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&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&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&ARG database is publicly available at: https://www.norman-network.com/nds/bacteria/.

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.

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 blaVIM-2 or blaNDM-1 genes were compared. The blaVIM-2 gene was selected because, although frequent, it is almost restricted to this species and blaNDM-1 gene due to its wide interspecies distribution. A group of genomes harbouring the genes blaVIM-2 (n = 116) or blaNDM-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 blaVIM-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 blaNDM-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 blaVIM-2+ and blaNDM-1+. The blaVIM-2 gene was frequently inserted in the Tn402-like and Tn21 transposons family and rarely in IS6100, while blaNDM-1 gene was preferentially flanked by ISAba125 and bleMBL 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.

Dissemination of Metallo-ß-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-ß-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.

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.

A rationale for the high limits of quantification of antibiotic resistance genes in soil.

The determination of values of abundance of antibiotic resistance genes (ARGs) per mass of soil is extremely useful to assess the potential impacts of relevant sources of antibiotic resistance, such as irrigation with treated wastewater or manure application. Culture-independent methods and, in particular, quantitative PCR (qPCR), have been regarded as suitable approaches for such a purpose. However, it is arguable if these methods are sensitive enough to measure ARGs abundance at levels that may represent a risk for environmental and human health. This study aimed at demonstrating the range of values of ARGs quantification that can be expected based on currently used procedures of DNA extraction and qPCR analyses. The demonstration was based on the use of soil samples spiked with known amounts of wastewater antibiotic resistant bacteria (ARB) (Enterococcus faecalis, Escherichia coli, Acinetobacter johnsonii, or Pseudomonas aeruginosa), harbouring known ARGs, and also on the calculation of expected values determined based on qPCR. The limits of quantification (LOQ) of the ARGs (vanA, qnrS, blaTEM, blaOXA, blaIMP, blaVIM) were observed to be approximately 4 log-units per gram of soil dry weight, irrespective of the type of soil tested. These values were close to the theoretical LOQ values calculated based on currently used DNA extraction methods and qPCR procedures. The observed LOQ values can be considered extremely high to perform an accurate assessment of the impacts of ARGs discharges in soils. A key message is that ARGs accumulation will be noticeable only at very high doses. The assessment of the impacts of ARGs discharges in soils, of associated risks of propagation and potential transmission to humans, must take into consideration this type of evidence, and avoid the simplistic assumption that no detection corresponds to risk absence.

Antibiotic resistance in wastewater treatment plants: Tackling the black box.

Wastewater is among the most important reservoirs of antibiotic resistance in urban environments. The abundance of carbon sources and other nutrients, a variety of possible electron acceptors such as oxygen or nitrate, the presence of particles onto which bacteria can adsorb, or a fairly stable pH and temperature are examples of conditions favouring the remarkable diversity of microorganisms in this peculiar habitat. The wastewater microbiome brings together bacteria of environmental, human and animal origins, many harbouring antibiotic resistance genes (ARGs). Although numerous factors contribute, mostly in a complex interplay, for shaping this microbiome, the effect of specific potential selective pressures such as antimicrobial residues or metals, is supposedly determinant to dictate the fate of antibiotic resistant bacteria (ARB) and ARGs during wastewater treatment. This paper aims to enrich the discussion on the ecology of ARB&ARGs in urban wastewater treatment plants (UWTPs), intending to serve as a guide for wastewater engineers or other professionals, who may be interested in studying or optimizing the wastewater treatment for the removal of ARB&ARGs. Fitting this aim, the paper overviews and discusses: i) aspects of the complexity of the wastewater system and/or treatment that may affect the fate of ARB&ARGs; ii) methods that can be used to explore the resistome, meaning the whole ARB&ARGs, in wastewater habitats; and iii) some frequently asked questions for which are proposed addressing modes. The paper aims at contributing to explore how ARB&ARGs behave in UWTPs having in mind that each plant is a unique system that will probably need a specific procedure to maximize ARB&ARGs removal.

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 Te0 in the cytoplasm as needle-like black precipitates. Interestingly, photosynthetic cultures of R. capsulatus can also generate Te0 nanoprecipitates (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 Te0 to 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.

Extracellular production of tellurium nanoparticles by the photosynthetic bacterium Rhodobacter capsulatus.

The toxic oxyanion tellurite (TeO3(2-)) is acquired by cells of Rhodobacter capsulatus grown anaerobically in the light, via acetate permease ActP2 and then reduced to Te(0) in the cytoplasm as needle-like black precipitates. Interestingly, photosynthetic cultures of R. capsulatus can also generate Te(0) nanoprecipitates (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 600-700 nm 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 Te(0) to 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.