Antimicrobial resistance and resistance gene determinants in clinical Escherichia coli from different animal species in Switzerland

Antimicrobial susceptibility testing was performed on a total of 581 clinical Escherichia coli isolates from diarrhea and edema disease in pigs, from acute mastitis in dairy cattle, from urinary tract infections in dogs and cats, and from septicemia in laying hens collected in Switzerland between 1999 and 2001. Among the 16 antimicrobial agents tested, resistance was most frequent for sulfonamides, tetracycline, and streptomycin. Isolates from swine presented significantly more resistance than those from the other animal species. The distribution of the resistance determinants for sulfonamides, tetracycline, and streptomycin was assessed by hybridization and PCR in resistant isolates. Significant differences in the distribution of resistance determinants for tetracycline (tetA, tetB) and sulfonamides (sulII) were observed between the isolates from swine and those from the other species. Resistance to sulfonamides could not be explained by known resistance mechanisms in more than a quarter of the sulfonamide-resistant and sulfonamide-intermediate isolates from swine, dogs and cats. This finding suggests that one or several new resistance mechanisms for sulfonamides may be widespread among E. coli isolates from these animal species. The integrase gene (intI) from class I integrons was detected in a large proportion of resistant isolates in association with the sulI and aadA genes, thus demonstrating the importance of integrons in the epidemiology of resistance in clinical E. coli isolates from animals.

Human CD8+ T lymphocyte subsets that express HLA class I-specific inhibitory receptors represent oligoclonally or monoclonally expanded cell populations.

A small percentage of human T lymphocytes, predominantly CD8+ T cells, express receptors for HLA class 1 molecules of natural killer type (NK-R) that are inhibitory for T-cell antigen receptor (TCR)-mediated functions. In the present study, it is demonstrated that the various NK-R molecules typically expressed by NK cells are also expressed on periheral blood T lymphocytes. These CD3+ NK-R+ cells have a cell surface phenotype typical of memory cells as indicated by the expression of CD45RO and CD29 and by the lack of CD28 and CD45RA. Furthermore, by the combined use of anti-TCR V beta-specific antibodies and a semiquantitative polymerase chain reaction assay, the TCR repertoire in this CD3+ NK-R+ cell subset was found to be skewed; in fact, one or two V beta families were largely represented, and most of the other V beta s were barely detected. In addition, analysis of recombinant clones of the largely represented V beta families demonstrated that these V beta s were oligoclonally or monoclonally expanded.

Isolation and characterization of integron-containing bacteria without antibiotic selection

The emergence of antibiotic resistance among pathogenic and commensal bacteria has become a serious problem worldwide. The use and overuse of antibiotics in a number of settings are contributing to the development of antibiotic-resistant microorganisms. The class 1 and 2 integrase genes (intI1 and intI2, respectively) were identified in mixed bacterial cultures enriched from bovine feces by growth in buffered peptone water (BPW) followed by integrase-specific PCR. Integrase-positive bacterial colonies from the enrichment cultures were then isolated by using hydrophobic grid membrane filters and integrase-specific gene probes. Bacterial clones isolated by this technique were then confirmed to carry integrons by further testing by PCR and DNA sequencing. Integron-associated antibiotic resistance genes were detected in bacteria such as Escherichia coli, Aeromonas spp., Proteus spp., Morganella morganii, Shewanella spp., and urea-positive Providencia stuartii isolates from bovine fecal samples without the use of selective enrichment media containing antibiotics. Streptomycin and trimethoprim resistance were commonly associated with integrons. The advantages conferred by this methodology are that a wide variety of integron-containing bacteria may be simultaneously cultured in BPW enrichments and culture biases due to antibiotic selection can be avoided. Rapid and efficient identification, isolation, and characterization of antibiotic resistance-associated integrons are possible by this protocol. These methods will facilitate greater understanding of the factors that contribute to the presence and transfer of integron-associated antibiotic resistance genes in bacterial isolates from red meat production animals.

The crystal structure of a bacterial Class II ketol-acid reductolsomerase: Domain conservation and evolution

Ketol-acid reductoisomerase (KARI; EC 1.1.1.86) catalyzes two steps in the biosynthesis of branched-chain amino acids. Amino acid sequence comparisons across species reveal that there are two types of this enzyme: a short form (Class 1) found in fungi and most bacteria, and a long form (Class 11) typical of plants. Crystal structures of each have been reported previously. However, some bacteria such as Escherichia coli possess a long form, where the amino acid sequence differs appreciably from that found in plants. Here, we report the crystal structure of the E. coli enzyme at 2.6 A resolution, the first three-dimensional structure of any bacterial Class 11 KARI. The enzyme consists of two domains, one with mixed alpha/beta structure, which is similar to that found in other pyridine nucleotide-dependent dehydrogenases. The second domain is mainly alpha-helical and shows strong evidence of internal duplication. Comparison of the active sites between KARI of E. coli, Pseudomonas aeruginosa, and spinach shows that most residues occupy conserved positions in the active site. E. coli KARI was crystallized as a tetramer, the likely biologically active unit. This contrasts with P. aeruginosa KARI, which forms a dodecamer, and spinach KARI, a dimer. In the E. coli KARI tetramer, a novel subunit-to-subunit interacting surface is formed by a symmetrical pair of bulbous protrusions.

Subtypes of Illicit Drug Users: A Latent Class Analysis of Data From an Australian Twin Sample

This article applies methods of latent class analysis (LCA) to data on lifetime illicit drug use in order to determine whether qualitatively distinct classes of illicit drug users can be identified. Self-report data on lifetime illicit drug use (cannabis, stimulants, hallucinogens, sedatives, inhalants, cocaine, opioids and solvents) collected from a sample of 6265 Australian twins (average age 30 years) were analyzed using LCA. Rates of childhood sexual and physical abuse, lifetime alcohol and tobacco dependence, symptoms of illicit drug abuse/dependence and psychiatric comorbidity were compared across classes using multinomial logistic regression. LCA identified a 5-class model: Class 1 (68.5%) had low risks of the use of all drugs except cannabis; Class 2 (17.8%) had moderate risks of the use of all drugs; Class 3 (6.6%) had high rates of cocaine, other stimulant and hallucinogen use but lower risks for the use of sedatives or opioids. Conversely, Class 4 (3.0%) had relatively low risks of cocaine, other stimulant or hallucinogen use but high rates of sedative and opioid use. Finally, Class 5 (4.2%) had uniformly high probabilities for the use of all drugs. Rates of psychiatric comorbidity were highest in the polydrug class although the sedative/opioid class had elevated rates of depression/suicidal behaviors and exposure to childhood abuse. Aggregation of population-level data may obscure important subgroup differences in patterns of illicit drug use and psychiatric comorbidity. Further exploration of a 'self-medicating' subgroup is needed.

Proteção da comunidade aquática em efluente tratado por tanques de estabilização de resíduos no rio São Lourenço em São Lourenço da Serra, Vale do Ribeira, estado de São Paulo

O tratamento de esgotos de pequenas cidades por lagoas de estabilização é uma maneira simples, eficiente e de baixo custo. Os esgotos são uma fonte de contaminação das águas e solos e, conseqüentemente, contribuem para a transmissão de doenças, além de serem uma ameaça à preservação do meio ambiente. Surge a necessidade de investigar as condições dos efluentes lançados nos cursos d´água. O presente trabalho tem como objetivo realizar uma investigação da qualidade das águas residuárias tratadas por lagoas de estabilização de uma estação de tratamento de esgoto localizadas no município de São Lourenço da Serra no Vale da Ribeira no Estado de São Paulo e verificar os riscos sanitários e a comunidade aquática no Rio São Lourenço da Serra. Foram realizadas amostragens para avaliar o conjunto de lagoas anaeróbia e facultativa da estação de tratamento de esgoto do Município de São Lourenço da Serra. Os parâmetros utilizados foram pH, temperatura do ar e da água, condições climáticas, demanda bioquímica de oxigênio e nitrogênio amoniacal, bactérias termotolerantes, pigmentos fotossintéticos e comunidade zooplanctônica. Verificou-se que s sistemas de lagoa anaeróbia e facultativa foram eficientes na produção de efluente e apresentou alguns valores de acordo com a Resolução Conama nº 357, que estabelecem os valores limites para lançamento em corpos d´água. O rio São Lourenço está localizado em uma área de proteção ambiental. Os dados são comparados aos limites estabelecidos na Classe 1 e 2 e demonstram processo de eutrofização, colocando em risco à biodiversidade aquática e a saúde da população

Sphericity of apatite particles determined by gas permeability through packed beds

Because shape is an assessment of the three-dimensional form of a particle, it may be described in terms of sphericity (Psi), which is a measure of how closely a particle approaches a spherical configuration. In this study, Darcy`s law and the Kozeny-Carman model for fluid flow through porous media were applied to packed beds to determine the sphericity (Psi) of apatite particles. The beds were composed of glass spheres or particles of apatite (igneous from Brazil and sedimentary from the United States) of three classes of size (Class 1: -297 +210 mu m; Class 2: -210 +149 mu m; Class 3: -149 +105 mu m). Glass spheres were used to validate the model because of its known sphericity (Psi = 1.00). Apatite particles, either igneous or sedimentary, showed very close values for particle sphericity (Psi approximate to 0.6). Observations on particle images conducted by scanning electron microscopy illustrated that igneous (Psi = 0.623) and sedimentary (Psi = 0.644) particles of apatite of Class 2 predominantly exhibit elongated shape. The close value of particle sphericity (Psi approximate to 0.6) showed by either igneous or sedimentary apatite may be justified by the similarity in particle shape.

Spatial Modeling of a Soil Fertility Index using Visible-Near-Infrared Spectra and Terrain Attributes

Our objective was to develop a methodology to predict soil fertility using visible near-infrared (vis-NIR) diffuse reflectance spectra and terrain attributes derived from a digital elevation model (DEM). Specifically, our aims were to: (i) assemble a minimum data set to develop a soil fertility index for sugarcane (Sarcharum officinarum L.) (SFI-SC) for biofuel production in tropical soils; (ii) construct a model to predict the SFI-SC using soil vis-NIR spectra and terrain attributes; and (iii) produce a soil fertility map for our study area and assess it by comparing it with a green vegetation index (GVI). The study area was 185 ha located in sao Paulo State, Brazil. In total, 184 soil samples were collected and analyzed for a range of soil chemical and physical properties. Their vis-NIR spectra were collected from 400 to 2500 nm. The Shuttle Radar Topographic Mission 3-arcsec (90-m resolution) DEM of the area was used to derive 17 terrain attributes. A minimum data set of soil properties was selected to develop the SFI-SC. The SFI-SC consisted of three classes: Class 1, the highly fertile soils; Class 2, the fertile soils; and Class 3, the least fertile soils. It was derived heuristically with conditionals and using expert knowledge. The index was modeled with the spectra and terrain data using cross-validated decision trees. The cross-validation of the model correctly predicted Class 1 in 75% of cases, Class 2 in 61%, and Class 3 in 65%. A fertility map was derived for the study area and compared with a map of the GVI. Our approach offers a methodology that incorporates expert knowledge to derive the SFI-SC and uses a versatile spectro-spatial methodology that may be implemented for rapid and accurate determination of soil fertility and better exploration of areas suitable for production.

Natural regeneration of Atlantic Forest species in the understory of Eucalyptus saligna Smith. in a former forest production unit at the Parque das Neblinas, Bertioga, SP

This study aimed at characterizing the potential for natural regeneration of native vegetation in the under-story of an earlier Eucalyptus saligna Smith production stand. The study was carried out at the Parque das Neblinas, Bertioga municipality, SP, in a 45 ha third rotation stand; which had been abandoned 15 years ago for natural regeneration to occur. The sampling was done in 24 plots of 20 x 40 m. The sampled area was of 19,200 m(2), with inventory made of 100% of the eucalyptus trees. All regeneration trees with a height >= 1.30 m and DBH >= 5.0 cm were measured, as well as adult individuals with DBH >= 5.0 cm; surveyed in two size classes. 1,417 individuals of E. saligna were measured, with a density of 738,02 individuals/ha and a basal area of 22.69 m(2)/ha. Among 2,763 natural regeneration individuals, 111 species belonged to 66 genera and 34 botanical families. The species represented 43.7% of the tree richness of neighboring native forest fragments. The total estimated density and the basal area were respectively 1,052.6 individuals/ha and 6.4 m(2)/ha of autochthonous trees with DBH >= 5.0 cm (Class 1); while for regeneration there were 3,864.58 individuals/ha, and 2.76 m(2)/ha of individuals with a height >= 1.30 m and DBH <5.0 cm (Class 2). Shannon diversity (H`) was 2.83 and 3.68, respectively, for Classes 1 and 2, and the corrected species richness for a 1000-individual sample (R(1000)) were 75.6 and 87.29 (Fisher`s a index) for the same classes. The majority of the species (34.84%) was typical from the understory of wet tropical forest and had zoochoric fruit dispersal (67.57%). The results indicate that, under these conditions, a eucalyptus forest is able to provide adequate regeneration niches for native vegetation, and may represent a sink habitat for local populations.

Mice expressing the E7 oncogene of HPV16 in epithelium show central tolerance, and evidence of peripheral anergising tolerance, to E7-encoded cytotoxic T-lymphocyte epitopes

In order to derive mice which expressed both the E7 open reading frame transgene of human papillomavirus type 16 in skin and MHC class 1 restriction elements for several E7-encoded cytotoxic T-lymphocyte (CTL) epitopes, K14.HPV16E7 mice which express E7 in basal keratinocytes were crossed to the F1 generation with A2.1 K-b transgenic mice which express the MHC binding cleft domains of human HLA A*0201, and murine H-2(b). F1 mice (denoted K14E7xA2.1) expressed E7 in the thymus at least as early as 2-5 days before birth. Immunisation of FVBxA2.1 control mice (transgenic for HLA A*0201 and H-2(b) but not for E7), with two HLA A*0201-restricted epitopes of E7 and one H-2(b)-restricted CTL epitope of E7, gave strong primary CTL responses recognising epitope-pulsed or constitutively E7-expressing syngeneic target cells. In contrast, in immunised K14E7xA2.1 mice, the CTL responses to the H-2(b) epitope and one of the HLA A*0201 CTL epitopes were strongly down-regulated, and to the other HLA A*0201 epitope, completely abolished, as demonstrated by percentage specific killing by bulk splenocyte cultures in cyrotoxicity assays, and by CTL precursor frequency analysis, In thymus-transplanted bone marrow radiation chimeras in which the immune system of K14E7xA2.1 mice was replaced by a FVBxA2.1 immune system, specific immunisation did not result in reemergence of strong E7-directed CTL responses. In agreement with these in vitro findings, specific immunisation failed to significantly alter the course of E7-associated tumour development in K14E7xA2.1 mice. These data are consistent with a model of central deletional CTL tolerance to E7-encoded epitopes recognised in the context of two distinct MHC class 1 restriction elements, and with the possibility of peripheral T-cell anergy maintained by expression of E7 in the skin. (C) 1998 Academic Press.

Papillomavirus virus-like particles for the delivery of multiple cytotoxic T cell epitopes

Chimeric papillomavirus (PV) virus-like particles (VLPs) based on the bovine papillomavirus type 1 (BPV-1) L1 protein were constructed by replacing the 23-carboxyl-terminal amino acids of the BPV1 major protein L1 with an artificial polytope minigene, containing known CTL epitopes of human PV16 E7 protein, HIV IIIB gp120 P18, Nef, and reverse transcriptase (RT) proteins, and an HPV16 E7 linear B epitope. The CTL epitopes were restricted by three different MHC class 1 alleles (H-2(b), H-2(d), HLA-A*0201). The chimeric L1 protein assembled into VLPs when expressed in SF-9 cells by recombinant baculovirus. After immunization of mice with polytope VLPs in the absence of adjuvant, serum antibodies were detected which reacted with both polytope VLPs and wild-type BPV1L1 VLPs, in addition to the HPV16E7 linear B cell epitope. CTL precursors specific for the HPV16 E7, HIV P18, and RT CTL epitopes were also detected in the spleen of immunized mice. Polytope VLPs can thus deliver multiple B and T epitopes as immunogens to the MHC class I and class II pathways, extending the utility of VLPs as self-adjuvanting immunogen delivery systems. (C) 2000 Academic Press.

Role of cytokine gene polymorphisms in acute rejection and renal impairment after liver transplantation

Although immunosuppressive regimens are effective, rejection occurs in up to 50% of patients after orthotopic liver transplantation (OLT), and there is concern about side effects from long-term therapy. Knowledge of clinical and immunogenetic variables may allow tailoring of immunosuppressive therapy to patients according to their potential risks. We studied the association between transforming growth factor-beta, interleukin-10, and tumor necrosis factor alpha (TNF-alpha) gene polymorphisms and graft rejection and renal impairment in 121 white liver transplant recipients. Clinical variables were collected retrospectively, and creatinine clearance was estimated using the formula of Cockcroft and Gault. Biallelic polymorphisms were detected using polymerase chain reaction-based methods. Thirty-seven of 121 patients (30.6%) developed at least 1 episode of rejection. Multivariate analysis showed that Child-Pugh score (P =.001), immune-mediated liver disease (P =.018), normal pre-OLT creatinine clearance (P =.037), and fewer HLA class 1 mismatches (P =.038) were independently associated with rejection, Renal impairment occurred in 80% of patients and was moderate or severe in 39%, Clinical variables independently associated with renal impairment were female sex (P =.001), pre-OLT renal dysfunction (P =.0001), and a diagnosis of viral hepatitis (P =.0008), There was a significant difference in the frequency of TNF-alpha -308 alleles among the primary liver diseases. After adjustment for potential confounders and a Bonferroni correction, the association between the TNF-alpha -308 polymorphism and graft rejection approached significance (P =.06). Recipient cytokine genotypes do not have a major independent role in graft rejection or renal impairment after OLT, Additional studies of immunogenetic factors require analysis of large numbers of patients with appropriate phenotypic information to avoid population stratification, which may lead to inappropriate conclusions.

Limitations of HLA-transgenic mice in presentation of HLA-restricted cytotoxic T-cell epitopes from endogenously processed human papillomavirus type 16 E7 protein

We investigated the use of mice transgenic for human leucocyte antigen (HLA) A*0201 antigen-binding domains to test vaccines composed of defined HLA A*0201-restricted cytotoxic T-lymphocyte (CTL) epitopes of human papillomavirus (HPV) type 16 E7 oncoprotein. HPV is detected in >90% of cervical carcinomas. HPV16 E7 oncoprotein transforms cells of the uterine cervix and functions as a tumour-associated antigen to which immunotherapeutic strategies may be directed. We report that although the HLA A*0201 E7 epitope peptides function both to prime for E7 CTL responses, and to sensitize target cells for E7-directed CTL killing in situations where antigen processing is not required, the epitopes are not processed out of either endogenously expressed or immunization-introduced E7, by the mouse antigen-processing and presentation machinery. Thus (1) CTL induced by HLA A*0201 peptide immunization killed E7 peptide-pulsed target cells, but did not kill target cells expressing whole E7; (2) immunization with whole E7 protein did not elicit CTL directed to HLA A*0201-restricted E7 CTL epitopes; (3) HLA A*0201-restricted CTL epitopes expressed in the context of a DNA polytope vaccine did not activate E7-specific T cells either in 'conventional' HLA A*0201 transgenic (A2.1K(b) ) mice, or in HHD transgenic mice in which expression of endogenous H-2 class 1 is precluded; and (4) HLA A*0201 E7 peptide epitope immunization was incapable of preventing the growth of an HLA A*0201- and E7-expressing tumour. There are generic implications for the universal applicability of HLA-class 1 transgenic mice for studies of human CTL epitope presentation in murine models of human infectious disease where recognition of endogenously processed antigen is necessary. There are also specific implications for the use of HLA A2 transgenic mice for the development of E7-based therapeutic vaccines for cervical cancer.

Pediculus capitis infestation according to sex and social factors in Argentina

OBJECTIVE: To determine the intensity of Pediculus capitis infestation (abundance) among Argentinean schoolchildren. Children's sex and social stratum were analyzed as modifiers of the general prevalence and degree of parasitism. METHODS: The study included 1,370 schoolchildren (692 girls, 678 boys) from 26 schools of the province of La Rioja (21 public schools, five private schools). Classic prevalence was obtained as the percentage of children with nits and/or lice. Moreover, five degrees of parasitism were classified: 0) children with no signs of pediculosis; 0+) children with evidence of past infestation; 1) children with a recent infestation and low probability of active parasitism; 2) children with a recent infestation and high probability of active parasitism; 3) children with mobile lice (active pediculosis). RESULTS: The general prevalence was 61.4% (girls: 79%; boys: 44%, p<0.001). Private schools showed lower prevalence than public schools (p=0.02), especially due to the low prevalence in boys. Fifty percent of children were classified in classes 0 and 0+, 22% in class 1; and 28% in grades 2 and 3. The proportion of children in grade 3 was higher in public schools than in private schools. There were significant sexual differences in the intensity of parasitism for grades 2 and 3, where girls' rates exceeded twice those of boys'. CONCLUSIONS: Sex and social stratum are important modifiers of P. capitis general prevalence and degree of infestation. The classification of children by intensity of infestation allowed a more precise delimitation of this condition, which is especially important for disease surveillance and application of control measures.

Two Novel CMY-2-Type β-Lactamases Encountered in Clinical Escherichia Coli Isolates

BACKGROUND: Chromosomally encoded AmpC β-lactamases may be acquired by transmissible plasmids which consequently can disseminate into bacteria lacking or poorly expressing a chromosomal bla AmpC gene. Nowadays, these plasmid-mediated AmpC β-lactamases are found in different bacterial species, namely Enterobacteriaceae, which typically do not express these types of β-lactamase such as Klebsiella spp. or Escherichia coli. This study was performed to characterize two E. coli isolates collected in two different Portuguese hospitals, both carrying a novel CMY-2-type β-lactamase-encoding gene. FINDINGS: Both isolates, INSRA1169 and INSRA3413, and their respective transformants, were non-susceptible to amoxicillin, amoxicillin plus clavulanic acid, cephalothin, cefoxitin, ceftazidime and cefotaxime, but susceptible to cefepime and imipenem, and presented evidence of synergy between cloxacilin and cefoxitin and/or ceftazidime. The genetic characterization of both isolates revealed the presence of bla CMY-46 and bla CMY-50 genes, respectively, and the following three resistance-encoding regions: a Citrobacter freundii chromosome-type structure encompassing a blc-sugE-bla CMY-2-type -ampR platform; a sul1-type class 1 integron with two antibiotic resistance gene cassettes (dfrA1 and aadA1); and a truncated mercury resistance operon. CONCLUSIONS: This study describes two new bla CMY-2-type genes in E. coli isolates, located within a C. freundii-derived fragment, which may suggest their mobilization through mobile genetic elements. The presence of the three different resistance regions in these isolates, with diverse genetic determinants of resistance and mobile elements, may further contribute to the emergence and spread of these genes, both at a chromosomal or/and plasmid level.