British squirrels infected with leprosy With the exception of armadillos in the Americas, leprosy infections are considered almost exclusively restricted to humans. Avanzi et al. examined warty growths on the faces and extremities of red squirrels in the British Isles and found that two species of leprosy-causing organisms were to blame (see the Perspective by Stinear and Brosch). Mycobacterium leprae in the southern population of Brownsea Island squirrels originated from a medieval human strain. M. lepromatosis was found in red squirrels from elsewhere in the United Kingdom and Ireland. Human leprosy is proving hard to eradicate, despite available drugs. Perhaps other wildlife species are also reservoirs for this stubborn disease. Science , this issue p. 744 ; see also p. 702

Leprosy is a chronic human disease caused by the yet-uncultured pathogen Mycobacterium leprae. Although readily curable with multidrug therapy (MDT), over 200,000 new cases are still reported annually. Here, we obtain M. leprae genome sequences from DNA extracted directly from patients’ skin biopsies using a customized protocol. Comparative and phylogenetic analysis of 154 genomes from 25 countries provides insight into evolution and antimicrobial resistance, uncovering lineages and phylogeographic trends, with the most ancestral strains linked to the Far East. In addition to known MDT-resistance mutations, we detect other mutations associated with antibiotic resistance, and retrace a potential stepwise emergence of extensive drug resistance in the pre-MDT era. Some of the previously undescribed mutations occur in genes that are apparently subject to positive selection, and two of these (ribD, fadD9) are restricted to drug-resistant strains. Finally, nonsense mutations in the nth excision repair gene are associated with greater sequence diversity and drug resistance.

Studying ancient DNA allows us to retrace the evolutionary history of human pathogens, such as Mycobacterium leprae, the main causative agent of leprosy. Leprosy is one of the oldest recorded and most stigmatizing diseases in human history. The disease was prevalent in Europe until the 16th century and is still endemic in many countries with over 200,000 new cases reported annually. Previous worldwide studies on modern and European medieval M. leprae genomes revealed that they cluster into several distinct branches of which two were present in medieval Northwestern Europe. In this study, we analyzed 10 new medieval M. leprae genomes including the so far oldest M. leprae genome from one of the earliest known cases of leprosy in the United Kingdom—a skeleton from the Great Chesterford cemetery with a calibrated age of 415–545 C.E. This dataset provides a genetic time transect of M. leprae diversity in Europe over the past 1500 years. We find M. leprae strains from four distinct branches to be present in the Early Medieval Period, and strains from three different branches were detected within a single cemetery from the High Medieval Period. Altogether these findings suggest a higher genetic diversity of M. leprae strains in medieval Europe at various time points than previously assumed. The resulting more complex picture of the past phylogeography of leprosy in Europe impacts current phylogeographical models of M. leprae dissemination. It suggests alternative models for the past spread of leprosy such as a wide spread prevalence of strains from different branches in Eurasia already in Antiquity or maybe even an origin in Western Eurasia. Furthermore, these results highlight how studying ancient M. leprae strains improves understanding the history of leprosy worldwide.

Mycobacterium lepromatosis is an uncultured human pathogen associated with diffuse lepromatous leprosy and a reactional state known as Lucio's phenomenon. By using deep sequencing with and without DNA enrichment, we obtained the near-complete genome sequence of M. lepromatosis present in a skin biopsy from a Mexican patient, and compared it with that of Mycobacterium leprae, which has undergone extensive reductive evolution. The genomes display extensive synteny and are similar in size (∼3.27 Mb). Protein-coding genes share 93% nucleotide sequence identity, whereas pseudogenes are only 82% identical. The events that led to pseudogenization of 50% of the genome likely occurred before divergence from their most recent common ancestor (MRCA), and both M. lepromatosis and M. leprae have since accumulated new pseudogenes or acquired specific deletions. Functional comparisons suggest that M. lepromatosis has lost several enzymes required for amino acid synthesis whereas M. leprae has a defective heme pathway. M. lepromatosis has retained all functions required to infect the Schwann cells of the peripheral nervous system and therefore may also be neuropathogenic. A phylogeographic survey of 227 leprosy biopsies by differential PCR revealed that 221 contained M. leprae whereas only six, all from Mexico, harbored M. lepromatosis. Phylogenetic comparisons indicate that M. lepromatosis is closer than M. leprae to the MRCA, and a Bayesian dating analysis suggests that they diverged from their MRCA approximately 13.9 Mya. Thus, despite their ancient separation, the two leprosy bacilli are remarkably conserved and still cause similar pathologic conditions. Mycobacterium lepromatosis | genome sequencing | Mycobacterium leprae | comparative genomics | reductive evolution

Mycobacterium leprae (M. leprae) is a human pathogen and the causative agent for leprosy, a chronic disease characterized by lesions of the skin and peripheral nerve damage. Zoonotic transmission of M. leprae to humans by nine-banded armadillos (Dasypus novemcinctus) has been shown to occur in the southern United States, mainly in Texas, Louisiana, and Florida. Nine-banded armadillos are also common in South America, and residents living in some areas in Brazil hunt and kill armadillos as a dietary source of protein. This study examines the extent of M. leprae infection in wild armadillos and whether these New World mammals may be a natural reservoir for leprosy transmission in Brazil, similar to the situation in the southern states of the U.S. The presence of the M. leprae-specific repetitive sequence RLEP was detected by PCR amplification in purified DNA extracted from armadillo spleen and liver tissue samples. A positive RLEP signal was confirmed in 62% of the armadillos (10/16), indicating high rates of infection with M. leprae. Immunohistochemistry of sections of infected armadillo spleens revealed mycobacterial DNA and cell wall constituents in situ detected by SYBR Gold and auramine/rhodamine staining techniques, respectively. The M. leprae-specific antigen, phenolic glycolipid I (PGL-I) was detected in spleen sections using a rabbit polyclonal antibody specific for PGL-I. Anti-PGL-I titers were assessed by ELISA in sera from 146 inhabitants of Belterra, a hyperendemic city located in western Pará state in Brazil. A positive anti-PGL-I titer is a known biomarker for M. leprae infection in both humans and armadillos. Individuals who consumed armadillo meat most frequently (more than once per month) showed a significantly higher anti-PGL-I titer than those who did not eat or ate less frequently than once per month. Armadillos infected with M. leprae represent a potential environmental reservoir. Consequently, people who hunt, kill, or process or eat armadillo meat are at a higher risk for infection with M. leprae from these animals.

Background: Recent advances in sequencing have facilitated large-scale analyses of the metagenomic composition of different samples, including the environmental microbiome of air, water, and soil, as well as the microbiome of living humans and other animals. Analyses of the microbiome of ancient human samples may provide insights into human health and disease, as well as pathogen evolution, but the field is still in its very early stages and considered highly challenging. Results: The metagenomic and pathogen content of Egyptian mummified individuals from different time periods was investigated via genetic analysis of the microbial composition of various tissues. The analysis of the dental calculus' microbiome identified Red Complex bacteria, which are correlated with periodontal diseases. From bone and soft tissue, genomes of two ancient pathogens, a 2200-year-old Mycobacterium leprae strain and a 2000-yearold human hepatitis B virus, were successfully reconstructed. Conclusions: The results show the reliability of metagenomic studies on Egyptian mummified individuals and the potential to use them as a source for the extraction of ancient pathogen DNA.

BackgroundSince leprosy is both treated and controlled by multidrug therapy (MDT) it is important to monitor recurrent cases for drug resistance and to distinguish between relapse and reinfection as a means of assessing therapeutic efficacy. All three objectives can be reached with single nucleotide resolution using next generation sequencing and bioinformatics analysis of Mycobacterium leprae DNA present in human skin.MethodologyDNA was isolated by means of optimized extraction and enrichment methods from samples from three recurrent cases in leprosy patients participating in an open-label, randomized, controlled clinical trial of uniform MDT in Brazil (U-MDT/CT-BR). Genome-wide sequencing of M. leprae was performed and the resultant sequence assemblies analyzed in silico.Principal findingsIn all three cases, no mutations responsible for resistance to rifampicin, dapsone and ofloxacin were found, thus eliminating drug resistance as a possible cause of disease recurrence. However, sequence differences were detected between the strains from the first and second disease episodes in all three patients. In one case, clear evidence was obtained for reinfection with an unrelated strain whereas in the other two cases, relapse appeared more probable.Conclusions/SignificanceThis is the first report of using M. leprae whole genome sequencing to reveal that treated and cured leprosy patients who remain in endemic areas can be reinfected by another strain. Next generation sequencing can be applied reliably to M. leprae DNA extracted from biopsies to discriminate between cases of relapse and reinfection, thereby providing a powerful tool for evaluating different outcomes of therapeutic regimens and for following disease transmission.

Molecular drug susceptibility testing was performed on skin biopsies from 24 leprosy patients from Guinea-Conakry for the first time. We identified primary drug resistance in 4 cases and a dapsone-resistant cluster caused by the same strain. Primary transmission of drug-resistant Mycobacterium leprae, including a rifampicin-resistant strain, is reported.