Rodent control to fight plague : field assessment of methods based on rat density reduction

Research funding: Directorate General for International Relations and Strategy. Grant Number: 2018‐SB‐024‐18SSEOC049‐PMG7‐SSA5‐IPMMADAGASCAR ACKNOWLEDGMENTS: We are especially grateful to the health authorities and the population in Miantso and Ankazobe for allowing us to do this work and being so helpful. We thank the staff of the Plague Unit, Institut Pasteur de Madagascar, for helping with the field and laboratory work, especially Alain Berthin Rakotoarisoa and Andrianiaina Parfait Rakotonindrainy. This work was supported by a Directorate General for International Relations and Strategy grant (2018‐SB‐024‐18SSEOC049‐PMG7‐SSA5‐IPMMADAGASCAR) covering the project “Développement de contre‐mesures médicales à la peste à Madagascar” with scientific support of IRBA (French Armed Forces Biomedical Research Institute), within the framework of French MoD's involvement in G7 Global partnership. The French Agency for International Technical Expertise (AFETI) ensures the proper financial execution of the project and contributes to the implementation of cooperation actions under the control of the Directorate General for International Relations and Strategy. This research was also funded in part by the Wellcome Trust [095171/Z/10/Z] and the Institut Pasteur de Madagascar. For the purpose of Open Access, the authors have applied a CC BY public copyright license to any Author Accepted Manuscript version arising from this submission. K.S. was supported by the Biotechnology and Biological Sciences Research Council (BBSRC) under the EastBio DTP (grant number BB/M010996/1).Peer reviewedPublisher PD

Origin and mobility of Iron Age Gaulish groups in present-day France revealed through archaeogenomics

The Iron Age period occupies an important place in French history, as the Gauls are regularly presented as the direct ancestors of the extant French population. We documented here the genomic diversity of Iron Age communities originating from six French regions. The 49 acquired genomes permitted us to highlight an absence of discontinuity between Bronze Age and Iron Age groups in France, lending support to a cultural transition linked to progressive local economic changes rather than to a massive influx of allochthone groups. Genomic analyses revealed strong genetic homogeneity among the regional groups associated with distinct archaeological cultures. This genomic homogenisation appears to be linked to individuals’ mobility between regions as well as gene flow with neighbouring groups from England and Spain. Thus, the results globally support a common genomic legacy for the Iron Age population of modern-day France that could be linked to recurrent gene flow between culturally differentiated communities

Bacterial DNA diagenesis and metagenomic analyses of past bacterial pathologies

Cette étude a pour objet la mise en évidence de traces d'ADN bactérien pathogène dans des échantillons animaux et humains anciens, et ainsi améliorer les connaissances sur l'évolution des maladies au cours du temps. En parallèle, nous avons étudié les phénomènes de dégradation de l'ADN dans le sol sur des cadavres de souris enterrées après avoir été contaminées par des bactéries non pathogènes. Cette étude des processus taphonomiques s'est étalée sur trois ans et a permis de montrer une disparition rapide des bactéries simulantes, remplacé par l'ADN des bactéries du sol, qui colonisent rapidement la dépouille et dégradent tant l'ADN endogène (murin) qu'exogène (bactérien). Cette disparition rapide explique la grande difficulté à mettre en évidence des pathogènes dans des échantillons anciens, à de rares exceptions près. Notre étude n'a pas permis de détecter d'agents pathogènes particuliers dans les échantillons que nous avons étudié, mais nous avons mis en évidence l'intérêt d'analyser certains types de restes pour accéder à une information génétique préservée. Le tartre dentaire indique est un bon indicateur de la flore buccale de l'hôte et les kystes calcifiés assurent une bonne préservation de l'ADN endogène, moins soumis à contamination et digestion par les bactéries de l'environnement. Les kystes présentent en règle générale une teneur en ADN endogène supérieure à tous les autres tissus étudiés.The aim of this study was the identification of pathogenic bacterial DNA traces in ancient animal and human samples, and thus improve knowledge of past diseases that affect humankind over time. In parallel, we studied the DNA degradation phenomena in the soil on the buried corpses of mice after being contaminated by non-pathogenic bacteria. This study of taphonomic processes was spread over three years and has shown a rapid disappearance of simulant bacteria, replaced with the DNA of soil bacteria that colonize the body quickly after burial and degrade both the endogenous DNA (murine) that exogenous (bacteria). This quick degradation can explain the high difficulty to detect and identify bacterial pathogens in old samples, with very few exceptions. Despite the fact in our study we were not able to detect specific pathogens in the samples we have studied, we have shown the interest to analyze certain types of remnants to access preserved and informative genetic data. Dental calculus is a good indicator of the oral flora of the host and calcified cysts ensure good preservation of the endogenous DNA, less subject to contamination and digestion by bacteria from the environment. Cysts generally have an endogenous DNA content higher than all other tissues examined

Polyamino-isoprenyl derivatives as antibiotic adjuvants and motility inhibitors for 1 Bordetella bronchiseptica porcine pulmonary infection treatment 2 3

International audienceThe spreading of multidrug-resistant bacteria and the lack of novel antibiotic molecules leave clinicians and veterinarians with very limited options to treat bacterial infections, especially those caused by Gram-negative pathogens. To reduce the selection of antibiotic resistance mechanisms and their transfer to human pathogens, veterinary pharmaceutical companies have dramatically decreased the number of antibiotics used. Among all the investigated alternate solutions, chemosensitizers, which decrease the amount of the used drugs, appear to be one of the most promising strategies. In this study, we reported that polyamino-isoprenyl derivatives can potentiate florfenicol activity against veterinary sensitive reference strains as well as clinical isolates. These molecules induce inner membrane depolarization and subsequently inhibit efflux pumps by collapsing the proton-motive force (PMF). Considering that Bordetella bronchiseptica rotor flagellum is highly PMF dependent and that flagellar motility represents an important factor involved in colonization, we monitored the swimming and swarming motilities of bacteria and showed a strong inhibition in the presence of the lead selected compound. Taken together, our results suggest that this class of molecules are able to increase treatment efficacy and decrease drug consumption

Selection and Validation of a Multilocus Variable-Number Tandem-Repeat Analysis Panel for Typing Shigella spp.▿ †

The Shigella genus has historically been separated into four species, based on biochemical assays. The classification within each species relies on serotyping. Recently, genome sequencing and DNA assays, in particular the multilocus sequence typing (MLST) approach, greatly improved the current knowledge of the origin and phylogenetic evolution of Shigella spp. The Shigella and Escherichia genera are now considered to belong to a unique genomospecies. Multilocus variable-number tandem-repeat (VNTR) analysis (MLVA) provides valuable polymorphic markers for genotyping and performing phylogenetic analyses of highly homogeneous bacterial pathogens. Here, we assess the capability of MLVA for Shigella typing. Thirty-two potentially polymorphic VNTRs were selected by analyzing in silico five Shigella genomic sequences and subsequently evaluated. Eventually, a panel of 15 VNTRs was selected (i.e., MLVA15 analysis). MLVA15 analysis of 78 strains or genome sequences of Shigella spp. and 11 strains or genome sequences of Escherichia coli distinguished 83 genotypes. Shigella population cluster analysis gave consistent results compared to MLST. MLVA15 analysis showed capabilities for E. coli typing, providing classification among pathogenic and nonpathogenic E. coli strains included in the study. The resulting data can be queried on our genotyping webpage (http://mlva.u-psud.fr). The MLVA15 assay is rapid, highly discriminatory, and reproducible for Shigella and Escherichia strains, suggesting that it could significantly contribute to epidemiological trace-back analysis of Shigella infections and pathogenic Escherichia outbreaks. Typing was performed on strains obtained mostly from collections. Further studies should include strains of much more diverse origins, including all pathogenic E. coli types

Coprolites, Paleogenomics and Bone Content Analysis

Coprolites are fossil scats and provide indirect witness of the activity of past animals of a given area, whether or not fossil bones of these animals are present in the site. The shape, size, inclusions and geo- and bio-chemical composition are criteria for identification of the animal that left the coprolite. Unit II from Azokh 1 has yielded two complete undamaged coprolites one of which contained partially digested fossil bones. Taphonomic and taxonomic indications from this coprolite could not conclusively identify the origin of the coprolites. Analysis of targeted mitochondrial DNA, performed on one of the coprolites, has provided evidence for the presence of hyena DNA, but this finding was not supported by further investigation using next-generation high throughput sequencing. The most parsimonious interpretation of the results of the genetic analyses is that the highly sensitive PCR assay reveals contamination of the coprolite with minute amounts of modern brown hyena DNA presumably originating from brown hyena scats sampled recently in South Africa.Peer Reviewe

Analysis of Ancient DNA in Microbial Ecology.

International audienceThe development of next-generation sequencing has led to a breakthrough in the analysis of ancient genomes, and the subsequent genomic analyses of the skeletal remains of ancient humans have revolutionized the knowledge of the evolution of our species, including the discovery of a new hominin, and demonstrated admixtures with more distantly related archaic populations such as Neandertals and Denisovans. Moreover, it has also yielded novel insights into the evolution of ancient pathogens. The analysis of ancient microbial genomes allows the study of their recent evolution, presently over the last several millennia. These spectacular results have been attained despite the degradation of DNA after the death of the host, which results in very short DNA molecules that become increasingly damaged, only low quantities of which remain. The low quantity of ancient DNA molecules renders their analysis difficult and prone to contamination with modern DNA molecules, in particular via contamination from the reagents used in DNA purification and downstream analysis steps. Finally, the rare ancient molecules are diluted in environmental DNA originating from the soil microorganisms that colonize bones and teeth. Thus, ancient skeletal remains can share DNA profiles with environmental samples and identifying ancient microbial genomes among the more recent, presently poorly characterized, environmental microbiome is particularly challenging. Here, we describe the methods developed and/or in use in our laboratory to produce reliable and reproducible paleogenomic results from ancient skeletal remains that can be used to identify the presence of ancient microbiota

Owl pellets: A wise DNA source for small mammal genetics

Pellets of raptors are an important source for the study of the taxonomy, phylogeography and ecological diversity of small vertebrates. Since birds of prey are efficient collecting agents for both rare species and those reluctant to enter traps, they offer an important complement to traditional trapping efforts in the field. The possibility of using bones and teeth recovered from pellets as a convenient alternative DNA source for genetic studies requires a more complete understanding of the DNA preservation of these partially digested remains. It is not yet clear, for example, to which degree DNA is preserved and how heterogeneous DNA preservation is in the different skeletal remains found in pellets since systematic quantitative analyses are missing. Here, we attempt to improve this knowledge by quantifying the degradation of DNA during the digestion process of raptors using 62 individual rodent bones and teeth from different pellets. We show a very high variability in DNA preservation between bones. Interestingly, variability between bones within the same pellet is much higher than that found between pellets, which has major consequences for genetic studies as it implies that bones from the same pellet that could belong to different individuals/species should be tested individually when multiple markers are analyzed. We find that neither the identity of the skeletal part nor microscopic observation of the degree of digestion are useful proxies for DNA preservation, and we thus recommend the use of screening strategies to identify the most suitable bones to include in analyses. We conclude that pellets of owls, whether freshly collected or from natural history collections, can be a practical noninvasive source for genetic studies of small vertebrates provided the design of the study takes into account the short DNA molecule length, the low quantity of DNA preserved and the heterogeneity in DNA preservation.The DNA analyses were funded by the project ANR‐PEX 004 – MOHMIE, the SEM analyses by the project CGL2010‐19825.Peer Reviewe

Transcriptomic studies and assessment of Yersinia pestis reference genes in various conditions

Abstract Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is a very sensitive widespread technique considered as the gold standard to explore transcriptional variations. While a particular methodology has to be followed to provide accurate results many published studies are likely to misinterpret results due to lack of minimal quality requirements. Yersinia pestis is a highly pathogenic bacterium responsible for plague. It has been used to propose a ready-to-use and complete approach to mitigate the risk of technical biases in transcriptomic studies. The selection of suitable reference genes (RGs) among 29 candidates was performed using four different methods (GeNorm, NormFinder, BestKeeper and the Delta-Ct method). An overall comprehensive ranking revealed that 12 following candidate RGs are suitable for accurate normalization: gmk, proC, fabD, rpoD, nadB, rho, thrA, ribD, mutL, rpoB, adk and tmk. Some frequently used genes like 16S RNA had even been found as unsuitable to study Y. pestis. This methodology allowed us to demonstrate, under different temperatures and states of growth, significant transcriptional changes of six efflux pumps genes involved in physiological aspects as antimicrobial resistance or virulence. Previous transcriptomic studies done under comparable conditions had not been able to highlight these transcriptional modifications. These results highlight the importance of validating RGs prior to the normalization of transcriptional expression levels of targeted genes. This accurate methodology can be extended to any gene of interest in Y. pestis. More generally, the same workflow can be applied to identify and validate appropriate RGs in other bacteria to study transcriptional variations