A relevant research line in Infectious Diseases focuses the understanding of how pathogens evolve in response to the host immune responses and drug pressures. This relies on the advances of the Whole-genome sequencing (WGS) associated procedures, which now allow the direct capture of pathogen “fingerprint” during human infection. While this encompasses non-straightforward multi-step experiments, those insights become even more relevant for non-cultivable pathogens, for which scientific advances are naturally delayed. One striking example stands for the bacterium Treponema pallidum, the causative agent of syphilis, an old human disease. As the application of common microbiological techniques (e.g., genetic manipulation and antibiotic testing) is extremely limited, little is known about how T. pallidum mediates survival and virulence during syphilis.

Taking advantage of multiple clinical samples available at the Portuguese National Institute of Health, our microbial genomics group dared to implement a strategy to understand how the T. pallidum genome is shaped during syphilis. We were aware that this would be risky (obstacles were a priori known: putative low bacterial load within clinical specimens, pathogen fragility and high genome %GC content), as revealed by a long-lasting project marked by some unsuccessful attempts. We tried multiple experiment designs applying immunomagnetic pathogen enrichment (with pathogen- and host-specific monoclonal antibodies) and random multiple displacement amplification before WGS, but they revealed unbalanced genome coverage and lack of specificity. The success was finally reached after optimizing a cutting-edge strategy relying on selective enrichment of the target DNA through hybridization with RNA oligonucleotides, an approach that had never been applied before for non-cultivable bacterial pathogens. We were fortunate to fully recover and sequence multiple high quality T. pallidum genomes directly from clinical samples. Our findings, published in Nature Microbiology on 17^th October 2016 (http://www.nature.com/articles/nmicrobiol2016190), demonstrate that this extremely genomic conserved bacterium takes advantage of the hypermutability of very few and specific loci to generate multiple variants within the same patient.

While our results complement previous data derived from important studies performed using the rabbit model of infection, it also open avenues for studies aiming, not only at understanding the worldwide molecular epidemiology of syphilitic strains, but also at decoding important pathogen traits such as the invasion of the central nervous system and transplacental transmission.