Thread by @richdavisphd

Antibiotic testing. Let's go.

Tentatively calling this #tweetorial "Antibiotic testing: why and how and huh?"

Important caveat: this is a HUGE topic. I’m mostly going to focus on 1) basic rationale and 2) laboratory methods. And 3) try not to say anything wrong/misleading!

Question #1: Why would a person want to know if bacteria A is resistant to antibiotic B?

And the answer is ...

All are legit answers! But it's very situation dependent and you'd have different reasons for each. Let’s go through them.
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Hard to say if A (so you can treat with that abx) is the most common, but it of course make sense!

Patient has Enterococcus (of some species) in their blood: I want to treat with vancomycin. Ergo, I should try to grow the bacteria in the presence of vancomycin.
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… But, let’s go with that example of Enterococcus some more.

Vancomycin resistant Enterococcus (VRE) is a serious hospital acquired infection (HAI). Which is a pretty big deal (PBD).
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This is an answer B situation. You test bc you want to know if this is VRE.

Enterococcus species against vanc can trigger an investigation into a possible VRE outbreak in a hospital setting. cdc.gov/hai/organisms/…
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Let's KEEP going with Enterooccus and vancomycin and talk about option C: wanting to IDENTIFY the bacteria.

In our system, 99% of Enterococcus faecalis is vancomycin sensitive. So a vanc resistant enterococcus? More likely to be E. faceium, E. cassioflavus or E. galinarum.

Here’s a better example for option C: “helps you identify the bacteria.”

There are several different species of rapidly growing mycobacterium.

Sometimes conventional identification methods (e.g. MALDI) can have some trouble calling them to the species level. #tweetorial

… However, there are can be distinct antibiotic susceptibility patterns among the rapid growing mycobacterium.

For instance, an isolate gets called as M. fortuitum.

But upon testing it looks resistant to cefoxitin and trimethoprim-sulfa and susceptible to clarithromycin. ...

A wary technologist knows that looks wrong: M. fortuitum is (fortuitously… get it?) usually only RESISTANT to clarithromycin and SUSCEPTIBLE to the others.

This looks like M. immunogenum’s resistance pattern. They should re-test the identification.
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Final option, D: test the antibiotic susceptibility because you just want to know!

Characterizing the antibiotic susceptibility profiles of unusual bugs can be very helpful! ... IF you get the information out there.

What if your bacteria is identified as something not seen in humans? You have no guidelines to treat it with!

You may turn to PubMed and see if something’s published.

If someone’s tested it before against a battery of antibiotics, that can help you know what to start with.

So that's the WHY of antibiotic resistance testing (there are other reasons and examples we didn't go into).

But for the second part of this #tweetorial: how do you actually DO this?

#AntibioticResistance

#AntibioticResistance Testing Method 1: Disk diffusion (Kirby-Bauer)

Small paper disk w/ antibiotics in it. Streak agar plate with a known concentration of bacteria. Put disks down. The closer to the disks the bacteria grows = the more resistant to the antibiotic effect it is.

#AntibioticResistance Test Method 2: Microbroth dilution

Antibotics are put into a well in smaller dilutions. Put known concentration of bacteria into the wells. Look to see the LOWEST concentration where no bacteria grows. More resistant = growth at higher concentrations.

#AntibioticResistance Test Method 3: antibiotic-containing agar

Good for screening test. An agar is impregnated with the antibiotic (e.g. oxacillin - MRSA, ertapenem – Carbapenem Resistance). If an organism grows, it’s likely a resistant isolate.

#AntibioticResistance Test Method 4: E-test

Instead of being in a disk, the antibiotic is in a strip with high- to low-concentration of the antibiotic. The growth of the bacteria next to the strip can tell you the inhibitory concentration.

#AntibioticResistance Test Method 5: inhibition assay

A modification of the disk method.

Step 1: incubate a disk w/ a bacteria for 2-4hrs.
Step 2: see if that disk can still inhibit a different bacteria (E. coli) on a plate!

see: twitter.com/search?q=cim%2…

#AntibioticResistance Test Method 6: disk plus an added enzyme inhibitor

You have a disk (a beta lactam drug alone) and a disk PLUS a beta-lactamASE inhibitor. If inhibitor makes bacteria more susceptible, the beta-lactamase was in the bacteria. see:

https://twitter.com/richdavisphd/status/981680315562213376

All of the above #AntibioticResistance methods 1-6 involve GROWING bacteria in the presence of the antibiotic indicating if it's resistant.

But other methods include looking for presence of a protein or gene that PREDICTS resistance.

One benefit: these can be faster! (esp PCR)

#AntibioticResistance Testing Method #7: presence of penicillin binding protein 2a.

Why is MRSA resistant to methycillin/oxacillin? Bc the beta-lactam drug can't bind to the bacteria's mutant penicillin binding protein PBP2a

Lateral flow test: a band forms if PBP2a is present.

#AntibioticResistance Testing Method #8: presence of the resistance mechanism gene.

This is a HUGE category. MRSA. Carabapenemase. M. tuberculosis resistance genes. There are hundreds of potential resistance mechanism-encoding genes. One benefit: (usually) faster than culture!

In the case of, for example, blood stream infections, there's a direct relationship between how fast you can start treating with the RIGHT antibiotics and patient outcomes.

One rapid PCR test, Biofire's BCID filmarray, includes some major #AntibioticResistance targets.

Question: if it's so fast, why don't we do ALL resistance testing by PCR?

Answers:
1) it can cost a lot!
2) many bacteria have multiple resistance mechanisms
3) in a mixed infection, you can get confounding results!
4) many #AntibioticResistant mechanism genes remain unknown

Last point: there are LOTS of additional "methods" (really small modifications of these major points) I didn't cover today (e.g. high-level aminoglycoside testing, penicillinase "sandy beach" test, cefoxitin screen).

This was by no means comprehensive. #ASMClinMicro #tweetorial

Resources for antibiotic UTILIZATION (if you're a physician or a laboratorian who wants to know what drugs treat what bugs):

1) @IDstewardship's website training.idstewardship.com
2) The Sanford Abx guide: sanfordguide.com/products/print…
3) Your nearest ID pharmacist!
#AntibioticResistance

Resources re: diagnostic testing methods for #AntibioticResistance

CLSI @CLSI_LabNews M100 manual (accessible for free) is a wealth of information clsi.org/standards/prod…

If there are other antibiotic resources PLEASE comment and I'll add them to this thread!

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A point that went unstated here: to minimize inappropriate antibiotic use, you have to know you're treating with something effective!

That'll do it for me! Thanks for following along!

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