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ARS Frye on Developing an Array to Detect Antimicrobial Resistance Genes

Jonathan Frye
Research Service

Name: Jonathan Frye

Title: Microbiologist, Agricultural Research Service

Professional Background: 2003 — present, microbiologist, Bacterial Epidemiology and Antimicrobial Resistance Research Unit, Agricultural Research Service, Athens, Ga.

Education: 2000 — PhD, microbiology, University of Georgia; 1993 — BS, biology, East Carolina University.

A team of microbiologists at the Agricultural Research Service in Athens, Ga., has developed a microarray that detects more than 100 antimicrobial-resistance genes in bacteria, according to a recent ARS report.

Their work appears in this month's issue of Agricultural Research, and shows how some scientists are using microarray technology to determine which bacteria are becoming resistant to antibiotics and how bacteria continue to develop resistance to new antibiotics in an effort to combat this phenomenon.

To learn more about the team's methodology and objectives, BioArray News spoke with ARS microbiologist Jonathan Frye this week.

What is your background and how did you wind up at ARS?

I did a bachelor's degree in biology at East Carolina University. I worked as a technician for a few years at the University of North Carolina, Chapel Hill. I came down to the University of Georgia to get my PhD and I was there from 1995 to 2000. Then I went out to California to do a post-doc with a scientist named Michael McClelland [currently the director of molecular genetics at the Sidney Kimmel Cancer Center]. He is the person who sequenced the Salmonella genome. We developed a whole Salmonella DNA microarray. While I was working there I got contacted by scientists here at ARS that wanted to collaborate to detect antimicrobial-resistance genes. So eventually a position came up and I have been here since 2003.

How serious is the problem of bacterial pathogens developing a resistance to antibiotics?

It can be easily characterized as a major problem. Lots of infections are becoming resistant to most of the antibiotics we use to treat them. Currently worldwide, there are strains of multi-drug-resistant [Mycobacterium] tuberculosis, for example, that are resistant to any treatment with any antibiotics. People who contract these diseases usually die. We don't have tuberculosis as bad in the US, but that is just an example of how bad drug resistance can become.

We do have drug resistance in the US, in enteric bacteria, like Escherichia coli and Salmonella, and also there's some drug resistance in bacteria that cause things like sore throat or wound infection — that would be Streptococcus or Staphylococcus. So it is a major concern that we are going to lose our ability to effectively use antibiotics to treat diseases that we've been capable of treating for past 50 years.

What kind of projects are being undertaken to remedy this?

This is a major issue. There's lots of research going on, both in the scientific setting and in the clinical setting. There are a lot of people that are very interested in figuring out what's causing this problem and how we can prevent it from getting any worse. There are thousands of papers published in this field every year. [Let me] give you an example of what we're doing here in my unit. My unit is called the Bacterial Epidemiology and Antimicrobial Resistance Research Unit. Part of what our unit does is participate in the National Antimicrobial Resistance Monitoring System.

It is the major system for looking at resistance in bacteria in the US, and we handle the animal end of that, being the United States Department of Agriculture. Meanwhile, we also collaborate with the FDA, and we collaborate with the Centers for Disease Control, and they handle the human samples while we handle the animal samples. We assay those samples for resistance to clinical antibiotics. Together these data go into databases. These are available on the web for the public to view and for other scientists to look at.

There are other large monitoring systems; I know the UK has one. Denmark has something called DanMap, which is where they monitor. I am sure Asia has its own monitoring system. It's a major concern in the world.

What are the difficulties with regards to this project in determining which genes create this resistance to antibiotics?

Probably the first problem in designing a DNA microarray to detect the genes is deciding which genes to design probes for and attach to your chip. The way that I've done that is by building on the results of other scientists out there. Going through and searching the published literature and finding the genes that are the most likely to be ones that I would expect to find in my sample. The first chip that we constructed had about 100 genes on it. That's how we did that. I found genes for Salmonella and E. coli and I asked my fellow [ARS] scientists Mark England and Charlene Jackson — their specialties were Campylobacter and Enterococcus — so I asked them 'What genes would you expect to find?' When I had those genes available, I went to the database and downloaded the sequence and designed probes for them.

The second part of the chip, which I am constructing as we speak, is going to have almost 800 genes on it. It's going to be designed to detect all the genes that I can find in the [National Center for Biotechnology Information]'s genome database. And I went through and searched for all genes that were annotated as being associated with antimicrobial resistance. And that gave me about 5,000 or 6,000 genes. And after that I had to narrow it down. So I went through and I actually had to search the annotation of each gene. One problem with the non-redundant database is that it is completely redundant for any horizontally exchanged gene. And resistance genes are often horizontally exchanged. So if you have an E. coli ampicillin-resistant gene, and it's also in Salmonella, then when they deposit both of those in the database they are considered different genes, even if they are completely identical in sequence.

That must have been a lot of work …

Well, it took months. I was lucky enough to have a technician, and a grad student, and a student aide. Mostly the student aide and I sat down and searched through these files. I told him what to look for in the annotation, which genes to keep based on the information in the annotation. And he gave me that list. And then I used the Blast algorithm, which is available for free from NCBI, to Blast each probe that I designed against the database, and to determine that it was designed to detect just the gene I wanted and was unique to just that gene. And then I had to go through and eliminate all the ones that were identical. That I physically did by myself.

I read in an article [released by ARS and written by Sharon Durham] that you are actually getting your samples for this project from slaughterhouses. How did these samples impact your work?

All of the samples that we get, that we assay for resistance, come from farms and slaughter facilities. So the animal health inspection service will give us samples, and that's from healthy animals, and then the slaughter samples come from [the USDA's Food Safety Initiative] — their job is to go and inspect slaughter facilities — and so they would send us things like carcass swabs from slaughtered animals. And we also get a lot of our samples from vets. Eight veterinary labs send us their samples. So we would assay all of those for antimicrobial resistance. Of course, what I am looking at is that we know we can assay pretty quickly for their resistance, but what I am interested in learning is what genes are determining their genetic resistance. So what I'll do is take the same samples where we have determined their phenotype, and then I'll [use] the microarray to try to determine their genotype.

Is this your array or is someone else spotting it?

No, no, no. It's all mine. But I had a lot of help. I had the first set of arrays spotted back out with my old post-doc advisor in California, just because they had a spotter up and running. Now we have a spotter here, and it appears to work, and I will be using it for my future chips.

What are the future chips? Where is this going now?

The future array will contain 800 to 1,000 genes that will cover the known, unique antimicrobial resistant genes that I could find. In theory it should cover most of what we know.

Ideally, what will be done with that chip?

First, of course, testing. It needs to be tested and validated to show the array is working well. The first array has worked very well, and a paper has been accepted at the International Journal of Antimicrobial Agents. My guess is that it will be published next year. We expect the testing to go forward relatively quickly with this new array, and then we'll have to test it to see if it's detecting most of the genes. The chip will probably go through many [changes]. Once we find out the shortcomings of this chip, we'll add things to the new chip. For instance, we might add probes to detect plasmids because they often carry these resistant genes, or transposons or integrons in phage, because they also carry these resistant genes. So it's a building process. [For example], take the Salmonella array we did out in California. Since then we've added specific genes so that the array covers five different types of Salmonella. Arrays require a lot of upkeep.

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