Urban market gardening and rodent-borne pathogenic Leptospira in arid zones: a case study in Niamey, Niger

Leptospirosis essentially affects human following contact with rodent urine-contaminated water. As such, it was mainly found associated with rice culture, recreational activities and flooding. This is also the reason why it has mainly been investigated in temperate as well as warm and humid regions, while arid zones have been only very occasionally monitored for this disease. In particular, data for West African countries are extremely scarce. Here, we took advantage of an extensive survey of urban rodents in Niamey, Niger, in order to look for rodent-borne pathogenic[i] Leptospira[/i] species presence and distribution across the city. To do so, we used high throughput bacterial 16S-based metabarcoding, [i]lipL32[/i] gene-targeting RT-PCR, rrs gene sequencing and VNTR typing as well as GIS-based multivariate spatial analysis. Our results show that leptospires seem absent from the core city where usual [i]Leptospira[/i] reservoir rodent species (namely [i]R. rattus[/i] and [i]M. natalensis[/i]) are yet abundant. On the contrary, [i]L. kirschneri[/i] was detected in [i]Arvicanthis niloticus[/i] and [i]Cricetomys gambianus[/i], two rodent species that are restricted to irrigated cultures within the city. Moreover, the VNTR profiles showed that rodent-borne leptospires in Niamey belong to previously undescribed serovars. Altogether, our study points towards the importance of market gardening in maintain and circulation of leptospirosis within Sahelian cities. In Africa, irrigated urban agriculture constitutes a pivotal source of food supply, especially in the context of the ongoing extensive urbanization of the continent. With this in mind, we speculate that leptospirosis may represent a zoonotic disease of concern also in arid regions that would deserve to be more rigorously surveyed, especially in urban agricultural settings

Heme rescues a two-component system Leptospira biflexa mutant

<p>Abstract</p> <p>Background</p> <p>Heme is typically a major iron source for bacteria, but little is known about how bacteria of the <it>Leptospira </it>genus, composed of both saprophytic and pathogenic species, access heme.</p> <p>Results</p> <p>In this study, we analysed a two-component system of the saprophyte <it>Leptospira biflexa</it>. <it>In vitro </it>phosphorylation and site-directed mutagenesis assays showed that Hklep is a histidine kinase which, after autophosphorylation of a conserved histidine, transfers the phosphate to an essential aspartate of the response regulator Rrlep. Hklep/Rrlep two-component system mutants were generated in <it>L. biflexa</it>. The mutants could only grow in medium supplemented with hemin or δ-aminolevulinic acid (ALA). In the pathogen <it>L. interrogans</it>, the <it>hklep </it>and <it>rrlep </it>orthologous genes are located between <it>hemE </it>and <it>hemL </it>genes, which encode proteins involved in heme biosynthesis. The <it>L. biflexa hklep </it>mutant could be complemented with a replicative plasmid harbouring the <it>L. interrogans </it>orthologous gene, suggesting that these two-component systems are functionally similar. By real-time quantitative reverse transcription-PCR, we also observed that this two-component system might influence the expression of heme biosynthetic genes.</p> <p>Conclusion</p> <p>These findings demonstrate that the Hklep/Rrlep regulatory system is critical for the <it>in vitro </it>growth of <it>L. biflexa</it>, and suggest that this two-component system is involved in a complex mechanism that regulates the heme biosynthetic pathway.</p

A public database for the new MLST scheme for Treponema pallidum subsp. pallidum : surveillance and epidemiology of the causative agent of syphilis.

International audienceTreponema pallidum subsp. pallidum is the causative agent of syphilis, a sexually transmitted disease with worldwide prevalence. Several different molecular typing schemes are currently available for this pathogen. To enable population biology studies of the syphilis agent and for epidemiological surveillance at the global scale, a harmonized typing tool needs to be introduced. Recently, we published a new multi-locus sequence typing (MLST) with the potential to significantly enhance the epidemiological data in several aspects (e.g., distinguishing genetically different clades of syphilis, subtyping inside these clades, and finally, distinguishing different subspecies of non-cultivable pathogenic treponemes). In this short report, we introduce the PubMLST database for treponemal DNA data storage and for assignments of allelic profiles and sequencing types. Moreover, we have summarized epidemiological data of all treponemal strains (n = 358) with available DNA sequences in typing loci and found several association between genetic groups and characteristics of patients. This study proposes the establishment of a single MLST of T. p. pallidum and encourages researchers and public health communities to use this PubMLST database as a universal tool for molecular typing studies of the syphilis pathogen

One Health field approach applied to Leptospirosis: A systematic review and meta-analysis across humans, animals and the environment

Background: Leptospirosis is a neglected zoonosis transmitted through urine of infected hosts or contaminated environments. The transmission of bacteria between humans, animals, and the environment underscores the necessity of a One Health approach. Methods: We conducted a systematic review to identify significant findings and challenges in One Health research on leptospirosis, focusing on studies involving sampling in at least two of the three compartments: human, animal, and environment. We searched in PubMed, Web of Science, Medline, Scopus, and ScienceDirect from January 1, 1918 to December 31, 2022. We assessed risk of bias in studies using Joanna Briggs Institute (JBI) tools and performed a meta-analysis to identify links between One Health compartments. Results: Out of 1,082 leptospirosis studies with sampling, 102 multicompartmental studies conducted between 1972 and 2022 were included: 70 Human-Animal, 18 Animal-Environment, 4 Human-Environment, and 10 across all compartments. Various methodological weaknesses were identified, from study design to statistical analysis. Meta-regressions identified positive associations between human and animal seroprevalences, particularly with livestock and with wild non-rodent animals, and a link between the environmental positivity rate and domestic animal seroprevalence. Our analysis was constrained by the limited number of studies included and by the quality of protocols. Conclusions: This 50-year overview of One Health field approach to leptospirosis highlights the critical need for more robust, well-supported One Health research to clarify the transmission dynamics and identify risk factors of zoonoses

Gene silencing based on RNA-guided catalytically inactive Cas9 (dCas9): a new tool for genetic engineering in Leptospira

International audienceLeptospirosis is a worldwide zoonosis caused by pathogenic bacteria of the genus Leptospira, which also includes free-living saprophyte strains. Many aspects of leptospiral basic biology and virulence mechanisms remain unexplored mainly due to the lack of effective genetic tools available for these bacteria. Recently, the type II CRISPR/Cas system from Streptococcus pyogenes has been widely used as an efficient genome engineering tool in bacteria by inducing double-strand breaks (DSBs) in the desired genomic targets caused by an RNA-guided DNA endonuclease called Cas9, and the DSB repair associated machinery. In the present work, plasmids expressing heterologous S. pyogenes Cas9 in L. biflexa cells were generated, and the enzyme could be expressed with no apparent toxicity to leptospiral cells. However, L. biflexa cells were unable to repair RNA-guided Cas9-induced DSBs. Thus, we used a catalytically dead Cas9 (dCas9) to obtain gene silencing rather than disruption, in a strategy called CRISPR interference (CRISPRi). We demonstrated complete gene silencing in L. biflexa cells when both dCas9 and single-guide RNA (sgRNA) targeting the coding strand of the β-galactosidase gene were expressed simultaneously. Furthermore, when the system was applied for silencing the dnaK gene, no colonies were recovered, indicating that DnaK protein is essential in Leptospira. In addition, flagellar motor switch FliG gene silencing resulted in reduced bacterial motility. To the best of our knowledge, this is the first work applying the CRISPRi system in Leptospira and spirochetes in general, expanding the tools available for understanding leptospiral biology

Leptospirosis—Improving Healthcare Outcomes for a Neglected Tropical Disease

Leptospirosis is a globally distributed zoonotic disease transmitted from animal reservoirs to humans. It is particularly common in tropical regions of Africa, Asia, and Central and South America during heavy rainfall when bacterial spirochetes are released from soil into areas of flooding. Despite causing &gt;1 million severe cases, 58 900 deaths, and 2.9 million disability-adjusted life-years annually—exceeding established neglected tropical diseases—leptospirosis remains underrecognized as a neglected tropical disease. It affects occupational groups like farmers due to high prevalence in livestock and is spread by rodents in urban settings that have poor sanitation and infrastructure. Although effectively treated with inexpensive antibiotics, neglect of leptospirosis research and development has led to a lack of awareness and unavailability of preventive and diagnostic approaches. This review covers the geographic prevalence, disproportionate impacts on marginalized communities, and opportunities for improving social, economic, and healthcare burdens for patients with leptospirosis

Etiology of Severe Non-malaria Febrile Illness in Northern Tanzania: A Prospective Cohort Study.

The syndrome of fever is a commonly presenting complaint among persons seeking healthcare in low-resource areas, yet the public health community has not approached fever in a comprehensive manner. In many areas, malaria is over-diagnosed, and patients without malaria have poor outcomes. We prospectively studied a cohort of 870 pediatric and adult febrile admissions to two hospitals in northern Tanzania over the period of one year using conventional standard diagnostic tests to establish fever etiology. Malaria was the clinical diagnosis for 528 (60.7%), but was the actual cause of fever in only 14 (1.6%). By contrast, bacterial, mycobacterial, and fungal bloodstream infections accounted for 85 (9.8%), 14 (1.6%), and 25 (2.9%) febrile admissions, respectively. Acute bacterial zoonoses were identified among 118 (26.2%) of febrile admissions; 16 (13.6%) had brucellosis, 40 (33.9%) leptospirosis, 24 (20.3%) had Q fever, 36 (30.5%) had spotted fever group rickettsioses, and 2 (1.8%) had typhus group rickettsioses. In addition, 55 (7.9%) participants had a confirmed acute arbovirus infection, all due to chikungunya. No patient had a bacterial zoonosis or an arbovirus infection included in the admission differential diagnosis. Malaria was uncommon and over-diagnosed, whereas invasive infections were underappreciated. Bacterial zoonoses and arbovirus infections were highly prevalent yet overlooked. An integrated approach to the syndrome of fever in resource-limited areas is needed to improve patient outcomes and to rationally target disease control efforts

The sigma factor σ54 is required for the long-term survival of Leptospira biflexa in water

Leptospira spp. comprise both pathogenic and free-living saprophytic species. Little is known about the environmental adaptation and survival mechanisms of Leptospira. Alternative sigma factor, σ54 (RpoN) is known to play an important role in environmental and host adaptation in many bacteria. In this study, we constructed an rpoN mutant by allele exchange, and the complemented strain in saprophytic L. biflexa. Transcriptome analysis revealed that expression of several genes involved in nitrogen uptake and metabolism, including amtB1, glnB-amtB2, ntrX and narK, were controlled by σ54 . While wild-type L. biflexa could not grow under nitrogen-limiting conditions but was able to survive under such conditions and recover rapidly, the rpoN mutant was not. The rpoN mutant also had dramatically reduced ability to survive long-term in water. σ54 appears to regulate expression of amtB1, glnK-amtB2, ntrX and narK in an indirect manner. However, we identified a novel nitrogen-related gene, LEPBI_I1011, whose expression was directly under the control of σ54 (herein renamed as rcfA for RpoN-controlled factor A). Taken together, our data reveal that the σ54 regulatory network plays an important role in the long-term environmental survival of Leptospira spp

Neutrophil extracellular traps are involved in the innate immune response to infection with <i>Leptospira</i>

NETosis is a process by which neutrophils extrude their DNA together with bactericidal proteins that trap and/or kill pathogens. In the present study, we evaluated the ability of Leptospira spp. to induce NETosis using human ex vivo and murine in vivo models. Microscopy and fluorometric studies showed that incubation of human neutrophils with Leptospira interrogans serovar Copenhageni strain Fiocruz L1-130 (LIC) resulted in the release of DNA extracellular traps (NETs). The bacteria number, pathogenicity and viability were relevant factors for induction of NETs, but bacteria motility was not. Entrapment of LIC in the NETs resulted in LIC death; however, pathogenic but not saprophytic Leptospira sp. exerted nuclease activity and degraded DNA. Mice infected with LIC showed circulating NETs after 2 days post-infection (dpi). Depletion of neutrophils with mAb1A8 significantly reduced the amount of intravascular NETs in LIC-infected mice, increasing bacteremia at 3 dpi. Although there was a low bacterial burden, scarce neutrophils and an absence of inflammation in the early stages of infection in the kidney and liver, at the beginning of the leptospiruric phase, the bacterial burden was significantly higher in kidneys of neutrophil-depleted-mice compared to non-depleted and infected mice. Surprisingly, interstitial nephritis was of similar intensity in both groups of infected mice. Taken together, these data suggest that LIC triggers NETs, and that the intravascular formation of these DNA traps appears to be critical not only to prevent early leptospiral dissemination but also to preclude further bacterial burden.Instituto de Biotecnologia y Biologia MolecularFacultad de Ciencias ExactasFacultad de Ciencias Médica