COCCUS AUREUS(MRSA)✦This plate illustrates the ef fect of (clockwise from top outer right) Nitrofurantoin (F/M300) Nor- floxacin (NOR 10), Oxacillin (OX 1), Sulfisoxazole (G 0.25), Ticarcillin (TIC 75), Trimethoprim-Sulfamethoxazole (SXT), Tetracycline (TE 30), Ceftizoxime (ZOX 30), Ciprofloxacin (CIP 5), and (inner circle from right) Penicillin (P 10), Vancomycin (VA 30), and Trimethoprim (TMP 5) on Methicillin-resistant Staphylococcus aureus.
7-5 MCFARLANDSTANDARDS✦This is a comparison of a McFarland turbidity standard to three broths having var ying degrees of turbidity. Each of the 11 McFarland standards (0.5 to 10) contains a specific percentage of precipitated barium sulfate to produce turbidity. In the Kirby-Bauer procedure, the test culture is diluted to match the 0.5 McFarland standard (roughly equivalent to 1.5 x 108cells per mL) before inoculating the plate. Comparison is made visually by placing a card with sharp black lines behind the tubes. Tube 3 is the 0.5 McFarland standard. Notice that the turbidity of Tube 2 matches the McFarland standard exactly, whereas Tubes 1 and 4 are too turbid and too clear, respectively.
1 2 3 4
The disks, which contain a specified amount of the antimicrobial agent (printed on the disk) are dispensed onto the inoculated plate and incubated at 35Ⳳ2°C (Figure 7-6). After 16 to 18 hours of incubation, the plates are removed and the clear zones are measured (Figure 7-7).
✦ Application
Antimicrobial susceptibility testing is a standardized method that is used to measure the effectiveness of anti - biotics and other chemotherapeutic agents on pathogenic microorganisms. In many cases, it is an essential tool in prescribing appropriate treatment.
✦ In This Exercise
You will test the susceptibility of Escherichia coliand Staphylococcus aureusstrains to penicillin, chloram- phenicol, trimethoprim, and ciprofloxacin. These anti - biotics were chosen because they exhibit different modes of action on bacterial cells (Table 7-3).
✦ Materials
Per Student Group
✦two Mueller-Hinton agar plates
✦penicillin, chloramphenicol, trimethoprim, and ciprofloxacin antibiotic disks (and/or other disks as available)
✦antibiotic disk dispenser or forceps for placement of disks
✦small beaker of alcohol (for sterilizing forceps)
✦two sterile cotton swabs
✦one metric ruler
✦sterile saline (0.85%)
✦sterile transfer pipettes
✦one McFarland 0.5 standard with card
✦black, nonreflective poster board (8.5"⳯11")
✦two Trypticase Soy Agar (or Nutrient Agar) plates (for optional procedure)
✦fresh broth cultures of:
⽧Escherichia coli
⽧Staphylococcus aureus(BSL-2)
✦ Medium Recipe
Mueller-Hinton II Agar
⽧Beef extract 2.0 g
⽧Acid hydrolysate of casein 17.5 g
⽧Starch 1.5 g
⽧Agar 17.0 g
⽧Distilled or deionized water 1.0 L pH ⳱7.2–7.4 at 25°C
7-6 DISKDISPENSER✦This antibiotic disk dispenser is used to deposit disks uniformly on a Mueller-Hinton agar plate.
7-7 MEASURING THEANTIMICROBIALSUSCEPTIBILITYZONES✦A metric ruler is used to measure the diameter of each clearing, in millimeters (mm).
Procedure
Lab One
1 Gently mix the E. coliculture and the McFarland standard until they reach their maximum turbidity.
2 Holding the culture and McFarland standard up- right in front of you, place the card behind them so you can see the black line(s) through the liquid in the tubes. As you can see in Figure 7-5, the line becomes distorted by turbidity in the tubes. Use the black line to compare the turbidity level of the two tubes. Dilute the broth with sterile saline until it appears to have the same level of turbidity as the standard. If it is not turbid enough, incubate it until it reaches that level.
3 Repeat Steps 1 and 2 with the S. aureus culture.
4 Dip a sterile swab into the E. coli broth. As you remove it, press and rotate the cotton tip against the side of the tube to remove excess broth.
5 Inoculate a Mueller-Hinton plate with E. coliby streaking the entire surface of the agar three times with the swab. Your goal is confluent growth, so make the streaks right next to each other. When you have covered the surface, rotate the plate 1/3 turn and repeat the streaking of the inoculum already on the plate, using the same technique to produce confluent growth. Then rotate the plate another 1/3 turn and repeat.
6 Using a fresh sterile swab, inoculate the other plate with S. aureusin the same fashion to produce con- fluent growth.
7 Label the plates with the organisms’ names, your name, and the date.
8 Apply the penicillin, chloramphenicol, ciprofloxacin, and trimethoprim disks to the agar surface of each plate. You can apply the disks either singly using sterile forceps, or with a dispenser (Figure 7-6). Be sure to space the disks sufficiently (4 to 5 cm) to prevent overlapping zones of inhibition. Also keep them away from the edge of the plate.
9 Press each disk gently with alcohol-flamed forceps so it makes full contact with the agar surface.
10 Invert the plates and incubate them aerobically at 35Ⳳ2°C for 16 to 18 hours. Have a volunteer in the group remove and refrigerate the plates at the appropriate time.
Lab Two
1 Remove the plates from the incubator (or refrigera- tor). Hold the plate over the black, nonreflective posterboard and examine the plate with reflected light. The edge of a zone is where no growth is visible to the naked eye. Measure the diameter of each zone of inhibition in millimeters (Figure 7-7).
2 Using Table 7-4 and those provided with your anti - biotic disks (if you used additional antibiotics), record your results in the table provided on the Data Sheet.
Antibiotic Cellular Target Resistance Mechanism
Chloramphenicol Prevents peptide bond formation during translation 1. Poor uptake of drug 2. Inactivation of drug
Ciprofloxacin Inter feres with DNA replication 1. Altered target
2. Poor uptake of drug Trimethoprim Inhibits purine and pyrimidine synthesis 1. Altered target Penicillin Inhibits cross-linking of the cell wall’s peptidoglycan One or more of:
1. Altered target 2. Poor uptake of drug 3. Production of -lactamases
TABLE 7-3 Antibiotic Targets and Resistance Mechanisms ✦Not all antibiotics affect cells in the same way. Some attack the bacterial cell wall, and others inter fere with biosynthesis reactions. Resistance mechanisms can be broken down into three main categories:
(a) altered target such that the antibiotic no longer can interact with the cellular process, (b) an alteration in how the drug is taken into the cell, and (c) enzymatic destruction of the drug.
Optional Procedure Beginning with Lab Two
1 Obtain two TSA or NA plates. With your marking pen, divide the plates into four sectors (or more if you used more antibiotics).
2 Label each sector with an antibiotic. It is easiest if you label in the same order as these are found on the MH plates.
3 Label one plate E. coliand the other S. aureus.
4 Using a sterile loop for each transfer, obtain a sample from each antibiotic’s zone of inhibition on the E. coli plate, and inoculate the corresponding sector on the TSA (or NA) plate. If there is no zone for a particular antibiotic, no transfer is necessary.
5 Repeat with the S. aureusMH plate.
6 Incubate the plates aerobically at 35Ⳳ2° for 24–48 hours.
Lab Three
1 Remove the plates from the incubator and examine each sector for growth.
2 Record your observations and answer the questions on the Data Sheet.
References
Clinical Laboratory Standards Institute (CLSI). 2009. Performance Stan- dards for Antimicrobial Disk Susceptibility Tests; Approved Standard—
10th Ed. CLSI document M02–A10. Wayne, PA.
Collins, C. H., Patricia M. Lyne, and J. M. Grange. 1995. Page 128 in Collins and Lyne’s Microbiological Methods, 7th ed. Butterworth- Heinemann, UK.
Ferraro, Mary Jane, and James H. Jorgensen. 2003. Chapter 15 in Manual of Clinical Microbiology, 8th ed., edited by Patrick R. Murray, Ellen Jo Baron, James. H. Jorgensen, Michael A. Pfaller, and Robert H. Yolken, ASM Press, Washington, DC.
Forbes, Betty A., Daniel F. Sahm, and Alice S. Weissfeld. 2002. Pages 236–240 in Bailey & Scott’s Diagnostic Microbiology,11th ed. Mosby- Yearbook, St. Louis, MO.
Koneman, Elmer W., Stephen D. Allen, William M. Janda, Paul C.
Schreckenberger, and Washington C. Winn, Jr. 1997. Pages 818–822 in Color Atlas and Textbook of Diagnostic Microbiology, 5th ed. J.B. Lippin- cott Company, Philadelphia, PA.
Mims, Cedric, Hazel M. Dockrell, Richard V. Goering, Ivan Roitt, Derek Wakelin, and Mark Zuckerman. 2004. Chapter 33 in Medical Microbiol- ogy, 3rd ed. Mosby, Philadelphia, PA.
Zimbro, Mary Jo, and David A. Power, editors. 2003. Page 376 in Difco™ and BBL™ Manual—Manual of Microbiological Culture Media.
Becton Dickinson and Company, Sparks, MD.
Antibiotic Organism(s) Code Potency Susceptible Intermediate Resistant
Chloramphenicol Enterobacteriaceae C 30 30 àg 욷18 13–17 울12
andStaphylococcus
Ciprofloxacin Enterobacteriaceae CIP 5 5 àg 욷21 16–20 울15
and Staphylococcus
Trimethoprim Enterobacteriaceae TMP 5 5 àg 욷16 11–15 울10
and Staphylococcus
Penicillin Staphylococcus P 10 10 U 욷29 울28
This char t includes the antibiotics used in this exercise and contains data provided by the Clinical and Laborator y Standards Institute (CLSI). Permission to use por tions (specifically Tables 2A and 2C) of M100-S19 (Per formance Standards for Antimicrobial Susceptibility Testing; Nineteenth Informational Supplement) has been granted by CLSI. The interpretive data are valid only if the methodology in M02-A10 (Per formance Standards for Antimicrobial Disk Susceptibility Tests—10th edition; Approved Stan- dard) is followed. CLSI frequently updates the interpretive tables through new editions of the standard and supplements. Users should refer to the most recent editions.
The current standard may be obtained from CLSI, 940 West Valley Road, Suite 1400, Wayne, PA 19087. Contact also may be made via the Web site (www.CLSI.org), email (customerser vice@clsi.org) and by phone (1.877.447.1888).
7-4 Clinical Biofilms
✦ Theory
According to Elvers and Lappin-Scott (2004), a biofilm is a “Complex association or matrix of microorganisms and microbial products attached to a surface.” Devel - opment begins with formation of a conditioning film composed of biomolecules and particles from the envi- ronment on the surface. This results in the surface becoming more hydrophilic and negatively charged.
Following this comes attachment of planktonic micro - organisms to the surface. The precise mechanism varies depending on the amount of fluid movement and other factors, however biofilm formation invariably depends upon the microbial community itself through secretion of an extracellular polysaccharide (glycocalyx). As more microbes become attached, they change the composition of the biofilm’s chemistry, providing a suitable environ- ment for still other microorganisms. Eventually, the biofilm gets thick enough that detachment, which can be the result of erosion, abrasion, or simple breakage, becomes a factor. At some point a steady state is reached in which addition to the biofilm is compensated by loss from the biofilm.
Biofilms occur in natural environments, but also are formed in industrial and medical settings. It is the medi - cal with which this lab is concerned. Indwelling devices, such as needles and catheters, are common locations for biofilm development. Reduced susceptibility of the biofilm community to antimicrobics (by a factor of 100–1000 times compared to their planktonic counter- parts!) with natural detachment make these biofilms problematic in the production of nosocomial infections.
✦ Application
Staphylococcus aureus andS. epidermidisare notorious for forming biofilms on invasive medical devices resulting in nosocomial infections.
✦ In This Exercise
You will grow a mixed culture of Staphylococcus aureus andS. epidermidisfor one week in a test tube, stain it, and observe for evidence of a biofilm.
✦ Materials
Per Student Group
✦overnight broth cultures of Staphylococcus aureus (BSL-2) and S. epidermidis
✦two tubes of TSB enriched with 1% glucose
✦phosphate buffered saline (PBS), pH 7.3
✦0.1% crystal violet
✦deionized water bottle
✦test tube rack
✦receptacle for culture media disposal
✦ Medium, Stain, and Reagent Recipes
Tryptic Soy Broth Plus 1% Glucose
⽧Tryptic Soy Broth 1 L
⽧Glucose 10 g
Crystal Violet Stain (0.1%)
⽧Gram Crystal Violet 0.1 mL
⽧dH2O 99.9 mL
10x Phosphate Buffered Saline (PBS)
⽧Na2HPO4, anhydrous, reagent grade 12.36 g
⽧NaH2PO4•H2O, reagent grade 1.80 g
⽧NaCl, reagent grade 85.00 g
Dissolve ingredients in distilled water to a final volume of 1 L
Working Solution (0.01 M Phosphate, pH 7.6)
⽧Stock solution 100 mL
⽧dH2O 900 mL
Procedure
Day 1
1 Heavily inoculate one TSBⳭ1% Glucose tube with both Staphylococcus aureus andS. epidermidis.
Label this tube with the names of the organisms.
2 Label the second tube “control” and do not inocu- late it.
3 Incubate the tubes for 24–48 hours at 35Ⳳ2°C.
Day 2
1 Decant the broth from each tube into the receptacle designated for disposal. Be sure not to spill or drip culture when pouring. If you do, clean it up with your lab’s disinfectant and wash your hands with your lab’s antiseptic.
2 Gently rinse each tube with PBS twice and pour it into the receptacle designated for disposal.
3 Air dry the tubes in an inverted position.
4 Stain both tubes with 0.1% crystal violet solution for ten minutes at room temperature. Be sure to add more stain than the original volume of broth in the tubes.
5 Gently wash the tubes with dH2O to remove unbound crystal violet.
6 Air dry the tubes in an inverted position.
7 Compare your results with Figure 7-8. You are looking for a purple film adhering to the inside of the experimental tube. Ignore any dark ring at the surface. Record your observations and answer the questions on the Data Sheet.
References
Elvers, Karen T. and Hilary M. Lappin-Scott. 2004. Biofilms and Bio - fouling, Chapter 12 in The Desk Encyclopedia of Microbiology,Moselio Schaechter, Ed. Elsevier Academic Press, 525 B Street, Suite 1900, San Diego, CA 92101-4495, USA.
Hirshfield, Irvin N., Subit Barua, and Paramita Basu. 2009. Overview of Biofilms and Some Key methods for Their Study. Chapter 42 in Practical Handbook of Microbiology, 2nd ed., Edited by Emanuel Goldman and Lorrence H. Green. CRC Press, Taylor, and Francis Group, Boca Raton, FL.
7-8 A STAPHYLOCOCCALBIOFILM✦The faint purple is the biofilm stained with dilute cr ystal violet. The tube on the right is from an uninoculated control.
✦ Theory
Epidemiologyis the study of the causes, occurrence, transmission, distribution, and prevention of diseases in a population. The Centers for Disease Control and Prevention (CDC) in Atlanta, GA, is the national clear- inghouse for epidemiological data. The CDC receives reports related to the occurrence of 26 notifiable diseases (Table 7-5) from the United States and its territories, and compiles the data into tabular form, available in the publication Morbidity and Mortality Weekly Report (MMWR).
Two important disease measures that epidemiologists collect are morbidity(sickness) and mortality (death).
Morbidity relative to a specific disease is the number of susceptible people who have the disease within a defined population during a specific time period. It usually is expressed as a rate. Because population size fluctuates constantly, it is conventional to use the population size at the midpoint of the study period. Also, the units for the rate fraction are “cases per person” and usually are small decimal fractions. To make the calculated rate more “user-friendly,” it is multiplied by some power of 10 (“K”) to achieve a value that is a whole number.
Thus, a morbidity rate of 0.00002 is multiplied by 100,000 (105) so it can be reported as 2 cases per 100,000 people rather than 0.00002 cases per person. Morbidity rate is calculated using the following equation:
number of existing cases in a time period
Morbidity Rate ⳱ ⳯K
size of at-risk population at midpoint of time period Mortality, also expressed as a rate, is the number of people who die from a specific disease out of the total population afflicted with that disease in a specified time period. It, too, is multiplied by a factor “K” so the rate can be reported as a whole number of cases. The equa- tion is:
number of deaths due to a disease in a time period
Mortality Rate ⳱ ⳯K
number of people with that disease in the time period Minimally, an epidemiological study evaluates morbidity or mortality data in terms of person (age, sex, race, etc.), place,and time. Sophisticated analyses
require training in biostatistics, but the simple epidemio- logical calculation you will be doing can be performed with little mathematical background. You will calculate incidence rate, which is the occurrence of new cases of a disease within a defined population during a specific period of time. As before, “K” is some power of 10 so the rate can be reported as a whole number of cases.
number of new cases in a time period
Incidence Rate ⳱ ⳯K
size of at-risk populations at midpoint of time period Because our focus is microbiology, we will deal only with infectious diseases—those caused by biological agents such as bacteria and viruses. Noninfectious dis- eases, such as stroke, heart disease, and emphysema, also are studied by epidemiologists but are not within the scope of microbiology.
✦ Application
An understanding of the causes and distribution of diseases in a population is useful to health-care providers in a couple of ways. First, awareness of what diseases are prevalent during a certain period of time aids in diagnosis. Second, an understanding of the disease, its causes, and transmission can be useful in implementing strategies for preventing it.
✦ In This Exercise
First you will choose a disease from the list of notifiable diseases in Table 7-5. Then you will collect data for the United States over the past two years. Once you have collected data, you will construct graphs illustrating the cumulative totals and incidence values for each year.
✦ Materials
✦a computer with Internet access or access to printed copies of Morbidity and Mortality Weekly Report.
7-5 Morbidity and Mortality Weekly Report (MMWR) Assignment
Procedure
1 Go to the CDC Web site http://www.cdc.gov. Then follow these links. (Note:Web sites often are re vised, so if the site doesn’t match this description exactly, it still is probably close. Improvise, and you’ll find what you need. These links are current as of November 2009):
⽧ Click on MMWR under “Publications” near the bottom of the home page.
⽧ Click on “State Health Statistics” in the menu bar at the left. This will drop down a new menu.
⽧ Click on “Morbidity Tables.”
⽧ Read the “Note” below the menu window on this page. It describes how the data in the tables are collected and why the numbers are provisional.
⽧ Select MMWR Week 1 of the most recent com- plete MMWR year, and then click on “Submit.”
⽧ Table II is divided into 9 parts. Upon your first visit, examine the 9 parts and choose a disease you would like to work on.1
On your Data Sheet, record the name of the dis- ease you have selected and write the cumulative number of cases in the United States for Week 1 of the two years you are studying. For instance, if you are studying 2009 and 2008, you would take the numbers from the columns entitled Cum 2009 and Cum 2008. Record these on the Data Sheet.
Using the years 2008 and 2009 as examples, you will find that the 2008 number reported for a par- ticular week in the 2009table might differ from the reported number in the same week of the 2008 table. This is a result of corrections in re- ported 2008 numbers in the 2009 table. Don’t fret. This is out of your control! Just record the numbers as given.
⽧ Return to the page with the MMWR Week and MMWR Year and continue the process for each week through MMWR Week 52. (Alternatively, you can just change the week number in the URL and press return.2) Record the cumulative totals on the Data Sheet.
2 Answer the questions and complete the activities on the Data Sheet.
Chlamydia Rabies, animal
Coccidioidomycosis Rocky Mountain spotted fever
Cr yptosporidiosis Salmonellosis
Girardiasis Shiga toxin-producing E. coli(STEC)
Gonorrhea Shigellosis
Haemophilus influenzae, Streptococcal disease, invasive, Group A invasive, all ages and serotypes
Hepatitis A Streptococcus pneumoniae, invasive disease, age <5 years
Hepatitis B Streptococcus pneumoniae,invasive disease, drug resistant, all ages Legionellosis Streptococcus pneumoniae,invasive disease, drug resistant, <5 years
Lyme Disease Syphilis, primar y and secondar y
Malaria Varicella (chickenpox)
Meningococcal diseases, West Nile Virus—neuroinvasive) invasive, all serogroups
Per tussis West Nile Virus—non-neuroinvasive)
TABLE 7-5 NOTIFIABLEDISEASES IN THEUNITEDSTATES ANDITSTERRITORIESPOSTED INTABLEII OFMMWR (AS OFNOVEMBER2009.)✦ Diseases highlighted in orange should not be selected because you won’t have enough information to complete the calculations required for this assignment. Some of the more “interesting diseases” (i.e.,AIDS and tuberculosis) are listed in Table IV, but are repor ted only quar terly and so don’t lend themselves to this assignment.
1Table I lists the occurrence of infrequently reported notifiable diseases (< 1000 cases in the previous year). Your instructor may allow you to choose a disease from this table in addition to Table II.
2Thanks to Joe Montes, Jr. for this shortcut tip!