Deep sequencing of New World screw-worm transcripts to discover genes involved in insecticide resistance

Background: The New World screw-worm (NWS), Cochliomyia hominivorax, is one of the most important myiasis-causing flies, causing severe losses to the livestock industry. In its current geographical distribution, this species has been controlled by the application of insecticides, mainly organophosphate (OP) compounds, but a number of lineages have been identified that are resistant to such chemicals. Despite its economic importance, only limited genetic information is available for the NWS. Here, as a part of an effort to characterize the C. hominivorax genome and identify putative genes involved in insecticide resistance, we sampled its transcriptome by deep sequencing of polyadenylated transcripts using the 454 sequencing technology. Results: Deep sequencing on the 454 platform of three normalized libraries (larval, adult male and adult female) generated a total of 548,940 reads. Eighteen candidate genes coding for three metabolic detoxification enzyme families, cytochrome P450 monooxygenases, glutathione S transferases and carboxyl/cholinesterases were selected and gene expression levels were measured using quantitative real-time polymerase chain reaction (qRT-PCR). Of the investigated candidates, only one gene was expressed differently between control and resistant larvae with, at least, a 10-fold down-regulation in the resistant larvae. The presence of mutations in the acetylcholinesterase (target site) and carboxylesterase E3 genes was investigated and all of the resistant flies presented E3 mutations previously associated with insecticide resistance. Conclusions: Here, we provided the largest database of NWS expressed sequence tags that is an important resource, not only for further studies on the molecular basis of the OP resistance in NWS fly, but also for functional and comparative studies among Calliphoridae flies. Among our candidates, only one gene was found differentially expressed in resistant individuals, and its role on insecticide resistance should be further investigated. Furthermore, the absence of mutations in the OP target site and the high frequency of mutant carboxylesterase E3 indicate that metabolic resistance mechanisms have evolved predominantly in this species.

Atenolol blunts blood pressure increase during dynamic resistance exercise in hypertensives

center dot Dynamic resistance exercise promotes a sizeable increase in blood pressure during its execution in non medicated hypertensives. WHAT THIS STUDY ADDS center dot Atenolol not only decreases blood pressure level but also mitigates the increase of blood pressure during dynamic resistance exercise in hypertensive patients. An increase in blood pressure during resistance exercise might be at least in part attributed to an increase in cardiac output. AIMS This study was conducted to determine whether atenolol was able to decrease BP level and mitigate BP increase during dynamic resistance exercise performed at three different intensities in hypertensives. METHODS Ten essential hypertensives (systolic/diastolic BP between 140/90 and 160/105 mmHg) were blindly studied after 6 weeks of placebo and atenolol. In each phase, volunteers executed, in a random order, three protocols of knee-extension exercises to fatigue: (i) one set at 100% of 1 RM; (ii) three sets at 80% of 1 RM; and (iii) three sets at 40% of 1 RM. Intra-arterial radial blood pressure was measured throughout the protocols. RESULTS Atenolol decreased systolic BP maximum values achieved during the three exercise protocols (100% = 186 +/- 4 vs. 215 +/- 7, 80% = 224 +/- 7 vs. 247 +/- 9 and 40% = 223 +/- 7 vs. 252 +/- 16 mmHg, P < 0.05). Atenolol also mitigated an increase in systolic BP in the first set of exercises (100% = +38 +/- 5 vs. +54 +/- 9; 80% = +68 +/- 11 vs. +84 +/- 13 and 40% = +69 +/- 7 vs. +84 +/- 14, mmHg, P < 0.05). Atenolol decreased diastolic BP values and mitigated its increase during exercise performed at 100% of 1 RM (126 +/- 6 vs. 145 +/- 6 and +41 +/- 6 vs. +52 +/- 6, mmHg, P < 0.05), but not at the other exercise intensities. CONCLUSIONS Atenolol was effective in both reducing systolic BP maximum values and mitigating BP increase during resistance exercise performed at different intensities in hypertensive subjects.

Novel sources of witches` broom resistance (causal agent Moniliophthora perniciosa) from natural populations of Theobroma cacao from the Brazilian Amazon

Witches` broom is a severe disease of Theobroma cacao L. (cacao), caused by the basidiomycete Moniliophthora perniciosa. The use of resistant cultivars is the ultimate method of control, but there are limited sources of resistance. Further, resistance from the most widely used source (`Scavina 6`) has been overcome after a few years of deployment. New sources of resistance have been intensively searched for in the Amazon basin. Here, we evaluated for witches` broom resistance, cacao accessions from various natural cacao populations originally collected in the Brazilian Amazon. Resistance of 43 families was evaluated under nursery and/or field conditions by artificial or natural infection, respectively, based on disease incidence. Screening for resistance by artificial inoculation under nursery conditions appeared to be efficient in identifying these novel resistance sources, confirmed by natural field evaluation over a nine-year period. The increase in natural field infection of `Scavina 6` was clearly demonstrated. Among the evaluated families with the least witches` broom incidence, there were accessions originally collected from distinct river basins, including the Jamari river (`CAB 0371`; `CAB 0388`; `CAB 0392`; and `CAB 0410`); Acre (`CAB 0169`); Javari (`CAB 0352`); Solimes (`CAB 0270`); and from the Purus river basin, the two most outstanding resistant accessions, `CAB 0208` and `CAB 0214`. The large genetic diversity found in cacao populations occurring at river basins from Acre and Amazonas states, Brazil, increased the chance that the selected resistant accessions would be genetically more dissimilar, and represent distinct sources of resistance to M. perniciosa from `Scavina 6`.

Progeny evaluation for resistance to Phaeosphaeria leaf spot in tropical maize

Maize breeding programmes in Brazil and elsewhere seek reliable methods to identify genotypes resistant to Phaeosphaeria leaf spot. The area under the disease progress curve (AUDPC) is an accurate method to evaluate the severity of foliar diseases. However, at least three data points are required to calculate the AUDPC, which is unfeasible when there are thousands of genotypes to be assessed. The aim of this work was to estimate the heritability of disease resistance, evaluate disease severity at different times using a nine-point scale in comparison to the AUDPC, and establish the most suitable phenological period for disease assessment. A repeated experiment was conducted in a 11 x 11 lattice experimental design with three replications. Disease assessments were carried out at flowering, 15 and 30 days post-anthesis for the parental lines DS95, DAS21, the F1 generation and 118 F2:3 progenies. Then, the AUDPC was obtained and results compared with the single-point evaluations used to calculate it. Individual and joint analyses of variance were conducted to obtain heritabiliy estimates. The assessments performed after the flowering stage gave higher estimates of heritability and correlation with AUDPC. We concluded that one assessment between the 15th and 30th day after flowering could provide enough information to distinguish maize genotypes for their resistance to Phaeosphaeria leaf spot under tropical conditions.

Inheritance of resistance to Puccinia psidii G. Winter in a eucalyptus interspecific hybrid progeny evaluated under conditions of natural infection

Inheritance of resistance to Puccinia psidii G. Winter in a eucalyptus interspecific hybrid progeny evaluated under conditions of natural infection Rust caused by the fungus Puccinia psidii is currently the most important disease of eucalyptus. It is widely disseminated in Brazil, and causes serious damage in nurseries and plantation areas. The identification of resistant germplasm along with knowledge of the genetic basis of resistance heredity are the first requirements for the success of breeding programs aiming to develop resistant varieties. Earlier studies carried out under controlled conditions suggested a monogenic control as well as the participation of at least two genes promoting resistance to rust. The goal of this study was to evaluate the resistance to P. psidii under field conditions in fourteen progenies from controlled crosses and self-crosses among four hybrid clones of Eucalyptus grandis Hill ex Maiden x Eucalyptus urophylla ST Blake that contrast for resistance to the fungus. Results indicated that resistance could be explained by one locus with main effects and at least three different alleles. However, loci with minor effects may influence the resistance, since variation on severity classes was observed. Differences in segregation of resistance between reciprocal crosses were not observed, indicating absence of cytoplasmic effects.

Monooxygenases play only a minor role in resistance to synthetic pyrethroids in the cattle tick, Boophilus microplus

We investigated the role of monooxygenases in resistance to synthetic pyrethroids (SPs) in the cattle tick, Boophilus microplus. We found that monooxygenases play only a minor role in resistance to SPs in both resistant and susceptible strains of B. microplus. We blocked the monooxygenases with piperonyl butoxide (PBO) and simultaneously applied the SPs, flumethrin and cypermethrin to larval B. microplus. PBO increased the effect of flumethrin (synergism ratios 2.7-8.9) more than it increased the effect of cypermethrin (synergism ratios 1.9-3.1). Of the four strains tested, Parkhurst, which is resistant to SPs, was the least affected by the addition of PBO (synergism ratios after cypermethrin was applied 1.9; after flumethrin 2.7) whereas N.R.F.S., the strain susceptible to SPs, was the most affected by synergism between PBO and SPs (synergism ratio after cypermethrin was applied 3.1; after flumethrin 8.9). We hypothesize that B. microplus lacks monooxygenases capable of conferring resistance to SPs because it and its recent ancestors were blood-feeders rather than herbivores.

Resistance exercise and glycemic control in women with gestational diabetes mellitus

OBJECTIVE: The objective of the study was to evaluate the effect of a resistance exercise program with an elastic band on insulin requirement and glycemic control in patients with gestational diabetes mellitus (GDM). STUDY DESIGN: Sixty-four patients with gestational diabetes mellitus were randomly assigned into 2 groups: an exercise group (EG; n = 32) and a control group not submitted to the exercise program (CG; n = 32). RESULTS: A significant reduction in the number of patients who required insulin was observed in the EG (7/32) compared with the CG group (18/32) (P = .005). The percentage of time spent within the proposed target glucose range (of at least 80% of weekly measurements below the limits preestablished for the disease) was significantly higher in EG compared with the CG group (EG = 0.63 +/- 0.30; CG = 0.41 +/- 0.31; P = .006). CONCLUSION: The resistance exercise program was effective in reducing the number of patients with GDM who required insulin and in improving capillary glycemic control in this population.

Ovary colonization by Claviceps africana is related to ergot resistance in male-sterile sorghum lines

Ergot, caused by Claviceps africana, has emerged as a serious threat to sorghum hybrid seed production worldwide. In the absence of gene-for-gene-based qualitative resistance in commercial cultivars, varieties with high pollen production that can escape ergot infection are preferred. Recent demonstration of differences in ergot susceptibility among male-sterile lines has indicated the presence of partial resistance. Using chitin-specific fluorescin-isothiocyanate-conjugated wheat germ agglutin and callose-specific aniline blue, this study investigated the process of sorghum ovary colonization by C. africana. Conidia germinated within 24 h after inoculation (a.i.); the pathogen was established in the ovary by 79 h a.i., and at least half of the ovary was converted into sphacelial tissue by 120 h a.i. Changes in fungal cell wall chitin content and strategic callose deposition in the host tissue were associated with penetration and invasion of the ovary. The rate of ovary colonization differed in three male-sterile lines that also differed in ergot susceptibility. This work demonstrates a possible histological basis for partial resistance in male-sterile sorghum lines that could lay the foundation for variety improvement through further breeding and selection.

The role of the efflux mechanisms in multidrug resistance in Mycobacterium tuberculosis

Abstract The emergence of multi and extensively drug resistant tuberculosis (MDRTB and XDRTB) has increased the concern of public health authorities around the world. The World Health Organization has defined MDRTB as tuberculosis (TB) caused by organisms resistant to at least isoniazid and rifampicin, the main first-line drugs used in TB therapy, whereas XDRTB refers to TB resistant not only to isoniazid and rifampicin, but also to a fluoroquinolone and to at least one of the three injectable second-line drugs, kanamycin, amikacin and capreomycin. Resistance in Mycobacterium tuberculosis is mainly due to the occurrence of spontaneous mutations and followed by selection of mutants by subsequent treatment. However, some resistant clinical isolates do not present mutations in any genes associated with resistance to a given antibiotic, which suggests that other mechanism(s) are involved in the development of drug resistance, namely the presence of efflux pump systems that extrude the drug to the exterior of the cell, preventing access to its target. Increased efflux activity can occur in response to prolonged exposure to subinhibitory concentrations of anti-TB drugs, a situation that may result from inadequate TB therapy. The inhibition of efflux activity with a non-antibiotic inhibitor may restore activity of an antibiotic subject to efflux and thus provide a way to enhance the activity of current anti-TB drugs. The work described in this thesis foccus on the study of efflux mechanisms in the development of multidrug resistance in M. tuberculosis and how phenotypic resistance, mediated by efflux pumps, correlates with genetic resistance. In order to accomplish this goal, several experimental protocols were developed using biological models such as Escherichia coli, the fast growing mycobacteria Mycobacterium smegmatis, and Mycobacterium avium, before their application to M. tuberculosis. This approach allowed the study of the mechanisms that result in the physiological adaptation of E. coli to subinhibitory concentrations of tetracycline (Chapter II), the development of a fluorometric method that allows the detection and quantification of efflux of ethidium bromide (Chapter III), the characterization of the ethidium bromide transport in M. smegmatis (Chapter IV) and the contribution of efflux activity to macrolide resistance in Mycobacterium avium complex (Chapter V). Finally, the methods developed allowed the study of the role of efflux pumps in M. tuberculosis strains induced to isoniazid resistance (Chapter VI). By this manner, in Chapter II it was possible to observe that the physiological adaptation of E. coli to tetracycline results from an interplay between events at the genetic level and protein folding that decrease permeability of the cell envelope and increase efflux pump activity. Furthermore, Chapter III describes the development of a semi-automated fluorometric method that allowed the correlation of this efflux activity with the transport kinetics of ethidium bromide (a known efflux pump substrate) in E. coli and the identification of efflux inhibitors. Concerning M. smegmatis, we have compared the wild-type M. smegmatis mc2155 with knockout mutants for LfrA and MspA for their ability to transport ethidium bromide. The results presented in Chapter IV showed that MspA, the major porin in M. smegmatis, plays an important role in the entrance of ethidium bromide and antibiotics into the cell and that efflux via the LfrA pump is involved in low-level resistance to these compounds in M. smegmatis. Chapter V describes the study of the contribution of efflux pumps to macrolide resistance in clinical M. avium complex isolates. It was demonstrated that resistance to clarithromycin was significantly reduced in the presence of efflux inhibitors such as thioridazine, chlorpromazine and verapamil. These same inhibitors decreased efflux of ethidium bromide and increased the retention of [14C]-erythromycin in these isolates. Finaly, the methods developed with the experimental models mentioned above allowed the study of the role of efflux pumps on M. tuberculosis strains induced to isoniazid resistance. This is described in Chapter VI of this Thesis, where it is demonstrated that induced resistance to isoniazid does not involve mutations in any of the genes known to be associated with isoniazid resistance, but an efflux system that is sensitive to efflux inhibitors. These inhibitors decreased the efflux of ethidium bromide and also reduced the minimum inhibitory concentration of isoniazid in these strains. Moreover, expression analysis showed overexpression of genes that code for efflux pumps in the induced strains relatively to the non-induced parental strains. In conclusion, the work described in this thesis demonstrates that efflux pumps play an important role in the development of drug resistance, namely in mycobacteria. A strategy to overcome efflux-mediated resistance may consist on the use of compounds that inhibit efflux activity, restoring the activity of antimicrobials that are efflux pump substrates, a useful approach particularly in TB where the most effective treatment regimens are becoming uneffective due to the increase of MDRTB/XDRTB.

Resistance of Streptococcus pneumoniae to antimicrobials in São Paulo, Brazil: clinical features and serotypes

To study resistance to antimicrobials, serotypes and clinical features of S. pneumoniae in S. Paulo, Brazil, 50 patients with a positive culture were evaluated: 7 were considered carriers and 43 had pneumococcal infections. Pneumonia and meningitis were the most commom infections. Mortality was 34% and underlying diseases were present in 70%. Relative resistance to penicillin occurred in 24% and complete resistance was not detected. Resistance to tetracycline was 32% and to sulfamethoxazole/trimethoprim 32%; one strain had intermediate susceptibility to erythromycin; no resistance was present for chloramphenicol, rifampin or vancomycin. Resistance to at least one of the drugs tested occurred in 62%. Results by the E-test for penicillin were similar to those by the agar dilution method. There were 24 different serotypes and 74% of the strains belonged to the 23-valent vaccine including all the penicillin-resistant strains. In this study S. pneumoniae caused severe infections and presented a high resistance rate to commonly used antimicrobials. Routine surveillance of resistance and the use of vaccination, as well as the restriction of inappropriate use of antimicrobials, are recommended in São Paulo, Brazil.

Epidemiology and antimicrobial resistance of B. fragilis group organisms isolated from clinical specimen and human intestinal microbiota

Epidemiological aspects and the antimicrobial susceptibility profile of the Bacteroides fragilis group isolated from clinical and human intestinal specimens were examined in this study. B. fragilis group strains were isolated from 46 (37%) of 124 clinical specimens and the source of the samples was: Blood culture (3), intraabdominal infection (27), brain abscess (2), soft tissue infection (17), respiratory sinus (3), pleural aspirate (9), breast abscess (3), surgical infected wound (22), pelvic inflammatory disease (22), chronic otitis media (9) and miscellaneous (7). Intraabdominal and soft tissue infections were responsible for more than half of the clinical isolates. Susceptibility to penicillin, cefoxitin, tetracycline, metronidazole, chloramphenicol and clindamycin was examined. All isolates were susceptible to metronidazole and chloramphenicol. For clindamycin and cefoxitin the resistance rates observed were 21.7% and 10.9% respectively. Susceptibility profiles varied among the different species tested. A total of 37 species of B. fragilis group isolated from intestinal microbiota of individuals who had no antimicrobial therapy for at least 1 month before the sampling was also examined. All strains were also susceptible to chloramphenicol and motronidazole and the resistance rates to clindamycin and cefoxitin were 19.4% and 5.4% respectively. A few institutions, in Brazil, have monitored the antimicrobial susceptibility of B. fragilis group strains isolated from anaerobic infections. The resistance rates to cefoxitin and clindamycin and the variation in susceptibility patterns among the species isolated in this study emphasize the need for monitoring of susceptibility patterns of B. fragilis group organisms isolated, especially at our University Hospitals.

Epidemiological characterization, antimicrobial resistance and virulence mechanisms of human and animal streptococci

Dissertação para obtenção do Grau de Doutor em Biologia

PREVALENCE OF DRUG RESISTANCE AND VIRULENCE FEATURES IN Salmonella spp. ISOLATED FROM FOODS ASSOCIATED OR NOT WITH SALMONELLOSIS IN BRAZIL

Salmonella is the most common etiological agent of cases and outbreaks of foodborne diarrheal illnesses. The emergence and spread of Salmonella spp., which has become multi-drug resistant and potentially more pathogenic, have increased the concern with this pathogen. In this study, 237 Salmonella spp., associated or not with foodborne salmonellosis in Brazil, belonging mainly to serotype Enteritidis, were tested for antimicrobial susceptibility and the presence of the virulence genes spvC, invA, sefA and pefA. Of the isolates, 46.8% were sensitive to all antimicrobials and 51.9% were resistant to at least one antimicrobial agent. Resistance to more than one antimicrobial agent was observed in 10.5% of the strains. The highest rates of resistance were observed for streptomycin (35.9%) and nalidixic acid (16.9%). No strain was resistant to cefoxitin, cephalothin, cefotaxime, amikacin, ciprofloxacin and imipenem. The invA gene was detected in all strains. Genes spvC and pefA were found in 48.1% and 44.3% of strains, respectively. The gene sefA was detected in 31.6% of the strains and only among S. Enteritidis. Resistance and virulence determinants were detected in Salmonella strains belonging to several serotypes. The high rates of antibiotic-resistance in strains isolated from poultry products demonstrate the potential risk associated with the consumption of these products and the need to ensure good food hygiene practices from farm to table to reduce the spread of pathogens relevant to public health.

Antimicrobial Resistance of Shigella spp. isolated in the State of Pará, Brazil

INTRODUCTION: Shigella spp. are Gram-negative, nonsporulating, rod-shaped bacteria that belong to the family Enterobacteriaceae and are responsible for shigellosis or bacillary dysentery, an important cause of worldwide morbidity and mortality. METHODS: We studied the antibiotic resistance profiles of 122 Shigella spp. strains (81 S. flexneri, 41 S. sonnei, 1 S. boydii) isolated from patients (female and male from 0 to 80 years of age) presenting diarrhea in different districts of the State of Pará, in the North of Brazil. The antibiotic resistance of the strains, isolated from human fecal samples, was determined by the diffusion disk method and by using the VITEK-2 system. RESULTS: The highest resistance rate found was the resistance rate to tetracycline (93.8%), followed by the resistance rate to chloramphenicol (63.9%) and to trimethoprim/sulfamethoxazole (63.1%). Resistance to at least three drugs was more common among S. flexneri than S. sonnei (39.5% vs. 10%). Six (4.9%) strains were susceptible to all the antibiotics tested. All strains were susceptible to cefotaxime, ceftazidime, ciprofloxacin, nalidixic acid and nitrofurantoin. CONCLUSIONS: High rates of multidrug resistance in Shigella spp. are a serious public health concern in Brazil. It is extremely important to continuously monitor the antimicrobial resistances of Shigella spp. for effective therapy and control measures against shigellosis.

Multidrug resistance genes, including blaKPC and blaCTX-M-2, among Klebsiella pneumoniae isolated in Recife, Brazil

INTRODUCTION: The prevalence of cephalosporins and carbapenem-resistant Klebsiella pneumoniae strains is rising in Brazil, with potential serious consequences in terms of patients' outcomes and general care. METHODS: This study characterized 24 clinical isolates of K. pneumoniae from two hospitals in Recife, Brazil, through the antimicrobial susceptibility profile, analyses of β-lactamase genes (blaTEM, blaSHV,blaCTX-MblaKPC, blaVIM, blaIMP, and blaSPM), plasmidial profile and ERIC-PCR (Enterobacterial repetitive intergenic consensus-polymerase chain reaction). RESULTS: ERIC-PCR and plasmidial analysis grouped the isolates in 17 and 19 patterns, respectively. Six isolates from one hospital presented the same pattern by ERIC-PCR, indicating clonal dissemination. All isolates presented blaSHV, 62.5% presented blaCTX-M-2, 29% blaTEM, and 41.7% blaKPC. Metallo-β-lactamase genes blaand blawere not detected. Eleven isolates were identified carrying at least 3 β-lactamase studied genes, and 2 isolates carried blaSHVblaTEM, blaCTX-M-2 and blaKPC simultaneously. CONCLUSIONS: The accumulation of resistance genes in some strains, observed in this study, imposes limitations in the therapeutic options available for the treatment of infections caused by K. pneumoniae in Recife, Brazil. These results should alert the Brazilian medical authorities to establish rigorous methods for more efficiently control the dissemination of antimicrobial resistance genes in the hospital environment.