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With so many antibiotics available, not to mention so much antibiotic resistance in bacteria, how is a doctor supposed to choose which drug to prescribe? In this lesson, learn about tests to determine which antibiotics will be most effective against a microbe.

Drug Susceptibility

With all you’ve learned about bacteria and other microbes, it might seem almost like they’re supernatural beings or something. They have virulence factors that help them set up infections better, they have ways to hide from our immune system, and they even have tons of ways to resist the antibiotics and other drugs that we throw at them.

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This lesson is about finding microbes’ weak spots: finding out which antibiotics a particular microbe is especially susceptible to and exactly how much of a drug is needed so that we can destroy it and all its little buddies as soon as possible.

Diffusion Tests

As we’ve learned in other lessons in this chapter, different kinds of drugs work better for different kinds of organisms. If the organism causing an infection is known, doctors can choose an appropriate drug pretty easily. But often, the organism is unknown. Or it could be a type of bacteria that is often antibiotic-resistant. In these cases, it’s a good idea to test which antibiotics it is susceptible to.The first way we’ll talk about is called the disk diffusion test / Kirby-Bauer test.

First, you take your microbe of choice – for example, one that you’ve cultured out of a patient’s infected lung. You spread a large amount of this microbe all over an agar plate in a Petri dish. There should be enough bacteria there that they’d cover the entire plate after you let them incubate at body temperature for a while.But before you incubate them, you need to place a few paper disks that contain known amounts of various antibiotics on the plate. The drugs are going to diffuse gradually out of the disks and into the agar, hence the name ‘diffusion test.’ The further away from a disk you get, the lower the concentration of antibiotic will be there.So let’s fast forward until after we’ve incubated the bacteria on the plate with the antibiotic disks.

What we’ll see if an antibiotic worked against our organism is a so-called zone of inhibition. This is an empty area on the plate surrounding an antibiotic disk. It’s empty because all of the bacteria that were there were killed or were unable to grow because of the action of the antibiotic. In general, the bigger the zone of inhibition, the more effective that drug was because it could work at the lower concentrations found further away from the disk. If there is no zone of inhibition, the organism was not susceptible to that antibiotic.One caveat to this method is that some drugs aren’t very soluble, meaning they don’t dissolve well in the agar plate. That means the antibiotics won’t be able to diffuse very far away from the disks.

So the zone of inhibition might look misleadingly small, even if the drug was effective.There’s also a more advanced version of a diffusion test that provides more detailed information to the microbiologist. It’s called the E test. The E stands for ‘epsilometer.’ In this test, you put plastic strips onto the plate of bacteria instead of the disks.

The key thing that makes the E test better than the Kirby-Bauer test is that the plastic strips have a precise, known gradient of antibiotic concentrations on them. So when you see the zones of inhibition, you will also know the minimal inhibitory concentration (MIC) of the antibiotic that works on the microbe in question. That is, you’ll know the lowest amount of antibiotic that can be used to inhibit your bug.Knowing the MIC allows physicians to avoid promoting antibiotic resistance by not using a high enough dose to control the bacteria. Also, they can minimize the toxic side effects that their patients might get from taking higher doses than necessary.

Broth Dilution Tests

The diffusion methods we’ve learned about so far can only tell us whether a drug inhibits a microbe, not whether it’s bactericidal or bacteriostatic. Remember that bactericidal antibiotics kill bacteria directly, while bacteriostatic antibiotics just stop bacteria from growing.

The broth dilution test overcomes this problem. Several different dilutions of a drug are made into liquid media in a special plate that has many sample wells. Then the same amount of bacteria is added to each well. Then we have to incubate again to let the bacteria grow and multiply.

After incubation, the wells with no bacterial growth had antibiotic concentrations that were above the MIC of the drug. Now comes the best part: to check if the drug was bacteriostatic or bactericidal, you can culture a sample from those wells in media without the drug.If there’s growth in this fresh, antibiotic-free media, you know that the drug was only bacteriostatic since it did not kill the bacteria in the well – they were still alive and able to replicate once they were away from that pesky antibiotic.

However, if nothing grows in the media without antibiotics, you know those little bugs were already completely dead because the antibiotic was bactericidal.From these results, a microbiologist can determine the minimal bactericidal concentration (MBC) – that is, the lowest concentration of the drug that can be used to actually kill the bacteria instead of just stop their growth.

Time- vs. Concentration-Dependent Drugs

Once you’ve figured out which drug your patient’s bacteria are most susceptible to, as well as the MIC and maybe the MBC, you’re good to go, right? Actually, there are a couple other considerations to think about when it comes to antibiotic effectiveness. For example, some antibiotics are time-dependent and some are concentration-dependent.Time-dependent antibiotics work better the longer they stay above the MIC in a patient’s body.

With these drugs, it doesn’t help to increase the dose of the antibiotic, but it’s very important that doses are not missed and that the antibiotic is kept above the MIC for a long enough period of time, usually days. Beta-lactam antibiotics, like penicillins and cephalosporins, are time-dependent. Their concentrations should be kept above the MIC for at least 80% of the time of treatment.On the other hand, concentration-dependent antibiotics aren’t so time-sensitive, but they work better the higher concentration they reach. Often, drug concentrations that are 10 times the MIC work best. It seems not to affect the treatment much if the drug concentration dips below the MIC for a period of time during treatment as long as high concentrations have been reached at some point.

Aminoglycosides are concentration-dependent antibiotics; they can often be given at a high dose only once a day.What’s the main point of all this? Knowing whether antibiotics are time- or concentration-dependent is crucial so that doctors can determine the right doses to fight a patient’s infection.

Drug Synergy and Antagonism

There is still one last consideration when it comes to antibiotic effectiveness. You may remember that some drugs, like sulfa drugs and trimethoprim, have so-called synergistic effects.

Drug synergy is when drugs work better together than they do on their own. There can be many reasons for this.In the case of sulfa drugs and trimethoprim, they synergize because they both inhibit bacterial enzymes in the same metabolic pathway, so it’s like a one-two punch.

Another interesting example of drug synergy is penicillin and streptomycin. The penicillin weakens the bacterial cell wall, making it easier for the streptomycin to get in and inhibit the bacterial ribosome.On the other hand, drugs can also make each other less effective. Drug antagonism is when drugs, well, antagonize each other and don’t work as well together as they do alone. One example of this is penicillin and tetracycline.

This is because tetracycline, being bacteriostatic, stops bacteria from growing. And penicillin is only effective against actively growing cells. So the two just don’t work well together.

Testing for drug synergy and antagonism is really important because knowing whether drugs are synergistic, antagonistic or neither helps doctors know whether or not two drugs can be taken together.

Lesson Summary

In this lesson, we’ve learned a lot about antibiotic effectiveness. First, we learned a few different ways to test which antibiotics a microbe is susceptible to.

In the disk diffusion test and the E test, antibiotics diffuse from special filter disks or strips through an agar medium and create a zone of inhibition if the drug is effective against the bacteria. The E test allows more precise measurement of the minimal inhibitory concentration (MIC).The broth dilution test is another way to find the MIC of a drug in liquid medium.

Bacteria are incubated in different concentrations of the drug, and wherever they don’t grow, the concentration is above the MIC. Moreover, then you can culture bacteria from the inhibited wells on a fresh medium without antibiotics. If they grow, you know the drug was bacteriostatic, and if they don’t grow, you know it was bactericidal and actually killed the bacteria. In this way, you can also determine the minimum bactericidal concentration (MBC) using the broth dilution test.Next, we learned that some drugs are time-dependent, meaning that it’s most important for them to be above the MIC in a patient’s body for the entire dosing period. Other drugs are concentration-dependent, meaning that it’s most important for them to reach very high concentrations, perhaps 10 times the MIC, and less important how long the concentration stays that high.

Finally, we heard about drug synergy and drug antagonism. Some drugs work better together than on their own because their activities synergize. And it’s better to use other drugs alone, because they decrease each other’s effectiveness if they are used together.

Learning Outcomes

When this video lesson is completed, you should be able to:

  • Differentiate between the types of diffusion tests
  • Explain why the broth dilution test is used
  • Describe time-dependent antibiotics and concentration-dependent antibiotics
  • Define drug synergy and drug antagonism

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