Over expression of AdeABC and AcrAB-TolC efflux systems confers tigecycline resistance in clinical isolates of Acinetobacter baumannii and Klebsiella pneumoniae

Abstract: INTRODUCTION: Due to the wide use of tigecycline in the treatment of severe infections caused by multidrug-resistant (MDR) bacteria, clinical resistance to tigecycline has increased in recent years. Here, we investigated the relationship between tigecycline resistance and the expression of efflux pumps. METHODS: Clinical isolates of Acinetobacter baumannii and Klebsiella pneumoniae were consecutively collected from hospitalized patients in three hospitals. The minimum inhibitory concentration (MIC) of tigecycline was determined using the broth microdilution method. Expression levels of efflux pump genes and regulators were examined by quantitative real-time reverse transcription polymerase chain reaction. The correlations between tigecycline MICs and gene expression levels were analyzed. RESULTS: Overall, 1,026 A. baumannii and 725 K. pneumoniae strains were collected. Most strains were isolated from sputum. The tigecycline resistance rate was 13.4% in A. baumannii isolates and 6.5% in K. pneumoniae isolates. Overexpression of AdeABC and AcrAB-TolC efflux systems was observed found in clinical tigecycline-resistant isolates. The tigecycline MIC had a linear relationship with the adeB expression level in A. baumannii isolates, but not with the acrB expression level in K. pneumoniae isolates. There were significant linear trends in the overexpression of ramA as the tigecycline MIC increased in K. pneumoniae isolates. CONCLUSIONS: Tigecycline resistance in A. baumannii and K. pneumoniae was strongly associated with the overexpression of efflux systems. More studies are needed to elucidate whether there are other regulators that affect the expression of adeB in A. baumannii and how ramA affects the expression of acrB in K. pneumoniae.

Soil CO2 efflux in central Amazonia: environmental and methodological effects

Soil respiration plays a significant role in the carbon cycle of Amazonian rainforests. Measurements of soil respiration have only been carried out in few places in the Amazon. This study investigated the effects of the method of ring insertion in the soil as well as of rainfall and spatial distribution on CO2 emission in the central Amazon region. The ring insertion effect increased the soil emission about 13-20% for sandy and loamy soils during the firsts 4-7 hours, respectively. After rainfall events below 2 mm, the soil respiration did not change, but for rainfall greater than 3 mm, after 2 hours there was a decrease in soil temperature and respiration of about 10-34% for the loamy and sand soils, with emissions returning to normal after around 15-18 hours. The size of the measurement areas and the spatial distribution of soil respiration were better estimated using the Shuttle Radar Topographic Mission (SRTM) data. The Campina reserve is a mosaic of bare soil, stunted heath forest-SHF and tall heath forest-THF. The estimated total average CO2 emissions from the area was 3.08±0.8 µmol CO2 m-2 s-1. The Cuieiras reserve is another mosaic of plateau, slope, Campinarana and riparian forests and the total average emission from the area was 3.82±0.76 µmol CO2 m-2 s-1. We also found that the main control factor of the soil respiration was soil temperature, with 90% explained by regression analysis. Automated soil respiration datasets are a good tool to improve the technique and increase the reliability of measurements to allow a better understanding of all possible factors driven by soil respiration processes.

Cellular cholesterol efflux mediated by HDL isolated from subjects with low HDL levels and coronary artery disease

OBJECTIVE: The aim of this study was to verify whether HDL particles isolated from patients with coronary artery disease (CAD) and low HDL-C had diminished ability to promote cholesterol efflux from cultured cells compared with HDL isolated from subjects without CAD and with normal HDL-C. METHODS: Smooth muscle cells isolated from human aortas cultured and radiolabeled with ³H-cholesterol were loaded with cholesterol and incubated with increasing concentrations of HDL isolated from 13 CAD patients with low HDL-C (CAD group) or from 5 controls without CAD (C group). Efflux of cellular cholesterol was measured by cellular depletion of radiolabeled cholesterol and by the appearance of ³H-cholesterol into experimental medium expressed as a percentage of total labeled cholesterol. RESULTS: Cholesterol efflux increased with the amount of HDL present in the medium, and no difference was found between groups at various HDL protein concentrations: efflux was 28 ± 6.3% (C) and 25.5 ± 8.9% (CAD) with 25 mg/mL; 34 ± 4.3% (C) and 31.9 ± 6.6% (CD) with 50 mg/mL and 39.5 ± 3.5% (C) and 37.1 ± 4.4% (CAD) with 100 mg/mL, HDL. CONCLUSION: Because the HDL fraction of CAD patients with low HDL-C have normal ability to extract cholesterol from cells of the vessel wall, it is suggested that low HDL-C atherogenicity should be ascribed to diminished concentrations of HDL particles rather than to the qualitative properties of the HDL fraction.

Carbon dioxide efflux in a rhodic hapludox as affected by tillage systems in southern Brazil

Agricultural soils can act as a source or sink of atmospheric C, according to the soil management. This long-term experiment (22 years) was evaluated during 30 days in autumn, to quantify the effect of tillage systems (conventional tillage-CT and no-till-NT) on the soil CO2-C flux in a Rhodic Hapludox in Rio Grande do Sul State, Southern Brazil. A closed-dynamic system (Flux Chamber 6400-09, Licor) and a static system (alkali absorption) were used to measure soil CO2-C flux immediately after soybean harvest. Soil temperature and soil moisture were measured simultaneously with CO2-C flux, by Licor-6400 soil temperature probe and manual TDR, respectively. During the entire month, a CO2-C emission of less than 30 % of the C input through soybean crop residues was estimated. In the mean of a 30 day period, the CO2-C flux in NT soil was similar to CT, independent of the chamber type used for measurements. Differences in tillage systems with dynamic chamber were verified only in short term (daily evaluation), where NT had higher CO2-C flux than CT at the beginning of the evaluation period and lower flux at the end. The dynamic chamber was more efficient than the static chamber in capturing variations in CO2-C flux as a function of abiotic factors. In this chamber, the soil temperature and the water-filled pore space (WFPS), in the NT soil, explained 83 and 62 % of CO2-C flux, respectively. The Q10 factor, which evaluates CO2-C flux dependence on soil temperature, was estimated as 3.93, suggesting a high sensitivity of the biological activity to changes in soil temperature during fall season. The CO2-C flux measured in a closed dynamic chamber was correlated with the static alkali adsorption chamber only in the NT system, although the values were underestimated in comparison to the other, particularly in the case of high flux values. At low soil temperature and WFPS conditions, soil tillage caused a limited increase in soil CO2-C flux.

Methane efflux in rice paddy field under different irrigation managements

Paddy rice fields may contribute to methane (CH4) emission from soil due to anaerobic conditions after flooding. Alternatives to continuous flooding irrigation in rice have been developed to mitigate CH4 efflux into the atmosphere. This study aims to investigate the effects of irrigation managements in the CH4 efflux during the rice growing season. An experiment was carried out at in Santa Maria, Rio Grande do Sul State, Brazil, during 2007/08 and 2009/10 growing seasons. The treatments were continuous flooding and intermittent irrigation in 2007/08 and continuous flooding, intermittent irrigation and flush irrigation in 2009/10. Intermittent irrigation is effective in mitigating CH4 efflux from rice fields when climatic conditions enable water absence during cultivation, but its efficiency depends on the electrochemical soil conditions during the flooding cycles.

Survey of sandfly vectors of leishmaniasis in Marambaia Island, municipality of Mangaratiba, State of Rio de Janeiro, Brazil

IntroductionThe influx and efflux of military personnel in the possible endemic areas of leishmaniasis provided the impetus for research on the sandflies on Marambaia Island.MethodsSandflies were collected with light traps installed in the 3 ecotypes of 3 areas chosen for their particular landscape aspects.ResultsIn 2009, were collected 32,006 specimens of sandflies belonging to 13 species. The species that showed highest density were Nyssomyia intermedia and Migoneimyia migonei.ConclusionsN. intermedia and M. migonei are the principal vectors of American tegumentary leishmaniasis (ATL) in Brazil; thus, extension studies related to the eco-epidemiology of these species of Marambaia Island are essential.

The effect of nitric oxide combined with fluoroquinolones against Salmonella enterica serovar Typhimurium in vitro

Two regulons, soxRS and marRAB, are associated with resistance to quinolones or multiple antibiotic in Salmonella enterica serovar Typhimurium. These regulons are activated by nitric oxide and redox-cycling drugs, such as paraquat and cause on activation of the acrAB-encoded efflux pump. In this study, we investigated the effect of nitric oxide (NO) alone and in combination with ofloxacin, ciprofloxacin, and pefloxacin against S. typhimurium clinical isolates and mutant strains in vitro. We did not observe synergistic effect against clinical isolates and SH5014 (parent strain of acr mutant), while we found synergistic effect against PP120 (soxRS mutant) and SH7616 (an acr mutant) S. typhimurium for all quinolones. Our results suggest that the efficiencies of some antibiotics, including ofloxacin, ciprofloxacin, and pefloxacin are decreased via activation of soxRS and marRAB regulons by NO in S. enterica serovar Typhimurium. Further studies are warranted to establish the interaction of NO with the genes of Salmonella and, with multiple antibiotic resistance.

Metallo-β-lactamase-production in meropenem-susceptible Pseudomonas aeruginosa isolates: risk for silent spread

The aim of this study was to characterize two metallo-β-lactamases (MBLs)-producing Pseudomonas aeruginosa clinical isolates showing meropenem susceptibility. Antimicrobial susceptibility was assessed by automated testing and Clinical and Laboratory Standards Institute agar dilution method. MBL production was investigated by phenotypic tests. Molecular typing was determined by pulsed field gel electrophoresis (PFGE). MBL-encoding genes, as well as their genetic context, were identified by polymerase chain reaction (PCR) and sequencing. The location of blaIMP-16 was determined by plasmid electrophoresis, Southern blot and hybridization. Transcriptional levels of blaIMP-16, mexB, mexD, mexF, mexY, ampC and oprD were determined by semi-quantitative real time PCR. The P. aeruginosa isolates studied, Pa30 and Pa43, showed imipenem and meropenem susceptibility by automated testing. Agar dilution assays confirmed meropenem susceptibility whereas both isolates showed low level of imipenem resistance. Pa30 and Pa43 were phenotypically detected as MBL producers. PFGE revealed their clonal relatedness. blaIMP-16 was identified in both isolates, carried as a single cassette in a class 1 integron that was embedded in a plasmid of about 60-Kb. Pa30 and Pa43 overexpressed MexAB-OprM, MexCD-OprJ and MexXY-OprM efflux systems and showed basal transcriptional levels of ampC and oprD. MBL-producing P. aeruginosa that are not resistant to meropenem may represent a risk for therapeutic failure and act as silent reservoirs of MBL-encoding genes.

Evaluation of the role of ATP-binding cassette transporters as a defence mechanism against temephos in populations of Aedes aegypti

The role of ATP-binding cassette (ABC) transporters in the efflux of the insecticide, temephos, was assessed in the larvae of Aedes aegypti. Bioassays were conducted using mosquito populations that were either susceptible or resistant to temephos by exposure to insecticide alone or in combination with sublethal doses of the ABC transporter inhibitor, verapamil (30, 35 and 40 μM). The best result in the series was obtained with the addition of verapamil (40 μM), which led to a 2x increase in the toxicity of temephos, suggesting that ABC transporters may be partially involved in conferring resistance to the populations evaluated.

Complete genome sequence of a clinical Bordetella pertussis isolate from Brazil

There has been a resurgence in the number of pertussis cases in Brazil and around the world. Here, the genome of a clinical Bordetella pertussis strain (Bz181) that was recently isolated in Brazil is reported. Analysis of the virulence-associated genes defining the pre- and post-vaccination lineages revealed the presence of the prn2-ptxS1A-fim3B-ptxP3 allelic profile in Bz181, which is characteristic of the current pandemic lineage. A putative metallo-β-lactamase gene presenting all of the conserved zinc-binding motifs that characterise the catalytic site was identified, in addition to a multidrug efflux pump of the RND family that could confer resistance to erythromycin, which is the antibiotic of choice for treating pertussis disease.

Mutational and acquired carbapenem resistance mechanisms in multidrug resistant Pseudomonas aeruginosa clinical isolates from Recife, Brazil

An investigation was carried out into the genetic mechanisms responsible for multidrug resistance in nine carbapenem-resistant Pseudomonas aeruginosaisolates from different hospitals in Recife, Brazil. Susceptibility to antimicrobial agents was determined by broth microdilution. Polymerase chain reaction (PCR) was employed to detect the presence of genes encoding β-lactamases, aminoglycoside-modifying enzymes (AMEs), 16S rRNA methylases, integron-related genes and OprD. Expression of genes coding for efflux pumps and AmpC cephalosporinase were assessed by quantitative PCR. The outer membrane proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The blaSPM-1, blaKPC-2 and blaGES-1 genes were detected in P. aeruginosaisolates in addition to different AME genes. The loss of OprD in nine isolates was mainly due to frameshift mutations, premature stop codons and point mutations. An association of loss of OprD with the overexpression of MexAB-OprM and MexXY-OprM was observed in most isolates. Hyper-production of AmpC was also observed in three isolates. Clonal relationship of the isolates was determined by repetitive element palindromic-PCR and multilocus sequence typing. Our results show that the loss of OprD along with overexpression of efflux pumps and β-lactamase production were responsible for the multidrug resistance in the isolates analysed.

Long-term C-CO2 emissions and carbon crop residue mineralization in an oxisol under different tillage and crop rotation systems

Soil C-CO2 emissions are sensitive indicators of management system impacts on soil organic matter (SOM). The main soil C-CO2 sources at the soil-plant interface are the decomposition of crop residues, SOM turnover, and respiration of roots and soil biota. The objectives of this study were to evaluate the impacts of tillage and cropping systems on long-term soil C-CO2 emissions and their relationship with carbon (C) mineralization of crop residues. A long-term experiment was conducted in a Red Oxisol in Cruz Alta, RS, Brazil, with subtropical climate Cfa (Köppen classification), mean annual precipitation of 1,774 mm and mean annual temperature of 19.2 ºC. Treatments consisted of two tillage systems: (a) conventional tillage (CT) and (b) no tillage (NT) in combination with three cropping systems: (a) R0- monoculture system (soybean/wheat), (b) R1- winter crop rotation (soybean/wheat/soybean/black oat), and (c) R2- intensive crop rotation (soybean/ black oat/soybean/black oat + common vetch/maize/oilseed radish/wheat). The soil C-CO2 efflux was measured every 14 days for two years (48 measurements), by trapping the CO2 in an alkaline solution. The soil gravimetric moisture in the 0-0.05 m layer was determined concomitantly with the C-CO2 efflux measurements. The crop residue C mineralization was evaluated with the mesh-bag method, with sampling 14, 28, 56, 84, 112, and 140 days after the beginning of the evaluation period for C measurements. Four C conservation indexes were used to assess the relation between C-CO2 efflux and soil C stock and its compartments. The crop residue C mineralization fit an exponential model in time. For black oat, wheat and maize residues, C mineralization was higher in CT than NT, while for soybean it was similar. Soil moisture was higher in NT than CT, mainly in the second year of evaluation. There was no difference in tillage systems for annual average C-CO2 emissions, but in some individual evaluations, differences between tillage systems were noticed for C-CO2 evolution. Soil C-CO2 effluxes followed a bi-modal pattern, with peaks in October/November and February/March. The highest emission was recorded in the summer and the lowest in the winter. The C-CO2 effluxes were weakly correlated to air temperature and not correlated to soil moisture. Based on the soil C conservation indexes investigated, NT associated to intensive crop rotation was more C conserving than CT with monoculture.

Organic acid adsorption and mineralization in oxisols with different textures

Organic acids play an important role in the nutritional conditions of plants. Their relevance is related to their formation dynamics, mineralization rate and adsorption by soil colloids. This study was carried out to evaluate the dynamics of mineralization and adsorption of organic acid (acetic acid - AA, citric acid - CA and humic acid - HA) applied to the soil. Samples of two Oxisols were used: Rhodic Haplustox (LV) and Typic Haplustox (LVA). The mineralization experiment was arranged in a 2 x 3 x 5 factorial design, based on the factors: two soils (LV and LVA) x three organic acid (OA) types (AA, CA and HA) x five OA rates (0, 1, 2, 4, and 8 mmol dm-3). Organic carbon mineralization in samples was measured by the C-CO2 efflux, produced by the microbial activity, in a 30-day (measurements after 4, 8, 12, 21, and 30 days) and in a 4-day experiment (measured after 24, 48, 72 and 96 h). Organic acid adsorption was tested in a 2 x 2 x 5 x 4 factorial design, with the factors and levels: two Oxisols; two organic acids (AA and CA); five OA rates (0, 1, 2, 4, and 8 mmol dm-3) and four adsorption periods (6, 24, 48, and 72 h). The C-CO2 production of soil treated with CA was highest. In the adsorption experiment, the affinity of CA to soil adsorption sites was greatest. The adsorption of organic acids to soils may be an important mechanism by which bioavailability and thus mineralization capacity by microbial activity are reduced.

Antimicrobial resistance of Staphylococcus spp. from small ruminant mastitis in Brazil

The study aimed to determine the antimicrobial resistance patterns and to identify molecular resistance markers in Staphylococcus spp. (n=210) isolated from small ruminant mastitis in Brazil. The antimicrobial resistance patterns were evaluated by the disk diffusion test and by detection of the presence of mecA, blaZ, ermA, ermB, ermC and msrA genes by PCR. The efflux pump test was performed using ethidium bromide and biofilm production was determined by Congo red agar test along with PCR for detection of the icaD gene. The isolates were most resistant to amoxicillin (50.0%), streptomycin (42.8%), tetracycline (40.4%), lincomycin (39.0%) and erythromycin (33.8%). Pan-susceptibility to all tested drugs was observed in 71 (33.8%) isolates and 41 Staphylococcus isolates were positive for the efflux pump. Although phenotypic resistance to oxacillin was observed in 12.8% of the isolates, none harbored the mecA gene. However, 45.7% of the isolates harbored blaZ indicating that beta-lactamase production was the main mechanism associated with staphylococci resistance to beta-lactams in the present study. The other determinants of resistance to antimicrobial agents ermA, ermB, ermC, and msrA were observed in 1.4%, 10.4%, 16.2%, and 0.9% of the isolates, respectively. In addition, the icaD gen was detected in 32.9% of the isolates. Seventy three isolates (54 from goats and 19 from sheep) were negative for all resistance genes tested and 69 isolates presented two or more resistance genes. Association among blaZ, ermA, ermB, ermC and efflux pump were observed in 17 isolates, 14 of which originated from goats and three from sheep. The data obtained in this study show the resistance of the isolates to beta-lactamics, which may be associated with the use of antimicrobial drugs without veterinary control.