Polymyxin B Heteroresistance and Adaptive Resistance in Multidrug- and Extremely Drug-Resistant Acinetobacter baumannii.

Acinetobacter baumannii is an emerging pathogen associated with nosocomial infections and multidrug resistance. Polymyxin B has been used to treat infections caused by multidrug-resistant (MDR) A. baumannii but an increase in polymyxin B resistance has been observed. We aimed to determine the diversity, antimicrobial susceptibility, presence of polymyxin B heteroresistance and adaptive resistance in 72 A. baumannii clinical isolates from two public hospitals in Rio de Janeiro. The isolates were identified by sequencing of rpoB gene. Determination of the genetic diversity of isolates was performed by pulsed-field gel electrophoresis and oxacillinases genes were detected by polymerase chain reaction. The polymyxin B heteroresistance was analyzed by population analysis profile and adaptive resistance was evaluated after serial daily passages of isolates in broth containing increasing polymyxin B concentrations. The results showed that 49% of the isolates were collected from respiratory system and 62% were MDR, while 35% were extensively drug resistant. Additionally, all the isolates carried blaOXA-23-like, blaOXA-51-like genes and ISAba1, while 1% had blaOXA-24-like gene. The association of ISAba1-blaOXA-23 was found in 96% of the isolates. Polymyxin B heteroresistance was found in 36% of the isolates and polymyxin B adaptive resistance was not found in the isolates. Our study demonstrated the high resistance to antimicrobials used in clinical practice and the spread of oxacillinases genes and insertion sequence (IS). We also reported the presence of heteroresistance to polymyxin B used as a last-resort therapy for MDR A. baumannii.

Comparative feeding kinematics of tropical hylid tadpoles.

Anuran larvae, which are otherwise simple in shape, typically have complex keratinized mouthparts (i.e. labial teeth and jaw sheaths) that allow them to graze upon surfaces. The diversity in these structures among species presumably reflects specializations that allow for maximal feeding efficiency on different types of food. However, we lack a general understanding of how these oral structures function during feeding. We used high-speed digital imaging (500 Hz) to observe tadpoles of six species from the anuran family Hylidae grazing on a standardized food-covered substrate. Tadpoles of these species vary in the number of labial tooth rows, belong to two different feeding guilds (benthic and nektonic), and inhabit ponds and streams. We confirmed that the labial teeth in these species serve two functions: anchoring the mouth to the substrate and raking material off of the substrate. In general, tadpoles with a larger maximum gape or those with fewer labial tooth rows opened and closed their mouths slower than tadpoles with smaller gape or more tooth rows. Nektonic feeding tadpoles released each of their tooth rows proportionally earlier in the gape cycle compared with benthic feeding tadpoles. Lastly, we found some support for the idea that deformation of the jaw sheaths during a feeding cycle is predictable based on tadpole feeding guild. Collectively, our data show that anatomical (e.g. number of labial teeth) and ecological features (e.g. feeding guild) of tadpoles significantly influence how tadpoles open and close their mouths during feeding.

Adaptation and validation of a method for evaluating the bactericidal activity of ethyl alcohol in gel format 70% (w/w).

Ethyl alcohol 70% (w/w) is a broad-spectrum bactericidal agent that is mandatorily marketed as a gel in Brazil since 2002. However, there is no method described for the microbiological quality control of surface disinfectants in the form of gel, which makes it impossible to monitor the effectiveness of these products by the Health Surveillance. Thus, the present study aimed to adapt an analytical method and validate it so that it is possible to evaluate the bactericidal activity of ethyl alcohol-based disinfectants at 70% (w/w) in gel form. The proposed analytical method is an adaptation of a method developed by the Association of Official Analytical Chemists (AOAC), which is currently used to evaluate the bacterial activity of disinfectants in the form of spray and aerosol. The method consisted of challenging the disinfectant by putting it in contact with 60 carriers - previously contaminated with the test microorganism - during the contact time established by the manufacturer. The bacteria used in this work were Staphylococcus aureus CBRVS 00039 ATCC 6538, Salmonella enterica CBRVS 00028 ATCC 10708, and Pseudomonas aeruginosa CBRVS 00025 ATCC 15442. The parameters chosen for the validation of the method were: matrix effect, robustness, and repeatability. The experiments performed with a disinfectant matrix showed that there is no interference of the gel on the effectiveness of alcohol. Satisfactory results regarding the evaluation of bactericidal activity were obtained for the three microorganisms tested when the volume of 100 µL of disinfectant was applied. The method also showed good repeatability, as it proved to be robust with modification of incubation times, equipment and analysts. The development and validation of this method were extremely important for the quality monitoring of these products, besides being the first method described for the evaluation of gel disinfectants.

Determination of the Pharmacokinetics and Pharmacodynamics of Isoniazid, Rifampicin, Pyrazinamide and Ethambutol in a Cross-Over Cynomolgus Macaque Model of Mycobacterium tuberculosis Infection.

Innovative cross-over study designs were explored in non-human primate (NHP) studies to determine the value of this approach for the evaluation of drug efficacy against tuberculosis (TB). Firstly, the pharmacokinetics (PK) of each of the drugs Isoniazid (H), Rifampicin (R), Pyrazinamide (Z) and Ethambutol (E), that are standardly used for the treatment of tuberculosis, was established in the blood of macaques after oral dosing as a monotherapy or in combination. Two studies were conducted to evaluate the pharmacokinetics and pharmacodynamics of different drug combinations using cross-over designs. The first employed a balanced, three-period Pigeon design with an extra period; this ensured that treatment by period interactions and carry-over could be detected comparing the treatments HR, HZ and HRZ using H37Rv as the challenge strain of Mycobacterium tuberculosis (M. tb). Although the design accounted for considerable variability between animals, the three regimens evaluated could not be distinguished using any of the alternative endpoints assessed. However, the degree of pathology achieved using H37Rv in the model during this study was less than expected. Based on these findings, a second experiment using a classical AB/BA design comparing HE with HRZ was conducted using the M. tb Erdman strain. More extensive pathology was observed, and differences in computerized tomography (CT) scores and bacteriology counts in the lungs were detected, although due to the small group sizes, clearer differences were not distinguished. Type 1 T helper (Th1) cell response profiles were characterized using the IFN-γ ELISPOT assay and revealed differences between drug treatments that corresponded to decreases in disease burden. Therefore, the studies performed support the utility of the NHP model for the determination of PK/PD of TB drugs, although further work is required to optimize the use of cross-over study designs.

Influence of substrate orientation on tadpoles' feeding efficiency.

In nature, tadpoles encounter food on substrates oriented at different angles (e.g. vertically along stems, horizontally on the bottom of the pond). We manipulated the orientation of food-covered surfaces to test how different orientations of surfaces affect tadpoles' feeding efficiency. We studied taxa that differed in the oral morphology of their larvae and position in the water column. We hypothesized that species would differ in their ability to graze upon surfaces at different orientations and that differences in the tadpoles' feeding ability would result in different growth rates. The orientation of food-covered surfaces did not affect the growth rate of bottom-dwelling tadpoles (whose growth rate varied only between species). Among midwater tadpoles, some species appear to have a generalist strategy and experienced a high relative growth rate on numerous substrate orientations, whereas others achieved high growth rates only on flat substrates (i.e. at 0° and 180°). We conclude that oral morphology constrains tadpoles' ability to feed at different substrate orientations, and this could lead to niche partitioning in structurally complex aquatic environments. Because physical parameters of the environment can affect tadpoles' growth rate, characterizing these features might help us better understand how competition structures tadpole assemblages.

Assessing Pharmacodynamic Interactions in Mice Using the Multistate Tuberculosis Pharmacometric and General Pharmacodynamic Interaction Models.

The aim of this study was to investigate pharmacodynamic (PD) interactions in mice infected with Mycobacterium tuberculosis using population pharmacokinetics (PKs), the Multistate Tuberculosis Pharmacometric (MTP) model, and the General Pharmacodynamic Interaction (GPDI) model. Rifampicin, isoniazid, ethambutol, or pyrazinamide were administered in monotherapy for 4 weeks. Rifampicin and isoniazid showed effects in monotherapy, whereas the animals became moribund after 7 days with ethambutol or pyrazinamide alone. No PD interactions were observed against fast-multiplying bacteria. Interactions between rifampicin and isoniazid on killing slow and non-multiplying bacteria were identified, which led to an increase of 0.86 log10 colony-forming unit (CFU)/lungs at 28 days after treatment compared to expected additivity (i.e., antagonism). An interaction between rifampicin and ethambutol on killing non-multiplying bacteria was quantified, which led to a decrease of 2.84 log10 CFU/lungs at 28 days after treatment (i.e., synergism). These results show the value of pharmacometrics to quantitatively assess PD interactions in preclinical tuberculosis drug development.

Differential effects of temperature on the feeding kinematics of the tadpoles of two sympatric anuran species.

Temperature impacts ectotherm performance by influencing many biochemical and physiological processes. When well adapted to their environment, ectotherms should perform most efficiently at the temperatures they most commonly encounter. In the present study, we tested how differences in temperature affects the feeding kinematics of tadpoles of two anuran species: the benthic tadpole of Rhinella schneideri and the nektonic tadpole of Trachycephalus typhonius. Benthic and nektonic tadpoles have segregated distributions within ponds and thus tend to face different environmental conditions, such as temperature. Muscle contractile dynamics, and thus whole organism performance, is primarily temperature dependent for ectotherms. We hypothesized that changes in mean temperatures would have differential effects on the feeding kinematics of these two species. We conducted a laboratory experiment in which we used high-speed videography to record tadpoles foraging at cold and warm temperatures. In general, tadpoles filmed at warm temperatures opened their jaws faster, attained maximum gape earlier, and exhibited shorter gape cycles than tadpoles in cold temperatures, irrespective of species. We also found species x temperature interactions regarding the closing phase velocity, and the percentage of time it takes tadpoles to achieve maximum gape and to start closing their jaws. These interactions could indicate that these two co-occurring species differ in their sensitivity to differences in water temperature and have temperature-dependent feeding strategies that maximize feeding performance in their preferred environment.

Tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide and N-benzyl-6',7'-dihydrospiro[piperidine-4,4'-thieno[3,2-c]pyran] analogues with bactericidal efficacy against Mycobacterium tuberculosis targeting MmpL3.

Mycobacterium tuberculosis is a major human pathogen and the causative agent for the pulmonary disease, tuberculosis (TB). Current treatment programs to combat TB are under threat due to the emergence of multi-drug and extensively-drug resistant TB. As part of our efforts towards the discovery of new anti-tubercular leads, a number of potent tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide (THPP) and N-benzyl-6',7'-dihydrospiro[piperidine-4,4'-thieno[3,2-c]pyran] (Spiro) analogues were recently identified against Mycobacterium tuberculosis and Mycobacterium bovis BCG through a high-throughput whole-cell screening campaign. Herein, we describe the attractive in vitro and in vivo anti-tubercular profiles of both lead series. The generation of M. tuberculosis spontaneous mutants and subsequent whole genome sequencing of several resistant mutants identified single mutations in the essential mmpL3 gene. This 'genetic phenotype' was further confirmed by a 'chemical phenotype', whereby M. bovis BCG treated with both the THPP and Spiro series resulted in the accumulation of trehalose monomycolate. In vivo efficacy evaluation of two optimized THPP and Spiro leads showed how the compounds were able to reduce >2 logs bacterial cfu counts in the lungs of infected mice.