Rescue of Mycobacterium bovis DNA Obtained from Cultured Samples during Official Surveillance of Animal TB: Key Steps for Robust Whole Genome Sequence Data Generation.

Epidemiological surveillance of animal tuberculosis (TB) based on whole genome sequencing (WGS) of Mycobacterium bovis has recently gained track due to its high resolution to identify infection sources, characterize the pathogen population structure, and facilitate contact tracing. However, the workflow from bacterial isolation to sequence data analysis has several technical challenges that may severely impact the power to understand the epidemiological scenario and inform outbreak response. While trying to use archived DNA from cultured samples obtained during routine official surveillance of animal TB in Portugal, we struggled against three major challenges: the low amount of M. bovis DNA obtained from routinely processed animal samples; the lack of purity of M. bovis DNA, i.e., high levels of contamination with DNA from other organisms; and the co-occurrence of more than one M. bovis strain per sample (within-host mixed infection). The loss of isolated genomes generates missed links in transmission chain reconstruction, hampering the biological and epidemiological interpretation of data as a whole. Upon identification of these challenges, we implemented an integrated solution framework based on whole genome amplification and a dedicated computational pipeline to minimize their effects and recover as many genomes as possible. With the approaches described herein, we were able to recover 62 out of 100 samples that would have otherwise been lost. Based on these results, we discuss adjustments that should be made in official and research laboratories to facilitate the sequential implementation of bacteriological culture, PCR, downstream genomics, and computational-based methods. All of this in a time frame supporting data-driven intervention.

Systematic SARS-CoV-2 S-gene sequencing in wastewater samples enables early lineage detection and uncovers rare mutations in Portugal.

As the COVID-19 pandemic reached its peak, many countries implemented genomic surveillance systems to track the evolution and transmission of SARS-CoV-2. Transition from the pandemic to the endemic phase prioritized alternative testing strategies to maintain effective epidemic surveillance at the population level, with less intensive sequencing efforts. One such promising approach was Wastewater-Based Surveillance (WBS), which offers non-invasive, cost-effective means for analysing virus trends at the sewershed level. From 2020 onwards, wastewater has been recognized as an instrumental source of information for public health, with national and international authorities exploring options to implement national wastewater surveillance systems and increasingly relying on WBS as early warning of potential pathogen outbreaks. In Portugal, several pioneer projects joined the academia, water utilities and Public Administration around WBS. To validate WBS as an effective genomic surveillance strategy, it is crucial to collect long term performance data. In this work, we present one year of systematic SARS-CoV-2 wastewater surveillance in Portugal, representing 35 % of the mainland population. We employed two complementary methods for lineage determination - allelic discrimination by RT-PCR and S-gene sequencing. This combination allowed us to monitor variant evolution in near-real-time and identify low-frequency mutations. Over the course of this year-long study, spanning from May 2022 to April 2023, we successfully tracked the dominant Omicron sub-lineages, their progression and evolution, which aligned with concurrent clinical surveillance data. Our results underscore the effectiveness of WBS as a tracking system for virus variants, with the ability to unveil mutations undetected via massive sequencing of clinical samples from Portugal, demonstrating the ability of WBS to uncover new mutations and detect rare genetic variants. Our findings emphasize that knowledge of the genetic diversity of SARS-CoV-2 at the population level can be extended far beyond via the combination of routine clinical genomic surveillance with wastewater sequencing and genotyping.

Widespread circulation and transmission risk of Mycobacterium avium subsp. paratuberculosis at the livestock-wildlife-environment interface in a Mediterranean agro-forestry farmstead.

Mycobacterium avium subsp. paratuberculosis (MAP) is the etiological agent of paratuberculosis, a chronic infection affecting ruminants and other species worldwide. Information on the ecological factors that increase infection risk at the livestock-wildlife-environment interface remains scarce. Thus, this work aimed at determining which factors modulate the exposure of a mammal community within a Mediterranean agro-forestry farmstead to MAP. Through field, molecular and ecological modeling approaches, MAP prevalence, distribution and spatial risk at the livestock-wildlife-environment was estimated in the study area by screening 436 samples (cattle, n = 150; wildlife, n = 206; soil, n = 80). Using molecular detection of IS900 as proxy, MAP was identified in ten wild mammal species. Being a central prey of mesocarnivores in Portugal, the high prevalence of MAP in the wild rabbit (19%) may be related with red fox's (22%). MAP was also detected in cattle managed in the farmstead (animal and herd prevalence, 54% and 100%) and in soil (44%), which may perpetuate intraspecies and interspecies transmission. Wildlife diversity showed a positive influence on MAP presence in wild mammals, while wildlife abundance showed a negative effect. Land use variables exerted distinct degrees of impact upon MAP detection in specific groups of mammals: mixed forest cover showed positive influence on carnivores, and shrubland showed positive effect on wild rabbits. The prevalence of MAP in cattle showed a negative influence on the detection of MAP in lagomorph, which may stem from wild rabbit lower density and avoidance of cattle areas. Based on explanatory variables, the spatial prediction of MAP occurrence in wildlife indicated two hotspots with increased exposure risk but future studies are needed to confirm this projection. This work represents the most comprehensive molecular survey of MAP occurrence and determinants in Mediterranean agroecosystems leveraging the principles and tools of community ecology, debating potential biological and ecological effects underlying MAP transmission.

Genomic epidemiology sheds light on the emergence and spread of Mycobacterium bovis Eu2 Clonal Complex in Portugal.

ABSTRACTAnimal tuberculosis (TB) remains a serious concern for animal and human health. Mycobacterium bovis circulates in multi-host systems, dominated by the European 2 clonal complex (Eu2) in Iberia. In this work, we use genomic epidemiology to infer the emergence, spread, and spatiotemporal patterns of Eu2 in the official epidemiological risk area of animal TB in Portugal. Phylogenetic analysis of 144 M. bovis whole-genome sequences from cattle, wild boar, and red deer, representing the 2002-2021 period, distinguished three Eu2 clades that evolved independently. The major Eu2 clade underwent phylodynamic inferences to estimate the time and location of outbreaks, host transitions, and spatial diffusion as well. The origin of this Eu2 clade was attributed to the red deer population in the Castelo Branco district, near the border with Spain. Most host transitions were intraspecific (80%), while interspecific transmissions between wildlife species (wild boar-red deer), and between wild boar and cattle, were highly supported. Phylogeographic reconstruction evidenced that most transitions (82%) occur within municipalities, highlighting local transmission corridors.Our study indicates that M. bovis continues to spread at the cattle-wildlife interface within the animal TB hotspot area, possibly driven by the foraging behaviour of wild boar near agricultural lands. Red deer seems to be an important driver of TB within wildlife hosts, while the wild boar links the multi-host wildlife community and livestock. This work highlights the value of combining genomic epidemiology with phylodynamic inference to resolve host jumps and spatial patterns of M. bovis, providing real-time clues about points of intervention.

Exploring the correlations between epi indicators of COVID-19 and the concentration of pharmaceutical compounds in wastewater treatment plants in Northern Portugal.

The COVID-19 pandemic caused by the SARS-CoV-2 virus led to changes in the lifestyle and human behaviour, which resulted in different consumption patterns of some classes of pharmaceuticals including curative, symptom-relieving, and psychotropic drugs. The trends in the consumption of these compounds are related to their concentrations in wastewater systems, since incompletely metabolised drugs (or their metabolites back transformed into the parental form) may be detected and quantified by analytical methods. Pharmaceuticals are highly recalcitrant compounds and conventional activated sludge processes implemented in wastewater treatment plants (WWTP) are ineffective at degrading these substances. As a results, these compounds end up in waterways or accumulate in the sludge, being a serious concern given their potential effects on ecosystems and public health. Therefore, it is crucial to evaluate the presence of pharmaceuticals in water and sludge to assist in the search for more effective processes. In this work, eight pharmaceuticals from five therapeutic classes were analysed in wastewater and sludge samples collected in two WWTP located in the Northern Portugal, during the third COVID-19 epidemic wave in Portugal. The two WWTP demonstrated a similar pattern with respect to the concentration levels in that period. However, the drugs loads reaching each WWTP were dissimilar when normalising the concentrations to the inlet flow rate. Acetaminophen (ACET) was the compound detected at highest concentrations in aqueous samples of both WWTP (98. 516 µg L - 1 in WWTP2 and 123. 506 µg L - 1in WWTP1), indicating that this drug is extensively used without the need of a prescription, known of general public knowledge as an antipyretic and analgesic agent to treat pain and fever. The concentrations determined in the sludge samples were below 1.65 µg g - 1 in both WWTP, the highest value being found for azithromycin (AZT). This result may be justified by the physico-chemical characteristics of the compound that favour its adsorption to the sludge surface through ionic interactions. It was not possible to establish a clear relationship between the incidence of COVID-19 cases in the sewer catchment and the concentration of drugs detected in the same period. However, looking at the data obtained, the high incidence of COVID-19 in January 2021 is in line with the high concentration of drugs detected in the aqueous and sludge samples but prediction of drug load from viral load data was unfeasible.

Unlocking environmental contamination of animal tuberculosis hotspots with viable mycobacteria at the intersection of flow cytometry, PCR, and ecological modelling.

Mycobacterium bovis, a member of the Mycobacterium tuberculosis complex (MTBC), circulates in multi-host mammal communities. While interactions between different host species are mainly indirect, current knowledge postulates interspecific transmission is favored by animal contact with natural substrates contaminated with droplets and fluids from infected animals. However, methodological constraints have severely hampered monitoring of MTBC outside its hosts and the subsequent validation of this hypothesis. In this work, we aimed to evaluate the extent to which environmental contamination with M. bovis occurs in an endemic animal TB setting, taking advantage of a new real-time monitoring tool we recently developed to quantify the proportion of viable and dormant MTBC cell fractions in environmental matrices. Sixty-five natural substrates were collected nearby the International Tagus Natural Park region, in the epidemiological TB risk area in Portugal. These included sediments, sludge, water, and food deployed at unfenced feeding stations. The tripartite workflow included detection, quantification, and sorting of different M. bovis cell populations: total, viable, and dormant. Real-time PCR targeting IS6110 to detect MTBC DNA was performed in parallel. The majority of samples (54 %) contained metabolically active or dormant MTBC cells. Sludge samples had a higher burden of total MTBC cells and a high concentration of viable cells (2.3 × 104 cells/g). Ecological modelling informed by climate, land use, livestock and human disturbance data suggested eucalyptus forest and pasture cover as potential major factors affecting the occurrence of viable MTBC cells in natural matrices. Our study demonstrates, for the first time, the widespread environmental contamination of animal TB hotspots with viable MTBC bacteria and with dormant MTBC cells that are able to recover metabolic activity. Further, we show that viable MTBC cell load in natural substrates is superior to the estimated minimum infective dose, providing real-time insights into the potential magnitude of environmental contamination for indirect TB transmission.

Knowledge, attitudes, and practices of municipal veterinary practitioners towards echinococcosis.

Cystic echinococcosis (CE) and alveolar echinococcosis (AE) are among the most relevant zoonoses in Europe. According to the European Food Safety Authority (EFSA), Echinococcus granulosus sensu lato causing CE is the most significant foodborne parasite in South-Western Europe, followed by Echinococcus multilocularis, the etiological agent of AE. Among the challenges and opportunities highlighted in the literature to combat these diseases are the need to evaluate and increase awareness of stakeholders. In Portugal, Municipal Veterinary Practitioners (MVP) are the animal health authority at the municipality level, playing a crucial role in diagnosis, prevention and control of infectious diseases in animals, helping to mitigate transmission to humans. However, their knowledge, attitudes, and practices (KAP) towards CE, as well as awareness of AE expansion across Europe, remain overlooked. In this work, a cross-sectional study was conducted for the first time in Portugal to bridge knowledge gaps concerning CE and AE, using an online self-administered questionnaire collecting information on the municipal kennel, KAP towards CE, and understanding of AE epidemiology. Eighty-three MVP from mainland and islands completed the questionnaire, with highest representability from the central-northern region. MVP had, on average, a medium to high level of knowledge of CE but acquaintance with AE was insufficient, although echinococcosis was frequently mentioned as target of health education sessions (77.0%). A high rate (60%) of reported entries into municipal kennels of stray dogs originating from countries with AE endemic areas was registered, suggesting that the presence of these potentially AE-infected stray dogs pose public health risks. Most kennels did not perform routine coprological analysis or faecal matter disinfection after dog internal deworming. The lack of proper training and well-conceived written plans of infection control and prevention were evidenced in several kennels. Altogether, our findings highlight the need to update knowledge and practice of MVP under the One Health approach, through reinforced education, training and communication involving all stakeholders.

A walk on the wild side: Wild ungulates as potential reservoirs of multi-drug resistant bacteria and genes, including Escherichia coli harbouring CTX-M beta-lactamases.

Extended-spectrum ß-lactamases (ESBL)-producing Enterobacterales have been classified as critical priority pathogens by the World Health Organization (WHO). ESBL are universally distributed and, in 2006, were firstly reported on a wild animal. Understanding the relative contributions of wild animals to ESBL circulation in the environment is urgently needed. In this work, we have conducted a nationwide study in Portugal to investigate the occurrence of bacteria carrying clinically significant antimicrobial resistance genes (ARG), using widely distributed wild ungulates as model species. A total of 151 antimicrobial resistant-Enterobacterales isolates were detected from 181 wild ungulates: 50% (44/88) of isolates from wild boar (Sus scrofa), 40.3% (25/62) from red deer (Cervus elaphus), 41.4% (12/29) from fallow deer (Dama dama) and 100% (2/2) from mouflon (Ovis aries subsp. musimon). Selected isolates showed a diversified resistance profile, with particularly high values corresponding to ampicillin (71.5%) and tetracycline (63.6%). Enterobacterales strains carried blaTEM, tetA, tetB, sul2, sul1 or dfrA1 ARG genes. They also carried blaCTX-M-type genes, which are prevalent in human infections, namely CTX-M-14, CTX-M-15 and CTX-M-98. Strikingly, this is the first report of CTX-M-98 in wildlife. Almost 40% (n = 59) of Enterobacterales were multi-drug resistant. The diversity of plasmids carried by ESBL isolates was remarkable, including IncF, K and P. This study highlights the potential role of wild ungulates as environmental reservoirs of CTX-M ESBL-producing E. coli and in the spill-over of AMR bacteria and their determinants. Our findings suggest that wild ungulates are useful as strategic sentinel species of AMR in terrestrial environments, especially in response to potential sources of anthropogenic pollution, providing early warning of potential risks to human, animal and environmental health.

A high-risk carbapenem-resistant Pseudomonas aeruginosa clone detected in red deer (Cervus elaphus) from Portugal.

Pseudomonas aeruginosa is a ubiquitous bacterium, successfully exploiting a variety of environmental niches due to its remarkable metabolic versatility. The World Health Organization classifies P. aeruginosa as a "priority pathogen" due to its a great ability to overcome the action of antimicrobials, including carbapenems. Hitherto, most studies have focused on clinical settings from humans, but much less on animal and environmental settings, particularly on wildlife. In this work, we report the isolation of a carbapenem-resistant Pseudomonas aeruginosa strain recovered from the faeces of a red deer adult female sampled in a humanized area. This isolate was obtained during a nationwide survey on antimicrobial resistance in wildlife aimed to determine the occurrence of carbapenem-resistant bacteria among 181 widely distributed wild ungulates. This P. aeruginosa isolate was found to be a high-risk clone, belonging to the sequence type (ST) 274. The genomic analysis of P. aeruginosa isolate UP4, classified this isolate as belonging to serogroup O3, which was also found to harbour the genes blaPAO, blaPDC-24, blaOXA-486 (encoding resistance to beta-lactams), aph(3')-IIb (aminoglycosides resistance), fosA (fosfomycin resistance) and catB7 (chloramphenicol resistance). Antimicrobial susceptibility screening, according to EUCAST, showed resistance to imipenem and intermediate resistance to meropenem and doripenem. To our knowledge, this is the first description of carbapenem-resistant P. aeruginosa in deer in Europe. Our results highlight the importance of wild ungulates either as victims of human activity or amplifiers of AMR, either way with potential impacts on animal, human and ecosystem health, since excretion of AMR bacteria might directly or indirectly contaminate other animals and the surrounding environment, perpetuating the spill-over and chain dissemination of AMR determinants.

Antimicrobial resistance in commensal Staphylococcus aureus from wild ungulates is driven by agricultural land cover and livestock farming.

Staphylococcus aureus is a human pathobiont (i.e., a commensal microorganism that is potentially pathogenic under certain conditions), a nosocomial pathogen and a leading cause of morbidity and mortality in humans. S. aureus is also a commensal and pathogen of companion animals and livestock. The dissemination of antimicrobial resistant (AMR) S. aureus, particularly methicillin-resistant (MRSA), has been associated to its ability for establishing new reservoirs, but limited attention has been devoted to the role of the environment. To fill this gap, we aimed to characterize animal carrier status, AMR phenotypes, predominant clonal lineages and their relationship with clinical and food-chain settings, as well as to find predictors of AMR occurrence. Nasal swabs (n = 254) from wild boar (n = 177), red deer (n = 54) and fallow deer (n = 23) hunted in Portugal, during the season 2019/2020, yielded an overall carrier proportion of 35.8%, ranging from 53.7% for red deer and 32.2% for wild boar to 21.7% for fallow deer. MRSA from wild boar and phenotypically linezolid-resistant S. aureus from wild boar and red deer were isolated, indicating that resistance to antimicrobials restricted to clinical practice also occurs in wildlife. The most prevalent genotypes were t11502/ST2678 (29.6%) and t12939/ST2678 (9.4%), previously reported in wild boar from Spain. Clonal lineages reported in humans and livestock, like CC1, CC5 or CC8 (19.1%) and ST425, CC133 or CC398 (23.5%), respectively, were also found. The sequence type ST544, previously restricted to humans, is described in wildlife for the first time. We also identified that land use (agricultural land cover), human driven disturbance (swine abundance) and host-related factors (sex) determine resistance occurrence. These findings suggest that antibiotics used in clinical settings, agriculture and livestock farming, spill over to wildlife, leading to AMR emergence, with potential biological, ecological, and human health effects. This work is one of the most comprehensive surveys in Europe of S. aureus occurrence and determinants among widely distributed wild ungulates.

Stalking Mycobacterium bovis in the total environment: FLOW-FISH & FACS to detect, quantify, and sort metabolically active and quiescent cells in complex matrices.

Mycobacterium bovis causes tuberculosis (TB) at the human-wildlife-livestock interface. Environmental persistence of M. bovis excreted by infected hosts may cause indirect transmission to other animals. However, methodological constrains hamper assessment of M. bovis viability and molecular signature in environmental matrices. In this work, an innovative, modular, and highly efficient single-cell workflow combining flow cytometry (FLOW), fluorescence in situ hybridization (FISH), and fluorescence-activated cell sorting (FACS) was developed, allowing detection, quantification, and sorting of viable and dormant M. bovis cells from environmental matrices. Validation with spiked water and sediments showed high efficiency (90%) of cell recovery, with high linearity between expected and observed results, both in cell viability evaluation (r2 =0.93) and FISH-labelled M. bovis cells quantification (r2 ≥0.96). The limit of detection was established at 105 cells/g of soil in the cell viability step and 102 cells/g of soil in the taxonomical labelling stage. Moreover, FACS efficiency attained noteworthy recovery yield (50%) and purity (60% viable cells; 70% taxonomically labelled M. bovis). This new methodology represents a huge step for M. bovis assessment outside the mammal host, offering the rapid quantification of M. bovis cell load and cell viability, including viable but non-culturable cells, and further downstream cell analyses after FACS. Subsequent environmental data integration with the clinical component will expand knowledge on transmission routes, promising new paths in TB research and an intervention tool to mitigate the underlying biohazard.

Discrimination and surveillance of infectious severe acute respiratory syndrome Coronavirus 2 in wastewater using cell culture and RT-qPCR.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA has been extensively detected in raw wastewater in studies exploring wastewater-based epidemiology (WBE) for early warning purposes. Nonetheless, only a few limited studies investigated the presence of SARS-CoV-2 in treated wastewaters to determine the potential health risks across the water cycle. The detection of SARS-CoV-2 has been done mostly by RT-qPCR and ddPCR, which only provides information on the presence of nucleic acids rather than information on potential infectivity. In this study, we set to develop and evaluate the use of viability RT-qPCR for the selective discrimination and surveillance of infectious SARS-CoV-2 in secondary-treated wastewater. Enzymatic (nuclease) and viability dye (Reagent D) pretreatments were applied to infer infectivity through RT-qPCR using porcine epidemic diarrhea virus (PEDV) as a CoV surrogate. Infectivity tests were first performed on PEDV purified RNA, then on infectious and heat-inactivated PEDV, and finally on heat inactivated PEDV spiked in concentrated secondary-treated wastewater. The two viability RT-qPCR methods were then applied to 27 secondary-treated wastewater samples positive for SARS-CoV-2 RNA at the outlet of five large urban wastewater treatment plants in Portugal. Reagent D pretreatment showed similar behavior to cell culture for heat-inactivated PEDV and both viability RT-qPCR methods performed comparably to VERO E6 cell culture for SARS-CoV-2 present in secondary-treated wastewater, eliminating completely the RT-qPCR signal. Our study demonstrated the lack of infectious SARS-CoV-2 viral particles on secondary-treated wastewater through the application of two pretreatment methods for the rapid inference of infectivity through RT-qPCR, showing their potential application in environmental screening. This study addressed a knowledge gap on the public health risks of SARS-CoV-2 across the water cycle.

Recovery of SARS-CoV-2 from large volumes of raw wastewater is enhanced with the inuvai R180 system.

Wastewater-based epidemiology (WBE) for severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2) is a powerful tool to complement syndromic surveillance. Although detection of SARS-CoV-2 in raw wastewater may be prompted with good recoveries during periods of high community prevalence, in the early stages of population outbreaks concentration procedures are required to overcome low viral concentrations. Several methods have become available for the recovery of SARS-CoV-2 from raw wastewater, generally involving filtration. However, these methods are limited to small sample volumes, possibly missing the early stages of virus circulation, and restrained applicability across different water matrices. The aim of this study was thus to evaluate the performance of three methods enabling the concentration of SARS-CoV-2 from large volumes of wastewater: i) hollow fiber filtration using the inuvai R180, with an enhanced elution protocol and polyethylene glycol (PEG) precipitation; ii) PEG precipitation; and iii) skimmed milk flocculation. The performance of the three approaches was evaluated in wastewater from multiple wastewater treatment plants (WWTP) with distinct singularities, according to: i) effective volume; ii) percentage of recovery; iii) extraction efficiency; iv) inhibitory effect; and v) the limits of detection and quantification. The inuvai R180 system had the best performance, with detection of spiked control across all samples, with average recovery percentages of 68% for porcine epidemic diarrhea virus (PEDV), with low variability. Mean recoveries for PEG precipitation and skimmed milk flocculation were 9% and 14%, respectively. The inuvai R180 enables the scalability of volumes without negative impact on the costs, time for analysis, and recovery/inhibition. Moreover, hollow fiber ultrafilters favor the concentration of different microbial taxonomic groups. Such combined features make this technology attractive for usage in environmental waters monitoring.

A wastewater-based epidemiology tool for COVID-19 surveillance in Portugal.

The presence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in wastewater produced interest in its use for sentinel surveillance at a community level and as a complementary approach to syndromic surveillance. With this work, we set the foundations for wastewater-based epidemiology (WBE) in Portugal by monitoring the trends of SARS-CoV-2 RNA circulation in the community, on a nationwide perspective during different epidemiological phases of the pandemic. The Charité assays (E_Sarbecco, RdRP, and N_Sarbecco) were applied to monitor, over 32-weeks (April to December 2020), the dynamics of SARS-CoV-2 RNA at the inlet of five wastewater treatment plants (WWTP), which together serve more than two million people in Portugal. Raw wastewater from three Coronavirus disease 2019 (COVID-19) reference hospitals was also analyzed during this period. In total, more than 600 samples were tested. For the first weeks, detection of SARS-CoV-2 RNA was sporadic, with concentrations varying from 103 to 105 genome copies per liter (GC/L). Prevalence of SARS-CoV-2 RNA increased steeply by the end of May into late June, mainly in Lisboa e Vale do Tejo region (LVT), during the reopening phase. After the summer, with the reopening of schools in mid-September and return to partial face-to-face work, a pronounced increase of SARS-CoV-2 RNA in wastewater was detected. In the LVT area, SARS-CoV-2 RNA load agreed with reported trends in hotspots of infection. Synchrony between trends of SARS-CoV-2 RNA in raw wastewater and daily new COVID-19 cases highlights the value of WBE as a surveillance tool, particularly after the phasing out of the epidemiological curve and when hotspots of disease re-emerge in the population which might be difficult to spot based solely on syndromic surveillance and contact tracing. This is the first study crossing several epidemiological stages highlighting the long-term use of WBE for SARS-CoV-2.

When FLOW-FISH met FACS: Combining multiparametric, dynamic approaches for microbial single-cell research in the total environment.

In environmental microbiology, the ability to assess, in a high-throughput way, single-cells within microbial communities is key to understand their heterogeneity. Fluorescence in situ hybridization (FISH) uses fluorescently labeled oligonucleotide probes to detect, identify, and quantify single cells of specific taxonomic groups. The combination of Flow Cytometry (FLOW) with FISH (FLOW-FISH) enables high-throughput quantification of complex whole cell populations, which when associated with fluorescence-activated cell sorting (FACS) enables sorting of target microorganisms. These sorted cells may be investigated in many ways, for instance opening new avenues for cytomics at a single-cell scale. In this review, an overview of FISH and FLOW methodologies is provided, addressing conventional methods, signal amplification approaches, common fluorophores for cell physiology parameters evaluation, and model variation techniques as well. The coupling of FLOW-FISH-FACS is explored in the context of different downstream applications of sorted cells. Current and emerging applications in environmental microbiology to outline the interactions and processes of complex microbial communities within soil, water, animal microbiota, polymicrobial biofilms, and food samples, are described.

Mapping the scientific knowledge of antimicrobial resistance in food-producing animals.

Antimicrobial resistance (AMR) can be highlighted as one of the most significant health concerns among the last decades, for which antimicrobial drug use in food-producing animals has contributed as one of the major drivers. Food-producing animals are one of the most important and rapidly expanding commercial agricultural sectors worldwide but there is currently limited knowledge on the temporal and geographical distribution of scientific research on antimicrobial resistance in food-producing animals. We provide a global overview of the spatial and temporal trends of scientific knowledge on AMR in food-producing animals. Peer-reviewed papers of AMR on food-producing animals were retrieved from the Web of Science, systemized and dissected. The final validated dataset contained 1341 occurrences observations covering the 1957-2018 period. There has been a shift of research efforts, both geographically and temporally, emphasizing regional differences in food animal production and changing practices in the food production industry. It becomes evident that many regions have been poorly surveyed, wherein intensified sampling and testing efforts should be most valuable. This systematization of knowledge will be crucial in helping to determine how to optimally allocate limited resources available for AMR monitor and control, aiding in the prediction where the threat of new resistant infections will be greatest. AMR research in food-producing animals in developing countries is markedly growing, reflecting changes in food animals production systems but also posing a particularly significant threat, not only due to intensive animal production, but also exacerbated by poor sanitation. We highlight that the use of antibiotics in food producing animals is pervasive, calling for urgent action. These findings raise the possibility to finetuning key priorities on AMR global issues.

Genome-wide estimation of recombination, mutation and positive selection enlightens diversification drivers of Mycobacterium bovis.

Genome sequencing has reinvigorated the infectious disease research field, shedding light on disease epidemiology, pathogenesis, host-pathogen interactions and also evolutionary processes exerted upon pathogens. Mycobacterium tuberculosis complex (MTBC), enclosing M. bovis as one of its animal-adapted members causing tuberculosis (TB) in terrestrial mammals, is a paradigmatic model of bacterial evolution. As other MTBC members, M. bovis is postulated as a strictly clonal, slowly evolving pathogen, with apparently no signs of recombination or horizontal gene transfer. In this work, we applied comparative genomics to a whole genome sequence (WGS) dataset composed by 70 M. bovis from different lineages (European and African) to gain insights into the evolutionary forces that shape genetic diversification in M. bovis. Three distinct approaches were used to estimate signs of recombination. Globally, a small number of recombinant events was identified and confirmed by two independent methods with solid support. Still, recombination reveals a weaker effect on M. bovis diversity compared with mutation (overall r/m = 0.037). The differential r/m average values obtained across the clonal complexes of M. bovis in our dataset are consistent with the general notion that the extent of recombination may vary widely among lineages assigned to the same taxonomical species. Based on this work, recombination in M. bovis cannot be excluded and should thus be a topic of further effort in future comparative genomics studies for which WGS of large datasets from different epidemiological scenarios across the world is crucial. A smaller M. bovis dataset (n = 42) from a multi-host TB endemic scenario was then subjected to additional analyses, with the identification of more than 1,800 sites wherein at least one strain showed a single nucleotide polymorphism (SNP). The majority (87.1%) was located in coding regions, with the global ratio of non-synonymous upon synonymous alterations (dN/dS) exceeding 1.5, suggesting that positive selection is an important evolutionary force exerted upon M. bovis. A higher percentage of SNPs was detected in genes enriched into "lipid metabolism", "cell wall and cell processes" and "intermediary metabolism and respiration" functional categories, revealing their underlying importance in M. bovis biology and evolution. A closer look on genes prone to horizontal gene transfer in the MTBC ancestor and included in the 3R (DNA repair, replication and recombination) system revealed a global average negative value for Taijima's D neutrality test, suggesting that past selective sweeps and population expansion after a recent bottleneck remain as major evolutionary drivers of the obligatory pathogen M. bovis in its struggle with the host.

Emergence of colistin resistance genes (mcr-1) in Escherichia coli among widely distributed wild ungulates.

The environment is considered a major reservoir of antimicrobial resistant microorganisms (AMR) and antimicrobial resistance genes (ARG). Colistin, a "last resort" antibiotic, is used for the treatment of severe infections caused by multidrug-resistant Gram-negative bacteria. The global dissemination of mobile colistin resistance genes (mcr) in natural and non-natural environments is a major setback in the fight against antimicrobial resistance. Hitherto, there is a limited number of studies screening this resistance determinant in bacteria from wildlife. In this study, we describe for the first time the detection of plasmid-mediated colistin resistance in Escherichia coli from wild ungulates in Portugal, which are also widely distributed across Europe. This information is critical to identify the importance of ungulates in the dissemination of resistant bacteria, and their corresponding genes, across the environment. Here, 151 resistant-Enterobacteriaceae isolated from 181 samples collected from different wild ungulate species throughout Portugal were screened for mcr genes. Four mcr-1-positive Escherichia coli were detected from four fallow deer individuals that were sampled in the same hunting ground. These four isolates harboured mcr-1-related IncP plasmids belonging to sequencing types ST155, ST533 and ST345 (n = 2), suggesting bacterial and/or plasmid circulation. All mcr-1-positive E. coli also showed other resistance phenotypes, including MDR, including the B1 commensal phylogenetic profile. All mcr-1-positive E. coli show additional resistance phenotypes, including MDR, including the B1 commensal phylogenetic profile. Our findings are upsetting, highlighting the global dissemination of colistin resistance genes in the whole ecosystem, which, under the One Health framework, emphasizes the urgent need for effective implementation of AMR surveillance and control in the human-animal-environment interfaces.

Whole Genome Sequencing Refines Knowledge on the Population Structure of Mycobacterium bovis from a Multi-Host Tuberculosis System.

Classical molecular analyses of Mycobacterium bovis based on spoligotyping and Variable Number Tandem Repeat (MIRU-VNTR) brought the first insights into the epidemiology of animal tuberculosis (TB) in Portugal, showing high genotypic diversity of circulating strains that mostly cluster within the European 2 clonal complex. Previous surveillance provided valuable information on the prevalence and spatial occurrence of TB and highlighted prevalent genotypes in areas where livestock and wild ungulates are sympatric. However, links at the wildlife-livestock interfaces were established mainly via classical genotype associations. Here, we apply whole genome sequencing (WGS) to cattle, red deer and wild boar isolates to reconstruct the M. bovis population structure in a multi-host, multi-region disease system and to explore links at a fine genomic scale between M. bovis from wildlife hosts and cattle. Whole genome sequences of 44 representative M. bovis isolates, obtained between 2003 and 2015 from three TB hotspots, were compared through single nucleotide polymorphism (SNP) variant calling analyses. Consistent with previous results combining classical genotyping with Bayesian population admixture modelling, SNP-based phylogenies support the branching of this M. bovis population into five genetic clades, three with apparent geographic specificities, as well as the establishment of an SNP catalogue specific to each clade, which may be explored in the future as phylogenetic markers. The core genome alignment of SNPs was integrated within a spatiotemporal metadata framework to further structure this M. bovis population by host species and TB hotspots, providing a baseline for network analyses in different epidemiological and disease control contexts. WGS of M. bovis isolates from Portugal is reported for the first time in this pilot study, refining the spatiotemporal context of TB at the wildlife-livestock interface and providing further support to the key role of red deer and wild boar on disease maintenance. The SNP diversity observed within this dataset supports the natural circulation of M. bovis for a long time period, as well as multiple introduction events of the pathogen in this Iberian multi-host system.