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.

A low dose of benzo(a)pyrene during prepuberty in male rats generated immediate oxidative stress in the testes and compromised steroidogenic enzymes/proteins.

The prepubertal period is crucial for sexual development and any alterations can interfere with the reproductive system in adulthood. The aim of this study was to evaluate how Benzo(a)pyrene (BaP) can affect the testes during the prepubertal period. Juvenile male Wistar rats were divided into a control (corn oil + DMSO) and a BaP-group (0.1 µg/kg/day), exposed to BaP for 31 days (gavage), and all parameters were evaluated on postnatal day (PND) 54. Leukocyte counts were decreased. Histological analyses of the testes revealed that height and seminiferous tubules diameters (STDs) were reduced, tubular dynamics were altered, and Leydig cell atrophy was evident in the BaP-group. The testosterone concentration was decreased while FSH levels increased within the BaP-exposed group. Steroidogenic enzymes in the testes were decreased, but steroidogenic acute regulatory protein was not altered. The expression of gstp1 and ckit enzymes was decreased. Reduced glutathione (GSH) and superoxide dismutase (SOD) were increased, whereas malondialdehyde (MDA) was decreased in the testes. In conclusion, BaP or its metabolites causes low systemic toxicity; however, it adversely influences testicular function by disrupting the hormonal axis, unbalancing testicular antioxidative, and blocking the action of the steroidogenic mechanisms.

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.

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.

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.

Empathy and Coping in Allied Health Sciences: Gender Patterns.

This study aimed to examine the patterns of associations between empathy and coping among undergraduate men and women studying at Allied Health Sciences. This cross-sectional study is part of a larger longitudinal study conducted in an Allied Health Sciences School. Participants were 183 undergraduate students from 12 training programs (e.g., Physiotherapy, Occupational Therapy, Speech Therapy). Their mean age was 20.79 years (SD = 2.64), and they were in their first, third, and fourth years of school. The instruments were the Brief-COPE and the Interpersonal Reactivity Index (IRI). Empathy correlated with coping strategies in both genders, though showing different patterns of association. First, distinct coping strategies were associated with the same empathy dimension (perspective taking) among women (positive reframing and self-blame) and among men (active coping). Second, the same three coping strategies appeared in both genders (seeking emotional or instrumental support and resorting to religion) but associated with different empathy dimensions (cognitive empathy among women and mostly emotional empathy among men). Third, among women (but not among men), two coping strategies (positive reframing and behavioral disengagement) were each simultaneously correlated with cognitive and emotional empathy in opposite directions. Fourth, emotional empathy correlated, only among women, with several coping strategies considered to be maladaptive (behavioral disengagement, denial and substance use). Among men, only one significant coping strategy was considered to be maladaptive (behavioral disengagement) and it was negatively correlated with cognitive empathy (perspective taking). Unlike in women, relationships between the empathic dimension of fantasy and coping strategies were non-significant among men. These distinct patterns of associations emerged despite significant differences in empathy by gender (fantasy, personal distress and empathic concern) and in coping strategies (instrumental support, emotional support, religion and venting). These results support the idea that the display of empathy might be associated with gender differences in the underlying empathy dimensions and in the coping strategies used to deal with stress in the undergraduate programs of Allied Health Sciences.

The hard numbers of tuberculosis epidemiology in wildlife: A meta-regression and systematic review.

Tuberculosis (TB) is a widespread disease that crosses the human and animal health boundaries, with infection being reported in wildlife, from temperate and subtropical to arctic regions. Often, TB in wild species is closely associated with disease occurrence in livestock but the TB burden in wildlife remains poorly quantified on a global level. Through meta-regression and systematic review, this study aimed to summarize global information on TB prevalence in commonly infected wildlife species and to draw a global picture of the scientific knowledge accumulated in wildlife TB. For these purposes, a literature search was conducted through the Web of Science and Google Scholar. The 223 articles retrieved, concerning a 39-year period, were submitted to bibliometric analysis and 54 publications regarding three wildlife hosts fulfilled the criteria for meta-regression. Using a random-effects model, the worldwide pooled TB prevalence in wild boar is higher than for any other species and estimated as 21.98%, peaking in Spain (31.68%), Italy (23.84%) and Hungary (18.12%). The pooled prevalence of TB in red deer is estimated at 13.71%, with Austria (31.58%), Portugal (27.75%), New Zealand (19.26%) and Spain (12.08%) positioning on the top, while for European badger it was computed 11.75%, peaking in the UK (16.43%) and Ireland (22.87%). Despite these hard numbers, a declining trend in wildlife TB prevalence is apparent over the last decades. The overall heterogeneity calculated by multivariable regression ranged from 28.61% (wild boar) to 60.92% (red deer), indicating that other unexplored moderators could explain disease burden. The systematic review shows that the most prolific countries contributing to knowledge related with wildlife TB are settled in Europe and Mycobacterium bovis is the most reported pathogen (89.5%). This study provides insight into the global epidemiology of wildlife TB, ascertaining research gaps that need to be explored and informing how should surveillance be refined.

Global trends of epidemiological research in livestock tuberculosis for the last four decades.

Animal tuberculosis (TB) caused by Mycobacterium tuberculosis complex (MTC) bacteria remains as one of the most significant infectious diseases of livestock, despite decades of eradication programmes and research efforts, in an era where the livestock sector is among the most important and rapidly expanding commercial agricultural segments worldwide. This work provides a global overview of the spatial and temporal trends of reported scientific knowledge of TB in livestock, aiming to gain insights into research subtopics within the animal TB epidemiology domain and to highlight territorial inequalities regarding data reporting and research outputs over the years. To deliver such information, peer-reviewed reports of TB studies in livestock were retrieved from the Web of Science and Google Scholar, systematized and dissected. The validated data set contained 443 occurrence observations, covering the 1981-2020 period (39 years). We highlight a clear move towards transdisciplinary areas and the One Health approach, with a global temporal increase in publications combining livestock with wildlife and/or human components, which reflect the importance of non-prototypical hosts as key to understanding animal TB. It becomes evident that cattle is the main host across works from all continents; however, many regions remain poorly surveyed. TB research in livestock in low-/middle-income countries is markedly growing, reflecting changes in animal husbandry, but also mirroring the globalization era, with a marked increase in international collaboration and capacitation programmes for scientific and technological development. This review gives an overview of the most prolific continents, countries and research fields in animal TB epidemiology, clearly outlining knowledge gaps and key priority topics. The estimated growth trend of livestock production until 2050, particularly in Asia and Africa, in response to human population growth and animal-protein demand, will require further investment in early surveillance and adaptive research to accommodate the higher diversity of livestock species and MTC members and raising the possibility to fine-tune funding schemes.

Long-term molecular surveillance provides clues on a cattle origin for Mycobacterium bovis in Portugal.

Animal tuberculosis (TB), caused by Mycobacterium bovis, is maintained in Portugal in a multi-host system, with cattle, red deer and wild boar, playing a central role. However, the ecological processes driving transmission are not understood. The main aim of this study was thus to contribute to the reconstruction of the spatiotemporal history of animal TB and to refine knowledge on M. bovis population structure in order to inform novel intervention strategies. A collection of 948 M. bovis isolates obtained during long-term surveillance (2002-2016, 15 years) of cattle (n = 384), red deer (n = 303) and wild boar (n = 261), from the main TB hotspot areas, was characterized by spoligotyping and 8 to 12-loci MIRU-VNTR. Spoligotyping identified 64 profiles and MIRU-VNTR distinguished 2 to 36 subtypes within each spoligotype, enabling differentiation of mixed or clonal populations. Common genotypic profiles within and among livestock and wildlife in the same spatiotemporal context highlighted epidemiological links across hosts and regions, as for example the SB0119-M205 genotype shared by cattle in Beja district or SB0121-M34 shared by the three hosts in Castelo Branco and Beja districts. These genomic data, together with metadata, were integrated in a Bayesian inference framework, identifying five ancestral M. bovis populations. The phylogeographic segregation of M. bovis in specific areas of Portugal where the disease persists locally is postulated. Concurrently, robust statistics indicates an association of the most probable ancient population with cattle and Beja, providing a clue on the origin of animal TB epidemics. This relationship was further confirmed through a multinomial probability model that assessed the influence of host species on spatiotemporal clustering. Two significant clusters were identified, one that persisted between 2004 and 2010, in Beja district, with Barrancos county at the centre, overlapping the central TB core area of the Iberian Peninsula, and highlighting a significant higher risk associated to cattle. The second cluster was predominant in the 2012-2016 period, holding the county Rosmaninhal at the centre, in Castelo Branco district, for which wild boar contributed the most in relative risk. These results provide novel quantitative insights beyond empirical perceptions, that may inform adaptive TB control choices in different regions.

Estimates of the global and continental burden of animal tuberculosis in key livestock species worldwide: A meta-analysis study.

Zoonotic animal tuberculosis (TB) is a One Health paradigm infectious disease, caused by Mycobacterium tuberculosis complex bacteria, that affects different host species with varying levels of management. In most developed countries, official surveillance and control strategies support the longitudinal reporting of herd and/or animal prevalence. However, for under resourced countries without surveillance plans, this information may be obtained from cross-sectional studies only. The objective of this meta-analysis was to perform a worldwide estimate of the overall prevalence of animal TB in different livestock species whose importance in production systems varies according to the region of the world. The ISI's Web of Science and Google Scholar were searched combining keywords and related database-specific subject terms to identify relevant cohort or cross-sectional work published in this topic. A total of 443 articles were retrieved, screened, and a final set of 182 references included. Potential sources of variation were investigated using subgroup analyses and meta-regression. Prevalence estimates in five mammalian host groups were stratified according to host species, host characteristics, anatomical localization of lesions, sample size, geographical location, and diagnostic tests. The multivariable meta-regression analysis accounted for a range between 0% (farmed wild boar) and 68.71% (camelids) of the overall observed heterogeneity, indicating that the pondered predictors partially explain the observed variability. Differences in the overall prevalence of TB across hosts were small, with most groups showing values around 10%, except farmed wild boar (41%). The sample size emerged as an important moderator, with small size studies leading to the overestimation of prevalence. TB prevalence rates were very heterogeneous across continents and depended on the host, with lower values (below 10%) in Africa and Asia, while North America (33.6%, cattle), Europe (51%, goats), and South America (85.7%, pigs) exhibited higher rates, possibly related to greater densities of specific host groups managed on more intensive production systems. Stratification by diagnostic tests evidenced heterogeneous prevalence rates depending on the host group, possibly reflecting differences in test performance across different hosts. Results from this study highlight different TB burden scenarios, pinpointing host groups and diagnostics that should be prioritized in surveillance systems in different regions, thus providing policy-relevant information to catalyse TB control in settings with lower installed capacity and better resource allocation at the human-animal-environment interface.

Non-Tuberculous Mycobacteria: Molecular and Physiological Bases of Virulence and Adaptation to Ecological Niches.

Non-tuberculous mycobacteria (NTM) are paradigmatic colonizers of the total environment, circulating at the interfaces of the atmosphere, lithosphere, hydrosphere, biosphere, and anthroposphere. Their striking adaptive ecology on the interconnection of multiple spheres results from the combination of several biological features related to their exclusive hydrophobic and lipid-rich impermeable cell wall, transcriptional regulation signatures, biofilm phenotype, and symbiosis with protozoa. This unique blend of traits is reviewed in this work, with highlights to the prodigious plasticity and persistence hallmarks of NTM in a wide diversity of environments, from extreme natural milieus to microniches in the human body. Knowledge on the taxonomy, evolution, and functional diversity of NTM is updated, as well as the molecular and physiological bases for environmental adaptation, tolerance to xenobiotics, and infection biology in the human and non-human host. The complex interplay between individual, species-specific and ecological niche traits contributing to NTM resilience across ecosystems are also explored. This work hinges current understandings of NTM, approaching their biology and heterogeneity from several angles and reinforcing the complexity of these microorganisms often associated with a multiplicity of diseases, including pulmonary, soft-tissue, or milliary. In addition to emphasizing the cornerstones of knowledge involving these bacteria, we identify research gaps that need to be addressed, stressing out the need for decision-makers to recognize NTM infection as a public health issue that has to be tackled, especially when considering an increasingly susceptible elderly and immunocompromised population in developed countries, as well as in low- or middle-income countries, where NTM infections are still highly misdiagnosed and neglected.

Animal tuberculosis: Impact of disease heterogeneity in transmission, diagnosis and control.

Animal tuberculosis (TB) in terrestrial mammals is mainly caused by Mycobacterium bovis. This pathogen is adapted to a wide range of host species, representing a threat to livestock, wildlife and human health. Disease heterogeneity is a hallmark of multi-host TB and a challenge for control. Drivers of animal TB heterogeneity are very diverse and may act at the level of the causative agent, the host species, the interface between mycobacteria and the host, community of hosts, the environment and even policy behind control programmes. In this paper, we examine the drivers that seem to contribute to this phenomenon. We begin by reviewing evidence accumulated to date supporting the consensus that a complex range of genetic, biological and socio-environmental factors contribute to the establishment and maintenance of animal TB, setting the grounds for heterogeneity. We then highlight the complex interplay between individual, species-specific and community protective factors with risk/maintenance variables that include animal movements and densities, co-infection and super-shedders. We finally consider how current interventions should seek to consider and explore heterogeneity in order to tackle potential limitations for diagnosis and control programmes, simultaneously increasing their efficacy.

Polyclonal infection as a new scenario in Mycobacterium caprae epidemiology.

Portugal is one of the European Union countries with an ongoing eradication program for bovine tuberculosis (TB), which does not include systematic goat testing. However, surveillance in small ruminants is increasingly important, since goat and sheep can harbour Mycobacterium caprae and be an infection source to cattle with impact in the success of bovine TB control. Furthermore, the information regarding the epidemiology and biology of M. caprae is quite limited comparing to the cognate bovine-adapted ecotype, M. bovis. In this work, we applied spoligotyping and MIRU-VNTR (Mycobacterial Interspersed Repetitive Units-Variable Number of Tandem Repeats) to M. caprae obtained between 2003 and 2014 from 55 animal hosts, including goat (n = 29), cattle (n = 21), sheep (n = 1) and wild boar (n = 4) from Portugal. The molecular analysis revealed a unique spoligotyping pattern (SB0157) and 24 MIRU types. Genotyping of serial M. caprae from herds with recurrent outbreaks enabled further discrimination of epidemiologically related isolates, supporting a clonal structure in Portugal and denoting the emergence of clonal diversity at the herd level, more apparent for MIRU4. Results suggest a founder effect and adaptive genotypic divergence, paving the way for sympatric speciation. Double allele findings at MIRU4 in over 20 % of infected animals indicates that co-infection and in vivo microevolution may be frequent in the goat-adapted ecotype. While polyclonal infection appears common in M. caprae epidemiology, the functional significance of subtle genotypic variations remains to be disclosed, namely at the interface with the host, to expand knowledge on the epidemiology and biology of this neglected ecotype.

Ecological drivers of Mycobacterium avium subsp. paratuberculosis detection in mongoose (Herpestes ichneumon) using IS900 as proxy.

Mycobacterium avium subsp. paratuberculosis (MAP) is the etiological agent of Johne's disease or paratuberculosis, a chronic infection affecting domestic ruminants worldwide. Despite sporadic reports of MAP occurrence in non-ruminants, information on the risk factors predisposing for infection is still scarce and evidence of transmission paths linking the livestock-wildlife-environment interfaces also remains lacking. In this study, we predicted that environmental, host-related, land use and human driven disturbance factors would modulate carnivore exposure to MAP. To test these hypotheses, we performed a retrospective survey, based on microbiological and molecular methods, in mainland Portugal including five sympatric species from the Herpestidae, Canidae, Viverridae, and Mustelidae families (n = 202) and examined 16 variables as putative predictors of MAP occurrence. Molecular evidence of MAP using IS900 as proxy was demonstrated in 7.43% (95%CI: 4.55-11.9) of surveyed carnivores, the highest proportions being registered for red fox (Vulpes vulpes) (10%; 95%CI: 4.0-23) and Egyptian mongoose (Herpestes ichneumon) (6.0%; 95%CI: 3.2-11). We demonstrate that important species of the Mediterranean carnivore guild, such as stone marten (Martes foina) and common genet (Genetta genetta), may also be exposed to MAP, being this the first time that occurrence in genet is reported. The high proportion of DNA-positive specimens, concurrent with the apparent lack of gastro-enteric lesions and molecular confirmation of IS900 in feces, argue for the presence of subclinical carriers that occasionally shed bacteria, potentially aiding as source of infection to susceptible species and possibly contributing for environmental contamination. Achievement of MAP isolation would prove beyond any doubt that MAP is present in this wildlife population. Ecological modelling results suggested that the probability of MAP infection using IS900 as proxy in mongoose is positively associated with higher altitude and temperature stability, as well as with lower annual rainfall. Density of livestock farms was found not to be a significant predictor, which may indicate that the livestock-wildlife interface is probably not important as an infection route for mongoose.

Biodegradation of antibiotics: The new resistance determinants - part I.

History shows that the discovery of, and the resistance to, antibiotics go hand in hand. While knowledge of resistance mechanisms, their impact and distribution is vast, over the years, the topic of antibiotic degradation has often been overlooked and regarded as being discrete from the research on resistance. As a result, understanding of the degradation of antibiotics and the impact of antibiotic degraders on the environment and human health are, for most classes, neither thoroughly documented nor understood. Current information on the biodegradation of antibiotics is described in two review articles. This first part focuses on sulfonamides, trimethoprim, aminoglycosides, amphenicols and tetracyclines. Detailed metabolic and molecular aspects as well as the role of the degraders in natural microbial communities are discussed. An integrated analysis of the accumulated data indicates that appreciation of the interplay between resistance and degradation is quite fragmented, and closing this gap will require novel experimental approaches.

Biodegradation of antibiotics: The new resistance determinants - part II.

Antibiotic residues are widespread in the environment and their presence is known to contribute to the propagation of antibiotic resistance. Nevertheless, knowledge on processes involved in their degradation is scattered. This second part of a two part review aims at compiling knowledge on the (bio-) degradation of antibiotics, focusing on ß-lactams, macrolides, quinolones and ionophores, as well as some less common classes. Detailed metabolic and molecular aspects are discussed, as well as the role of antibiotic degraders in natural microbial communities. This exercise led to the conclusion that among the classes analyzed, the majority of antibiotics are prone to microbial cleavage or transformation.

Comparative genomics reveals a novel genetic organization of the sad cluster in the sulfonamide-degrader 'Candidatus Leucobacter sulfamidivorax' strain GP.

BACKGROUND: Microbial communities recurrently establish metabolic associations resulting in increased fitness and ability to perform complex tasks, such as xenobiotic degradation. In a previous study, we have described a sulfonamide-degrading consortium consisting of a novel low-abundant actinobacterium, named strain GP, and Achromobacter denitrificans PR1. However, we found that strain GP was unable to grow independently and could not be further purified. RESULTS: Previous studies suggested that strain GP might represent a new putative species within the Leucobacter genus (16S rRNA gene similarity < 97%). In this study, we found that average nucleotide identity (ANI) with other Leucobacter spp. ranged between 76.8 and 82.1%, further corroborating the affiliation of strain GP to a new provisional species. The average amino acid identity (AAI) and percentage of conserved genes (POCP) values were near the lower edge of the genus delimitation thresholds (65 and 55%, respectively). Phylogenetic analysis of core genes between strain GP and Leucobacter spp. corroborated these findings. Comparative genomic analysis indicates that strain GP may have lost genes related to tetrapyrrole biosynthesis and thiol transporters, both crucial for the correct assembly of cytochromes and aerobic growth. However, supplying exogenous heme and catalase was insufficient to abolish the dependent phenotype. The actinobacterium harbors at least two copies of a novel genetic element containing a sulfonamide monooxygenase (sadA) flanked by a single IS1380 family transposase. Additionally, two homologs of sadB (4-aminophenol monooxygenase) were identified in the metagenome-assembled draft genome of strain GP, but these were not located in the vicinity of sadA nor of mobile or integrative elements. CONCLUSIONS: Comparative genomics of the genus Leucobacter suggested the absence of some genes encoding for important metabolic traits in strain GP. Nevertheless, although media and culture conditions were tailored to supply its potential metabolic needs, these conditions were insufficient to isolate the PR1-dependent actinobacterium further. This study gives important insights regarding strain GP metabolism; however, gene expression and functional studies are necessary to characterize and further isolate strain GP. Based on our data, we propose to classify strain GP in a provisional new species within the genus Leucobacter, 'Candidatus Leucobacter sulfamidivorax'.

Bioaugmentation of membrane bioreactor with Achromobacter denitrificans strain PR1 for enhanced sulfamethoxazole removal in wastewater.

Achromobacter denitrificans strain PR1, previously found to harbour specific degradation pathways with high sulfamethoxazole (SMX) degradation rates, was bioaugmented into laboratory-scale membrane bioreactors (MBRs) operated under aerobic conditions to treat SMX-containing real domestic wastewater. Different hydraulic retention times (HRTs), which is related to reaction time and loading rates, were considered and found to affect the SMX removal efficiency. The availability of primary substrates was important in both bioaugmented and non-bioaugmented activated sludge (AS) for cometabolism of SMX. High HRT (24 h) resulted in low food to microorganism ratio (F/M) and low SMX removal, due to substrate limitation. Decrease in HRT from 24 h to 12 h, 6 h and finally 4 h led to gradual increases in primary substrates availability, e.g. organic compounds and ammonia, resulted in increased SMX removal efficiency and degradation rate, and is more favorable for high-rate wastewater treatment processes. After inoculation into the MBRs, the bioaugmentation strain was sustained in the reactor for a maximum of 31 days even though a significant decrease in abundance was observed. The bioaugmented MBRs showed enhanced SMX removal, especially under SMX shock loads compared to the control MBRs. The results of this study indicate that re-inoculation is required regularly after a period of time to maintain the removal efficiency of the target compound.