CRISPR-Cas is a form of adaptive sequence-specific immunity in microbes. This system offers unique opportunities for the study of coevolution between bacteria and their viral pathogens, bacteriophages. A full understanding of the coevolutionary dynamics of CRISPR-Cas requires knowing the magnitude of the cost of resisting infection. Here, using the gram-positive bacterium Streptococcus thermophilus and its associated virulent phage 2972, a well-established model system harbouring at least two type II functional CRISPR-Cas systems, we obtained different fitness measures based on growth assays in isolation or in pairwise competition. We measured the fitness cost associated with different components of this adaptive immune system: the cost of Cas protein expression, the constitutive cost of increasing immune memory through additional spacers, and the conditional costs of immunity during phage exposure. We found that Cas protein expression is particularly costly, as Cas-deficient mutants achieved higher competitive abilities than the wild-type strain with functional Cas proteins. Increasing immune memory by acquiring up to four phage-derived spacers was not associated with fitness costs. In addition, the activation of the CRISPR-Cas system during phage exposure induces significant but small fitness costs. Together these results suggest that the costs of the CRISPR-Cas system arise mainly due to the maintenance of the defence system. We discuss the implications of these results for the evolution of CRISPR-Cas-mediated immunity.

Cryptococcal meningitis is a severe opportunistic infection in HIV-infected patients. In Ivory Coast, despite the availability of antiretroviral treatment (ART), this infection is still prevalent. The study investigates the genetic diversity of 363 clinical isolates of Cryptococcus from 61 Ivorian HIV-positive patients, the occurrence of mixed infections and the in vitro antifungal susceptibility of the isolates. Serotyping was performed via LAC1 and CAP64 gene amplification. Genotyping was performed using the phage M13 core (GACA) and (GTG) primers and restriction fragment length polymorphism analysis of the URA5 gene. By PCR fingerprinting, the presence of the three serotypes were demonstrated among the 363 isolates in the population studied: A (n=318; 87.6%), AD (n=40; 11%) and B (n=4; 1.1%). Using PCR fingerprinting with primers M13 (GACA) and (GTG) , we grouped the isolates into 56 molecular subtypes. We observed a high frequency (39.3%) of mixed infections, with up to two different genotypes per sample. None of the isolates were resistant to amphotericin B. Only 0.3% and 1.1% of the isolates were resistant to fluconazole and flucytosine respectively. This study revealed the high genetic diversity among Cryptococcus isolates, the occurrence of mixed infections and a high antifungal susceptibility for the majority of Ivorian cryptococcal isolates.

Introduction:Cryptococcal meningitis (CM) may be caused by several species of Cryptococcus. Case presentation: We describe a fatal case of CM in a HIV-positive patient from Ivory Coast infected by Cryptococcus neoformans VNI and Cryptococcus deuterogattii. Isolates were recovered from cerebrospinal fluid (CSF) prior to systemic antifungal treatment. Six isolates were studied (the entire culture plus five isolated colonies from it). Serotyping was performed via LAC 1 and CAP 64 gene amplification. Genotyping was performed using restriction fragment length polymorphism (RFLP) analysis of the URA5 gene, (GACA)4, (GTG)5 and M13 PCR fingerprinting. URA5-RFLP analysis identified the original culture with two different molecular type combinations. However, URA5-RFLP profiles of the five colonies isolated from the original sample revealed two different species. Four colonies were identified as C. deuterogattii and the last isolate as C. neoformans VNI. The in vitro susceptibility profile was determined using the standard method according to the CLSI M27-A3 protocol. The isolates were susceptible to the tested antifungals (fluconazole, flucytosine and amphotericin B). Treatment with fluconazole (1200  mg day−1) was initiated; however, the patient died 17 days after the onset of antifungal therapy.Conclusion: This is the first reported case of mixed infection with C. neoformans and C. deuterogattii in a HIV-positive patient.

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Mutations allowing pathogens to escape host immunity promote the spread of infectious diseases in heterogeneous host populations and can lead to major epidemics. Understanding the conditions favoring these evolutionary emergences is key for the development of durable control strategies against pathogens. Here we compare the durability of three different control strategies: (i) a mixing strategy where the host population results from the mix of two single-resistant genotypes, (ii) a pyramiding strategy where host resistance is due to a single double-resistant genotype, (iii) a combining strategy where host resistance is due to a mix between a single-resistant genotype and a double-resistant genotype. First, we use evolutionary epidemiology theory to clarify the interplay between demographic stochasticity and evolutionary dynamics to show that the pyramiding strategy always yields lower probability of evolutionary emergence. Second, we confirm experimentally these predictions using virulent bacteriophages introduced in bacterial populations where we can manipulate the diversity and the depth of CRISPR immunity. Our work shows that pyramiding multiple defenses into the same individual host and avoiding mixing with single-defense strategies is a robust way to protect individuals and populations against pathogen evolutionary emergence. These results have practical implications for the optimal deployment of host resistance in agriculture and biotechnology but also for the optimal use of vaccination against human pathogens.

Genotypes exhibiting an increased mutation rate, called hypermutators, can propagate in microbial populations because they can have an advantage due to the higher supply of beneficial mutations needed for adaptation. Although this is a frequently observed phenomenon in natural and laboratory populations, little is known about the influence of parameters such as the degree of maladaptation, stress intensity, and the genetic architecture for adaptation on the emergence of hypermutators. To address this knowledge gap, we measured the emergence of hypermutators over ~1,000 generations in experimental Escherichia coli populations exposed to different levels of osmotic or antibiotic stress. Our stress types were chosen based on the assumption that the genetic architecture for adaptation differs between them. Indeed, we show that the size of the genetic basis for adaptation is larger for osmotic stress compared to antibiotic stress. During our experiment, we observed an increased emergence of hypermutators in populations exposed to osmotic stress but not in those exposed to antibiotic stress, indicating that hypermutator emergence rates are stress type dependent. These results support our hypothesis that hypermutator emergence is linked to the size of the genetic basis for adaptation. In addition, we identified other parameters that covaried with stress type (stress level and IS transposition rates) that might have contributed to an increased hypermutator provision and selection. Our results provide a first comparison of hypermutator emergence rates under varying stress conditions and point towards complex interactions of multiple stress-related factors on the evolution of mutation rates.