Literature Review on the Characterization of Carbapenem Resistant Escherichia Coli for Antibiotic Resistance

Human beings are closely related to the earth and all its components, which includes the flora, fauna, from the largest of beings to the smallest of creature. While we can experience our association with the seemingly visible world, surely we can’t ignore the role of microorganisms in health and well-being. They are even present in many body parts like gut playing vital role as gut flora. Bacteria are group of most primitive microorganisms which are present almost every part of the earth ranging from lakes to human skin or intestine. They may be beneficial or pathogenic to the human beings depending on their species or strain. Bacterial pathogen causes diseases. Research works in the last few decades has led to the discovery of antibiotics to inhibit and counter bacterial infections and diseases. Bacteria are classified in accordance with their different shapes as cocci, bacilli, vibro, spirilla. Based on their different nutritional habit they can be autotrophs, heterotrophs or chemotrophs etc.). They possess different type of flagella and their number varies (atrichos, monotrichous, amphitrichous etc.). Their response to varied degree of temperature keeps them in different classes (thermophilic, mesophilic, psychrophilic etc.). Based on their ability to retain few strains which is due to the composition of their cell wall they are called as gram positive or gram negative.

Gram negative bacteria

Gram negative bacteria cannot retain the crystal violet stain used in gram staining method due to its cell wall structure which has a thin peptidoglycan layer, has outer membrane containing lipopolysaccharides, and characterized by absence of teichoic acid. The presence of two membrane regulate the movement of substance in and out of the cell and helps maintaining shape and structural integrity of the cell. Cocci like Neisseria gonorrhoeae cause many sexually transmitted diseases (STDs) and respiratory diseases (Haemophilus influenzae). Many gram negative bacilli like Klebsiella pneumoniae, Pseudomonas aeruginosa are causative agent for many respiratory Problems and urinary tract infection by Escherichia coli and some bacteria like Helicobacter pylori, salmonella typhi causing gastrointestinal problems are extensively reported worldwide. Resistant gram negative bacterial infections treatment is challenging and can lead to high morbidity and mortality rates.

The ESKAPE pathogens which includes Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species are the principal cause of nosocomial infections all over the earth.Escherichia coliTheoder Escherich, a German pediatrician discovered Escherichia coli and hence the genus name is named after him. E. coli is a gram negative Facultative anaerobic, motile, Rod shaped, coliform, flagellated bacteria which don’t produce any spore and Commonly located in the lower intestinal region of endotherms. Usually they are harmless and sometimes even benefit the host by producing vitamin K2 and stops the pathogenic bacteria to invade the intestine. However, some strains are pathogenic in nature and contain disease causing genes.

Phylogenetic classification of Escherichia coli Domain Bacteria Kingdom Bacteria Phylum Proteobacteria Class Gamma proteobacteria Order Enterobacteriales Family Enterobacteriaceae Genus Escherichia Species Coli Phylogenetic classification of E. coli. Response of E. coli to different biochemical tests are the characteristic feature for its identification. It shows negative results for gram staining, oxidase test, Voges-proskauer, citrate and urease tests. It shows positive result for catalase test, methyl red test and indole test due to its ability to produce indole ring. It can ferment glucose, lactose, mannitol and beta galactosidase (ONPG test) but cannot ferment inositol.The closest genus to E. coli is shigella which also belongs to the same family Enterobacteriaceae. It can be differentiated from E. coli in MacConkey agar. This medium is a selective as well as differential media which selects gram negative bacteria over gram positive bacteria and differentiates lactose fermenting (E. coli) and non-lactose fermenting bacteria (shigella). Appearance of pink coloration indicates the presence of lactose fermenting E. coli which has used the lactose available in the medium and produced acids resulting in the lowered pH below 6.8 causing reversal of colour of the medium.

Antibiotics

Antibiotics are drugs used in the treatment and prevention of bacterial infections. Antibiotics were a huge discovery in 20th century and many bacterial diseases were under control until recently the antibiotic resistance came into the account. The fate of modern medicine relies onefficacious antibiotics. Antibiotics can be classified in different ways –Based on Mechanism of action-

Drug inactivation – bacteria produce enzymes like aminoglycoside-modifying enzymes, Beta-lactamases, or chloramphenicol acetyltransferases that irreversibly modify and inactivate antibiotics.

Alternation of target binding site – Ribosomal protection protein protect the bacterial cell from antibiotics that target ribosome of the cell to inhibit translation. The ribosomal protection protein binds to the bacteria cell’s ribosome which in turn changes its conformational shape. This permits the bacterial cell to continue synthesizing the bacterial proteins while preventing the antibiotics from binding to the ribosome. The target for the antibiotic is altered in this example.

Alteration of metabolic pathway of the target cell.iv. By reducing drug accumulation – due to decrease in drug permeability or increase in efflux pumps for the drug in the cell wall which pump out the drug and nullify its effect.

Basis of their spectrum of activity – can be broad spectrum or narrow spectrum. Can also be grouped on the basis of Weather they either kill the bacteria (Bactericidal) or inhibit (Bacteriostatic) the bacterial growth.

On the basis of the mechanism of action they follow to attack host they are of following types

β-lactams and β-lactamase inhibitors – Cell wall synthesis inhibitors

• Cephalosporins –inhibits cell wall synthesis.
• Aminoglycosides – Inhibits protein synthesis.
• Tetracyclines – Inhibits protein synthesis
• Macrolides –Inhibits protein synthesis
• Fluoroquinolones –DNA synthesis inhibitor
• Lipopeptides – inhibitors of Protein, DNA and RNA synthesis
• Sulphonamides –Folic acid synthesis inhibitor
• Macrolides –Inhibits protein synthesis, etc.

Antibiotic resistance

The ability of the bacteria to overcome the effect of antibiotics to which they were sensitive previously. Antibiotic resistance arises when bacteria undergo modification in response to the use of antibiotics to survive. It occurs naturally, however overuse of antibiotics stimulate and quicken the process. Resistance may arise. Naturally, by random genetic mutation or by One species acquiring mutation from other either by horizontal gene transfer, (transformation, transduction, or conjugation). Overuse of antibiotics increase selective pressure in bacterial populations resulting in survival of only resistance strains, which increases exponentially.

The world is exhausted of antibiotics. More than 20 new classes of antibiotics were marketed between 1940 and 1962. Since then, only two new classes of antibiotics have entered the market. With the emergence of resistant bacteria until 10–20 years ago not enough analogues are reaching the market to tackle the problem of antibiotic resistance, particularly among gram-negative bacteria.World health organization considers this as a huge threat towards human health and launched many programs like The Global Antimicrobial Resistance Surveillance System (GLASS), Global Antibiotic Research and Development Partnership (GARDP). β-lactam antibioticsSince the discovery of benzyl-penicillin in the 1920s, thousands of new penicillin derivatives and related β-lactam classes have been discovered. Each new class of βlactam has been developed either to increase the spectrum of or to address specific resistance mechanisms that have emerged in the targeted bacterial population.

These are the class of antibiotics with broad spectrum activity which have a β-lactam ring in their molecular structure. Bacteria develop resistance to these antibiotics by synthesizing an enzyme β-lactamase which possess hydrolytic activity & attacks the β-lactam ring.They work by inhibiting the cell wall synthesis of bacteria. The final step in the synthesis of the peptidoglycan later of bacterial cell wall is aided by transpeptidases known as penicillin-binding proteins (PBPs). β-Lactam antibiotics have similar structures to D-alanyl-D-alanine, the terminal amino acid residues on the precursor N-acetylglucosamine (NAG) & N-acetlymuramicacid (NAM) peptide subunits of the nascent peptidoglycan layer. This structural analogy between them facilitates binding of β-Lactam antibiotics to the active site of PBPs. Prevents the Peptidoglycan layer cross linking and blocks cell wall synthesis.β-lactamasesBacteria develop resistance to these antibiotics by synthesizing an enzyme β-lactamase which possess hydrolytic activity & attacks the β-lactam ring.

Depending on their action profile, β-lactamases could be classified as

1. Penicillinases – inactivate penicillin but do not degrade cephalosporin, aztreonam, or carbapenem.2
2. Cephalosporinases – they inactivate cephalosporins and aminopenicillins, but do not affect other (carboxy-penicillins and ureido-penicillins), aztreonam, and carbapenems.
3. Extended-spectrum b-lactamases (ESBL) -inactivate all β-lactams except carbapenems-ESBL are inhibited by clavulanic acid.
4. Carbapenemases- inactivate mainly carbapenems. The constant use of β-lactam class of antibiotics resulted in a drastic situation. β-lactamases genes have undergone mutations and are no longer vulnerable against newly developed β-lactam antibiotics. These enzymes are now known as extended-spectrum β-lactamases (ESBLs).

Carbapenem

Among the β-lactams currently available, they have the broadest range of activity and uniqueness of carbapenem is advantageous due to their relatively resistance to hydrolysis by most β-lactamases. In some cases, they act as “slow substrates” or inhibitors of βlactamases, and still target penicillin binding proteins to inhibit the cell wall synthesis and kill the bacteria. From the studies carried out on the previous carbapenems, the C1Position carbon was reported to be playing a major role in the potency and range of carbapenems and in their permanence against β-lactamases. There are different types of carbapenem due to their structural difference.

Types of carbapenem

Carbapenem resistance In Enterobacteriaceae, carbapenem resistance emerges due to two main mechanisms. Firstly, by gain of genes for carbapenemase that encode for enzymes capable of degrading carbapenems, or secondly by a decrease in the uptake of antibiotics due to porin expression in association with overexpression of β- lactamases that has very weak affinity for carbapenems.Carbapenem resistance can also be without the activity of carbapenemase. The outer membrane in gram negative bacteria which protects the cell from heavy metal and detergents, contains porins which are specific to this membrane. Porins form hydrophilic channels to selectively permit the uptake of essential nutrients and other compounds, including antibiotics. Hence changes in its number or activity like mutation in the gatekeeping loop, loss of porin expression, shift in the types of porins can help acquiring carbapenem resistance. Carbapenemase mediated resistance can be due three major ambler classes-Class A carbapenemase-Three major enzyme types namely IMI, SME, and KPC enzymes come under class A carbapenemases. Out of which KPC enzymes are currently have greater medical significance. All three enzyme types hydrolyze a broad variety of β-lactams which includes penicillins, cephalosporins carbapenems, and aztreonam. The first KPC producing strain was identified in 1996 in the Eastern USA. Till date 11 more KPC variants of it have been determined.

Class B carbapenemase-Class B β- lactamases exhibit a broad spectrum of hydrolytic activity including all penicillins, cephalosporins, and carbapenems, however it cannot hydrolyse monobactam aztreonam. Commercially available b-lactamase inhibitors like clavulanic acid, tazobactam, sulbactam cannot hinder their activity. Class B β-lactamases are metallo-β-lactamases that require zinc for its activity and are comprise of VIMs, IMPs, and the new NDM group. NDM (New Delhi metallo-β-lactamase) is one of the most clinically important carbapenemases. NDM-1 is endemic in the Indian subcontinent and was first detected in 2008 in K. pneumoniae and E. coli in a patient returning to Sweden from India and now it is globally spread.Class D carbapenemase-Class D β-lactamases, also named OXAs for oxacillinases, it includes about 232 enzymes. Based on their amino acid sequence, class D carbapenemases were reorganized into 12 subgroups namely OXA-48, OXA-23, OXA-24/40, OXA-51, OXA-58, OXA-134a, OXA-143, OXA-211, OXA-213, OXA-214, OXA-229, and OXA- 235. OXA-48 is the most potent class D carbapenemase for imipenem and is one of the most common class D carbapenemases. From K. pneumoniae isolates recovered in Turkey in 2003, the first OXA-48 producer was reported. Since then OXA-48 producing strains have been extensively reported as sources of nosocomial outbreaks in Turkey, Southern Europe and Africa.