Microbial Resistance

1. N.B. Due to a seminar, the Monday morning help session on December 5th will be changed to the following: Help Sessions in room M245: Monday, December 5th, 10-11 am & 2-3 pm. Tuesday, December 6th, 11 am-noon & 2-3 pm.

2. 02 December 2005 M.S. Peppler Dept of MMI mark.peppler@ualberta.ca 1-69 Medical Sciences Building Microbial Resistance

3. Microbial Resistance Objectives. After today’s session you will understand: 1. How efflux pumps allow bacteria to resist the action of some metals and antibiotics . 2. How R plasmids contribute to the growing problem of bacterial resistance. 3. The impact of bacterial resistance on health care.

4. Objective 1a. Microbial resistance and efflux pumps. Previously we discussed resistance by spontaneous mutation and by acquired genes to alter targets. Another mechanism is to (c) acquire genes coding for efflux pumps: protein systems which pump the antibiotic OUT of the cell as fast as it diffuses IN. e.g. tetracyclines, chloramphenicol. Background: Bacteria have an array of cell membrane transport systems normally used to take up essential nutrients (amino acids, sugars, etc.) and to excrete wastes (e.g. organic acid byproducts). We now know that many transport protein complexes confer antibiotic resistance (and sometimes metalR) on the bacterium by actively transporting toxic chemicals (i.e. antibiotics) OUT of the cell.

5. Objective 1b. Microbial resistance and efflux pumps. The actual pumping requires • Membrane-spanning transport proteins (often a trimer spanning CM, periplasm, OM • Energy (PMF or ATP). Some efflux pumps are relatively specific, and confer resistance to only one type of antibiotic and its structural analogues (e.g. tetracycline pump) some are more general, and confer the phenotype of MULTIDRUG RESISTANCE ("MDR") so that the bacteria become MULTIPLY ANTIBIOTIC RESISTANT ("MAR") The molecular basis for broad substrate specificity of efflux protein systems is unclear at present: seems to be based more on charge, hydrophobicity, etc. rather than structure.

6. Objective 1c. Microbial resistance and efflux pumps. What is the "normal" role of these pumps? Not yet certain for all cases… …but many of these pumps fall into a family of transporters called RND proteins: (metal Resistance/Nodulation/cell Division), presumably having natural roles: e.g., an E. coli pump also transports bile salts (found in intestine): "natural" role may be efflux of bile salts. e.g., tet pump may also act in Na+/H+antiporter. Some also may be naturally involved in heavy metal efflux (see following table).

7. Objective 1d. Microbial resistance and efflux pumps.

8. Objective 1e. Microbial resistance and efflux pumps. Structure of a notorious efflux pump. Multidrug resistance among Gram-negative bacteria is conferred by three-component membranepumps that expel diverse antibiotics from the cell. These efflux pumps consist of an inner membrane transporter such as the AcrB proton antiporter, an outer membrane exit duct of the TolC family, and a periplasmic protein known as the adaptor. TolC MexA AcrB Data from Higgins et al., Structure of the periplasmic component of a bacterial drug efflux pump PNAS 2004,10: 9994-9999.

9. Objective 1f. Microbial resistance and efflux pumps. N.B. Efflux pumps can be acquired IN ADDITION TO other resistance mechanisms (e.g., intrinsic, penicillinases, etc.) Many "natural" efflux pumps are chromosomally encoded, but some (particularly clinical isolates of pathogens) are plasmid-borne and can be horizontally transferred.

10. Objective 2a. Microbial resistance and R plasmids. • R PLASMIDS • Have origins BEFORE antibiotic era: • e.g., soil bacteria (responsible for resistance to naturally • produced antibiotics in situ?). • e.g., antibiotic-producing strains usually have a form of resistance to the antibiotic they produce. • e.g., Franklin expedition • (Pb-soldered tins: link to heavy metal resistance). • Have multiple drug resistance: • e.g., plasmid R100 has genes for resistance to sulfonamides, streptomycin, spectinomycin, fusidic acid, chloramphenicol, tetracycline, and mercury; • i.e., can carry combinations of resistance mechanisms. • Have a broad bacterial host range: • e.g., found in E. coli, Klebsiella, Proteus, Salmonella, Shigella.

11. Objective 2b. Microbial resistance and R plasmids. M. Teuber et al. (2003) Internat J Food Microbiol 88: 325–329. Fig. 1. Genomic structure of the 50 237 bp conjugative multiresistance plasmid pRE25 of Enterococcus faecalis isolated from a raw meat sausage (GenBank accession number X92945). The basic structure comprising orf ’s 6 to 40 is very similar to identical to the Inc18 family broad host range plasmid pIP501 from Streptococcus agalactiae and pSM19035 from Streptococcus pyogenes. The transfer region ranges from orf 24 (nickase) to orf 40 containing 10 genes for membrane spanning proteins. Resistance genes orf 10 and 14 code for a chloramphenicol acetyltransferase and a RNA-methylase, respectively, like in pIP501. This basic streptococcal plasmid has been obviously upgraded with the help of E. faecium IS elements (orf 41, 51, 54, 55, 56, 3, and 5) to contain another antibiotic resistance gene-assembly (orf 44, 45, 46) coding for an aminoglycoside 6-adenylyltransferase, a streptothricin acetyltransferase, and an aminoglygoside phosphotransferase type III. In addition, pRE25 contains information for 3 replication proteins (orf 1, 6, and 11), two resolvases (orf 8, 53) and 2 ATPases (orf 16 and 48 with 98 to 100% amino acid identities with Clostridium perfringens/difficile proteins). The plasmid can be experimentally transferred by conjugation to Lactococcus lactis, Listeria innocua, and other enterococci. It is a demonstration that resistance genes are freely floating between commensal and pathogenic bacteria found in humans, animals and food (modified from F. Schwarz et al. 2001). Color code: dark red: aa identities (95 – 100%) with Inc18 plasmid coded proteins (streptococcal module). Blue: enterococcal region (module). White: no homologies in data bases. Black: IS-elements. Blue bar: nucleotide sequences with a 98 – 100% identity to sequences in a vancomycin-resistant E. faecium whose genome has been completely sequenced in the USA (GenBank accession number NZ_AAAK01000000).

12. Objective 3a. Microbial resistance and health care. Nosocomial (hospital-acquired) infections: Refers to any infection acquired in hospital (e.g., P. aeruginosa infections of patients undergoing burn therapy; immunocompromised patients picking up opportunistic pathogens). Hospitals are the perfect environment for the breeding of antibiotic-resistantstrains because of • continuous exposure to antibiotics, i.e., selective pressure for resistance. • debilitated/ immunocompromised hosts i.e., opportunistic pathogens can flourish.

13. Objective 3b. Microbial resistance and health care. The dawn of “SUPERBUGS”: Vancomycin-resistant Enterococcus (VRE) Enterococcus is part of natural flora of human gut but is now a concern now because of more invasive procedures, more immunocompromised people. But the big worry: The potential for spread of resistance genes to other, pathogenic, Gram +ve bacteria like methicillin-resistant Staph. aureus ("MRSA"). Currently, vancomycin is the only treatment for MRSA. When this is taken away by resistance, we could return to a situation with Staph. akin to the pre-antibiotic era.

14. Objective 3c. Microbial resistance and health care. • Causes of increasing frequency of bacterial resistance to antibiotics: • 1.Inappropriate (medical) use of antibiotics: • - wrong dosage, wrong antibiotic. • - incorrect use by patient, • e.g., stop taking antibiotic before all pathogens are killed (i.e. enrich for resistance, then remove bacteriostatic agent) • - using antibiotics for viral diseases.

15. Objective 3d. Microbial resistance and health care. Causes of increasing frequency of bacterial resistance to antibiotics: 2.Non-medical uses of antibiotics - supplement in animal feeds. e.g. chickens: prophylactic use. e.g. feed lots for cattle: growth boost. Consequence: • Enriches for resistant bacteria in animal gut. • We consume (inadvertently) the resistant bacteria. • Our gut bacteria acquire resistance (e.g., through transfer of R plasmids). NOTE: neither we nor the cow become ‘resistant’, the bacterial microflora do; meat does NOT pass on antibiotics!!

16. Objective 3e. Microbial resistance and health care. • WHAT TO DO? • (1) Don't request antibiotics for viral diseases. • (2) Finish your prescription, following directions. • (3) Find new antibiotics (takes years, $100s of Millions). • (4) Educate the public about responsible antibiotic use. • (5) Restricting the use of antibiotics in animal feed has reduced the incidence of resistant community-acquired infections in Denmark and Germany.* • *A lesson learned? • The original wisdom stated that only non-therapeutic veterinary antibiotics could be used for animals, e.g., apramycin and viomycin. • However, avoparcin, chemically related to vancomycin, was used in animal feeds in Denmark and Germany which resulted in vancomycinR gut flora in cows and vancomycinR enterococci found in supermarket meat products.

17. Next time: Biowarfare: Bioterror.