Resistant strains of the bacteria Staphylococcus aureus are the scourge of hospitals worldwide, frequently sickening and killing patients who were admitted to overcome other ills. And until now, scientists have not been able to closely track the transmission and mutation patterns of single strains.

A new project, using high-throughput, whole-genome sequencing, has begun to demystify MRSA (methicillin-resistant Staphylococcus aureus), revealing how the bacteria tend to spread among patients—and continents. Results of the project were published online January 21 in the journal Science.

The researchers took as their main subject a common strain of the bacteria known as ST239. "We knew that this strain was widespread," said Sharon Peacock, of the Department of Medicine at the University of Cambridge in England, in a conference call with reporters January 20. "But we had no idea how transmission was occurring." Given that MRSA has been found worldwide, scientists assumed it was capable of traveling between continents, but just how and where various subtypes were spreading remained obscured by low-resolution data.

Using older analysis techniques, such as multilocus sequence typing (MLST), most isolates of a MRSA strain appeared to have the same genetic profile. Scientists would typically sample DNA sequences in six or seven genes across the whole MRSA genome (which contains some 3,000 genes). At this rough resolution, subtle genetic changes would often go undetected, the researchers behind the new study explained. More rapid, whole-genome sequencing, however, enabled researcher to see "very precise differences" that occur on the level of single-nucleotide changes, Stephen Bentley, from the Wellcome Trust Sanger Institute (WTSI) in Cambridge, and senior study author, said during the teleconference. For instance, dozens of MRSA isolates collected across the globe (from Asia, Australia, Europe, and North and South America) between 1982 and 2003 had appeared identical using the MLST approach, but with the newer, whole-genome analysis, each proved to be genetically distinct.

The more thorough process, when applied to MRSA, has created "a leap in understanding," which allowed the researchers to construct a rough genetic evolutionary tree for the ST239 strain of MRSA. "It allows us to estimate the date of emergence and trace how it subsequently spread across the world," Simon Harris, also of WTSI, said during the call. This strain in particular appears to have emerged in Europe in the 1960s—a time when antibiotic use was on the rise in that part of the world.

Mutations on the move
In comparing the genetic sequences of the bacteria samples that had been collected on various continents, the team found that the isolates tended to be strongly clustered geographically, with closely related forms throughout South America, whereas others were more common in Asia. This finding "suggests that intercontinental transmission is a rare event," Harris said. The paper describing the project highlights some of these apparently infrequent infections, describing one instance, for example, in which a Brazilian line of MRSA cropped up in a Portugal hospital, showing that the infection does occasionally get transmitted between continents.

All of these conclusions are made possible by the newly detailed picture of the genetic changes that the bacterium undergoes across generations. This process of genetic mutation is occurring at the rate of about one core base pair every six weeks, the researchers deduced. It may sound plodding, but this rate is "far faster than was previously thought," Harris said. This bacterium's substitution rate is, the authors pointed out, about 1,000 times more rapid than the estimated rate for Escherichia coli.

Beyond the speed of mutations, their accumulation seemed to boost the most problematic aspect of the MRSA—its antibiotic resistance. And mutations that provide resistance appear to have happened on multiple occasions in various lines of a single strain, as Harris explained: "Mutations that confer resistance are occurring around the world." In particular, the resistance appears specifically tuned to popular antibiotics, which put "an immense selective pressure" on the bacteria, Harris said. In fact, some 29 percent of convergent mutations "can be directly related to evolution of resistance to antibiotic drugs currently in use, confirming clinical practice as a major driver of pathogen evolution," the researchers concluded in the study.
By studying the origins and transmission patterns of MRSA, researchers hope to be able to recommend more effective ways to stem the spread of this aggressive form of staph—not just within hospitals but also among them and within other settings. With the higher-resolution analysis, "you can see if strains are being transmitted from patient to patient or being brought into the hospital," Peacock said. Highly diverse samples collected over just seven months from a hospital in Thailand indicate that "there [were] multiple introductions," Harris said. At this point it is not clear where these introductions are likely coming from, whether it is from workers, patients or visitors who have picked up the infection in other health care or community settings, but it is clear that the map of transmission has expanded beyond wards and hospital walls.

"We think about MRSA in two distinct boxes: hospital-acquired MRSA and community-acquired MRSA," Peacock said. Increasingly, the two are overlapping, and Peacock noted that the community-acquired side appears to be growing even more prevalent. Although this analysis was conducted on hospital-based cases, an assay could also be done on community-based MRSA, she said.

Putting high throughput to work
One of the main goals in learning more about MRSA mutation and transmission patterns is to improve policies that might stop—or at least slow—the spread of this deadly infection. It will allow researchers to find breaks in the prevention chain, said Peacock, who added that, "we're not going to stop [it] by any amount of hand washing." Nor will this new analysis stop MRSA in its tracks, she noted, but "this tool will provide extra ammunition to identify routes of MRSA transmission."

The project, although it provided a new look into the dynamics of MRSA, was not designed to provide a comprehensive map or history of the infection. Rather, "this was a proof of principle," Harris explained. And as such, the researchers said they anticipated more advances in the field to follow soon.

Where it once took years to piece together bacterial genomes, full genomes can be sequenced "in a matter of weeks," Bentley noted. The technology used by these researchers allowed them to process 96 samples at once. A decade ago sequencing a genome for a mycobacterium that causes tuberculosis took three years and cost about $800,000. Today, sequencing these MRSA isolates took four to six weeks and cost about $300 each. Nevertheless, a $300 test is hardly practical for routine use, and as Bentley noted, the turnaround time is "still not a practical timescale for use in a health care setting." With both cost and processing time decreasing, however, the researchers said they expected the technology to become more widely available—and eventually integrated into basic hospital laboratories for rapid on-site testing.

In the meantime, such high-powered sequencing is already being put to use to study other microbial threats to human health. "This technology can be useful for any organism," Bentley said. Researchers are in the process of applying it to study the organisms at work in pneumonia, diarrhea, tuberculosis and meningitis.

But putting these new capabilities to work will not simply be a matter of scaling-up past or current projects, Bentley noted, adding: "It's going to require scientists to do some real innovative thinking to fully exploit this technology."