sub-inhibitory concentrations of different antibiotics elicit certain phenotypic and even metabolic observed changes in specific bacterial species; changes that, in most cases, are beneficial to the target organism or community.
Basically, antibiotic compounds play an important role of effector molecules in their natural environments, then their mechanism of action could be seen as using their specific targets as signal relays. For example, tetracyclines binding to the ribosome cause a breakdown in translation that ultimately lead to cell death at high concentrations; but at sub-inhibitory concentrations, the binding to the ribosomes causes a temporary stall in translation that leads to an mRNA build up thereby stabilizing mRNA transcript concentrations in target cells. (Fajardo et al., 2008). Therefore, the action of antibiotic is actually an interesting way of using ribosomes, DNA, RNA, carbohydrates (peptidoglycan) as signal effector receptors and transducers .If antibiotic compounds play the role of effector molecules in their natural environments, then their mechanism of action could be seen as using their specific targets as signal relays. An example, is when tetracyclines binding to the ribosome cause a breakdown in translation that ultimately lead to cell death at high concentrations; but at sub-inhibitory concentrations, the binding to the ribosomes causes a temporary stall in translation that leads to an mRNA build up thereby stabilizing mRNA transcript concentrations in target cells .
2.9.1 CLASSES AND MODE OF ACTION OF ANTIBIOTICS
Bacterial cells are surrounded by a cell wall that is made of peptidoglycan layer, which consists of a long sugar polymer. The peptidoglycan layer undergoes cross-linking of the glycan strands by the action of transglycosidases, which serves as an enzymes and the peptide chains which extend from the sugars in the polymers and form cross links, one peptide to another (Kahne et al., 2005).The D-alanyl-alanine portion of peptide chain is cross linked by glycine residues in the presence of penicillin binding proteins (PBPs).This cross-linking strengthens the cell wall. ?-lactams and the glycopeptides inhibit cell wall synthesis.
?-lactam agents which are the PBPs has a primary agent. It has been hypothesized that the ?-lactam ring mimics the D-alanyl D-alanine portion of peptide chain that is normally bound by PBP. The PBP interacts with ?-lactam ring and are not available for the synthesis of new peptidoglycan. When the peptidoglycan layer lysis it lead to disruption of bacterium.
The glycopeptides binds to D-alanyl D-alanine portion of peptide side chain of the precursor peptidoglycan subunit. Vancomycin is a large drug molecules that prevents binding of this D-alanyl subunit with the PBP, and hence inhibits cell wall synthesis.(Grundmann et al., 2016).
• Inhibitors of protein biosynthesis
Bacterial DNA information was first used to synthesize an RNA molecule referred to as messenger RNA (m-RNA) through a process known as transcription. Then, the macromolecular structure called ribosome synthesizes proteins present in m-RNA, a process called translation. Protein biosynthesis is catalyzed by ribosomes and cytoplasmic factors. The bacterial 70S ribosome is composed of two ribo-nucleoprotein subunits, the 30S and 50S subunits. (Yoneyana et al., 2016). Antimicrobials inhibit protein biosynthesis by targeting the 30S or 50S subunit of the bacterial ribosome. (Johnston et al., 2002).
– Inhibitors of 30S subunit Aminoglycosides
The aminoglycosides (AG’s) are positively-charged molecules which attach to the OM which is negatively charged leading to formation of large pores, and thus allow antibiotic penetration inside the bacterium. The main target of action is bacterial ribosome; to enter, there it must pass through cytoplasmic membrane requiring energy dependent active bacterial transport mechanism, which requires oxygen and an active proton motive force. For these reasons, AG work in aerobic conditions and have poor activity against anaerobic bacteria. These AG have synergism with those antibiotics, which inhibit cell wall synthesis (such as ?-lactam and glycopeptides) as it allows greater penetration of AG within the cell and at low dosages. AG’s interact with the 16S r-RNA of the 30S subunit near the A site through hydrogen bonds. They cause misreading and premature termination of translation of mRNA.
Tetracycline’s, such as tetracycline, chlortetracycline, doxycycline, or minocycline, act upon the conserved sequences of the 16S r-RNA of the 30S ribosomal subunit to prevent binding of t-RNA to the A site. (Yoneyana et al., 2006)
• Inhibitors of 50S subunit
This antibiotics interacts with the conserved sequences of the peptidyl transferase cavity of the 23S r-RNA of the 50S subunit, then it tends inhibits the protein synthesis by preventing binding of t-RNA to the A site of the ribosome. (Yoneyana et al., 2006).
These affect the early stage of protein synthesis, namely translocation, by targeting the conserved sequences of the peptidyl transferase center of the 23S r-RNA of the 50S ribosomal subunit. (Yoneyana et al., 2006).
This results in a premature detachment of incomplete peptide chains. Macrolides, lincosamides, and streptogramins B shows similar mechanism of action.
Linezolid is a recently approved member of novel class of antibiotic of this group which is completely synthetic. Oxazolidinones interfere with protein synthesis at several stages:
(i) Inhibit protein synthesis by binding to 23Sr RNA of the 50S subunit
(ii) Suppress 70S inhibition and interact with peptidyl-t-RNA. (Lambert, 2005).
• Inhibitors of DNA replication
The fluoroquinolones that is (FQ) inhibit the enzyme bacterial DNA gyrase, which nicks the double-stranded DNA, when a negative supercoils is introduced and then resealed then the nicked ends. This is necessary to prevent excessive positive supercoiling of the strands when they separate to permit replication or transcription. The DNA gyrase consists of two subunit. A subunit carries out the nicking of DNA, B subunit introduces negative supercoils, and then A subunit reseal the strands. The FQ’s bind to A subunit with high affinity and interfere with its strand cutting and resealing function. In Gram-positive bacteria, the major target of action is topoisomerase IV which nicks and separate’s daughter DNA strand after DNA replication. Greater affinity for this enzyme may confer higher potency against Gram-positive bacteria. In place of DNA gyrase or topoisomerase IV, mammalian cells possess topoisomerase II, which has very low affinity for FQ-hence low toxicity to cells. ( Higgins et al., 2003)
• Folic Acid Metabolism Inhibitors
-Sulfonamides and Trimethoprim
Each of these drugs inhibits distinct steps in folic acid metabolism. A combination of sulpha drugs and trimethoprim acting at distinct steps on the same biosynthetic pathway shows synergy and a reduced mutation rate for resistance. (Yoneyana et al., 2006).
Sulfonamides inhibit dihydropteroate synthase in a competitive manner with higher affinity for the enzyme than the natural substrate, p-amino benzoic acid. Agents such as trimethoprim act at a later stage of folic acid synthesis and inhibit the enzyme dihydrofolate reductase.
2.9.2 MECHANISM OF ANTIBIOTIC
2.7.1. Inactivation of Antibiotics
There are three major enzymes that inactivate antibiotics such as ?-lactamases, aminoglycoside-modifying enzymes, and chloramphenicol acetyltransferases (AACs).
?-lactamases hydrolyze nearly all ?-lactams that have ester and amide bond, for examples, penicillins, cephalosporins, monobactams, and carbapenems. About 300 ?-lactamases are known till date. ?-lactamases are broadly prevalent enzymes that are classified using two main classification systems: Ambler (structural) and Bush–Jacoby–Medeiros (functional). (Alebshun, et tal., 2007) Ambler classification system is described below:
a. Class A ?-lactamases: Also referred as penicillinase; these are clavulanic acid susceptible. Two commonly encountered Class A ?-lactamases found in members of Enterobacteriaceae are designated as TEM-1, SHV-1. These are penicillinase with little or no activity against cephalosporin. ( Rice et al., 2003).These are progenitors of extended-spectrum ?-lactamases (ESBL). ESBL are enzymes that have changed substrate profile because of amino-acid substitution allowing hydrolysis of most cephalosporins. ESBL are resistant to penicillins, third-generation cephalosporins (e.g., ceftazidime, cefotaxime, ceftriaxone), aztreonam, cefamandole, cefoperazone, but are sensitive to methoxy-cephalosporins, e.g., cephamycins and carbapenems and are inhibited by inhibitors of ?-lactamases, e.g., clavulanic acid, sulbactam, or tazobactam.