New methods for detecting antibiotic resistant bacteria and preventing their spread

Methicillin resistant Staphylococcus aureus (MRSA) bacteria have emerged in the early 1980's in numerous health care institutions around the world. The main transmission mechanism within hospitals and healthcare facilities is through the hands of health care workers. Resistant to several antibiotics, the MRSA is one of the most feared pathogens in the hospital setting since it is very difficult to eradicate with the standard treatments. There are still a limited number of anti-MRSA antibiotics but the first cases of resistance to these compounds have already been reported and their frequency is likely to increase in the coming years. Every year, the MRSA infections result in major human and financial costs, due to the high associated mortality and expenses related to the required care. Measures towards a faster detection of resistant bacteria and establishment of appropriate antibiotic treatment parameters are fundamental. Also as part as infection prevention, diminution of bacteria present on the commonly touched surfaces could also limit the spread and selection of antibiotic resistant bacteria. During my thesis, projects were developed around MRSA and antibiotic resistance investigation using innovative technologies. The thesis was subdivided in three main parts with the use of atomic force microscopy AFM for antibiotic resistance detection in part 1, the importance of the bacterial inoculum size in the selection of antibiotic resistance in part 2 and the testing of antimicrobial surfaces creating by sputtering copper onto polyester in part 3. In part 1 the AFM was used two different ways, first for the measurement of stiffness (elasticity) of bacteria and second as a nanosensor for antibiotic susceptibility testing. The stiffness of MRSA with different susceptibility profiles to vancomycin was investigated using the stiffness tomography mode of the AFM and results have demonstrated and increased stiffness in the vancomycin resistant strains that also paralleled with increased thickness of the bacterial cell wall. Parts of the AFM were also used to build a new antibiotic susceptibility-testing device. This nano sensor was able to measure vibrations emitted from living bacteria that ceased definitively upon antibiotic exposure to which they were susceptible but restarted after antibiotic removal to which they were resistant, allowing in a matter of minute the assessment of antibiotic susceptibility determination. In part 2 the inoculum effect (IE) of vancomycin, daptomycin and linezolid and its importance in antibiotic resistance selection was investigated with MRSA during a 15 days of cycling experiment. Results indicated that a high bacterial inoculum and a prolonged antibiotic exposure were two key factors in the in vitro antibiotic resistance selection in MRSA and should be taken into consideration when choosing the drug treatment. Finally in part 3 bactericidal textile surfaces were investigated against MRSA. Polyesters coated after 160 seconds of copper sputtering have demonstrated a high bactericidal activity reducing the bacterial load of at least 3 logio after one hour of contact. -- Au cours des dernières décennies, des bactéries multirésistantes aux antibiotiques (BMR) ont émergé dans les hôpitaux du monde entier. Depuis lors, le nombre de BMR et la prévalence des infections liées aux soins (IAS) continuent de croître et sont associés à une augmentation des taux de morbidité et de mortalité ainsi qu'à des coûts élevés. De plus, le nombre de résistance à différentes classes d'antibiotiques a également augmenté parmi les BMR, limitant ainsi les options thérapeutiques disponibles lorsqu'elles ont liées a des infections. Des mesures visant une détection plus rapide des bactéries résistantes ainsi que l'établissement des paramètres de traitement antibiotiques adéquats sont primordiales lors d'infections déjà présentes. Dans une optique de prévention, la diminution des bactéries présentes sur les surfaces communément touchées pourrait aussi freiner la dissémination et l'évolution des bactéries résistantes. Durant ma thèse, différents projets incluant des nouvelles technologies et évoluant autour de la résistance antibiotique ont été traités. Des nouvelles technologies telles que le microscope à force atomique (AFM) et la pulvérisation cathodique de cuivre (PCC) ont été utilisées, et le Staphylococcus aureus résistant à la méticilline (SARM) a été la principale BMR étudiée. Deux grandes lignes de recherche ont été développées; la première visant à détecter la résistance antibiotique plus rapidement avec l'AFM et la seconde visant à prévenir la dissémination des BMR avec des surfaces crées grâce à la PCC. L'AFM a tout d'abord été utilisé en tant que microscope à sonde locale afin d'investiguer la résistance à la vancomycine chez les SARMs. Les résultats ont démontré que la rigidité de la paroi augmentait avec la résistance à la vancomycine et que celle-ci corrélait aussi avec une augmentation de l'épaisseur des parois, vérifiée grâce à la microscopie électronique. Des parties d'un AFM ont été ensuite utilisées afin de créer un nouveau dispositif de test de sensibilité aux antibiotiques, un nanocapteur. Ce nanocapteur mesure des vibrations produites par les bactéries vivantes. Après l'ajout d'antibiotique, les vibrations cessent définitivement chez les bactéries sensibles à l'antibiotique. En revanche pour les bactéries résistantes, les vibrations reprennent après le retrait de l'antibiotique dans le milieu permettant ainsi, en l'espace de minutes de détecter la sensibilité de la bactérie à un antibiotique. La PCC a été utilisée afin de créer des surfaces bactéricides pour la prévention de la viabilité des BMR sur des surfaces inertes. Des polyesters finement recouverts de cuivre (Cu), connu pour ses propriétés bactéricides, ont été produits et testés contre des SARMs. Une méthode de détection de viabilité des bactéries sur ces surfaces a été mise au point, et les polyesters obtenus après 160 secondes de pulvérisation au Cu ont démontré une excellente activité bactéricide, diminuant la charge bactérienne d'au moins 3 logio après une heure de contact. En conclusion, l'utilisation de nouvelles technologies nous a permis d'évoluer vers de méthodes de détection de la résistance antibiotique plus rapides ainsi que vers le développement d'un nouveau type de surface bactéricide, dans le but d'améliorer le diagnostic et la gestion des BMR.

Unusual spread of a penicillin-susceptible methicillin-resistant Staphylococcus aureus clone in a geographic area of low incidence.

We describe the unusual spread of a penicillin-susceptible methicillin-resistant Staphylococcus aureus (MRSA) clone in hospitals in western Switzerland, where the incidence of MRSA is usually low. During a 2-year period, this clone had been responsible for several outbreaks and had been isolated from >156 persons in 21 institutions. Molecular typing by pulsed-field gel electrophoresis (PFGE) demonstrated that all of these isolates belonged to the same clone. In 1 of the outbreaks, involving 30 cases, the clone was responsible for at least 17 secondary cases. In contrast, during the period of the latter outbreak, 9 other patients harboring different MRSA strains, as assessed by PFGE, were hospitalized in the same wards, but no secondary cases occurred. These observations suggest that this clone, compared with other MRSA strains, had some intrinsic factor(s) that contributed to its ability to disseminate and could thus be considered epidemic.

The spread of incompatibility-inducing parasites in sub-divided host populations.

BACKGROUND:Maternally transmitted symbionts have evolved a variety of ways to promote their spread through host populations. One strategy is to hamper the reproduction of uninfected females by a mechanism called cytoplasmic incompatibility (CI). CI occurs in crosses between infected males and uninfected females and leads to partial to near-complete infertility. CI-infections are under positive frequency-dependent selection and require genetic drift to overcome the range of low frequencies where they are counter-selected. Given the importance of drift, population sub-division would be expected to facilitate the spread of CI. Nevertheless, a previous model concluded that variance in infection between competing groups of breeding individuals impedes the spread of CI.RESULTS:In this paper we derive a model on the spread of CI-infections in populations composed of demes linked by restricted migration. Our model shows that population sub-division facilitates the invasion of CI. While host philopatry (low migration) favours the spread of infection, deme size has a non-monotonous effect, with CI-invasion being most likely at intermediate deme size. Individual-based simulations confirm these predictions and show that high levels of local drift speed up invasion but prevent high levels of prevalence across the entire population. Additional simulations with sex-specific migration rates further show that low migration rates of both sexes are required to facilitate the spread of CI.CONCLUSION:Our analyses show that population structure facilitates the invasion of CI-infections. Since some level of sub-division is likely to occur in most natural populations, our results help to explain the high incidence of CI-infections across species of arthropods. Furthermore, our work has important implications for the use of CI-systems in order to genetically modify natural populations of disease vectors.

Accelerated MR imaging with spread spectrum encoding

We advocate the use of a novel compressed sensing technique for accelerating the magnetic resonance image acquisition process, coined spread spectrum MR imaging or simply s2MRI. The method resides in pre-modulating the signal of interest by a linear chirp, resulting from the application of quadratic phase profiles, before random k-space under-sampling with uniform average density. The effectiveness of the procedure is theoretically underpinned by the optimization of the coherence between the sparsity and sensing bases. The application of the technique for single coil acquisitions is thoroughly studied by means of numerical simulations as well as phantom and in vivo experiments on a 7T scanner. The corresponding results suggest a favorable comparison with state-of-the-art variable density k-space under-sampling approaches.

Extracapsular tumor spread and the risk of local, axillary and supraclavicular recurrence in node-positive, premenopausal patients with breast cancer.

BACKGROUND: Extracapsular tumor spread (ECS) has been identified as a possible risk factor for breast cancer recurrence, but controversy exists regarding its role in decision making for regional radiotherapy. This study evaluates ECS as a predictor of local, axillary, and supraclavicular recurrence. PATIENTS AND METHODS: International Breast Cancer Study Group Trial VI accrued 1475 eligible pre- and perimenopausal women with node-positive breast cancer who were randomly assigned to receive three to nine courses of classical combination chemotherapy with cyclophosphamide, methotrexate, and fluorouracil. ECS status was determined retrospectively in 933 patients based on review of pathology reports. Cumulative incidence and hazard ratios (HRs) were estimated using methods for competing risks analysis. Adjustment factors included treatment group and baseline patient and tumor characteristics. The median follow-up was 14 years. RESULTS: In univariable analysis, ECS was significantly associated with supraclavicular recurrence (HR = 1.96; 95% confidence interval 1.23-3.13; P = 0.005). HRs for local and axillary recurrence were 1.38 (P = 0.06) and 1.81 (P = 0.11), respectively. Following adjustment for number of lymph node metastases and other baseline prognostic factors, ECS was not significantly associated with any of the three recurrence types studied. CONCLUSIONS: Our results indicate that the decision for additional regional radiotherapy should not be based solely on the presence of ECS.

Using a Bayesian method to assign individuals to karyotypic taxa in shrew hybrid zones.

Individuals sampled in hybrid zones are usually analysed according to their sampling locality, morphology, behaviour or karyotype. But the increasing availability of genetic information more and more favours its use for individual sorting purposes and numerous assignment methods based on the genetic composition of individuals have been developed. The shrews of the Sorex araneus group offer good opportunities to test the genetic assignment on individuals identified by their karyotype. Here we explored the potential and efficiency of a Bayesian assignment method combined or not with a reference dataset to study admixture and individual assignment in the difficult context of two hybrid zones between karyotypic species of the Sorex araneus group. As a whole, we assigned more than 80% of the individuals to their respective karyotypic categories (i.e. 'pure' species or hybrids). This assignment level is comparable to what was obtained for the same species away from hybrid zones. Additionally, we showed that the assignment result for several individuals was strongly affected by the inclusion or not of a reference dataset. This highlights the importance of such comparisons when analysing hybrid zones. Finally, differences between the admixture levels detected in both hybrid zones support the hypothesis of an impact of chromosomal rearrangements on gene flow.

Cytogenetic and molecular relationships between Zarudny's rock shrew (Crocidura zarudnyi; Mammalia : Soricomorpha) and Eurasian taxa

We karyotyped and sequenced 1,140 base pairs of the mitochondrial DNA cytochrome b of a specimen of Zarudny's rock shrew (Crocidura zarudnyi) from Baluchestan, southeastern Iran, to clarify its cytogenetic and molecular relationships with other Eurasian species of Crocidura. According to the karyotype (2N = 40, FN = 50), Zarudny's rock shrew belongs to the group of the lesser white-toothed shrew (C. suaveolens), which is different from other known crocidurine karyotypes, considering the combination of the diploid and fundamental number of chromosomes. Molecular results revealed that C. zarudnyi is included in a monophyletic clade with the C. suaveolens group, where it is a sister taxon to the others (mean Kimura 2-parameter distance = 9.7%).

Genetic causes of transitions from sexual reproduction to asexuality in plants and animals.

The persistence of sexual reproduction in the face of competition from asexual invaders is more likely if asexual lineages are produced infrequently or have low fitness. The generation rate and success of new asexual lineages will be influenced by the proximate mechanisms underlying transitions to asexuality. As such, characterization of these mechanisms can help explain the distribution of reproductive modes among natural populations. Here, we synthesize the literature addressing proximate causes of transitions from sexual to asexual reproduction in plants and animals. In cyclical and facultatively asexual taxa, individual mutations can cause obligate asexuality. The evolution of asexuality in obligately sexual groups is more complex, requiring the simultaneous acquisition of two traits generally controlled by different genetic factors: unreduced gamete formation and spontaneous development of unfertilized gametes. At least three 'pre-adaptations' could favour transitions to obligate asexuality in obligate sexuals. First, linkage among loci affecting separate key components of asexuality facilitates its spread, with evidence for these linkage blocks in plants. Second, asexuality should evolve more readily in haplodiploids; support for this hypothesis comes from two examples where a single locus causes transitions to asexuality. Third, standing genetic variation for the production of unreduced gametes could facilitate transitions to asexuality, but whether the ability to produce unreduced gametes contributes to the evolution of obligate asexuality remains unclear. We close by reviewing the associations between asexuality, hybridization and polyploidy, and argue that current data suggest that hybridization is more likely to play a causal role in transitions to asexuality than polyploidy.

Gene-flow in a mosaic hybrid zone: is local introgression adaptive?

Genome-wide scans of genetic differentiation between hybridizing taxa can identify genome regions with unusual rates of introgression. Regions of high differentiation might represent barriers to gene flow, while regions of low differentiation might indicate adaptive introgression-the spread of selectively beneficial alleles between reproductively isolated genetic backgrounds. Here we conduct a scan for unusual patterns of differentiation in a mosaic hybrid zone between two mussel species, Mytilus edulis and M. galloprovincialis. One outlying locus, mac-1, showed a characteristic footprint of local introgression, with abnormally high frequency of edulis-derived alleles in a patch of M. galloprovincialis enclosed within the mosaic zone, but low frequencies outside of the zone. Further analysis of DNA sequences showed that almost all of the edulis allelic diversity had introgressed into the M. galloprovincialis background in this patch. We then used a variety of approaches to test the hypothesis that there had been adaptive introgression at mac-1. Simulations and model fitting with maximum-likelihood and approximate Bayesian computation approaches suggested that adaptive introgression could generate a "soft sweep," which was qualitatively consistent with our data. Although the migration rate required was high, it was compatible with the functioning of an effective barrier to gene flow as revealed by demographic inferences. As such, adaptive introgression could explain both the reduced intraspecific differentiation around mac-1 and the high diversity of introgressed alleles, although a localized change in barrier strength may also be invoked. Together, our results emphasize the need to account for the complex history of secondary contacts in interpreting outlier loci.

Residual antimalarials in malaria patients from Tanzania--implications on drug efficacy assessment and spread of parasite resistance.

Repeated antimalarial treatment for febrile episodes and self-treatment are common in malaria-endemic areas. The intake of antimalarials prior to participating in an in vivo study may alter treatment outcome and affect the interpretation of both efficacy and safety outcomes. We report the findings from baseline plasma sampling of malaria patients prior to inclusion into an in vivo study in Tanzania and discuss the implications of residual concentrations of antimalarials in this setting. In an in vivo study conducted in a rural area of Tanzania in 2008, baseline plasma samples from patients reporting no antimalarial intake within the last 28 days were screened for the presence of 14 antimalarials (parent drugs or metabolites) using liquid chromatography-tandem mass spectrometry. Among the 148 patients enrolled, 110 (74.3%) had at least one antimalarial in their plasma: 80 (54.1%) had lumefantrine above the lower limit of calibration (LLC = 4 ng/mL), 7 (4.7%) desbutyl-lumefantrine (4 ng/mL), 77 (52.0%) sulfadoxine (0.5 ng/mL), 15 (10.1%) pyrimethamine (0.5 ng/mL), 16 (10.8%) quinine (2.5 ng/mL) and none chloroquine (2.5 ng/mL). The proportion of patients with detectable antimalarial drug levels prior to enrollment into the study is worrying. Indeed artemether-lumefantrine was supposed to be available only at government health facilities. Although sulfadoxine-pyrimethamine is only recommended for intermittent preventive treatment in pregnancy (IPTp), it was still widely used in public and private health facilities and sold in drug shops. Self-reporting of previous drug intake is unreliable and thus screening for the presence of antimalarial drug levels should be considered in future in vivo studies to allow for accurate assessment of treatment outcome. Furthermore, persisting sub-therapeutic drug levels of antimalarials in a population could promote the spread of drug resistance. The knowledge on drug pressure in a given population is important to monitor standard treatment policy implementation.

Exotic taxa less related to native species are more invasive.

Some species introduced into new geographical areas from their native ranges wreak ecological and economic havoc in their new environment. Although many studies have searched for either species or habitat characteristics that predict invasiveness of exotic species, the match between characteristics of the invader and those of members of the existing native community may be essential to understanding invasiveness. Here, we find that one metric, the phylogenetic relatedness of an invader to the native community, provides a predictive tool for invasiveness. Using a phylogenetic supertree of all grass species in California, we show that highly invasive grass species are, on average, significantly less related to native grasses than are introduced but noninvasive grasses. The match between the invader and the existing native community may explain why exotic pest species are not uniformly noxious in all novel habitats. Relatedness of invaders to the native biota may be one useful criterion for prioritizing management efforts of exotic species.

Spread spectrum magnetic resonance imaging.

We propose a novel compressed sensing technique to accelerate the magnetic resonance imaging (MRI) acquisition process. The method, coined spread spectrum MRI or simply s(2)MRI, consists of premodulating the signal of interest by a linear chirp before random k-space under-sampling, and then reconstructing the signal with nonlinear algorithms that promote sparsity. The effectiveness of the procedure is theoretically underpinned by the optimization of the coherence between the sparsity and sensing bases. The proposed technique is thoroughly studied by means of numerical simulations, as well as phantom and in vivo experiments on a 7T scanner. Our results suggest that s(2)MRI performs better than state-of-the-art variable density k-space under-sampling approaches.

Flowering date of taxonomic families predicts phenological sensitivity to temperature: Implications for forecasting the effects of climate change on unstudied taxa.

PREMISE OF THE STUDY: Numerous long-term studies in seasonal habitats have tracked interannual variation in first flowering date (FFD) in relation to climate, documenting the effect of warming on the FFD of many species. Despite these efforts, long-term phenological observations are still lacking for many species. If we could forecast responses based on taxonomic affinity, however, then we could leverage existing data to predict the climate-related phenological shifts of many taxa not yet studied. METHODS: We examined phenological time series of 1226 species occurrences (1031 unique species in 119 families) across seven sites in North America and England to determine whether family membership (or family mean FFD) predicts the sensitivity of FFD to standardized interannual changes in temperature and precipitation during seasonal periods before flowering and whether families differ significantly in the direction of their phenological shifts. KEY RESULTS: Patterns observed among species within and across sites are mirrored among family means across sites; early-flowering families advance their FFD in response to warming more than late-flowering families. By contrast, we found no consistent relationships among taxa between mean FFD and sensitivity to precipitation as measured here. CONCLUSIONS: Family membership can be used to identify taxa of high and low sensitivity to temperature within the seasonal, temperate zone plant communities analyzed here. The high sensitivity of early-flowering families (and the absence of early-flowering families not sensitive to temperature) may reflect plasticity in flowering time, which may be adaptive in environments where early-season conditions are highly variable among years.