Crack the code - whole-genome sequencing

28 May 2015



Healthcare-acquired infections (HAIs) are a critical public health threat, but tools to identify the genetic makeup of perpetrating microorganisms might soon prove their worth in controlling outbreaks. Beryl Oppenheim, consultant microbiologist at Queen Elizabeth Hospital in Birmingham, talks to Natalie Healey about her team’s pioneering use of whole-genome sequencing to stop multidrug-resistant pathogens.


Back in 2011, at Queen Elizabeth Hospital in Birmingham, UK, a stubborn germ was proving impossible to quash. The bug persisted for several months, while the route of the outbreak remained undetected.

The perpetrating pathogen was Acinetobacter baumannii, a rod-shaped bacterium that gained notoriety during the Iraq War when it emerged in military treatment facilities. Then a big problem for veterans and soldiers, it soon spread to civilian hospitals as infected cadets were transported to mainstream wards. Affecting severely ill patients, particularly those with trauma and burns, it can trigger pneumonia and bloodstream infections.

Multidrug-resistant strains of A baumannii have been reported worldwide and are a common cause of hospital outbreaks because it is difficult to identify the routes of cross-infection quickly and accurately enough to eliminate it. Healthcare-acquired infections (HAIs) such as these are estimated to cost the UK £1 billion a year, affecting one in 15 patients at any given time.

"Acinetobacter is a problem organism because it's resistant to so many antibiotics," explains Queen Elizabeth's consultant microbiologist Beryl Oppenheim from the Surgical Reconstruction and Microbiology Research Centre, where she advises on infections in people with trauma or burns (which make people susceptible to Acinetobacter).

She explains that one reason for the pathogen's hardiness is its penchant for living on surfaces in biofilms - densely packed communities of microbial cells that surround themselves with secreted polymers. The configuration and chemicals provide armour against antimicrobial cleaning agents, which would usually be the first line of action. "You may think you're cleaning it, but it's really very difficult to get rid of," Oppenheim stresses.

"The outbreak [at Queen Elizabeth Hospital] didn’t follow the usual patterns because it wasn’t restricted to patients on the same ward. We had to think about other places they may have picked it up."

She explains that the outbreak at Queen Elizabeth started following the admittance of a military patient from Afghanistan who had blast industries. The soldier was carrying a novel strain of the bacterium that had not previously been observed in the region's hospitals, and which was resistant to multiple classes of antibiotic.

The infection-control team first sent a sample of the bug to the national reference library, which used pulse-field gel electrophoresis to generate a DNA fingerprint. It was numbered 27, marking the 27th time the hospital had sent over a new strain of A baumannii.

In the following weeks, the hospital saw increasing cases of isolates from the same strain. Every time the group thought the pathogen had been eliminated, more patients were struck down with number 27. A total of 51 people on different wards were affected. Oppenheim was stumped.

The outbreak carried on and on, and it seemed transmission was ongoing. It didn't follow the usual patterns because it wasn't restricted to patients on the same ward. We had to think about other places they may have picked it up, and of course, they might not have been there at the same time," she points out.

Forty weeks after the emergence, and following much head-scratching and little joy, Oppenheim and her team decided to try a novel technique for pinpointing the pathogen's hot spots. Collaborating with the University of Birmingham, which had access to the latest technology, the researchers trialled whole-genome sequencing - a technique tipped to revolutionise outbreak surveillance and investigation.

"With conventional tools, we would just culture a bacterium and know its name. Then, there are other molecular techniques where you can chop up the DNA of the bug and do some typing. But all of these things are going to take time, so when you're faced with a number of people appearing to have the same infection in a hospital, you have a lot of problems to deal with," says Oppenheim.

To solve these challenges, the team needed to trace how the bacterium had spread through the wards by working out how closely related each sample was to the next. Deciding which pathogens were part of a cluster and which were a coincidence was vital for the team to work out how the bug was infecting others.

"We often think along very simple routes - for example by establishing that two patients were on the same ward - but it's not always that easy," says Oppenheim.

Luckily, whole-genome sequencing can help in these situations by allowing researchers to rapidly read the genetic code of an organism down to the last nucleotide (adenine, thymine, guanine and cytosine) of the investigational species' genetic code.

"You culture the bacterium, then break it up to extract the DNA, and you get a list of all the nucleotides in all their orders," explains Oppenheim. "There are hundreds and thousands of them, so you need complex computer skills to analyse the different bacteria."

What the researchers end up with is a tree that shows how genetically similar each bacterium is. "It might show that two are absolutely identical or have one or two single nucleotide polymorphisms. Some could be hundreds or thousands apart and you can say: 'Well, these patients aren't in any way related.' It does always have its surprises. You always start with a hypothesis, but it might not be right," Oppenheim says.

In this case, the whole-genome sequencing information was paired with patient records that had catalogued which rooms they'd occupied and which equipment was used on them. The combination of data was the key to solving the mystery, with the results pointing to a few transmission spots in the hospital. These included an operating theatre and a specialised bed used for burns patients. Deep cleaning of these sites followed and new decontamination protocols were adopted. In May 2013, 80 weeks after it started, the outbreak was declared over.

"When sequencing technology first became available, the sequences were huge. They would take up a vast amount of space and it would cost thousands to do just one."

"It was getting the sequence data that confirmed our hypothesis that was vital. If you're going to close down an area or do some complex cleaning, it's good to know you've got your facts right. You don't want to take unnecessary control measures; you want to hone in on exactly what will prevent transmission," says Oppenheim.

She believes genetic sequencing will be essential for controlling infection outbreaks in the future. It's really a question of the equipment becoming more accessible.

"When sequencing technology first became available, the sequences were huge. They would take up a vast amount of space and it would cost thousands to do just one," she reveals.

Now, she and her team use a bench-based Illumina Miseq, which costs about £50 to do a thorough analysis of a bacterium. Technology is getting even more intricate, though, with sequencers emerging on the market that can fit in your hand. It means such research could be done anywhere and can benefit more than incidents related to A baumannii.

"People have looked at MRSA, which is obviously something the public worries about a lot. Clostridium difficile is another one," Oppenheim adds.

She was also part of another whole-genome sequencing effort to find the cause of an outbreak of Pseudomonas aeruginosa, which can be found in hospital water supplies and is particularly problematic for cystic fibrosis patients. When an outbreak of the similarly biofilm-loving bacterium occurred following the opening of the Queen Elizabeth burns ward, the source turned out to be the plumbing on a valve that mixed hot and cold water, which created the optimal environment for the growth of many bacteria.

Additionally, Oppenheim has worked on multiple drug-resistant enterococci, which is a problem bug that develops in liver-transplant settings. She believes use of whole-genome sequencing will be widespread in the next five to ten years, when the technology should be automated. There are currently only a few experts capable of conducting similar research, so much development needs to be done before every lab is doing it.

And, of course, for modern health services to take something like this on in a big way, an economic evaluation will be required. whole-genome sequencing will be relatively expensive, so it needs to prove its worth.

"I really believe the fine level of detail you get with whole-genome sequencing will give us a lot of information that will help us to understand the germs and act fast," Oppenheim concludes. "It all comes down to speed - if you want to stop transmission, you have to do it quickly."

Dr Beryl Oppenheim is consultant microbiologist at Queen Elizabeth Hospital, Birmingham. She is honorary secretary to the British Infection Association, assistant editor of the Journal of Hospital Infection and sits on a number of expert groups.


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