History of antibiotics:

In 1942, Selman Waksman coined the term “antibiotic” to substances which are produced by a microorganism and inhibit the growth of or kill other microorganisms even in low concentrations. Initially, the term chemotherapeutic agents were restricted to synthetic compounds. There has been a modification of the definition as many drugs are synthesized artificially. Hence, the term antibiotic is used to designate the antimicrobial agents or substances that are derived from natural or synthetic sources which attenuate the growth of or kill microorganisms by specific interactions with the bacterial targets causing no harm to the eukaryotic host who harbors the pathogen.¹

Various researches on antibiotics and its implications in treating dreadful infections began in the late 1800s. A French physician, Ernest Duchesne noted that a mould, from the genus Penicillium, inhibited the growth of bacteria.

But unfortunately, he failed to explain the factor which had antibacterial properties. Within a few years after his death, in 1928, a British scientist, Alexander Fleming accidentally found that one of his Staphylococci culture plates was contaminated by colonies of Penicillium, a mould. His earlier work on lysozyme, an antibacterial agent present in human tears helped him to realize that the mould secreted a similar substance that destroyed the bacterial colonies immediately surrounding the mould. He extracted the substance and named it as Penicillin.² Since he was unable to purify the substance and carry out clinical trials his work came to an end. Thus, penicillin was unavailable for therapeutic use until early 1940s. A team headed by Howard Walter Florey purified the substance and succeeded with clinical trials, thus proving its efficacy in treatment of dreadful infections. Howard Florey and Ernst Boris Chain produced penicillin in large quantities. This was a reliable and miraculously rapid drug for curing life- threatening infections during the Second World War.

In 1945, the Nobel Prize of Medicine and Physiology was awarded to Fleming, Florey and Chain for the discovery of penicillin and its clinical application.³ In 1949, x- ray crystallography helped in identifying the beta-lactam ring as the major component in the penicillin compound.⁴

With the development of penicillin, there has been a huge interest in the discovery of new antibiotics. Gerhard Domagk, in the year 1932 developed a synthetic compound, sulphonamide. Later, various drugs, both synthetic compounds and from natural sources were discovered. Some of the antibiotics obtained from microbes include streptomycin (1944), cephalosporin (1945), bacitracin (1945), chloramphenicol (1947), polymyxin (1947), tetracycline (1950), aminoglycoside and macrolides (1950s), vancomycin (1956) and so on. They were effective in treatment of bacterial pneumonia, syphilis, tuberculosis. But many of them for example, neomycin was too toxic, so the therapeutic use of these drugs had to be diminished. Hence, there began a new search for semi-synthetic and synthetic products with modifications to enhance their activity. In 1960, the first semi-synthetic drug was synthesized from penicillin compound, methicillin. The next synthetic drug was nalidixic acid (1962). All these drugs were effective against Staphylococcus spp., E. coli, H. influenza, S. Typhi, P. aeruginosa. Later in 1960s, there was development of first generation cephalosporins which led to the progression of second and third generation cephalosporins in 1970s and carbapenems in 1980s.⁵

Usage of these drugs in the field of medicine and surgery also improved clinical outcome. Subsequently, the rate of development of new antimicrobial agents dwindled, although many companies competed for research and development of newer antimicrobial drugs. But, in contrast, emerging and re-emerging infections, opportunistic infections caused by MDR organisms continue to rise. For an antimicrobial agent to be effective it should have the potency to enter the bacterial cell and accessibility to reach the target site. The reasons for their decline include their complexity and technical difficulty in discovering new drugs which target the thick cell wall of Gram-negative bacteria.⁶

Beta-lactam antibiotics:

With the discovery of penicillin and penicillin derivatives, the treatment of Gram-negative and Gram-positive bacterial infections had become an easy task with β-lactams. The β-lactam antibiotics are either from natural sources or semi-synthetic in origin. They are classified into four classical groups, based on the basic nucleus structure into penicillins, cephalosporins, carbapenems, and monobactams. They are further classified into subgroups based on the structure of the side chain. The structure of a β-lactam antibiotic consists of a β-lactam ring with one nitrogen and three carbon atoms.

Later on, modifications were made in order to enhance the biological activity of the antibiotic, to reduce its toxicity and to maintain stability against β-lactamases. The β-lactam ring, a four-membered structure is made to fuse with another ring consisting of about five to six atoms such that it forms a bicyclic ring structure.⁷ For example, in penicillins, the β-lactam ring is fused with thiazolidine, a five-membered ring. In cephalosporins, the β-lactam ring is fused with dihydrothiazine, a six-membered ring. In carbapenems, there is an additional ring similar to penicillins except the fact that the carbon atom replaces the sulphur atom. In monobactams, there are no fused rings. The four different classes of β-lactam antibiotics are described below in brief.

Penicillins:

Penicillins have a thiazolidine nucleus, β-lactam ring, a side chain at position C6. They are classified into Natural penicillins (penicillin G, penicillin V), penicillinase-resistant penicillins (methicillin, Oxacillin, cloxacillin), aminopenicillins (ampicillin, amoxicillin) and broad-spectrum penicillins (carboxypenicillins, ureidopenicillins).⁸

Cephalosporins:

In 1945, cephalosporin was discovered from Cephalosporium acremonium, a fungus. Cephalosporin-C was produced in large quantities from a mutant culture of C. acremonium. It was found to be 7- aminocephalosporanic acid.⁸ The first clinical application of cephalosporin in parenteral form was a modified type of cephalosporin-C, called cephalothin. Cephalosporins have a dihydrothiazine nucleus, a β-lactam ring, and sulphur atom at position 1. Cephamycin is derived from Actinomycetes and cefoxitin is the first semisynthetic cephamycin. It is classified into four generations based on the development of the antibiotic and the antimicrobial spectrum. Newer generation cephalosporins when compared to the previous generation have a greater action against gram negative bacteria.

The new generation cephalosporins with the mention of few of their examples are as follows; First generation cephalosporins- cephalothin, cefazolin. Second generation cephalosporins- ceftriaxone, cefoxitin, cefuroxime. Third generation cephalosporins- ceftazidime, cefotaxime, cefixime.Fourth generation cephalosporins- cefepime, cefpirome.

The fourth generation cephalosporins have a better penetration through porins in outer membrane of the cell wall. They are stable against hydrolyses by β-lactamases. In case of cephamycin, 7-alpha-methoxyl group is fused with the cephalosporin nucleus. This is responsible for the stability against class A β-lactamases.⁷

Carbapenems:

Carbapenems have a broad antimicrobial spectrum of activity among the β-lactam antibiotics. They are effective against Gram-negative, Gram-positive and anaerobic bacterial infections. They are the antibiotics of last resort for nosocomial infections and sepsis caused by MDR organisms. Carbapenems are broadly classified based on their origin into two types. They are,

· Natural - thienamycin.

· Synthetic - imipenem, meropenem, doripenem, ertapenem.

Thienamycin was the first carbapenem which was discovered from Streptomyces cattleya. Since thienamycin was unstable at a pH greater than 8 it was unsuitable for therapeutic use. The first carbapenem to be used clinically was imipenem. It is N-formimidoyl thienamycin, which is a stable compound. Carbapenems have a β-lactam ring and a thiazolidine ring which are fused together by nitrogen and tetrahedral carbon atoms. Imipenem consists of a thienamycin nucleus with a non-substituted group at position 1. Meropenem differs in having a methyl group in its chemical structure.^{9, 10}

Table 1: Indications of carbapenems¹⁰

Carbapenem	Indications
Imipenem	· Lower respiratory tract infections · UTI · Intra-abdominal infections · Gynecological infections · Bacterial septicemia · Bone and joint infections · Skin and soft-tissue infections · Endocarditis · Polymicrobial infections
Meropenem	· Nosocomial/community-acquired pneumonia · Septicemia · Skin and soft-tissue infections · Complicated intra-abdominal infections · Bacterial meningitis
Ertapenem	· Complicated intra- abdominal/UTI/skin and soft-tissue infections · Community acquired pneumonia · Acute pelvic infections · Prophylaxis for elective colorectal surgery

Monobactams:

It was discovered from Chromobacterium violaceum. It is the first monocyclic β-lactam antibiotic. In 1985, a compound called aztreonam was developed after modification in its chemical structure. It is the only drug which is used therapeutically. It is highly active against Enterobacteriaceae and Pseudomonas spp.¹¹

Polymyxins:

Polymyxins are cationic lipopeptide antimicrobial agents. There are five different polymyxin compounds (A-E). In 1949, polymyxin B was derived from Bacillus polymyxa and polymyxin E was derived from Bacillus polymyxa subspecies colistinus. Since 1959, clinically important polymyxins include polymyxin B and colistin (polymyxin E). They cause increased permeability of the cell membrane and favors diffusion of cellular components out of the cell by binding to phosphate moieties in the outer membrane of the cell wall.¹² The use of polymyxin A, C, D was withdrawn in the 1970s due to the risk of toxicity. With the emergence of MDR, they have been reintroduced for the infections caused by P. aeruginosa, A. baumannii and K. pneumoniae. However, there are reports which indicate the emergence of resistance to polymyxins due to a reduction in binding capacity with the outer membrane caused by an alteration in lipopolysaccharide (LPS). For the antimicrobial agent to exert its antimicrobial activity, it should have the ability to enter into the bacterial cell and reach the target site.¹³

The challenges of Gram-negative resistance:

Evolution of resistance:

On the other hand, unfortunately, bacteria have developed resistance mechanisms against most antibiotics thereby rendering them useless. Antibiotic resistance is defined as “the ability of a microorganism to resist the antibiotic pressure and survive”.¹⁴ It has to be noted that bacteria resistant to penicillin were isolated soon after the substance was discovered. Abraham and Chain noted that even before the introduction of penicillin into clinical use, the presence of enzyme penicillinase was reported from Escherichia coli (formerly Bacillus coli).¹⁵ As early as 1940s, it was observed that bacteria not only had the ability to become multidrug resistant but also the capacity to transfer the resistance to sensitive strains. Widespread use of antibiotics not only in humans but also in animals and in agriculture has induced a selection pressure in the history of evolution.¹⁶

Figure 1: Diagrammatic representation of number of β-lactamases reported since 1970¹

Some bacteria are intrinsically resistant to antibiotics with the resistant genes existing in the genome.¹ Lack of cell wall in Mycoplasma is an example of intrinsic resistance against cell wall acting antibiotics. There are chances for the susceptible bacteria to acquire antibiotic resistance genes and thus surviving the therapy. Acquired resistance is either by mutation or by transduction, transformation and conjugation.¹⁷ The most common mode of acquired resistance of β-lactamase genes is through plasmids, transposons and insertion sequences.¹

Plasmids are mobile genetic elements which transfer the genetic information, including resistance genes, between bacteria and capable of independent replication. Transposons are jumping genes that can transfer DNA from one site of the bacterial chromosome to another site or to a plasmid. Insertion sequence (IS) is a short DNA sequence which acts as a transposable element.^1,14

The three most common mechanisms of resistance include:

• Production of enzymes that hydrolyze the active site of the antibiotics and prevent binding to the target site

• Changes in the antibiotic target site, which reduce the affinity for antibiotics

• Porin loss or alterations of outer membrane proteins that reduce antibiotic permeability through the bacterial outer membrane and increased exportation of antibiotics through efflux pumps.

Bacteria also exhibit resistance to antibiotics by combination mechanisms. For example in K. pneumoniae, a combination of porin loss and plasmid-mediated AmpC β-lactamases confers resistance to imipenem.¹⁸

Figure 2: An overall representation of bacterial resistance mechanisms.¹⁶

Enzymatic inactivation or modification:

The chemical structure of most antibiotics is characterized by amide, ester bonds. Bacterial enzymes target these bonds and inactivate or modify them. Examples of bacterial enzymes responsible for resistance include β-lactamases, aminoglycoside modifying enzymes.¹⁹

Target alteration:

Target alteration by bacteria plays an important role in reduction of antibiotic affinity. Certain bacteria possess genes, for example, mecA gene found in methicillin resistant Staphylococcus aureus (MRSA) which causes alteration in penicillin binding proteins (PBP2a or PBP2’). PBPs are the targets for β-lactam antibiotics like penicillin and extended spectrum penicillins. Alteration of PBPs caused by certain Gram-negative bacteria, for example, P. aeruginosa contributes to the β-lactam antibiotic resistance.²⁰

Reduced permeability and active efflux:

Gram-negative bacterial outer membrane consists of porins, which are protein channels responsible for transport of nutrients into the cell. Alteration or loss of porins reduces the permeability to antibiotics thereby conferring resistance against β-lactams in organisms such as P. aeruginosa, K. pneumoniae, and A. baumannii.²¹

Membrane bound efflux pumps play an important role in resistance by expelling the antibiotics out of the bacteria. Some Gram-negative bacteria such as P. aeruginosa possess efflux pump mechanisms like MexAB-OprM, MexCD-OprJ, and MexXY-OprM which pumps out different groups of antibiotics.²²

There is an acceleration of antibiotic resistance during the past 70 years. This has been attributed to the extensive use of antibiotics in humans and animals. At an individual level, selection of pre-existing resistant subpopulations in the normal flora or at the infection site can contribute to the emergence of resistance, treatment failures and future infections with resistant strains but the susceptible strains die. At the community level, high antibiotic consumption can lead to high resistance rates.^1,17

Mechanisms of carbapenem resistance:

There are two prime mechanisms involved in phenotypic resistance to carbapenems in Gram-negative bacteria. They are production of carbapenemase or combination mechanisms involving production of broad-spectrum β-lactamases (ESBLs and AmpC β-lactamases) along with mutation in the structural genes.

Carbapenemases not only hydrolyze carbapenems but also other β-lactams such as penicillin, cephalosporins, and monobactams. However, MBL producers are susceptible to monobactams. They are either chromosomal or plasmid-mediated.²³ Porins are proteins on the outer cell membrane of Gram-negative bacteria which permit diffusion of substances such as growth requirements and antibiotics across the membrane. Mutations in the structural genes causing alterations or loss of porins on the outer cell membrane of Gram-negative bacteria in combination with production of other β-lactamases such as ESBLs and AmpC β-lactamases contribute to carbapenem resistance.^24,25,26 In Enterobacteriaceae, AmpC production is mainly due to inducible or de-repressed chromosomal genes which results in hyperproduction of these enzymes.²³

In case of Pseudomonas spp., resistance to carbapenem is due to increased efflux systems, carbapenem hydrolyzing enzymes- carbapenemases, decreased outer membrane permeability and alteration of PBPs.²⁷ In Acinetobacter spp., enzymatic inactivation especially β-lactamase production and altered receptors are common resistance mechanisms which act on β-lactams. They contain diverse genes encoding many β-lactamase enzymes.²¹

Table 2: Carbapenem resistance mechanism in Enterobacteriaceae^25,26

Resistance mechanism	Ambler/Bush-Jacoby classification	Genetic basis	Notable types
Carbapenemases
Serine	A/2f	Plasmid	KPC
	A/2f	Chromosomal	SME, IMI, NMC-A
	D/2df	Plasmid	OXA
MBLs	B/3	Plasmid	VIM, IMP, NDM
Other mechanisms (β-lactamases)
AmpC hyperproduction + porin deletion or alteration	C/1	Chromosomal
AmpC hyperproduction + porin deletion or alteration	C/1	Plasmid	CMY, FOX, ACT
ESBL production + porin deletion or alteration	A/2be	Plasmid	TEM, SHV, CTX-M

Carbapenemases:

Presently, more than 890 bacterial enzymes have been discovered, both chromosomal and plasmid-mediated which are extremely higher in number than the currently available antibiotics.¹² The common carbapenemases are the Klebsiella pneumoniae carbapenemases (KPC), Serratia marcescens enzyme (SME), Non-metallo-enzyme carbapenemase (NMC-A), imipenem-hydrolyzing β-lactamases (IMI), Guiana extended spectrum β-lactamases (GES), imipenemase (IMP), Verona integron-borne metallo-β-lactamase (VIM) and New Delhi metallo-β-lactamase (NDM).²⁵

Due to worldwide dissemination of genes encoding carbapenemase production, Carbapenem-resistant Enterobacteriaceae (CRE) has emerged. In this current situation of increasing resistance, it is important to identify the source and predisposing factors involved in colonization and infection with resistant bacteria to promote hygiene measures and tailor empirical therapy. In order to fight against resistant organisms, estimation of effective antibiotic options and rigorous infection control measures are essential. Hence, a better understanding on antibiotic stewardship and to optimize the use of existing antibiotics is the need of the hour.

REFERENCES:

1. Davies J, Davies D. Origins and evolution of antibiotic resistance. Microbiol Mol Biol Rev 2010;74:417-33.

2. Fleming A. On the antimicrobial action of cultures of a Penicillium with special reference to their use in the isolation of B. influenzae. The British Journal of Experimental Pathology 1929;10:226-236.

3. Chain E, et al. Penicillin as a chemotherapeutic agent. The Lancet, 1940;239:226-228.

4. Hodgkin DC. The X-ray analysis of the structure of penicillin, Advancement of science 1949;6:85-89.

5. Bentley R. Different roads to discovery; Prontosil (hence sulfa drugs) and penicillin (hence beta-lactams). J Ind Microbiol Biotechnol 2009;36:775-786.

6. Livermore DM. British Society for Antimicrobial Chemotherapy Working Party on The Urgent Need: Regenerating Antibacterial Drug Discovery and Development. Discovery research: the scientific challenge of finding new antibiotics. J Antimicrob Chemother 2011;66:1941-1944.

7. Essack SY. The development of β-lactam antibiotics in response to the evolution of β-lactamases. Pharmaceutical Research 2001.18:1391-1399.

8. Muniz CC, ZT, Esquivel GR, Fernandez FJ (7 A.D). Penicillin and cephalosporin production: A historical perspective. Revista Latinoamericana de Microbiologia 49:88-98.

9. Kesado T, Hashizume T, Asahi Y. Antibacterial activities of a new stabilized thienamycin, N-formimidoylthienamycin, in comparison with other antibiotics. Antimicrobial agents and chemotherapy, 1980.17(6):912-7.

10. Nicolau DP. Carbapenems: a potent class of antibiotics. Expert opinion on Pharmacotherapy 2008;9:23-37.

11. Sykes RB, Koster WH, Bonner DP. The new monobactams: chemistry and biology. Journal of clinical pharmacology 1988.28:113-9.

12. Yuan Z, Tam VH. Polymyxin B: a new strategy for multidrug-resistant Gram-negative organisms. Expert Opin Investig Drugs 2008;17:661-668.

13. Lim L.M, et al. Resurgence of colistin: a review of resistance, toxicity, pharmacodynamics, and dosing. Pharmacotherapy 2010;30:1279-1291.

14. Walsh C. Molecular mechanisms that confer antibacterial drug resistance. Nature 2000. 406:775-781.

15. Abraham EP, Chain E. An enzyme from bacteria able to destroy penicillin. Nature 1940;146:837-837.

16. Levy SB, Marshall B. Antibacterial resistance worldwide: causes, challenges and responses. Nature medicine 2004. 10:122-9.

17. Sykes R. The 2009 Garrod lecture: the evolution of antimicrobial resistance: a Darwinian perspective. The Journal of antimicrobial chemotherapy 2010. 65:1842-52.

18. Cao VT, et al. Emergence of imipenem resistance in Klebsiella pneumoniae owing to combination of plasmid-mediated CMY-4 and permeability alteration. The Journal of antimicrobial chemotherapy 2000;46:895-900.

19. Ramirez MS, Tolmasky ME. Aminoglycoside modifying enzymes. Drug resistance updates: reviews and commentaries in antimicrobial and anticancer chemotherapy, 2010;13:151-71.

20. Zamorano L, et al . Differential β-lactam resistance response driven by ampD or dacB (PBP4) inactivation in genetically diverse Pseudomonas aeruginosa strains. The Journal of antimicrobial chemotherapy, 2010;65:1540-2.

21. Hancock RE. Resistance mechanisms in Pseudomonas aeruginosa and other non-fermentative Gram-negative bacteria. Clinical infectious diseases: an official publication of the Infectious Diseases Society of America 1998;27:93-99.

22. Masuda N, et al. Substrate specificities of MexAB-OprM, MexCD-OprJ, and MexXY-oprM efflux pumps in Pseudomonas aeruginosa. Antimicrobial agents and chemotherapy 2000;44:3322-7.

23. Bush K, Jacoby GA. Updated functional classification of β-lactamases. Antimicrob Agents Chemother 2010;54:969-76.

24. Charrel RN, et al. Prevalence of outer membrane porin alteration in beta-lactam-antibiotic-resistant Enterobacter aerogenes. Antimicrob Agents Chemother 1996;40:2854-8.

25. Queenan AM, Bush K. Carbapenemases: the versatile β-lactamases. Clin Microbiology Rev. 2007;20:440–458.

26. Yong D, et al. Characterization of a new metallo-β-lactamase gene, bla_NDM-1, and a novel erythromycin esterase gene carried on a unique genetic structure in Klebsiella pneumoniae sequence type 14 from India. Antimicrob Agents Chemother 2009;53:5046-54.

27. Taneja N, et al. Paediatric urinary tract infections in a tertiary care center from north India. Indian J Med Sci 2010;131:101-5.

Received on 31.08.2015 Modified on 13.09.2015

Research J. Pharm. and Tech. 8(12): Dec., 2015; Page 1719-1724

DOI: 10.5958/0974-360X.2015.00309.1