Antibiotic Resistance Genes in Enterobacteriaceae

Enterobacteriaceae is a family of Gram-negative rod-shaped Gammaproteobacteria in the order Enterobacterales in the phylum Pseudomonadota. These bacteria are commensals of the intestine of animals, including humans; however, some are pathogenic to humans and even cause severe infections.

They are held accountable for several infectious diseases like urinary tract infections (UTIs), respiratory tract infections (RTIs), blood-stream infections (BSIs), gastrointestinal infections, wounds, and soft tissue infections. For the treatment of such Enterobacterales-associated infections, β-lactam antibiotics are the most preferred antibiotics. Besides β-lactams, tetracyclines, colistin, and aminoglycosides are also widely used in case β-lactams have lower sensitivity.

However, the emergence of antibiotic resistance has made most of the available and preferred treatments useless against Enterobacterales. Antibiotic resistantance genes such as Beta-lactamase genes (bla genes), colistin-resistant genes, tetracycline-resistant genes, and aminoglycoside-resistant genes are the most important antibiotic-resistant genes in Enterobacterales.

Some of the most important members of these antibiotic-resistant gene families are briefly described in this article.

Beta-lactamase (bla) Genes

• Beta-lactamase genes (bla genes) are the largest group of antibiotic-resistant genes found in Enterobacteriaceae and other bacteria that confer resistance against the β-lactam antibiotics. These genes encode for β-lactamase enzymes that hydrolyze the β-lactam ring of β-lactam antibiotics such as penicillins, cephalosporins, carbapenems, aztreonam, etc. 
• Mechanism of Conferring Resistance: bla genes encode for the synthesis of β-lactamase enzymes. These enzymes are capable of hydrolyzing β-lactam ring of β-lactam antibiotics. Hydrolysis of the β-lactam ring disrupts their molecular structure and the antibiotics can’t interrupt the process of crosslinking during bacterial cell wall synthesis. 
• Different types of bla genes code for different variants of β-lactamase enzymes, and each of them may have different activity toward different members of β-lactam antibiotic class. Some can hydrolyze β-lactam ring of penicillins only, while some can hydrolyze the β-lactam ring of penicillins, cephalosporins, monobactams, carbapenems, and even β-lactamase inhibitors.

Some of the most important bla genes found in clinical isolates of Enterobacteriaceae are listed below.

1. bla_TEM Genes

• bla_TEM genes are antibiotic-resistant genes in the bla gene class which confer resistance against β-lactam antibiotics, mostly ampicillin and penicillins, to the bacteria containing the genes. bla_TEM gene family contains the most prevalent bla genes in Enterobacteriaceae. 
• TEM is the abbreviated form of Temoniera, an Athenian patient from which this gene and its gene product were isolated. TEM-1 enzyme encoded by the bla_TEM-1 gene was the first plasmid-mediated β-lactamase enzyme identified in 1963. 
• Currently, there are 170 types of TEM β-lactamases each encoded by a variant of the bla_TEM gene. The bla_TEM-1 gene is the most commonly isolated variant in Enterobacteriaceae. bla_TEM-1 gene variants are referred to as Extended Spectrum Beta-Lactamases and show hydrolyzing activity against a wide range of β-lactam antibiotics like penicillins and cephalosporins. They are mainly reported in ESBL-producing E. coli and K. pneumoniae strains. Other variants of bla_TEM genes are also reported to encode enzymes that can hydrolyze aztreonam, monobactam, the 3^rd and 4^th generation cephalosporins, and some β-lactamase inhibitors. 

Mechanism of Conferring Resistance of bla_TEM Genes

• bla_TEM genes are β-lactamase enzyme encoding genes. They encode for TEM- β-lactamase enzymes, Class-A β-lactamases. These enzymes are capable of hydrolyzing β-lactam ring of aminopenicillins (like amoxicillin and ampicillin) and early cephalosporins (mostly the first and second-generation cephalosporins).

Detection Method of bla_TEM Genes

• The presence of bla genes can be phenotypically presumed by performing antimicrobial sensitivity testing using different groups of beta-lactam antibiotics, but, for the identification of actual genes involved in the process, molecular detection methods are very effective.  PCR, DNA probe method, DNA microarray, etc. can be used. Polymerase chain reaction (PCR) remains superior over all available molecular detection methods and is widely used in clinical and research laboratories. 
• Primers that are used in some research to identify bla_TEM genes are:

Nucleic acid sequencing of the isolated TEM gene must be done in order to classify the gene up to the level of its gene variants. 

2. bla_CTX-M Genes

• bla_CTX-M gene is another family of bla genes that encode for CTX-M type β-lactamase synthesis. bla_CTX-M genes are identified predominantly in ESBL strains of E. coli and Klebsiella pneumoniae globally. These are found in the plasmid of Salmonella enterica serovar Typhimurium, E. coli, Klebsiella spp., and Proteus spp. These genes are usually found in the larger plasmid, IncFII plasmids, containing other antibiotic-resistance genes. These genes are capable of horizontal gene transfer and hence the bla_CTX-M genes can be easily disseminated in other Enterobacterales. 
• CTX-M is an abbreviated form of cefotaximase and Munich (CTX for cefotaximase and M for Munich) as CTX-M type β-lactamases are capable of hydrolyzing cefotaxime and first identified in E. coli isolated from ear discharge of a 4-month child in 1989 in Munich, Germany. This enzyme was called CTX-M-1, and the gene encoding the enzyme is bla_CTX-M-1. 
• Currently, 172 CTX-M types enzymes are identified suggesting that there are more than 172 variants of bla_CTX-M genes. bla_CTX-M-1 and bla_CTX-M-15 genes are the most predominant types of the bla_CTX-M gene family. 

Mechanism of Conferring Resistance of bla_CTX-M Genes

• bla_CTX-M genes encode for the synthesis of CTX-M (cefotaximase ) beta-lactamase enzymes. These enzymes hydrolyze the β-lactam ring of oxyimino-thiazolyl cephalosporins (like the 3^rd and the 4^th generation cephalosporins such as cefotaxime, ceftazidime, ceftriaxone, etc.). Most of them have greater activity against Cefotaxime, while some have higher activity against ceftazidime. 

Detection Method of bla_CTX-M Genes

• PCR remains the superior method for the detection of bla_CTX-M genes. Some primers that can be used for their detection are:

CTX-M consensus primers can be used to differentiate the identified bla_CTX-M genes into several variants. However, PCR alone can’t be enough to promptly differentiate the bla_CTX-M genes, hence, nucleic acid sequencing of the amplicons must be done for complete identification of the gene variants. 

3. bla_SHV Genes

• bla_SHV gene family is another group of bla genes that encodes for the synthesis of SHV-type β-lactamases that can hydrolyze cephalosporins and other β-lactam antibiotics. These genes were originally found in chromosomal DNA; however, they are now widely reported to be found in the plasmid DNA of several clinically isolated Enterobacterales. These genes are mainly found in Klebsiella pneumoniae, E. coli, Shigella spp., and Enterobacter cloacae.  
• SHV is the abbreviated form of the ‘Sulfhydryl variant’. 
• The first bla_SHV gene was identified in the early 1970s in E. coli and was called the bla_{SHV -1} gene. Since then, more than 215 bla_SHV gene variants are identified. These variants encode several SHV-type beta-lactamase enzymes. Some of them have activity against a wide variety of cephalosporins (including the oxyimino cephalosporins), penicillins, and monobactam; hence are called ESBL SHV variants. Whereas, others are active against penicillins, ampicillin, and earlier cephalosporins. These are called non-ESBL SHV variants. 
• bla_SHV-1, bla_SHV-11, and bla_SHV-2 are the most predominantly reported variants of the bla_SHV genes.  

Mechanism of Conferring Resistance of bla_SHV Genes

• bla_SHV genes encode for SHV-type beta-lactamase enzymes that hydrolyze the beta-lactam ring of several susceptible beta-lactam antibiotics. They are mainly responsible for ampicillin and amoxicillin resistance in Klebsiella and E. coli. But, they can even hydrolyze the beta-lactam ring of the extended cephalosporins, aztreonam, monobactam, and even carbapenems.   

Detection Method of bla_SHV Genes

• PCR is the most helpful tool to isolate, amplify and identify the bla_SHV genes. Nucleic acid sequencing of the isolated bla_SHV gene is done to know which variant it is. The primers that can be used to amplify the bla_SHV genes are: 

4. bla_OXA Genes

• bla_OXA genes (Oxacillinase genes) are another type of bla genes that encode for the production of Oxacillinase enzymes. These genes have been reported in E. coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, etc. 
• Most of the bla_OXA genes are plasmid-encoded while some are also found in chromosomes and transposons of several Gram-negative bacteria.
• Some bla_OXA genes encode for ESBLs and carbapenemases, while some are responsible for non-ESBL Oxacillinases. Currently, more than 127 variants of OXA enzymes, each with several sub-variants, are discovered globally. Accordingly, more than 127 bla_OXA variants, each with multiple sub-variants are estimated to exist. 
• These genes encode for OXA-type enzymes that can primarily hydrolyze isoxazolylpenicillines like oxacillin, cloxacillin, dicloxacillin, etc., and other similar penicillins. Some variants can also hydrolyze cephalosporins, aztreonam, carbapenems, etc.
•   bla_OXA-1 types are most common in Enterobacterales and bla_OXA-48 gene variants are the most important variants as they are ESBL type and can even hydrolyze carbapenems. 

Mechanism of Conferring Resistance of bla_OXA Genes

• bla_OXA genes encode for class-D beta-lactamases called the Oxacillinases enzymes. These enzymes hydrolyze the beta-lactam ring of oxacillin (isoxazolylpenicillines) and other susceptible beta-lactam antibiotics. 

Detection Method of bla_OXA Genes

• PCR and gene sequencing methods are the primary methods for the detection and identification of the bla_OXA genes. The primers that can be used during the process are:

5. bla_IMP Genes

• bla_IMP (Imipenemase) genes are another group of bla genes that code for the synthesis of Imipenemases enzymes in bacteria. These genes are found in a wide variety of carbapenem-resistant species of Gram-negative bacteria including a variety of Enterobacterales (mainly, several strains of E.coli, Klebsiella pneumoniae, and Serratia marcescens).    
• Most of these genes are found in the integron of plasmid or chromosomal DNA and hence can be transferable with related bacteria. 
• 19 different types of Imipenemase enzymes are clearly known to date suggesting about at least 19 different variants of the bla_IMP genes. 

Mechanism of Conferring Resistance of bla_IMP Genes

• bla_IMP genes encode for Imipenemase enzymes, Metallo-beta-lactamase enzymes capable of hydrolyzing broad-spectrum beta-lactam antibiotics like broad-spectrum penicillins and cephalosporins, monobactam, and carbapenems. Enzymes coded by the bla_IMP genes can hydrolyze the beta-lactam ring of carbapenems making them clinically very important bla genes.

Detection Method of bla_IMP Genes

• PCR method is used to detect the presence of the bla_IMP genes in suspected bacteria. Some of the PCR primers used in the process are: 

6. bla_VIM Genes

• bla_VIM genes are a group of bla genes that encodes for the production of VIM (Verona integron encoded Metallo-beta-lactamase) type beta-lactamase enzymes. These genes are plasmid-encoded genes reported in several members of Enterobacteriaceae, primarily Klebsiella pneumoniae, E. coli, Serratia marcescens, Citrobacter spp., etc. Besides, they are very important in carbapenem-resistant strains of Acinetobacter baumannii and Pseudomonas aeruginosa. 
• More than 40 types of VIM enzymes are described indicating the presence of more than 40 variants (46 reported variants) of the bla_VIM genes. The bla_VIM-2 and bla_VIM-1 type genes are the two most commonly reported variants of the bla_VIM genes. 

Mechanism of Conferring Resistance of bla_VIM Genes

• bla_VIM genes encode for Verona integron-encoded Metallo-beta-lactamase enzymes. These enzymes can hydrolyze the beta-lactam ring of penicillins, extended-spectrum cephalosporins, carbapenems, and other beta-lactam antibiotics.    

Detection Method of bla_VIM Genes

• PCR is the widely used tool for the detection of the bla_VIM genes. The followings are some of the primers that can be used during PCR of bla_VIM genes. 

7. bla_NDM Genes

• bla_NDM genes are novel types of bla genes that are responsible for the production of NDM (New Delhi Metallo-beta-lactamase) type beta-lactamase enzymes. These genes are plasmid-encoded genes found in the transposon region of several large plasmids making them readily disseminated in a wide range of bacteria. They are reported in 20 different types of plasmid, extensively in IncFIB, IncA/C, IncFII, IncX3, IncL/M, and IncH type plasmids, and also in bacterial chromosomes. 
• The first bla_NDM gene detected was the bla_NDM-1 gene found in E. coli and Klebsiella pneumoniae of a Swedish patient in 2008. Since then this gene is globally reported in several Enterobacterales, Pseudomonas aeruginosa, and Acinetobacter baumannii. 
• 29 different variants of the bla_NDM genes are known, but the bla_NDM-1 type gene is most commonly reported. These genes are most prevalent in East Asia, South East Asia, and South Asia, but are also frequently reported in Europe and the USA. 

Mechanism of Conferring Resistance of bla_NDM Genes

• The bla_NDM genes encode for New Delhi Metallo-beta-lactamase enzymes capable of hydrolyzing beta-lactam ring of penicillins, extended-spectrum cephalosporins, aztreonam, carbapenems, and other beta-lactams. These enzymes are Metallo-beta-lactamases having two zinc ions on the active site that helps in hydrolytically cleaving the beta-lactam rings of carbapenems.  

Detection Method of bla_NDM Genes

• The molecular detection method using the PCR technique is the most widely used gene detection method used to identify and amplify the bla_NDM genes. The primers that can be used during the process are tabulated below. 

8. bla_AmpC Genes

• bla_AmpC genes are a group of antibiotic-resistant (bla) genes that encode for AmpC-beta-lactamase enzymes. These genes were originally considered to be chromosomal genes; however, diverse types of plasmid-mediated bla_AmpC genes (pMAmpC genes) are discovered since the first detection of pMAMPC in 1989. 
• These genes are among less studied bla genes but have very great importance in conferring resistance against -methoxy-β-lactam antibiotics, extended-spectrum cephalosporins, and monobactam. Additionally, the AmpC-type beta-lactamases encoded by the bla_AmpC genes are poorly inhibited by several beta-lactamase inhibitors, so they are very potent Cephalosporinases.  
• These genes are extensively reported in Enterobacterales, Pseudomonas, and Acinetobacter species. Among the Enterobacterales, E. coli, Klebsiella spp. (mainly K. pneumoniae and K. oxytoca), Citrobacter spp., Enterobacter spp., Proteus spp., and Serratia spp. are mostly found to have the bla_AmpC genes. 

Mechanism of Conferring Resistance of bla_AmpC Genes

• These genes encode for AmpC-type beta-lactamase enzymes, a class of Cephalosporinases, which hydrolyze the beta-lactam ring of earlier as well as extended-spectrum cephalosporins. These enzymes hydrolyze the beta-lactam ring of most of the cephalosporins; hence making them ineffective against the bacteria containing the bla_AmpC genes. 

Detection Method of bla_AmpC Genes

• The PCR is the superior tool used in the detection of the bla_AmpC genes. Some of the primers used during the PCR amplification processes are:

Other bla Genes

Besides the 8 most common and clinically very important bla genes described above, there are several types of the bla genes responsible for beta-lactam resistance in several species of the Enterobacteriaceae family. Some of them are:

aac(6′)-Ib Genes

• The Aminoglycoside 6′-N-Acyltransferase type Ib genes (aac(6′)-Ib genes) are the antibiotic-resistant genes that confer resistance against aminoglycoside antibiotics. Its variant the aac(6′)-Ib-cr gene also confers resistance to fluoroquinolones to some extent. Most of the aac(6′)-Ib genes isolated from pathogens are reported in the plasmid in the transposons; however, these genes are also present as chromosomal genes in a few bacteria. 
• Among the aminoglycoside acetyltransferase (aac) genes that enzymatically modify the aminoglycosides, the aac(6′)-Ib genes are the most prevalent and clinically important acc genes. Over 50 variants of the aminoglycoside 6′-N-acyltransferase type Ib enzymes are known indicating the presence of more than 50 variants of the aac(6′)-Ib genes. 
• The aac(6′)-Ib genes are the major genes in Enterobacterales like E. coli, Serratia marcescens, Proteus mirabilis, and Enterobacter spp. and in Pseudomonas aeruginosa. In such bacteria, the aac(6′)-Ib genes are mainly provide higher resistance against amikacin, kanamycin, tobramycin, etc., and comparatively lower resistance against gentamycin and streptomycin. 

Mechanism of Conferring Resistance of aac(6′)-Ib Genes

• The aac(6′)-Ib genes encode for the aminoglycoside 6′-N-acyltransferase type Ib enzyme that causes acylation of the aminoglycoside antibiotics. These genes are capable of acetylation at the multiple sites of aminoglycosides using the acetyl CoA as a substrate donor. These acetylated aminoglycosides are inactivated and can’t disturb the protein synthesis (amino-acid elongation step) process in bacteria.  

Detection Method of aac(6′)-Ib Genes

• As in the detection of other antibiotic-resistant genes, the PCR remains the most important tool to detect the aac(6′)-Ib genes. Some of the primers used for the amplification of the aac(6′)-Ib genes are tabulated below. 

aph(3’) Genes

• Aminoglycoside phosphotransferase type 3 genes (the aph(3’) genes) are antibiotic-resistant genes that phosphorylate the aminoglycosides and confer resistance against such antibiotics to the bacteria-harboring such genes.  These genes are primarily plasmid-encoded genes; however, some variants of the aph(3’) genes are also reported in a few bacteria. 
• The aph(3’)-Ib type genes are found in Enterobacterales like E. coli, Klebsiella pneumoniae, K. quasipneumoniae, and Serratia marcescens. This gene encodes for aminoglycoside phosphotransferase type Ib enzyme in these bacteria making them resistant to aminoglycoside antibiotics like gentamicin, kanamycin, and tobramycin. 

Mechanism of Conferring Resistance of aph(3’) Genes

• The aph(3’) genes code for the production of aminoglycoside 3-phosphotransferase (APH(3’)) enzymes that phosphorylate aminoglycosides making them unable to affect the bacterial protein synthesis process. 
• The APH(3’) enzyme, also called the aminoglycoside kinase,  catalyzes the transfer of phosphate from an ATP molecule to the 3’ hydroxyl group of aminoglycosides like Kanamycin through a dissociative mechanism. Thus phosphorylated aminoglycosides lose their ability to disturb the peptide elongation process.   

Detection Method of aph(3’) Genes

• The molecular detection method using a PCR is the most important tool in identifying the aph(3’)-Ib type genes. Primers that can be used in the process are:

Mobilized Colistin Resistance (mcr) Genes

• Mobilized colistin resistance genes (mcr genes) are a class of antibiotic-resistant genes that confer resistance to the bacteria against polymixin E (colistin) antibiotic. These are plasmid-encoded antibiotic-resistance genes (except the mcr-6 genes that are reported in bacterial chromosomes) and can be easily transferred to other bacteria of clinical concern. So far, these genes are most commonly reported in E. coli and Klebsiella pneumoniae, but considering the ability of the mcr genes to rapidly transfer from one bacterium to another, it is very likely that several other pathogenic bacteria also harbor these genes. 
• To date, 10 different mcr gene types (mcr-1 to mcr-10), each with multiple variants, have been known. Among these mcr gene types, the mcr-1 type genes are the most predominant. The mcr-1 gene was first identified in November 2015 in China in E. coli and K. pneumoniae. A total of 27 different variants of the mcr-1 gene have been recorded so far in a dozen of pathogenic bacterial strains.
• The mcr genes are reported in E. coli, Klebsiella pneumoniae, K. oxytoca, Salmonella enterica, Enterobacter spp., Shigella spp., Citrobacter freundii, Cronobacter spp., and Aeromonas spp.  

Mechanism of Conferring Resistance

• The mcr genes confer resistance by modifying the antibiotic (colistin) target site. Colistin binds to the lipopolysaccharide and phospholipid of the bacterial cell membrane and replaces the Ca²⁺ and Mg⁺² leading to the disruption of the cell membrane and leaking of the internal contents causing bacteria to die.  
• The mcr genes encode for Mobilized Colistin Resistant proteins (MCR proteins). These proteins are phosphoethanolamine (PEA) transferase enzymes that catalyze the addition of the phosphoethanolamine moiety from a phospholipid found in the bacterial membrane, called the phosphatidylethanolamine, to the head of lipid A. This modification of lipid A reduces the affinity of colistin to the lipopolysaccharide (LPS) rending the bacterium resistance against colistin.   

Detection Method

• Although several methods have been devised to detect colistin resistance in bacteria, the detection of the mcr genes primarily depends on the PCR method. For the PCR, different primers have been used, some of which are tabulated below. 

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