more common in the last 20 years. Recently, a dramatic increase has been reported in the frequency of blaCTX-M types β-lactamases-producing bacteria, which replaced the predominant types in the past, such as blaTEM and blaSHV [4]. blaCTX-M carrier, E. coli, can disseminate these genes in the community and hospitals from intestinal flora and cause infection [5]. Sudan is one of the many developing countries suffering from irrational use of antibiotics, where 63% of prescriptions contain antibiotics, and various forms of irrational cephalosporins usage are noticed [6, 7]. In Sudan, there is no regulation or system to govern antimicrobial use in humans or animals [8]. Resistance to cephalosporins and production of ESBLs genes in hospitals and environment have been reported in previous studies [9–11]. For the first time, this study aimed to detect blaCTX-M, blaSHV, and blaTEM genes and their subtypes among ESBLs-producing Enterobacteriaceae in different regions of Sudan. 2. Materials and Methods A total of 975 clinical samples (n = 985; urine = 951, wound swab = 17, high vaginal swab = 12, pus = 3, sputum = 2) were collected from four different regions of Sudan – the Haj Alsafi Teaching Hospital in Khartoum (the capital of Sudan); Wad Madani Teaching Hospital in Wad Madani City (the second city in Sudan); Wad Alabass Hospital in Sennar State, Southern Sudan; and Abu Rugba village (remote village) in White Nile State. All samples were collected randomly from symptomatic patients (hospitalized or outpatients) during the study period and cultured on Chromogenic agar media (Liofilchem Co. Italy). Biochemical tests were applied for bacterial identification [12]. Escherichia coli (ATCC 25922), Staphylococcus aureus (ATCC 29213), and K. pneumonia (ATCC 700603) were used as quality-control strains. DOI 10.18502/sjms.v16i1.8933 Page 6 Sudan Journal of Medical Sciences Hisham N Altayb et al 2.1. Antimicrobial susceptibility testing The antimicrobial susceptibility of the selected bacteria was done by the disc-diffusion technique [13]. The following antibiotic discs were used: amikacin (30 μg), ceftazidime (30 μg), cefotaxime (30 μg), cefepime (30 μg), chloramphenicol (30 μg), meropenem (10 μg), and nalidixic acid (30 μg) (Liofilchem Co. Italy). 2.2. Phenotypic detection of ESBLs Phenotypic screening of ESBLs was done according to CLSI guidelines, the following discs were used: cefotaxime (30 μg), ceftazidime (30 μg), and cefepime (30 μg) discs with and without clavulanic acid. Phenotypically, ESBLs-positive isolates showed an increase of ≥5 mm in the zone around discs with clavulanic acid discs compared to the area around the disc without clavulanic acid [13]. 2.3. Identification of extended-spectrum 𝛽-lactamase genes All positive isolates with phenotypic confirmatory tests were subjected to molecular screening to detect β-lactamases genes using a conventional PCR machine. DNA isolation was done by the guanidine hydrochloride method, according to Sabeel et al. [14]. PCR was carried out using primer sequences presented in Table 1 (Metabion, Germany) for blaCTX-M, blaTEM, and blaSHV genes [15, 16]. A reaction volume of 25 μl containing 5 µl Master Mix (iNtRON Biotechnology, Seongnam, Korea), 2 µl DNA, 0.6 μl of each primer, and 16.8 µl DW was used. The PCR steps were firstly subjected to 94°C for 5 min, then 30 cycles (94°C for 45 sec, 57°C for 45 sec, 72°C for 60 sec), and final elongation at 72°C for 5 min. PCR products were run at 2% agarose gel for bands detection by UV Transilluminator. Control positive (obtained from previously sequenced blaCTX-M, blaTEM, and blaSHV genes) and control negative (containing DW, primers, and Master Mix) were used. Sanger sequencing was achieved for both directions of DNA products by Macrogen Company (Seoul, Korea). DNA sequencing was performed for 25 blaCTX-M (7 from Sinnar, 5 from Khartoum, 1 from White Nile, and 12 from Gizera), 3 blaSHV, and 4 blaTEM genes. DOI 10.18502/sjms.v16i1.8933 Page 7 Sudan Journal of Medical Sciences Hisham N Altayb et al 2.4. Statistical analysis Data were analyzed by the statistical package for social science (SPSS) version 16, using the Chi-square test. A P-value < 0.05 was considered significant. 3. Results While 68% (671/985) of the cultured samples showed a significant growth, 32% (314/985) showed no growth. Of the 671 isolates, 305 (45.4%) were Enterobacteriaceae isolates (E. coli = 177, K. pneumoniae = 81, C. freundii complex = 11, Enterobacter species = 19, P. mirabilis = 11, P. vulgaris = 6); 133 (19.7%) were Enterococcus species; 177 (26.3%) were S. aureus; 19 (2.8%) were Pseudomonas aeruginosa; and 37 (5.5%) were yeast cells. More than 45% (139/305) of all Enterobacteriaceae isolates were resistant to cefo- taxime, 39.3% (120/305) to ceftazidime, and 13.4% (41/305) to cefepime. In addition, 92 (30.2%) isolates were resistant to ciprofloxacin, 79 (25.9%) to the nalidixic acid, 21 (6.9%) to meropenem, 99 (32.5%) to chloramphenicol, and 54 (17.7%) to amikacin. There was a statistically significant (P-value < 0.05) association between resistance to third- generation cephalosporins and resistance to other antibiotics used in this study (P-value = 0.001 for all except for chloramphenicol [P = 0.014] and amikacin [P = 0.017]) (Table 2). 3.1. Phenotypic confirmatory test of ESBLs The occurrence of ESBLs genes was confirmed phenotypically in >41% (128/305) of the isolates; 54% (54/100) were in Gizera State (Madani), 63.8% (23/36) in Khartoum State (Haj Alsafi Hospital), 52.1% (48/92) in Sinnar State (Wad Alabas), and 1.3% (1/77) in White Nile State (Abu Rugba Village) (Table 3). There was a statistically significant (P-value < 0.05) association between ESBLs genes and the different regions of Sudan. 3.2. Genotyping of genes Of the 128 isolates that showed ESBLs phenotype, 89% (114/128) were positive for ESBLs genes by PCR; 75% (96/128) were positive for blaCTX-M genes (Figure 1), 42% (40/96) of which were in Gizera, 33% (32.96) in Sinnar, 24% (23/96) in Khartoum, and 1% (1/96) in White Nile; 61% (78/128) were positive for blaTEM genes (Figure 2), and 38% (48/128) were positive for blaSHV genes (Figure 3; Table 4). Twenty-four isolates possessed only DOI 10.18502/sjms.v16i1.8933 Page 8 Sudan Journal of Medical Sciences Hisham N Altayb et al blaCTX-M genes, seven only blaTEM genes, and one only blaSHV gene. Twenty-six isolates possessed the three genes together, seven harbored blaTEM and blaSHV genes, twenty- nine possessed both blaCTX-M and blaTEM genes, and ten harbored blaCTX-M and blaSHV genes. Moreover, 14 isolates (11%) gave negative results for the three genes. 3.3. DNA sequencing blaCTX-M−15 represented 78.3% (18/23) of blaCTX-M genes, blaCTX-M−14 13.1% (3/23), blaCTX-M−27 4.3% (1/23), and blaCTX-M−98 4.3% (1/23). Out of the five blaTEM genes (971 bp), three isolates showed 100% identity with blaTEM−1 (KM598665), while one isolate (isolate-29) showed 99% identity with blaTEM−1 of E. coli from China (AFI61435). DNA sequencing was also done for three blaSHV genes (797 bp); two isolates showed 100% identity (one with blaSHV−28 [ACZ97629] and the other with blaSHV−1 [ACZ97625.1]). The third isolate showed 98% identity with blaSHV−1 (ACZ97624). TABLE 1: Primer sequences used in the detection of genes. Target Primer name Sequence Product size (bp) Annealing Temp. CTX-M MA-1 F MA-2 R SCSATGTGCAGYACC AGTAA CCGCRATATGRT TGGTGGTG 550 57°C TEM C-F D-R TCGGGGAAATGTGCGCG TGCTTAATCAGTGAGGCA CC 971 55°C SHV OS-5-F OS-6-R TTATCTCCCTGTTAGCCACC GATTTGCTGATTTCGCTCGG 797 55°C TABLE 2: The association between antibiotic resistance and ESBLs-producing and non-ESBLs-producing bacteria. Anti-microbial agent ESBL producers Resistant isolates Non-ESBL producers Resistant isolates P-value Ciprofloxacin 66 (72%) 26 (28.2%) 0.001 Nalidixic acid 61 (77%) 18 (22.7%) 0.001 Meropenem 17 (81%) 4 (19%) 0.001 Chloramphenicol 70 (71%) 29 (29.2%) 0.014 Amikacin 43 (80%) 11 (20%) 0.017 DOI 10.18502/sjms.v16i1.8933 Page 9 Sudan Journal of Medical Sciences Hisham N Altayb et al TABLE 3: Phenotypic confirmatory test of ESBLs-producing isolates in different regions of Sudan. Region Confirmatory test P-value CTX/CLA CAZ/CLA CPM/CLA Khartoum (n = 36) 66 (72%) 19 (52.7%) 21 (58.3) 0.001 Gizera (n = 100) 61 (77%) 45 (45%) 44 (44) 0.001 Sinnar (n = 92) 17 (81%) 43 (46.7%) 44 (47.8) 0.0001 White Nile (n = 77) 70 (71%) 1 (1.3%) 1 (1.3) 0.0001 Total (n = 305) 43 (80%) 108 (35.4%) 110 (36) 0.0001 CAZ: ceftazidime; CTX: cefotaxime; CPM: cefepime; CLA: clavulanic acid; ESBLs: extended spectrum beta-lactamases TABLE 4: Frequency of CTX-M TEM and SHV genes among ESBLs-producing Enterobacteriaceae isolates. Isolate CTX-M TEM SHV E. coli (n = 75) 63 (84%) 42 (56%) 24 (32%) K. pneumoniae (n = 34) 21 (62%) 28 (82%) 20 (59%) Enterobacter sp. (n = 7) 5 (71%) 4 (57%) 1 (14%) P. mirabilis (8) 4 (50%) 3 (38%) 2 (25%) P. vulgaris (1) 1 (100%) 0 (0%) 1 (100%) C. freundii complex (n = 3) 2 (66%) 1 (33%) 0 (0%) Total (n = 128) 96 (75%) 78 (61%) 48 (38%) Figure 1: PCR amplification of CTX-M genes. Lane 1 DNA marker (100–1500 bp), lane 2 positive control, lane 3 negative control, lanes 4–10 were positive for CTX-M genes (550 bp). 4. Discussion Several studies exhibited that the prevalence of ESBL-producing bacteria is a serious problem of global public health, and their distribution can be varied according to geographic region, country, and studied institution [17, 18]. DOI 10.18502/sjms.v16i1.8933 Page 10 Sudan Journal of Medical Sciences Hisham N Altayb et al Figure 2: Amplification of TEM genes. Lane 1 DNA marker (100–1500bp), lane 2 positive control, lanes 3–5 were positive for TEM (971 bp), lanes 6–10 are negative samples. Figure 3: PCR amplification of SHV genes separated. Lane 1 DNA marker: MW 100–1500 bp, lane 2 positive control, lanes 3–9 were positive for SHV genes (797 bp), lane 10 is negative control. In this study, we report the increasing rates of ESBLs-producing Enterobacteriaceae compared to other previous study conducted in Khartoum State by Mekki et al. [19], we recorded ESBLs production among Klebsiella sp. and E. coli isolated as 53% in 2010. Moreover, Ahmed et al. [20] had recorded ESBLs production among Enterobac- teriaceae sp. as 59.6% in 2013. This finding in the Sinnar State is higher than that previously reported in 2012 by Hamedelnil and Eltayeb [21], who reported that 36% of 133 isolates were ESBLs producers. In the current study, we report that resistance to cefotaxime (45%) is higher than that to ceftazidime (39.3%) and cefepime (13.4%). We also observed a high ceftazidime- resistant rate (39.3%) within CTX-M-positive isolates, which may be because we report high frequencies of blaCTX-M genes in our isolates. Previous reports indicated several types of blaCTX-M genes exhibiting an increased enzymatic activity against ceftazidime DOI 10.18502/sjms.v16i1.8933 Page 11 Sudan Journal of Medical Sciences Hisham N Altayb et al [22, 23]. A high resistance rate was observed in this study to ciprofloxacin, nalidixic acid, meropenem, chloramphenicol, and amikacin within ESBLs-producing isolates. There was statistically significant (P-value < 0.05) association between resistance to third- generation cephalosporins and these antibiotics (P-value = 0.001, 0.001, 0.001, 0.014, and 0.017, respectively). The possible cause for this phenomenon may be that ESBLs are encoded on mobile plasmids, facilitating its transmission from one organism to another [24]. Furthermore, 89% (114/128) of ESBLs-positive isolates by phenotypic detection were also positive by PCR; blaCTX-M genes were positive in 75% (96/128), blaTEM genes in 61% (78/128), and blaSHV genes in 38% (48/128). This finding agrees with Hamedelnil and Eltayeb [21] and Omar et al. [20], who reported that blaCTX-M genes were the most dominant genes followed by blaTEM and blaSHV genes. High frequencies of blaSHV genes (67.4%) were reported in other studies; Feizabadi et al. [25] reported genes encoding the ESBLs, including blaSHV and blaCTX-M among 89 K. pneumonia isolates by PCR. There were 14 isolates (11%) that gave negative results by PCR but positive by phe- notypic test for ESBLs; this may be due to other ESBLs genes that were not covered by our primers sets. Many studies have confirmed the presence of other ESBLs genes in Enterobacteriaceae like blaVEB-1, blaOXA, and blaPER or may be due to the presence of another mechanism of resistance [26, 27]. Furthermore, 19 ESBLs-positive isolates harbored only blaTEM and blaSHV genes; some of these genes were blaTEM-1, and blaSHV-1 and ESBLs phenomenon may arise from another mechanism of resistance. In the present study, we observed that the cephalosporin-resistance rate and produc- tion of ESBLs genes were higher in urban cities compared to rural areas. In Khartoum, 64% (23/36) of the isolates were resistant to cephalosporin, 54% (54/100) in Madani (Gizera State), and 51% (48/94) in Wad Alabass locality (Sinnar State), while in rural areas such as Abu Rugba village, they were much lower (2.6 % [2/76]). This difference may be because cities are more crowded than rural areas, which facilitates ESBLs spread. Also, antibiotic consumption is higher in cities than in rural areas due to easy access to hospitals and pharmacies, and this is observed in the result of Abu Rugba village, where there is no pharmacy or hospital. Unfortunately, in Sudan, cephalosporins and other antibiotics are sold as over-the-counter medication, explaining the overuse of antibiotics [6, 7]. DOI 10.18502/sjms.v16i1.8933 Page 12 Sudan Journal of Medical Sciences Hisham N Altayb et al Limitations Limited resources prevented us from sequencing all amplified genes, and we selected some samples from different regions to be used as control strains to give us a general idea about the common gene subtypes. 5. Conclusions This study has shown a high prevalence of ESBLs-producing bacteria in different regions of Sudan, especially in big cities than in rural areas. blaCTX-M genes are the most domi- nant genes in ESBLs-producing isolates. This alarming situation of explosive spreading of ESBLs genes, especially blaCTX−𝑀-producing isolates, highlights the need for their epidemiological monitoring. Integrated and regular management of antibiotic consump- tion needs to be monitored in our society to limit their spread. Acknowledgments The authors are thankful to Hassuna Eltayeb from Abu Dhabi Media Co. for the language editing of the manuscript. They also acknowledge Dr. Hiba Salah-Eldin and Dr. Saher M Bakhiet for their support. Ethical considerations The study protocol was approved by the Ethical Research Committee of Sudan Uni- versity of Science and Technology (SUST/DSR/1EC/EA2/2014), Sudan. Because we collected the remaining medical samples with limited data, participant consent was waived. Competing interests None. Availability of data and material All sequencing data were uploaded on GenBank and assigned accession numbers found in the supplementary file. DOI 10.18502/sjms.v16i1.8933 Page 13 Sudan Journal of Medical Sciences Hisham N Altayb et al Funding None. Authors’ contributions HNA, MAM, NME, and MMM contributed to the study design; HNA did the experiments; HNA and NME contributed to data analysis; HNA, MAM, NME, and MMM contributed to manuscript writing; all approved the final version of this article. References [1] Kapoor, D., Kalia, S., and Kalia, A. (2019). 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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. DOI 10.18502/sjms.v16i1.8933 Page 16 Introduction Materials and Methods Antimicrobial susceptibility testing Phenotypic detection of ESBLs Identification of extended-spectrum -lactamase genes Statistical analysis Results Phenotypic confirmatory test of ESBLs Genotyping of genes DNA sequencing Discussion Limitations Conclusions Acknowledgments Ethical considerations Competing interests Availability of data and material Funding Authors' contributions References