Heteroresistance to penicillin in Streptococcus pneumoniae

Heteroresistance to beta-lactam antibiotics has been mainly described for staphylococci, for which it complicates diagnostic procedures and therapeutic success. This study investigated whether heteroresistance to penicillin exists in Streptococcus pneumoniae. Population analysis profile (PAP) showed the presence of subpopulations with higher penicillin resistance in four of nine clinical pneumococcal strains obtained from a local surveillance program (representing the multiresistant clones ST179, ST276, and ST344) and in seven of 16 reference strains (representing the international clones Spain(23F)-1, Spain(9V)-3, Spain(14)-5, Hungary(19A)-6, South Africa(19A)-13, Taiwan(23F)-15, and Finland(6B)-12). Heteroresistant strains had penicillin minimal inhibitory concentrations (MICs) (for the majority of cells) in the intermediate- to high-level range (0.19-2.0 mug/ml). PAP curves suggested the presence of subpopulations also for the highly penicillin-resistant strains Taiwan(19F)-14, Poland(23F)-16, CSR(19A)-11, and CSR(14)-10. PAP of bacterial subpopulations with higher penicillin resistance showed a shift toward higher penicillin-resistance levels, which reverted upon multiple passages on antibiotic-free media. Convergence to a homotypic resistance phenotype did not occur. Comparison of two strains of clone ST179 showed a correlation between the heteroresistant phenotype and a higher-penicillin MIC and a greater number of altered penicillin-binding proteins (PBP1a, -2b, and -2x), respectively. Therefore, heteroresistance to penicillin occurs in international multiresistant clones of S. pneumoniae. Pneumococci may use heteroresistance to penicillin as a tool during their evolution to high penicillin resistance, because it gives the bacteria an opportunity to explore growth in the presence of antibiotics before acquisition of resistance genes.

Techniques of population analysis.

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Molecular analysis of methicillin-resistant Staphylococcus aureus dissemination among healthcare professionals and/or HIV patients from a tertiary hospital

Abstract INTRODUCTION: Methicillin-resistant Staphylococcus aureus (MRSA) is a nosocomial pathogen in community settings. MRSA colonized individuals may contribute to its dissemination; the risk of MRSA infection is increased in human immunodeficiency virus/acquired immune deficiency syndrome (HIV/AIDS) patients, although the prevalence of colonization in this group is not well established. The present study addressed this issue by characterizing MRSA isolates from HIV/AIDS patients and their healthcare providers (HCPs) to determine whether transmission occurred between these two populations. METHODS: A total of 24 MRSA isolates from HIV-infected patients and five from HCPs were collected between August 2011 and May 2013. Susceptibility to currently available antimicrobials was determined. Epidemiological typing was carried out by pulsed-field gel electrophoresis, multilocus sequence typing, and Staphylococcus cassette chromosome (SCCmec) typing. The presence of heterogeneous vancomycin-intermediate Staphylococcus aureus (hVISA) and heterogeneous daptomycin-resistant Staphylococcus aureus (hDRSA) was confirmed by population analysis profile. Isolates characterized in this study were also compared to isolates from 2009 obtained from patients at the same hospital. RESULTS: A variety of lineages were found among patients, including ST5-SCCmecII and ST30-SCCmecIV. Two isolates were Panton-Valentine leukocidin-positive, and hVISA and hDRSA were detected. MRSA isolates from two HCPs were not related to those from HIV/AIDS patients, but clustered with archived MRSA from 2009 with no known relationship to the current study population. CONCLUSIONS: ST105-SCCmecII clones that colonized professionals in 2011 and 2012 were already circulating among patients in 2009, but there is no evidence that these clones spread to or between HIV/AIDS patients up to the 7th day of their hospitalization.

Population analysis of susceptibility to propolis in reference strains of Staphylococcus aureus and Escherichia coli

Susceptibility to several ethanolic extracts of propolis (EEP) concentrations was tested with the population analysis technique in reference strains of Staphylococcus aureus (ATCC 25923) and Escherichia coli (ATCC 35218). The results of these tests showed that all bacteria were killed by EEP concentrations approximately equal or higher (2.0% v/v - S. aureus; 10.0% v/v - E. coli) than the respective minimal inhibitory concentrations (MIC). Regarding the susceptibility to propolis, there was a homogeneity of data with the respective time kill curves showing a clear bactericidal effect during 6 to 9 h of exposition.

Population analysis of Scomberomorus cavalla (Cuvier, 1829) (Perciformes, Scombridae) from the Northern and Northeastern coast of Brazil

Scomberomorus cavalla é uma espécie de peixe pelágico amplamente distribuído na costa oeste do Atlântico, e uma diminuição no seu nível de captura tem sido verificada nos E.U.A e Golfo do México, comparada com os níveis alcançados pela espécie no passado. Da mesma forma, em algumas áreas do Brasil, há indícios de sobre-exploração. Entretanto, não existem estudos moleculares que visam o manejo deste importante item. Desta forma, no presente estudo, foram seqüenciados 380 pares de bases nucleotídicas da região da Alça-D do DNA mitocondrial de amostras provenientes de desembarque em Macapá, Bragança e Fortaleza. As análises filogenéticas e populacionais revelaram que há apenas uma população panmítica e baixos níveis de variabilidade genética foram observados. Estes resultados, assim como a observada sobre-exploração de S. cavala, representam dados muito importantes para o estabelecimento do manejo deste estoque a fim de prevenir um colapso ou risco de extinção no futuro.

Large-Scale Population Analysis Challenges the Current Criteria for the Molecular Diagnosis of Fascioscapulohumeral Muscular Dystrophy

Facioscapulohumeral muscular dystrophy (FSHD) is a common hereditary myopathy causally linked to reduced numbers (<= 8) of 3.3 kilobase D4Z4 tandem repeats at 4q35. However, because individuals carrying D4Z4-reduced alleles and no FSHD and patients with FSHD and no short allele have been observed, additional markers have been proposed to support an FSHD molecular diagnosis. In particular a reduction in the number of D4Z4 elements combined with the 4A(159/161/168)PAS haplotype (which provides the possibility of expressing DUX4) is currently used as the genetic signature uniquely associated with FSHD. Here, we analyzed these DNA elements in more than 800 Italian and Brazilian samples of normal individuals unrelated to any FSHD patients. We find that 3% of healthy subjects carry alleles with a reduced number (4-8) of D4Z4 repeats on chromosome 4q and that one-third of these alleles, 1.3%, occur in combination with the 4A161PAS haplotype. We also systematically characterized the 4q35 haplotype in 253 unrelated FSHD patients. We find that only 127 of them (50.1%) carry alleles with 1-8 D4Z4 repeats associated with 4A161PAS, whereas the remaining FSHD probands carry different haplotypes or alleles with a greater number of D4Z4 repeats. The present study shows that the current genetic signature of FSHD is a common polymorphism and that only half of FSHD probands carry this molecular signature. Our results suggest that the genetic basis of FSHD, which is remarkably heterogeneous, should be revisited, because this has important implications for genetic counseling and prenatal diagnosis of at-risk families.

Evaluation of antimicrobial strategies against biomaterial-associated infections: impact of the surrounding environmental conditions

Dissertação de mestrado em Bioengenharia

A low affinity penicillin-binding protein 2x is required for heteroresistance in Streptococcus pneumoniae

Heteroresistance to penicillin in Streptococcus pneumoniae is the ability of subpopulations to grow at a higher antibiotic concentration than expected from the minimal inhibitory concentration (MIC). This may render conventional resistance testing unreliable and lead to therapeutic failure. We investigated the role of the primary β-lactam resistance determinants, penicillin binding proteins PBP2b and PBP2x and secondary resistance determinant PBP1a in heteroresistance to penicillin. Transformants containing PBP genes from heteroresistant strain Spain(23F)2349 in non-heteroresistant strain R6 background were tested for heteroresistance by population analysis profiling (PAP). We found that pbp2x, but not pbp2b or pbp1a alone, conferred heteroresistance to R6. However, a change of pbp2x expression is not observed and therefore expression does not correlate with an increased proportion of resistant subpopulations. Additional ciaR disruption mutants which have been described to mediate PBP-independent β-lactam resistance revealed no heteroresistant phenotype by PAP.We also showed, that the highly resistant subpopulations (HOM*) of transformants containing low affinity pbp2x undergo an increase in resistance upon selection on penicillin plates which partially reverts after passaging on selection-free medium. Shotgun proteomic analysis showed an upregulation of phosphate ABC transporter subunit proteins pstS, phoU, pstB and pstC in these highly resistant subpopulations.In conclusion, the presence of low affinity pbp2x enables certain pneumococcal colonies to survive in the presence of beta lactams. Upregulation of phosphate ABC transporter genes may represent a reversible adaption to antibiotic stress.

Analysis of the DF508 mutation in a Brazilian cystic fibrosis population: comparison of pulmonary status of homozygotes with other patients

Sixty-one cystic fibrosis patients admitted for check-up or antibiotic treatment were enrolled for genetic and clinical evaluation. Genetic analysis was performed on blood samples stored on neonatal screening cards using PCR techniques to determine the presence of DF508 mutations. Clinical evaluation included Shwachman and Chrispin-Norman scores, age at onset of symptoms and diagnosis, spirometry, awake and sleep pulse oximetry, hyponychial angle measurement and presence of chronic Pseudomonas aeruginosa colonization. Eighteen patients (29.5%) were homozygous for the DF508 mutation, 26 (42.6%) had one DF508 mutation and 17 (27.9%) were noncarriers, corresponding to a 50.8% prevalence of the mutation in the whole population. Analysis by the Kruskal-Wallis test for comparison of genetic status with continuous variables or by the chi-square test and logistic regression for dichotomous variables showed no significant differences between any two groups for a = 0.05. We conclude that genetic status in relation to the DF508 mutation is not associated with pulmonary status as evaluated by the above variables

Population pharmacokinetic analysis of mycophenolic acid in renal transplant recipients following oral administration of mycophenolate mofetil

Aim To develop a population pharmacokinetic model for mycophenolic acid in adult kidney transplant recipients, quantifying average population pharmacokinetic parameter values, and between- and within-subject variability and to evaluate the influence of covariates on the pharmacokinetic variability. Methods Pharmacokinetic data for mycophenolic acid and covariate information were previously available from 22 patients who underwent kidney transplantation at the Princess Alexandra Hospital. All patients received mycophenolate mofetil 1 g orally twice daily. A total of 557 concentration-time points were available. Data were analysed using the first-order method in NONMEM (version 5 level 1.1) using the G77 FORTRAN compiler. Results The best base model was a two-compartment model with a lag time (apparent oral clearance was 271 h(-1), and apparent volume of the central compartment 981). There was visual evidence of complex absorption and time-dependent clearance processes, but they could not be successfully modelled in this study. Weight was investigated as a covariate, but no significant relationship was determined. Conclusions The complexity in determining the pharmacokinetics of mycophenolic acid is currently underestimated. More complex pharmacokinetic models, though not supported by the limited data collected for this study, may prove useful in the future. The large between-subject and between-occasion variability and the possibility of nonlinear processes associated with the pharmacokinetics of mycophenolic acid raise questions about the value of the use of therapeutic monitoring and limited sampling strategies.

Analysis of population pharmacokinetic data using NONMEM and WinBUGS

The aim of this report is to describe the use of WinBUGS for two datasets that arise from typical population pharmacokinetic studies. The first dataset relates to gentamicin concentration-time data that arose as part of routine clinical care of 55 neonates. The second dataset incorporated data from 96 patients receiving enoxaparin. Both datasets were originally analyzed by using NONMEM. In the first instance, although NONMEM provided reasonable estimates of the fixed effects parameters it was unable to provide satisfactory estimates of the between-subject variance. In the second instance, the use of NONMEM resulted in the development of a successful model, albeit with limited available information on the between-subject variability of the pharmacokinetic parameters. WinBUGS was used to develop a model for both of these datasets. Model comparison for the enoxaparin dataset was performed by using the posterior distribution of the log-likelihood and a posterior predictive check. The use of WinBUGS supported the same structural models tried in NONMEM. For the gentamicin dataset a one-compartment model with intravenous infusion was developed, and the population parameters including the full between-subject variance-covariance matrix were available. Analysis of the enoxaparin dataset supported a two compartment model as superior to the one-compartment model, based on the posterior predictive check. Again, the full between-subject variance-covariance matrix parameters were available. Fully Bayesian approaches using MCMC methods, via WinBUGS, can offer added value for analysis of population pharmacokinetic data.

Variation in cervical cancer screening by region, socio-economic, migrant and Indigenous status in women in New South Wales

The purpose of this study was to estimate the extent of association of cervical screening in NSW women with socio-economic status (SES), rurality, and proportions of non-English speaking background (NESB) and Indigenous status. Data on women who had at least one Pap test over two years (January 1998-December 1999) were obtained from the NSW Pap test Register. Each local government area (LGA) was allocated to categories of population proportions of NESB and Indigenous status, a rurality classification based on population density and remoteness, and to an SES quintile. The odds ratios (OR) of having a Pap test were estimated and confounding adjusted by multiple logistic regression analysis. Implied Pap test rates in urban NESB and in rural Indigenous women were estimated from the modelled estimates. The adjusted OR for a Pap test in large rural centres (1.14) was significantly higher than those for metropolitan or capital city residents (0.9 and 1.0 respectively). Adjusted OR for a Pap test in other rural centres (0.73) and other remote areas (0.64) were significantly lower than those for metropolitan or capital city residents. In urban populations the lowest OR were in areas with both low SES and high proportion of NESB. The lowest OR for Pap screening in rural populations occurred in the most remote areas with the highest proportion of Indigenous women. For urban NESB women the biennial Pap test rate was estimated as 50%, and for rural Indigenous women 29%, compared with the NSW average of 59%.

Cell homeostasis in a Leishmania major mutant overexpressing the spliced leader RNA is maintained by an increased proteolytic activity

Although several stage-specific genes have been identified in Leishmania, the molecular mechanisms governing developmental gene regulation in this organism are still not well understood. We have previously reported an attenuation of virulence in Leishmania major and L braziliensis carrying extra-copies of the spliced leader RNA gene. Here, we surveyed the major differences in proteome and transcript expression profiles between the spliced leader RNA overexpressor and control lines using two-dimensional gel electrophoresis and differential display reverse transcription PCR, respectively. Thirty-nine genes related to stress response, cytoskeleton, proteolysis, cell cycle control and proliferation, energy generation, gene transcription, RNA processing and post-transcriptional regulation have abnormal patterns of expression in the spliced leader RNA overexpressor line. The evaluation of proteolytic pathways in the mutant revealed a selective increase of cysteine protease activity and an exacerbated ubiquitin-labeled protein population. Polysome profile analysis and measurement of cellular protein aggregates showed that protein translation in the spliced leader RNA overexpressor line is increased when compared to the control line. We found that L major promastigotes maintain homeostasis in culture when challenged with a metabolic imbalance generated by spliced leader RNA surplus through modulation of intracellular proteolysis. However, this might interfere with a fine-tuned gene expression control necessary for the amastigote multiplication in the mammalian host. (c) 2010 Elsevier Ltd. All rights reserved.

Population pharmacokinetics of lamotrigine

The present study estimated the population pharmacokinetics of lamotrigine in patients receiving oral lamotrigine therapy with drug concentration monitoring, and determined intersubject and intrasubject variability. A total of 129 patients were analyzed from two clinical sites. Of these, 124 patients provided spare data (198 concentration-time points); nine patients (four from a previous group plus five from the current group) provided rich data (431 points). The population analysis was conducted using P-PHARM (TM) (SIMED Scientific Software, Cedex, France), a nonlinear mixed-effect modeling program. A single exponential elimination model (first-order absorption) with heteroscedastic weighting was used. Apparent clearance (CL/F) and volume of distribution (V/F) were the pharmacokinetic parameters estimated. Covariate analysis was performed to determine which factors explained any of the variability associated with lamotrigine clearance. Population estimates of CL/F and V/F for lamotrigine generated in the final model were 2.14 +/- 0.81 L/h and 78.1 +/- 5.1 L/kg. Intersubject and intrasubject variability for clearance was 38% and 38%, respectively. The covariates of concomitant valproate and phenytoin therapy accounted for 42% of the intersubject variability of clearance. Age, gender, clinic site, and other concomitant antiepileptic drugs did not influence clearance. This study of the population pharmacokinetics of lamotrigine in patients using the drug clinically provides useful data and should lead to better dosage individualization for lamotrigine.

Population pharmacokinetics of tacrolimus in adult kidney transplant recipients

Objectives: The aims of this study were to investigate the population pharmacokinetics of tacrolimus in adult kidney transplant recipients and to identify factors that explain variability. Methods: Population analysis was performed on retrospective data from 70 patients who received oral tacrolimus twice daily. Morning blood trough concentrations were measured by liquid chromatography-tandem mass spectrometry. Maximum likelihood estimates were sought for apparent clearance (CL/F) and apparent volume of distribution (V/F), with the use of NONMEM (GloboMax LLC, Hanover, Md). Factors screened for influence on these parameters were weight, age, gender, postoperative day, days of tacrolimus therapy, liver function tests, creatinine clearance, hematocrit fraction, corticosteroid dose, and potential interacting drugs. Results. CL/F was greater in patients with abnormally low hematocrit fraction (data from 21 patients only), and it decreased with increasing days of therapy and AST concentrations (P