Full Resistance Research Articles

Pneumococcal carriage among sickle cell disease patients in Accra, Ghana: Risk factors, serotypes and antibiotic resistance.

BackgroundPneumococcal carriage is the precursor for development of pneumococcal disease, and is also responsible for transmission of the organism from person-to-person. Individuals with Sickle Cell Disease (SCD) are more likely to develop invasive disease with S. pneumoniae compared to their healthy counterparts and the presentation of disease in the former is usually abrupt and severe. In Africa, little is known about the pneumococcus in relation to people with SCD Sickle Cell Disease (SCD). The aim of the study was to investigate the epidemiology of pneumococcal carriage among SCD patients including the carriage prevalence, risk factors, serotypes and antibiotic resistance.MethodThis was a cross sectional study involving 402 SCD patients recruited from Korle Bu Teaching Hospital and Princess Marie Louis Hospital in Accra from October 2016 to March 2017. The study subjects included 202 children of the age groups: ≤5 years (94), >5–9 years (75), ≥10–13 years (33) and 200 adults of the age groups: 14–20 years (46), 21–40 years (112), 41–60 years (25), ≤ 61 years (17). Nasopharyngeal (NP) swabs were collected from the study participants as well as epidemiological data on demographic, household and clinical features. The NP specimens were cultured for S. pneumoniae and the isolates were serotyped by latex agglutination. Antimicrobial susceptibility tests of the isolates were done by the disc diffusion test and E-test.ResultsPrevalence of S. pneumoniae carriage among children and adult SCD patients enrolled in the study were 79/202 (39.1%; 95% CI: 32.3 to 46.2) and 20/200 (10.0%; 95% CI: 6.2 to 15.0) respectively. Risk factors associated with pneumococcal carriage were age (OR = 1.137; 95% CI: 1.036–1.248; p = 0.007) and runny nose (OR = 5.371; 95% CI: 1.760–16.390; p = 0.003). Overall, twenty-six pneumococcal serotypes were isolated from the study participants and the predominant serotype was 6B (10.6%), followed by 23B (8.2%). Among the children, serotype coverage of the 13-valent Pneumococcal Conjugate Vaccine, which is currently used in Ghana was 32.4%. Prevalence of penicillin resistance among the pneumococcal isolates was 37.4% (37/99) and all the penicillin-resistant isolates exhibited intermediate penicillin resistance with the exception of one isolate that showed full resistance and was susceptible to ceftriaxone. Prevalence of resistance to the other antibiotics ranged from 2.5% (levofloxacin) to 85% (cotrimoxazole). Multidrug resistance occurred among 34.3% (34/99) of the pneumococcal isolates.ConclusionPneumococcal carriage was four-fold higher in SCD children than adults and was characterized by predominance of non-vaccine serotypes and considerable level of multidrug resistance, though penicillin, cefotaxime and levofloxacin resistance appeared to be very low.

Antimicrobial resistance and molecular characteristics of Neisseria gonorrhoeae isolates from men who have sex with men

ObjectivesTo analyze the susceptibility of Neisseria gonorrhoeae isolates to penicillin (Pen), cefixime (Cfm), ceftriaxone (Cro), tetracycline (Tet), ciprofloxacin (Cip), azithromycin (Azm), and spectinomycin (Spt), and to verify the presence of mutations in resistance genes. MethodsAntibiotic susceptibility testing was performed by Etest method on 30 N. gonorrhoeae isolates collected from the MSM (men who have sex with men) population. PCR and DNA sequencing were performed to identify mutations within the penA, mtrR, gyrA, and parC genes in intermediately resistant and fully resistant isolates. ResultsN. gonorrhoeae isolates showed intermediate or full resistance to Pen (73%), Cfm (3%), Tet (60%), Cip (37%), and Azm (13%). One isolate with resistance to Cfm presented a penicillin-binding protein 2 (PBP2) mosaic XXXIV. All isolates with intermediate or full resistance to Pen (except at PBP2 mosaic) presented a D345a in PBP2. All Cip-resistant isolates had an S91F in the gyrA gene together with mutations in the parC gene. All intermediate or fully resistant isolates to substrates of the MtrCDE efflux pump had an A39T or G45D mutation in the mtrR gene or an adenine deletion within the mtrR promoter. One isolate presented a Neisseria meningitidis-like mtrR promoter sequence. ConclusionsThe results of this study are consistent with the findings of other studies and reinforce the importance of the expedient development of new therapeutic options.

A Detailed Full-Cell Model of a 2018 Commercial PERC Solar Cell in Quokka3

An unprecedented detailed model of a full-size passivated emitter and rear cell (PERC) solar cell design, as manufactured at a current Trina Solar production-line during ramp-up, is presented. Combining a newly proposed multidomain approach with the multiscale skin-concept of Quokka3, the 15.6 cm × 15.6 cm three-dimensional cell geometry including the details of the emitter skins can thoroughly be solved within a single simulation. The multidomain approach uses an inner and two edge domains as irreducible symmetry elements, each containing the unequal front and rear pitch, the dashed rear contacts, as well as part of the busbars and consequently the full finger resistance. The full-cell current density is then determined by simple area-averaging, opposed to the more complicated common approach of coupling it with a distributed network model. The multiscale skin approach enables to model all emitter parts of the PERC cell in detail (accounting for dopant profiles, front surface recombination, Fermi-Dirac statistics, etc.), whereas the other skin regions can still be described by their lumped properties, i.e., Rsheet and J0/Seff. A complete set of carefully established electrical and optical input parameters as well as a detailed loss breakdown is presented, providing fellow researchers with a point of reference for modeling a state-of-the-art PERC solar cell in 2018.

Identifying key factors of the transmission dynamics of drug-resistant malaria

Development of resistance to malaria treatments remains a great threat to continued malaria burden reduction and elimination. Quantifying the impact of key factors which increase the emergence and spread of drug resistance can guide intervention strategies. Whilst modelling provides a framework to understand these factors, we show that a simple of model with a sensitive–resistant dichotomy leads to incorrectly focusing on reducing the treatment rate as a means to prevent resistance. Instead we present a model that considers the development of resistance within hosts as a scale, and we then quantify the number of resistant infections that would arise from a single sensitive infection. By including just one step before full resistance, the model highlights that disrupting this development is more effective than reducing treatment rate. This result is compounded when the model includes the more realistic scenario of several intermediary steps. An additional comparison to transmission probabilities, where resistant infections are less likely to be transmitted (cost of resistance), confirms that preventing the establishment of resistance is more effective than controlling the spread. Our work strongly advocates for further studies into within-host models of resistance, including the potential of combination therapies to disrupt emergence.

Fracture mechanics of nacre-like materials using discrete-element models: Effects of microstructure, interfaces and randomness

Biological materials such as nacre, bone and spider silk boast unusual combinations of stiffness, strength and toughness. Behind this performance is the staggered arrangement of stiff and elongated inclusions embedded in a softer and more deformable matrix. In this study, we use the discrete element method (DEM) to simulate large fracture models of staggered composites (up to 450,000 tablets). The models explore the combined effects of tablet arrangement, interface properties and statistical variations on fracture mechanics: crack deflection, crack bridging, volumetric process zones of different size and shapes, transient and steady-state crack propagation regimes, full crack resistance curves. We find that moderate statistical variations in the microstructure increases toughness because the crack gets pinned into tougher regions. However, higher statistical variations generate very weak regions which can be activated far from the main crack, leading to discontinuous cohesive zones, sparse process zones, and an overall decrease in toughness. These results suggest an important rule for the design of nacre-like materials: microstructural randomness should be minimized to achieve the highest combinations of properties.

Quadriceps Weakness and Risk of Knee Cartilage Loss Seen on Magnetic Resonance Imaging in a Population-based Cohort with Knee Pain.

To determine whether baseline quadriceps weakness predicts cartilage loss assessed on magnetic resonance imaging (MRI). Subjects aged 40-79 with knee pain (n = 163) were recruited from a random population sample and examined for quadriceps weakness with manual isometric strength testing, using a 3-point scoring system (0 = poor resistance, 1 = moderate resistance, 2 = full resistance), which was dichotomized as normal (grade 2) versus weak (grade 0/1). MRI of the more symptomatic knee was obtained at baseline and at mean of 3.3 years. Cartilage was graded 0-4 on MRI. Exponential regression analysis was used to evaluate whether quadriceps weakness was associated with whole knee cartilage loss, and in secondary analyses with compartment-specific cartilage loss, adjusted for age, sex, body mass index, Western Ontario and McMaster Universities Osteoarthritis Arthritis Index pain score, and baseline MRI cartilage score. Of 163 subjects, 54% were female, with a mean age of 57.7 years. Quadriceps weakness was seen in 11.9% of the subjects. Weakness was a predictor of whole knee cartilage loss (HR 3.48, 95% CI 1.30-9.35). Quadriceps weakness was associated with cartilage loss in the medial tibiofemoral (TF) compartment (HR 4.60, 95% CI 1.25-17.02), while no significant association was found with lateral TF (HR 1.53, 95% CI 0.24-9.78) or patellofemoral compartment (HR 2.76, 95% CI 0.46-16.44). In this symptomatic, population-based cohort, quadriceps weakness predicted whole knee and medial TF cartilage loss after 3 years. To our knowledge, this is the first study to show that a simple clinical examination of quadriceps strength can predict the risk of knee cartilage loss.

High-level artemisinin-resistance with quinine co-resistance emerges in P. falciparum malaria under in vivo artesunate pressure

BackgroundHumanity has become largely dependent on artemisinin derivatives for both the treatment and control of malaria, with few alternatives available. A Plasmodium falciparum phenotype with delayed parasite clearance during artemisinin-based combination therapy has established in Southeast Asia, and is emerging elsewhere. Therefore, we must know how fast, and by how much, artemisinin-resistance can strengthen.MethodsP. falciparum was subjected to discontinuous in vivo artemisinin drug pressure by capitalizing on a novel model that allows for long-lasting, high-parasite loads. Intravenous artesunate was administered, using either single flash-doses or a 2-day regimen, to P. falciparum-infected humanized NOD/SCID IL-2Rγ−/−immunocompromised mice, with progressive dose increments as parasites recovered. The parasite’s response to artemisinins and other available anti-malarial compounds was characterized in vivo and in vitro.ResultsArtemisinin resistance evolved very rapidly up to extreme, near-lethal doses of artesunate (240 mg/kg), an increase of > 3000-fold in the effective in vivo dose, far above resistance levels reported from the field. Artemisinin resistance selection was reproducible, occurring in 80% and 41% of mice treated with flash-dose and 2-day regimens, respectively, and the resistance phenotype was stable. Measuring in vitro sensitivity proved inappropriate as an early marker of resistance, as IC50 remained stable despite in vivo resistance up to 30 mg/kg (ART-S: 10.7 nM (95% CI 10.2–11.2) vs. ART-R30: 11.5 nM (6.6–16.9), F = 0.525, p = 0.47). However, when in vivo resistance strengthened further, IC50 increased 10-fold (ART-R240 100.3 nM (92.9–118.4), F = 304.8, p < 0.0001), reaching a level much higher than ever seen in clinical samples. Artemisinin resistance in this African P. falciparum strain was not associated with mutations in kelch-13, casting doubt over the universality of this genetic marker for resistance screening. Remarkably, despite exclusive exposure to artesunate, full resistance to quinine, the only other drug sufficiently fast-acting to deal with severe malaria, evolved independently in two parasite lines exposed to different artesunate regimens in vivo, and was confirmed in vitro.ConclusionP. falciparum has the potential to evolve extreme artemisinin resistance and more complex patterns of multidrug resistance than anticipated. If resistance in the field continues to advance along this trajectory, we will be left with a limited choice of suboptimal treatments for acute malaria, and no satisfactory option for severe malaria.

Strain gauges debonding fault detection for structural health monitoring

Physical redundancy is a common approach in applications with safety-critical systems. But that is not always possible in some structural health monitoring (SHM) applications whether for economic, spatial, or safety reasons. Many SHM sensor validating applications only rely on analytical redundancy. In such circumstances, sensor faults and structural damage need to be assuredly discriminated. A self-diagnosis strain sensor operating in a continuous online SHM scenario is considered. The sensor is based on a full electric resistance strain gauge Wheatstone bridge. The state of the art shows that such a sensor has not yet been developed. The loop current step response (LCSR) is a well-known method to detect strain gauge debonding. However, applying the LCSR method to a full strain gauge Wheatstone bridge has some limitations analyzed in this paper. Two new methods for detecting strain gauge debonding are proposed and evaluated. These methods are based on consistency checking of the strain gauges grids temperature measurements—employing an array of (a) digital contact temperature sensors or (b) quasi-contact microelectromechanical system thermopile sensors. The experimental results reveal that both methods are suitable for application in an SHM self-diagnosis sensor scenario. However, the quasi-contact measuring method showed to be more sensitive to the strain gauge grid debonding fault though.

Activation of Caenorhabditis elegans Levamisole-Sensitive and Mammalian Nicotinic Receptors by the Antiparasitic Bephenium.

Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels involved in neuromuscular transmission. In nematodes, muscle nAChRs are targets of antiparasitic drugs. Bephenium is an anthelmintic compound whose molecular action in the free-living nematode Caenorhabditis elegans, which is a model for anthelmintic drug discovery, is poorly known. We explored the effect of bephenium on C. elegans locomotion and applied single-channel recordings to identify its molecular target, mechanism of action, and selectivity between mammalian and C. elegans nAChRs. As in parasites, bephenium paralyzes C. elegans A mutant strain lacking the muscle levamisole-sensitive nAChR (L-AChR) shows full resistance to bephenium, indicating that this receptor is the target site. Bephenium activates L-AChR channels from larvae muscle cells in the micromolar range. Channel activity is similar to that elicited by levamisole, appearing mainly as isolated brief openings. Our analysis revealed that bephenium is an agonist of L-AChR and an open-channel blocker at higher concentrations. It also activates mammalian muscle nAChRs. Opening events are significantly briefer than those elicited by ACh and do not appear in activation episodes at a range of concentrations, indicating that it is a very weak agonist of mammalian nAChRs. Recordings in the presence of ACh showed that bephenium acts as a voltage-dependent channel blocker and a low-affinity agonist. Molecular docking into homology-modeled binding-site interfaces represent the binding mode of bephenium that explains its partial agonism. Given the great diversity of helminth nAChRs and the overlap of their pharmacological profiles, unraveling the basis of drug receptor-selectivity will be required for rational design of anthelmintic drugs.

Experimental Investigations into the Mechanical, Tribological, and Corrosion Properties of Hybrid Polymer Matrix Composites Comprising Ceramic Reinforcement for Biomedical Applications.

Hybrid polymer matrix composites (HPMC) are prominent material for the formation of biomaterial and offer various advantages such as low cost, high strength, and the fact that they are easy to manufacture. However, they are associated with low mechanical (low hardness) and tribological properties (high wear rate). The average hip joint load fluctuates between three to five times of the body weight during jumping and jogging and depends on various actions relating to body positions. Alternate bone and prosthesis material plays a critical role in attaining strength as it determines the method of load transferred to the system. The material property called modulus of elasticity is an important design variable during the selection of the geometry and design methodology. The present work is demonstrated on how to improve the properties of high-density polyethylene (HDPE) substantially by the addition of bioceramic fillers such as titanium oxide (TiO2) and alumina (Al2O3). The volume fractions of Al2O3 and TiO2 are limited to 20% and 10%, respectively. Samples were fabricated as per ASTM standards using an injection moulding machine and various properties such as mechanical (tensile, flexural, and impact), tribological (hardness, wear), and corrosion including SEM, density, and fractography analysis studied. Experimental results revealed that an injection moulding process is suitable for producing defect-free mould HPMC. HPMC comprising 70% HDPE/20% Al2O3/10% TiO2 has proved biocompatible and a substitute for biomaterial. A substantial increase in the mechanical and tribological properties and full resistance to corrosion makes HPMC suitable for use in orthopaedic applications such as human bone replacement, bone fixation plates, hip joint replacement, bone cement, and bone graft in bone surgery.

Registration of ‘CP 09‐1430’ Sugarcane

‘CP 09‐1430’ (Reg. No. CV‐178; PI 686940) sugarcane (a complex hybrid of Saccharum spp.) was developed and released (6 June 2016) through cooperative research conducted by the USDA–ARS Sugarcane Field Station, Canal Point, the University of Florida, and the Florida Sugarcane League, Inc., for use on organic muck soils. The clone results from CP cross #06‐0576 (CPCL 00‐6756 × CP 01‐2390) and possesses the following agronomic traits: high cane and sucrose yields, high economic index, and acceptable commercial recoverable sucrose on muck soils. It germinates well, particularly under wet planting conditions. Results of replicated trials on muck soils (2012, five locations; 2013, two locations) included 19 total harvests for the three crop stages. Yield comparisons with check cultivars (‘CP 72‐2086’, ‘CP 78‐1628’, and ‘CP 89‐2143’) indicated CP 09‐1430 had significantly higher means than CP 89‐2143 (P = 0.10) for the following: stalk weight (1.38 kg stalk−1), second ratoon cane yield (129.4 Mg ha−1), mean cane yield of three crops (146.4 Mg ha−1), sugar yield (plant cane [21.9 Mg ha−1], second ratoon [13.1 Mg ha−1], and overall mean for the three crop harvests [17.6 Mg ha−1]); and the economic index (US$3137 ha−1). CP 09‐1430 is susceptible to brown rust, scald, and ratoon stunting disease and is moderately resistant to orange rust, smut, and Sugarcane yellow leaf virus, with full resistance to mosaic. It is moderately resistant to freezing temperatures.

Characterization of culturable airborne bacteria and antibiotic susceptibility profiles of indoor and immediate-outdoor environments of a research institute in Ghana

Background: The study was conducted to determine the bacterial composition and antibiotic susceptibility profiles of a research institute at the University of Ghana where workers and students spend about 70-85% of their lives in indoor and immediate-outdoor environments. This is imperative as one-third of the recognized infectious diseases are transmitted through airborne-route. Furthermore, the increasing rate of bacterial antimicrobial resistance associated with such environments poses serious public health challenges. Methods: A total of 42 airborne samples were collected from eight major sites at the Department of Biochemistry, Cell and Molecular Biology (BCMB), using passive bacterial sampling techniques. Standard phenotypic microbiological procedures were used to characterize the isolates. Antibiotic susceptibility profiles were determined using standard disk diffusion method and guidelines of Clinical and Laboratory Standards Institute (CLSI). Results: Four groups of bacterial isolates were identified from the total samples collected with Gram positive bacilli as the most common. All the isolates showed resistance to beta lactam and sulfonamide classes of antibiotics with full resistance (100%) to ampicillin and penicillin. In total, seven different anti-biotypes were observed with the highest susceptibility displayed towards tetracycline and gentamycin. Significantly, the various air sampling sites of the institute indicated the presence of bacteria with the majority showing multiple antibiotics resistance. Conclusions: Although the recovery of bacteria from supposed sterile environments calls for attention, the observed low contamination rate as compared to the WHO standard suggests a minimum risk of exposure of students and workers to airborne microbial contamination.

Characterization of culturable airborne bacteria and antibiotic susceptibility profiles of indoor and immediate-outdoor environments of a research institute in Ghana.

Background: The study was conducted to determine the bacterial composition and antibiotic susceptibility profiles of a research institute at the University of Ghana where workers and students spend about 70-85% of their lives in indoor and immediate-outdoor environments. This is imperative as one-third of the recognized infectious diseases are transmitted through airborne-route. Furthermore, the increasing rate of bacterial antimicrobial resistance associated with such environments poses serious public health challenges. Methods: A total of 42 airborne samples were collected from eight major sites at the Department of Biochemistry, Cell and Molecular Biology (BCMB), using passive bacterial sampling techniques. Standard phenotypic microbiological procedures were used to characterize the isolates. Antibiotic susceptibility profiles were determined using standard disk diffusion method and guidelines of Clinical and Laboratory Standards Institute (CLSI). Results: Four groups of bacterial isolates were identified from the total samples collected withGram positivebacilli as the most common. All the isolates showed resistance tobeta lactamand sulfonamideclasses of antibiotics with full resistance (100%) to ampicillin and penicillin. In total, seven different anti-biotypes were observed with the highest susceptibility displayed towards tetracycline and gentamycin. Significantly, the various air sampling sites of the institute indicated the presence of bacteria with the majority showing multiple antibiotics resistance. Conclusions: Although the recovery of bacteria from supposed sterile environments calls for attention, the observed low contamination rate as compared to the WHO standard suggests a minimum risk of exposure of students and workers to airborne microbial contamination.

Critical Role of Supporting Electrolyte Selection on Flow Battery Cost: Combined Effects of Cell Materials and Operation

With commercial development of redox flow batteries (RFBs) underway and given the importance of capital cost, we analyze RFB supporting electrolyte charge carrier, membrane, open-circuit voltage (OCV), cell performance, and active species size as possible major cost drivers. Physics-based and techno-economic modeling includes a porous electrode model and accounts for chemical and material selection and amounts, stack and tank sizing, and flow-dependent pumping power.1 The 1D porous electrode model, validated in a prior study,2 couples mass transfer, charge transfer, and ohmic transport to estimate the electrode contribution to full cell area specific resistance (ASR). Primary findings for a 5-h discharge of an aqueous RFB are that present costs are in general $10 ~ 100/kWh lower with 1.4 V versus 1.0 V OCV, size selective separators (SSS) versus ion exchange membranes (IEM), as shown in Figure 1a. Large active species size alone would increase costs slightly due to higher viscosity and thus higher pumping cost, reduced mass transfer, and higher mass-based electrolyte cost versus lower molecular weight actives. However, large actives enable SSS use, and the slight incremental cost is more than offset by cost reductions from the high conductivity and inherent low cost of SSS membranes. In the future, the costs of different scenarios converge assuming significant cost reductions in the stack, membranes, and unit cost less materials. In Figure 1b, multiple options, led by H+/IEM with small active species and OCV > 1.3 V, can meet a Joint Center for Energy Storage Research target adjusted to $100/kWh for 5-h storage. Implications of the work here are: 1) recommendations on the path to enable low cost aqueous RFBs, and 2) a modeling tool and opportunity to quantify cost implications of additional factors held constant in the present study. One recommendation is the further exploration of IEMs using H+ or Na+ and SSS, as these membranes have relatively favorable likelihoods to enable system cost targets. We also suggest development work towards redox couples and conditions for high OCV, which enhances electrolyte energy content and stack power. Subsequently, engineering effort and adoption of RFBs is needed to enable significant reduction in cost through high volume manufacturing of stacks, membranes, and overall RFB systems. Lastly, one can use the present model to explore the effect of various additional parameters on cost. Such parameters include: the cell components (e.g., electrode properties, membrane thickness) affecting ASR, the operation (e.g., flow rate, V-I operating point) affecting ASR, and the application (e.g., discharge duration) affecting power-related cost (e.g., stack) versus energy-related cost (e.g., electrolyte volume, tank size). Acknowledgments This work was supported by the Joint Center for Energy Storage Research (JCESR), an Energy Innovation Hub funded by the United States Department of Energy. JDM acknowledges additional support from the NSF Graduate Research Fellowship.

Rapid Optimization of Antibiotic Therapy for Multidrug-Resistant Gram-Negative Infections Using Nanopore Whole Genome Sequencing

Background: Standard antimicrobial susceptibility testing (AST) approaches lead to delays in the selection of optimal antimicrobial therapy. We sought to determine the accuracy of antimicrobial resistance (AMR) determinants identified by Nanopore whole genome sequencing in predicting AST results. Methods: Using a cohort of 40 clinical Klebsiella pneumoniae isolates, three separate sequencing and analysis pipelines were performed: (1) a real-time Nanopore analysis approach that identified acquired AMR genes, (2) an assembly-based Nanopore approach that identified acquired AMR genes and chromosomal mutations, and (3) a Pilon-corrected Nanopore-Illumina hybrid approach. The Pilon-corrected assemblies served as the reference standard to determine the accuracy of Nanopore sequencing results. Findings: With the real-time analysis approach, full annotation of acquired AMR genes occurred within 8 hours of subcultured isolates. Assemblies sufficient for full resistance gene and single nucleotide polymorphism annotation were available within 14 hours from subcultured isolates. The overall agreement of genotypic results and anticipated AST results for the 40 K. pneumoniae isolates was 75% (range 30-95%) and 91% (range 80-98%) for the real-time approach and the assembly approach, respectively. Evaluating the patients contributing the 40 isolates, the real-time approach and assembly approach could substantially shorten the median time to effective antibiotic therapy by 20 hours and 26 hours, respectively, compared to standard AST. Interpretations: Nanopore sequencing offers a rapid approach to both accurately identify resistance mechanisms as well as to predict AST results. Bioinformatics improvements enabling real-time alignment coupled with rapid extraction and library preparation will further enhance the accuracy and workflow of the Nanopore real-time approach. Funding Statement: The work was supported by funding from the National Institutes of Health K23-AI127935 awarded to PDT, a Canadian Institutes of Health Research fellowship awarded to YF, an R01-HG009190 awarded to WT, an R01-HG006677 awarded to MCS, and an R21-AI130608 awarded to PJS. Declaration of Interests: PDT has received research funding from Merck, outside of the submitted work. KCC has received research funding from GenMark, Inc. and Singulex outside of the submitted work. WT has two patents (8,748,091 and 8,394,584) licensed to Oxford Nanopore. WT, YF, PJS have received travel funds from Oxford Nanopore Technologies. PJS has received grants and personal fees from Accelerate Diagnostics, grants from BD Diagnostics, Inc., grants from bioMerieux, Inc., grants from Check-Points Diagnostics, BV, grants from Hardy Diagnostics, personal fees from Roche Diagnostics, personal fees from Opgen Inc, outside the submitted work. All other authors declare no competing interests. Ethics Approval Statement: The Johns Hopkins University School of Medicine Institutional Review Board approved this study, with a waiver of informed consent.

MntC-Dependent Manganese Transport Is Essential for Staphylococcus aureus Oxidative Stress Resistance and Virulence.

Staphylococcus aureus is a human pathogen that has developed several approaches to evade the immune system, including a strategy to resist oxidative killing by phagocytes. This resistance is mediated by production of superoxide dismutase (SOD) enzymes which use manganese as a cofactor. S.aureus encodes two manganese ion transporters, MntABC and MntH, and a possible Nramp family manganese transporter, exemplified by S.aureus N315 SA1432. Their relative contributions to manganese transport have not been well defined in clinically relevant isolates. For this purpose, insertional inactivation mutations were introduced into mntC, mntH, and SA1432 individually and in combination. mntC was necessary for full resistance to methyl viologen, a compound that generates intracellular free radicals. In contrast, strains with an intact mntH gene had a minimal increase in resistance that was revealed only in mntC strains, and no change was observed upon mutation of SA1432 in strains lacking both mntC and mntH Similarly, MntC alone was required for high cellular SOD activity. In addition, mntC strains were attenuated in a murine sepsis model. To further link these observations to manganese transport, an S.aureus MntC protein lacking manganese binding activity was designed, expressed, and purified. While circular dichroism experiments demonstrated that the secondary and tertiary structures of this protein were unaltered, a defect in manganese binding was confirmed by isothermal titration calorimetry. Unlike complementation with wild-type mntC, introduction of the manganese-binding defective allele into the chromosome of an mntC strain did not restore resistance to oxidative stress or virulence. Collectively, these results underscore the importance of MntC-dependent manganese transport in S.aureus oxidative stress resistance and virulence.IMPORTANCE Work outlined in this report demonstrated that MntC-dependent manganese transport is required for S.aureus virulence. These study results support the model that MntC-specific antibodies elicited by a vaccine have the potential to disrupt S.aureus manganese transport and thus abrogate to its virulence.

HIV-1 Antiretroviral Resistance in Cuba, 2009-2014.

INTRODUCTION By the end of 2017, there were more than 28,000 individuals living with HIV in Cuba, over 80% receiving antiretroviral therapy, which dramatically reduces viral replication, improves immune status and decreases risk of transmission. These results could be jeopardized by emergence of HIV-1 drug resistance. In 2009, a test for HIV-1 genotypic resistance was introduced in routine clinical practice in Cuba. OBJECTIVE Investigate antiretroviral resistance and its relation to subtype distribution in HIV-1 treatment-naive and previously treated patients in Cuba. METHODS Resistance and HIV-1 subtype distribution were determined in 342 antiretroviral treatment-naive patients and 584 previously treated for HIV-1 whose blood specimens were sent to the Pedro Kouri Tropical Medicine Institute during 2009-2014. Transmitted drug resistance was determined using the Calibrated Population Resistance Tool v.6. Drug resistance analysis was conducted using the algorithm Rega v9.1.0. RESULTS Prevalence of transmitted drug resistance was 11.4%, and 41% of mutated viruses exhibited dual-class resistance to nucleoside reverse transcriptase inhibitor and non-nucleoside reverse transcriptase inhibitor. Overall, 84.9% of patients had ≥1 resistance mutation, 80% had ≥1 nucleoside reverse transcriptase inhibitor mutation, 71.4% had ≥1 non-nucleoside reverse transcriptase inhibitor mutation and 31.7% had ≥1 protease inhibitor mutation. K65R and K101E mutations were significantly more frequent in subtype C, L210W in CRF19_cpx, and M47V/I in CRF BGs (20, 23, 24). Full class resistance to nucleoside reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors, protease inhibitors and multidrug resistance were detected in 21.2%, 32.4%, 8% and 4.1% of patients, respectively. Average percentage resistance to nucleoside reverse transcriptase inhibitor, protease inhibitor, full class resistance to nucleoside reverse transcriptase inhibitor, protease inhibitor and multidrug resistance increased in patients failing two or more regimens. Nevertheless, after 2011, a declining trend was observed in the frequency of multidrug resistance and full class resistance to nucleoside reverse transcriptase inhibitors and protease inhibitors. CONCLUSIONS Detected levels of transmitted drug resistance highlight the need for a national surveillance study in treatment-naive patients. Resistance prevalence is high in previously treated patients but appears to be decreasing over time. The frequency of resistance mutations in recombinant forms of HIV in Cuba needs further study. KEYWORDS Antiretroviral therapy, highly active antiretroviral therapy, HIV, anti-HIV agents, drug resistance, multiple drug resistance, Cuba.

Abstract 4038: A functional genomics approach to the identification of genes involved in resistance to Oxaliplatin in colorectal cancer

Abstract Currently, the first line therapy for metastatic colorectal cancer (CRC) is the FOLFOX regimen, which includes Oxaliplatin in combination with Fluorouracil and Leucovorin. Responses to FOLFOX range from overt regressions (35%) to full resistance (25%), and eventually all patients become resistant. Indeed, the molecular mechanisms of Oxaliplatin resistance remain mostly to be understood. To identify genes involved in sensitivity/resistance to Oxaliplatin, we designed an integrated approach including forward genetic screens and computational analyses. The pipeline includes: (i) stable transduction of CRC cells with pooled shRNA libraries; (ii) Oxaliplatin treatment for six weeks to select a drug-resistant subpopulation; (iii) identification of shRNA constructs enriched in the Oxaliplatin-resistant subpopulation by next-generation sequencing analysis. We focused the shRNA libraries targeting phosphatases and ubiquitin conjugation pathway genes. The first are involved in several biological processes, reversing the biochemical activity of kinases and typically acting as feedback loop regulators. The second direct proteins to degradation by the proteasome regulating homeostasis, cell cycle, and DNA repair pathways. HCT116 CRC cells, markedly sensitive to Oxaliplatin, were transduced in duplicate with both shRNA libraries. After Oxaliplatin treatment for six weeks, resistant populations emerged from the library-transduced cells, but not from control-transduced cells. To reveal shRNA constructs responsible of such phenotype, we extracted genomic DNA from transduced cells and, upon amplification of shRNA constructs by targeted PCR and next-generation sequencing, we compared the repertoire of shRNA sequences in HCT116 cells selected with Oxaliplatin with that of cells grown in standard medium. Screening hits were then prioritized for functional characterization, resulting in a phosphatase (CDC25C) and an ubiquitin ligase candidate (DCAF17). In conclusion, we successfully setup a functional genomic screening for acquired resistance to Oxaliplatin in CRC. Specific shRNA constructs from both the phosphatase and ubiquitin ligase libraries were enriched after Oxaliplatin treatment, highlighting candidate genes whose loss of function could potentially drive resistances to Oxaliplatin. Citation Format: Federica Invrea, Francesca Cavicchioli, Consalvo Petti, Rosalia Russo, Claudio Isella, Enzo Medico. A functional genomics approach to the identification of genes involved in resistance to Oxaliplatin in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4038.

Postexercise hypotension and autonomic modulation response after full versus split body resistance exercise in trained men.

To investigate the effects of full versus split body resistance exercise on postexercise hypotension and autonomic modulation in trained men. Sixteen resistance recreationally trained males (age, 24.9±5.3 years) performed three randomized trials: upper body (UB), lower body (LB), and full body (FB) conditions. Blood pressure and heart rate variability were collected at rest, immediately postexercise, post-10, and post-30 min during recovery. For systolic blood pressure, delta (30 min minus rest) was lower for the FB condition compared to the UB (−10.1±7.4 mmHg [FB] vs. −3.3±12.6 mmHg [LB] vs. −1.9±8.1 mmHg [UB], P=0.004). For diastolic blood pressure (−8.2±10.9 mmHg [FB] vs. −1.5±9.8 mmHg [LB] vs. −8.7±11.4 mmHg [UB], P=0.038) and mean blood pressure delta during recovery (−11.7±14 mmHg [FB] vs. −2.2±10.6 mmHg [LB] vs. −5.2±6.8 mmHg [UB], P=0.045), there were statistically significant lower values in the FB condition in relation to the LB condition. Regarding heart rate variability, there were no significant differences between conditions, however, the square root of the mean squared difference between adjacent RR intervals presented an increase until post-30 during recovery (P<0.001), and there was a higher values of low frequency and lower values of high frequency across time (P<0.001). Postexercise hypotension was influenced by the amount of muscle mass involved in a bout of resistance exercise, with the FB condition inducing lower systolic blood pressure in relation to the UB after exercise, as well as a great decrease in postexercise diastolic and mean blood pressure compared with the LB. The autonomic modulation response was similar between conditions during recovery.

Development of user-friendly markers for disease resistance to black root rot of tobacco through genotyping by sequencing

Black root rot (BRR), a disease caused by the hemibiotrophic fungus Thielaviopsis basicola, seriously compromises yield and leaf quality in tobacco (Nicotiana tabacum). Full resistance to black root rot, conferred by the resistance to BRR 1 (RBRR1) locus from Nicotiana debneyi Domin, was transferred to a burley tobacco cultivar through interspecific hybridization. Some undesirable traits potentially caused by linkage drag restrict wider application of RBRR1 in flue-cured tobacco. Therefore, user-friendly molecular markers are needed to assist selection for resistance to black root rot and to break the unfavorable linkage. Genotyping by sequencing (GBS) is a rapid and robust approach for reduced representation sequencing of multiplexed genomic DNA samples that combines genome-wide molecular marker discovery with genotyping. In the present study, we used GBS to identify single-nucleotide polymorphisms (SNPs) linked to the RBRR1 locus, and PCR-based assays for detection of these SNPs were also developed. Sequence analysis of the SNP markers suggested that RBRR1 is located on chromosome 17, providing a basis for map-based cloning of this valuable gene. Co-dominant CAPS markers that co-segregate with the disease-resistant phenotype offer user-friendly tools for tobacco breeding and variety improvement. Furthermore, tested with diverse N. tabacum germplasm, SS192650 displayed 100% selection accuracy for resistance to BRR, suggesting that this marker can be used in diverse tobacco populations.