• An international collaboration has uncovered the first strain of an ongoing process of evolution, which may be spawning more dangerous strains over time
• We need alternatives to antibiotics to control the spread of these infections

In 2007, an international collaboration, led by Octavie Lunguya in the Democratic Republic of the Congo (DRC) and Jan Jacobs in Belgium, led to the establishment of a surveillance program to monitor bloodstream infections in the DRC. This year, that program has proven critical in capturing the early evolution of a dangerous new agent of disease.

Across Africa, Salmonella is the most common non-malarial cause of bloodstream infections. Around two thirds of cases are caused by “non-typhoidal Salmonella (NTS)”, types of Salmonella that usually cause food poisoning. But Salmonella is evolving, and some strains are capable of causing more than food poisoning.

Globally, non-typhoidal Salmonella bloodstream infections affect 3.4 million people, and cause 681,316 deaths annually. It’s one of only two causes of child mortality (along with AIDS) that result in a higher rate of death today than in 1990.

The DRC is a resource-poor nation that spans 2.3 million square kilometres. It is a challenging environment for providing healthcare. Patients pay for their own care, and it’s estimated that only 30 per cent of people have access to a regular healthcare system, while 40 per cent self-medicate. Access to antibiotics in the DRC is limited by both price and method of administering the treatment. Diagnosing bloodstream infections is time-consuming, costly and challenging, but crucial for designing strategies to reduce the rates of these infections and deciding how to treat them effectively.

As part of my work here at the Sanger Institute, I was able to help characterise the samples that had been collected during the surveillance study in the DRC. In this work, we identified the world’s first extensively drug resistant strain of invasive non-typhoidal Salmonella (iNTS).

The study began with the setup of a program for surveillance of bloodstream infections in the DRC. Blood samples from people infected with salmonella that had concerning levels of antibiotic resistance were collected by participating hospitals and health centres. The samples were sent to the Institut National de Recherche Biomédicale (INRB) in Kinshasa, DRC, which coordinated the project. Then they were shipped to the Institute of Tropical Medicine (ITM) in Antwerp, Belgium, where they were studied in the lab, and some were sent here to the Sanger Institute for further study.

The major cause of iNTS infections is a strain of Salmonella Typhimurium called ST313. Through DNA sequencing, we found a new strain of salmonella ST313 and named it lineage II.1. It made up 10 per cent of the samples that were collected in the project. Using the accumulation of DNA changes over time as a way to trace the history of this strain, we can estimate that lineage II.1 emerged in 2004, and started to take off around 2012.

Levels of antibiotic resistance appear to have increased over time as this lineage has evolved. Currently the recommended treatment for iNTS is one of two remaining drugs, ciprofloxacin or ceftriaxone. But, lineage II.1 is now resistant to ceftriaxone, making this the first “extensively drug resistant” iNTS. One strain of the 51 samples of lineage II.1 we looked at also showed an increased tolerance of ciprofloxacin, the last available drug in the DRC to treat these infections. If this resistance continues to rise, these infections may no longer be treatable in the DRC.

iNTS is Salmonella that doesn’t cause typhoid, but invades its human host – it moves from the gut into the bloodstream. It has been around for a long time, but the growing problem of these invasive infections has only gained recognition in the last decade. The disease usually affects children under five suffering from malnutrition and malaria, and adults with HIV.

Since the emergence of AIDS in the 1980s, a change has been occurring in Salmonella, a common stomach bug, and the mortality rate associated with infections has increased. It’s thought that HIV, which has a profound impact on the way the human body interacts with and responds to bacteria, on top of malnutrition and malaria in sub-Saharan Africa, has created a unique niche^[1]. It is a niche in which bacteria that would normally be met with an intense immune response can now stay in the human body for longer and become better adapted to infecting it.

These bacteria have been able to take advantage of a young, immunocompromised population for a long time, so why the sudden rise in disease now? The timing and pattern of movement of HIV and iNTS suggest that invasive salmonella may be following the spread of vulnerable populations across Africa. It’s thought that the rise of the HIV epidemic likely drove the spread and success of these invasive strains through an adult population that is internationally mobile^[2].

Antibiotic-resistant Salmonella Typhimurium ST313 strains have caused several epidemics in sub-Saharan Africa, and have required the poorest health services in the world to turn to more expensive antibiotics for treatment.

ST313 is thought to be moving along a continuum of infection styles seen in Salmonella, changing from a strain that can infect a broad range of hosts and cause relatively mild disease to one that’s specialised to a smaller range of hosts but can cause more life-threatening illness. We looked closer at the DNA of this strain, to see how genetically different it was from related samples we’d seen in the past. It had accumulated genetic changes that we’ve also seen in Salmonella Typhi (which causes typhoid) and other invasive salmonella, which make them different from Salmonella that cause mild stomach upset.

For the first time in a real outbreak scenario, I got the opportunity to test a machine learning model I’ve been developing for the last four years. We used the algorithm to look for characteristic patterns of change in the DNA of salmonella that cause invasive infections. The algorithm outputs an “invasiveness index”, which gives an indication of whether strains are shifting along this continuum over time.

We see an upwards shift in invasiveness index with each new wave of ST313, mirrored by a shift in the way these bacteria behave in the lab. An example is the strains’ ability to form biofilms — complex communities of bacteria that are better at surviving tough conditions like those faced when salmonella transmit via the environment. As you can see in the image above, the appearance of these communities of bacteria appears to be changing with each successful new lineage, to look more like the bacteria that cause typhoid and less like their close relatives that cause food poisoning.

While not conclusive, these parallel lines of evidence suggest that the bacteria are behaving more like invasive strains.

Because these bacteria look like they’re adapting to an invasive lifestyle, and are slowly gaining resistance to more antibiotics, preventing infections is crucial. There are long-term interventions we can take to make infections less likely, but there are also solutions available now that can have an impact.

A better understanding of transmission

We still don’t really understand how this disease spreads. Knowing this would help us prioritise interventions such as improving sanitation and nutrition, developing vaccines, or controlling risk factors by reducing rates of malaria infections and administering antiretrovirals to treat HIV.

Better diagnosis of infections

The symptoms of invasive Salmonella infection can indicate a range of diseases. Diagnosing these infections is time-consuming, expensive, and needs the right equipment. Local partners in this surveillance study discovered this strain from limited sampling in the DRC. We have no idea what’s going on in the rest of the DRC, and won’t know unless better infrastructure is in place to make these diagnoses. It’s also possible the disease may currently be underestimated in other areas of the world like Southeast Asia and Latin America, where there is less reporting of the causes of bloodstream infections.

Identifying and tracking dangerous new strains

Identifying these new invasive strains can be tricky. It’s hard to tell how invasive a strain is, especially in humans. The ability of a machine learning algorithm to flag newer strains of ST313 as more invasive gives us a promising indication that it could be used to identify other dangerous new strains as they appear.

We’ve seen major changes occurring in these bacteria on a timescale of decades, that have allowed them to become more competitive and dangerous. Being able to recognise a new strain as it appears means we can mount a response to contain its spread. It also helps us to understand and target the upstream factors that lead to their appearance and success. DNA sequencing is becoming a cheaper, more viable option for public health. Our work illustrates the insight we gain into a complex disease like bloodstream infections by forming collaborative networks and examining the DNA of the agents causing disease.

To see more about what we do, visit our website; to learn more about what we’re doing to track the global spread of antibiotic resistance, see the Global Health Research Unit.