Outline This week s plans Growth curve analysis Thursday Introduction to the food lab Introduction to Kirby Bauer and Antibiotics Group projects--hand back of feedback, revised versions due Thursday 1 This week Tuesday: Bring in food sample and perform food lab (ex. 9) Carry out cross streak with soil microbe Streak Nitrogen-free enrichments Thursday: Analysis of antibiotics using Kirby Bauer Count food lab plates Examine soil/cross streak plates Revised group project due Growth curve lab due (ex 8) 2 For ex 8: 1. Present your results as (1) Table of OD 550 readings at each time; (2) Semi-log plots constructed by hand; (3) Hand-fitted slopes of your plots. Do both plus and minus antibacterial agent (can be on same graph). You must do it by hand, but can also do it by excel. The slope = generation or doubling time 2. Include one-two paragraphs discussing your results that includes the two generation times (both plus and minus antibacterial). Also discuss why you got your specific outcome, including how your antibacterial compound functions. Discuss how you think your antibacterial should affect OD550 and compare that to your actual data. Be sure to include references for where you got your information about the compound. How to get doubling time from graph Culture must be in exponential growth phase to only take the points from that part Plot the log of the cell densities time Slope will give the generation or doubling time Can determine by just examining graph 3. Perform a Pubmed search for your antibacterial, and then do a second one comprised of your antibacterial+a bacterium you are interested in. Select one paper, and summarize the findings in one of the figures or tables. Include your search terms. 3 4 1
Tuesday we will analyze food for microbes Each group of four needs to bring in a food, don t forget! You need 20 grams. Can drop it off (label the bag) in the classroom fridge Foodborne disease Food-associated infectious agents E. coli O157:H7, Campylobacter, Salmonella Usually proper cooking will kill these agents Agents that multiply in improperly stored food. Some may produce toxins that cause the illness. Listeria, Staphylococcus, Clostridia botulinum 5 6 Many of these agents are covered by FDA testing requirements E.coli O157:H7 Genetically different from lab E. coli but also gramnegative, motile, rod Has extra set of genes Got it s name from a typing scheme 1) In this scheme, researchers use antibodies to highly variable surface antigens. They have a bank of these antibodies, and basically just say that a given strain reacts with, for example, Antibody #26. 2) Two antigens are tested a) one is the O Antigen, aka LPS b) one is the H Antigen, aka Flagellin» O157 means it has O-antigen #157» H7: means it has flagellum #7 Also called EHEC, for Enterohemoragic E. coli 7 8 2
EHEC Disease 1. 1. You ingest bacteria in contaminated food Has a very low infectious dose of ~ 10 2. Bacteria gets to your intestine and multiply. You get abdominal cramping, nausea, bloody diarrhea Other E. coli s cause diarrhea, but usually it is not bloody 3. Bacteria Adhere tightly to your cells The type of adherence looks similar to another E. coli, called EPEC which is better studied (these notes are based on EPEC) Cup-like pedestal under the E. coli. Attaching and Effacing. Adhering bacteria can deliver proteins directly to the host cell 9 EHEC Disease 2 4. EHEC Produces a Toxin called VeroToxin Being able to produce this toxin is the main difference between EPEC and EHEC Highly related to Shiga Toxin (STX) from Shigella Protein Toxin that is made by E. coli in the intestine but gets into the blood, and can travel to the kidneys Cleaves host cell ribosomal RNA and stops protein synthesis Our cells have receptors for Verotoxin, but only intestinal and kidney cells Kidney disease is called Hemoyltic Uremia Syndrome gene for verotoxin is carried by a temperate bacteriophage integrated into the genome. Can be passed readily from strain-strain; EHEC probably was created when an EPEC acquired the phage from a Shigella http://upload.wikimedia.org/wikipedia/commons/3/30/shiga_toxin_stx1_1dm0.png 10 EHEC is a commensal of cattle Recent EHEC Outbreaks FIG. 2. Mean counts of tissue-associated E. coli O157:H7 on five regions of the rectum defined by distance from the RAJ (in centimeters). Error bars indicate upper and lower 95% confidence intervals. From Infection and Immunity, March 2003, p. 1505-1512, Vol. 71, Lymphoid Follicle-Dense Mucosa at the Terminal Rectum Is the Principal Site of Colonization of Enterohemorrhagic Escherichia coli O157:H7 in the Bovine Host. Naylor, et al. Resides in region of intestine called rectoanal junction Is shed in feces, and also ends up on hair Current strategy is to remove coat and clean the carcass in a separate area from subsequent processing Cook meat well 11 Many foods can be contaminated--beef, cookie dough, spinach. On-going Multistate Outbreak of E. coli O157:H7 Infections Associated with Beef from National Steak and Poultry http://www.cdc.gov/ecoli/outbre aks.html There are testing standards but it is challenging when infection can be by 10 organisms. Typically PCR-based No major outbreaks recently, although several smaller ones. Links on web page 12 3
Food Lab-Overview Each group of four is bringing in food item You will need 20 g, which is about 20 ml of a wet food, or ~ 1 ounce Try to minimize hand exposure Food Lab-safety Blender food up in the biosafety cabinet Will have sign up sheet to make it all go smoothly After blendering, wait a few minutes before you open up the blender to let aerosols sink We will plate the blended food in two ways: Mix with molten nutrient agar to disperse the bacteria througout the agar Streak some of the food onto EMB agar (same as used for water lab) to look for E. coli 13 14 Microbiological analysis of food Goal is to calculate the CFU/g 100 20 g 10-1 1 ml 10-3 180 ml 99 ml 100 20 g Food Lab-data analysis Lab Report Due 11/9 10-1 1 ml 10-3 180 ml 99 ml 0.1 ml 1 ml 0.1 ml The 10 4 plate has: 10 CFU/0.1 ml 100 CFU/ml This sample was diluted by 100: 100 CFU*100 = 10 4 CFU/ml There was 200 ml total in this bo7le 0.1 ml 1 ml 0.1 ml 100 CFU 10 CFU Slide courtesy Chad Saltikov 10-2 plate 10-3 plate 10-4 plate 10 4 CFU/ml * 200ml = 2x10 6 CFU/bottle 20 g of food/bo7le 2x10 6 CFU/bottle*(bottle/20g) = 1x10 5 CFU/g 16 4
Soil Lab: Azotobacter enrichment results Soil Lab: clearing-zone colony follow up Examine Azotobacter flask for gummy growth Streak gummy growth onto Azotobacter plates for single colonies Cross streak Tuesday Examine Thursday 17 Kirby-Bauer Analysis of response to antibacterial compounds We need overnight grown cultures for Thursday so Weds before 5 pm: Head to the lab. Plates will be set up on 229, labeled UNKNOWN 1, 2, etc Select two single colonies from two different plates Inoculate each one into a tube of sterile broth, using loops or sterile sticks Place tubes in 37ºC incubator with shaking on (same one as used for Water durham tubes) Each group should have two tubes 19 18 Kirby Bauer Swab each of your cultures + E. coli control onto Mueller Hinton Agar Add antibiotics to each sterile disk and place disk on plate Template at end of exercise to help with placing discs. Need all 8 on each plate. Incubate until Next day at 37ºC (staff will grab all plates) X http://us.123rf.com/400wm/400/400/lovleah/lovleah0806/lovleah 080600075/3235971.jpg 20 5
Antibiotics target crucial bacterial processes Kirby Bauer: Results Measure and record diameter of clearing zone Note whether zones are totally or partially clear, or even contain small colonies that may be resistant mutants 21 New Antibiotics are needed: 1) Resistance 2) More choices 23 22 Antibiotic Resistance is serious 24 6
New Antibiotics Almost all antibiotics come from microbes Streptomyces spp: streptomycin, kanamycin, other mycins, tetracycline Penicllium molds produce penicillins Search for new includes Chemically synthesizing variants/analogs Random screening of chemicals Brock quotes that 7 million chemicals have to be screened to identify one useful drug, lasting 10-25 years from start to approval. Computer-aided design to predict small molecules that bind to targets HIV protease inhibitors Random screening of natural products Bacteriophages 25 Example: Platensimycin Found in a screen of 250,000 natural product extracts from 83,000 potential antibiotic producing strains at Merck Screened against Staphylococcus aureus, but started with a crippled strain that had a defect in lipid synthesis Goal was to get an antibiotic that targeted this pathway as that would be unique Crippled the strain using anti-sense RNA against a known enzyme in the pathway to basically make the strain more sensitive to a second block on this pathway Review article on web site, in readings portion High throughput screening here at UCSC 26 Example: Daptomycin/Cubicin Found recently by screening soil samples, new antibiotic that makes membranes lose their PMF. Works against gm-positives like Staph. Group projects Specific feedback handed back today; revision due Thursday Thursday revision should include a complete materials lists for everything: Amount of media, recipes Other things like swabs Equipment like shakers, blenders, specs 27 28 7