Fate of pseudomonas aeruginosa and blaVIM in soil under selective pressure by copper and zinc

info:eu-repo/semantics/publishedVersio

Insight into phylogenomic bias of blaVIM-2 or blaNDM-1 dissemination amongst carbapenem resistant Pseudomonas aeruginosa

Objectives: Pseudomonas aeruginosa (P. aeruginosa) are ubiquitous opportunistic pathogens that combine intrinsic and acquired multidrug resistance phenotypes. Due to different types of acquired genes, carbapenem resistance has been expanding in this species. This study hypothesised that the spread of carbapenem resistance among P. aeruginosa is influenced by phylogenomic features, being distinct for different genes. Methods: To test this hypothesis, the genomes of P. aeruginosa harbouring bla VIM-2 or bla NDM-1 genes were compared. The bla VIM-2 gene was selected because, although frequent, it is almost restricted to this species and bla NDM-1 gene due to its wide interspecies distribution. A group of genomes harbouring the genes bla VIM-2 (n = 116) or bla NDM-1 (n = 27), available in GenBank, was characterised based on core phylogenomic analysis, functional categories in the accessory genome and mobile genetic elements flanking the selected genes. Results: Most bla VIM-2 gene hosts belonged to multilocus sequence types (ST) ST111 (n = 32 of 116) and ST233 (n = 27 of 116) and were reported in Europe (n = 75 of 116). The bla NDM-1 gene hosts were distributed by different STs (ST38, ST773, ST235, ST357 and ST654), frequently from Asia (n = 11 of 27). Significant differences in the prevalence of functional protein/enzyme annotations per number of accessory genomes were observed between bla VIM-2+ and bla NDM-1+. The bla VIM-2 gene was frequently inserted in the Tn402-like and Tn21 transposons family and rarely in IS6100, while bla NDM-1 gene was preferentially flanked by ISAba125 and ble MBL genes or associated with IS91 insertion sequence. Conclusion: The hypothesis that carbapenem resistance gene acquisition is not random among phylogenomic lineages was confirmed, suggesting the importance of phylogeny in the dissemination of antibiotic resistance genes.info:eu-repo/semantics/publishedVersio

Measurement of the impact of antibiotic resistance discharge in wastewater and in soil : ecological aspects

Antibiotic resistance represents a serious threat to human health and a relevant environmental contaminant. Antibiotic-resistant bacteria (ARB) and harboured antibiotic resistance genes (ARGs) were described in different settings, mainly in clinical contexts, but also in wastewater treatment plants or agricultural soil. In the environment, the general pollution context may favour the persistence or proliferation of ARB due to multiple genetic characteristics. Monitoring of ARGs and ARB in the environment plays an important role in unveiling sources and paths of dissemination. To address these issues this thesis explored three topics based on antibiotic resistant Pseudomonas aeruginosa and on soil: i) the biases that may be imposed by the high limits of quantification of ARGs in soil; ii) the survival of an exogenous ubiquitous bacterial strain (blaVIM+ P. aeruginosa) in soil and the possible effects of metal salts; and iii) the inferred interplay between phylogeny and accessory genome in distinct genotypes of carbapenem resistance (blaVIM+ or blaNDM+ P. aeruginosa). The first work (chapter 3) aimed to assess the limit of quantification (LOQ) for ARGs (vanA, qnrS, blaTEM, blaOXA, blaIMP, blaVIM) in soil, based on the hypothesis that low doses of ARB and ARGs in soil are not quantifiable with current qPCR techniques, mainly due to DNA extraction procedures. To determine the LOQ, microcosms (10 g of agricultural soil, potting soil, sand, fallow soil and compost) were spiked with wastewater isolated ARB doses ranging from 102 to 107 CFU/g of dry soil. These spiked ARB harboured respectively the vancomycin resistance gene vanA (E. faecalis), quinolone resistance gene qnrS (Escherichia coli), and β-lactam resistance genes blaTEM (E. coli), blaOXA (E. coli, Acinetobacter johnsonii), blaIMP (A. johnsonii), blaVIM (P. aeruginosa) based on which the LOQs were determined. The microcosms were sampled to enumerate bacterial colony forming units (CFU), and extract DNA for ARGs quantification by qPCR. The LOQ was determined to be 104 copies of ARG per g of dry soil, independently of the soil type. Below this limit, it was not possible to quantify ARGs even when the respective host ARB could be cultivated. The results support the hypothesis that LOQ values are relatively high and may suggest the absence of ARGs in situations in which these may represent a threat. The second work (chapter 4) aimed at assessing the metal impact on the survival of a hospital effluent blaVIM+ P. aeruginosa and on the soil microbial community’s diversity. Microcosms were prepared with agricultural soil non-amended or amended with copper and zinc sulfate or nitrate aged for one month, and spiked with known doses of blaVIM+ P. aeruginosa. The ARB survival was monitored based on CFU enumeration and quantification of selected genes - extracytoplasmic function sigma factor, ecf, Verona Integron–encoded Metallo-β-Lactamase, blaVIM, class 1 integron- integrase gene, intl1, over 30 days. In addition, the microbial community composition (V3-V4 16S rRNA gene amplicon sequencing) was analysed. Over this period, the P. aeruginosa content (CFUs/g dry soil) and ecf and blaVIM (copy number/g dry soil) decreased in all the tested conditions but was still quantifiable after 30 days. This confirms the ARB persistence in soil along the 30 days, excluding the hypothesis of ARG loss during this period. Microbiome analysis revealed a clear influence of metals in the bacterial community diversity, independent of the metal type and salts nature. This study permitted to conclude that the metal amendment affects the soil microbial quality but has a negligible impact on the exogenous bacteria survival. These results highlight the importance of considering microbial interaction and characteristics, such as metal tolerance, in the assessment of ARB persistence in the environment. In chapter 5 the genomes of blaVIM-2+ or blaNDM-1+ P. aeruginosa strains were compared. The ARG blaVIM-2 is mostly observed in Pseudomonas species, while blaNDM-1 is distributed among distinct genera and orders. The work focused on phylogenetic distribution and genomic features of a dataset of 116 blaVIM-2+ and 27 blaNDM-1+ genomes, from 38 countries. The selected genomes were annotated and the core sequence multilocus sequence typing (MLST) were determined. The blaVIM-2+ and blaNDM-1+ genomes were analysed using a comparative genome approach to assess the core and accessory genes, later used to determine the bacteria antibiotic resistance and functional profile. To describe the blaVIM-2 and blaNDM-1 genomic environment, the flanking regions were annotated through sequence comparison. The phylogenetic and geographic distribution analyses suggested a worldwide distribution of the strains belonging to several STs with cases of endemism. The blaVIM-2 + and blaNDM-1 + accessory genomes presented different antibiotic resistance and functional profiles, regardless the majority of the ARGs and proteins families were shared. Interestingly, the copresence of blaVIM-2 and blaNDM-1 and other carbapenems resistance genes in different genomes was observed. The genomic environments of the two ARGs were different, being blaVIM-2 associated with distinct transposons structures (Tn21, Tn402-like mostly) and blaNDM-1 to different elements (ISAba125 and bleMBL and IS91). This work emphasized the importance of considering different approaches to tackle the spread of carbapenem resistant bacteria evaluating the phylogeny, and geographical distribution but mostly the genomic characteristics of the strains. Our works aimed to indicate possible weaknesses to be improved in antibiotic resistance monitoring and highlight the ARB phylogenetic role and genetic characteristics favouring the ARGs spread. In particular, the experimental work evidences the possible survival of ARB in soil, mostly in extremely polluted conditions. Moreover, the survival and presence of these ARB in soil could avoid the quantification by molecular biology methods due to their high LOQ. The study of the ARB genomic characteristics may be useful to prevent the adaptation to environments and to find additional biomarkers for their monitoring.A resistência a antibióticos representa uma séria ameaça para a saúde humana e um contaminante ambiental relevante. As bactérias resistentes a antibióticos (ARB) e os genes de resistência a antibióticos (ARGs) foram descritos em diferentes contextos, principalmente em casos clínicos, mas também em estações de tratamento de águas residuais ou solos agrícolas. No ambiente, o contexto geral da poluição pode favorecer a persistência ou a proliferação de ARB devido a múltiplas características genéticas. A monitorização de ARGs e de ARB no ambiente pode ter um papel importante na identificação de fontes e vias de disseminação. Para abordar estas questões, esta tese explorou três temas baseados em Pseudomonas aeruginosa resistente a antibióticos e em solo: i) as limitações que podem existir devido aos elevados limites de quantificação de ARGs no solo; ii) a sobrevivência de uma estirpe bacteriana exógena ubíqua (blaVIM+ P. aeruginosa) no solo e os possíveis efeitos de sais de metais; e iii) a relação que poderá existir entre a filogenia e o genoma acessório em diferentes genótipos de resistência a carbapenemos (blaVIM+ ou blaNDM+ P. aeruginosa). O primeiro trabalho (capítulo 3) visou avaliar o limite de quantificação (LOQ) de ARGs (vanA, qnrS, blaTEM, blaOXA, blaIMP, blaVIM) em solo, baseado na hipótese de que doses baixas de ARB e ARGs no solo não são quantificáveis com as atuais técnicas de qPCR, principalmente devido aos procedimentos de extração de DNA. Para determinar o LOQ, microcosmos (10 g de solo agrícola, substrato, areia, solo pousio e composto) foram inoculados com ARB isoladas de águas residuais em doses que variam de 102 a 107 CFU/g de solo seco. As ARB usadas como inóculo continham respetivamente o gene de resistência a vancomicina vanA (E. faecalis), o gene de resistência a quinolonas qnrS (Escherichia coli), e os genes de resistência a β-lactâmicos blaTEM (E. coli), blaOXA (E. coli, Acinetobacter johnsonii), blaIMP (A. johnsonii), blaVIM (P. aeruginosa) com base nos quail os LOQs foram determinados. Os microcosmos foram amostrados para enumerar unidades de formação de colónias bacterianas (CFU), e extrair DNA para quantificação de ARGs por qPCR. O LOQ foi determinado como sendo de 104 cópias de ARG por g de solo seco, independentemente do tipo de solo. Abaixo deste limite, não foi possível quantificar os ARGs mesmo quando o respetivo hospedeiro ARB foi detetado por cultivo. Os resultados apoiam a hipótese de que os valores de LOQ são relativamente elevados e podem sugerir a ausência de ARGs em situações em que estes possam representar um perigo. O segundo trabalho (capítulo 4) visou avaliar o impacto de metais na sobrevivência de um isolado de P. aeruginosa proveniente de efluente hospitalar blaVIM+ e na diversidade da comunidade microbiana do solo. Microcosmos foram preparados com solo agrícola ou neste suplementado com sulfato ou nitrato de cobre e zinco envelhecidos durante um mês, e inoculados com doses conhecidas de P. aeruginosa blaVIM+. A sobrevivência da ARB foi monitorizada com base na enumeração das CFU e quantificação de genes selecionados - extracytoplasmic function sigma factor, ecf, Verona Integron–encoded Metallo-β-Lactamase, blaVIM, class 1 integron-integrase gene, intl1, ao longo de 30 dias. Além disso, foi analisada a composição da comunidade microbiana (sequenciação de amplicão V3-V4 do gene 16S rRNA). Durante este período, a abundância de P. aeruginosa (CFUs/g solo seco) e ecf e blaVIM-2 (número de cópia/g de solo seco) diminuiu em todas as condições testadas, mas foi ainda assim quantificável após 30 dias. Estes resultados confirmam a persistência da ARB no solo ao longo dos 30 dias, excluindo a hipótese de perda do ARG durante este período. A análise do microbioma revelou uma clara influência dos metais na diversidade da comunidade bacteriana, independentemente do tipo de metal ou sal. Este estudo permitiu concluir que a suplementação com metais afeta a qualidade microbiana do solo, mas tem um impacto negligenciável na sobrevivência das bactérias exógenas. Estes resultados destacam a importância de considerar a interação e características microbianas, como a tolerância a metais, na avaliação da persistência da ARB no ambiente. No capítulo 5 foram comparados os genomas de estirpes de P. aeruginosa blaVIM-2+ ou blaNDM-1+. O ARG blaVIM-2 é observado principalmente em espécies de Pseudomonas, enquanto blaNDM-1 é distribuído entre géneros e ordens distintos. O trabalho centrou-se na distribuição filogenética e características genómicas de um conjunto de 116 genomas blaVIM-2+ e 27 genomas blaNDM-1+, de 38 países. Os genomas selecionados foram anotados e as sequências do core genome multilocus sequence type (MLST) foram determinadas. Os genomas blaVIM-2+ e blaNDM-1+ foram analisados usando uma abordagem comparativa do genoma para avaliar os genes do genoma core e acessório, mais tarde usados para determinar a resistência a antibióticos e o perfil funcional das bactérias. Para descrever o ambiente genómico de blaVIM-2 e blaNDM-1, as regiões onde se integravam foram anotadas através da comparação de sequências. As análises da distribuição filogenética e geográfica sugeriram uma distribuição mundial das estirpes pertencentes aos vários STs com casos de endemismo. Os genomas acessórios de blaVIM-2+ e blaNDM-1+ apresentaram diferentes perfis de resistência a antibióticos e funcionais, independentemente da maioria dos ARGs e das famílias de proteínas terem sido partilhados. Curiosamente, a presença simultânea de blaVIM-2 e blaNDM-1 e outros genes de resistência a carbapenemos foi observada em diferentes genomas. Os ambientes genómicos dos dois ARGs eram distintos, sendo blaVIM-2 associado a diferentes transposões (Tn21, Tn402-like na sua maioria) e blaNDM-1 a diferentes sequências de inserção (ISAba125 e bleMBL e IS91). Este trabalho enfatizou a importância de considerar diferentes abordagens para combater a propagação de bactérias resistentes a carbapenemos que avalim a filogenia, e a distribuição geográfica, mas principalmente as características genómicas das estirpes. Os nossos trabalhos visavam indicar possíveis limitações a serem melhoradas na monitorização da resistência aos antibióticos e destacar o papel da filogenia das ARB e das características genéticas que favorecem a propagação dos ARGs. Em particular, o trabalho experimental evidencia a possível sobrevivência de ARB no solo, principalmente em condições de elevada poluição. Além disso, a sobrevivência e a presença destas ARB no solo pode não ser possível de quantificar por métodos de biologia molecular devido ao seu elevado LOQ. O estudo das características genómicas das ARB pode ser útil para prevenir a adaptação aos ambientes e para encontrar mais biomarcadores para a sua monitorização

Effect of copper and zinc as sulfate or nitrate salts on soil microbiome dynamics and blaVIM-positive pseudomonas aeruginosa survival

The exposure of soil to metals and to antibiotic resistant bacteria may lead to the progressive deterioration of soil quality. The persistence of antibiotic resistant bacteria or antibiotic resistance genes in soil can be influenced by the microbial community or by soil amendments with metal salts. This work assessed the effect of soil amendment with copper and zinc, as sulfate or nitrate salts, on the fate of a carbapenem-resistant (blaVIM+) hospital effluent isolate of Pseudomonas aeruginosa (strain H1FC49) and on the variations of the microbial community composition. Microcosms with soil aged or not with copper and zinc salts (20 mM), and inoculated with P. aeruginosa H1FC49 were monitored at 0, 7, 14 and/or 30 days, for community composition (16S rRNA gene amplicon) and strain H1FC49 persistence. Data on culturable P. aeruginosa, quantitative PCR of the housekeeping gene ecf, and the presumably acquired genes blaVIM+ and integrase (intI1), and community composition were interpreted based on descriptive statistics and multivariate analysis. P. aeruginosa and the presumably acquired genes, were quantifiable in soil for up to one month, in both metal-amended and non-amended soil. Metal amendments were associated with a significant decrease of bacterial community diversity and richness. The persistence of P. aeruginosa and acquired genes in soils, combined with the adverse effect of metals on the bacterial community, highlight the vulnerability of soil to both types of exogenous contamination.info:eu-repo/semantics/acceptedVersio

The impact of HPP-assisted biocontrol approach on the bacterial communities’ dynamics and quality parameters of a fermented meat sausage model

Traditional foods are increasingly valued by consumers, whose attention and purchase willingness are highly influenced by other claims such as ‘natural’, ‘sustainable’, and ‘clean label’. The purpose of the present study was to evaluate the impact of a novel non-thermal food processing method (i.e., HPP-assisted biocontrol combining mild high hydrostatic pressure, listeriophage Listex, and pediocin PA-1 producing Pediococcus acidilactici) on the succession of bacterial communities and quality of a fermented sausage model. A comparative analysis of instrumental color, texture, and lipid peroxidation revealed no significant differences (p > 0.05) in these quality parameters between non- and minimally processed fermented sausages throughout 60-day refrigerated storage (4 °C). The microbiota dynamics of biotreated and untreated fermented sausages were assessed by 16S rRNA next-generation sequencing, and the alpha and beta diversity analyses revealed no dissimilarity in the structure and composition of the bacterial communities over the analyzed period. The innovative multi-hurdle technology proposed herein holds valuable potential for the manufacture of traditional fermented sausages while preserving their unique intrinsic characteristics.info:eu-repo/semantics/publishedVersio

Reprint of "Extracellular production of tellurium nanoparticles by the photosynthetic bacterium Rhodobacter capsulatus"

The toxic oxyanion tellurite (TeO32-) is acquired by cells of Rhodobacter capsulatus grown anaerobically in the light, via acetate permease ActP2 and then reduced to Te0in the cytoplasm as needle-like black precipitates. Interestingly, photosynthetic cultures of R. capsulatus can also generate Te0nanoprecipitates (TeNPs) outside the cells upon addition of the redox mediator lawsone (2-hydroxy-1,4-naphtoquinone). TeNPs generation kinetics were monitored to define the optimal conditions to produce TeNPs as a function of various carbon sources and lawsone concentration. We report that growing cultures over a 10 days period with daily additions of 1mM tellurite led to the accumulation in the growth medium of TeNPs with dimensions from 200 up to 600-700nm in length as determined by atomic force microscopy (AFM). This result suggests that nucleation of TeNPs takes place over the entire cell growth period although the addition of new tellurium Te0to pre-formed TeNPs is the main strategy used by R. capsulatus to generate TeNPs outside the cells. Finally, X-ray photoelectron spectroscopy (XPS) and Fourier transform infrared (FT-IR) analysis of TeNPs indicate they are coated with an organic material which keeps the particles in solution in aqueous solvents

Dissemination of Metallo-β-Lactamase-Producing <i>Pseudomonas aeruginosa</i> in Serbian Hospital Settings: Expansion of ST235 and ST654 Clones

This nationwide study aimed to investigate the molecular characteristics of metallo-β-lactamase (MBL)-producing Pseudomonas aeruginosa in Serbia, underlying resistance mechanisms, the genetic context of detected MBL genes, and the clonal relationship between isolates harboring genes-encoding MBL. Overall, 320/5334 isolates collected from 2018 to 2021 were identified as P. aeruginosa. Carbapenem-resistant P. aeruginosa (CRPA) were screened for the presence of blaVIM, blaIMP, and blaNDM, genes whereas MBL-positive isolates were tested for the presence of the blaCTX-M-2, blaPER, blaTEM, blaSHV, blaVEB, and blaGES. Multilocus sequence typing and phylogenomic analysis were performed for P. aeruginosa-producing MBL. The majority of the P. aeruginosa isolates were recovered from the lower respiratory tract (n = 120; 37.5%) and wound specimens (n = 108; 33.75%). CRPA isolates accounted for 43.1% (n = 138) of the tested isolates, 31 out of them being blaNDM-1-positive (22.5%). The colistin resistance rate was 0.3%. MLST analysis revealed the occurrence of ST235 (n = 25) and ST654 (n = 6), mostly confined to Serbia. The distribution of beta-lactamase-encoding genes in these isolates suggested clonal dissemination and possible recombination: ST235/blaNDM-1, ST235/blaNDM-1/blaPER-1, ST654/blaNDM-1, ST654/blaNDM-1/blaPER-1, and ST654/blaNDM-1/blaGES-5. High-risk clones ST235 and ST654 identified for the first time in Serbia, are important vectors of acquired MBL and ESBL and their associated multidrug resistance phenotypes represent a cause for considerable concern

Dissemination of Metallo-β-Lactamase-Producing Pseudomonas aeruginosa in Serbian Hospital Settings: Expansion of ST235 and ST654 Clones

This nationwide study aimed to investigate the molecular characteristics of metallo-β-lactamase (MBL)-producing Pseudomonas aeruginosa in Serbia, underlying resistance mechanisms, the genetic context of detected MBL genes, and the clonal relationship between isolates harboring genes-encoding MBL. Overall, 320/5334 isolates collected from 2018 to 2021 were identified as P. aeruginosa. Carbapenem-resistant P. aeruginosa (CRPA) were screened for the presence of blaVIM, blaIMP, and blaNDM, genes whereas MBL-positive isolates were tested for the presence of the blaCTX-M-2, blaPER, blaTEM, blaSHV, blaVEB, and blaGES. Multilocus sequence typing and phylogenomic analysis were performed for P. aeruginosa-producing MBL. The majority of the P. aeruginosa isolates were recovered from the lower respiratory tract (n = 120; 37.5%) and wound specimens (n = 108; 33.75%). CRPA isolates accounted for 43.1% (n = 138) of the tested isolates, 31 out of them being blaNDM-1-positive (22.5%). The colistin resistance rate was 0.3%. MLST analysis revealed the occurrence of ST235 (n = 25) and ST654 (n = 6), mostly confined to Serbia. The distribution of beta-lactamase-encoding genes in these isolates suggested clonal dissemination and possible recombination: ST235/blaNDM-1, ST235/blaNDM-1/blaPER-1, ST654/blaNDM-1, ST654/blaNDM-1/blaPER-1, and ST654/blaNDM-1/blaGES-5. High-risk clones ST235 and ST654 identified for the first time in Serbia, are important vectors of acquired MBL and ESBL and their associated multidrug resistance phenotypes represent a cause for considerable concern.</jats:p

Making waves: The NORMAN antibiotic resistant bacteria and resistance genes database (NORMAN ARB&ARG)–An invitation for collaboration to tackle antibiotic resistance

With the global concerns on antibiotic resistance (AR) as a public health issue, it is pivotal to have data exchange platforms for studies on antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs) in the environment. For this purpose, the NORMAN Association is hosting the NORMAN ARB &amp;ARG database, which was developed within the European project ANSWER. The present article provides an overview on the database functionalities, the extraction and the contribution of data to the database. In this study, AR data from three studies from China and Nepal were extracted and imported into the NORMAN ARB &amp;ARG in addition to the existing AR data from 11 studies (mainly European studies) on the database. This feasibility study demonstrates how the scientific community can share their data on AR to generate an international evidence base to inform AR mitigation strategies. The open and FAIR data are of high potential relevance for regulatory applications, including the development of emission limit values / environmental quality standards in relation to AR. The growth in sharing of data and analytical methods will foster collaboration on risk management of AR worldwide, and facilitate the harmonization in the effort for identification and surveillance of critical hotspots of AR. The NORMAN ARB &amp;ARG database is publicly available at: https://www.norman-network.com/nds/bacteria/