Metabolic reprogramming during Candida albicans planktonic-biofilm transition is modulated by the transcription factors Zcf15 and Zcf26.

Candida albicans is a commensal of the human microbiota that can form biofilms on implanted medical devices. These biofilms are tolerant to antifungals and to the host immune system. To identify novel genes modulating C. albicans biofilm formation, we performed a large-scale screen with 2,454 C. albicans doxycycline-dependent overexpression strains and identified 16 genes whose overexpression significantly hampered biofilm formation. Among those, overexpression of the ZCF15 and ZCF26 paralogs that encode transcription factors and have orthologs only in biofilm-forming species of the Candida clade, caused impaired biofilm formation both in vitro and in vivo. Interestingly, overexpression of ZCF15 impeded biofilm formation without any defect in hyphal growth. Transcript profiling, transcription factor binding, and phenotypic microarray analyses conducted upon overexpression of ZCF15 and ZCF26 demonstrated their role in reprogramming cellular metabolism by regulating central metabolism including glyoxylate and tricarboxylic acid cycle genes. Taken together, this study has identified a new set of biofilm regulators, including ZCF15 and ZCF26, that appear to control biofilm development through their specific role in metabolic remodeling.

Perturbation and resilience of the gut microbiome up to 3 months after ß-lactams exposure in healthy volunteers suggest an important role of microbial ß-lactamases.

BACKGROUND: Antibiotics notoriously perturb the gut microbiota. We treated healthy volunteers either with cefotaxime or ceftriaxone for 3 days, and collected in each subject 12 faecal samples up to day 90. Using untargeted and targeted phenotypic and genotypic approaches, we studied the changes in the bacterial, phage and fungal components of the microbiota as well as the metabolome and the ß-lactamase activity of the stools. This allowed assessing their degrees of perturbation and resilience. RESULTS: While only two subjects had detectable concentrations of antibiotics in their faeces, suggesting important antibiotic degradation in the gut, the intravenous treatment perturbed very significantly the bacterial and phage microbiota, as well as the composition of the metabolome. In contrast, treatment impact was relatively low on the fungal microbiota. At the end of the surveillance period, we found evidence of resilience across the gut system since most components returned to a state like the initial one, even if the structure of the bacterial microbiota changed and the dynamics of the different components over time were rarely correlated. The observed richness of the antibiotic resistance genes repertoire was significantly reduced up to day 30, while a significant increase in the relative abundance of ß-lactamase encoding genes was observed up to day 10, consistent with a concomitant increase in the ß-lactamase activity of the microbiota. The level of ß-lactamase activity at baseline was positively associated with the resilience of the metabolome content of the stools. CONCLUSIONS: In healthy adults, antibiotics perturb many components of the microbiota, which return close to the baseline state within 30 days. These data suggest an important role of endogenous ß-lactamase-producing anaerobes in protecting the functions of the microbiota by de-activating the antibiotics reaching the colon. Video Abstract.

A CO2 sensing module modulates ß-1,3-glucan exposure in Candida albicans.

Microbial species capable of co-existing with healthy individuals, such as the commensal fungus Candida albicans, exploit multifarious strategies to evade our immune defenses. These strategies include the masking of immunoinflammatory pathogen-associated molecular patterns (PAMPs) at their cell surface. We reported previously that C. albicans actively reduces the exposure of the proinflammatory PAMP, ß-1,3-glucan, at its cell surface in response to host-related signals such as lactate and hypoxia. Here, we show that clinical isolates of C. albicans display phenotypic variability with respect to their lactate- and hypoxia-induced ß-1,3-glucan masking. We have exploited this variability to identify responsive and non-responsive clinical isolates. We then performed RNA sequencing on these isolates to reveal genes whose expression patterns suggested potential association with lactate- or hypoxia-induced ß-1,3-glucan masking. The deletion of two such genes attenuated masking: PHO84 and NCE103. We examined NCE103-related signaling further because NCE103 has been shown previously to encode carbonic anhydrase, which promotes adenylyl cyclase-protein kinase A (PKA) signaling at low CO2 levels. We show that while CO2 does not trigger ß-1,3-glucan masking in C. albicans, the Sch9-Rca1-Nce103 signaling module strongly influences ß-1,3-glucan exposure in response to hypoxia and lactate. In addition to identifying a new regulatory module that controls PAMP exposure in C. albicans, our data imply that this module is important for PKA signaling in response to environmental inputs other than CO2.IMPORTANCEOur innate immune defenses have evolved to protect us against microbial infection in part via receptor-mediated detection of "pathogen-associated molecular patterns" (PAMPs) expressed by invading microbes, which then triggers their immune clearance. Despite this surveillance, many microbial species are able to colonize healthy, immune-competent individuals, without causing infection. To do so, these microbes must evade immunity. The commensal fungus Candida albicans exploits a variety of strategies to evade immunity, one of which involves reducing the exposure of a proinflammatory PAMP (ß-1,3-glucan) at its cell surface. Most of the ß-1,3-glucan is located in the inner layer of the C. albicans cell wall, hidden by an outer layer of mannan fibrils. Nevertheless, some ß-1,3-glucan can become exposed at the fungal cell surface. However, in response to certain specific host signals, such as lactate or hypoxia, C. albicans activates an anticipatory protective response that decreases ß-1,3-glucan exposure, thereby reducing the susceptibility of the fungus to impending innate immune attack. Here, we exploited the natural phenotypic variability of C. albicans clinical isolates to identify strains that do not display the response to ß-1,3-glucan masking signals observed for the reference isolate, SC5314. Then, using genome-wide transcriptional profiling, we compared these non-responsive isolates with responsive controls to identify genes potentially involved in ß-1,3-glucan masking. Mutational analysis of these genes revealed that a sensing module that was previously associated with CO2 sensing also modulates ß-1,3-glucan exposure in response to hypoxia and lactate in this major fungal pathogen of humans.

The pathogenic and colonization potential of Candida africana.

The Candida albicans population displays high genetic diversity illustrated by 18-well differentiated genetic clusters. Cluster 13, also known as Candida africana, is an outlying cluster and includes strains first described as atypical C. albicans isolates of vaginal origin, showing apparent tropism for the female genital tract. In our study, we combined in vitro, and in vivo models to explore the colonization and pathogenic potential of C. africana. We report that C. africana has similar fitness to C. albicans when it comes to colonization of the oral and vaginal mucosa, however it has decreased fitness in gastro-intestinal colonization and systemic infection. Interestingly, despite high population homogeneity, our in vitro data highlighted for the first time a variability in terms of growth rate, biofilm formation and filamentation properties between C. africana strains. Overall, our data lays the foundations for exploring specific features of C. africana that might contribute to its apparent niche restriction.

Unveiling Candida albicans intestinal carriage in healthy volunteers: the role of micro- and mycobiota, diet, host genetics and immune response.

Candida albicans is a commensal yeast present in the gut of most healthy individuals but with highly variable concentrations. However, little is known about the host factors that influence colonization densities. We investigated how microbiota, host lifestyle factors, and genetics could shape C. albicans intestinal carriage in 695 healthy individuals from the Milieu Intérieur cohort. C. albicans intestinal carriage was detected in 82.9% of the subjects using quantitative PCR. Using linear mixed models and multiway-ANOVA, we explored C. albicans intestinal levels with regard to gut microbiota composition and lifestyle factors including diet. By analyzing shotgun metagenomics data and C. albicans qPCR data, we showed that Intestinimonas butyriciproducens was the only gut microbiota species whose relative abundance was negatively correlated with C. albicans concentration. Diet is also linked to C. albicans growth, with eating between meals and a low-sodium diet being associated with higher C. albicans levels. Furthermore, by Genome-Wide Association Study, we identified 26 single nucleotide polymorphisms suggestively associated with C. albicans colonization. In addition, we found that the intestinal levels of C. albicans might influence the host immune response, specifically in response to fungal challenge. We analyzed the transcriptional levels of 546 immune genes and the concentration of 13 cytokines after whole blood stimulation with C. albicans cells and showed positive associations between the extent of C. albicans intestinal levels and NLRP3 expression, as well as secreted IL-2 and CXCL5 concentrations. Taken together, these findings open the way for potential new interventional strategies to curb C. albicans intestinal overgrowth.

Metagenomics and metabolomics approaches in the study of Candida albicans colonization of host niches: a framework for finding microbiome-based antifungal strategies.

In silico and experimental approaches have allowed an ever-growing understanding of the interactions within the microbiota. For instance, recently acquired data have increased knowledge of the mechanisms that support, in the gut and vaginal microbiota, the resistance to colonization by Candida albicans, an opportunistic fungal pathogen whose overgrowth can initiate severe infections in immunocompromised patients. Here, we review how bacteria from the microbiota interact with C. albicans. We show how recent OMICs-based pipelines, using metagenomics and/or metabolomics, have identified bacterial species and metabolites modulating C. albicans growth. We finally discuss how the combined use of cutting-edge OMICs-based and experimental approaches could provide new means to control C. albicans overgrowth within the microbiota and prevent its consequences.

A Clinical Study Provides the First Direct Evidence That Interindividual Variations in Fecal ß-Lactamase Activity Affect the Gut Mycobiota Dynamics in Response to ß-Lactam Antibiotics.

Antibiotics disturb the intestinal bacterial microbiota, leading to gut dysbiosis and an increased risk for the overgrowth of opportunistic pathogens. It is not fully understood to what extent antibiotics affect the fungal fraction of the intestinal microbiota, the mycobiota. There is no report of the direct role of antibiotics in the overgrowth in healthy humans of the opportunistic pathogenic yeast Candida albicans. Here, we have explored the gut mycobiota of 22 healthy subjects before, during, and up to 6 months after a 3-day regimen of third-generation cephalosporins (3GCs). Using ITS1-targeted metagenomics, we highlighted the strong intra- and interindividual diversity of the healthy gut mycobiota. With a specific quantitative approach, we showed that C. albicans prevalence was much higher than previously reported, with all subjects but one being carriers of C. albicans, although with highly variable burdens. 3GCs significantly altered the mycobiota composition and the fungal load was increased both at short and long term. Both C. albicans relative and absolute abundances were increased but 3GCs did not reduce intersubject variability. Variations in C. albicans burden in response to 3GC treatment could be partly explained by changes in the levels of endogenous fecal ß-lactamase activity, with subjects characterized by a high increase of ß-lactamase activity displaying a lower increase of C. albicans levels. A same antibiotic treatment might thus affect differentially the gut mycobiota and C. albicans carriage, depending on the treated subject, suggesting a need to adjust the current risk factors for C. albicans overgrowth after a ß-lactam treatment. IMPORTANCE Fungal infections are redoubtable healthcare-associated complications in immunocompromised patients. Particularly, the commensal intestinal yeast Candida albicans causes invasive infections in intensive care patients and is, therefore, associated with high mortality. These infections are preceded by an intestinal expansion of C. albicans before its translocation into the bloodstream. Antibiotics are a well-known risk factor for C. albicans overgrowth but the impact of antibiotic-induced dysbiosis on the human gut mycobiota-the fungal microbiota-and the understanding of the mechanisms involved in C. albicans overgrowth in humans are very limited. Our study shows that antibiotics increase the fungal proportion in the gut and disturb the fungal composition, especially C. albicans, in a subject-dependent manner. Indeed, variations across subjects in C. albicans burden in response to ß-lactam treatment could be partly explained by changes in the levels of endogenous fecal ß-lactamase activity. This highlighted a potential new key factor for C. albicans overgrowth. Thus, the significance of our research is in providing a better understanding of the factors behind C. albicans intestinal overgrowth, which might lead to new means to prevent life-threatening secondary infections.

A conserved regulator controls asexual sporulation in the fungal pathogen Candida albicans.

Transcription factor Rme1 is conserved among ascomycetes and regulates meiosis and pseudohyphal growth in Saccharomyces cerevisiae. The genome of the meiosis-defective pathogen Candida albicans encodes an Rme1 homolog that is part of a transcriptional circuitry controlling hyphal growth. Here, we use chromatin immunoprecipitation and genome-wide expression analyses to study a possible role of Rme1 in C. albicans morphogenesis. We find that Rme1 binds upstream and activates the expression of genes that are upregulated during chlamydosporulation, an asexual process leading to formation of large, spherical, thick-walled cells during nutrient starvation. RME1 deletion abolishes chlamydosporulation in three Candida species, whereas its overexpression bypasses the requirement for chlamydosporulation cues and regulators. RME1 expression levels correlate with chlamydosporulation efficiency across clinical isolates. Interestingly, RME1 displays a biphasic pattern of expression, with a first phase independent of Rme1 function and dependent on chlamydospore-inducing cues, and a second phase dependent on Rme1 function and independent of chlamydospore-inducing cues. Our results indicate that Rme1 plays a central role in chlamydospore development in Candida species.

Large-scale genome mining allows identification of neutral polymorphisms and novel resistance mutations in genes involved in Candida albicans resistance to azoles and echinocandins.

BACKGROUND: The genome of Candida albicans displays significant polymorphism. Point mutations in genes involved in resistance to antifungals may either confer phenotypic resistance or be devoid of phenotypic consequences. OBJECTIVES: To catalogue polymorphisms in azole and echinocandin resistance genes occurring in susceptible strains in order to rapidly pinpoint relevant mutations in resistant strains. METHODS: Genome sequences from 151 unrelated C. albicans strains susceptible to fluconazole and caspofungin were used to create a catalogue of non-synonymous polymorphisms in genes involved in resistance to azoles (ERG11, TAC1, MRR1 and UPC2) or echinocandins (FKS1). The potential of this catalogue to reveal putative resistance mutations was tested in 10 azole-resistant isolates, including 1 intermediate to caspofungin. Selected mutations were analysed by mutagenesis experiments or mutational prediction effect. RESULTS: In the susceptible strains, we identified 126 amino acid substitutions constituting the catalogue of phenotypically neutral polymorphisms. By excluding these neutral substitutions, we identified 22 additional substitutions in the 10 resistant strains. Among these substitutions, 10 had already been associated with resistance. The remaining 12 were in Tac1p (n = 6), Upc2p (n = 2) and Erg11p (n = 4). Four out of the six homozygous substitutions in Tac1p (H263Y, A790V, H839Y and P971S) conferred increases in azole MICs, while no effects were observed for those in Upc2p. Additionally, two homozygous substitutions (Y64H and P236S) had a predicted conformation effect on Erg11p. CONCLUSIONS: By establishing a catalogue of neutral polymorphisms occurring in genes involved in resistance to antifungal drugs, we provide a useful resource for rapid identification of mutations possibly responsible for phenotypic resistance in C. albicans.

Within-Host Genomic Diversity of Candida albicans in Healthy Carriers.

Genomic variations in Candida albicans, a major fungal pathogen of humans, have been observed upon exposure of this yeast to different stresses and experimental infections, possibly contributing to subsequent adaptation to these stress conditions. Yet, little is known about the extent of genomic diversity that is associated with commensalism, the predominant lifestyle of C. albicans in humans. In this study, we investigated the genetic diversity of C. albicans oral isolates recovered from healthy individuals, using multilocus sequencing typing (MLST) and whole genome sequencing. While MLST revealed occasional differences between isolates collected from a single individual, genome sequencing showed that they differed by numerous single nucleotide polymorphisms, mostly resulting from short-range loss-of-heterozygosity events. These differences were shown to have occurred upon human carriage of C. albicans rather than subsequent in vitro manipulation of the isolates. Thus, C. albicans intra-sample diversity appears common in healthy individuals, higher than that observed using MLST. We propose that diversifying lineages coexist in a single human individual, and this diversity can enable rapid adaptation under stress exposure. These results are crucial for the interpretation of longitudinal studies evaluating the evolution of the C. albicans genome.

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.

Systematic gene overexpression in Candida albicans identifies a regulator of early adaptation to the mammalian gut.

Candida albicans is part of the human gastrointestinal (GI) microbiota. To better understand how C. albicans efficiently establishes GI colonisation, we competitively challenged growth of 572 signature-tagged strains (~10% genome coverage), each conditionally overexpressing a single gene, in the murine gut. We identified CRZ2, a transcription factor whose overexpression and deletion respectively increased and decreased early GI colonisation. Using clues from genome-wide expression and gene-set enrichment analyses, we found that the optimal activity of Crz2p occurs under hypoxia at 37°C, as evidenced by both phenotypic and transcriptomic analyses following CRZ2 genetic perturbation. Consistent with early colonisation of the GI tract, we show that CRZ2 overexpression confers resistance to acidic pH and bile salts, suggesting an adaptation to the upper sections of the gut. Genome-wide location analyses revealed that Crz2p directly modulates the expression of many mannosyltransferase- and cell-wall protein-encoding genes, suggesting a link with cell-wall function. We show that CRZ2 overexpression alters cell-wall phosphomannan abundance and increases sensitivity to tunicamycin, suggesting a role in protein glycosylation. Our study reflects the powerful use of gene overexpression as a complementary approach to gene deletion to identify relevant biological pathways involved in C. albicans interaction with the host environment.

Gene flow contributes to diversification of the major fungal pathogen Candida albicans.

Elucidating population structure and levels of genetic diversity and recombination is necessary to understand the evolution and adaptation of species. Candida albicans is the second most frequent agent of human fungal infections worldwide, causing high-mortality rates. Here we present the genomic sequences of 182 C. albicans isolates collected worldwide, including commensal isolates, as well as ones responsible for superficial and invasive infections, constituting the largest dataset to date for this major fungal pathogen. Although, C. albicans shows a predominantly clonal population structure, we find evidence of gene flow between previously known and newly identified genetic clusters, supporting the occurrence of (para)sexuality in nature. A highly clonal lineage, which experimentally shows reduced fitness, has undergone pseudogenization in genes required for virulence and morphogenesis, which may explain its niche restriction. Candida albicans thus takes advantage of both clonality and gene flow to diversify.

Infection of Ixodes ricinus by Borrelia burgdorferi sensu lato in peri-urban forests of France.

Lyme borreliosis is the most common tick-borne disease in the northern hemisphere. In Europe, it is transmitted by Ixodes ticks that carry bacteria belonging to the Borrelia burgdorferi sensu lato complex. The objective of this work was to explore eco-epidemiological factors of Lyme borreliosis in peri-urban forests of France (Sénart, Notre-Dame and Rambouillet). We investigated whether the introduction of Tamias sibiricus in Sénart could alter the density of infected ticks. Moreover, the density and tick infection were investigated according to the tree species found in various patches of Sénart forest. For this purpose, ticks were sampled during 3 years. In the Sénart forest, the density of nymph and adult ticks showed no significant difference between 2008, 2009 and 2011. The nymph density varied significantly as a function of the month of collection. Regarding the nymphs, a higher rate of infection and infected density were found in 2009. Plots with chipmunks (C) presented a lower density of both nymphs and adult ticks than plots without chipmunks (NC) did. A higher rate of infection of nymphs with Borrelia was seen in C plots. The prevalence of the various species of Borrelia was also found to vary between C and NC plots with the year of the collect. The presence of chestnut trees positively influenced the density of both nymphs and adults. The infected nymph density showed a significant difference depending on the peri-urban forest studied, Sénart being higher than Rambouillet. The prevalence of Borrelia species also differed between the various forests studied. Concerning the putative role that Tamias sibiricus may play in the transmission of Borrelia, our results suggest that its presence is correlated with a higher rate of infection of questing ticks by Borrelia genospecies and if its population increases, it could play a significant role in the risk of transmission of Lyme borreliosis.

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

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.

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.

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.

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

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.

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 ß-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.

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.