ANTIMICROBIAL RESISTANCE Prof. Khalifa Sifaw Ghenghesh

Each class of antimicrobial agent has a unique mode of action. Some antibiotics differ in their action on Gram+ve and Gram-ve bacteria due to the difference in the cell wall structure of such bacteria. Other antibiotics are active on both types of bacteria >> Broad spectrum antibiotics.

Site of action of some antimicrobial agents Agent Site of action ------------------------------------------------------------------- Penicillins Cell wall Cephalosporins Cell wall Glycopeptides Cell wall Tetracyclines Ribosome Aminogylcosides Ribosome Macrolides Ribosome Rifamycins RNA synthesis Sulphonamides Folate metabolism Quinolones DNA synthesis

Antimicrobial agents can be classified to: Bacteriostatic: Inhibits growth and multiplication of bacteria. Bactericidal: Kills bacteria.

Bacterial Mechanisms of Antibiotic Resistance

1. Production of enzymes that destroy the antibiotic before it reaches its target. b-lactamases: In G -ve: b-lactam drug enters the cell through the porin channels where it encounters the b-lactamases in the periplasmic space. b-lactamases destroy the b-lactam molecules before they reach their PBP targets. In G +ve: b-lactamases excreted extracellularly. Thus b-lactam drugs are inactivated outside the cell.

2. The cell wall becomes impermeable to antibiotics. G –ve bacteria may become resistant to b-lactam drugs by developing permeability barriers. This caused by altered porin channels that no longer allow the entrance and passage of drug molecules into the cell.

3. Alteration of the target site by mutation PBP alteration through mutation in G+ve bacteria. The b-lactam can no longer bind to it and the cell becomes resistant to the drug. Also occurs in G –ve bacteria.

4. Possession of an efflux pump Efflux pump is a channel in the cell that actively exports tetracycline molecules and other drugs out of the cell as fast as they are transported or diffuse into the cell. The antibiotic can not reach its target.

5. Alteration of specific metabolic pathways By mutation or acquisition of genes encoding an alternative metabolic pathway so that the drug can not exert an effect.

Intrinsic Resistance Escherichia coli intrinsically resistant to vancomycin because vancomycin is too large to pass through porin channels. Gram +ve bacteria have no porins and thus are not intrinsically resistant to vancomycin.

Acquired Resistance Susceptible bacteria can acquire resistance to antibiotics by: 1. Genetic mutation: During multiplication of G -ve bacteria one cell undergoes a mutation to streptomycin for example. When the population of cells is exposed to streptomycin only the mutant streptomycin-resistant cell survives. This resistant cell now multiplies and a streptomycin-resistant population arises.

2. Acquisition of resistance: By Conjugation: When two bacterial cells are in close proximity a bridge-like structure (Pilus) forms between them. If one of the cell carries a plasmid encoding ampicillin resistance gene this plasmid can be transferred to the other cell. By Transformation: Gram-ve bacteria encounter a fragment of DNA contains a gentamicin-resistance gene. This gene has been released from a Gr +ve bacteria during cell lysis. The DNA fragment is taken into the cell during transformation. The gene is incorporated into host’s chromosome by recombination.

Some examples of b-lactamases in Enterobacteriaceae Plasmid mediated TEM-1, TEM-2 and SHV-1. Confer resistance to ampicillin and other penicllins. Inhibited by calvulanic acid. As level of expression of broad spectrum b-lactamases, increases resistance to other b-lactams occurs.

Extended Spectrum Beta-Lactamases (ESBLs) Mutations of the genes encoding TEM-1, TEM-2 and SHV-1 b-lactamases results in the production of b-lactamases known as ESBLs. Found in E. coli, Klebsiella pneumoniae and other G –ve bacilli. Hydrolyse: All penicillins All cephalosporins (except cephramycin) and aztreonam. Inhibited by b-lactamase inhibitors (e.g clavulanic acid).

Resistance to antimicrobial drugs is a major problem that inflicts the whole world. The problem is still worse in developing countries where lack of antimicrobial-resistance surveys and control policies are the norm.

Methicillin-resistance (MRSA) among Staphylcoccus aureus isolated from different cities in Libya City Source No % MRSA tested ------------------------------------------------------------------------------- Tripoli and Clinical samples 218 25 Misulata Benghazi Nares 238 24 (HCW, Pts, GP) Misurata Nares 601 26 Daw et al. 1996; El-Gadi 2000; Ghenghesh and Sanalla (2001)

Resistance of Shigella isolated from children with and without diarrhea in Tripoli to antibiotics (1992-1993). Antibiotic % resistant (n=11) ------------------------------------------------------------------------------------- Ampicillin 36 Ceftriaxone 0.0 Chloramphenicol 27 Ciprofloxacin 0.0 Gentamicin 0.0 Kanamycin 9 Nalidixic acid 0.0 Norfloxacin 0.0 Streptomycin 91 Trimethoprim- sulphamethoxazole 64 -----------------------------------------------------------------------------------Ghenghesh et al 1997.

Antibiotic % resistant (n=215) Resistance of enterobacteria isolated from different sources in Tripoli to antibiotics (1991-1993) Antibiotic % resistant (n=215) ------------------------------------------------------------------ Ampicillin 58 Chloramphenicol 26 Gentamicin 06 Kanamycin 29 Tetracycline 66 Trimethoprim- 28 sulphamethoxazole Ghenghesh et al. 1994.

Resistance of Escherichia coli isolated from urinary tract infections in Benghazi to antibiotics (1996) Antibiotic Hospital acquired Community acquired (n=62)* (n=148) ------------------------------------------------------------------------------------- Ampicillin 52(84)* 111(75) Carbenicillin 53(85) 117(79) Cephaloridine 22(35) 53(36) Chloramphenicol 37(60) 67(45) Gentamicin 19(31) 27(18) Nalidixic acid 3(5) 15(10) Nitrofurantoin 4(6) 10(7) Tetracycline 45(73) 121(82) Trimethoprim 52(84) 120(81) sulphamethoxazole ------------------------------------------------------------------------------------*(%); Tobgi et al 2001

Resistance of Salmonella species isolated from children with diarrhoea in Zliten (2001) to antibiotics. Antibiotic No. (%) resistant: (n=23) ------------------------------------------------------------------------------------- Ampicillin 23 (100) Amoxicillin+calvulanic acid 22 (95.7) Cefoxitin 20 (87) Gentamicin 18 (78.3) Doxycycline 21 (91.3) Chloramphenicol 22 (95.7) Nalidixic acid 1 (4.3) Norfloxacin 0 (0.0) Trimethoprim-sulphamehtoxazole 1 (4.3) Ghenghesh et al. 2002

Resistance of different bacterial pathogens isolated from ice cream in Tripoli % resistant Antibiotic Gram-negative Gram-positive (n=48) (n=67) ------------------------------------------------------------------------------------- Ampicillin 83 90 Amoxicillin- 40 45 clavulanic acid Cefuroxime 25 NT Ciprofloxacin 0.0 6 Gentamicin 0.0 12 Tetracycline 19 24 TMP-SMZ 12.5 25 Ghenghesh et al. 2003; NT=not tested

-------------------------------------------------------------- ESBLs-producing Escherichia coli and Klebsiella pneumoniae isolated from different clinical samples in Tripoli Organism No % ESBLs tested -------------------------------------------------------------- E. coli 383 8.6 K. pneumoniae 209 15.3 Total 592 9.3 Gebreel and Ghenghesh 2005.

THE PROBLEM The high prevalence of resistant bacteria in Libya seems to be related to antibiotic usage Easy availability without prescription at drug stores, Injudicious use in hospitals, and Uncontrolled use in animal husbandry.

CONCLUSION The problem of antibiotic resistance is very serious in Libya, as it appears to be on the increase, particularly with the emergence of resistance to newer drugs that include the fluoroquinolones (e.g. ciprofloxacin) among the clinically important bacterial species.

RECOMMENDATIONS It is urgently required: To ban the sale of antibiotics without prescription, To use antibiotics more judiciously in hospitals by intensive teaching of the principles of the use of antibiotics, and To establish better control measures of nosocomial infections. Regulation of antimicrobials for other than human use is also required. These issues are not easy to address and require the collective action of health authorities, the pharmaceutical community, health care providers, and consumers