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PacBio Long Reads Assess Antibiotic Resistance Plasmids in Wake of Enterobacteriaceae Outbreak

NEW YORK (GenomeWeb) – A new long-read sequencing study in Science Translational Medicine has started to untangle the complexity behind plasmid-mediated antibiotic resistance in hospital-acquired infections involving Enterobacteriaceae pathogens.

Researchers from the National Institutes of Health and Pacific Biosciences used long-read, PacBio single-molecule real-time (SMRT) sequencing to profile antibiotic resistance enzyme-containing plasmids present in gram-negative Enterobacteriaceae isolates collected at an NIH hospital during and after a 2011 outbreak of antibiotic resistant Enterobacteriaceae.

Their results revealed extensive diversity in the background plasmids carrying these resistance genes — even within the same human host, in one case — with many of the resistance-related plasmids apparently stemming from independent sources within or beyond the hospital environment.

"We're starting to see the patterns that we just couldn't see," co-corresponding author Julie Segre, with the National Human Genome Research Institute, told GenomeWeb Daily News. "Now what were looking at is a map of the [resistance plasmid diversity]."

Bacterial plasmids containing sequences that code for a carbapenemase enzymes have been implicated in the development of Enterobacteriaceae bacteria that can resist a class of antibiotics called carbapenems — drugs typically used for cases that do not respond to conventional antibiotic interventions.

The presence of carbapenem-resistant Enterobacteriaceae appears to be on the rise in US hospitals and long-term care facilities over the past decade or so, the study's authors noted, prompting concern over the potential impacts of tricky-to-treat infections stemming from these bugs as well as the interactions between these resistance plasmid-carrying pathogens and other microbes.

In an effort to characterize carbapenemase enzyme-coding plasmids carried by Enterobacteriaceae — and their potential movement between bacterial species — the researchers did whole-genome sequencing on clinical and hospital environment isolates of Enterobacteriaceae species such as Klebsiella pneumoniae, K. oxytoca, and Escherichia coli that contained one or more carbapenem resistance plasmids.

The team focused its attention on carbapenemase enzyme-based resistance involving the so-called KPC gene. Enterobacteriaceae bugs with KPC-containing plasmid sequences were behind an outbreak involving 18 patients at an NIH clinical center in 2011, the researchers explained, including six individuals who succumbed to related bloodstream infections.

Carbapenem resistance in Enterobacteriaceae can also stem from carriage of a plasmid containing another carbapenemase enzyme encoded by the NDM1 gene, Segre explained, which is more prevalent in Southeast Asia at the moment. That plasmid was not detected through surveillance and screening at the NIH center during or after the 2011 outbreak.

For their new analysis, researchers collected samples from almost 1,100 patients at the NIH clinical center in the years following the carbapenem-resistant Enterobacteriaceae outbreak.

These included perirectal, throat, and/or groin swabs collected twice weekly from patients in the hospital's intensive care unit and a high-risk medical ward during 2012 and 2013, along with monthly surveillance samples collected in other hospital wards and samples collected from newly admitted individuals (including some transferred from other medical centers).

When they screened more than 14,200 patient samples, together with hundreds of samples collected in the hospital environment, the team tracked down 10 patients carrying KPC-positive plasmid-containing Enterobacteriaceae. Two more individuals tested positive for KPC Enterobacteriaceae at other centers after being discharged from the NIH hospital.

The team had previously encountered problems with trying to fully sequence and track KPC-containing plasmids using short-read sequencing techniques, Segre explained, in part because the plasmids that house resistance genes also tend to contain large, mobile genetic elements.

In the case of the carbapenem resistance plasmids considered in the current study, for example, the KPC gene is typically flanked by Tn4401 transposons that stretch out up to 10,000 bases, she noted, which presents a challenge when trying to characterize multiple plasmids per bacterial cell with short reads.

To get around this issue, the researchers decided to take a crack at using PacBio's RSII to generate long, single-molecule reads that would allow them to look at both bacterial genome sequences and sequences from plasmids carried by the cell.

Using this approach, they sequenced the isolates from all 12 of the KPC-positive patients identified in 2012 and 2013, as well as two isolates collected during the 2011 outbreak and six environmental surveillance samples at the hospital.

Together, these sequences represented species from the Klebsiella, Enterobacter, Citrobacter, and Pantoea genera, along with plasmids spanning between 9,300 and 379,000 bases apiece, making it possible for the team to identify potential transmission events involving specific plasmids and microbial species.

Just one of the cases detected from swabs collected in 2012 and 2013 could be directly linked to the 2011 outbreak based on shared genetic features, the researchers reported. Instead, their results indicated that many of the carbapenem resistance plasmids had been introduced to the center independently, with no clear epidemiological ties to the earlier infections.

Somewhat unexpectedly, the sequence data also pointed to relatively infrequent transfer of the resistance plasmids horizontally from one isolate to another.

For instance, the team found that one individual from the 2011 outbreak had been infected with both K. pneumoniae and Enterobacter cloacae, each carrying the KPC gene in distinct plasmid backgrounds that had not been traded between the bugs.

While horizontal plasmid transfer was "less prevalent" than anticipated in the current analysis, though, there is evidence that it can happen in healthcare settings in general, Segre explained. "We did not observe it in patients, but we did observe it in the [hospital] environment."

Such patterns are prompting questions about the mechanisms that help or hinder plasmid movement between different microbes — something Segre and her colleagues plan to look at more closely in their follow-up studies.

"We still see these plasmids on the move," Segre said, noting that genomic studies "are now allowing us to do the types of foundational biological experiments that we want to do" to understand these processes.

In particular, the researchers are attempting to mimic conditions in a hospital biofilm-containing sink drain or patient to figure out what conditions might prompt the KPC-containing plasmids to move to a new microbial host.

"We can now do it in the context of fully sequenced genomes because we now know every plasmid and what it carries," Segre explained. "We're now doing a study that I think is fully informed."

At the NIH clinical center where the study was done, active surveillance for carbapenem-resistant Enterobacteriaceae is continuing both for newly admitted patients and patients in intensive care and other hospital wards.

The price of sequencing was roughly $1,200 per sample for the current study, though Segre noted that it may be possible to do wholesale sequencing on high-throughput, short-read instruments once complete reference genomes have been generated for all of the antibiotic resistance gene-containing plasmids.

To that end, she and her colleagues are keen to begin establishing a set of resistance-related plasmid reference genomes that can be used as part of a national surveillance effort aimed at curbing the spread of antibiotic resistance within hospital settings and beyond.