Microbial Genomics

Author: scottbeatson

New paper: Tackling a hospital superbug outbreak with multiple genomic technologies

First author Dr Leah Roberts loading bacterial DNA on to an Oxford Nanopore MinION for rapid sequencing.

Integrating multiple genomic technologies to investigate an outbreak of carbapenemase-producing Enterobacter hormaechei

Leah W. Roberts, Patrick N. A. Harris, Brian M. Forde, Nouri L. Ben Zakour, Elizabeth Catchpoole, Mitchell Stanton-Cook, Minh-Duy Phan, Hanna E. Sidjabat, Haakon Bergh, Claire Heney, Jayde A. Gawthorne, Jeffrey Lipman, Anthony Allworth, Kok-Gan Chan, Teik Min Chong, Wai-Fong Yin, Mark A. Schembri, David L. Paterson, Scott A. Beatson

What is it about?

We applied multiple genomic technologies to help inform infection control responses to a superbug outbreak at a Brisbane hospital. The cause of the outbreak was a type of multidrug resistant bacteria known as CPE that is resistant to most commonly prescribed antibiotics. CPE do not usually cause problems for healthy people, but for critically-ill patients an infection with CPE can leave doctors with very limited options for treatment. This article shares our experience over three years characterising and monitoring the outbreak using a range of genomic approaches.

Why is it important?

In 2015, integrating bacterial genomics during a hospital outbreak was still a rarity. As a microbial genomics group we were familiar with applying the latest DNA sequencing technologies to examine how bacteria evolved and caused disease, but we had relatively little experience in applying this knowledge in the clinic. In fact, this was our first attempt to communicate genomic analyses to clinical colleagues while an outbreak was still in progress.

Most readers will not have heard of Enterobacter hormaechei before. The type we characterised during the initial outbreak is a superbug resistant to most antibiotics, including carbapenems, hence the CPE moniker (carbapenem-producing Enterobacteriaceae). Hospital staff were keen to use every means necessary to characterise the outbreak bacterial strain and prevent the outbreak from getting worse. Our paper offers a candid account of this process from first recognition of the CPE outbreak in 2015 to identification of a suspected source within the hospital plumbing nearly three years later.

During the outbreak we integrated multiple genomic approaches (using Illumina, PacBio and Nanopore technologies) to address clinical questions and challenges as they arose. These included: 

-linking new CPE patient isolates to an historical isolate from the same ward two years prior, confirming a hospital source for the outbreak; 

-identifying the context of drug resistance genes on a broad-host range plasmid using PacBio long-read sequencing; 

-discovering the same plasmid is carried by unrelated bacteria from different hospitals in Queensland (and some other parts of Australia and Asia); 

-ruling out transmission of the original outbreak strain to a patient in a different ward using a rapid Nanopore sequencing;

-identifying the original outbreak strain in water samples using metagenomics, implicating the hospital plumbing as an unexpected reservoir. 

Some of these methods are now considered routine. On the other hand, integrating these technologies altogether in a single outbreak investigation is uncommon. Ultimately we hope that presenting the entire investigation together with a ‘warts-and-all’ account of our experience integrating each genomic approach will be of general interest to anyone now implementing genomics in the clinical setting.

This work in perspective:

At the heart of our story is the transformation of bacterial genomics from an academic pursuit to a powerful investigative tool for the healthcare professional. Collaborations between our microbial genomics group at The University of Queensland (UQ) and David Paterson’s clinical microbiology group at the UQ Centre for Clinical Research provided the means to translate our basic knowledge of genome sequencing and analysis direct to healthcare professionals. First author Leah Roberts carried out much of this work as a PhD student, often working alongside her clinical counterpart in the Paterson lab, Patrick Harris (UQCCR). Credit is due to Leah not just for driving the genomic analyses reported in our Nature Communications paper, but for her success bridging the gap between basic genomic science and applied clinical science. Following the award of her PhD last year, this is a theme she now continues in her role as an EBPOD Post-doctoral Fellow at EMBL-EBI in Cambridge, United Kingdom. 

The interdisciplinary collaborations between clinical scientists, genome scientists, molecular microbiologists, and healthcare professionals that developed during our study proved vital in recent efforts to establish bacterial genomics in the Queensland healthcare system. With the aim of transitioning to a State-wide service in 2021, we are now piloting regular genomic reporting about multidrug resistant bacteria to Queensland hospitals. This State-funded Queensland Genomics project draws on many of our experiences first developed during the CPE outbreak reported here. 

Our paper outlines a number of scenarios where genomic reporting appeared to have the potential to positively impact patient outcomes (such as preventing onward transmission of the outbreak strain to other patients) or economic outcomes (such as avoiding unnecessary quarantine procedures).  Speculation aside, this single study alone is insufficient to support evidence-based medicine in this area. There is an urgent need for more cross-disciplinary studies, including clinical trials and economic modelling, to measure the true impact of introducing bacterial genomics into the infection control workflow of healthcare facilities.

Further resources:

CPE/CRE factsheets have been developed by several authorities including one for families by Children’s Health Queensland here, and another by the Centers for Disease Control and Prevention here.

Clinical questions related to the outbreak should be directed to Patrick Harris (co-corresponding author) or David Paterson, both at UQCCR.

I am happy to answer any other questions about this work (s.beatson@uq.edu.au). For further updates including links to new commentaries about our work aimed at the general public or other scientists please follow @beatsonlab on twitter.

Lineage-Specific Methyltransferases Define the Methylome of the Globally Disseminated Escherichia coli ST131 Clone


Escherichia coli sequence type 131 (ST131) is a clone of uropathogenic E. coli that has emerged rapidly and disseminated globally in both clinical and community settings. Members of the ST131 lineage from across the globe have been comprehensively characterized in terms of antibiotic resistance, virulence potential, and pathogenicity, but to date nothing is known about the methylome of these important human pathogens. Here we used single-molecule real-time (SMRT) PacBio sequencing to determine the methylome of E. coli EC958, the most-well-characterized completely sequenced ST131 strain. Our analysis of 52,081 methylated adenines in the genome of EC958 discovered three m6A methylation motifs that have not been described previously. Subsequent SMRT sequencing of isogenic knockout mutants identified the two type I methyltransferases (MTases) and one type IIG MTase responsible for m6A methylation of novel recognition sites. Although both type I sites were rare, the type IIG sites accounted for more than 12% of all methylated adenines in EC958. Analysis of the distribution of MTase genes across 95 ST131 genomes revealed their prevalence is highly conserved within the ST131 lineage, with most variation due to the presence or absence of mobile genetic elements on which individual MTase genes are located.

Forde BM, Phan MD, Gawthorne JA, Ashcroft MM, Stanton-Cook M, Sarkar S, Peters KM, Chan KG, Chong TM, Yin WF, Upton M, Schembri MA, Beatson SA

Read the full publication here.

Transfer of scarlet fever-associated elements into the group A Streptococcus M1T1 clone


The group A Streptococcus (GAS) M1T1 clone emerged in the 1980s as a leading cause of epidemic invasive infections worldwide, including necrotizing fasciitis and toxic shock syndrome. Horizontal transfer of mobile genetic elements has played a central role in the evolution of the M1T1 clone, with bacteriophage-encoded determinants DNase Sda1 and superantigen SpeA2 contributing to enhanced virulence and colonization respectively. Outbreaks of scarlet fever in Hong Kong and China in 2011, caused primarily by emm12 GAS, led to our investigation of the next most common cause of scarlet fever, emm1 GAS. Genomic analysis of 18 emm1 isolates from Hong Kong and 16 emm1 isolates from mainland China revealed the presence of mobile genetic elements associated with the expansion of emm12 scarlet fever clones in the M1T1 genomic background. These mobile genetic elements confer expression of superantigens SSA and SpeC, and resistance to tetracycline, erythromycin and clindamycin. Horizontal transfer of mobile DNA conferring multi-drug resistance and expression of a new superantigen repertoire in the M1T1 clone should trigger heightened public health awareness for the global dissemination of these genetic elements.

Ben Zakour NL, Davies MR, You Y, Chen JH, Forde BM, Stanton-Cook M, Yang R, Cui Y, Barnett TC, Venturini C, Ong CL, Tse H, Dougan G, Zhang J, Yuen KY, Beatson SA, Walker MJ

Read the full publication here.

Hospital-Wide Eradication of a Nosocomial Legionella pneumophila Serogroup 1 Outbreak

Background. Two proven nosocomial cases of Legionella pneumonia occurred at the Wesley Hospital (Brisbane, Australia) in May 2013. To trace the epidemiology of these cases, whole genome sequence analysis was performed on Legionella pneumophila isolates from the infected patients, prospective isolates collected from the hospital water distribution system (WDS), and retrospective patient isolates available from the Wesley Hospital and other local hospitals.

Results. The 2011 and 2013 L. pneumophila patient isolates were serogroup 1 and closely related to all 2013 hospital water isolates based on single nucleotide polymorphisms and mobile genetic element profiles, suggesting a single L. pneumophila population as the source of nosocomial infection. The L. pneumophila population has evolved to comprise 3 clonal variants, each associated with different parts of the hospital WDS.

Conclusions. This study provides an exemplar for the use of clinical and genomic epidemiological methods together with a program of rapid, effective remedial biofilm, plumbing and water treatment to characterize and eliminate a L. pneumophila population responsible for nosocomial infections.

Bartley PB, Ben Zakour NL, Stanton-Cook M, Muguli R, Prado L, Garnys V, Taylor K, Barnett TC, Pinna G, Robson J, Paterson DL, Walker MJ, Schembri MA, Beatson SA. Clin Infect Dis. 2015 Oct 13. pii: civ870.

See the full publication here.

Global Dissemination of a multidrug resistant Escherichia coli clone

What is the study about?

We examined the DNA of a particular type of multidrug resistant E. coli called ST131. This type of bacteria was relatively unheard of until about 5 years ago but is now one of the most common causes of urinary tract and bloodstream infections. We were able to track the global emergence of ST131 by analysing a collection of isolates from all over the world. We found that they all evolved from a common ancestor quite recently and were resistant to multiple antibiotics.

Why is it important?

Urinary tract infections affect more than 150 million people around the world every year and about 50% of all women experience a urinary tract infection in their lifetime. ST131 are resistant to most antibiotics commonly used to treat urinary tract infections so it is important to be able to identify this type of E. coli early and treat the infection with an antibiotic that will work. Carbapenems are one of the few remaining types of antibiotics that are effective in treating ST131 but resistance to this antibiotic is increasing.

What should we be worried about?

Carbapenem resistance is an emerging problem, as the gene responsible is able to be easily transferred between bacteria. In the last couple of years carbapenem resistance genes have been reported in a small number of ST131 bacteria. The concern is that this gene will spread throughout the ST131 population, which is already the most successful *E. coli* clone to date. Not only would this limit treatment options for ST131 even further, it would also increase the chances that these resistance genes would be passed to other types of pathogenic bacteria.

How does our study help?

Our study provides a framework for understanding how these bacteria have evolved and spread around the globe. The genetic data that we produced can be used to develop new screening methods to ensure that ST131 are identified early in patients so that the best antibiotic treatment can be used. Ongoing surveillance of ST131 will also be important for tracking increased antibiotic resistance in this type of E. coli. In the longer term this work gives us the opportunity to develop new vaccines or therapies for preventing ST131. The fact that all ST131 are descended from a single ancestor means they may share an
Achilles’ heel that we can target.

How did we do the study?

We collected ST131 isolates from around the world from the year 2000 to 2011. We used “next generation” DNA sequencing to determine the genome of each isolate – the genome is the collection of all genes in an organism. Each E. coli has about 5000 genes, so we studied about half a million genes using bioinformatics, a discipline that merges biology and computing. The software that we developed to do this analysis and the data that we produced are now freely available to the rest of the scientific community.

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