The modern rise of an ancient disease

Historical figures who are believed to have had syphilis

Syphilis is an ancient disease. It is believed to have first swept through Europe in a pandemic in the 15^th Century, though it likely existed well before that too. Historical figures including Henry VIII, Genghis Khan, Al Capone and Schubert are suspected to have had it. Following the widespread availability of antibiotics from the 1940s, syphilis had all but disappeared in the UK by the 1980s – but now it’s back.

Since 2000, rates of syphilis infection have been skyrocketing in the UK, Europe and the USA, along with other sexually transmitted diseases. Its rise is thought to be down to changing sexual behaviours. Untreated, syphilis can cause brain damage and eventually death.

Syphilis is caused by the spiral-shaped bacterium Treponema pallidum. Since its discovery in 1905it has been impossible to grow the bacteria in a laboratory, hampering efforts to study it. Only in the last year have scientists found a way to grow it outside the body, using a specially designed sustenance and a carefully controlled environment[1]. And a technique developed in 2016 to purify its DNA[2] directly from patient swabs has enabled Sanger scientists to study its genome.

Their findings shed light on the evolution of this ancient bacteria and the rise of antibiotic resistance, and have ramifications for the treatment of syphilis and related conditions today.

Spiral-shaped Treponema pallidum bacteria seen with a scanning electron microscope

Sequencing syphilis

Dr Mathew Beale, a postdoctoral researcher at the Sanger Institute, led recent explorations into the genomics of T. pallidum. He spoke to us about the difficulty of working with the bacteria.

“Because it wasn’t possible to grow T. pallidum in the lab, all of our samples for DNA sequencing were previously taken directly from patient swabs. But over 95% of the DNA in those samples is going to be human DNA, not bacterial; the DNA we’re after is never present in high amounts.”

He described the new method for purifying the bacterial DNA as a bit like fishing. RNA, a molecule similar to DNA, is used as ‘bait’. RNA molecules are specially designed to stick to the DNA sequences of interest, in this case from T. pallidum. The RNA is also stuck to microscopic magnetic beads and so once it has stuck to its target DNA, it can be drawn out.

“My previous research group at UCL had pioneered the DNA purification technique for a number of viruses; I was very familiar with the technology. When my new group leader at the Sanger Institute, Professor Nick Thomson, suggested I look into projects on syphilis the potential was clear: we would refine the sequencing method, and with the help of an enthusiastic group of clinical and scientific collaborators, start to unravel the complex evolutionary relationships between the bacterium and human host.”

“Our early research with a small number of samples collected by Dr Michael Marks gave us hints on how to alter the parameters to improve yield and efficiency of the sequencing. We established thresholds for the minimum amount of bacterial DNA we needed to piece together a genome.

“In September 2017, with some trepidation, I sent our largest batch of DNA for sequencing yet – over 60 samples from close collaborators Professor Sheila Lukehart and Dr Christina Marra at the University of Washington in Seattle. Would the thresholds we had established be appropriate, or was this a colossal waste of effort and resource? By early January 2018 I had an answer. The sequencing had worked spectacularly well, with 62 of 64 samples

1930s syphilis awareness health campaign in America

Evolution

The team sequenced a total of 73 bacterial genomes from the UK and USA, collected over the last 16 years. They analysed these together with 49 previously published sequences.

Because DNA changes occur at a known and predictable rate over time, the ancestral relationships between different sequences can be established. The team were able to construct a more detailed evolutionary tree of T. pallidum than ever before, mapping how it has evolved.

T. pallidum was already known to have diverged into two ‘lineages’ known as SS14 and Nichols. They found Nichols was more common in the UK than previous research would suggest, whereas SS14 was dominant in the USA. The team found that SS14 can be divided even further into ‘sub-lineages’.

“This was interesting enough from a genetics perspective, but is pretty dry. The really interesting finding came when I decided to look at the emergence of antibiotic resistance,” said Mathew.

Antibiotic resistance

Previous work had shown that a very large proportion of T. pallidum was resistant to macrolides, a class of antibiotics. These broad acting antibiotics are commonly used to treat other STIs like gonorrhoea and chlamydia.

“One possibility was that the increase in syphilis rates in many countries was driven by macrolide resistance. Perhaps use of macrolides meant that resistant strains were able to survive and flourish.

“I wanted to see if the genetic indicator of macrolide resistance in our sequences had increased over time; in our genomic data it did, but this was not the most surprising finding. We found that each sub-lineage actually acquired the same resistance independently of one another. At the same time, we found several sub-lineages that were treatable with the drugs. The two types, resistant and non-resistant syphilis, co-exist – if there was constant pressure from macrolides, we would expect to only see the resistant bacteria,” Mathew said.

To uncover what was going on, they used mathematical modelling to rebuild the ancestral origins of the different sub-lineages.

“All of the sub-lineages we studied had their origins between the late 1980s and the very early 2000s… Something happened around the 1990s that caused the simultaneous selection of macrolide-resistant strains and

HIV

During the 1990s, before the development of highly active antiretroviral therapy to treat HIV, many people with it were given large doses of antibiotics to prevent the possibility of life-threatening infections. In many cases people took these antibiotics, including macrolides, for years.

There was also an increase in safe-sex practises and a reduction in STI transmission in many countries. This would have resulted in less syphilis in circulation and a bottleneck in the species.

“These two factors together could have been enough to generate the macrolide-resistant lineages we see today. With the introduction of effective HIV treatment, persistent antibiotic treatment became unnecessary in the early 2000s. The fact that not all sub-lineages in our analysis are resistant to macrolides suggests that the selection pressure has been reduced, and this, combined with changing sexual behavioural practices may explain the changing patterns of resistance we observe.”

Yaws

The team’s research is relevant for other diseases too. A very close relative of Treponema pallidum (a subspecies called Treponema pallidum pertenue) causes a disease called Yaws. Yaws is described as a tropical infection of the skin, bones and joints. It can cause permanent physical damage. Infected children can suffer facial disfigurement and be stigmatised by their communities.

The World Health Organisation (WHO) is running an eradication programme – treatment is with a single dose of a macrolide antibiotic. “Our findings are relevant for the treatment of yaws. There is a real possibility that yaws-causing bacteria can become resistant to macrolides.”

Syphilis Today

Syphilis is effectively treated with benzathine benzylpenicillin (a form of penicillin), not macrolides. At the moment, the bacteria do not show any signs of becoming resistant to penicillin. However, this specific form of penicillin used is only manufactured in three places in the entire world, and there are often shortages.

Mathew explained why: “There is declining demand for producing this drug because many other bacteria, including Neisseria gonorrhoea and Staphylococcus aureus, are often resistant to it. So it’s becoming uneconomical to make it. Plus there seems to be a push for ‘new’ drugs; it’s seen as old.”

Shortages of benzathine benzylpenicillin result in suboptimal treatment for syphilis. As an alternative, macrolides are sometimes prescribed, despite the bacteria’s resistance to them. This, in turn, results in increased spread of syphilis.

“What’s worrying is there is no going back. Once this bacteria has evolved resistance to macrolides, it appears to stay resistant,” Mathew said.

While another pandemic on the scale of the one in Renaissance Europe is unlikely, the increasing rates, combined with the threat of antibiotic resistance, mean syphilis continues to be a problem across the globe. Recent public health campaigns have focused on raising awareness and encouraging people at risk to get tested. Genomic analysis of the bacteria which cause the disease is vital too. The Sanger Institute team plan to continue genomic sequencing in the UK and beyond – aiding research into syphilis, its treatment and the wider issues of antibiotic resistance.

Modern-day syphilis awareness campaign in Canada