Longitudinal Study Detects the Co-Carriage of ESBL and mcr-1 and -4 Genes in Escherichia coli Strains in a Portuguese Farrow-to-Finish Swine Herd.

Cephalosporins and polymyxins are employed in antimicrobial protocols to control and treat neonatal infections and post-weaning diarrhoea in swine operations. We conducted a longitudinal study to evaluate the colonization and transmission of antibiotic-resistant Escherichia coli in sows and their piglets in a farrow-to-finish operation, focusing on characterization of Extended Spectrum Beta-Lactamase (ESBL) and mcr genes, virulence traits and genetic relatedness. A total of 293 E. coli isolates were obtained from faecal samples collected in five time points. At birth blaCTX-M-1group cluster was detected in E. coli isolates from 9 sows and 49 piglets (73.41%), while in the following four' piglets sampling moments it was detected in 91.8%, 57.6%, 71.4% and 97.4%. The gene mcr-1 was detected in E. coli from one sow and from three piglets from different litters at birth and increased in the first weeks of piglet life (68.85%, 100%, 90% and 8.1%). A new mcr-4 allele, mcr-4.7, was identified in 3.28%, 28.57%, 7.5% of E. coli isolates. Most mcr-positive E. coli isolates (96,7%) carried blaCTX-M-1Group genes and 93,33% carried both mcr-4 and mcr-1. CTX-M-1 and CTX-M-32 were the most predominant ESBLs. Plasmids belonged to IncI1, IncF and IncN groups. Most isolates belong to phylogenetic group B1; PAI IV536 marker was detected in nine isolates. The strains were kept in the different stages of the piglets' life. The use of ceftiofur and colistin may explain the high prevalence and co-selection of blaCTX-M-1Group and mcr-1 and/or -4 genes, contributing to the maintenance of resistant and virulent isolates throughout the pig life cycle that may reach the food chain.

Forest phenoclusters for Argentina based on vegetation phenology and climate.

Forest biodiversity conservation and species distribution modeling greatly benefit from broad-scale forest maps depicting tree species or forest types rather than just presence and absence of forest, or coarse classifications. Ideally, such maps would stem from satellite image classification based on abundant field data for both model training and accuracy assessments, but such field data do not exist in many parts of the globe. However, different forest types and tree species differ in their vegetation phenology, offering an opportunity to map and characterize forests based on the seasonal dynamic of vegetation indices and auxiliary data. Our goal was to map and characterize forests based on both land surface phenology and climate patterns, defined here as forest phenoclusters. We applied our methodology in Argentina (2.8 million km2 ), which has a wide variety of forests, from rainforests to cold-temperate forests. We calculated phenology measures after fitting a harmonic curve of the enhanced vegetation index (EVI) time series derived from 30-m Sentinel 2 and Landsat 8 data from 2018-2019. For climate, we calculated land surface temperature (LST) from Band 10 of the thermal infrared sensor (TIRS) of Landsat 8, and precipitation from Worldclim (BIO12). We performed stratified X-means cluster classifications followed by hierarchical clustering. The resulting clusters separated well into 54 forest phenoclusters with unique combinations of vegetation phenology and climate characteristics. The EVI 90th percentile was more important than our climate and other phenology measures in providing separability among different forest phenoclusters. Our results highlight the potential of combining remotely sensed phenology measures and climate data to improve broad-scale forest mapping for different management and conservation goals, capturing functional rather than structural or compositional characteristics between and within tree species. Our approach results in classifications that go beyond simple forest-nonforest in areas where the lack of detailed ecological field data precludes tree species-level classifications, yet conservation needs are high. Our map of forest phenoclusters is a valuable tool for the assessment of natural resources, and the management of the environment at scales relevant for conservation actions.

Diversity of metal and antibiotic resistance genes in Enterococcus spp. from the last century reflects multiple pollution and genetic exchange among phyla from overlapping ecosystems.

Arsenic (As), mercury (Hg), and copper (Cu) are among the major historical and contemporary metal pollutants linked to global anthropogenic activities. Enterococcus have been considered indicators of fecal pollution and antibiotic resistance for years, but its largely underexplored metallome precludes understanding their role as metal pollution bioindicators as well. Our goal was to determine the occurrence, diversity, and phenotypes associated with known acquired genes/operons conferring tolerance to As, Hg or Cu among Enterococcus and to identify their genetic context (381 field isolates from diverse epidemiological and genetic backgrounds; 3547 enterococcal genomes available in databases representing a time span during 1900-2019). Genes conferring tolerance to As (arsA), Hg (merA) or Cu (tcrB) were used as biomarkers of widespread metal tolerance operons. Different variants of metal tolerance (MeT) genes (13 arsA, 6 merA, 1 tcrB) were more commonly recovered from the food-chain (arsA, tcrB) or humans (merA), and were shared with 49 other bacterial taxa. Comparative genomics analysis revealed that MeT genes occurred in heterogeneous operons, at least since the 1900s, with an increasing accretion of antibiotic resistance genes since the 1960's, reflecting diverse antimicrobial pollution. Multiple MeT genes were co-located on the chromosome or conjugative plasmids flanked by elements with high potential for recombination, often along with antibiotic resistance genes. Phenotypic analysis of some isolates carrying MeT genes revealed up to 128× fold increase in the minimum inhibitory concentrations to metals. The main distribution of functional MeT genes among Enterococcus faecium and Enterococcus faecalis from different sources, time spans, and clonal lineages, and their ability to acquire diverse genes from multiple taxa bacterial communities places these species as good candidates to be used as model organisms in future projects aiming at the identification and quantification of bioindicators of metal polluted environments by anthropogenic activities.

Spatial distribution of wood volume in Brazilian savannas.

Here we model and describe the wood volume of Cerrado Sensu Stricto, a highly heterogeneous vegetation type in the Savanna biome, in the state of Minas Gerais, Brazil, integrating forest inventory data with spatial-environmental variables, multivariate regression, and regression kriging. Our study contributes to a better understanding of the factors that affect the spatial distribution of the wood volume of this vegetation type as well as allowing better representation of the spatial heterogeneity of this biome. Wood volume estimates were obtained through regression models using different environmental variables as independent variables. Using the best fitted model, spatial analysis of the residuals was carried out by selecting a semivariogram model for generating an ordinary kriging map, which in turn was used with the fitted regression model in the regression kriging technique. Seasonality of both temperature and precipitation, along with the density of deforestation, explained the variations of wood volume throughout Minas Gerais. The spatial distribution of predicted wood volume of Cerrado Sensu Stricto in Minas Gerais revealed the high variability of this variable (15.32 to 98.38 m3 ha-1) and the decreasing gradient in the southeast-northwest direction.

Molecular characterization of carbapenemases of clinical Acinetobacter baumannii-calcoaceticus complex isolates from a University Hospital in Tunisia.

The aim of this study was to identify the carbapenemases from clinical carbapenem-resistant Acinetobacter baumannii-calcoaceticus complex (CRABC) isolates and to assess their potential dissemination by conjugation and natural transformation. CRABC (n = 101) were collected consecutively from inpatients of the University Hospital of Monastir, Tunisia, from 2013 to 2016. Antimicrobial susceptibility was determined by the disk diffusion method and E-test. Carbapenemase-encoding genes were screened by PCR. Genotyping was performed by Pasteur MLST scheme. Isolates were resistant to all beta-lactams, fluoroquinolones and aminoglycosides while 80 and 90% were susceptible to tigecycline and colistin, respectively. Resistance and intermediate resistance to imipenem were 87 and 13%, respectively. The genes blaOXA-24-like, blaOXA-58-like, blaOXA-143-like, blaOXA-48-like, blaVIM, blaIMP, and blaKPC were not found. The blaOXA-51-like and blaOXA-23-like genes were present in 100 and 82.17% isolates, respectively. One isolate (< 1%) carried blaNDM-1 and blaOXA-51-like and belonged to Sequence Type 85 (ST85). Absence of transconjugants suggests a chromosomal location of NDM-1 determinant. The blaNDM-1 gene was inserted in a truncated form of Tn125, which may explain the absence of blaNDM-1 carrier-transformants. To our knowledge, this is the first report of the finding of NDM-positive A. baumannii in Tunisian territory. The study shows that despite the low prevalence and potential spread of NDM-1 enzyme among CRABC, continuous regional antimicrobial resistance surveillance and improved infection control measures are required in Tunisia to prevent further dissemination.

Water supply and feed as sources of antimicrobial-resistant Enterococcus spp. in aquacultures of rainbow trout (Oncorhyncus mykiss), Portugal.

The role of European fish farms in the spread of antimicrobial-resistance in the environment and food chain, as well as possible sources of their contamination by clinically relevant antimicrobial-resistance bacteria is scarcely known. This study aimed to assess the contribution of Portuguese rural trout farms on dispersion of Enterococcus with antimicrobial-resistance and putative virulence genes in the environment and food chain, as well as to identify farms contamination sources. We also assessed the presence of Enterococcus with low-levels of antimicrobial-resistance using epidemiological cut-offs (ECOFFs). Enterococcus spp. (n=391) from water/sediment recovered upstream, within and downstream trout tanks, feed, trout (2 aquacultures; no antibiotic use) and marketed trout (8 supermarkets) showed variable resistance to tetracycline, erythromycin, ciprofloxacin, chloramphenicol, quinupristin-dalfopristin, nitrofurantoin or aminoglycosides. Antimicrobial-resistance rates were similar among upstream, within and downstream trout tank samples (P>0.05), positioning water-supplying aquacultures as a source of multidrug-resistant (MDR) strains. Nevertheless, predominance of MDR E. faecium in feed, trout tanks and trout comparing to upstream samples, suggests feed as an additional aquaculture contamination source. The observation of E. faecium and E. faecalis susceptible to ampicillin and gentamicin by clinical breakpoints but with low-levels of resistance to those antimicrobials by ECOFFs breakpoints is of concern, as they might evolve throughout secondary genetic events to resistance levels with human clinical impact. Multiple MDR clones carrying copper tolerance (tcrB/cueO), putative virulence or other genes often associated with clinical strains (e.g. E. faecium with IS16/ptsD/sgrA) were observed, some in distinct samples (e.g. upstream and within trout tanks). They included major human and animal Enterococcus lineages, suggesting human and non-aquatic animal origins. The results highlight the need to define the maximum acceptance level of antimicrobial-resistance genes/bacteria to assess water quality and to monitor antimicrobial-resistance strains on feed, essential requirements to maintain a sustainable aquaculture production.

Object-based change detection using semivariogram indices derived from NDVI images: The environmental disaster in Mariana, Brazil / Detecção de mudanças baseada em objetos utilizando indices do semivariograma derivados de imagens NDVI: O disastre ambiental em Mariana, Brasil

ABSTRACT Object-based change detection is a powerful analysis tool for remote sensing data, but few studies consider the potential of temporal semivariogram indices for mapping land-cover changes using object-based approaches. In this study, we explored and evaluated the performance of semivariogram indices calculated from remote sensing imagery, using the Normalized Differential Vegetation Index (NDVI) to detect changes in spatial features related to land cover caused by a disastrous 2015 dam failure in Brazil's Mariana district. We calculated the NDVI from Landsat 8 images acquired before and after the disaster, then created objects by multiresolution segmentation analysis based on post-disaster images. Experimental semivariograms were computed within the image objects and semivariogram indices were calculated and selected by principal component analysis. We used the selected indices as input data to a support vector machine algorithm for classifying change and no-change classes. The selected semivariogram indices showed their effectiveness as input data for object-based change detection analysis, producing highly accurate maps of areas affected by post-dam-failure flooding in the region. This approach can be used in many other contexts for rapid and accurate assessment of such land-cover changes.

RESUMO Recentemente, variáveis geoestatísticas derivadas de imagens de sensoriamento remoto ganharam espaço dentre os procedimentos de detecção de mudanças, porém, o potencial temporal destas variáveis para o mapeamento das mudanças baseado na análise por objetos ainda é pouco estudado. Neste estudo, o desempenho de um conjunto de índices calculados de semivariogramas derivados de imagens NDVI bitemporais para detectar mudanças na cobertura do solo foi analisado e avaliado. O município de Mariana foi selecionado para teste e validação da metodologia devido ao grande impacto ocasionado pelo desastre. O processo iniciou-se com a aquisição de imagens Landsat 8 antes e após o desastre e o cálculo do NDVI. Os objetos foram criados através da segmentação em multiresolução baseada na imagem pós-desastre. Os semivariogramas experimentais foram gerados dentro de cada objeto e os índices foram extraídos e selecionados através da análise de componentes principais. Os índices selecionados foram utilizados como dados de entrada para o algoritmo support vector machines para a classificação de áreas de mudança e não mudança. Os índices selecionados se mostraram efetivos para a detecção de mudanças, indicando a possibilidade de utilização para a detecção de mudanças baseada em objetos, resultando em um mapa precisos das áreas inundadas afetadas pelo desastre. Esta abordagem pode ser usada em muitos outros contextos para uma avaliação rápida e precisa de tais mudanças na cobertura do solo.

Co-diversification of Enterococcus faecium Core Genomes and PBP5: Evidences of pbp5 Horizontal Transfer.

Ampicillin resistance has greatly contributed to the recent dramatic increase of a cluster of human adapted Enterococcus faecium lineages (ST17, ST18, and ST78) in hospital-based infections. Changes in the chromosomal pbp5 gene have been associated with different levels of ampicillin susceptibility, leading to protein variants (designated as PBP5 C-types to keep the nomenclature used in previous works) with diverse degrees of reduction in penicillin affinity. Our goal was to use a comparative genomics approach to evaluate the relationship between the diversity of PBP5 among E. faecium isolates of different phylogenomic groups as well as to assess the pbp5 transferability among isolates of disparate clonal lineages. The analyses of 78 selected E. faecium strains as well as published E. faecium genomes, suggested that the diversity of pbp5 mirrors the phylogenomic diversification of E. faecium. The presence of identical PBP5 C-types as well as similar pbp5 genetic environments in different E. faecium lineages and clones from quite different geographical and environmental origin was also documented and would indicate their horizontal gene transfer among E. faecium populations. This was supported by experimental assays showing transfer of large (≈180-280 kb) chromosomal genetic platforms containing pbp5 alleles, ponA (transglycosilase) and other metabolic and adaptive features, from E. faecium donor isolates to suitable E. faecium recipient strains. Mutation profile analysis of PBP5 from available genomes and strains from this study suggests that the spread of PBP5 C-types might have occurred even in the absence of a significant ampicillin resistance phenotype. In summary, genetic platforms containing pbp5 sequences were stably maintained in particular E. faecium lineages, but were also able to be transferred among E. faecium clones of different origins, emphasizing the growing risk of further spread of ampicillin resistance in this nosocomial pathogen.

Co-transfer of resistance to high concentrations of copper and first-line antibiotics among Enterococcus from different origins (humans, animals, the environment and foods) and clonal lineages.

OBJECTIVES: We studied the occurrence of diverse copper (Cu) tolerance genes from Gram-positive bacteria and their co-transfer with antibiotic resistance genes among Enterococcus from diverse sources. METHODS: Enterococcus (n = 922) of several species and from human, animal, environment and food samples were included. Antimicrobial and CuSO4 susceptibility and conjugation assays were performed by standard procedures, bacterial screening of Cu and antibiotic resistance genes by PCR, and clonality by PFGE/multilocus sequence typing. RESULTS: tcrB and cueO genes occurred in 15% (n = 137/922) and 14% (n = 128/922) of isolates, respectively, with the highest occurrence in piggeries (P < 0.05). They were more frequent among Enterococcus faecium (tcrB: 23% versus 8% in Enterococcus faecalis and 12% in other species; cueO: 25% versus 5% and 9%, respectively; P < 0.05). A correlation between phenotypic and genotypic assays was observed for most E. faecium (CuSO4 MIC50 = 24 mM in tcrB/cueO(+) versus CuSO4 MIC50 = 12 mM in tcrB/cueO(-)), but not for other species. Co-transfer of Cu tolerance (associated with tcrB, cueO or an unknown mechanism) with erythromycin, tetracycline, vancomycin, aminoglycosides or ampicillin resistance was demonstrated. A variety of PFGE types was detected among isolates carrying Cu tolerance mechanisms, some identified in sequence types (STs) often linked to human infections (E. faecium from ST18 and ST78 clonal lineages and E. faecalis clonal complex 2). CONCLUSIONS: Cu tolerance might contribute to the selection/maintenance of multidrug-resistant Enterococcus (including resistance to first-line antibiotics used to treat enterococcal infections) due to the use of Cu compounds (e.g. antiseptics/animal feed supplements). The distribution of the multicopper oxidase cueO and the co-transfer of ampicillin resistance along with Cu tolerance genes are described for the first time.

Spread of multidrug-resistant Enterococcus to animals and humans: an underestimated role for the pig farm environment.

OBJECTIVES: The aim of this study was to discover the potential role of the pig farm environment in the spread of multidrug-resistant (MDR) Enterococcus strains, including high-risk clones, to animals and humans. METHODS: Enterococcus isolates were recovered from a variety of samples (n = 82; swine, feed/medicines/antiseptics and pig farm facilities) from six Portuguese farms, most using antibiotics. Antimicrobial susceptibility/conjugation assays were performed by standard procedures, bacterial identification/screening of antibiotic resistance genes were performed by PCR and clonality was determined using PFGE/multilocus sequence typing. RESULTS: Enterococcus isolates resistant to antibiotics (n= 473) were recovered from samples of different origin (swine, feed/antiseptics, animal residues and pig farm facilities), but only the clinically relevant species Enterococcus faecium (n = 171) and Enterococcus faecalis (n = 78) were included for further comprehensive molecular analysis. Isolates resistant to vancomycin, ampicillin, tetracyclines, erythromycin and aminoglycosides were better recovered in Slanetz-Bartley medium with these antibiotics present than in media not supplemented with antibiotics (P < 0.05). E. faecium was more frequently resistant to ampicillin, ciprofloxacin or nitrofurantoin and E. faecalis to tetracyclines, chloramphenicol or aminoglycosides (P < 0.05). Glycopeptide and erythromycin resistance rates were similar in both species. The transfer of resistance to several antibiotics, including vancomycin and ampicillin, was demonstrated. Clones associated with human infections were detected in different samples from the same farm [E. faecium from sequence type (ST) 78 lineage and E. faecalis ST16; manure, waste lagoons, faeces and drinking water] and in geographically distant farms [E. faecium clonal complex (CC) 5; E. faecalis CC21 and ST16]. CONCLUSIONS: The pig farm environment has an underestimated potential role in the transmission of MDR Enterococcus to animals and, possibly, to humans. The continuous contact of swine with MDR Enterococcus by different routes (e.g. feed, dust, air and rooms) might decrease the impact of restrictive antibiotic use policies and reinforces the need for different and preliminary interventions at the husbandry management level.

Different genetic supports for the tet(S) gene in Enterococci.

The diversity of tet(S) genetic contexts of 13 enterococci from human, animal, and environmental samples from different geographical areas is reported. The tet(S) gene was linked to either CTn6000 variants of chromosomal location or composite platforms flanked by IS1216 located on plasmids (∼40 to 115 kb). The comparative analysis of all tet(S) genetic elements available in the GenBank databases suggests that CTn6000 might be the origin of a variety of tet(S)-carrying platforms that were mobilized to different plasmids.