A Review of Extended Spectrum β -Lactamases: Definition and Types

Extended-spectrum β -lactamases (ESBLs) are defined as those bacterial enzymes which are capable to hydrolyze most beta-lactam antibiotics, including penicillins, cephalosporins, and the monobactam aztreonam and especially expanded spectrum cephalosporins such as ceftriaxone, cefotaxime, ceftazidime. Worldwide, ESBLs are considered to be a serious threat, especially in hospitalized and immunocompromised patients. There is a growing prevalence and dissemination of ESBLs in bacterial isolates all over the world. Individuals at high risk are those exposed to bacterial species harboring ESBLs as they result in treatment failure in many cases. Thus, there is an urgent need to detect ESBLs producers with the formulation of strategic initiatives that participate in controlling their prevalence and dissemination. The current review aims to illustrate the importance of


Introduction:
It is well established that antimicrobials are employed for treating and preventing microbial, particularly bacterial, diseases in both humans and animals. They are produced from synthetic, natural, or semi-synthetic origins and inhibit or kill microbial cells [1], [2]. Antimicrobial agents have saved millions of lives worldwide in the past years by treating infections and preventing them. Unfortunately, the emergence of antimicrobial resistance has accompanied their introduction and usage in the medical field posing a growing global health challenge and threat [3]. Antimicrobial abuse, misuse, and overuse in different sectors and fields (medical, veterinary, agricultural, and industrial) are reported as the leading causes of what we can call an antimicrobial resistance pandemic [2], [3]. Deaths resulting from multidrug-resistant bacterial infections are expected to rise from seven hundred thousand to more than10 million per year, and the cost may exceed  [3], [4], [5], [6].
Bacterial possession of β -lactamases is considered as the most prevalent mechanism used to overcome the fatal effect of β -lactams through the breaking of the β -lactam ring which is crucial for the bactericidal action of the drug [7], [8], [9]. The resistance to β -lactams developed even before the discovery of the first antibiotic, penicillin. Penicillinase (the first β -lactamase known) was characterized in Escherichia coli (previously Bacillus coli) before the use of penicillin medically [10]. Chromosomally mediated β -lactamases are naturally occurring in many Gram-negative bacteria. Such enzymes are believed to be originated from bacterial penicillin binding proteins (PBPs), as their sequence share some homology with them. It is presumed that this evolution was likely because of the co-occurrence of soil microorganisms which produce β -lactam antibiotics and exert their selective pressure in the surrounding environment [11]. The enzyme TEM-1, a plasmid-encoded enzyme, was reported in the 1960s [12]. This enzyme was initially characterized in E. coli recovered from a Greek patient called Temoniera [13]. Being transposon and plasmid-encoded has assisted the dissemination of TEM-1 to other bacteria. Within several years, this enzyme had been spread globally in some species of the Enterobacteri-aceae, Haemophilus influenza, Pseudomonas aeruginosa, and Neisseria gonorrhoeae [7], [14]. SHV-1 (sulfhydryl variable) is another plasmid-encoded enzyme found commonly in E. coli and Klebsiella pneumoniae strains. SHV-1 was firstly detected in 1979. In general, it is chromosomally mediated in K. pneumoniae strains, however, it is commonly located on plasmid in the isolates belonging to E. coli [15].
In an attempt to overcome the new and growing challenge imposed by β -lactamases, many semi-synthetic β -lactam antimicrobials have been introduced that were specially manufactured to resist β -lactamase activity. Though, new variants have evolved with each new class causing resistance to those antimicrobials. The selective pressure imposed by the random use and abuse of the antimicrobials in health settings has participated in the emergence of new β -lactamases with expanded hydrolytic capabilities. Oxyimino-cephalosporins was one of the new antimicrobials introduced and became broadly used for the management of serious diseases, especially those caused by multi-resistant Gram-negative bacilli in the 1980s [7], [14]. Not surprisingly, the emergence of the β -lactamase variants capable of destroying oxyimino-cephalosporins was quick. SHV-2 was the first enzyme of this group, it was firstly described in a Klebsiella ozaenae strain recovered in Germany [16]. Due to their extended range of action and hydrolytic activity, particularly towards oxyimino-cephalosporins, these enzymes were termed extended-spectrum β -lactamases (ES-BLs). At present, several hundred variants of ESBLs have been characterized. These enzymes have been reported globally in different bacterial species especially those belonging to Enterobacterales and P. aeruginosa [7], [14], [17]. This review aims to give a simple definition of ESBL types and their impact on the global threat of antimicrobial resistance.

ESBLs:
ESBL enzymes represent an important category of serine enzymes that belong to class A of Ambler's molecular scheme and 2be subgroup of the functional scheme of Bush [18], [19]. They are widely disseminated in nature and categorized into numerous groups Table 1. ESBL-producing bacterial strains are distinguished by having the power to resist and hydrolyze numerous β -lactam agents i.e., penicillins, first cephalosporins, aztreonam (a monobactam), and oxyimino-βlactams, such as ceftazidime, cefotaxime, with no ability to hydrolyze carbapenems or cephamycins. However, they are affected by clavulanic acid, tazobactam, and sulbactam [9]. The largest subset of 2be subgroup was a result of mutations that lead to substitutions in amino acid sequences of TEM-1, SHV-1, and TEM-2 that expanded their action to include oxyimino-β -lactams and decreased, in return, their hydrolytic power for cephaloridine and benzylpenicillin [9] [14].
Subsequently, the rapidly proliferated Cefotaxime-hydrolyzing β -lactamase from Munich (CTX-M) enzymes were functionally similar to SHV and TEM enzymes and were re-lated to the chromosomally encoded β -lactamases of Kluyvera [14]. The majority of these variants attack cefotaxime more rapidly than ceftazidime (hence the name), and a number of them can attack cefepime as well. Contrary to the SHV or TEM enzymes, CTX-M variants are inhibited by tazobactam more readily than clavulanic acid [20], [21]. Furthermore, other types of ESBLs are also present, they are less common and unrelated to CTX-M, SHV, or TEM. Examples of these enzymes are SFO-1, BEL-1, TLA-1, BES-1, TLA-2, and members of the families VEB and PER. Typically, 2be subgroup enzymes remain susceptible to clavulanic acid, this characteristic is usually employed by clinical laboratories in the detection test for ESBLs [9], [22]. Additionally, active site extension which permits the activity increase against oxyimino drugs may also make the enzymes more susceptible to inhibitors such as tazobactam and clavulanic acid [23]. Generally, ESBLs are sensitive to cephamycins, and the majority of ESBLs-producing bacteria are sensitive to cefotetan and cefoxitin. Nevertheless, it has been documented that strains expressing ESBLs can show resistance to cephamycins because of the loss of a porin protein in the outer membrane [24], [25], [26].
β -lactamase variety has numerous reasons, the serine enzymes are very ancient once. It is estimated that they have been developing and evolving for almost 2 billion years, even before the bacterial divergence into Gram-positive and Gramnegative species [27]. They have been found in different bacterial species living in varied environments and hence are exposed to various selective pressures. Furthermore, ESBL genes have used the horizontal gene transfer mechanisms, i.e., conjugation and transduction, to transfer to new bacterial hosts and to become part of multi-resistance transposable elements now spreading in clinical and environmental isolates [14], [17]. In consequence, it is unfortunately true expectation that these enzymes will persist and continue to develop. ESBL-expressing strains, usually correlated with antimicrobial-resistant infections, are continuously reported with increasing rates worldwide which represents a universal threat facing the control and treatment of hospital-and community-acquired bacterial infections, especially those caused by Gram-negative bacilli with multiple drug-resistance such as Klebsiella spp., Pseudomonas aeruginosa, and Escherichia coli. which will limit therapeutic options and may lead to treatment failure [6], [9].

Types of ESBLs:
Many ESBL enzymes are derived from the original SHV or TEM β -lactamases and categorized in several groups with different designations Table 2 [18], [23] The ESBLs phenotype usually evolved as a consequence of point mutations at selected loci. SHV and TEM enzymes are most common in bacterial strains belonging to Klebsiella pneumoniae and Escherichia coli. As well, they have also been detected in

TEMs:
These enzyme variants are originated from TEM-1 enzyme, which was plasmid-mediated and firstly reported in the early 1960s [12]. It was initially characterized in an E. coli strain recovered in Greece from a local patient named Temoneira [13]. TEM-1 is considered the most frequently expressed enzyme in Gram-negative strains. It has been reported that almost 90% of ampicillin-resistant E. coli isolates are possessing this enzyme [8]. In addition, TEM-1 is also one of the major mechanisms used in resisting penicillin and ampicillin increasingly seen in N. gonorrhoeae and H. influenza clinical isolates. This enzyme can hydrolyze the first cephalosporins such as cephaloridine, cephalothin, and penicillins. TEM-2, as the first variant of TEM, has one substitution (glutamine for lysine at position 39) in comparison to the parent enzyme [28]. Although changing the isoelectric point (pI) from (5.4) to (5.6), it has no effect to alter the substrate profile. However, TEM-2 acted as the originator of several TEM variants with extended-spectrum activity [7], [14]. TEM-3, originally reported in K. pneumoniae in France in the late 1980s, was the first extended-spectrum TEM enzyme reported [29]. In the beginning, the enzyme was known as CTX-1, as it was more active against cefotaxime [30]. At present, nearly 243 various TEM variants have been reported and characterized, some of them are inhibitor-resistant, but most of them are ESBLs.
Amino acid alterations usually take place at a few specific and known number of positions [7]. These alterations result in numerous modifications in the enzyme phenotype, i.e., the ability to attack certain antimicrobials like cefotaxime and ceftazidime, or changing the enzyme isoelectric points (usual range: 5.2 -6.5). Several residues are particularly significant for generating the extended spectrum phenotype when alterations take place at that positions. For example, arginine substitution to either histidine or serine at position 164, glutamate to lysine at position 104, glutamate to lysine at position 240, and glycine to serine at position 238. It is noteworthy that among these substitutions, the alteration of glycine to serine and glutamate to lysine appear to have the most influence on the production of the extended-spectrum phenotype of the enzyme [14]. Furthermore, newer TEM enzymes show subtle alterations in their profiles. For instance, TEM-184 with the following substitutions: glutamate to lysine (position 6), glutamic acid substitution to lysine (position 104), isoleucine to valine (position 127), arginine substitution to serine (position 164), and methionine to threonine (position 182) can hydrolyze aztreonam more efficiently than cefotaxime or ceftazidime [31]. Although the analyzing of bacteria genomes through Whole Genome Sequencing (WGS) participated in detecting and discovering so many new TEM variants, only a few of them are phenotypically characterized. Nevertheless, network analysis with computer modeling has enabled researchers to predict whether a specific enzyme sequence has the probability to fit in the functional groups 2be (extendedspectrum), 2br (inhibitor resistant), or 2b (the original broad spectrum) [32].
Interestingly, it has been documented that the occurrence of TEM enzymes was regional to some geographical areas. For example, TEM-10 was the most prevailing enzyme in the USA [33]. In contrast, TEM-3 was infrequently described in the USA, but it was very common in France [34]. On the other hand, the TEM-26 variant was characterized in different bacterial species worldwide [34], [35], [36], [37]. At the present, as the CTX-M enzymes came to be the prevalent ESBLs globally, TEM variants have become infrequently reported. In a recent study screening bacterial isolates in Europe for ESBLs, these enzymes were characterized in no more than 1% of ESBL-expressing Klebsiella spp. and Escherichia coli [14], [38].

SHVs:
Sulfhydryl variable (SHV) β -lactamases were firstly reported as chromosomally determined enzymes in the strains of K. pneumoniae [8]. The first variant with ESBL phenotype (designated as SHV-2) was characterized in 1985 in an isolate of K. ozaenae recovered in Germany, this variant is varied from the SHV-1 enzyme by only one substitution (glycine to serine, position 238) [47]. Like TEM variants, most of the SHV-variants have substitutions at positions 238 (glycine to serine) and 240 (lysine to glutamine) [8]. Interestingly, both of these alterations are resemble of those found in the TEM variants. Serine substitution seems to be important for the effective breakdown of ceftazidime, while lysine substitution (position 240) is important for the effective cefotaxime hydrolysis as well [47]. The significance of amino acid substitutions concerning the phenotypic alterations in substrate profile has been studied and analyzed by mathematical modeling [48]. The SHV-1 enzyme is frequently characterized in bacterial isolates belonging to K. pneumoniae and it is the mechanism used in more than 20% of ampicillin resistance (usually plasmid-encoded) in these strains [49]. Noteworthy, SHV enzymes are more prevalent in clinical bacterial isolates than other types of ESBLs [50]. In many isolates of K. pneumoniae, blaSHV-1 has been found to be integrated into the chromosomal DNA [8]. Although the hypothesis of being part of transposable elements like plasmids, the SHV-1 encoding gene has never been characterized as so [51]. Contrary to the TEM enzymes, there are few variants of the SHV-1 enzyme. Additionally, the alterations that have been detected in the blaSHV gene to produce the variants take place in fewer positions in comparison to TEM enzyme, many of these variants express the ESBL phenotype. Though, a single variant, SHV-10, is documented to have the IR (Inhibitor Resistant) characteristics, it seems this variant has been resulted from the SHV-5 enzyme containing an extra alteration in amino acid sequence at position 130 as glycine replaced by serine [52].
In the latest European surveillance, 3.1%-17.0% of the clinical strains belonging to K. pneumoniae were found to harbor SHV-ESBLs [38]. Nevertheless, in a clinical study investigated clinically recovered ceftazidime resistant bacteria, SHV enzymes were infrequently detected and were only presented in isolates that also harbored a carbapenemase or a plasmid-encoded AmpC [62]. Although SHV and TEM enzymes are still reported, it seems that the influence of their occurrence amongst clinical strains is insignificant [14].

CTX-Ms:
Afterward, a new group of plasmid-encoded enzymes named CTX-M, which favorably attack cefotaxime has developed. This group of enzymes is differing from the SHV or TEM enzymes as they show approximately 40% homology with the both enzymes [63]. Initially, CTX-M was first reported in the late 1980s and their numbers are continuously growing as more than 128 variants of this enzyme have been detected worldwide [14], [46], [63]]. These enzymes are distinguished from others by hydrolyzing cefotaxime more effectively than ceftazidime, and cephalothin more efficiently than benzylpenicillin, they attack cefepime as well [63]. Unlike SHV and TEM ESBLs, no point mutation is occurred in CTX-M enzyme. It is believed that this enzyme was firstly described in the Kluyvera spp chromosome [14], [64]. The term CTX-M (standing for cefotaximase from Munich, Table 2 was firstly used in a German study [65]. Nevertheless, CTX-Ms that recognized in other areas were given diverse designations, like Toho-1 (Japan), MEN-1 (France), and FEC-1 (Japan) [21]. Outbreaks in different countries were followed and presenting an alarm for the potential threat that these enzymes could represent. Additionally, these variants have been documented as the most prevalent ESBL enzymes, instead of SHV and TEM. Variants of CTX-M have been described amongst various members of the enteric bacteria, Acinetobacter isolates and P. aeruginosa strains [66], [67], [68]. Furthermore, bacterial strains harboring CTX-M genes have been identified in the community and public health establishments, the environment, food products, livestock, and the companion animals [69].
As mentioned previously, the CTX-M enzymes preferentially hydrolyze cephaloridine or cephalothin (in comparison to benzylpenicillin) and cefotaxime (in comparison to ceftazidime) [63], [70]. As for ceftazidime, although these enzymes have a minor effect on it, they could not provide the required hydrolysis activity to make the strains clinically resistant to the antimicrobial. Serine (found in all CTX-M variants at position 237) is believed to have a critical effect in the extended-spectrum action of these enzymes [63]. Likewise as proposed by molecular modeling studies, the arginine residue (at position 276) which is equivalent in position to arginine 244 in SHV or TEM ESBLs, may participate in the enzymatic hydrolysis of oxyimino-cephalosporins [71]. Additionally, CTX-M ESBLs have an extra unique characteristic of being inhibited more efficiently by tazobactam in comparison to clavulanic acid or sulbactam [63], [70], [72], [73].  [75], [76], [77], [78]. CTX-M-25, CTX-M-2, and CTX-M-8 are prevailing in their own groups. Interestingly, it is documented by the analysis and studying of the CTX-M-2 gene to be originated from Kluyvera spp. Additional investigations had confirmed that this group has resulted from the Kluyvera ascorbate KLUA-1 enzyme [79], [80]. Likewise, CTX-M-134 (CTX-M-1 group) is documented to be resulting from the Kluyvera cryocrescens KLUC-1 enzyme and the CTX-M-9 has a resemble like characteristics with the Kluyvera georgiana KLUG-1 enzyme [81], [82]. Accordingly, an early divergence from a common ancestor may be suggested based on the evolutionary distances among these groups [7], [83]. Noteworthy, these variants also showed basic resemblance and enzymatic activities with various class A enzymes that were described in bacterial strains recovered from the environment, such as Rahnella aquatilis and Erwinia persicina [84], [85].

M-27 and CTX-M-15 are good examples of that. CTX-M-15
is a derivative from CTX-M-3 with only one amino acid alteration (aspartic acid to glycine at position 240) [86]. This substitution is responsible for the enzymatic accommodation to ceftazidime molecule, which has a larger size compared to cefotaxime [87], [88]. Similarly, CTX-M-27 has the same amino acid (position 240) which is believed to be responsible for the increased MIC values for ceftazidime in comparison to its originator CTX-M-14 [89]. Recently, a new variant (CTX-M-33) with a substitution of aspartic acid to serine (at position 109) compared with CTX-M-15 was described in a K. pneumoniae clinical isolate [90]. This enzyme has showed decreased hydrolytic activity against ceftazidime and elevated hydrolysis against meropenem. Interestingly, the isolate also had impaired permeability resulting in an elevated MIC for meropenem [90]. CTX-M enzymes are widely distributed and continuously emerging globally. However, even though alternative options are still available for bacterial strains carrying them alone, the concurrent occurrence of CTX-M ESBLs with other resistance mechanisms (i.e. impermeability) in the same isolate could affect the action of carbapenems or other newer agents and limit it [1].
Bacterial strains expressing various types of CTX-M enzymes have been reported from different parts of the globe, although they mostly have been related to outbreaks recorded in eastern Europe [70], [90], [91], [92], Japan, and South America [72], [93]. Furthermore, these enzymes also have been reported in bacterial strains isolated from immigrated patients in western Europe [94]. For instance, a clinical strain of Enterobacter cloacae with CTX-M-3 was isolated in France in 1998 [95]. Numerous laboratories and institutions in the outbreaks areas had documented the prevalence of CTX-M variants in the recovered isolates in comparison to other types of ESBLs [73]. Remarkably, some of these enzymes have been described in Salmonella enterica clinical isolates as well [70], [91], [94], [96], [97]. S. enterica strains possessing CTX-M enzymes were responsible for large outbreaks that occurred in both eastern Europe and South America. Additionally, these strains were documented to have multiple CTX-M enzymes. Consequently, it is questionable thing that a single origin for the existence and tendency of CTX-M enzymes within S. enterica can be existed [7], [14].

OXAs:
OXA β -lactamase is a growing group of ESBLs belonging to the functional group 2d and Ambler class D [18]. These enzymes, which display resistance to cephalothin and ampicillin, are well known for their specific hydrolysis of cloxacillin and oxacillin and poor inhibition by clavulanic acid [9], [18]. Generally, OXA enzymes show variability in amino acid sequences and substrate profiles. Nevertheless, many OXA enzymes have been reported to hydrolyze cephems, and/or monobactams as well as cephalosporins. Therefore, these enzymes are now classified as subgroup 2de [9]. Whether or not OXA enzymes with expanded-spectrum activity are considered as ESBLs is still questionable [98]. Many scientists do not agree with applying the ESBLs designation to oxacillinases because the OXA variants are grouped in the 2de subgroup and not in the 2be, in addition to their resistance to inhibition by clavulanate [14].
As documented by a current review, 27 oxacillinases have been characterized as ESBLs. The substrate profile includes the new cephalosporins (3 rd and/or 4 th generation drugs) in addition to the early ones and penicillins [99]. OXA enzymes have been described mostly in bacterial isolates belonging to P. aeruginosa in addition to Acinetobacter baumannii strains, and not in K. pneumoniae and E. coli as other ESBLs. For instance, OXA-21 was reported in an isolate of Acinetobacter baumannii as the first occurrence of OXA enzymes in this species [100]. The majority of OXA ESBL variants originate from OXA-2 and OXA-10 (PSE-2). The variants originated from OXA-10 are OXA-16, -14, -13, -11, -17, -28, and -19 [101], while those derived from OXA-2 include OXA-15, -53, -34, -141, -32, -36, -161, -226, and -210, most of these variants are described in P. aeruginosa [99]. OXA-14 originated from the OXA-10 enzyme and differs from it by one substitution, OXA-16 and -11 differ by two, and OXA-19 and -13 differ by nine. Interestingly, amongst these enzymes, the ESBL variants have one of these amino acids alterations: aspartate for glycine (amino acid position: 157), or asparagine for serine (amino acid position: 73). Particularly, the aspartate for glycine substitution may be critical for ceftazidime resistance [102]. Either substitutions could be essential to display the extended spectrum phenotype. On the other hand, the OXA-17 variant shows resistance to ceftriaxone and cefotaxime but displays only minimal hydrolysis activity against ceftazidime [103]. Additionally, although the traditional OXA enzymes were known for their resistance to inhibition by clavulanate, the OXA-18 variant was characterized to be affected and inhibited by it [7], [103].
Although OXA-1 and OXA-30 are not considered as ES-BLs, their capabilities to destroy cefepime have been documented [104], [105], [106]. These two variants were firstly reported to be different by one substitution; but it was revised later that these enzymes were actually the same [107]. Noteworthy, the OXA-1 enzyme accompanied by porin deficiency has been detected in false-ESBL phenotype-expressing E. coli strains [108]. Furthermore, an OXA-31 variant described in a P. aeruginosa strain had (3) alterations in comparison to OXA-1 and also showed a hydrolytic effect towards cefepime [109]. As well, OXA-405 and OXA-163 (derivatives from OXA- 48) have also been reported to show hydrolytic effect towards the new cephalosporins in addition to the carbapenemase activity characteristic of OXA-48-like enzymes [14], [110], [111], [112].

Inhibitor Resistant β -Lactamases:
Inhibitor-resistant enzymes were firstly discovered in the early 1990s. Although these enzymes don not have the distinguished ESBL phenotype, they are regularly discussed with them because they are usually originated from the traditional SHV or TEM enzymes. They are categorized into the functional group 2br Table 1 [9], [18]. The inhibitor-resistant variants derived from TEM enzymes are often not inhibited by sulbactam and clavulanic acid, but they remain sensitive to tazobactam and avibactam [112], [113], [114], [115], [116]. It is suggested that the mutations conferring resistance to sulbactam and clavulanate are also reduce the enzyme efficiency of hydrolyzing some cephalosporins like cephalothin and penicillins [115], [117].
Inhibitor-resistant TEM variants have been detected mostly in E. coli clinical strains, and infrequently in P. mirabilis, K. pneumoniae, Citrobacter freundii, and Klebsiella oxytoca isolates [118], [119]. They have mainly been described in France and in some other countries within the Europe continent [115]. Although these enzymes are infrequently detected, the variant TEM-30 was characterized in a K. pneumoniae strain with numerous KPC-producing bacteria isolated from a carbapenemresistant Enterobacteriaceae outbreak occurred in New York [113]. As documented by nucleotide sequencing data, the majority of these enzymes originated from TEM-1 and were previously known as " Inhibitor Resistant TEM, IRT", but now they are renamed using numerical TEM designations with 19 distinct inhibitor-resistant TEM variants known and documented so far [14], [120]. The common alterations in these enzymes are methionine at position 69, serine at position 130, arginine at position 244, arginine at position 275, and asparagine at position 276 [121]. These alterations in amino acids sequence are different from those described in the ESBL variants. Several variants of the SHV-1 and OHIO-1 have been reported to be resistant to inhibitors, such as SHV-107, -56, and -49 characterized in clinical isolates of K. pneumoniae in Europe [52], [116], [122], [123], [124].
In a laboratory experiment, mutants containing common substitutions for both extended spectrum and inhibitor resistant phenotypes have been constructed. These variants possessed either the IRT or the ESBL phenotype, but not both [125]. Additionally, a few TEM variants with both the inhibitor resistance and the ESBL phenotype have been reported [121]. Interestingly, these enzymes could be detected with ESBLs-screening methods depending on the clavulanic acid inhibition principle. For instance, TEM-50 enzyme with alterations characteristic of both the inhibitor-resistant and the ESBL phenotypes was reported in 1997. This variant was not affected by clavulanic acid, and displayed a minor resistance to 3 rd . generation cephalosporins [126]. TEM-152 is another variant of such enzymes, it was described in an E. coli isolate recovered from a French patient [127]. This mutant harbored the following amino acid substitutions: arginine for histidine (position164) and glutamine for lysine (position 240) which characterize the ESBLs phenotype, in addition to methionine for valine (position 69) and asparagine for aspartic acid (position 276) which characterize the inhibitor-resistant variant TEM-36. This variant efficiently hydrolyzes ceftazidime with 50% susceptibility to clavulanic acid. This could specify the emergence likelihood of a new subgroup with a complicated profile that shares features of inhibitor-resistant and ESBL enzymes. Interestingly, these complex enzymes are susceptible to avibactam, therefore the combination of a new β -lactamase inhibitor like ceftazidime/avibactam may represent an alternative therapy to face and treat bacterial diseases caused by strains with one of these variants [127]. It is expected that the dissemination of SHV-or TEM-type IR-variants is undervalued due to the absence of a specific phenotypic screening test that could be used routinely by laboratories to identify and detect the occurrence of these enzymes [14], [128], [129].

Other ESBLs:
There are several extended-spectrum β -lactamases have been described that are not part of the well-known groups of βlactamases Table 3. The PER-1 (Pseudomonas Extended Resistant) enzyme was firstly characterized in a P. aeruginosa isolate which was resistant to 3 rd generation cephalosporins and inhibited by clavulanate [130], [131]. Furthermore, this enzyme could hydrolyze many penicillins as well as cephalosporins including ceftazidime, cefoperazone, cefalotin, cefuroxime, and ceftriaxone, but not oxacillin, imipenem, and cephamycins. Soon after, it was also reported among other bacterial strains belonging to A. baumannii and S. enterica Typhimurium [132], [133], [134]. This enzyme is most commonly described in Turkey and Mediterranean countries in up to 60% of A. baumannii isolates that are ceftazidime-resistant [134], [135], [136]. Interestingly, the PER-1 enzyme was plasmid-mediated in several nosocomial strains of S. enterica Typhimurium, which might suggest the spread and acquisition of the resistance plasmid in the hospital setting [133]. Consequently, PER-2 was characterized in another S. enterica Typhimurium strain from Argentina with 86.4% homology with the original PER-1 enzyme [137]. Since after, PER variants have been reported in different species of Enterobacterales as well as Aeromonas spp. and A. baumannii isolates. PER-1 and PER-2 enzymes are the most common variants of the PER group, they have been characterized by their susceptibility to avibactam in comparison to other class A enzymes [138], [139]. Noteworthy, a recent analysis has documented that A. baumannii strains expressing PER variants can show increased minimum inhibitory concentrations against the siderophore cephalosporin, cefiderocol [140]. * pI : isoelectric point, CAZ: ceftazidime, CTX: cefotaxime, ATM: aztreonam.
VEB-1 is related to some extent to PER-1, the abbrevia- tion stands for Vietnamese Extended-spectrum β -lactamase Table 2, it was firstly described in a local E. coli strain from Vietnam [141]. Subsequently, this enzyme was described in a local P. aeruginosa isolate from a Thai patient [142]. VEB-1 displayed high resistance to aztreonam and ceftazidime Table 3, but only moderate susceptibility for cefotaxime and no activity against imipenem. Additionally, it was susceptible to clavulanic acid but not to avibactam [128], [143]. VEB variants were identified in different bacterial species including members of Enterobacterales, Achromobacter xylosoxidans, Vibrio spp., A. baumannii, and P. aeruginosa [144], [145], [146]. The Guiana Extended-Spectrum β -lactamases (abbreviated GES) are the most prevailing enzymes among the infrequently detected ESBLs. The GES-1 gene is not closely associated with other plasmid-encoded enzymes, even so, it has some homology (36%) with a carbenicillin-hydrolyzing enzyme identified in Proteus mirabilis Table 3 [7], [147]. These enzymes are more commonly described in A. baumannii and P. aeruginosa isolates. Noteworthy, they have been initially reported among Enterobacterales species [66], [148], [149], [150], [151]. Furthermore, these enzymes are known for their acquisition of one or two substitutions in amino acid sequences and expanding the substrate profiles to include carbapenems [12], [112].
GES-1 enzyme was firstly identified in 1998 in a strain of K. pneumoniae recovered from a French patient in French Guiana [147]. Concurrently, the IBC-1 enzyme was described in a strain of E. cloacae recovered in Greece [152]. Subsequently, the variants IBC-2 and GES-2 were characterized in clinically isolated strains of P. aeruginosa [153], [154]. Later, IBC-2 was renamed and given the designation GES-8 and IBC-2, GES-7. Remarkably, GES-2 differed from GES-1 in a single alteration in amino acid sequence (glycine for asparagine at position 170) and could hydrolyze carbapenems to some extent [153]. Therefore, the subsequent characterized GES variants were grouped into two categories: those with the ESBL phenotype, and those with modest activity against carbapenems. The original GES variants hydrolyze cephalosporins and penicillins, but not aztreonam [147]. They are affected by tazobactam, clavulanic acid, and newer inhibitors like vaborbactam, relebactam, and avibactam [138], [155]. Notably, GES-1 hydrolyzes ceftazidime more effi-ciently than cefotaxime. The following substitutions; glutamine for lysine (position 104), or glycine for alanine or serine (position 243) that noticed in the latterly characterized GES enzymes have displayed a hydrolytic activity against aztreonam and cephalosporins [14], [156].
Finally, numerous additional ESBLs enzymes were identified in the chromosomes of Enterobacteriaceae and other non-fermentative bacteria in different countries including Iraq [157], [158], [159], [160], [161], [162]. For instance, the OXY enzymes in Klebsiella oxytoca strains are undoubtedly the predominant resistance mechanism detected in this species [163], [164]. Moreover, there are other infrequently detected ESBLs have also been characterized in clinical bacterial isolates, but their incidence is somehow limited. An example of these enzymes is the SFO-1 variant, which was initially identified in an isolate of Serratia fonticola. This enzyme is a transferable variant, its production can be induced and enhanced by imipenem [165]. The plasmid encoding the gene blaSFO-1 also encodes ampR (regulatory gene) which is essential for expression induction of class C β -lactamases. Nevertheless, contrary to class C enzymes, SFO-1 is unable to attack cephamycins and is easily affected by clavulanate [164]. CME-1 is another related enzyme, that was identified in an isolate of Chryseobacterium meningosepticum [166]. Additional examples are; the TLA-1 enzyme described in an E. coli strain recovered from the Tlahuicas group (Mexican indigenous people), TLA-2 which was identified in Germany with 51% homology to TLA-1, BES-1 which was described in Brazil, and BEL-1 in Belgium Table 3 [144], [167], [168]. All of these enzymes display resistance to 3 rd generation cephalosporins, particularly ceftazidime, cefotaxime, and aztreonam. Additionally, they display limited resemblances to the chromosomally-encoded cephalosporinases described in Bacteroides spp. from which they might be originated [7], [166].

Conclusions:
Undoubtedly, the ESBL-producing bacteria are of great concern to the medical field as they are directly connected with an elevated mortality and morbidity rates. Additionally, it is time consuming to identify them, and subsequently their infections are difficult to treat. It is documented that the overuse and misuse of the broad-spectrum cephalosporins in the veterinary and health settings participated in the development and dissemination of ESBLs. Current therapy options for bacterial strains expressing the ESBLs enzymes is restricted to the broad-spectrum antimicrobials such as carbapenems. Nevertheless, there have already been therapeutic failure reports of these drugs as well. Bacterial strains conferring ESBLs would represent a serious challenge for clinicians and clinical microbiologists as we are heading into the next quarter of the 21 st century. Taking into account the globally rising prevalence rates of ESBLs-producing strains, and the lack of alternative therapy, the future is extremely concerning. Thus there is a crucial need for instant documentation and suitable strategy to control and reduce the occurrence of ESBLs. Controlling the use of broad-spectrum agents in different aspects and inspecting the environmental contamination are essential. Therefore, urgent and continuous works are mandatory to develop reliable, quicker, and cost-effective diagnostic tools as well as new active alternative antimicrobials for dealing with such ESBLs producing bacterial strains.
Data Availability Statement: All of the data supporting the findings of the presented study are available from corresponding author on request.

Declarations:
Conflict of interest: The authors declare that they have no conflict of interest.
Ethical approval: The manuscript has not been published or submitted to another journal, nor is it under review.