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Multi-Drug Resistance

            The ability of microorganisms to develop resistance to different antimicrobial agents has long been a dilemma to clinical practice. This is compounded by the remarkable capacity of bacteria to acquire and transfer this resistance through genetic elements which leads to the development of multi-drug resistant stains. The emergence of multi-drug resistance has slowly limited the treatment options for even relatively common infections. The spread of diseases associated with resistant strains have been increasing over the past few decades that the World Health Organization has considered this problem a major threat to global public health (World Health Organization, 2001 cited in Ahmed, Furuta, Shimomura, Kasama & Shimamoto, 2006). Understanding the molecular and genetic basis involved in this process will provide significant advantage in the treatment of diseases caused by multi-drug resistant pathogens. Further, remedies can be initiated to

Mechanisms of Bacterial Resistance to Antimicrobial Agents

            There are several ways by which bacteria can counteract the toxic effects of various antimicrobial agents. These include the enzymatic inactivation the drug compound caused by the production of specific enzymes which hydrolysis the antibiotic. Bacteria can also acquire resistance by alteration of the target site of the drug which decreases its affinity to that drug. Other instances of bacterial resistance are caused by the inability of the antimicrobial agent to penetrate to its site of action. Furthermore, the active efflux of the drug out of the bacteria cell through the use bacterial membrane proteins also render drug resistance to the bacteria (Stoitsova, Braun, Ullrich and Weingart, 2008). In addition, bacteria can not only acquire any of these mechanisms but also propagate this ability throughout the species through exogenous genes using mobile genetic elements such as plasmids and transposons (Rowe-Magnus et al, 2002 cited in Ahmed, Furuta, Shimomura, Kasama & Shimamoto, 2006).

Enzymatic Inactivation

            One of the major defenses of bacteria against antibiotics is the synthesis of various enzymes which deactivates specific classes of drugs. Among these enzymes, the ?-lactamases hold the most significance since it confers resistance to a major class of antibiotics called ?-lactams. ?-lactam antibiotics are antimicrobial agents that share a common chemical structure known as the ?-lactam ring. These antibiotics include penicillins and cephalosporins. These are frequently prescribed drugs and bacterial resistance to these antibiotics has considerable impact in the treatment of bacterial infections. Bacteria which produce ?-lactamases can destroy ?-lactam antibiotics enzymatically. Most gram-positive bacteria produce large quantities of ?-lactamases while gram-negative bacteria elaborate only small amounts of these enzymes. In order to extend the effectiveness of ?-lactam antibiotics, ?-lactamase inhibitors such as clavulanate are combined with certain ?-lactam antibiotics in the treatment of serious infections. New ?-lactam antibiotics have also been developed which were designed to resist hydrolysis by ?-lactamases. Because of their extended spectrum against ?-lactamase-producing bacteria, their overuse has lead to the emergence of new variants of ?-lactamases which confer resistance to these antibiotics. The first of these ?-lactamases capable of inactivating the newer ?-lactams is known as SHV-2 and was isolated from a single strain of Klebsiella ozaenae. These ?-lactamases were later identified as extended-spectrum ?-lactamases or ESBLs because of their action on extended-spectrum ?-lactams (Bradford, 2001).

Throughout the use of penicillin, a plasmid-mediated ?-lactamase known as penicillinase have lead to the emerging resistance of Staphylococcus aureus to penicillin. This beta-lactamase later spread to other species of Staphylococci and other bacteria such as Enterobacteriaceae, Pseudomonas aeruginosa, Haemophilus influenzae, and Neisseria gonorrhoeae (Bradford, 2001).

Alteration of Drug-binding Site

            Another mechanism used by bacteria to develop antimicrobial resistance involved the alteration of the target site which the drug has to bind in order to exert an effect that will be damaging to the bacteria. Variations or changes at these target sites prevent the drug from binding to the bacteria which leads to resistance. In the case of ?-lactam antibiotics, their target sites are collectively known as penicillin-binding proteins or PBPs (Spratt, 1980 cited in Hardman et al, 1996). Microorganism may be intrinsically resistant to ?-lactams due to structural differences in their PBPs. However, ?-lactam antibiotics inhibit many different PBPs in a single bacterium which requires many PBPs to have decreased affinity for ?-lactams in order for a bacterium to be resistant. Altered PBPs with decreased affinity for ?-lactam antibiotics are acquired by homologous recombination between PBP genes of different bacterial species.

Reduced Drug Uptake.

            Even if the drug is already bound with the cell surface of the bacteria, some bacteria has impermeable cell membrane that prevents the entry of the drug into the bacterial cell. In the case of Gram-negative bacteria which have an outer membrane and a capsule, only some hydrophilic antibiotics can diffuse through the barrier using aqueous channels in the outer membrane composed of proteins called porins. Mutation that causes bacteria to be deficient of these transport proteins required for the entry of a particular drug into the cell will render the bacteria to become resistant to that drug (Hardman et al, 1996).

Multidrug resistance efflux pumps

            Another survival mechanism employed by bacteria to defend against the harmful effects of antimicrobial agents is through the use of multidrug resistance efflux pumps. These efflux proteins facilitate the extrusion of a broad range of structurally unrelated toxins including antimicrobials out of the cell (Blackmore et al, 2001 cited in Brown, Swanson & Allen, 2007). A study on plant-associated bacteria showed that induced mutations on bacterial multidrug efflux pumps resulted in less virulence of the mutants compared to the wild-type strains. This was due the increased sensitivity of the mutants to antimicrobial compounds endogenously produced the plants. However, the restoration of the genes coding for the efflux proteins also restored the virulence of the mutated bacteria. This study demonstrated the function of multidrug efflux pumps in the protection of bacteria from the toxic effects of antimicrobial compounds (Brown, Swanson & Allen, 2007).

Development of Multi-Drug Resistance

            Resistance may be acquired by a mutation and passed vertically by selection to daughter cells. More commonly, resistance is acquired by horizontal transfer of resistance determinants from a donor cell, often of another bacterial species, by transformation, transduction, or conjugation. Resistance that is acquired by horizontal transfer can become rapidly and widely disseminated either by clonal spread of the resistant strain itself or by further genetic exchanges between the resistant strain and other susceptible strains. In many instances, individual bacterial strains show resistances to several antibiotics. In gram-negative bacteria, such multi-drug resistance is specified by extrachromosomal elements known as drug-resistance factors or R factors (Hentges, 1995). A recent study showed that horizontal transfer occurred through multi-drug resistant R plasmids which were found in Escherichia coli isolated from feces of cattle and humans (Oppegaard, Steinum, & Wasteson, 2001). The results indicate the multi-drug resistance persists in coliform bacteria from various host due to the horizontal transfer of R plamids. The investigators suggest that the occurrence of multi-drug resistance in farm inhabitants from coliform bacteria of farm animals may be due to the excessive use of antimicrobial agents.

            Drug-resistant plasmids are also found in gram-positive organisms which are associated with the production of enzymes that confer resistance to specific antibiotics. These plasmids are not usually called R plasmids. An example of this is penicillinase plasmids which are associated with penicillinase production. These plasmids also control resistance to other non-?-lactam antibiotics such as erythromycin. Unlike most R factors, penicillinase plasmids are not transferred by conjugation although their transduction has been reported (Hentges, 1995).

Mutation

            Mutation occurring in a previously susceptible cell and antibiotic selection of this resistant mutant are the molecular basis for resistance to some drugs. Mutations may occur in the gene encoding the target protein, altering its structure so that it can no longer bind to the drug. This protein may be involved in drug transport. Mutations may also occur in a regulatory gene which leads to the expression of an inactivating enzyme. Any large population of antibiotic-susceptible bacteria is likely to contain some mutants that are relatively resistant to the drug. There are no evidence that these mutations are the result of exposure to the particular drug but rather such mutations are the result of random events that confer a selective advantage to the cell upon reexposure to the drug.

Transduction

            Transduction occurs by the intervention of a bacteriophage, which is a virus that infects bacteria, that contains bacterial DNA incorporated within its protein coat. If this genetic material includes a gene for drug resistance, the newly infected bacterial cell may become resistant to the antimicrobial agent and capable of passing this trait on to its progeny. Transduction is particularly important in the transfer of antibiotic resistance among strains of S.aureus, where some phages can carry plasmids that code for penicillinase, while others transfer genes encoding resistance to erythromycin, tetracycline, or chloramphenicol.

Transformation

            This method of transferring resistance involves the acquisition of DNA which encodes for drug resistance that is free in the environment into the bacterial cell. Transformation is the molecular basis of penicillin resistance in pneumococci and Neisseria. Penicillin-resistant pneumococci produce altered penicillin-binding proteins that have low-affinity binding of penicillin. Nucleotide sequence analysis of the genes encoding these altered proteins indicates that they are mosaics in which blocks of foreign DNA from an unknown, but probably closely related, species of streptococcus have been imported and incorporated into the resident penicillin-binding protein gene by homologous recombination.

Conjugation

            The passage of genes from cell to cell by direct contact through a sex pilus or bridge is termed conjugation. This is now recognized as an extremely important mechanism for spread of antibiotic resistance, since DNA that encodes for resistance to multiple drugs may be so transferred. Genetic transfer by conjugation occurs predominantly among gram-negative bacilli, and resistance is conferred on a susceptible cell as a single event (Hardman et al, 1996). A study showed that acquisition of resistance to cephalosporins from E.coli to Salmonella enterica may occur through conjugation (Poppe et al, 2005). The study used E.coli with a large conjugative plasmid encoding a certain ß-lactamase which was given to animal subjects. The result showed that the subspecies of Salmonella isolated from the animals not only acquired the plasmid coding for the ß-lactamase but also other drug resistance genes present in the E.coli as well. The study showed that resistance to extended-spectrum cephalosporins can be transferred horizontally from E. to S.enterica through conjugation. The study also highlights the concern of acquiring drug resistant bacteria from foods through transfer of resistance determinants from food-borne bacteria to the intestinal flora. A human study showed that these transfer is possible through conjugative elements. The study revealed that extensive resistance gene transfer occurs among Bacteroide species in human colon (Shoemaker, Vlamakis, Hayes & Salyers, (2001). The results also showed that the transfer also occurred between Bacteroides species, which are Gram-negative, and Gram-positive bacteria in the colon. The results suggest that intestinal flora, which are previously sensitive to antibiotics, may be rendered resistant due to conjugation with ingested multidrug resistant bacteria.

            Gram-positive bacteria such as enterococci also contain broad host range conjugative plasmids which are involved in the transfer and spread of resistance genes among gram-positive organisms (Hardman et al, 1996).

Integrons

            Another mechanism of disseminating multidrug resistance involves genetic elements known as integrons. These elements can acquire and exchange exogenous DNA known as gene cassettes through recombination (Stokes & Hall, 1989 cited in Ahmed et al, 2006). Most of these gene cassettes that are isolated within integrons code for resistance determinants. The acquisition and dissemination of resistance determinants by integrons have lead to the rapid emergence of various drug-resistant Gram-negative bacteria (Rowe-Magnus et al., 2002 cited by Ahmed et al, 2006). A study on the genetic basis of multidrug resistance in Shigella showed that integrons play an essential role in antimicrobial resistance of Shigella to a number of antibiotics including ampicillin, tetracycline and ciprofloxacin (Ahmed et al, 2006). Integrons acquire and transfer antibiotic resistance gene cassettes through an enzyme known as integrase. There are different classes of integrons that have been identified although classes 1 and 2 integrons are the most common in Gram-negative bacteria (White et al., 2001 cited in Ahmed 2006).

Bacteria with Major Drug Resistance

Gram-positive Bacteria

            There are several Gram-positive bacteria that are best known for their antibiotic resistance because they are commonly isolated in patients who have received multiple courses of antibiotics or in patients who have prolonged hospital stays. These include Enterococcus, S.aureus and Streptococcus pneumoniae.

            Enterococcus. Enterococci are well established as etiologic agents of endocarditis and urinary tract infections and some of its members are common causes of nosocomial infections. Enterococci resistance to aminoglycoside and ?-lactam antibiotics have already been established which made treatment for infections caused by enterococci particularly challenging (Murray, 2000). The treatment of choice for enterococcal infection is vancomycin. However, with emergence of vancomycin-resistant enterococci especially with the species E. faecium, enterococcal infections are alarmingly difficult to treat. The mechanism of resistance us due to the gene expression of mobile genetic elements which synthesizes enzymes that produce cell call precursors with low affinity to vancomycin (Murray 2000).

            Salmonella aureus. S.aureus is the most virulent of the staphylococci and is responsible for localized and diffused skin infections such as the common sty, boils, carbuncles and impetigo. Certain strains of S.aureus produce toxins that cause toxic shock syndrome and scalded skin syndrome. There are strains of S.aureus that have developed resistance to ?-lactam antibiotics such as methicillin that are effective against ?-lactamase-producing organism. They are known Methicillin-resistant S.aureus or MRSA and are clinically significant since they cause infections that are difficult to treat. These strains have acquired methicillin resistance through chromosomal modifications leading to PBPs with low affinity to ?-lactamase-resitant ?-lactam antibiotics (Mallorquí-Fernández et al, 2004). The standard treatment for MRSA infections are glycopeptide antibiotics such as vancomycin although the treatment outcomes with these antibiotics are already compromised due to problems of increasing resistance (Gould, 2007).

            Streptococcus pneumoniae. S.pneumoniae is the most common cause of pneumonia, otitis media and meningitis (Chen et al, 1999). Penicillin G has been the drug of choice for streptococcal infections. For resistant infections, vancomycin therapy has been effective. However, multi-drug resistance of S.pneumoniae, not only to these drugs but also with other antibiotics such as fluoroquinolones, is increasingly prevalent (Chen et al, 1999). A study which evaluated the clinical impact of this prevalence in hospital patients revealed that patients with resistant streptococcal infections were 58% more likely to die than patients with nonresistant infections (Foster, Heller & Young 2001). This showed that infections associated with multidrug-resistant S.pneumoniae have lead to treatment failures with fatal outcome. Another study suggests that the use of conjugate vaccines against S.pneumoniae is imperative to avoid multidrug-resistant S.pneumoniae infections. This is due to the fact that there are only limited number serotypes associated with resistant infections and these vaccines offer protection even against the most resistant strains of S.pneumoniae (Whitney et al, 2000).

Gram-negative bacteria

Gram-negative bacteria include aerobic and anaerobic cocci and bacilli which cause a variety of infections like dysentery, typhoid fever and gonorrhea. Several of these bacteria have been known to have multi-drug resistance which makes medical management of infections caused by these bacteria difficult.

Enterobacteriaceae. Escherichia coli belong to this family of bacteria which causes mostly intestinal infections although E.coli is also known as the most common cause of urinary tract infections and is also a major cause of neonatal meningitis (Sader et al, 2004; Thielman et al, 2004). Multi-drug resistance demonstrated by these bacteria is due to production of extended-spectrum ?-lactamases which render most ?-lactam antibiotics ineffective against E.coli infection (Bedenica et al, 2005). Standard treatment includes the use of co-trimoxazole and quinolones, however, resistance to these antibiotics have lead to treatment failures in clinical practice (Karaca et al, 2005).

Neisseria gonorrhea. Neisseria gonorrhea is one of the most common etiologic agents of sexually transmitted diseases. It is a penicillinase-producing organism which causes gonorrhea. The multi-drug resistance exhibited by the bacteria is due to resistance genes which reside on nonconjugative plasmids (Djajakusumaha et al, 1998). Studies have shown that N.gonorrhea have developed resistance to a multiple antimicrobials including tetracycline, co-trimoxazole and thiamphenicol (Guyot et al, 1998;Djajakusumaha et al, 1998).

Mycobacterium tuberculosis

            M.tuberculosis is an acid-fast bacillus which belongs to a group of bacteria that stains poorly with Gram-stain. This bacteria cause tuberculosis, which is among the leading cause of death worldwide. Multidrug-resistant tuberculosis is defined as resistance to isoniazid and rifampicin, which constitutes the first-line standard treatment (Mitnick, 2008). Infection caused by multidrug-resistant strains is associated with unfavorable treatment outcome. Second-line drugs such as fluoroquinolones and amikacin play essential roles in the effective treatment for multidru-resistant tuberculosis. Recently, resistance to these drugs as well has been reported in many countries and has now been known as extensively multidrug-resistant tuberculosis.

Conclusion

            Multidrug resistance is indeed a growing health issue which threatens our ability to effective treat infections. The inappropriate use of antibiotics has been the major problem which facilitates the emergence of multidrug resistance. As more pathologic organisms become less susceptible to many antimicrobial agents, intense pressure to use newer drugs to cure resistant infection has further increased the potential for resistant organisms to develop resistance to these drugs. Health policies worldwide should be geared to curb the inappropriate antibiotic use.

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