Escherichia coli in Saudi Arabia: An Overview of Antibiotic-Resistant Strains

Escherichia coli (E. coli) bacterial strains are considered as the most important human pathogens. Health issues are increasing in complexity owing to the persistent emergence of antibiotic resistant E. coli strains, which have been isolated and detected worldwide, including Saudi Arabia. A review of the prevalent strains resistant to the standard antibiotics used in a local region is critical and may be beneficial at the national and international levels. Treatment for E. coli infection has been highly difficult by the rise of resistance to most first-line antibiotics. The present study aimed to update the scientific information regarding E. coli strains, which have the ability to resist the standard drugs used to treat bacterial infections in Saudi Arabia. The data retrieved from https://scholar.google.com and Saudi Digital Library (https://sdl.edu.sa/) indicate that E. coli strains isolated from several sources in Saudi Arabia show resistance to almost all antibiotics, except 5th generation cephalosporins (ceftraolin and ceftobiprole), which no isolate in Saudi Arabia has been recorded to resist. Based on the results of the present study, we conclude and recommend that integrated monitoring and management of the antibiotics may reduce the health risks associated with antibiotic resistant E. coli strains.

Escherichia coli(E. coli)is a coliform rod-shaped, Gram-negative, facultative, non-spore forming bacteriumof the genus Escherichia and family Enterobacteriaceae,which includes over 53 genera and 210 species (Jenkins et al., 2017;Tenaillon et al., 2010).According to a study on drug resistances it is predicted that ten million people may die from antibiotic-resistant diseases each year by 2050 if no precautions are taken to tackle the issue, among that more than three million will lose their lives to one bacterial infection by antibiotic resistant E. coli ( O'Neill J., 2016).TheE. coli strains are considered as one of the few microbes that have the skill to be adapted to numerous biofunctions. These bacteria can colonize the healthy intestinal tract of several mammals including humans. They are used as an important bio-tool in several biotechnological applications. Furthermore, they have virulence factors which cause numerous diseases in humans and animals, and affect a wide range of bio-cellular processes (Kaper et al., 2004). Although E. coli strains inhabit the gastrointestinal tract of healthy humans, it is considered as one of the most pathogenic microorganisms isolated from humans. E. coli is a very versatile bacterium that can modify easily from one bio-activity to another.
Highly variable mutation rates have been reported in commensal and pathogenic E. coli strains (Matic et al., 1997).
Some strains of E. coli cause infections in the urinary, intestinal,and respiratory tractsalong with other diseases. The sources of pathogenic E. coli strains include contaminated water and food, and itmay be transmitted through direct contact with infected people and animals or non-direct contact. Pathogenic E. coli strains may cause enteric/diarrheal illness, urinary tract diseases (UTDs) or sepsis/meningitis (Kaper et al., 2004). According to the Centers for Disease Control and Prevention (CDC) (https://www.cdc.gov/ ecoli/general/index.html, December 1, 2014), pathogenic E. coli strains can be classified into six pathotypic strains as follows: Shiga toxinproducing E. coli (STEC), enterotoxigenic E. coli (ETEC), enteropathogenic E. coli (EPEC), enteroaggregative E. coli (EAEC), enter invasiveE. coli (EIEC), and diffusely adherent E. coli (DAEC). Illnesses resulting from bacterial infections caused by pathogenic E. coli strains involve diarrhea, inflammation of the kidney (pyelonephritis), inflammation of the colon (dysentery), and hemolytic-uremic syndrome. Extreme response to such infectionsmay lead to tissue damage, organ failure, and death (Donnenberg, 2013). These bacterial strains may develop resistance mechanisms to inhibit the effects of antibiotics and there are confirmed scientific evidences reporting that these bacterial strains can also disseminate the resistance genes to other bacteria (Morris et al., 1998).
The treatment for infectious diseases caused by E. coli strains must not include antibiotics that can replicate the risk of severe complications such as hemolytic uremia. The misdiagnosis of E. coli infection and misuse of antibiotics for treatment, may lead to the emergence of antibiotic resistant E. coli strains. A number of pathogenic and non-pathogenic E. coli strains have developed the ability to resist the standard antibiotics through numerous mechanisms,whichwe have discussed in this study. In Saudi Arabia, antimicrobial drug resistance genes, including β-lactam (bla SHV ), gentamicin (aac(3)-IV), streptomycin ( aadA1), tetracyclines (tet(A),tet(B)), chloramphenicol (catA1,cmlA), erythromycin (ere(A)), and sulfonamide (sul1) resistance genes, have been detected among E. coli isolates(Abo-Amer et al., 2018). The present review aimed to update several scientific concepts related to antibiotic resistant E. coli strains. Antibiotic resistant E. coli strains.
In general, the pure bacterial isolates are considered resistant to a specific antibiotic if the minimum inhibitory concentration (MIC) (mg/L) of the antibiotics is greater than the breakpoint (mg/L). The standard assays define MIC as the lowest concentration of the antibiotic,which has the ability to inhibit the bacterial growth, using the standard methods such as two-fold macro-dilution serials, two-fold micro-dilution serials, or E-test protocol.
The MIC breakpoints are determined by several health organizations, such as the European Committee on Antimicrobial Susceptibility Test (EUCAST) and Clinical Laboratory and Standards Institute (CLSI), based on clinical and pharmacokinetic studies (Kuper et al., 2009;Reller et al., 2009). The bacterial strains, which show resistance to most standard antibiotics,are often known as superbugs.The MIC breakpoints (mg/L) and zone diameter breakpoints (mm) for most pathogenic microorganisms, including E. coli, are available in breakpoint tables for interpretation of MICs and zone diameters, Version 9.0, valid from 2019-01-01, http://www. eucast.org/clinical_breakpoints/). The breakpoints are alsoupdated regularly by CLSI (https://clsi. org/media/2270/clsi_astnewsupdate_june2018_ final.pdf). The methods used to determine MICs and MIC breakpoints must adhere to the procedures approved by the international committees on antimicrobial susceptibility testing, such as performance standards for antimicrobial susceptibility testing. 28th ed. CLSI supplement M100.
Antibiotic resistance pattern among E. coli isolates in Saudi Arabia has been evaluated using a group of standard antibiotics listed in Table 1. Figure 1 shows the percentage of antibioticsused for the treatment of urinary tract infections caused by Gram-negative bacilli, including E. coli isolates, in Buraidah Central Hospital from 1/8/2016 to 1/1/2017.

β-lactam antibiotic resistant E. coli strains
Chemically, the presence of a β-lactam ring is sufficient to distinguish between the molecular structures of β-lactam antibiotics and those of the others. The β-lactam antibiotics are considered as the most widely prescribed group among all antibiotics, and include a large group of antibiotics such as penicillins (penicillin G and penicillin V, ampicillin, carbenicillin, oxacillin, piperacillin, ticarcillin , dicloxacillin, nafcillin, amoxicillin, and ampicillin), cephalosporins (cephalothin, cefoxitin, cefuroxime, ceftriaxone, cefotaxime, cefepime, ceftaroline,fosamil, and ceftolozane), monobactams (aztreonam, tigemonam, nocardicin A, and tabtoxin), carbapenems (imipenem, ertapenem, doripenem, and meropenem), and carbacephems. The β-lactam antibiotics perform a specific biological activity to inhibit bacterial cell wall biosynthesis (Demain and Elander, 1999;Elander, 2003) The β-lactam antibiotics act by penetrating the bacterial outer membrane through protein membrane channelscalled porins, to bind with penicillin-binding proteins (PBPs). Modifications in porinsmay reduce the permeability of bacterial cell membrane and β-lactam antibiotic resistance. The primary strategy followed by β-lactam antibioticresistant bacterial strains is the production of β-lactamase enzymes that biochemically disrupt the β-lactam ring, leading to the inactivation ofthe antibiotic (Bush and Bradford, 2016;Féria et al., 2002).

Ampicillin resistant E. coli strains
Ampicillin (aminobenzylpenicillin) is a â-lactam and broad-spectrum antibacterial agent that can be produced from penicillin using semisynthetic methods. Ampicillin inhibits the cell wall biosynthesis of Gram-negative and Grampositive bacteria as well as aerobic and anaerobic bacteria. The biochemical functions of specific proteins, called PBPs, located inside the bacterial cell wall are hampered by ampicillin. Ampicillin is considered as a bacteriolytic agent,which can interfere with autolytic enzyme inhibitors such as the autolysin inhibitor. The amino group present in the chemical structure of ampicillin facilitates the passage of ampicillin through the outer membrane of pathogenic Gram-negative bacteria causing irreversible inhibition of transpeptidase enzyme, leading to bacterial lysis during the binary fission stage.
The major mechanism of resistance to β-lactam antibiotics depends on the disruption of these compounds by the β-lactamases, which destroy the amide bond of the lactam ring(Munita and Arias, 2016). Although TEM, SHV, and OXAtype β-lactamases have been detected in E. coli strains resistant to ampicillin, TEM is considered as a major β-lactamase enzyme responsible for resistance to ampicillin .
Ampicillin resistant E. coli strains have been isolated and identified from patients and several environmental sources over the last 20 years in Saudi Arabia. The following table summarizes the most important findings.

Oxacillin-resistant E. coli
Oxacillin is a β-lactam antibiotic witha narrow-spectrum activity against penicillin-and methicillin-resistant bacterial strains. It generally is described as β-lactam antibiotic resistance to penicillinase enzyme. Oxacillin may obstruct chemical transpeptidation reaction in bacterial cell walls, leading to the inhibition of peptidoglycan synthesis, which in turn causes bacterial cell autolysis (Nadarajah et al., 2006). One of the studies carried out in Saudi Arabia has reported that E. coli isolates are the most predominant among uropathogenic bacteria (N=632) and concluded that all E. coli strains have no ability to resist oxacillin (Ali, 2018).
These findings were confirmed in a study (Alharbi et al., 2018), which showed that E. coli strains (N=227) isolated from wounds did not resist oxacillin. In 2004, Shobrak and Abo-Amer reported that all E. coli strains (N=82) isolated from migratory and non-migratory wild birds were resistant strains to oxacillin (Shobrak and Abo-Amer, 2014).

Piperacillin-resistant E. coli
Piperacillin is β-lactam antibiotic with broad-spectrum activity, classified as ureidopenicillin antibiotics, which are a class of penicillins used to treat Pseudomonas aeruginosa. Piperacillin prohibits the 3ed and final phase of the synthesis of the microbial cell wall, and is believed to inhibit autolysin inhibitors in microbial cell lysis stages. The E. coli strains can hydrolyze piperacillin via ampC and TEM-1 β-lactamase mediated in the chromosome or plasmid (Kadima and Weiner, 1997;Schechter et al., 2018). Combinations of piperacillin-tazobactam are often used to avoid these problems; nevertheless, piperacillin-tazobactam resistant E. coli strains

Imipenem-resistant E. coli
Imipenem (C 12 H 17 N 3 O 4 S) is one of the β-lactam carbapenem antibiotics with the ability to resist β-lactamase. It has wide spectrum activity against aerobic, anaerobic, Gram-positive, and Gram-negative pathogenic bacteria. It can be used as a combination (imipenem-cilastatin) or triple-antibiotic (imipenem-cilastatin-relebactam) product. It is reported that bacterial resistance to carbapenem antimicrobial agents (imipenem and meropenem) leads to limiting therapeutic choices.
There are generally two methods employed by E. coli strains to resist carbapenem antibiotics; producing β-lactamase enzyme or reducing the permeability of bacterial cells, and strains may sometimes use both these ways.   , also known as anhydrotetracycline or deschlorobiomycin, is a bacteriostatic broad spectrum antibiotic that can act against an extensive range of pathogenic microbes including Gram-positive and Gram negative bacteria, chlamydiae, mycoplasmas, rickettsiae, and protozoan parasites (Chopra and Roberts, 2001). It is secondary metabolic products follows the polyketide antibiotics produced by some of the actinomycetes bacteria "Streptomyces spp.". In general, the tetracyclines can inhibit the biosynthesis of bacterial proteins by preventing the combination of aminoacyl-tRNA with the acceptor site, in the bacterial ribosome. The biological activity of tetracycline may include the 30S, 50S bacterial ribosomal subunit, and the cytoplasmic membrane.
The outer membrane of Gram-negative bacteria is traversed by the tetracyclines through the OmpF and OmpC porin pathways (Chopra et al., 1992). The bacteria can resist the tetracyclines by exporting tetracycline from the bacterial cell by efflux proteins, which are encoded by thetet efflux genes, protection of bacterial ribosomes by cytoplasmic proteins, or inactivation of tetracycline through enzymatic modification (Ref.). The misuse of tetracycline compounds has been confirmed in Saudi Arabia. Several poultry products have been screened to detect the residues of tetracycline agents, the results indicate that the level of the tetracycline residues has reached over the maximum residue limit in some tested samples (Al- Ghamdi et al., 2000).

Aminoglycosides resistant E. coli
The aminoglycosides are natural (gentamicin and tobramycin) or semisynthetic (derivatives of natural antibiotics such as amikacin), broad-spectrum, bactericidal antimicrobial agents which are generally introduced for the treatment of Gram-negative infections in humans (Germovsek et al., 2017;Krause et al., 2016). Aminoglycoside antibiotic-resistant bacterial strainscan fight the antibiotics derived from aminoglycosides,using various strategies including modification of target sites by biosynthesis of specific enzymes, as well as mutation in bacterial chromosome and efflux pump (Krause et al., 2016;Rosenberg et al., 2000). Aminoglycoside antibiotic-resistant E. coli strains have been detected in patients, individuals, and food products in Saudi Arabia (Al Ghamdi et al., 1999;Alharbi et al., 2018;Ali, 2018).

Gentamicin
Gentamicin (C 21 H 43 N 5 O 7 ) is a secondary metabolite produced by Micromonosporapurpurea (a saprophytic, filamentous, aerobic, sporeforming, and Gram-positive bacterium, which can be isolated from the soil). In general, it is used for the treatment of bacterial infections caused by bacterial strains susceptible to antibiotics, including E. coli strains. Gentamicin is classified as an aminoglycoside antimicrobial agent with Patients (Ineta et al., 2018) broad-spectrum activity against Gram-positive and Gram-negative bacteria. It can obstruct the synthesis of bacterial proteins through interaction with the prokaryotic 30S ribosomal subunit, leading to misinterpretation of transfer ribonucleic acid (t-RNA) (Yoshizawa et al., 1998). In general, the bacteria acquire aminoglycoside resistance via three potential mechanisms: 1) alteration in bacterial cell permeability (reducing uptake), 2) modification at sites in the ribosomal subunit, and 3) synthesis of specific enzymes having the ability to modify the chemical structure of aminoglycosides (Yoshizawa et al., 1998)

Streptomycin
Streptomycin (C 21 H 39 N 7 O 12 ) is chemically classified as an aminoglycoside antimicrobial agent that can be produced by Streptomyces griseus,which is frequently isolated from the soil. Streptomycin has the ability to irreversibly bindto the 30S ribosomal subunit proteins and 16S rRNA. The interaction between streptomycin and decoding area in16S rRNA of the 30S ribosomal subunit (site near nucleotide 1400). The principle of interaction is the capacity of streptomycin to bind with a single amino acid of the 30S ribosomal protein S12 and four nucleotides of 16S rRNA, which lead to mRNA misreading. In bacteria, genetically acquiredstreptomycin resistance is frequently due to genetic alteration in rpsLgene,which encodes the ribosomal protein S12 (Springer et al., 2001).
In Saudi Arabia, E. coli strains resistant to gentamicin have been identified and isolated from inpatients, outpatients, animals, and foods. Spontaneous streptomycin resistant E coli strains have a genetic alteration in several sites in 30S ribosomal protein S12 including Lys42, Lys87, Pro90, and Gly9 (Chumpolkulwong et al., 2004). In Saudi Arabia, the E coli strains resistant to streptomycin have been characterized and streptomycin-resistance genes have been detected (Abo-Amer et al., 2018). Streptomycin-resistant E. coli strains have been isolated from numerous sources including raw chicken meat, wastewater, as well as in patient samples (skin, blood, urine, stool, and respiratory tract) (   study performed in Taif, Saudi Arabia, more than 48% of the streptomycin-resistant E. coli strains (N=119) were isolated from retail raw chicken meat (Al Johani et al., 2010).
In Riyadh, Saudi Arabia, it has been reported that all isolates of E. coli(N=200) detected in thefeces of broiler chickens are resistant to streptomycin (Al-Arfaj et al., 2015). Streptomycin-resistant strains have been found to be the preponderant strains among enterotoxigenic E. coli isolates (N=181) collected from patients with diarrhea (Willshaw et al., 1995).

Tobramycin
Tobramycin (C 18 H 37 N 5 O 9 ) is a narrow spectrum aminoglycoside antimicrobial agent, which can interact with microbial 30S and 50S ribosome,thereby preventing the formation of 70S ribosome complex. It is widely used to treat microbial infections caused by Gram-negative bacteria. The intracellular concentration of tobramycin is critical for its action. Active transport of tobramycin through the bacterial membrane is a significant mechanismthat helps to increase tobramycin concentration inside the bacterial cell. The bacterial strains generally gain resistance to tobramycin through one or more of the three strategies mentioned above (physiological or genetic alteration in cell permeability, mutation at the ribosomal binding sites, or synthesis of enzymes having the ability to modify the aminoglycoside) (Islam et al., 2009). In Saudi Arabia, 67% of the E. coli strains (N=1116) isolated from patients were tobramycin-resistant strains (Kader and Kumar, 2004). In 2015,57% of E. coli strains (N=130) detected in pilgrims (patients) admitted in Makkah, Saudi Arabia were tobramycin-resistant (Haseeb et al., 2016).In King Fahd Hospital University, Clinical Microbiology Department, Al-Khobar, Saudi Arabia (Al-Zahrani and Akhtar, 2005), more than 75% of the E. coli strains (N=48) depicted the ability to resist tobramycin.  (Ali, 2018).It has been reported that neomycin is one of the less-used drugs among the 44 antibiotic drugs used to treat urinary tract infections (N=339) (Alsohaim et al., 2019).
Bothextended spectrum β-lactamase (ESB) E. coli or non-ESB E. coli strains show resistance to synergic action produced from sulfamethoxazole and trimethoprim (cotrimoxazole) among clinical isolates (Al-Otaibi and Bukhari, 2013). In fact, it is believed that the resistance to antibiotics is increasing continuously;however, sometimes the opposite occurs and the strains show susceptibility to the same antibiotics to which they were resistant in the past (Table).

ConClusion
Antibiotics are frequently used for therapy of infected humans and animals. Treatment for E. coli infection has been highly difficult by the rise of resistance to most first-line antibiotics. The data showed the prevalence of E. coli strains in Saudi Arabia, which can resist all antibiotic groups including β-lactams, cephalosporins, aminoglycosides (except fifth generation), macrolides, chloramphenicol, quinolones, flouroquiolones, nitrofurans, sulfonaides, pyrimidine analogues, semisynthetic lincosamides, glycopeptides, and tetracycline antibiotics. The integrated monitoring and management of the antibiotics used to treat infections caused by E. coli must be applied to reduce health hazards.

ACknowledgments
The author wishes to thank Dr. Jamal M. Khaled and Dr. Shine Kadaikunnan for the helpful discussions on the topic. This project was funded by the King Abdulaziz City for Science and Technology (KACST), Kingdom of Saudi Arabia (Award number: LGP-36-7).