Co-selection of antibiotic and heavy metal resistance in freshwater bacteria

Antibiotic resistant bacteria are found in most environments, especially in highly anthropized waters. A direct correlation between human activities (e.g., pollution) and spread and persistence of antibiotic resistant bacteria (ARB) and resistance genes (ARGs) within the resident bacterial communities appears more and more obvious. Furthermore, the threat posed for human health by the presence of ARB and ARGs in these environments is enhanced by the risk of horizontal gene transfer of resistance genes to human pathogens. Although the knowledge on the spread of antibiotic resistances in waters is increasing, the understanding of the driving factors determining the selection for antibiotic resistance in the environment is still scarce. Antibiotic pollution is generally coupled with contamination by heavy metals (HMs) and other chemicals, which can also promote the development of resistance mechanisms, often through co-selecting for multiple resistances. The co-selection of heavy metal resistance genes and ARGs in waters, sediments, and soils, increases the complexity of the ecological role of ARGs, and reduces the effectiveness of control actions. In this mini-review we present the state-of-the-art of the research on antibiotic- and HM-resistance and their connection in the environment, with a focus on HM pollution and aquatic environments. We review the spread and the persistence of HMs and/or ARB, and how it influences their respective gene co-selection. In the last chapter, we propose Lake Orta, a system characterized by an intensive HM pollution followed by a successful restoration of the chemistry of the water column, as a study-site to evaluate the spread and selection of HMs and antibiotic resistances in heavily disturbed environments

Does a barcoding gap exist in Prokaryotes? Evidences from species delimitation in Cyanobacteria

The amount of information that is available on 16S rRNA sequences for prokaryotes thanks to high-throughput sequencing could allow a better understanding of diversity. Nevertheless, the application of predetermined threshold in genetic distances to identify units of diversity (Operative Taxonomic Units, OTUs) may provide biased results. Here we tests for the existence of a barcoding gap in several groups of Cyanobacteria, defining units of diversity according to clear differences between within-species and among-species genetic distances in 16S rRNA. The application of a tool developed for animal DNA taxonomy, the Automatic Barcode Gap Detector (ABGD), revealed that a barcoding gap could actually be found in almost half of the datasets that we tested. The identification of units of diversity through this method provided results that were not compatible with those obtained with the identification of OTUs with threshold of similarity in genetic distances of 97% or 99%. The main message of our results is a call for caution in the estimate of diversity from 16S sequences only, given that different subjective choices in the method to delimit units could provide different results

Efeito da erosão extrínseca nas forças de adesão de uma peça de resina a esmalte: comparação in vitro de dois métodos de cimentação

Dissertação para obtenção do grau de Mestre no Instituto Superior de Ciências da Saúde Egas MonizObjetivo: O principal objetivo deste trabalho é estudar a resistência de adesão de uma peça de resina a esmalte erodido e não erodido, utilizando dois métodos de cimentação. Como objetivo secundário, pretendeu-se averiguar se existe correlação entre as forças de adesão e o tipo de fraturas ocorridas no teste de microtração e entre as forças de adesão e a espessura da camada de cimento.Materiais e Métodos: Nesta investigação in vitro serão utilizados quarenta incisivos inferiores de bovino, hígidos, que serão divididos aleatoriamente em dois grupos experimentais (n=20) segundo o protocolo de adesão. No primeiro grupo os dentes não serão sujeitos a qualquer protocolo de erosão, funcionando como grupo de controlo. No segundo, vão sofrer um processo de erosão ácida antes da cimentação simulando a erosão extrínseca provocada pela ingestão abusiva de refrigerantes. Cada grupo será depois dividido em dois subgrupos, variando entre eles o tipo de cimentação testado. Dez dentes serão usados para testar cimento resinoso, os outros dez para testar a cimentação com resina aquecida. Depois da cimentação, os dentes serão armazenados 24 horas num frasco com água destilada, numa estufa a 37º Celsius. Posteriormente serão seccionados por uma lâmina diamantada sob irrigação, na direção X e Y de forma a se obterem palitos com 0,9±0,2 milímetros (mm) para serem testados numa máquina de testes universal a uma velocidade de 0,5 milímetros por minuto (mm/min). As forças de adesão, variável dependente neste estudo, serão calculadas através da divisão da força aplicada no momento da fratura (N) do palito pela área da superfície aderida (mm2), e serão expressas em MPa. As forças de adesão serão analisadas estatisticamente para comparar os dois métodos de cimentação e estudar o efeito da pré-erosão extrínseca do esmalte dentário na adesão da resina.Resultados: As forças de adesão entre o esmalte e a resina não apresentaram diferenças estatisticamente significantes para os vários grupos estudados. Não se encontraram correlações entre as forças de adesão e o tipo de fratura, nem entre as forças de adesão e a espessura do cimento.Conclusões: A erosão do esmalte não influenciou a adesão da peça de resina em nenhum dos casos. Apresentando os dois sistemas adesivos valores similares de adesão

Presenza e distribuzione di batteri antibiotico-resistenti nelle acque del Lago Maggiore

Not availableRicerche sull\u27evoluzione del Lago Maggiore. Aspetti limnologici. Programma triennale 2013-2015. Campagna 2013. Presenza e distribuzione di batteri antibiotico-resistenti nelle acque del Lago Maggiore. Valutazione della presenza di geni di antibiotico-resistenza (ABR). Quantificazione dei geni di resistenza alle tetracicline

Zooplankton as refuge for invading bacteria

Nonetheless, studies on the impact of non-parasitic bacteria are still scarce. In this study we analysed the consequences of this colonisation on the distribution of potentially pathogenic and antibiotic resistant bacteria in freshwater systems. This is based on the hypothesis that potential pathogens have a selective advantage when growing in biofilms, as has been shown in clinical settings. Thus we postulate that attaching to animals might allow potential pathogens, e.g. deriving from the efflux of a waste water treatment plant (WWTP), to survive in freshwaters. Additionally the movement of the animal transports the bacteria away from the point-contamination to more pristine waters. We experimentally show that, if added to a natural bacterial community, Escherichia coli attaches to daphnids and derives particularly well in the gut of the animals, where they naturally do not form part of their microflora, whilst decreasing in abundance in the surrounding water. Moreover, the presence of daphnids decreased the abundance of a tetracycline resistance gene (tetA) in the surrounding water, whilst a higher number of tetA gene copies were detected in the animals. In addition we tested whether zooplankton species form a surface for the horizontal gene transfer, of e.g. antibiotic resistant genes, from one E. coli strain to another. In order to expand our hypothesis to natural environment we sampled Lake Maggiore at three points with increasing distance to an inflow of a WWTP and compared the microbial community on various zooplankton species as well as on stones and in the sediment. Whereas similar relationships have been described in marine systems, e.g. Vibrio cholera and Copepods, this is the first study that confronts the potential role of zooplankton in the spread of potential pathogens in freshwater systems

Antibiotics and pathogens, when the natural bacterial community resists!

The spread and persistence of antibiotic resistances (AR) in waters is a major threat for the environmental management, with direct risks for human health. Although antibiotic resistant bacteria (ARB) are found everywhere, their ecological success is related to anthropic impact, and to disturbed natural communities. In the same waters where AR can be a problem, other disturbances promoted by human activities can interfere with natural communities, e.g. the input of allochthonous (and potentially pathogenic) bacteria. The concomitant impact of those disturbances can have dramatic effects on the persistence and resistance of natural communities. With an experimental system on continuous cultures, we tested the impact on the microbial community from anthropized Lake Maggiore of enhanced tetracycline concentrations and of a subsequent invasion by a mixed population of E. coli. We measured the response in terms of bacterial and eukaryotic abundances, phenotypical distribution, bacterial community composition, spread and persistence of tetracycline resistance genes, and the fate of E. coli strains, by applying a number of techniques including flow-cytometry, microscopy, qPCR, and Illumina sequencing. We observed the preservation of AR genes, even when antibiotics were not in the system, suggesting a relatively small cost of maintenance of these genes, or their involvement in other genetic pathways. However, we did not see a general increase of e.g. tetA resistance genes, following the addition of antibiotics. In fact, it rather seemed that resistance to tetracycline was rather acquired by morphological changes and species interactions; we observed many more aggregated cells when the antibiotic was added. On the other hand, the invasion with E.coli seemed to generally promote the overall productivity of the systems without and with little antibiotic. Our results demonstrate that despite the heavy disturbance the natural microbial communities can develop a number of mechanisms of resistance in order to reduce the impact (e.g. aggregation), complicating the system modelling, thus reducing our forecasting possibilities

PET particles raise microbiological concerns for human health while tyre wear microplastic particles potentially affect ecosystem services in waters

Although abundant and chemically peculiar, tyre wear microplastic particles (TWP) and their impact on the microbial communities in water are largely understudied. We tested in laboratory based semi-continuous cultures the impact of TWP and of polyethylene terephthalate (PET) derived particles (following a gradient of relative abundance) on the pathobiome (the group of potential human pathogenic bacteria) of a freshwater microbial community exposed to contamination by the effluent of a urban wastewater treatment plant, for a period of 28 days. We could define the modulated impact of the two types of microplastic particles: while PET does not favour bacterial growth, it offers a refuge to several potential pathogens of allochthonous origin (from the treated sewage effluent), TWP act as an additional carbon source, promoting the development and the massive growth of a biofilm composed by fast-growing bacterial genera including species potentially harmful and competitive in abating biodiversity in surface waters. Our results demonstrate the different ecological role and impact on freshwater environments of TWP and PET particles, and the need to approach the study of this pollutant not as a whole, but considering the origin and the chemical composition of the different particles.Fil: Sathicq, María Belén. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Limnología "Dr. Raúl A. Ringuelet". Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Instituto de Limnología; ArgentinaFil: Sabatino, Raffaella. Consiglio Nazionale delle Ricerche; ItaliaFil: Di Cesare, Andrea. Consiglio Nazionale delle Ricerche; ItaliaFil: Eckert, Ester M.. Consiglio Nazionale delle Ricerche; ItaliaFil: Fontaneto, Diego. Consiglio Nazionale delle Ricerche; ItaliaFil: Rogora, Michela. Consiglio Nazionale delle Ricerche; ItaliaFil: Corno, Gianluca. Consiglio Nazionale delle Ricerche; Itali

Seasonality of the antibiotic resistance gene blaCTX-M in temperate Lake Maggiore

The beta lactamase gene blaCTX-M, responsible of the resistance to cephalosporins, has been detected in microbes from hospitals to open waters. We studied the seasonality and stability of blaCTX-M in Lake Maggiore over 3 years and the role of potential inputs of allochthonous bacteria and/or antibiotic pollution in promoting its occurrence. blaCTX-M was mainly present from January to July in the pelagic microbial community and the gene occurrence was significantly related to low water temperature. To evaluate its temporal stability in the bacterial community over a short period, we measured blaCTX-M daily over the course of 6 days. The gene was below the limit of quantification except for one sampling when its abundance peaked, suggesting a point contamination. The bacterial community of the lake in which blaCTX-M was detected suggests that at least two distinct bacterial populations contained the gene. The occurrence of known blaCTX-M containing genera and the occurrence of the gene, however, did not overlap. Furthermore, the experimental addition of cefotaxime to lake water incubations did not promote abundance of the gene. These data imply that blaCTX-M was present in the environmental microbial community. Increases of gene abundances were likely caused by environmental parameters other than antibiotic contamination

Lake water as reservoir of antibiotic resistance genes constitutively present within the natural microbial community

The fate of antibiotic resistance genes (ARGs) in environmental microbial community is largely unknown although ARGs can constitute a threat for human health. In order to understand the dynamics and the relations of ARGs with biotic and abiotic factors in aquatic environment, we sampled water for 1.5 year in four stations of Lake Maggiore; for one of them (Ghiffa), we also sampled water from the whole water column. Moreover, for Ghiffa station, limnological parameters and prokaryotic cell abundance were determined. Each water sample was filtered and processed for the presence of twelve ARGs by PCR and, for the positive ARGs, qPCR protocols were developed. In order to investigate the relations between ARGs profiles and microbial community composition, we also sampled water every day for one week in Pallanza station. The DNA recovered during both the 18 months\u27 (long term) and the week (short term) monitoring were analyzed for the presence and abundance of ARGs and for the microbial community composition by 16SrDNA Illumina sequencing. Four ARGs were detectable in all stations during the whole sampling campaign, for instance tet(A) and sulII genes resulted quantifiable in more than half of the samples, and their abundance was positively correlated to oxygen and negatively to chlorophyll a, while str(B) and blaCTXM were present but rarely quantifiable. The composition of the microbial community was stable during the week while a seasonality was observed during 1.5 year of analysis. Moreover a potential relation between the presence of blaCTXM and the abundance of Flavobacteria was observed. All together, these results suggest the Lake as reservoir of ARGs fixed within the natural microbial community, thus it can constitute a hazardous hotspot for the transmission of antibiotic resistance to human pathogens