Culture and Sensitivity
Culture and Sensitivity
History and Scientific Foundations
Antimicrobial Susceptibility Testing
Introduction
Culture and sensitivity in microbiology refers to laboratory techniques that allow a disease-causing microorganism to be identified, and that determine which antibiotics are sensitive to (effective against) the identified microorganism.
Physicians must take numerous important factors into account when deciding how to appropriately treat infectious disease. Broad patient-specific factors include the natural history of the infection and the strength of the patient's immune system. If antibiotic treatment of the disease is suitable, as in the case of bacterial, certain fungal, and some other microbial diseases, the type of antibiotics used may depend on ease of absorption, metabolism, ability to reach the infection site, and other factors. Microbe culturing and susceptibility testing offers information to help make appropriate decisions. The availability of such information from laboratory testing can be of life-saving assistance to doctors, but can also result in excessive reliance on testing when a simpler, broader approach to treatment may be more efficient.
History and Scientific Foundations
Most of the techniques of culturing microbes were developed in the mid- to late 1800s by Robert Koch, Paul Erlich, and Hans Christian Gram. Using some of these techniques, Louis Pasteur (1822–1895) developed the foundations of the modern science of infectious disease at that time.
The German physician Robert Koch (1843–1910) perfected a technique to distinguish different types of bacteria and grow pure cultures of these bacterial types, and in the process founded the science of bacteriology. He formalized the approach of determining whether a particular microbe caused a given disease in a set of rules now known as Koch's Postulates (1882), which supported the concept that a disease is caused by a specific microbe:
- The agent of an infectious disease must be present in every case of the disease.
- The agent must be isolated from the host and grown in vitro (pure culture) for several generations.
- The disease must be reproduced when a pure culture of the agent is inoculated into a healthy susceptible host.
- The same agent must be recovered once again from the experimentally infected host.
Koch developed a solid medium for bacterial growth using gelatin, later modified by other scientists to include a seaweed called agar to keep the medium solid at room temperature. The German bacteriologist Richard Julius Petri (1852–1921) developed a glass dish still used today that helps foster optimal bacterial growth.
In 1877, Koch also developed a technique for dryfixing thin films of bacterial culture on glass slides, staining them with aniline dyes, and recording the microscopic images on film. Dry-fixing continues to be a standard procedure in identifying various bacterial cultures. Different types of media are optimal for specific types of bacteria, and identification of a specific pathogen can still be a matter of clinical experience and judgment.
Microbe staining techniques were developed in 1839 by the German scientist Christian Gottfried Ehrenberg (1795–1876). These staining techniques depended on two properties of stains: chromogenicity (inclusion of groups of atoms that are color-forming) and the ability to dissociate into positively charged ions (cations) and negatively charged ions (anions). For example, when the common methylene blue dye is added to water, it dissociates into a chloride anion and a methylene blue cation, which is visible in solution and makes methylene blue a “cation dye.” Another common dye, eosin, dissociates into a sodium cation and a visible eosin anion and is an anion dye. Anionic dyes such as eosin interact with the cationic portions of the bacterial protein being identified, while the converse happens with cationic dyes.
WORDS TO KNOW
ANTIBIOTIC RESISTANCE: The ability of bacteria to resist the actions of antibiotic drugs.
ANTIBIOTIC SENSITIVITY: Antibiotic sensitivity refers to the susceptibility of a bacterium to an antibiotic. Bacteria can be killed by some types of antibiotics and not be affected by other types. Different types of bacteria exhibit different patterns of antibiotic sensitivity.
BROAD-SPECTRUM ANTIBIOTICS: Broad-spectrum anti-biotics are drugs that kill a wide range of bacteria rather than just those from a specific family. For, example, amoxicillin is a broad-spectrum antibiotic that is used against many common illnesses such as ear infections.
COHORTING: Cohorting is the practice of grouping persons with like infections or symptoms together in order to reduce transmission to others.
CULTURE AND SENSITIVITY: Culture and sensitivity refer to laboratory tests that are used to identify the type of microorganism causing an infection and compounds that the identified organism is sensitive and resistant to. In the case of bacteria, this approach permits the selection of antibiotics that will be most effective in dealing with the infection.
GRAM-NEGATIVE BACTERIA: All types of bacteria identified and classified as a group that does not retain crystal-violet dye during Gram's method of staining.
GRAM-POSITIVE BACTERIA: All types of bacteria identified and classified as a group that retains crystalviolet dye during Gram's method of staining.
INPATIENT: A patient who is admitted to a hospital or clinic for treatment, typically requiring the patient to stay overnight.
OUTPATIENT: A person who receives health care services without being admitted to a hospital or clinic for an overnight stay.
MINIMAL INHIBITORY CONCENTRATION (MIC): The minimal inhibitory concentration (MIC) refers to the lowest level of an antibiotic that prevents growth of the particular type of bacteria in a liquid food source after a certain amount of time. Growth is detected by clouding of the food source. The MIC is the lowest concentration of the antibiotic at which the no cloudiness occurs.
SEPSIS: Sepsis refers to a bacterial infection in the bloodstream or body tissues. This is a very broad term covering the presence of many types of microscopic disease-causing organisms. Sepsis is also called bacteremia. Closely related terms include septicemia and septic syndrome. According to the Society of Critical Care Medicine, severe sepsis affects about 750,000 people in the United States each year. However, it is predicted to rapidly rise to one million people by 2010 due to the aging U.S. population. Over the decade of the 1990s, the incident rate of sepsis increased over 91%.
The Gram stain, developed in 1884 and named after discoverer Hans Christian Gram (1853–1938) is in a different category from ionic stains. This technique involves first staining bacteria with gentian violet dye, then washing the stained bacteria with iodine solution, and then with ethyl alcohol. For “Gram-negative” bacteria, the second and third steps wash away the dye, while “Gram-positive” bacteria remain colored after washing with iodine and alcohol. The final step is to stain the Gram-negative bacteria with a reddish-pink dye that does not stain the Gram-positive bacteria. The Gram-positive bacteria will thus have violet structural features under the microscope, while the Gram-negative bacteria will have pinkish structural features. Whether a bacterium is Gram-positive or Gram-negative is often an indicator or whether the bacteria can be destroyed using a particular antibiotic.
Many antibiotics can kill Gram-positive bacteria, while Gram-negative bacteria resist common antibiotics. Gram-negative bacteria have an extra layer of polysaccharides, proteins, and phospholipids, which blocks many antibiotics from reaching the peptidoglycan cell wall. For example, penicillin works by attacking the cell wall, but is prevented from doing so by this extra layer, making the bacteria penicillin-resistant.
Antimicrobial Susceptibility Testing
The susceptibility methods used by clinical laboratories include the Kirby-Bauer disc diffusion susceptibility test, macrotube dilution susceptibility test, and the microtube dilution test. In the Kirby-Bauer test, disks containing antibiotics are placed over an agar plate inoculated with the organism. The size of the zone of inhibition indicates whether or not the organism is sensitive or resistant to the antibiotic at level normally used (doses). Laboratories report antibiotic sensitivities as “Susceptible,” “Intermediate,” or “Resistant” as defined by the National Committee on Clinical Laboratory Standards (NCCLS).
The minimal inhibitory concentration (MIC) is the lowest concentration of the antibiotic (mcg/ml) that will inhibit bacterial growth in vitro, and is correlated with the concentration of the antibiotic achievable in blood.
The MIC is traditionally determined using the macrotube dilution technique in which a standard inoculum is tested against serial dilutions of a particular antibiotic—a time-consuming process. A newer technique uses tiny wells in an automated plastic susceptibility card that are injected with standard dilutions of antimicrobial agents by the manufacturer. The laboratory adds a standard concentration of the organism to the card and the organism is automatically dispersed to all of the wells. After an incubation period of 12–24 hours, the card is machine- read for bacterial growth at hourly intervals and a growth curve for the isolate is calculated for each antibiotic on the plastic card. The antibiotics are grouped according to whether the organism being tested is Gram-positive or Gram-negative, and the antibiotics for the Gram-negative isolate are further grouped according to whether they can be used in an inpatient or outpatient setting depending on whether the patient is in the process of being admitted or discharged from the hospital.
Applications
Depending on MIC results, physicians may change the dosage of a particular antibiotic to be used in treatment, or choose a different antibiotic to treat the infection. For example, a blood-borne Escherichia coli infection tested with ampicillin may have a MIC of 2 mcg/ml (sensitive), multiplied by 2–4 times gives 4–8 mcg/ml as a potential peak level of the antibiotic in the blood, which is considerably less than an intravenous representative dose from the patient of 47 mcg/mg. Thus, ampicillin would be expected to provide adequate therapy for the patient.
In a different example of a leg wound tested with ampicillin, a higher MIC of, say, 16 mcg/ml would be correlated with a 32–64 mcg/ml peak blood concentration, which could fall over the range of the representative intravenous dose of 47 mcg/ml dose of bacteria from the patient. Furthermore, since the patient has a leg wound where the infection is in tissue rather than blood, the concentration of antibiotics in tissue will be lower than in blood. In this case, the physician would consider a higher ampicillin dose or a different antibiotic for treatment.
Impacts and Issues
Culturing and MIC testing are possible for most, but not all, types of bacteria-caused diseases. Infections for which cultures generally cannot be obtained include ear infections, sinusitis, and bronchitis, along with viral infections. For such infections there is a considerable risk of over-prescribing antibiotic treatments that are likely to be inappropriate and ineffective, and there are increasing calls for the distribution of procedures and new guidelines to address this issue.
GERMAN PHYSICIAN ROBERT KOCH (1843–1910)
Robert Koch pioneered principles and techniques in studying bacteria and discovered the specific agents that cause tuberculosis, cholera, and anthrax. As a pioneer in microbiology and public health, Koch aided legislation and changing prevailing attitudes about hygiene to prevent the spread of various infectious diseases. For his work on tuberculosis, Koch was awarded the Nobel Prize in 1905.
In the first paper he wrote on tuberculosis, Koch stated his lifelong goal: “I have undertaken my investigations in the interests of public health and I hope the greatest benefits will accrue therefrom.”
The timing and choice of antibiotics can be important in treating older adults. For example, in sepsis (a generalized infection in the blood due to microorganisms or toxins) most research suggests that starting with broad-spectrum antibiotics without culturing is beneficial because deaths and long hospital stays are reduced if the initial antibiotic treatment attacks and reduces the infectious agent. Delaying therapy initiation by four or more hours after hospital admission, as could happen with long laboratory testing, is associated with higher mortality. On the other hand, up to 75% of antibiotic use in long-term care may be inappropriate, so strict minimum criteria for initiating antibiotic treatment should be set.
The emergence of resistant bacteria has led to the reliance on the newer class of antibiotics (fluoroquinolones) for relatively routine infections such as community-acquired pneumonia (CAP) in spite of the potential for adverse effects. Over-utilization has, in turn, given rise to increasing fluoroquinolone resistance in some geographic regions. Current Infectious Disease Society of America guidelines advise keeping newer fluoroquinolones that are active against S. pneumoniae in reserve, while using other anti-biotics such as an advanced generation cephalosporin (e.g., cefotaxime) as initial therapy.
IN CONTEXT: REAL-WORLD RISKS
The medical dangers and escalating health care costs associated with antimicrobial resistance led to the formation of a special interagency task force tasked with developing effective plans to combat the problem. Formed in 1999, the Interagency Task Force on Antimicrobial Resistance is co-chaired by the Centers for Disease Control and Prevention (CDC), the Food and Drug Administration (FDA), and the National Institutes of Health (NIH), and also includes the Agency for Healthcare Research and Quality (AHRQ), the Centers for Medicare and Medicaid Services (CMS, formerly the Health Care Financing Administration [HCFA]), the Department of Agriculture (USDA), the Department of Defense (DoD), the Department of Veterans Affairs (DVA), the Environmental Protection Agency (EPA), and the Health Resources and Services Administration (HRSA).
One of the top priorities of the task force is to “conduct a public health education campaign to promote appropriate anti-microbial use as a national health priority.”
This example demonstrates the vicious circle that arises from physicians’ reliance upon specialized antibiotics that show in vitro (in the body) potency against given infections when treatment with broad-spectrum antibiotics would provide faster treatment yet would not lead to resistance to the specialized antibiotics.
Furthermore, over-relience on antibiotics in long-term care facilities and in hospitals can cause health care workers to disregard simple infection control activities such as handwashing, isolation, and cohorting (grouping) of infected patients, skin testing for tuberculosis, and immunization to prevent infection with resistant organisms in the first place.
See AlsoAntibacterial Drugs; Bacterial Disease; Resistant Organisms; Vancomycin-resistant Enterococci.
BIBLIOGRAPHY
Books
Ryan, Kenneth J. and C. George Ray. Sherris Medical Microbiology: an Introduction to Infectious Disease. New York: McGraw-Hill Medical, 2003.
Web Sites
National Center for Biotechnology Information.“Microbiologic Examination.” in Medical Microbiology, 4th ed., Samuel Baron, ed. <http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=mmed.section.5451> (accessed April 2, 2007).
University of Virginia Health Systems. “What is Microbiology?” <http://www.healthsystem.virginia.edu/uvahealth/adult_path/micro.cfm> (accessed April 2, 2007).
Kenneth T. LaPensee