Scrutinizing an emerging drug resistant Salmonella in Malawi

Benjamin Kumwenda is a biomedical science researcher and AESA-RISE Postdoctoral Fellow based at College of Medicine, University of Malawi. The AESA-RISE Postdoctoral Fellowship is an African Academy of Sciences (AAS) Programme that builds on the foundation of the Regional Initiative in Science and Education (RISE), which has for a decade prepared PhD and masters level scientists and engineers in Africa through competitively selected, university-based research and teaching networks, to respond to an urgent need to increase the number of researchers in Africa. AESA RISE is implemented through AESA (The Alliance for Accelerating Excellence in Science in Africa), a funding, agenda setting and programme implementing platform of the AAS in partnership with the African Union Development Agency (AUDA-NEPAD). AESA RISE is supported by the Carnegie Corporation of New York (CCNY).

Background

Salmonella is a bacterium which is a major cause of diarrhoea and systemic infection worldwide. The two main types (serovars) of Salmonella which cause diarrhoea are Typhimurium and Enteritidis. Salmonella serovars are classified into sequence types (ST) which are further sub-grouped into lineages based on the differences in their genetic features. Salmonella Typhimurium which causes diarrhoea is known as Non-Typhoidal Salmonella (NTS). Typhoidal Salmonella is responsible for typhoid fever. In Europe and other developed countries, NTS and Enteritidis cause diarrhoea which is usually self-healing but it is responsible for over 155,000 deaths worldwide annually.

In recent years, particularly in Africa, NTS has emerged as the most common bacterium isolated from the bloodstream of patients. In Africa, Salmonella bacteria enters human tissue, cells and body systems and cerebral spinal fluid (CSF), which are supposed to be free from microorganisms or any other foreign materials, by invading the lining of the small intestines. This is clinically presented as fever, a condition known as Salmonellosis. NTS that invades the human tissue and body systems is known as invasive Non-Typhoidal Salmonella (iNTS), which is the major cause of systemic infections. In Africa, iNTS which causes systemic infections mainly belongs to the sequence type 313 (ST313). The annual incidence of iNTS infections is 3.4 million worldwide and causes 388,000 deaths in Africa alone. Individuals who are HIV positive, infected with malaria, and children with severe anaemia and/or malnutrition are usually at high risk of being infected by iNTS.

The threat of iNTS disease has increased due to the rise of multi-drug resistance. This has required the use of more expensive antibiotics in some of the poorest health systems in the world, which has increased treatment costs due to limited and expensive therapeutic options. This work is a collective effort to improve diagnosis of Salmonella in order to inform clinical practice to reduce morbidity and mortality, as well as costs, due to Salmonella infection in Africa.

Description of the study

In 2010, at Queen Elizabeth Central Hospital (QECH), the largest referral hospital in Blantyre, Malawi, a high rate of Salmonella infection was observed among patients. Msefula et al., (2012) conducted an initial investigation using 27 Salmonella isolates in which a new group of strains was demonstrated to be genetically distinct from the previously known group (lineage 2). There are two distinct and tightly clustered ST313 lineages that have been identified in sub-Saharan Africa according to the work of Okoro et al., (2012): ST313 lineages 1 and 2, emerged approximately 52 and 35 years ago, respectively. The use of antibiotics such as chloramphenicol led to the rise of lineage 2 from lineage 1, around 2002. While lineage 1 is treatable with most antibiotics, lineage 2 carries genes on a plasmid responsible for multi-drug resistance to frontline drugs, such as chloramphenicol, cotrimoxazole and ampicillin.

This new group of Salmonella strains which diverged from lineage 2 was designated lineage 2.2 in the study. (This was because there was another study conducted in the Democratic Republic of Congo by Van Puyvelde, et al., (2019) which identified a different group of Salmonella strains branching off lineage 2 and was designated lineage 2.1.) The DRC lineage 2.1 was more resistant to azithromycin and had altered phenotypic properties, including biofilm formation and metabolic capacity. In Blantyre, Malawi, however, it was noted that the lineage 2.2 was causing more infections than lineage 2 and other lineages among patients at QECH. In addition, both lineages 2 and 2.2 had similar multidrug resistance profile. The reasons for the emergence of lineage 2.2 had been unclear until this study. Lineage 2.2 was causing more disease among patients than lineage 2, thereby complicating treatment of HIV-positive patients and patients whose immunity was compromised. This work resulted in a clear understanding of the emergence and abundance of lineage 2.2 Salmonella strains in Blantyre, Malawi. 

Technological advancements have allowed molecular biologists to isolate the genetic material from bacteria and determine its sequence and composition. This has led to the discovery of the genetic features which distinguish Salmonella isolates which cause diarrhoea from those which cause systemic infections. The S. Typhimurium ST313, to which lineage 2, 2.1 and 2.2 belong, is responsible for two-thirds of iNTS cases in Africa. It has distinct genetic features which are unique to Africa.

The first phase of this project compared the genetic material of the two bacteria strains from lineage 2 and 2.2. It further compared the differences in expression of genes in different infection stages in the body. To study genes responsible for infection in different infection stages, both Salmonella strains were grown in 16 infection-relevant conditions from the time the Salmonella pathogen was eaten with contaminated food in the mouth, to the time it entered human tissue, cells or body systems. The infection conditions mimicked the different stages and environment through which food passes during digestion in the body. For example, while food is still in the stomach, there is still enough oxygen available to maintain the Salmonella, but when it enters the small intestines and body tissues, oxygen is deprived. Salmonella then has to find other energy sources using different genes to survive. Different conditions are created due to the discharge of enzymes for digestion and other chemicals which also alters levels of acidity.

The second part of this study collected and analysed over 600 Salmonella bacteria strains. The aim was to investigate whether the genetic features which were observed in the representative strains in lineage 2 and 2.2 were consistent across all strains in their respective groups. The aim was to determine whether the observed genetic differences were real among the 27 bacteria strains, and not random occurrences. We also wanted to establish whether lineage 2.2, which was causing more infections among patients, continued to increase among patients over subsequent years. Genetic features which could be established across all strains in a lineage will inform the design of a technique that could quickly detect the different lineages. Genetic features will also be used to determine whether a new lineage had emerged in the environment.

Outcome of the study

This study revealed differences in genetic features and expression (activation) of genes in different infection stages in the humans, which explains the increase in lineage 2.2 infections among patients in Blantyre, Malawi. The genetic materials of the two representative strains differed with 27 mutation (SNPs) which occurred in genes that are responsible for carrying out metabolism, production of energy, formation and maintenance of the cell membrane and transportation of nutrients in and out of the cell.

The study found that genes which code for the flagella which propel Salmonella in the stomach were surprisingly highly deactivated in lineage 2.2. Genes which Salmonella uses to invade the human cells, tissues and body systems were, also surprisingly, highly activated in a condition which is supposed to deactivate the genes (NonSPI2). These findings suggest that the new Salmonella lineage 2.2 bacteria have different ways of infecting human hosts with which the human immune system is not familiar. The bacteria appears to hide away from the host immune system by reducing its movement within the stomach. It activates genes which are responsible for invasion under a condition where the human body doesn't expect the genes to be active, thereby taking the immune system off guard. Bacteria movements in the small intestine activates the immune system, which allows the body to produce watery fluid resulting in diarrhoea to remove the bacteria.

Lessons Learned

There is need for continuous research on prevailing bacteria to determine genetic changes to inform clinical practice. Salmonella for example, quickly responds to changes in the environment due to changes in treatment, resulting in comorbidities and damaging the health status of the population. This forces bacteria pathogens to adapt to the environmental changes by adjusting its genetic makeup. Such pathogens can therefore not be treated by old clinical techniques: effective treatment requires rapid diagnosis of infections. This will allow treatment that targets specific pathogens for quick response and recovery, thereby reducing death as well as decreasing costs due to treatment with expensive antibiotics that are effective for that strain.

Impact

Analysis of genetic changes over time has shed light on the evolution of these dangerous Salmonella pathogens. The identification of lineage 2.2-specific gene markers is being used to develop a laboratory technique which will enable the efficient distinction among different lineages in clinical laboratories, as has been done for other human pathogens. This will allow identification of Salmonella lineages which are prevalent in different geographic regions. Comparing geographical areas to lineages will reveal the physical characteristics, socioeconomic and cultural factors which promote the emergence of the lineages which is  key in controlling transmission. The rapid diagnosis of Salmonella lineages will inform clinical practice, treatment plans, transmission control, antimicrobial stewardship and influence policy on iNTS in developing countries.

This work has served to alert the Malawi Ministry of Health of the potential to treat Salmonella iNTS more effectively through analysis of genetic changes observed in the strains. At the end of this study, concrete recommendations will be made on Salmonella transmission control and clinical practice.