Distribution of Extended-Spectrum β-Lactamase (ESBL)-Encoding Genes among Multidrug-Resistant Gram-Negative Pathogens Collected from Three Different Countries

The incidence of Extended-spectrum β-lactamase (ESBL)-encoding genes (blaCTX-M and blaTEM) among Gram-negative multidrug-resistant pathogens collected from three different countries was investigated. Two hundred and ninety-two clinical isolates were collected from Egypt (n = 90), Saudi Arabia (n = 162), and Sudan (n = 40). Based on the antimicrobial sensitivity against 20 antimicrobial agents from 11 antibiotic classes, the most resistant strains were selected and identified using the Vitek2 system and 16S rRNA gene sequence analysis. A total of 85.6% of the isolates were found to be resistant to more than three antibiotic classes. The ratios of the multidrug-resistant strains for Egypt, Saudi Arabia, and Sudan were 74.4%, 90.1%, and 97.5%, respectively. Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa showed inconstant resistance levels to the different classes of antibiotics. Escherichia coli and Klebsiella pneumoniae had the highest levels of resistance against macrolides followed by penicillins and cephalosporin, while Pseudomonas aeruginosa was most resistant to penicillins followed by classes that varied among different countries. The isolates were positive for the presence of the blaCTX-M and blaTEM genes. The blaCTX-M gene was the predominant gene in all isolates (100%), while blaTEM was detected in 66.7% of the selected isolates. This work highlights the detection of multidrug-resistant bacteria and resistant genes among different countries. We suggest that the medical authorities urgently implement antimicrobial surveillance plans and infection control policies for early detection and effective prevention of the rapid spread of these pathogens.


Introduction
Widespread use of antibiotics is often accompanied by increased bacterial resistance [1]. This can lead to the emergence of new antibiotic-resistant mechanisms that adversely affecting our ability to treat diseases. Increased antimicrobial resistance to antibiotics leads to longer treatment periods and higher health care costs. Several reports have indicated that multi-drug resistance exists in different countries such as the United States [2,3] and European countries [4]. However, resistance to microbial agents is growing very rapidly in developing countries in Africa [5] and Asia [6]. Multidrug-resistant (MDR) bacteria  [15]).
The patterns of the susceptibility of antibiotic classes to pathogens isolated from our clinical samples are shown in Figure 2. Most strains showed multidrug-resistance phenotypes to at least three different antimicrobial classes. In the present study, 3.4% (n = 10) of all strains were sensitive to all antibiotic classes, while 4.5% (n = 13) were resistant to one antibiotic class, and 6.5% (n = 19) were resistant to two antibiotic classes. Surprisingly, 85.6% (n = 250) of the isolates were multidrug-resistant (MDR), out of which 2.8% (n = 7) were extensively-drug-resistant (XDR) (Figure 2). The distribution of antibiotic resistance varied among strains obtained from the three countries. Among the 90 strains obtained from Egypt, 4.4% (n = 4) were sensitive to all antibiotics, while 10% (n = 9) were resistant to one antibiotic class, and 11.1% (n = 10) were resistant to two antibiotic classes. A total of 74.5% (n = 67) strains were classified as MDR, of which 1.5% (n = 1) were XDR. On the other hand, of the 162 strains obtained from Saudi Arabia, only 2.5% (n = 4) were sensitive to all antibiotic classes, while 1.9% (n = 3) were resistant to one antibiotic class, and 5.6% (n = 9) were resistant to two classes. A total of 90% (n = 146) were classified as MDR, of which 2.7% (n = 4) were XDR. Among the 40 strains obtained from Sudan, no isolates were sensitive to all antibiotics, while 3.0% (n = 1) of the strains were resistant to only one class. A total of 97.5% (n = 39) of all strains were classified as MDR, of which 7.7% (n = 3) were XDR (Figure 3).   The distribution of antibiotic resistance varied among strains obtained from the three countries. Among the 90 strains obtained from Egypt, 4.4% (n = 4) were sensitive to all antibiotics, while 10% (n = 9) were resistant to one antibiotic class, and 11.1% (n = 10) were resistant to two antibiotic classes. A total of 74.5% (n = 67) strains were classified as MDR, of which 1.5% (n = 1) were XDR. On the other hand, of the 162 strains obtained from Saudi Arabia, only 2.5% (n = 4) were sensitive to all antibiotic classes, while 1.9% (n = 3) were resistant to one antibiotic class, and 5.6% (n = 9) were resistant to two classes. A total of 90% (n = 146) were classified as MDR, of which 2.7% (n = 4) were XDR. Among the 40 strains obtained from Sudan, no isolates were sensitive to all antibiotics, while 3.0% (n = 1) of the strains were resistant to only one class. A total of 97.5% (n = 39) of all strains were classified as MDR, of which 7.7% (n = 3) were XDR ( Figure 3).
Antibiotics 2021, 10, x FOR PEER REVIEW 6 of 19 Figure 3. The percentages of strains resistant to antimicrobial agents from 11 antibiotic classes from all bacterial strains isolated from Egypt, Sudan, and Saudi Arabia: 0 means the percentage of strains obtained from a country that were not resistant to any antibiotic group (sensitive to all groups); numbers 1-10 indicate the numbers of classes that the bacterial strains showed resistance to; 1 indicates the percentage of strains obtained from this country that showed resistance to only one class of antibiotic; 2 indicates the percentage of strains obtained from a country that showed resistance to two classes of antibiotics amongst the 11 well known antibiotic classes, etc.
The diversity of drug-resistance also varied according to strain. Of the 103 E. coli isolates, 1.9% (n =2) were sensitive to all antibiotic classes, while 2.9% (n =3) and 5.8% (n =6) of the E. coli strains were resistant to one or two classes, respectively. The percentage of E. coli strains that were MDR was 89.3% (n =92), of which 1.9% (n =1) were XDR. Of a total of 47 K. pneumoniae strains, 6.4% (n =3) and 4.3% (n =2) showed resistance to one or two classes, while no isolates were sensitive to all classes. A total of 89.3% (n =42) of the K. pneumoniae strains were MDR, of which 4.7% (n =2) were XDR. On the other hand, no P. meribalis strains were resistant to less than three classes, but all P. meribalis strains (n =14) were MDR, of which 7.1% (n =1) were XDR. Among the 83 P. aeruginosa strains, no strains were found to be sensitive to all classes, while 3.6% (n =3) were resistant to either one or two classes of antibiotics. A total of 92.8% (n =77) of P. aeruginosa strains were MDR, of which 3.9% (n =3) were XDR. For the 45 obtained S. aureus strains, 13.3% (n = 6) were found to be sensitive to all classes, while 8.9% (n =4) and 17.8% (n = 8) of the strains exhibited resistance to one or two antibiotic classes, respectively. A total of 60% (n = 27) were MDR, but no XDR strains were obtained ( Figure 4). Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae were chosen for further molecular characterization and study, as they were the most frequently identified strains in the three countries under study. Egypt % Saudia Arabia% Sudan % Figure 3. The percentages of strains resistant to antimicrobial agents from 11 antibiotic classes from all bacterial strains isolated from Egypt, Sudan, and Saudi Arabia: 0 means the percentage of strains obtained from a country that were not resistant to any antibiotic group (sensitive to all groups); numbers 1-10 indicate the numbers of classes that the bacterial strains showed resistance to; 1 indicates the percentage of strains obtained from this country that showed resistance to only one class of antibiotic; 2 indicates the percentage of strains obtained from a country that showed resistance to two classes of antibiotics amongst the 11 well known antibiotic classes, etc.

Number of antibiotic classes Percentage of resistance among isolates (%)
The diversity of drug-resistance also varied according to strain. Of the 103 E. coli isolates, 1.9% (n = 2) were sensitive to all antibiotic classes, while 2.9% (n = 3) and 5.8% (n = 6) of the E. coli strains were resistant to one or two classes, respectively. The percentage of E. coli strains that were MDR was 89.3% (n = 92), of which 1.9% (n = 1) were XDR. Of a total of 47 K. pneumoniae strains, 6.4% (n = 3) and 4.3% (n = 2) showed resistance to one or two classes, while no isolates were sensitive to all classes. A total of 89.3% (n = 42) of the K. pneumoniae strains were MDR, of which 4.7% (n = 2) were XDR. On the other hand, no P. meribalis strains were resistant to less than three classes, but all P. meribalis strains (n = 14) were MDR, of which 7.1% (n = 1) were XDR. Among the 83 P. aeruginosa strains, no strains were found to be sensitive to all classes, while 3.6% (n = 3) were resistant to either one or two classes of antibiotics. A total of 92.8% (n = 77) of P. aeruginosa strains were MDR, of which 3.9% (n = 3) were XDR. For the 45 obtained S. aureus strains, 13.3% (n = 6) were found to be sensitive to all classes, while 8.9% (n = 4) and 17.8% (n = 8) of the strains exhibited resistance to one or two antibiotic classes, respectively. A total of 60% (n = 27) were MDR, but no XDR strains were obtained ( Figure 4). Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae were chosen for further molecular characterization and study, as they were the most frequently identified strains in the three countries under study.

Molecular Characterization of Isolates
Molecular characterization of the two most significant strains obtained from each country was done with 16S rRNA gene fragments. These were chosen for further molecular identification and investigation of gene resistance based on their frequency among the Antibiotics 2021, 10, 247 9 of 18 three countries and their high resistance to the majority of antibiotic classes. A 16S rRNA gene fragment showed that one isolate belonged to the genus Escherichia and was identified as the Escherichia coli strain and the second isolate belonged to the genus Klebsiella and was identified as the Klebsiella pneumoniae strain for all countries under study. A phylogenetic analysis based on 16S rRNA gene sequencing and alignment revealed that the two isolates were clustered with Escherichia coli and Klebsiella pneumoniae strains. The first isolates were confirmed to be E. coli strains (reference no. NR_114042.1) with similarity levels of 99.3%, 99.6%, and 98.6% for the Egypt, Saudi Arabia, and Sudan isolates, respectively. The second isolates were confirmed to be K. pneumoniae strains (reference strain nos. NR_114715.1, NR_117686.1, and NR_119276.1) with similarity levels of 98.8%, 96.7%, and 96.9% for Egypt, Saudi Arabia, and Sudan isolates, respectively ( Figure 5). 98.8%, 96.7%, and 96.9% for Egypt, Saudi Arabia, and Sudan isolates, respectively ( Figure  5).

Frequency of Antibiotic Resistance
Our results showed that all selected strains were resistant to macrolides, nitrofuran, penicillins, tetracyclines, cephalosporins 1st, cephalosporins 2nd, cephalosporins 3rd, quinolones, aminoglycosides, sulfonamide, and carbapenem. These strains were positive for the presence of bla CTX-M and bla TEM genes. The predominant gene in this study was bla CTX-M (size 500 bp), which was found in all selected isolates (n = 6, 100%). The bla TEM gene (size 600 bp) was found in most of the selected isolates (n = 4, 66.7%). bla CTX-M was detected in E. coli and K. pneumoniae obtained from the three countries included in this study, while the bla TEM gene was not detected in the E. coli and K. pneumoniae strains obtained from Saudi Arabia and Egypt, respectively ( Figure 6). E. coli and K. pneumoniae obtained from the three countries included in this study, while the blaTEM gene was not detected in the E. coli and K. pneumoniae strains obtained from Saudi Arabia and Egypt, respectively ( Figure 6). The blaCTX-M gene (size 500 bp) was found in all selected isolates (n = 6). The blaCTX-M gene was detected in E. coli and K. pneumoniae obtained from the three countries included in this study; 1, 2, and 5 are codes for the selected E coli strains from Saudi Arabia, Egypt, and Sudan, respectively; 3, 4, and 6 are codes for the selected K. pneumoniae strains from Egypt, Saudi Arabia, and Sudan, respectively.

Discussion
Investigating the prevalence of antimicrobial resistance rates is important for creating empirical treatment strategies and evaluating the existing guidelines. The frequency and number of types of infection caused by Gram-negative bacteria have increased dramatically in the past few decades, with disparities between reports from different institutions and countries [16]. Infection with multidrug-resistant Gram-negative bacteria is a global problem that is correlated with increased morbidity and mortality rates [17]. Bacteria can obtain antibiotic resistance through mutational changes or by gaining resistance genes through horizontal gene transfer [18]. The prevalence of antimicrobial resistance is increasing worldwide, particularly in developing countries, because of greater access to antibiotic drugs. This increase in microbial drug resistance is mainly caused by the use of antimicrobials in humans and other animals [19].
In our previous study, a total of 292 different clinical isolates obtained from various discharged private laboratories from countries including Egypt, Saudi Arabia, and Sudan were evaluated to determine the pathogen distribution. In that study, the most frequently isolated organism was Escherichia coli, followed by Pseudomonas aeruginosa, Klebsiella pneumoniae, and Staphylococcus aureus. The least frequently isolated organism was Proteus mirabilis [15]. Our reported incidences were used to compare the prevalence of pathogens in different countries in the same region and to study multidrug resistance. Figure 6. The presence of bla CTX-M and bla TEM genes. bla CTX-M genes in multidrug-resistant isolates obtained from three different countries are shown. The DNA ladder is 50 bp and the bla TEM gene (size 600 bp) was found in most of the selected isolates (n = 4); it was not detected in E. coli and K. pneumoniae strains obtained from Saudi Arabia and Egypt, respectively. The bla CTX-M gene (size 500 bp) was found in all selected isolates (n = 6). The bla CTX-M gene was detected in E. coli and K. pneumoniae obtained from the three countries included in this study; 1, 2, and 5 are codes for the selected E coli strains from Saudi Arabia, Egypt, and Sudan, respectively; 3, 4, and 6 are codes for the selected K. pneumoniae strains from Egypt, Saudi Arabia, and Sudan, respectively.

Discussion
Investigating the prevalence of antimicrobial resistance rates is important for creating empirical treatment strategies and evaluating the existing guidelines. The frequency and number of types of infection caused by Gram-negative bacteria have increased dramatically in the past few decades, with disparities between reports from different institutions and countries [16]. Infection with multidrug-resistant Gram-negative bacteria is a global problem that is correlated with increased morbidity and mortality rates [17]. Bacteria can obtain antibiotic resistance through mutational changes or by gaining resistance genes through horizontal gene transfer [18]. The prevalence of antimicrobial resistance is increasing worldwide, particularly in developing countries, because of greater access to antibiotic drugs. This increase in microbial drug resistance is mainly caused by the use of antimicrobials in humans and other animals [19].
In our previous study, a total of 292 different clinical isolates obtained from various discharged private laboratories from countries including Egypt, Saudi Arabia, and Sudan were evaluated to determine the pathogen distribution. In that study, the most frequently isolated organism was Escherichia coli, followed by Pseudomonas aeruginosa, Klebsiella pneumoniae, and Staphylococcus aureus. The least frequently isolated organism was Proteus mirabilis [15]. Our reported incidences were used to compare the prevalence of pathogens in different countries in the same region and to study multidrug resistance.
In the current study, we evaluated antimicrobial susceptibility patterns, and the results showed that a small percentage (3.4%) of the total isolated organisms were sensitive to all classes of antibiotics and a small percentage (2.7%) were resistant to ten classes of a total of eleven classes (extensively drug-resistant). The most common level of resistance was resistance to five classes, followed by resistance to six, seven, and eight classes, respectively. We found that 85.6% of all isolates were resistant to three or more (≥3) antibiotic classes, so the majority of isolates were multidrug-resistant (MDR). The highest frequency of multidrug-resistant isolates was detected in Sudan with (97.5%) followed by Saudi Arabia (90.1%) and Egypt (74.4%), while 50.3% of all strains were resistant to six antibiotic classes or more (≥6): Percentages of 48.9%, 50.6% and 49.9% for Egypt, Saudi Arabia, and Sudan, respectively. This high frequency is comparable with those found in other reports. The highest percentage of organisms that are sensitive to all antibiotic classes was found in Egypt, and the lowest rate of resistance was found in Saudi Arabia, while no organisms were detected to be sensitive to all groups in Sudan. In contrast, Sudan had the highest percentage of strains that were resistant to nine and ten groups, followed by Saudi Arabia and Egypt.
Our findings are in agreement with previous studies conducted in Sudan, which showed that E. coli, K. pneumoniae, P. aeruginosa, S. aureus, and Proteus spp. are the most commonly encountered organisms, and most of these strains have been found to be highly resistant to several antibiotic classes [20]. Inconsistent with another study, we showed that most isolates were resistant to many antibiotics, and Gram-negative isolates were resistant to 67% of the examined antibiotics [21]. This is in contrast to earlier publications that demonstrated that the most common pathogen isolated from different clinical specimens was K. pneumoniae followed by P. aeruginosa and then E. coli, and about 22.3% were resistant to three or more classes of antibiotics [22].
Our findings agree with previous studies conducted in Egypt, which showed that 93.6% of the isolated Gram-negative bacteria were multidrug-resistant [23]. Other studies have reported that 71.1% of isolates studied exhibited multidrug-resistance [24], and 91% of all E. coli strains were resistant to three or more antibiotics [25]. In another study conducted on bacterial isolates in drinking water in Cairo, the majority of the tested strains (62.4 to 98%) exhibited multiple antibiotic resistance [26]. Previous data from two major hospitals in Makkah, KSA showed that 24.6% of E. coli strains, 34.4% of K. pneumoniae strains, and 52.7% of P. aeruginosa strains were resistant to ceftazidime (cephalosporin 3rd generation) [27]. They documented the increasing prevalence of extended-spectrum beta-lactamase (ESBL) producing isolates from Saudi Arabia; in some institutes, 29% of E. coli strains and 65% of K. pneumoniae strains were ESBL [28].
In the current study, K. pneumoniae strains showed inconstant resistance levels to all classes of antibiotics except carbapenem. K. pneumoniae strains were most commonly resistant to macrolides followed by penicillins and then cephalosporin 1st, but the results for other groups were variable. In Saudi Arabia and Sudan, the lowest number of strains were resistant to carbapenem, but in Egypt, resistance was least commonly observed for tetracycline. However, this is in contrast to earlier publications from Sudan that demonstrated that a high number of K. pneumoniae strains were resistant to penicillins [21], followed by cephalosporines [21,22,30], nitrofuran, carbapenem, and quinolones [21]. Resistance was least commonly found for carbapenem, quinolones [22], aminoglycosides [21,22], and tetracyclines [21]. Other reported studies showed complete sensitivity toward carbapenems [30,39]. Studies in Saudi Arabia showed a high degree of resistance of K. pneumoniae to penicillins and tetracyclines [39,40], cephalosporins, carbapenems, aminoglycosides, and nitrofuran [40]. Resistance was least commonly found toward aminoglycosides [39,40], carbapenems [40], and quinolones [39]. Previous studies in Egypt showed that K. pneumoniae were highly resistant to cephalosporins [41][42][43], followed by penicillins, carbapenem, quinolones, and aminoglycosides [41,42].
In the present study, P. aeruginosa strains showed inconstancy in their resistance level to all classes of antibiotics, except carbapenem. The pathogen that strains were most commonly resistant to varied from macrolides to cephalosporin among countries, and the pathogens associated with the highest level of sensitivity were carbapenem and tetracycline. Studies in Sudan have shown that P. aeruginosa strains are most frequently resistant toward penicillins [21], followed by cephalosporines [22,44], quinolones, nitrofuran, and tetracyclines [21], and aminoglycosides [44]. Resistance was least commonly found for carbapenem, aminoglycosides, and quinolones [22]. Other studies in Saud Arabia have shown frequent resistance of P. aeruginosa to penicillins and carbapenems [45,46] and a low frequency of resistance toward aminoglycosides and cephalosporins 4th generation [45,46]. In contrast with our findings, other studies in Egypt showed that P. aeruginosa most frequently exhibited resistance against carbapenems [47][48][49], followed by extended-spectrum beta-lactamases [49], penicillins, cephalosporins, macrolides, and tetracyclines [47]. Resistance was least frequently exhibited toward carbapenems [50]. Other studies showed widespread occurrence of Gram-negative bacteria with resistance to beta-lactam and carbapenem in Middle Eastern hospitals [51].
The frequencies and types of infections caused by extended-spectrum β-lactamase (ESBL) producing pathogens have increased dramatically in the past few decades with disparities between different regions and countries. Since the beginning of the new millennium, Gram-negative bacteria have become the most commonly isolated ESBL-producing bacteria worldwide with bla CTX-M ESBLs being the most frequently isolated type. Thus, we evaluated the prevalence of bla CTX-M and bla TEM resistant gene traits from clinical specimens within different countries. Resistant genes were characterized at the molecular level. In the current study, the selected isolates were found to be positive for the presence of bla CTX-M and bla TEM genes. The predominant gene identified in this study was bla CTX-M , which was found in all selected isolates (100%). The bla TEM gene was found in most isolates (66.7%), the bla CTX-M was detected in E. coli and K.pneumoniae for all countries included in our study, while the bla TEM gene was not detected in E. coli and K. pneumoniae strains isolated from Saudi Arabia and Egypt, respectively. Our findings are in agreement with previous studies that showed that bla CTX-M is more prevalent among tested strains of E. coli, K. pneumoniae, and P. mirabilis as compared with bla TEM [52]. A study in Egypt revealed 46 E. coli isolates and showed that all extended-spectrum beta-lactamase (ESBL) producers were positive for bla CTX-M and bla TEM genes [53]. Another study carried out on K. pneumoniae showed that bla CTX-M and bla TEM genes were detected in 52.8% and 53.3% of ESBL isolates, respectively [54,55]. In another study, bla CTX-M and bla TEM genes were detected in K. pneumoniae more frequently than E. coli, and bla TEM genes were detected more frequently than bla CTX-M [38]. In agreement with our findings, studies conducted in different regions of Sudan have shown that E. coli is the most predominant isolate with the bla CTX-M genotype, followed by K. pneumoniae [56,57]. In another study, the bla TEM gene was detected in 55.1% of E. coli strains and 58% of K. pneumoniae strains, but the bla CTX-M gene was detected in 71.4% of E. coli strains and 68.4% of K. pneumoniae strains [54]. Other studies conducted in Saudi Arabia detected both genes in a large number of K. pneumoniae strains isolated from two different hospitals. The results showed that the isolates contained the bla CTX-M and bla TEM genes [53,58].
Our results present alarming evidence of a severe spread of ESBL genes in the studied countries, especially the epidemiological bla CTX-M and bla TEM genes. There is potential for the dissemination of MDR strains in different regions. ESBLs are plasmid-mediated β-lactamases that have been recognized for their ability to hydrolyze different generations of cephalosporins (oxyimino-cephalosporin) and other antibiotic classes. These enzymes are repressed by β-lactamase inhibitors such as clavulanic acid and tazobactam. Public health efforts should focus on the correct use of antibiotics to limit their dissemination. In addition, further investigation of the molecular epidemiology of ESBLs in various clinical specimens is needed to obtain a better database of ESBL-producing pathogens present in different countries.

Study Design and Specimen Collection
A total of 292 clinical specimens were collected over the period from April 2015 to July 2016. Bacterial isolates of Gram-negative pathogens were obtained from various discharged private laboratories; the clinical specimens were collected from three countries (Egypt, Saudi Arabia, and Sudan). Of the 292 samples, 90 (30.8%) were from Egypt, 162 (55.5%) were from Saudi Arabia, and 40 (13.7%) were from Sudan. Bacterial strains were collected from different clinical specimens (urine (n = 168), vaginal swab (n = 43), ear swab (n = 22), blood (n = 19), abscess (n = 17), endotracheal tube (n = 8), sputum (n = 8), throat swab (n = 3), nasal swab (n = 3), and urethral swab (n = 1)). The study protocol was approved by the ethics committee, and the research followed the principles of the Declaration of Helsinki. All specimens were collected aseptically and transported to the microbiology laboratory, where they were immediately processed according to the standard microbiological procedures [59].

Isolation and Identification of Isolates
The samples were cultured by the streaking on blood agar [60], cysteine-lactoseelectrolyte-deficient agar [61], chocolate agar [62], MacConkey agar [63], and Thayer martin agar [64], and then incubated at a 37 • C for 24 hours. The bacterial colonies were purified and identified according to the colonies' morphology. The pathogenic isolates were identified basely by physiological and different biochemical tests [65].
Vitek2 system was used for the identification of isolates (bioMerieux, Vitek2 Compact System Version: 07.01, GN card), HCWW-Reference Lab. Microbiology Lab. GN card is based on 64 biochemical methods and newly developed substrates that measuring carbon source utilization, enzymatic activities, and resistance [66].

Molecular Identification of Bacterial Isolates
Molecular identification was performed by the extraction of genomic DNA using the modified method as previously described [69,70]. The 16S rRNA gene fragments were analyzed using forward and reverse universal primers as 27f (5 GAGTTTGATCACTGGCTCAG The extension was performed at 72 • C for 10 min. The PCR products were commercially sequenced at Sigma Company (Cairo, Egypt) using an ABI 3730xl DNA sequencer. Through BLASTN, The sequences were compared with sequences available at GenBank database. Multiple sequence alignments were then performed by ClustalX 1.8 software package and the phylogenetic tree was established using a neighbor-joining method of Kimura 2-parameter model to calculate genetic distance as the transitional and transversional substitution rates using MEGA (version 6.1) software. The level of confidence for each branch at 1000 repeats was tested by bootstrap analysis. Detection of chimeras (>3% diverged from the closest sequences) was conducted using Uchime2_NCBI tool. 16S rRNA gene sequences of all strains have been deposited in NCBI Gene Bank using Bankit program and assigned with accession numbers MT279577-MT279580.

Molecular Screening of Resistant Genes
Bacterial template DNA was prepared using the boiled lysate method for molecular screening of two different resistance genes (bla TEM and bla CTX-M genes). For bla TEM gene, the forward primer was bla TEM -F (5 -GCTCACCCAGAAACGCTGGT-3 ), and the reverse primer was bla TEM -R (5 -CCATCTGGCCCCAGTGCTGC-3 ). For bla CTX-M gene, the forward primer was bla CTXM-F (5 -SCSATGTGCAGYACCA GTAA-3 ), and reverse primer was bla CTX-M -R (5 -ACCAGAAYVAGCGGBGC-3 ). The oligonucleotide primers bla TEM was designed based on the nucleotide sequence of bla TEM gene reported in the National Center for Biotechnology Information (NCBI) Gene Bank database, while bla CTX-M primers were synthesized according to a previously published protocol [71]. The PCR mixture (25 µ) contained 1 µL of each primer (10 pmol/µL), 12.5 µL of 2× Taq PCR RED Master Mix (Ampliqon, Denmark), 3 µL of template DNA, and 7.5 µL of ultra pure water. The PCR conditions for amplification of the bla TEM and bla CTX-M genes were selected based on the properties of the primers and were as follows, initial denaturation for 5 min at 94 • C, 35 cycles of denaturation for 45 s at 94 • C, annealing for 30 s at 54 • C, and extension for 60 s at 72 • C with a final extension for 5 min at 72 • C; and initial denaturation for 3 min at 94 • C, 35 cycles of denaturation for 60 s at 94 • C, annealing for 30 s at 58 • C, and extension for 60 s at 72 • C with a final extension for 10 min at 72 • C, respectively. The PCR products were subjected to agarose gel (1.2%) electrophoresis in in borate buffer (TBE, Tris-Borate-EDTA) at 80 V for 2 h and was visualized by a UV illumination after ethidium bromide (0.5 µg/mL) staining. The molecular weight was estimated by comparing with 50 bp DNA Ladder (InVitrogen) marker.

Statistical Analysis
Collected data was analyzed using Statistical Package for Social Sciences (SPSS; Version 23) software to calculate frequencies and percentages for all categories of organisms and resistance of isolated organisms against different antibiotics among the three countries.

Conclusions
This work highlights the detection of multidrug-resistant bacteria and resistant genes in different countries. These bacteria and genes compromise the effectiveness of different antibiotic classes. Thus, implementation of antimicrobial surveillance plans and infection control policies by medical authorities to ensure early detection and effectively halt the rapid spread of these pathogens is required.
Author Contributions: All authors contributed to data analysis, drafting or revising the article, gave final approval of the version to be published, and agree to be accountable for all aspects of the work.

Informed Consent Statement:
The study protocol was approved by the ethics committee, and the research was conducted following the principles of the Declaration of Helsinki. All specimens were collected aseptically and transported to the microbiology laboratory, where they were immediately processed according to the standard microbiological procedures. We would like to confirm that this material is the authors' own original work, which has not been previously published elsewhere. The paper is not currently being considered for publication elsewhere. The paper reflects the authors' own research and analysis in a truthful and complete manner. The paper properly credits the meaningful contributions of co-authors and co-researchers. The results are appropriately placed in the context of prior and existing research. All authors have been personally and actively involved in substantive work leading to the manuscript and will hold themselves jointly and individually responsible for its content.

Data Availability Statement:
The datasets used and analyzed in the current study are available from the corre-sponding author upon reasonable request.