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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

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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)

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For ex 8:• 1. Present your results as (1) Table of OD550 readings at each time; (2)

Semi-log plots constructed by hand; (3) Hand-fitted slopes of yourplots. Do both plus and minus antibacterial agent (can be on samegraph).

– 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 thetwo generation times (both plus and minus antibacterial). Also discusswhy you got your specific outcome, including how your antibacterialcompound functions. Discuss how you think your antibacterial shouldaffect OD550 and compare that to your actual data. Be sure to includereferences for where you got your information about the compound.

• 3. Perform a Pubmed search for your antibacterial, and then do asecond one comprised of your antibacterial+a bacterium you areinterested in. Select one paper, and summarize the findings in one ofthe figures or tables. Include your search terms. 4

How to get doubling time fromgraph

• Culture must be inexponential growth phase toonly take the points from thatpart

• Plot the log of the celldensities time

• Slope will give the generationor doubling time

• Can determine by justexamining graph

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Tuesday we will analyze foodfor microbes

• Each group of four needs to bring in afood, don’t forget! You need 20 grams.

• Can drop it off (label the bag) in theclassroom fridge

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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. Somemay produce toxins that cause the illness.– Listeria, Staphylococcus, Clostridia botulinum

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Many of these agents are coveredby FDA testing requirements

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E.coli O157:H7• Genetically different from lab E. coli but also gram-

negative, motile, rod• Has extra set of genes• Got it’s name from a typing scheme

• 1) In this scheme, researchers use antibodies to highly variablesurface antigens. They have a bank of these antibodies, and basicallyjust 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

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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 toanother E. coli, called EPEC which isbetter studied (these notes are based onEPEC)

– Cup-like pedestal under the E. coli.Attaching and Effacing.

– Adhering bacteria can deliver proteinsdirectly to the host cell

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EHEC Disease 24. EHEC Produces a Toxin called

VeroToxin– Being able to produce this toxin is the

main difference between EPEC andEHEC

– Highly related to Shiga Toxin (STX)from Shigella

– Protein Toxin that is made by E. coli inthe intestine but gets into the blood, andcan travel to the kidneys

– Cleaves host cell ribosomal RNA andstops protein synthesis

– Our cells have receptors for Verotoxin,but only intestinal and kidney cells

Kidney disease is called Hemoyltic UremiaSyndrome

– gene for verotoxin is carried by atemperate bacteriophage integrated intothe genome.

Can be passed readily from strain-strain;EHEC probably was created when anEPEC acquired the phage from aShigella

http://upload.wikimedia.org/wikipedia/commons/3/30/Shiga_Toxin_Stx1_1DM0.png

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EHEC is a commensal ofcattle

• Resides in region ofintestine called recto-anal junction

• Is shed in feces, andalso ends up on hair

• Current strategy is toremove coat and cleanthe carcass in aseparate area fromsubsequent processing

• Cook meat wellFIG. 2. Mean counts of tissue-associated E. coli O157:H7 on fiveregions of the rectum defined by distance from the RAJ (incentimeters). Error bars indicate upper and lower 95% confidenceintervals. From Infection and Immunity, March 2003, p. 1505-1512,Vol. 71, Lymphoid Follicle-Dense Mucosa at the Terminal RectumIs the Principal Site of Colonization of EnterohemorrhagicEscherichia coli O157:H7 in the Bovine Host. Naylor, et al.

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Recent EHEC Outbreaks

• Many foods can becontaminated--beef, cookiedough, spinach.

• On-going” Multistate Outbreakof E. coli O157:H7 InfectionsAssociated with Beef fromNational Steak and Poultry

• http://www.cdc.gov/ecoli/outbreaks.html

• There are testing standards butit is challenging when infectioncan be by 10 organisms.Typically PCR-based

• No major outbreaks recently,although several smaller ones.Links on web page

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Food Lab-OverviewFood Lab-Overview

• Each group of four is bringing in fooditem– You will need 20 g, which is about 20 ml of

a wet food, or ~ 1 ounce• Try to minimize hand exposure…

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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 bacteriathrougout the agar

– Streak some of the food onto EMB agar (same as used forwater lab) to look for E. coli

Slide courtesy Chad Saltikov

Microbiological analysis offood

• Goal is to calculate the CFU/g

20 g

180 ml 99 ml

1 ml

0.1 ml 1 ml 0.1 ml

10 CFU100 CFU

10-310-1100

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Food Lab-data analysisLab Report Due 11/9

10 CFU/0.1 ml

The 10‐4 plate has:

100 CFU/ml

There was 200 ml total in thisbo7le

104CFU/ml * 200ml = 2x106 CFU/bottle

20 g180 ml 99 ml

1 ml

0.1 ml 1 ml 0.1 ml

10-310-1100

10-2 plate 10-3 plate 10-4 plate

This sample was diluted by 100:

100 CFU*100 = 104 CFU/ml

20 g of food/bo7le

2x106CFU/bottle*(bottle/20g) =1x105CFU/g

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Soil Lab: Azotobacterenrichment results

• ExamineAzotobacter flask forgummy growth

• Streak gummygrowth ontoAzotobacter platesfor single colonies

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Soil Lab: clearing-zone colonyfollow up

• Cross streak Tuesday• Examine Thursday

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Kirby-Bauer Analysis of response toantibacterial compounds

• We need overnight growncultures 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 orsterile sticks

– Place tubes in 37ºC incubatorwith shaking on (same one asused for Water durham tubes)

– Each group should have twotubes

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Kirby Bauer• Swab each of your

cultures + E. coli controlonto Mueller HintonAgar

• Add antibiotics to eachsterile disk and placedisk on plate

• Template at end ofexercise to help withplacing discs. Need all8 on each plate.

• Incubate until Next dayat 37ºC (staff will graball plates)

X

http://us.123rf.com/400wm/400/400/lovleah/lovleah0806/lovleah080600075/3235971.jpg

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Kirby Bauer: Results

• Measure and recorddiameter of clearingzone

• Note whether zonesare totally or partiallyclear, or evencontain smallcolonies that may beresistant mutants

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Antibiotics target crucialbacterial processes

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New Antibiotics are needed:1) Resistance

2) More choices

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Antibiotic Resistance isserious

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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 toidentify one useful drug, lasting 10-25 years from start toapproval.

– Computer-aided design to predict small molecules that bindto targets

• HIV protease inhibitors– Random screening of natural products– Bacteriophages

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Example: Platensimycin• Found in a screen of 250,000 natural product

extracts from 83,000 potential antibioticproducing strains at Merck

• Screened against Staphylococcus aureus, butstarted with a “crippled” strain that had a defectin 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 makethe strain more sensitive to a second block on thispathway

• Review article on web site, in readings portion• High throughput screening here at UCSC

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Example:Daptomycin/Cubicin

•Found recently by screening soilsamples, new antibiotic thatmakes membranes lose theirPMF. Works against gm-positiveslike Staph.

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Group projects

• Specific feedback handed back today;revision due Thursday

• Thursday revision should include acomplete materials lists for everything:– Amount of media, recipes– Other things like swabs– Equipment like shakers, blenders, specs