Natacha Sertour

First Isolation and Direct Evidence for the Existence of Large Small-Mammal Reservoirs of Leptospira sp. in Madagascar

Background Leptospirosis has long been a major public health concern in the southwestern Indian Ocean. However, in Madagascar, only a few, old studies have provided indirect serological evidence of the disease in humans or animals. Methodology/Principal Findings We conducted a large animal study focusing on small-mammal populations. Five field trapping surveys were carried out at five sites, from April 2008 to August 2009. Captures consisted of Rattus norvegicus (35.8%), R. rattus (35.1%), Mus musculus (20.5%) and Suncus murinus (8.6%). We used microbiological culture, serodiagnosis tests (MAT) and real-time PCR to assess Leptospira infection. Leptospira carriage was detected by PCR in 91 (33.9%) of the 268 small mammals, by MAT in 17 of the 151 (11.3%) animals for which serum samples were available and by culture in 9 of the 268 animals (3.3%). Rates of infection based on positive PCR results were significantly higher in Moramanga (54%), Toliara (48%) and Mahajanga (47.4%) than in Antsiranana (8.5%) and Toamasina (14%) (p = 0.001). The prevalence of Leptospira carriage was significantly higher in R. norvegicus (48.9%), S. murinus (43.5%) and R. rattus (30.8%) than in M. musculus (9.1%) (p<0.001). The MAT detected antibodies against the serogroups Canicola and Icterohaemorrhagiae. Isolates were characterized by serology, secY sequence-based phylogeny, partial sequencing of rrs, multi-locus VNTR analysis and pulsed field gel electrophoresis. The 10 isolates obtained from nine rats were all identified as species L. interrogans serogroup Canicola serovar Kuwait and all had identical partial rrs and secY sequences. Conclusions/Significance We present here the first direct evidence of widespread leptospiral carriage in small mammals in Madagascar. Our results strongly suggest a high level of environmental contamination, consistent with probable transmission of the infection to humans. This first isolation of pathogenic Leptospira strains in this country may significantly improve the detection of specific antibodies in human cases.

Genetic structure of marine Borrelia garinii and population admixture with the terrestrial cycle of Lyme borreliosis

Despite the importance of population structure for the epidemiology of pathogenic bacteria, the spatial and ecological heterogeneity of these populations is often poorly characterized. Here, we investigated the genetic diversity and population structure of the Lyme borreliosis (LB) spirochaete Borrelia garinii in its marine cycle involving colonial seabirds and different host races of the seabird tick Ixodes uriae. Multilocus sequence analyses (MLSA) on eight chromosomal and two plasmid loci (ospA and ospC) indicate that B. garinii circulating in the marine system is highly diverse. Microevolution in marine B. garinii seems to be mainly clonal, but recombination and selection do occur. Sequence types were not evenly distributed among geographic regions, with substantial population subdivision between Atlantic and Pacific Ocean basins. However, no geographic structuring was evident within regions. Results of selection analyses and phylogenetic discordance between chromosomal and plasmid loci indicate adaptive evolution is likely occurring in this system, but no pattern of host or vector-associated divergence was found. Recombination analyses showed evidence for population admixture between terrestrial and marine strains, suggesting that LB spirochaetes are exchanged between these enzootic cycles. Importantly, our results highlight the need to explicitly consider the marine system for a complete understanding of the evolutionary ecology and global epidemiology of Lyme borreliosis.

Severe Leptospirosis in Hospitalized Patients, Guadeloupe

We evaluated prognostic factors for leptospirosis in 168 consecutive hospitalized patients in Guadeloupe. Factors independently associated with severity included chronic hypertension or chronic alcoholism, late initiation of antibacterial therapy, abnormal chest auscultation results, icterus, oligoanuria, disorders of consciousness, elevated aspartate aminotransferase levels, hyperamylasemia, and Leptospira interrogans serovar Icterohemorrhagiae.

Synergy of the antibiotic colistin with echinocandin antifungals in Candida species

OBJECTIVES Candida albicans is the most prevalent fungal pathogen of humans, causing a wide range of infections from harmless superficial to severe systemic infections. Improvement of the antifungal arsenal is needed since existing antifungals can be associated with limited efficacy, toxicity and antifungal resistance. Here we aimed to identify compounds that act synergistically with echinocandin antifungals and that could contribute to a faster reduction of the fungal burden. METHODS A total of 38 758 compounds were tested for their ability to act synergistically with aminocandin, a β-1,3-glucan synthase inhibitor of the echinocandin family of antifungals. The synergy between echinocandins and an identified hit was studied with chemogenomic screens and testing of individual Saccharomyces cerevisiae and C. albicans mutant strains. RESULTS We found that colistin, an antibiotic that targets membranes in Gram-negative bacteria, is synergistic with drugs of the echinocandin family against all Candida species tested. The combination of colistin and aminocandin led to faster and increased permeabilization of C. albicans cells than either colistin or aminocandin alone. Echinocandin susceptibility was a prerequisite to be able to observe the synergy. A large-scale screen for genes involved in natural resistance of yeast cells to low doses of the drugs, alone or in combination, identified efficient sphingolipid and chitin biosynthesis as necessary to protect S. cerevisiae and C. albicans cells against the antifungal combination. CONCLUSIONS These results suggest that echinocandin-mediated weakening of the cell wall facilitates colistin targeting of fungal membranes, which in turn reinforces the antifungal activity of echinocandins.

Next-generation sequencing offers new insights into the resistance of Candida spp. to echinocandins and azoles

OBJECTIVES MDR Candida strains are emerging. Next-generation sequencing (NGS), which enables extensive and deep genome analysis, was used to investigate echinocandin and azole resistance in clinical Candida isolates. METHODS Six genes commonly involved in antifungal resistance (ERG11, ERG3, TAC1, CgPDR1, FKS1 and FKS2) were analysed using NGS in 40 Candida isolates (18 Candida albicans, 15 Candida glabrata and 7 Candida parapsilosis). The strategy was validated using strains with known sequences. Then, 8 clinical strains displaying antifungal resistance and 23 sequential isolates collected from 10 patients receiving antifungal therapy were analysed. RESULTS A total of 391 SNPs were detected, among which 6 coding SNPs were reported for the first time. Novel genetic alterations were detected in both azole and echinocandin resistance genes. A C. glabrata strain, which was resistant to echinocandins but highly susceptible to azoles, harboured an FKS2 S663P mutation plus a novel presumed loss-of-function CgPDR1 mutation. This isolate was from a patient with deep-seated and urinary candidiasis. Another C. glabrata isolate, with an MDR phenotype, carried a new FKS2 S663A mutation and a new putative gain-of-function CgPDR1 mutation (T370I); this isolate showed mutated (80%) and WT (20%) populations and was collected after 75 days of exposure to caspofungin from a patient who underwent complicated abdominal surgery. CONCLUSIONS This study shows that NGS can be used for extensive assessment of genetic mutations involved in antifungal resistance. This type of wide genome approach will become very valuable for detecting mechanisms of resistance in clinical strains subjected to multidrug pressure.

Differential expression of Ixodes ricinus salivary gland proteins in the presence of the Borrelia burgdorferi sensu lato complex

UNLABELLED In Europe, Ixodes ricinus is the main vector of Lyme borreliosis. Their salivary glands play a critical role in the biological success of ticks. To better understand the cross-talk between Borrelia burgdorferi and tick salivary glands, we analyzed protein expression in the salivary glands of I. ricinus adult ticks that were infected by various strains of the B. burgdorferi sl complex. iTRAQ allowed the identification of more than 120 proteins, providing the first proteomic data pertaining to I. ricinus salivary glands. Among these proteins, only 12 were modulated in the presence of various Borrelia strains. Most of them are up-regulated and are involved in cell defense and protein synthesis and processing. Down-regulated proteins are mostly implicated in the cytoskeleton. The DIGE analysis allowed us to identify 35 proteins and showed the down-regulation of 4 proteins. All 15 proteins were not modulated by all strains. Overall, these observations showed that the presence of Borrelia in tick salivary glands is a factor of stress for the protein machinery, and also that some Borrelia strains produce a dysregulation of cytoskeletal proteins. Interestingly, a protein from Borrelia, OspA, was found in infected salivary glands. The consequence of its presence in salivary glands is discussed. BIOLOGICAL SIGNIFICANCE Lyme borreliosis is still the most prevalent arthropod-borne disease in the temperate regions of the northern hemisphere. The geographical distribution of Lyme borreliosis is expanding, especially towards higher altitudes and latitudes. Human pathogenic spirochetes causing Lyme borreliosis belong to the B. burgdorferi sensu lato complex. They are extracellular pathogens transmitted to humans through the bite of Ixodes spp. ticks. The bioactive molecules present in tick saliva not only promote tick feeding, but also create an advantageous microenvironment at the tick bite site for survival and replication of Borrelia bacteria. Investigation of the tick-host-pathogen interface would provide new strategies to control tick-borne infections. We chose to analyze the interaction of several strains of the B. burgdorferi sensu lato complex with I. ricinus salivary glands. We also investigated the presence of bacterial proteins in salivary glands. For these purposes, we undertook a proteomic study implying the complementary approaches of iTRAQ and DIGE. Our study allowed identifying several salivary markers of infection that were shown to vary according to the strain. Moreover, OspA, a bacterial protein was shown to be expressed in salivary glands and may be implied in the pathogenicity of some Borrelia strains.

Comparative Population Genomics of the Borrelia burgdorferi Species Complex Reveals High Degree of Genetic Isolation among Species and Underscores Benefits and Constraints to Studying Intra-Specific Epidemiological Processes

Lyme borreliosis, one of the most frequently contracted zoonotic diseases in the Northern Hemisphere, is caused by bacteria belonging to different genetic groups within the Borrelia burgdorferi species complex, which are transmitted by ticks among various wildlife reservoirs, such as small mammals and birds. These features make the Borrelia burgdorferi species complex an attractive biological model that can be used to study the diversification and the epidemiology of endemic bacterial pathogens. We investigated the potential of population genomic approaches to study these processes. Sixty-three strains belonging to three species within the Borrelia burgdorferi complex were isolated from questing ticks in Alsace (France), a region where Lyme disease is highly endemic. We first aimed to characterize the degree of genetic isolation among the species sampled. Phylogenetic and coalescent-based analyses revealed clear delineations: there was a ∼50 fold difference between intra-specific and inter-specific recombination rates. We then investigated whether the population genomic data contained information of epidemiological relevance. In phylogenies inferred using most of the genome, conspecific strains did not cluster in clades. These results raise questions about the relevance of different strategies when investigating pathogen epidemiology. For instance, here, both classical analytic approaches and phylodynamic simulations suggested that population sizes and migration rates were higher in B. garinii populations, which are normally associated with birds, than in B. burgdorferi s.s. populations. The phylogenetic analyses of the infection-related ospC gene and its flanking region provided additional support for this finding. Traces of recombination among the B. burgdorferi s.s. lineages and lineages associated with small mammals were found, suggesting that they shared the same hosts. Altogether, these results provide baseline evidence that can be used to formulate hypotheses regarding the host range of B. burgdorferi lineages based on population genomic data.

Analysis of Repair Mechanisms following an Induced Double-Strand Break Uncovers Recessive Deleterious Alleles in the Candida albicans Diploid Genome

ABSTRACT The diploid genome of the yeast Candida albicans is highly plastic, exhibiting frequent loss-of-heterozygosity (LOH) events. To provide a deeper understanding of the mechanisms leading to LOH, we investigated the repair of a unique DNA double-strand break (DSB) in the laboratory C. albicans SC5314 strain using the I-SceI meganuclease. Upon I-SceI induction, we detected a strong increase in the frequency of LOH events at an I-SceI target locus positioned on chromosome 4 (Chr4), including events spreading from this locus to the proximal telomere. Characterization of the repair events by single nucleotide polymorphism (SNP) typing and whole-genome sequencing revealed a predominance of gene conversions, but we also observed mitotic crossover or break-induced replication events, as well as combinations of independent events. Importantly, progeny that had undergone homozygosis of part or all of Chr4 haplotype B (Chr4B) were inviable. Mining of genome sequencing data for 155 C. albicans isolates allowed the identification of a recessive lethal allele in the GPI16 gene on Chr4B unique to C. albicans strain SC5314 which is responsible for this inviability. Additional recessive lethal or deleterious alleles were identified in the genomes of strain SC5314 and two clinical isolates. Our results demonstrate that recessive lethal alleles in the genomes of C. albicans isolates prevent the occurrence of specific extended LOH events. While these and other recessive lethal and deleterious alleles are likely to accumulate in C. albicans due to clonal reproduction, their occurrence may in turn promote the maintenance of corresponding nondeleterious alleles and, consequently, heterozygosity in the C. albicans species. IMPORTANCE Recessive lethal alleles impose significant constraints on the biology of diploid organisms. Using a combination of an I-SceI meganuclease-mediated DNA DSB, a fluorescence-activated cell sorter (FACS)-optimized reporter of LOH, and a compendium of 155 genome sequences, we were able to unmask and identify recessive lethal and deleterious alleles in isolates of Candida albicans, a diploid yeast and the major fungal pathogen of humans. Accumulation of recessive deleterious mutations upon clonal reproduction of C. albicans could contribute to the maintenance of heterozygosity despite the high frequency of LOH events in this species. Recessive lethal alleles impose significant constraints on the biology of diploid organisms. Using a combination of an I-SceI meganuclease-mediated DNA DSB, a fluorescence-activated cell sorter (FACS)-optimized reporter of LOH, and a compendium of 155 genome sequences, we were able to unmask and identify recessive lethal and deleterious alleles in isolates of Candida albicans, a diploid yeast and the major fungal pathogen of humans. Accumulation of recessive deleterious mutations upon clonal reproduction of C. albicans could contribute to the maintenance of heterozygosity despite the high frequency of LOH events in this species.

Borrelia crocidurae meningoencephalitis, West Africa.

Borrelia crocidurae–associated relapsing fever is endemic to West Africa and is considered benign. We report 4 patients with B. crocidurae–associated neurologic symptoms; 2 of their cases had been misdiagnosed. Frequency and severity of this disease could be underestimated; molecular methods and serodiagnostic tests for Lyme disease might be helpful in its detection.

Infection Kinetics and Tropism of Borrelia burgdorferi sensu lato in Mouse After Natural (via Ticks) or Artificial (Needle) Infection Depends on the Bacterial Strain

Borrelia burgdorferi sl is a complex of pathogen bacteria transmitted to the host by Ixodes ticks. European Ixodes ricinus ticks transmit different B. burgdorferi species, pathogenic to human. Bacteria are principally present in unfed tick midgut, then migrate to salivary glands during blood meal and infect a new host via saliva. In this study, efficiency of transmission in a mouse model of three pathogen species belonging to the B. burgdorferi sl complex, B. burgdorferi sensu stricto (B31, N40, and BRE-13), B. afzelii (IBS-5), and B. bavariensis (PBi) is examined in order to evaluate infection risk after tick bite. We compared the dissemination of the Borrelia species in mice after tick bite and needle injection. Location in the ticks and transmission to mice were also determined for the three species by following infection kinetics. After inoculation, we found a significant prevalence in the brain for PBi and BRE-13, in the heart, for PBi, in the skin where B31 was more prevalent than PBi and in the ankle where both B31 and N40 were more present than PBi. After tick bite, statistical analyses showed that BRE-13 was more prevalent than N40 in the brain, in the bladder and in the inguinal lymph node. When Borrelia dissemination was compared after inoculation and tick bite, we observed heart infection only after tick inoculation of BRE-13, and PBi was only detected after tick bite in the skin. For N40, a higher number of positive organs was found after inoculation compared to tick bite. All European B. burgdorferi sl strains studied were detected in female salivary glands before blood meal and infected mice within 24 h of tick bite. Moreover, Borrelia-infected nymphs were able to infect mice as early as 12 h of tick attachment. Our study shows the need to remove ticks as early as possible after attachment. Moreover, Borrelia tropism varied according to the strain as well as between ticks bite and needle inoculation, confirming the association between some strains and clinical manifestation of Lyme borreliosis, as well as the role played by tick saliva in the efficiency of Borrelia infection and dissemination in vertebrates.