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Attempts to control bacterial pathogens have led to an increase in antibiotic-resistant cells and the genetic elements that confer resistance phenotypes. These cells and genes are disseminated simultaneously with the original selective agents via human waste streams. This might lead to a second, unintended consequence of antimicrobial therapy; an increase in the evolvability of all bacterial cells. The genetic variation upon which natural selection acts is a consequence of mutation, recombination and lateral gene transfer (LGT). These processes are under selection, balancing genomic integrity against the advantages accrued by genetic innovation. Saturation of the environment with selective agents might cause directional selection for higher rates of mutation, recombination and LGT, producing unpredictable consequences for humans and the biosphere. [ABSTRACT FROM AUTHOR]

One of the major stumbling blocks to understanding evolution is the difficulty in reconciling the emergence of complexity with the apparently undirected forces that drive evolutionary processes. This difficulty was originally framed as the "Watch and Watchmaker" argument and more recently revived by proponents of "intelligent design." Undergraduates in particular often attribute purpose and forethought as the driving force behind biological phenomena, and have difficulty understanding evolutionary processes. To demonstrate that complexity can arise solely through mutations that fix in populations via natural selection or drift, we can use analogies where processes can be observed across short time frames and where the key data are accessible to those without specialized biological knowledge. The evolution of language provides such an example. Processes of natural selection, mutation, genetic drift, acquisition of new functions, punctuated equilibria, and lateral gene transfer can be illustrated using examples of changing spellings, neologism, and acquisition of words from other languages. The examples presented in this article are readily accessible, and demonstrate to students that languages have dynamically increased in complexity, simply driven by the usage patterns of their speakers. [ABSTRACT FROM AUTHOR]

Cryptosporidium species generally lack distinguishing morphological traits, and consequently, molecular methods are commonly used for parasite identification. Various methods for Cryptosporidium identification have been proposed, each with their advantages and disadvantages. In this study, we show that capillary electrophoresis coupled with single-strand conformation polymorphism (CE-SSCP) is a rapid, simple and cost-effective method for the identification of Cryptosporidium species and genotypes. Species could be readily differentiated based on the SSCP mobility of amplified 18S rRNA gene molecules. Clones that differed by single-nucleotide polymorphisms could be distinguished on CE-SSCP mobility. Profiles of species known to have heterogenic copies of 18S rRNA gene contained multiple peaks. Cloning and sequencing of Cryptosporidium parvum, Cryptosporidium hominis, Cryptosporidium fayeri and Cryptosporidium possum genotype 18S rRNA gene amplicons confirmed that these multiple peaks represented type A and type B 18S rRNA gene copies. CE-SSCP provides a reliable and sensitive analysis for epidemiological studies, environmental detection and diversity screening. [ABSTRACT FROM AUTHOR]

Class 1 integrons carried by pathogens have acquired over 100 different gene cassettes encoding resistance to antimicrobial compounds, helping to generate a crisis in the management of infectious disease. It is presumed that these cassettes originated from environmental bacteria, but exchange of gene cassettes has surprisingly never been demonstrated outside laboratory or clinical contexts. We aimed to identify a natural environment where such exchanges might occur, and determine the phylogenetic range of participating integrons. Here we examine freshwater biofilms and show that families of cassettes conferring resistance to quaternary ammonium compounds ( qac) are found on class 1 integrons identical to those from clinical contexts, on sequence variants of class 1 integrons only known from natural environments, and on other diverse classes of integrons only known from the chromosomes of soil and freshwater Proteobacteria. We conclude that gene cassettes might be readily shared between different integron classes found in environmental, commensal and pathogenic bacteria. This suggests that class 1 integrons in pathogens have access to a vast pool of gene cassettes, any of which could confer a phenotype of clinical relevance. Exploration of this resource might allow identification of resistance or virulence genes before they become part of multi-drug-resistant human pathogens. [ABSTRACT FROM AUTHOR]

DNA sequencing, phylogenetic and mapping studies suggest that the class 1 integron found in pathogens arose when one member of the diverse family of environmental class 1 integrons became embedded into a Tn402 transposon. However, the timing of this event and the selective forces that first fixed the newly formed element in a bacterial lineage are still unknown. Biocides have a longer use in clinical practice than antibiotics, and a qac (quaternary ammonium compound) resistance gene, or remnant thereof, is a normal feature of class 1 integrons recovered from clinical isolates. Consequently, it is possible that the initial selective advantage was conferred by resistance to biocides, mediated by qac. Here, we show that diverse qac gene cassettes are a dominant feature of cassette arrays from environmental class 1 integrons, and that they occur in the absence of any antibiotic resistance gene cassettes. They are present in arrays that are dynamic, acquiring and rearranging gene cassettes within the arrays. The abundance of qac gene cassettes makes them a likely candidate for participation in the original insertion into Tn402, and as a source of a readily selectable phenotype. More broadly, the increasing use of qac and other biocides at the present time seems likely to promote the fixation of further novel genetic elements, with unpredictable and potentially adverse consequences for human health and agriculture.The ISME Journal (2009) 3, 209–215; doi:10.1038/ismej.2008.98; published online 16 October 2008 [ABSTRACT FROM AUTHOR]

Integrons are best known for assembling antibiotic resistance genes in clinical bacteria. They capture genes by using integrase mediated site-specific recombination of mobile gene cassettes. Integrons also occur in the chromosomes of many bacteria, notably β- and γ-Proteobacteria. In a survey of Xanthomonas, integrons were found in all 32 strains representing 12 pathovars of two species. Their chromosomal location was downstream from the acid dehydratase gene, ilvD, suggesting that an integron was present at this site in the ancestral xanthomonad. There was considerable sequence and structural diversity among the extant integrons. The majority of integrase genes were predicted to be inactivated by frameshifts, stop codons, or large deletions, suggesting that the associated gene cassettes can no longer be mobilized. In support, groups of strains with the same deletions or stop codons/frameshifts in their integrase gene usually contained identical arrays of gene cassettes. In general, strains within individual pathovars had identical cassettes, and these exhibited no similarity to cassettes detected in other pathovars. The variety and characteristics of contemporary gene cassettes suggests that the ancestral integron had access to a diverse pool of these mobile elements, and that their genes originated outside the Xanthomonas genome. Subsequent inactivation of the integrase gene in particular lineages has largely fixed the gene cassette arrays in particular pathovars during their differentiation and specialization into ecological niches. The acquisition of diverse gene cassettes by different lineages, within Xanthomonas has contributed to the species-genome diversity of the genus. The role of gene cassettes in survival on plant surfaces is currently unknown. [ABSTRACT FROM AUTHOR]

Horizontal gene transfer increases genetic diversity in prokaryotes to a degree not allowed by the limitations of reproduction by binary fission. The integron/gene cassette system is one of the most recently characterized examples of a system that facilitates horizontal gene transfer. This system, discovered in the context of multidrug resistance, is recognized in a clinical context for its role in allowing pathogens to adapt to the widespread use of antibiotics. Recent studies suggest that gene cassettes are common and encode functions relevant to many adaptive traits. To estimate the diversity of mobile cassettes in a natural environment, a molecular technique was developed to provide representative distributions of cassette populations at points within a sampling area. Subsequently, statistical methods analogous to those used for calculating species diversity were employed to assess the diversity of gene cassettes within the sample area in addition to gaining an estimate of cassette pool size. Results indicated that the number of cassettes within a 5 × 10-m sample area was large and that the overall mobile cassette metagenome was likely to be orders of magnitude larger again. Accordingly, gene cassettes appear to be capable of mobilizing a significant genetic resource and consequently have a substantial impact on bacterial adaptability. [ABSTRACT FROM AUTHOR]

Antibiotics at sub-inhibitory concentrations are often found in the environment. Here they could impose selective pressure on bacteria, leading to the selection and dissemination of antibiotic resistance, despite being under the inhibitory threshold. The goal of this study was to evaluate the effects of sub-inhibitory concentrations of gentamicin on environmental class 1 integron cassettes in natural river microbial communities. Gentamicin at sub-inhibitory concentrations promoted the integration and selection of gentamicin resistance genes (GmRG) in class 1 integrons after only a one-day exposure. Therefore, sub-inhibitory concentrations of gentamicin induced integron rearrangements, increasing the mobilization potential of gentamicin resistance genes and potentially increasing their dissemination in the environment. This study demonstrates the effects of antibiotics at sub-inhibitory concentrations in the environment and supports concerns about antibiotics as emerging pollutants. [ABSTRACT FROM AUTHOR]

Habitats with intermittent flooding, such as paddy soils, are crucial reservoirs in the global carbon pool; however, the effect of phage-host interactions on the biogeochemical cycling of carbon in paddy soils remains unclear. Hence, this study applied multiomics and global datasets integrated with validation experiments to investigate phage-host community interactions and the potential of phages to impact carbon sequestration in paddy soils. The results demonstrated that paddy soil phages harbor a diverse and abundant repertoire of auxiliary metabolic genes (AMGs) associated with carbon fixation, comprising 23.7% of the identified AMGs. The successful annotation of protein structures and promoters further suggested an elevated expression potential of these genes within their bacterial hosts. Moreover, environmental stressors, such as heavy metal contamination, cause genetic variation in paddy phages and up-regulate the expression of carbon fixation AMGs, as demonstrated by the significant enrichment of related metabolites (P < 0.05). Notably, the findings indicate that lysogenic phages infecting carbon-fixing hosts increased by 10.7% under heavy metal stress. In addition, in situ isotopic labeling experiments induced by mitomycin-C revealed that by increasing heavy metal concentrations, 13CO2 emissions from the treatment with added lysogenic phage decreased by approximately 17.9%. In contrast, 13C-labeled microbial biomass carbon content increased by an average of 35.4% compared to the control. These results suggest that paddy soil phages prominently influence the global carbon cycle, particularly under global change conditions. This research enhances our understanding of phage-host cooperation in driving carbon sequestration in paddy soils amid evolving environmental conditions. [ABSTRACT FROM AUTHOR]

Integrons are bacterial genetic elements that can capture mobile gene cassettes. They are mostly known for their role in the spread of antibiotic resistance cassettes, contributing significantly to the global resistance crisis. These resistance cassettes likely originated from sedentary chromosomal integrons, having subsequently been acquired and disseminated by mobilised integrons. However, their taxonomic and environmental origins are unknown. Here, we use cassette recombination sites (attCs) to predict the origins of those resistance cassettes now spread by mobile integrons. We modelled the structure and sequence homology of 1,978 chromosomal attCs from 11 different taxa. Using these models, we show that at least 27% of resistance cassettes have attCs that are structurally conserved among one of three taxa (Xanthomonadales, Spirochaetes and Vibrionales). Indeed, we found some resistance cassettes still residing in sedentary chromosomal integrons of the predicted taxa. Further, we show that attCs cluster according to host environment rather than host phylogeny, allowing us to assign their likely environmental sources. For example, the majority of β-lactamases and aminoglycoside acetyltransferases, the two most prevalent resistance cassettes, appear to have originated from marine environments. Together, our data represent the first evidence of the taxonomic and environmental origins of resistance cassettes spread by mobile integrons. Ghaly et al. use cassette recombination sites to predict the origins of antibiotic resistance cassettes spread by mobile integrons. They find that more than a quarter of all resistance cassettes have recombination sites that are structurally conserved among one of three taxa (Xanthomonadales, Spirochaetes and Vibrionales). [ABSTRACT FROM AUTHOR]

• Antibiotic pollution can increase rates of mutation and lateral transfer events and continue to exert selection pressure even at sub-inhibitory concentrations. • We conducted a literature survey on environmental concentrations of antibiotics. We collated 887 data points from 40 peer-reviewed papers. • In order to determine whether these concentrations were environmentally relevant we compared them to their minimum selective concentrations (1/4-1/230 of MIC) • We found that environmental concentrations of antibiotics often fall into this range and are likely to be influencing microbial ecology, and to be driving the selection of antibiotic resistant bacteria. Antibiotics are poorly metabolized, and can enter the environment via human waste streams, agricultural run-off and pharmaceutical effluent. We consequently expect to see a concentration gradient of antibiotic compounds radiating from areas of human population. Such antibiotics should be thought of as pollutants, as they can accumulate, and have biological effects. These antibiotic pollutants can increase rates of mutation and lateral transfer events, and continue to exert selection pressure even at sub-inhibitory concentrations. Here, we conducted a literature survey on environmental concentrations of antibiotics. We collated 887 data points from 40 peer-reviewed papers. We then determined whether these concentrations were biologically relevant by comparing them to their minimum selective concentrations, usually defined as between 1/4 and 1/230 of the minimum inhibitory concentration. Environmental concentrations of antibiotics surveyed often fall into this range. In general, the antibiotic concentrations recorded in aquatic and sediment samples were similar. These findings indicate that environmental concentrations of antibiotics are likely to be influencing microbial ecology, and to be driving the selection of antibiotic resistant bacteria. Image, graphical abstract [ABSTRACT FROM AUTHOR]

Societal Impact Statement: Plants and bacteria interact in complex ways that are crucial to the health and productivity of native vegetation and croplands. While the range of characterised plant‐beneficial bacterial traits continues to grow, key questions remain regarding the distribution and mobility of genes associated with these traits. This work explores the diversity of mobile genetic elements carried by bacteria associated with plant root surfaces, assessing their capacity to help shape plant–bacterial interactions. The significance of this work lies in the potential to contribute to new strategies for enhancing plant health, promoting sustainable agriculture and managing plant diseases in an era when we must respond to environmental change. Summary: Integrons are gene capture and expression systems that contribute to bacterial adaptation. Integron research has mainly focused on the role that these elements play in spreading antimicrobial resistance. However, their contribution to niche adaptation is potentially much broader because integrons can sample the vast repertoire of diverse functions encoded by integron gene cassettes. Integrons and gene cassettes have been identified in many bacterial lineages residing in soil and water across varied ecosystems, but there has been little investigation of integrons in plant‐associated bacteria.Bacteria and plants have complex, dynamic relationships that influence plant health and productivity. To investigate whether integrons contribute to adaptative processes in plant microbiomes, we examined gene cassette and microbial taxonomic profiles in rhizoplanes of four important crop species grown under controlled glasshouse conditions. We identified 38,546 unique gene cassettes, including elements carrying genes associated with antibiotic resistance, type II toxin–antitoxin systems and genes with putative functions associated with plant growth promotion, along with a larger set encoding genes of unknown functions.Rhizoplane microbiomes of different plant species showed more similarity in their community composition profiles than in their gene cassette profiles, with complex and distinct suites of gene cassettes associated with each plant species, suggesting that gene cassettes might have a role in specific plant–bacterial interactions.We show that rhizoplane microbiomes carry diverse integron gene cassettes that could play a role in establishing and maintaining rhizoplane communities. [ABSTRACT FROM AUTHOR]

Understanding that evolution progresses through generation of DNA variants followed by selection is a key learning outcome for biology students. We designed an integrated and innovative undergraduate laboratory exercise using Saccharomyces cerevisiae to demonstrate these principles. Students perform in vitro experimental evolution by repeatedly propagating large or small yeast colonies on a weekly basis. Small-colony variants known as petites arise by mutations that disrupt aerobic respiration. To demonstrate the effects of increased mutation rates, half of the selection lines are exposed to ultraviolet irradiation. To understand how the petite phenotype arises, polymerase chain reaction (PCR) is performed to examine mitochondrial DNA, while biochemical assays are used to assess the ability of petites to undergo aerobic respiration. This exercise demonstrates evolution by artificial selection over a suitably short timeframe and links the results to a critical biochemical process: the role of mitochondria in aerobic respiration and ATP production. By implementing these experiments, we successfully demonstrated that the frequencies of petite mutants in evolved populations varied according to the selection pressure we applied, and that petite mutants carried deletions in mitochondrial DNA as anticipated. Through an integrated learning context, this practical exercise promotes fundamental understanding of evolutionary processes and fosters critical thinking skills. [ABSTRACT FROM AUTHOR]

Objectives Our study aimed to sequence class 1 integrons in uncultured environmental bacterial cells in freshwater from suburban creeks and uncover the taxonomy of their bacterial hosts. We also aimed to characterize integron gene cassettes with altered DNA sequences relative to those from databases or literature and identify key signatures of their molecular evolution. Methods We applied a single-cell fusion PCR-based technique— e mulsion, p aired i solation and c oncatenation PCR (epicPCR)—to link class 1 integron gene cassette arrays to the phylogenetic markers of their bacterial hosts. The levels of streptomycin resistance conferred by the WT and altered aadA5 and aadA11 gene cassettes that encode aminoglycoside (3″) adenylyltransferases were experimentally quantified in an Escherichia coli host. Results Class 1 integron gene cassette arrays were detected in Alphaproteobacteria and Gammaproteobacteria hosts. A subset of three gene cassettes displayed signatures of molecular evolution, namely the gain of a regulatory 5′-untranslated region (5′-UTR), the loss of attC recombination sites between adjacent gene cassettes, and the invasion of a 5′-UTR by an IS element. Notably, our experimental testing of a novel variant of the aadA11 gene cassette demonstrated that gaining the observed 5′-UTR contributed to a 3-fold increase in the MIC of streptomycin relative to the ancestral reference gene cassette in E. coli. Conclusions Dissecting the observed signatures of molecular evolution of class 1 integrons allowed us to explain their effects on antibiotic resistance phenotypes, while identifying their bacterial hosts enabled us to make better inferences on the likely origins of novel gene cassettes and IS that invade known gene cassettes. [ABSTRACT FROM AUTHOR]

Background:Quantifying genetic diversity and metapopulation structure provides insights into the evolutionary history of a species and helps develop appropriate management strategies. We provide the first assessment of genetic structure in spinner sharks (Carcharhinus brevipinna), a large cosmopolitan carcharhinid, sampled from eastern and northern Australia and South Africa. Methods and Findings:Sequencing of the mitochondrial DNA NADH dehydrogenase subunit 4 gene for 430 individuals revealed 37 haplotypes and moderately high haplotype diversity (h = 0.6770 ±0.025). While two metrics of genetic divergence (ΦST and FST) revealed somewhat different results, subdivision was detected between South Africa and all Australian locations (pairwise ΦST, range 0.02717–0.03508, p values ≤ 0.0013; pairwise FST South Africa vs New South Wales = 0.04056, p = 0.0008). Evidence for fine-scale genetic structuring was also detected along Australia’s east coast (pairwise ΦST = 0.01328, p < 0.015), and between south-eastern and northern locations (pairwise ΦST = 0.00669, p < 0.04). Conclusions:The Indian Ocean represents a robust barrier to contemporary gene flow in C. brevipinna between Australia and South Africa. Gene flow also appears restricted along a continuous continental margin in this species, with data tentatively suggesting the delineation of two management units within Australian waters. Further sampling, however, is required for a more robust evaluation of the latter finding. Evidence indicates that all sampled populations were shaped by a substantial demographic expansion event, with the resultant high genetic diversity being cause for optimism when considering conservation of this commercially-targeted species in the southern Indo-Pacific. [ABSTRACT FROM AUTHOR]

The article presents a study which reported the discovery that integrons are present in 75 archaeal metagenome-assembled genomes from nine phyla and are particularly enriched among Asgard archaea . Topics discussed include genetic structure of archaea integrons, platforms for cross-domain gene transfer, and functional diversity of gene cassettes. An analysis of integron integrases (Intls), inserting cassette (attC) sites, and cassette-encoded proteins is also included.

[Display omitted] • Livestock restriction successfully reduces riverine antibiotic resistance at a watershed scale. • Animal waste reduction is key to decreasing ARG abundance in river. • Long-term surveillance is crucial for environmental monitoring. • The necessity of an integrated framework to effectively mitigate ARG spread. Natural environments play a crucial role in transmission of antimicrobial resistance (AMR). Development of methods to manage antibiotic resistance genes (ARGs) in natural environments are usually limited to the laboratory or field scale, partially due to the complex dynamics of transmission between different environmental compartments. Here, we conducted a nine-year longitudinal profiling of ARGs at a watershed scale, and provide evidence that restrictions on livestock farms near water bodies significantly reduced riverine ARG abundance. Substantial reductions were revealed in the relative abundance of genes conferring resistance to aminoglycosides (42%), MLSB (36%), multidrug (55%), tetracyclines (53%), and other gene categories (59%). Additionally, improvements in water quality were observed, with distinct changes in concentrations of dissolved reactive phosphorus, ammonium, nitrite, pH, and dissolved oxygen. Antibiotic residues and other pharmaceuticals and personal care products (PPCPs) maintain at a similarly low level. Microbial source tracking demonstrates a significant decrease in swine fecal indicators, while human fecal pollution remains unchanged. These results suggest that the reduction in ARGs was due to a substantial reduction in input of antibiotic resistant bacteria and genes from animal excreta. Our findings highlight the watershed as a living laboratory for understanding the dynamics of AMR, and for evaluating the efficacy of environmental regulations, with implications for reducing environmental risks associated with AMR on a global scale. [ABSTRACT FROM AUTHOR]

In the waters of southeast Australia, two species of sawshark—the common (Pristiophorus cirratus) and southern (Pristiophorus nudipinnis) sawshark—are frequent by-catch in commercial fisheries. While harvesting of both species is currently considered sustainable, there has been no investigation of whether P. cirratus and P. nudipinnis display genetically distinct populations throughout their ranges. Such information is necessary for effective management of these species in commercial fisheries. This study examined population structure in both sawshark species through analysis of two mitochondrial genes: cytochrome b (Cyt-b) and NADH dehydrogenase subunit 5 (ND5). Results indicated contrasting levels of population structure, with P. cirratus consisting of two, possibly three, genetically distinct populations with two mitochondrial lineages and P. nudipinnis consisting of a single population. Tests for population expansion also highlighted differences between the two species. Population expansion was detected for the entire P. nudipinnis population, whereas this was only the case for one mitochondrial lineage in P. cirratus. The entire P. cirratus population displayed signals of demographic stability. It is hypothesised that the opening and closing of Bass Strait during glacial-interglacial cycles played a major role in shaping the population structure and expansion signatures observed in this study. Mitochondrial data also suggest that patterned and uniform brown P. cirratus are the same species. Fisheries managers should consider adopting two management units in southern Australia—one along the east coast (for the eastern P. cirratus population) and one along the south coast (for the southern P. cirratus population and the single P. nudipinnis population). [ABSTRACT FROM AUTHOR]

Integrons are bacterial genetic elements that can capture, rearrange, and express mobile gene cassettes. They are best known for their role in disseminating antibiotic-resistance genes among pathogens. Their ability to rapidly spread resistance phenotypes makes it important to consider what other integron-mediated traits might impact human health in the future, such as increased virulence, pathogenicity, or resistance to novel antimicrobial strategies. Exploring the functional diversity of cassettes and understanding their de novo creation will allow better pre-emptive management of bacterial growth, while also facilitating development of technologies that could harness integron activity. If we can control integrons and cassette formation, we could use integrons as a platform for enzyme discovery and to construct novel biochemical pathways, with applications in bioremediation or biosynthesis of industrial and therapeutic molecules. Integron activity thus holds both peril and promise for humans. Integrons are DNA elements that helped drive the global antibiotic-resistance crisis. They capture and express mobile genes known as cassettes. Their activity holds both peril and promise for humans. The peril is the next wave of integron-mediated traits: those beyond resistance. What other cassette-encoded traits might impact human health? Cassettes encoding virulence and novel resistance mechanisms can be recovered from environmental samples around the globe. Such cassettes should be accessible to integron-carrying pathogens anywhere on the planet. The promise of integrons lies in their potential use in biotechnology. Harnessing their ability for genetic rearrangement, and their diverse gene cassettes, could facilitate enzyme discovery, and the construction of novel biochemical pathways. [ABSTRACT FROM AUTHOR]

The ability of fungi to weather a wide range of minerals influences plant nutrition and enhances global biogeochemical cycles of life-essential elements. The fungus-mineral interface plays a key role in weathering, but the specific mechanisms underlying these processes remain poorly understood. Here, we examined fungal-mineral weathering using hematite and Trichoderma guizhouense. We showed that hematite dissolution increased over cultivation time, with the formation of secondary minerals up to ∼3000 µm−2 at the interfaces after 66 h cultivation. Of the hematite associated with hyphae, approximately 15% was converted to the secondary mineral ferrihydrite. Importantly, superoxide radicals were detected at the hyphal tips and along the whole hyphae. During cultivation, a high concentration (∼1000 nM) of hydroxyl radical was also detected. Synchrotron radiation based spectromicroscopies at fungus-mineral interfaces suggest that fungus hyphae alter the local redox state of iron and thus are redox-active. These findings indicate that fungus-initiated catalytic reactions occur at hyphal-mineral interfaces, in view of the fact that superoxide does not diffuse far from the site of formation. Furthermore, these results also suggest that the catalytic reactions may serve as a new strategy for microbial iron uptake. Together, these findings constitute a significant step forward in understanding the ways that fungi make minerals available to biological systems. [ABSTRACT FROM AUTHOR]

Wastewater treatment plants (WWTPs) contain diverse antibiotic resistance genes (ARGs), and thus are considered as a major pathway for the dissemination of these genes into the environments. However, comprehensive evaluations of ARGs dynamic during wastewater treatment process lack extensive investigations on a broad spectrum of ARGs. Here, we investigated the dynamics of ARGs and bacterial community structures in 114 samples from eleven Chinese WWTPs using high-throughput quantitative PCR and 16S rRNA-based Illumina sequencing analysis. Significant shift of ARGs profiles was observed and wastewater treatment process could significantly reduce the abundance and diversity of ARGs, with the removal of ARGs concentration by 1–2 orders of magnitude. Whereas, a considerable number of ARGs were detected and enriched in effluents compared with influents. In particular, seven ARGs mainly conferring resistance to beta-lactams and aminoglycosides and three mobile genetic elements persisted in all WWTPs samples after wastewater treatment. ARGs profiles varied with wastewater treatment processes, seasons and regions. This study tracked the footprint of ARGs during wastewater treatment process, which would support the assessment on the spread of ARGs from WWTPs and provide data for identifying management options to improve ARG mitigation in WWTPs. [ABSTRACT FROM AUTHOR]

The integron platform allows the acquisition, expression, and dissemination of antibiotic resistance genes within gene cassettes. Wastewater treatment plants (WWTPs) contain abundant resistance genes; however, knowledge about the impacts of wastewater treatment on integrons and their gene cassettes is limited. In this study, by using clone library analysis and high-throughput sequencing, we investigated the abundance of class 1, 2, and 3 integrons and their corresponding gene cassettes in three urban WWTPs. Our results showed that class 1 integrons were most abundant in WWTPs and that wastewater treatment significantly reduced the abundance of all integrons. The WWTP influents harbored the highest diversity of class 1 integron gene cassettes, whereas class 3 integron gene cassettes exhibited highest diversity in activated sludge. Most of the gene cassette arrays detected in class 1 integrons were novel. Aminoglycoside, beta-lactam, and trimethoprim resistance genes were highly prevalent in class 1 integron gene cassettes, while class 3 integrons mainly carried beta-lactam resistance gene cassettes. A core class 1 integron resistance gene cassette pool persisted during wastewater treatment, implying that these resistance genes could have high potential to spread into environments through WWTPs. These data provide new insights into the impact of wastewater treatment on integron pools and highlight the need for surveillance of resistance genes within both class 1 and 3 integrons. IMPORTANCE Wastewater treatment plants represent a significant sink and transport medium for antibiotic resistance bacteria and genes spreading into environments. Integrons are important genetic elements involved in the evolution of antibiotic resistance. To better understand the impact of wastewater treatment on integrons and their gene cassette contexts, we conducted clone library construction and high-throughput sequencing to analyze gene cassette contexts for class 1 and class 3 integrons during the wastewater treatment process. This study comprehensively profiled the distribution of integrons and their gene cassettes (especially class 3 integrons) in influents, activated sludge, and effluents of conventional municipal wastewater treatment plants. We further demonstrated that while wastewater treatment significantly reduced the abundance of integrons and the diversity of associated gene cassettes, a large fraction of integrons persisted in wastewater effluents and were consequentially discharged into downstream natural environments. [ABSTRACT FROM AUTHOR]

The presence of integrons was assessed in gut bacteria isolated from wild-caught prawns. A pseudomonad was recovered that contained a Tn402-like class 1 integron with a complete transposition module and two gene cassettes. One cassette was identical to a previously described cassette from a chromosomal class 3 integron in Delftia tsuruhatensis. [ABSTRACT FROM AUTHOR]

• The abundance of intI1 increased 12-fold under STM stress. • The abundance of intI1 increased 29-fold under OTC stress. • Aminoglycoside resistance genes were enriched under increased STM stress. • Synteny between tet genes and intI1 led to multidrug resistance under OTC stress. Class 1 integrons are strongly associated with the dissemination of antibiotic resistance in bacteria. However, little is known about whether the presence of antibiotics affects the abundance of integrons and antibiotic resistance genes during biological wastewater treatment. To explore the roles of class 1 integrons in spreading antibiotic resistance genes in environmental compartments, the dynamics of integrons were followed in biofilm reactors treating synthetic wastewater respectively spiked with streptomycin (STM) and oxytetracycline (OTC). The relative abundance of the integron-integrase gene (intI1) increased 12 or 29-fold respectively when treated with STM or OTC, under incrementally increasing dosage regimes from 0 to 50 mg L−1. Significant increases in intI1 abundance initially occurred at an antibiotic dose of 0.1 mg L−1. At the beginning of the experiment, 51% to 64% of integrons carried no gene cassettes. In STM and OTC spiked systems, there was a significant increase in the proportion of integrons that contained resistance gene cassettes, particularly at intermediate and higher antibiotic concentrations. Gene cassettes encoding resistance to aminoglycosides, trimethoprim, beta-lactam, erythromycin, and quaternary ammonium compounds were all detected in the treated systems. Three tetracycline resistance genes (tetA, tetC, tetG) were significantly correlated with the abundance of intI1 (p < 0.01), despite no tet resistance being present as a gene cassette. Genome sequencing of isolates showed synteny between the tet resistance genes and intI1 , mediated through linkage to transposable elements including Tn 3 , IS 26 and IS CR3. Class 1 integrons appeared to be under positive selection in the presence of antibiotics, and might have actively acquired new gene cassettes during the experiment. [ABSTRACT FROM AUTHOR]

Objectives: This study aimed to identify novel fosfomycin resistance genes across diverse environmental samples, ranging in levels of anthropogenic pollution. We focused on fosfomycin resistance, and given its increasing clinical importance, explored the prevalence of these genes within different environmental contexts., Methods: Metagenomic DNA was extracted from wastewater and sediment samples collected from sites in India, Sweden, and Antarctica. Class 1 integron gene cassette libraries were prepared, and resistant clones were selected on fosfomycin-supplemented media. Long-read sequencing was performed followed by bioinformatics analysis to identify novel fosfomycin resistance genes. The genes were cloned and functionally characterized in E. coli, and the impact of phosphonoformate on the enzymes was assessed., Results: Four novel fosfomycin resistance genes were identified. Phylogenetic analysis placed these genes within the FosX family, a group of metalloenzymes that hydrolyse fosfomycin without thiol conjugation. The genes were subsequently renamed fosE2, fosI2, fosI3, and fosP. Functional assays confirmed that these genes conferred resistance to fosfomycin in E. coli, with MIC ranging from 32 μg/ml to 256 μg/ml. Unlike FosA/B enzymes, these FosX-like proteins were resistant to phosphonoformate inhibitory action. A fosI3 homolog was identified in Pseudomonas aeruginosa, highlighting potential clinical relevance., Conclusions: This study expands the understanding of fosfomycin resistance by identifying new FosX family members across diverse environments. The lack of phosphonoformate inhibition underscores the clinical importance of these poorly studied enzymes, which warrant further investigation, particularly in pathogenic contexts., Competing Interests: Declaration of competing interest The authors do not have any conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Giant clams ( Tridacna and Hippopus ) are large marine bivalves occupying tropical and subtropical reefs in the Indo-Pacific. Giant clam populations have declined in many areas of the Indo-Pacific and continue to be threatened by harvesting and environmental change. The small giant clam ( Tridacna maxima ) occurs throughout the Indo-Pacific and has been subject to several phylogeographic studies across its range. However, given its broad range, there are several areas where the genetic diversity and connectivity of T. maxima populations has not been characterised. Here, we analyse the mitochondrial marker cytochrome oxidase 1 (CO1) to examine the genetic diversity and connectivity of T. maxima in two regions: Australia's Coral Sea Marine Park and the Cook Islands. Samples were collected from 13 reefs within the Coral Sea Marine Park and ten islands within the Cook Islands archipelago. Tridacna maxima across the sampled region of the Coral Sea did not display any population structure, whereas significant population structure was detected for T. maxima within the Cook Islands. For the Cook Islands, most pairwise comparisons involving an island in the northern group (Manihiki) were significant, as were comparisons for Palmerston (a more centrally located island) and the southern islands, Rarotonga and Mangaia. Both regions displayed high haplotype diversities (> 0.90), indicating that they are important repositories of genetic diversity. Additional CO1 data from throughout T. maxima 's distribution showed that the Coral Sea clams belonged to the clade occurring in the South-Western Pacific Ocean, whilst those from the Cook Islands belonged to a unique clade found in the Central Pacific Ocean. This clade extended from Fiji in the west to French Polynesia in the east and the atolls of Palmyra and Tarawa (Kiribati) in the north. Our assessment of genetic diversity and population structure in these regions will assist with management decisions for the species., Competing Interests: The authors declare no conflicts of interest., (© 2024 The Author(s). Ecology and Evolution published by John Wiley & Sons Ltd.)

Xylem serves as a conduit linking soil to the aboveground plant parts and facilitating the upward movement of microbes into leaves and fruits. Despite this potential, the composition of the xylem microbiome and its associated risks, including antibiotic resistance, are understudied. Here, we cultivated tomatoes and analyzed their xylem sap to assess the microbiome and antibiotic resistance profiles following treatment with sewage sludge. Our findings show that xylem microbes primarily originate from soil, albeit with reduced diversity in comparison to those of their soil microbiomes. Using single-cell Raman spectroscopy coupled with D 2 O labeling, we detected significantly higher metabolic activity in xylem microbes than in rhizosphere soil, with 87% of xylem microbes active compared to just 36% in the soil. Additionally, xylem was pinpointed as a reservoir for antibiotic resistance genes (ARGs), with their abundance being 2.4-6.9 times higher than in rhizosphere soil. Sludge addition dramatically increased the abundance of ARGs in xylem and also increased their mobility and host pathogenicity. Xylem represents a distinct ecological niche for microbes and is a significant reservoir for ARGs. These results could be used to manage the resistome in crops and improve food safety.

Plant microbiomes play important roles in plant health and fitness. Bacterial horizontal gene transfer (HGT) can influence plant health outcomes, driving the spread of both plant growth-promoting and phytopathogenic traits. However, community dynamics, including the range of genetic elements and bacteria involved in this process are still poorly understood. Integrons are genetic elements recently shown to be abundant in plant microbiomes, and are associated with HGT across broad phylogenetic boundaries. They facilitate the spread of gene cassettes, small mobile elements that collectively confer a diverse suite of adaptive functions. Here, we analysed 5,565 plant-associated bacterial genomes to investigate the prevalence and functional diversity of integrons in this niche. We found that integrons are particularly abundant in the genomes of Pseudomonadales, Burkholderiales, and Xanthomonadales. In total, we detected nearly 9,000 gene cassettes, and found that many could be involved in plant growth promotion or phytopathogenicity, suggesting that integrons might play a role in bacterial mutualistic or pathogenic lifestyles. The rhizosphere was enriched in cassettes involved in the transport and metabolism of diverse substrates, suggesting that they may aid in adaptation to this environment, which is rich in root exudates. We also found that integrons facilitate cross-species HGT, which is particularly enhanced in the phyllosphere. This finding may provide an ideal opportunity to promote plant growth by fostering the spread of genes cassettes relevant to leaf health. Together, our findings suggest that integrons are important elements in plant microbiomes that drive HGT, and have the potential to facilitate plant host adaptation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Ghaly, Gillings, Rajabal, Paulsen and Tetu.)

Integrons are genetic platforms that capture, rearrange and express mobile modules called gene cassettes. The best characterized gene cassettes encode antibiotic resistance, but the function of most integron gene cassettes remains unknown. Functional predictions suggest that many gene cassettes could encode proteins that facilitate interactions with other cells and with the extracellular environment. Because cell interactions are essential for biofilm stability, we sequenced gene cassettes from biofilms growing on the surface of the marine macroalgae Ulva australis and Sargassum linearifolium . Algal samples were obtained from coastal rock platforms around Sydney, Australia, using seawater as a control. We demonstrated that integrons in microbial biofilms did not sample genes randomly from the surrounding seawater, but harboured specific functions that potentially provided an adaptive advantage to both the bacterial cells in biofilm communities and their macroalgal host. Further, integron gene cassettes had a well-defined spatial distribution, suggesting that each bacterial biofilm acquired these genetic elements via sampling from a large but localized pool of gene cassettes. These findings suggest two forms of filtering: a selective acquisition of different integron-containing bacterial species into the distinct biofilms on Ulva and Sargassum surfaces, and a selective retention of unique populations of gene cassettes at each sampling location.

Antimicrobial resistance is a major threat for public health. Plasmids play a critical role in the spread of antimicrobial resistance via horizontal gene transfer between bacterial species. However, it remains unclear how plasmids originally recruit and assemble various antibiotic resistance genes (ARGs). Here, we track ARG recruitment and assembly in clinically relevant plasmids by combining a systematic analysis of 2420 complete plasmid genomes and experimental validation. Results showed that ARG transfer across plasmids is prevalent, and 87% ARGs were observed to potentially transfer among various plasmids among 8229 plasmid-borne ARGs. Interestingly, recruitment and assembly of ARGs occur mostly among compatible plasmids within the same bacterial cell, with over 88% of ARG transfers occurring between compatible plasmids. Integron and insertion sequences drive the ongoing ARG acquisition by plasmids, especially in which IS26 facilitates 63.1% of ARG transfer events among plasmids. In vitro experiment validated the important role of IS26 involved in transferring gentamicin resistance gene aacC1 between compatible plasmids. Network analysis showed four beta-lactam genes (blaTEM-1, blaNDM-4, blaKPC-2, and blaSHV-1) shuffling among 1029 plasmids and 45 clinical pathogens, suggesting that clinically alarming ARGs transferred accelerate the propagation of antibiotic resistance in clinical pathogens. ARGs in plasmids are also able to transmit across clinical and environmental boundaries, in terms of the high-sequence similarities of plasmid-borne ARGs between clinical and environmental plasmids. This study demonstrated that inter-plasmid ARG transfer is a universal mechanism for plasmid to recruit various ARGs, thus advancing our understanding of the emergence of multidrug-resistant plasmids., (© The Author(s) 2024. Published by Oxford University Press on behalf of the International Society for Microbial Ecology.)

Plastic offers a new niche for microorganisms, the plastisphere. The ever-increasing emission of plastic waste makes it critical to understand the microbial ecology of the plastisphere and associated effects. Here, we present a global fingerprint of the plastisphere, analyzing samples collected from freshwater, seawater, and terrestrial ecosystems. The plastisphere assembles a distinct microbial community that has a clearly higher heterogeneity and a more deterministically dominated assembly compared to natural habitats. New coexistence patterns-loose and fragile networks with mostly specialist linkages among microorganisms that are rarely found in natural habitats-are seen in the plastisphere. Plastisphere microbiomes generally have a great potential to metabolize organic compounds, which could accelerate carbon turnover. Microorganisms involved in the nitrogen cycle are also altered in the plastisphere, especially in freshwater plastispheres, where a high abundance of denitrifiers may increase the release of nitrite (aquatic toxicant) and nitrous oxide (greenhouse gas). Enrichment of animal, plant, and human pathogens means that the plastisphere could become an increasingly mobile reservoir of harmful microorganisms. Our findings highlight that if the trajectory of plastic emissions is not reversed, the expanding plastisphere could pose critical planetary health challenges., Competing Interests: The authors declare no competing interests., (© 2023 The Author(s).)

Integrons are genetic elements, found among diverse bacteria and archaea, that capture and rearrange gene cassettes to rapidly generate genetic diversity and drive adaptation. Despite their broad taxonomic and geographic prevalence, and their role in microbial adaptation, the functions of gene cassettes remain poorly characterized. Here, using a combination of bioinformatic and experimental analyses, we examined the functional diversity of gene cassettes from different environments. We find that cassettes encode diverse antimicrobial resistance (AMR) determinants, including those conferring resistance to antibiotics currently in the developmental pipeline. Further, we find a subset of cassette functions is universally enriched relative to their broader metagenomes. These are largely involved in (a)biotic interactions, including AMR, phage defense, virulence, biodegradation, and stress tolerance. The remainder of functions are sample-specific, suggesting that they confer localised functions relevant to their microenvironment. Together, they comprise functional profiles different from bulk metagenomes, representing niche-adaptive components of the prokaryotic pangenome., Competing Interests: The authors declare that they have no competing interests., (© 2023 The Authors.)

Surveillance of antimicrobial resistance is essential for an effective One Health response. This study explores the efficacy of European honey bees ( Apis mellifera ) for biomonitoring antimicrobial resistance (AMR) in urban areas. Class 1 integrons ( intI1 ) are investigated as a universal AMR indicator, as well as associated cassette arrays and trace element contaminants at a city-wide scale. Class 1 integrons were found to be pervasive across the urban environment, occurring in 52% (75/144) of the honey bees assessed. The area of waterbodies within the honey bee's foraging radius was associated with intI1 prevalence, indicating an exposure pathway for future investigation to address. Trace element concentrations in honey bees reflected urban sources, supporting the application of this biomonitoring approach. As the first study of intI1 in honey bees, we provide insights into the environmental transfer of bacterial DNA to a keystone species and demonstrate how intI1 biomonitoring can support the surveillance of AMR.