28 Original ISSN 2413-0516 J Contemp Med Sci | Vol. 7, No. 1, January-February 2021: 28 – 33 Introduction The wrong use of antibiotics in medicine has been considered a principal start leading to the emergence of bacteria resist to many antibiotics (multidrug resistance).1,2 Because of the main sources for transmission of extended-spectrum β-lactamase (ESBL)-producing bacteria to humans are animals, hence some measures were raised recently to reduce antimicrobial agents use in husbandry of animal in Europe.3 This ESBLs may be transmitted either directly or indirectly by contaminated meat products consumption.4 ESBLs bacteria can hydrolyze cephalosporin and pen- icillin antibiotics by production of β-lactamases enzymes. So, Gram-negative bacteria in the intestine belonging to Enterobacteriaceae becomes resistant to these antibiotic classes when they possess an ESBL gene. ESBLs are prevalent globally with more than 1.5 billion people colonized Enterobacteriaceae especially with ESBLs.5 Most of this load falls on the develop- ing countries, but the pervasiveness of organisms producing ESBLs increased in the developed countries. There are many ESBLs groups with the same attitude but different in evolution. The largest ones are TEM and SHV β-lactamases mutants. Some critical amino acids were affected by the mutation resulting in enlargement in active sites and enable it to shield the ring of β-lactam by deflecting the oxy- imino substitutes.6 CTX-M enzymes is the second largest group. These are divided into five subgroups based on sequence homology. Enterobacteriaceae (especially E. coli) that produce the CTX-M enzymes have been identified to cause urinary tract causing infections.7,8 Many studies reported the ESBLs CTX-M-type is the most frequent ESBL type worldwide.6 ESBLs are enzymes leading to increase resistance to azt- reonam, cefotaxime, ceftazidime, cephalosporins, and penicil- lin, while clavulanic acid inhibits them. The CTX-M, TEM, and SHV are the three major types of ESBLs. The CTX-M is more prevalent than TEM and SHV has a distribution among a broad range of clinically important bacteria.7,8 The critical patients are susceptible to infection particu- larly, and the nature of causative agents and epidemiology can very extremely. The pathogens that are drug-resistant are con- sidered as a source of concern as they carry a higher mortality and morbidity, and are difficult to be routinely identified by laboratory assays. This leads to delay in diagnosis and finding the appropriate therapy by antimicrobial. There is also a rising worry regarding the new antibiotics deficiency,9 especially for ESBLs Gram-negative bacteria. Drug-resistant strains of Enterobacteriaceae are import- ant10; thus, the aim of the present study was to determine the Enterobacteriaceae strains among patients who are suffering from diarrhea, phenotypic, and genotypic characterization of ESBL-producing isolates in Jeddah, Saudi Arabia. Extended-spectrum β-lactamase Enterobacteriaceae from patients in Jeddah, Saudi Arabia: Antibiotic susceptibility and molecular approaches Wafa A. Alshehri1, Tarek A. A. Moussa2* 1 Department of Biology, College of Science, University of Jeddah, Jeddah, Saudi Arabia. 2 Botany and Microbiology Department, Faculty of Science, Cairo University, Giza, Egypt. *Correspondence to: Wafa A. Alshehri (E-mail: waalshehri@uj.edu.sa) (Submitted: 23 November 2020 – Revised version received: 05 December 2020 – Accepted: 19 December 2020 – Published online: 26 February 2021) Abstract Objective This study aimed to determine the Enterobacteriaceae strains among patients who are suffering from diarrhea, phenotypic, and genotypic characterization of extended-spectrum β-lactamase (ESBL)-producing isolates in Jeddah, Saudi Arabia. Methods Stool samples were collected and cultured to determine the different Enterobacteriaceae strains. PCR was done for the isolates to detect the different ESBLs genes and antibiotic susceptibility against different antibiotics. Results The total number of patients in this study was 200 (114 males [57%] and 86 females [43%]). The patients were categorized to teenagers (21, 10.5%), adults (92, 46%), middle age (25, 12.5%), and elderlies (25, 12.5%) according to age. Five Enterobacteriaceae strains were found: Enterobacter cloaca (7, 3.5%), Escherichia coli (111, 55.5%), Klebsiella pneumoniae (75, 37.5%), Proteus mirabilis (6, 3.0%), and Pseudomonas aeruginosa (1.0, 0.5%). P. aeruginosa was absent in all female patients under investigation. The response of the isolated (E. coli, K. pneumoniae, and E. cloaca) strains to ampicillin, cefotaxime, cefotaxime-clavulanate, ceftriaxone, cephalothin, chloramphenicol, ciprofloxacin, nalidixic acid, streptomycin, tetracycline, and trimethoprim-sulphamethoxazole was highly resistant, while the response was highly susceptible to ampicillin sulbactam, ceftazidime, ceftazidime-clavulanate, and imipenem. The most frequent gene was bla CTX-M (195) followed by bla TEM (149), bla SHV (73), and bla OXA (3), while the highest pair of genes in the same organism was bla TEM +bla CTX-M (134) followed by bla SHV +bla CTX-M (64), bla SHV +bla TEM (52), and the least pairs were bla TEM +bla OXA (3) and bla CTX-M +bla OXA (2). bla SHV +bla CTX-M +bla TEM was found in 44 organisms and bla CTX-M +bla TEM +bla OXA in 2 organisms only. The bla SHV +bla OXA , bla SHV +bla CTX-M +bla OXA , bla SHV +bla TEM +bla OXA , and bla CTX- M +bla TEM +bla OXA +bla SHV were not present in any organism under investigation. Conclusions In teenager group, there were no organism that contained bla OXA gene, while bla OXA was present in E. cloaca and P. mirabilis only. bla SHV gene was absent in E. coli but present in E. cloaca and K. pneumoniae. The most susceptible group to infection with Enterobacteriaceae was adults’ group, while teenage was more resist to infection. Keywords ESBLs, Antibiotics, Molecular, Virulence genes, Enterobacteriaceae 29 Original Extended-spectrum β-lactamase Enterobacteriaceae from patients in Jeddah, Saudi ArabiaWafa A. Alshehri, Tarek A. A. Moussa J Contemp Med Sci | Vol. 7, No. 1, January-February 2021: 28 – 33 Materials and Methods Sample collection and isolation Stool samples were collected from 200 diarrheal patients at different hospitals in Jeddah, Saudi Arabia. The samples were transferred to the laboratory within 2 h for isolation of bacte- ria. The samples were streaked on MacConkey agar with crys- tal violet plates (Difco, Detroit, MI, USA). After incubation at 37°C overnight, the colonies were picked up and transferred to new plates. The purified isolates were identified biochemically by the API 20E (Biomerieux, France). Antibiotic susceptibility and detection of ESBLs The susceptibility of isolated bacteria to antibiotics was tested by the Kirby-Bauer agar disc diffusion method. Susceptibility to ampicillin (AMP, 10 μg), amoxicillin/clavulanate (AMC, 30 μg), ampicillin sulbactam (SAM, 20 μg), cefotaxime (CTX, 30 μg), cefotaxime/clavulanate (CTL, 30 μg/10 μg), ceftazidime (CAZ, 30 μg), ceftazidime/clavulanate (CAL, 30/10 μg), ceftri- axone (CRO, 30 μg), cephalothin (CEF, 30 μg), chloramphen- icol (CHL, 30 μg), ciprofloxacin (CIP, 5 μg), imipenem (IPM, 10 μg), nalidixic acid (NAL, 10 μg), streptomycin (STR, 10 μg), tetracycline (TET, 30 μg), and trimethoprim-sulphamethox- azole (SXT, 25 μg) was determined according to the criteria of the Clinical Laboratory Standards Institute.11 All antibiotics were purchased from Oxoid (Italy). The double-synergy test was used to screen the ESBL activity of the isolated bacteria.12 Characterization of ESBL producing bacteria Genomic DNA was extracted from the ESBL producing bacteria according to the manufacturer instructions using mericon DNA Bacteria (Plus) Kit (Qiagen, Valencia, CA, USA). The encoding genes of β-lactamase enzymes was amplified using the primers as follows: blaCTX-M F-ATGTGCAGYACCAGTAARGTKATGGC, R-TGGGTRAARTARGTS ACCAGAAYCAGCGG (593 bp); blaOXA F-ACACAATACATATCAACTTCGC, R- AGTG TGTTTAGAATGGTGATC (813 bp); blaSHV F-CTTTATCGGCCCTCACTCAA, R-AGGTG CTCATCATGGGAAAG (327 bp); blaTEM F - C G C C G C A T A C A C T A T T C T C A G A A T G A , R-ACGCTCACCGGCTCCAGATTTAT (445 bp). PCR was then performed in total volume (50 µl) reaction mixture: DNA templet (50 ng), dNTPs (0.25mM), MgCl2 (1.5 mM), Pfu DNA polymerase (0.2 U), primers (50 pmol) and complete to 50 µl with distilled H2O. The temperature profile included an initial denaturation step at 95°C for 10 min, followed by 35 cycles of 95°C for 30 s, 55°C for 1 min, and 72°C for 1 min and a final extension step at 72°C for 7 min. Results In modern medicine, one of the greatest challenges is antimi- crobial resistance.13,14 The most used classes in the infection treatment caused by Gram-negative pathogenic bacteria are the combination of β-lactam/β-lactamase, cephalosporins, and fluoroquinolones inhibitor due to their safety, available in both oral forms and parenteral, and efficacy. The resistance to these agents would restrict the empiric treatment efficacy of Gram-negative infections and also, limit the options of their treatment. In this study, the total patient’s number was 200 (114 males [57%] and 86 females [43%]) (Fig. 1). The patients were categorized to teenagers 13–19 years (21, 10.5%), adults 20–40 years (92, 46%), middle age 41–59 years (25, 12.5%) and elder- lies 60 < years (25, 12.5%). It was found that the number of females was greater than males in teenager category only (15 vs 6), while the number of males was greater than females in the rest three categories (Table 1, Fig. 2). Five Enterobacteriaceae strains was found in the sam- ples collected where Enterobacter cloaca (7, 3.5%), Escherichia coli (111, 55.5%), Klebsiella pneumoniae (75, 37.5%), Proteus mirabilis (6, 3.0%), and Pseudomonas aeruginosa (1.0, 0.5%) (Table 2). E. coli and K. pneumoniae were the most dominant Enterobacteriaceae strains in the patients under investigation. The prevalence of E. coli was noticed among males of adults, middle age, and elderlies categories in comparing to the other four strains. P. aeruginosa was absent in all female patients under investigation, also it is noticed that the high prevalence of E. cloaca in both female and male in middle age category (Table 3, Fig. 3). Fig. 1 Percentage of male and female in the collected samples. (n=8665). Table 1. Age grouping of the patients suffering from diarrhea. Category Age group Total Female Male Number % Number % Number % Teenager 13-19 21 10.5 15 71.4 6.0 28.6 Adults 20-40 92 46.0 38 41.3 54 58.7 Middle Age 41-59 62 31.0 25 40.3 37 59.7 Elderlies 60 < 25 12.5 8.0 32.0 17 68.0 Total - 200 100 86 43.0 114 57.0 30 Original Extended-spectrum β-lactamase Enterobacteriaceae from patients in Jeddah, Saudi Arabia Wafa A. Alshehri, Tarek A. A. Moussa J Contemp Med Sci | Vol. 7, No. 1, January-February 2021: 28 – 33 Fig. 2 Percentage of male and female in the different category. Male: outer circle and female inner circle. Table 3. Distribution of ESBLs organisms among the group categories. Category E. coli K. pneumoniae E. cloacae P. aeruginosa P. mirabilis Female Male Female Male Female Male Female Male Female Male Teenager 8.0 3.0 7.0 3.0 0.0 0.0 0.0 0.0 0.0 0.0 Adults 19.0 34.0 15.0 16.0 1.0 1.0 0.0 1.0 2.0 2.0 Middle Age 14.0 18.0 8.0 18.0 2.0 2.0 0.0 0.0 0.0 1.0 Elderlies 5.0 10.0 3.0 5.0 0.0 1.0 0.0 0.0 0.0 1.0 Total 46.0 65.0 33.0 42.0 3.0 4.0 0.0 1.0 2.0 4.0 Total 111.0 75.0 7.0 1.0 6.0 Discussion E. coli (65%), Klebsiella spp. (25%) Pseudomonas (5%), Enterobacter spp. (4%), and Acinetobacter spp. (2%) were detected in three tertiary care hospitals samples in Lahore, Pakistan.15 A few Enterobacteriaceae species including Enterobacter aerogenes, E. cloacae, E. coli, K. pneumoniae, P mirabilis, and Serratia marcescens are responsible for most infections produced by this family.16,17 The crucial factor in increasing antimicrobial resistance was the production of β-Lactamase in Gram-negative bacteria, like ESBLs CTX-M and SHV and K. pneumoniae carbapenemase.14,18 The broad spreading of the ESBLs has left patients and clinicians with very limited options in the infections treatment caused by multidrug resistant (MDR) Enterobacteriaceae.19–21 The antibiotic susceptibility test was performed, and the results presented in Table 4. The response of the isolated E. coli strains to ampicillin (AMP), cefotaxime (CTX), cefotax- ime-clavulanate (CTL), ceftriaxone (CRO), cephalothin (CEF), chloramphenicol (CHL), ciprofloxacin (CIP), nalidixic acid (NAL), streptomycin (STR), tetracycline (TET), and trimetho- prim-sulphamethoxazole (SXT) was highly resistant while the response of the isolated E. coli strains to amoxicillin-clavulanic acid (AMC) was moderately susceptible, while to ampicillin sulbactam (SAM), ceftazidime (CAZ), ceftazidime-clavula- nate (CAL), and imipenem (IPM) was highly susceptible. The response of K. pneumoniae strains to AMP, CTX, CTL, CRO, CEF, CHL, CIP, STR, TET and SXT was highly resistant while the response to AMC and NAL was moderately susceptible, and to SAM, CAZ, CAL, and IPM was highly susceptible. The response of E. cloaca was highly resistant to AMP, AMC, CTX, CRO, CEF, CHL, NAL, STR, TET and SXT, while the response was moderately susceptible to CTL and CIP, and highly sus- ceptible to SAM, CAZ, CAL, and IPM (Table 4). Enterobacteriaceae isolates resistant to ceftazidime-avi- bactam were evaluated for the presence of ESBLs encod- ing genes.22,23 More than 99.9% of Enterobacteriaceae was inhibited by ceftazidime-avibactam. Only 82.2% of MDR Enterobacteriaceae and 64.2% of ceftriaxone-non-susceptible Table 2. ESBLs producing organisms detected in the collected samples. Isolate Total Female Male Number % Number % Number % Enterobacter cloacae 7.0 3.5 3.0 42.9 4.0 57.1 Escherichia coli 111.0 55.5 46.0 41.4 65.0 58.6 Klebsiella pneumoniae 75.0 37.5 33.0 44.0 42.0 56.0 Proteus mirabilis 6.0 3.0 2.0 33.3 4.0 66.7 Pseudomonas aeruginosa 1.0 0.5 0.0 0.0 1.0 100 Total 200 100 84 42.0 116 58.0 31 Original Extended-spectrum β-lactamase Enterobacteriaceae from patients in Jeddah, Saudi ArabiaWafa A. Alshehri, Tarek A. A. Moussa J Contemp Med Sci | Vol. 7, No. 1, January-February 2021: 28 – 33 Fig. 3 The number of the five isolated organisms in different categories. Table 4. The percentage of antimicrobial resistance ESBL producing isolates. Antibiotic Abb. Conc. (µg) E. coli (n = 111) K. pneumoniae (n = 75) E. cloacae (n = 7) n (%) n (%) n (%) Ampicillin AMP 10 111 (100) 75 (100) 7 (100) Amoxicillin-clavulanic acid AMC 30 44 (39.6) 32 (42.7) 7 (100) Ampicillin sulbactam SAM 20 11 (10) 11 (14.7) 0 (0.0) Cefotaxime CTX 30 100 (90) 64 (85.3) 7 (100) Cefotaxime-clavulanate CTL 30/10 88 (79.2) 53 (70.7) 3 (42.9) Ceftazidime CAZ 30 11 (10) 11 (14.7) 1 (14.3) Ceftazidime-clavulanate CAL 30/10 9 (8.1) 8 (10.7) 0.0 (0.0) Ceftriaxone CRO 30 111 (100) 75 (100) 7 (100) Cephalothin CEF 30 111 (100) 75 (100) 7 (100) Chloramphenicol CHL 30 111 (100) 74 (98.7) 7 (100) Ciprofloxacin CIP 5 83 (74.8) 64 (85.3) 4 (57.1) Imipenem IPM 10 11 (10) 11 (14.7) 0.0 (0.0) Nalidixic acid NAL 10 89 (80.1) 26 (34.7) 6 (85.7) Streptomycin STR 10 94 (84.7) 68 (90.7) 7 (100) Tetracycline TET 30 100 (90.1) 66 (88) 7 (100) Trimethoprim-sulphamethoxazole SXT 25 106 (95.5) 70 (93.3) 7 (100) 32 Original Extended-spectrum β-lactamase Enterobacteriaceae from patients in Jeddah, Saudi Arabia Wafa A. Alshehri, Tarek A. A. Moussa J Contemp Med Sci | Vol. 7, No. 1, January-February 2021: 28 – 33 K. pneumoniae isolates were susceptible to meropenem. Among E. cloacae strains (99.8%) were susceptible to ceftazi- dime-avibactam. Only 0.06% Enterobacteriaceae isolates were non-susceptible to ceftazidime-avibactam.24 A potent activity of ceftazidime-avibactam was shown against P. aeruginosa, where 97.1% isolates was susceptible, and 71.8% was inhib- ited of non-susceptible isolates to ceftazidime, meropenem, and piperacillin-tazobactam.22,23 Most of the E. coli (87.5%) and K. pneumoniae (75%) isolates were MDR and showed also MDR phenotypes.17,21–23 The most frequent gene was blaCTX-M (195) followed by blaTEM (149), blaSHV (73), and blaOXA (3). The blaCTX-M was the most widespread gene among the E. coli strains and in all age categories, while the rarest gene was blaOXA (Table 5). Only E. coli and K. pneumoniae were detected in teenagers. The presence of these genes in pairs in the same strain was detected, where the highest pair of genes in the same organism was blaTEM + blaCTX-M (134) (E. coli (74), K. pneumoniae (51) and E. cloaca (9)), followed by blaSHV + blaCTX-M (64) (K. pneu- moniae (63) and E. cloaca (1)), blaSHV + blaTEM (52) (K. pneu- moniae (51) and E. cloaca (1)) and the least pairs were blaTEM + blaOXA (3) (E. cloaca (2) and P. mirabilis (1)) and blaCTX-M + blaOXA (2) in E. cloaca only. The blaSHV + blaCTX-M + blaTEM was found in 44 organisms and blaCTX-M + blaTEM + blaOXA in two organisms only. The blaSHV + blaOXA, blaSHV + blaCTX-M + blaOXA, blaSHV + blaTEM + blaOXA and blaSHV + blaCTX-M + blaTEM + blaOXA was not found in any organism under investigation (Table 6). E. coli strains had 32.4% blaCTX-M, 81% blaTEM, and 16.2% blaSHV genes, while in K. pneumoniae strains had 41.1% blaCTX-M, 64.7% blaTEM, and 35.2% blaSHV genes. 24 PCR showed that blaCTX − M gene represented by 76% fol- lowed by 52% blaOXA, 28% blaTEM and 21% blaSHV were most predominant detected by CDST among ESBLs. The 78% of blaOXA, 65% of blaCTX − M − 1, and 57% of blaTEM genes were found on plasmids. Amplicon sequencing demonstrated that blaCTX − M − 15 (75%), blaOXA − 1 (49%) and blaTEM − 1B (34%) and 21 isolates carried three genes in them.15,25 Conclusion In conclusion, in teenager group, there were no organism that contained OXA gene, while OXA present in E. cloaca and P. mirabilis only. SHV gene was absent in E. coli but present in E. cloaca and K. pneumoniae in this study. The most sus- ceptible group to infection with Enterobacteriaceae was adults’ group, while teenage group was more resist to infection. Conflict of Interest None Table 5. The frequency occurrence of ESBL encoding genes in the isolated bacteria. Isolate bla CTX-M bla OXA bla SHV bla TEM Number % Number % Number % Number % E. cloacae 9.0 4.5 2.0 66.7 1.0 0.5 11.0 5.5 E. coli 111.0 57.0 0.0 0.0 0.0 0.0 78.0 39.0 K. pneumoniae 74.0 38.0 0.0 0.0 72.0 36.0 59.0 29.5 P. mirabilis 0.0 0.0 1.0 33.3 0.0 0.0 1.0 0.5 P. aeruginosa 1.0 0.5 0.0 0.0 0.0 0.0 0.0 0.0 Total 195 - 3.0 - 73 - 149 - Table 6. The prevalence of the coexistence of ESBL encoding genes in the isolated bacteria. Genes E. cloaca E. coli K. pneumoniae P. mirabilis P. aeruginosa Total bla TEM +bla CTX-M 9 74 51 0 0 134 bla SHV +bla CTX-M 1 0 63 0 0 64 bla SHV +bla TEM 1 0 51 0 0 52 bla TEM +bla OXA 2 0 0 1 0 3 bla CTX-M +bla OXA 2 0 0 0 0 2 bla SHV +bla OXA 0 0 0 0 0 0 bla SHV +bla CTX-M +bla TEM 1 0 43 0 0 44 bla CTX-M +bla TEM +bla OXA 2 0 0 0 0 2 bla SHV +bla CTX-M +bla OXA 0 0 0 0 0 0 bla SHV +bla TEM +bla OXA 0 0 0 0 0 0 bla SHV +bla CTX-M +bla TEM +bla OXA 0 0 0 0 0 0 33 Original Extended-spectrum β-lactamase Enterobacteriaceae from patients in Jeddah, Saudi ArabiaWafa A. Alshehri, Tarek A. A. Moussa J Contemp Med Sci | Vol. 7, No. 1, January-February 2021: 28 – 33 References 1. Collignon P, Aarestrup FM, Irwin R, McEwen S. Human deaths and third- generation cephalosporin use in poultry, Europe. 2013; 2. Chantziaras I, Boyen F, Callens B, Dewulf J. Correlation between veterinary antimicrobial use and antimicrobial resistance in food-producing animals: a report on seven countries. 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Available from: https://pubmed.ncbi.nlm.nih. gov/28827415 24. Pishtiwan AH, Khadija KM. Prevalence of blaTEM, blaSHV, and blaCTX-M genes among ESBL-producing Klebsiella pneumoniae and Escherichia coli isolated from thalassemia patients in Erbil, Iraq. Mediterr J Hematol Infect Dis. 2019;11(1). 25. Ali MMM, Mohamed ZK, Klena JD, Ahmed SF, Moussa TAA, Ghenghesh KS. Molecular characterization of diarrheagenic Escherichia coli from Libya. Am J Trop Med Hyg. 2012;86(5):866–71. This work is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported License which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. https://doi.org/10.22317/jcms.v7i1.920