Walsh, F. | bioRxiv Science

bioRxiv2026

Microbial community diversity predicts invasion resistance of freshwater biofilms against antibiotic-resistant bacteria

Rivers receive continuous inputs of antibiotic-resistant bacteria (ARB) from wastewater, agriculture, and other anthropogenic sources, yet it remains unclear whether the recipient ecological component and its microbial communities determine whether introduced ARB establish or disappear. Ecological invasion theory predicts that invasion success depends on biodiversity, community stability, and occupation of ecological niche space, but these mechanisms have rarely been evaluated together in natural microbial communities. Here, we challenged river biofilms collected from 20 sites in 12 European rivers across six countries with a model antibiotic-resistant Escherichia coli carrying a conjugative IncP-1 plasmid The invasion assays were carried out under standardized laboratory flume conditions. River biofilms differed markedly in their permissiveness to invasion despite identical invasion conditions. Higher bacterial diversity consistently accelerated invader loss rates, whereas communities containing more abundant and diverse close phylogenetic neighbours of the invader exhibited stronger exclusion during early biofilm establishment. Diversity loss during transition into the experimental system emerged as the strongest explanatory variable of invasion resistance prior to biofilm maturation, whereas Shannon diversity became the dominant predictor in mature communities. Integrating these complementary ecological dimensions substantially improved explanatory prediction of ARB persistence compared with individual predictors alone. Particularly invasion-resistant biofilms also exhibited distinct ecological community composition consistent with mature, structurally complex microbial assemblages. Together, our findings demonstrate that the establishment of ARB in the environment is not stochastic but can be predicted from measurable ecological properties of recipient microbiomes, highlighting microbial biodiversity and community organization as natural barriers to antimicrobial resistance dissemination.

microbiology↗

bioRxiv2026

A Novel Plasmid-Encoded Mobile Colistin Resistance Gene mcr-13.1 Detected in Escherichia coli Isolated from Grassland.

Plasmid-encoded mobile colistin resistance (mcr) genes have raised concern due to dissemination potential. While mcr variants are reported across One Health compartments, they remain unreported in grass. This study characterises a novel mcr variant (mcr-13.1), detected in Escherichia coli isolated from the grass phylosphere within an agricultural grassland. The two mcr-positive isolates were clonal copies isolated at timepoints eight weeks apart. They belonged to the serotype O17:H18 and were of the sequence type ST394. The E. coli were phenotypically susceptible to {beta}-lactams, aminoglycosides, quinolones, sulphonamides, phenicols, tetracyclines, diaminopyrimidine and colistin (Minimum Inhibitory Concentration (MIC) = 0.5 {micro}g/mL). The mcr-13.1 gene was encoded on an IncFIB plasmid. This plasmid was transferable by conjugation but the colistin MIC of the E. coli J53 transconjugant did not change (0.5 {micro}g/mL). Further, cloned pUC19::mcr-13.1 did not alter the colistin MIC for E. coli DH5 (0.25 {micro}g/mL). The translated amino acid sequence showed highest homology (82 %) to MCR-10.2 and MCR-10.4. Our findings identify grass as a previously unrecognised reservoir for E. coli carrying mobile mcr genes, reports the identification of the novel mcr-13.1 variant from this niche and demonstrates the importance of genomic screening in identifying mcr genes that would otherwise remain undetected.

microbiology↗

bioRxiv2024

Genomic analysis of antimicrobial resistant Escherichia coli isolated from manure and manured agricultural grasslands

Antimicrobial resistance (AMR) is a multifactorial issue involving an intertwining relationship between animals, humans and the environment. The environment can harbour bacteria that are pathogenic to human health, including Escherichia coli, an indicator of environmental faecal contamination. Through culture dependent approaches this study identified 46 E. coli isolates in porcine and bovine manure, non-manured and manured soil, and the phyllosphere of manured grass. The grass isolation highlights grass as an environmental reservoir for E. coli. Whole genome sequencing identified 11 different multi-locus sequence types. We also identified a diverse plasmidome with 23 different plasmid replicon types. The E. coli isolates were phenotypically antibiotic resistance, predominantly multidrug resistant. Additionally, whole genome sequencing identified 31 antibiotic resistance genes, and mutations in the gyrA, parC, and parE genes, conferring fluoroquinolone resistance. The main virulence genes were associated actin mediated locomotion (icsP/sopA), siderophore production and alginate production (algA), which suggest adaptation to survive in the non-gut environment or the UV environment of grass surfaces. These results suggest that E. coli in soils and grasses may adapt to their new environments evolving novel strategies. This study demonstrates grass as an understudied environmental niche of AMR E. coli, which directly links the environment to the grass grazing animal and vice-versa via the circular economy of manure application. Impact statementEscherichia coli is capable of surviving across biomes within One Health. This study sheds light on the genomic elements present in AMR E. coli in the understudied niche of agricultural grassland. Data summaryThe genome sequences have been deposited in Genbank. Bioproject number PRJNA1080214 and SRP491607 in the sequence read archive https://www.ncbi.nlm.nih.gov/sra/?term=SRP491607.

microbiology↗

bioRxiv2024

Plasmids from a complex biome exist as communities

Plasmids play a crucial role in the spread of antimicrobial resistance genes (ARGs) across One Health due to their ability to transfer a wide range of ARGs within and across bacterial species and biomes. We sequenced 173 circularised plasmids transferred from wastewater treatment plant (WWTP) effluent into Escherichia coli and subsequently characterised their genetic content. Multiple multidrug resistant plasmids were identified with a significant number of mega plasmids (>100Kb). Plasmids existing in isolation were rare and almost all existed with other plasmids. Our results suggest that positive epistasis promotes plasmid persistence in WWTP populations in a similar manner to that identified in vitro via infectious transmission, varying properties against plasmid community backgrounds, interactions with a range of other plasmids, source-sink spill-over transmission within the plasmid community rather than the host bacteria and compensatory mutations. We have demonstrated that the plasmid paradox solutions apply to plasmid communities in addition to plasmid host interactions. Our study identified that rather than existing as lone entities plasmids co-exist in small packs, the protection is afforded to the pack not by all members but by one or two and many plasmids coast within this pack as they contain no obvious advantage to the host. Our findings show that we need to enter a new paradigm and study plasmids in packs rather than as single entities in order to understand their transmission across One Health.

microbiology↗

bioRxiv2023

Improved workflow for untargeted metabolomics and NMR analysis of intracellular and extracellular metabolites isolated from Gram positive and Gram negative bacteria

Metabolomics and Nuclear Magnetic Resonance (NMR) spectroscopy have proven to be useful for characterising key metabolome fluctuations in bacteria during stress responses to various environmental agents and antibiotics. However, a number of impediments to current workflows have led to the reduced use of these techniques in microbial research. In this study, we address these limitations and in response have developed a novel 1H NMR-based untargeted metabolomics workflow. This method is suitable for use with various bacterial species, reducing the workload in comparison to previously established workflows. Our protocol is simple and reproducible and allows for the isolation of both intracellular (IC) and extracellular (EC) metabolites simultaneously from both Gram (+) and Gram (-) species. This method has been shown to produce consistent results for the ESKAPE pathogens Escherichia coli, Klebsiella pneumoniae, Enterococcus faecium and Staphylococcus aureus. By using these data as a baseline, future studies involving a myriad of stress conditions can be compared to identify key metabolome differences in each species and to determine the mechanisms utilised by bacteria to respond to stress.

microbiology↗

bioRxiv2023

Microbiome diversity: A barrier to the environmental spread of antimicrobial resistance?

BackgroundIn the environment, microbial communities are constantly exposed to invasion by antimicrobial resistant bacteria (ARB) and their associated antimicrobial resistance genes (ARGs) that were enriched in the anthroposphere. A successful invader has to overcome the biotic resilience of the habitat, which is more difficult with increasing biodiversity. The capacity to exploit resources in a given habitat is enhanced when communities exhibit greater diversity, reducing opportunities for invaders, leading to a lower persistence. In the context of antimicrobial resistance (AMR) dissemination, exogenous ARB reaching a natural community may persist longer if the biodiversity of the autochthonous community is low, increasing the chance of ARGs to transfer to community members. Reciprocally, high microbial diversity could serve as a natural long-term barrier towards invasion by ARB and ARGs. ResultsTo test this hypothesis, a sampling campaign across seven European countries was carried out to obtain 172 environmental samples from sites with low anthropogenic impact. Samples were collected from contrasting environments: stationary structured forest soils, or dynamic river biofilms and sediments. Microbial diversity and relative abundance of 27 ARGs and 5 mobile genetic element marker genes were determined. In soils, higher diversity, evenness and richness were all significantly negatively correlated with the relative abundance of the majority (>85%) of ARGs. Furthermore, the number of detected ARGs per sample was inversely correlated with diversity. However, no such effects were found for the more dynamic, regularly mixed rivers. Conclusions: In conclusion, we demonstrate that diversity can serve as barrier towards AMR dissemination in the environment. This effect is mainly observed in stationary, structured environments, where long-term, diversity-based resilience against invasion can evolve. Such barrier effects can in the future be exploited to limit the environmental proliferation of AMR.

microbiology↗

bioRxiv2022

Data based slurry treatment decision tree to minimize antibiotic resistance and pathogen transfer while maximizing nutrient recycling

Direct application of pig slurry to agricultural land, as a means of nutrient recycling, introduces pathogens, antibiotic resistant bacteria, or genes, to the environment. With global environmental sustainability policies mandating a reduction in synthetic fertilisation and a commitment to a circular economy it is imperative to find effective on-farm treatments of slurry that maximises its fertilisation value and minimises risk to health and the environment. We assessed and compared the effect of storage, composting, and anaerobic digestion on pig slurry microbiome, resistome and nutrient content. Shotgun metagenomic sequencing and HT-qPCR arrays were implemented to understand the dynamics across the treatments. Our results identified that each of the treatment methods had advantages and disadvantages, depending on the parameter measured. The data suggests that storage and composting are optimal for the removal of human pathogens and anaerobic digestion for the reduction in AMR genes and mobile genetic elements. The nitrogen content is increased in storage and AD and reduced in composting. Thus, depending on the requirement for increased or reduced nitrogen the optimum treatment varies. Combining the results indicates that composting provides the greatest gain by reducing risk to human health and the environment. Network analysis revealed reducing Proteobacteria and Bacteroidetes while increasing Firmicutes will reduce the AMR content. KEGG analysis identified no significant change in the pathways across all treatments. This novel study provides a data driven decision tree to determine the optimal treatment for best practice to minimise pathogen, AMR and excess or increasing nutrient transfer from slurry to environment. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=98 SRC="FIGDIR/small/481976v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@141af73org.highwire.dtl.DTLVardef@165bf42org.highwire.dtl.DTLVardef@ef86c5org.highwire.dtl.DTLVardef@1790fca_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2021

Comparative genomics and pangenomics of vancomycin resistant and susceptible Enterococcus faecium from Irish hospitals across 20 years

Enterococcus faecium has emerged as an important nosocomial pathogen, which is increasingly difficult to treat due to the genetic acquisition of vancomycin resistance. Ireland exhibits a recalcitrant vancomycin resistant bloodstream infection rate compared to other developed countries. A set of 28 vancomycin resistant isolates was sequenced to construct a dataset alongside 61 other publicly available Irish genomes. This dataset was extensively analysed using in-silico methodologies and uncovered distinct evolutionary, coevolutionary, and clinically relevant population trends. These results suggest that a stable (in terms of genome size, GC%, and number of genes), yet genetically diverse population (in terms of gene content) of Enterococcus faecium persist in Ireland with acquired resistance arising via plasmid acquisition (vanA) or to a lesser extent, chromosomal recombination (vanB). Population analysis described five clusters with one cluster partitioned into four clades which transcend isolation dates. Pangenomic and recombination analyses revealed an open (whole genome and chromosomal specific) pangenome illustrating a rampant evolutionary pattern. Comparative resistomics and virulomics uncovered distinct chromosomal and mobilomal propensity for multidrug resistance, widespread chromosomal point-mutation mediated resistance, and chromosomal harboured arsenals of virulence factors. Comparative phagomics revealed a core prophagome of three prophages throughout the dataset. Interestingly, a potential difference in biofilm formation strategies was highlighted by coevolutionary analysis, suggesting differential biofilm genotypes between vanA and vanB isolates. These results highlight the evolutionary history of Irish Enterococcus faecium isolates and may provide an insight into underlying infection dynamics in a clinical setting.

microbiology↗

bioRxiv2021

Global protein responses of multi-drug resistant plasmid containing Escherichia coli to ampicillin, cefotaxime, imipenem and ciprofloxacin.

Antimicrobial resistance (AMR) and multi-drug resistance (MDR) in pathogenic bacteria are frequently mediated by plasmids. However, plasmids do not exist in isolation but rather require the bacterial host interaction in order to produce the AMR phenotype. This study aimed to utilise mass spectrometry-based proteomics to reveal the plasmid and chromosomally derived protein profile of Escherichia coli under antimicrobial stress. This was achieved by comparing the proteomes of E. coli containing the MDR pEK499 plasmid, under ampicillin, cefotaxime, imipenem or ciprofloxacin stress with the proteomes of these bacteria grown in the absence of antimicrobial. Our analysis identified statistically significant differentially abundant proteins common to groups exposed to the {beta}-lactam antimicrobials but not ciprofloxacin, indicating a {beta}-lactam stress response to exposure from this class of drugs, irrespective of {beta}-lactam resistance or susceptibility. These include ecotin and free methionine-R-sulfoxide reductase. These data also identified distinct differences in the cellular response to each {beta}-lactam. Data arising from comparisons of the proteomes of ciprofloxacin-treated E. coli and controls detected an increase in the relative abundance of proteins associated with ribosomes, translation, the TCA-cycle and several proteins associated with detoxification and a decrease in the relative abundances of proteins associated with stress response, including oxidative stress. We identified changes in proteins associated with persister formation in the presence of ciprofloxacin but not the {beta}-lactams. The plasmid proteome differed across each treatment and did not follow the pattern of antimicrobial - AMR protein associations. For example, a relative increase in the amount of blaCTX-M-15 in the presence of cefotaxime and ciprofloxacin but not the other {beta}-lactams, suggesting regulation of the blaCTX-M-15 protein production. The proteomic data from the this study provided novel insights into the proteins produced from the chromosome and plasmid under different antimicrobial stresses. These data also identified novel proteins not previously associated with AMR or antimicrobials responses in pathogens, which may well represent potential targets of AMR inhibition.

microbiology↗

bioRxiv2021

statSuma: automated selection and performance of statistical comparisons for microbiome studies

There is a reproducibility crisis in scientific studies. Some of these crises arise from incorrect application of statistical tests to data that follow inappropriate distributions, have inconsistent equivariance, or have very small sample sizes. As determining which test is most appropriate for all data in a multicategorical study (such as comparing taxa between sites in microbiome studies), we present statsSuma, an interactive Python notebook (which can be run from any desktop computer using the Google Colaboratory web service) and does not require a user to have any programming experience. This software assesses underlying data structures in a given dataset to advise what pairwise or listwise statistical procedure would be best suited for all data. As some users may be interested in further mining specific trends, statSuma performs 5 different two-tailed pairwise tests (Students t-test, Welchs t-test, Mann-Whitney U-test, Brunner-Munzel test, and a pairwise Kruskal-Wallis H-test) and advises the best test for each comparison. This software also advises whether ANOVA or a multicategorical Kruskal-Wallis H-test is most appropriate for a given dataset and performs both procedures. A data distribution-vs-Gaussian distribution plot is produced for each taxon at each site and a variance plot between all combinations of 2 taxa at each site are produced so Gaussian tests and variance tests can be visually confirmed alongside associated statistical determinants.

microbiology↗

bioRxiv2021

uniForest: an unsupervised machine learning technique to detect outliers and restrict variance in microbiome studies

Isolation Forests is an unsupervised machine learning technique for detecting outliers in continuous datasets that does not require an underlying equivariant or Gaussian distribution and is suitable for use on small datasets. While this procedure is widely used across quantitative fields, to our knowledge, this is the first attempt to solely assess its use for microbiome datasets. Here we present uniForest, an interactive Python notebook (which can be run from any desktop computer using the Google Colaboratory web service) for the processing of microbiome outliers. We used uniForest to apply Isolation Forests to the Healthy Human Microbiome project dataset and imputed outliers with the mean of the remaining inliers to maintain sample size and assessed its prowess in variance reduction in both community structure and derived ecological statistics (-diversity). We also assessed its functionality in anatomical site differentiation (pre- and postprocessing) using principal component analysis, dissimilarity matrices, and ANOSIM. We observed a minimum variance reduction of 81.17% across the entire dataset and in alpha diversity at the Phylum level. Application of Isolation Forests also separated the dataset to an extremely high specificity, reducing variance within taxa samples by a minimum of 81.33%. It is evident that Isolation Forests are a potent tool in restricting the effect of variance in microbiome analysis and has potential for broad application in studies where high levels of microbiome variance is expected. This software allows for clean analyses of otherwise noisy datasets.

microbiology↗

bioRxiv2019

Antibiotic resistant and extended-spectrum β-lactamase producing faecal coliforms in wastewater treatment plant effluent

Wastewater treatment plants (WWTPs) provide optimal conditions for the maintenance and spread of antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs). In this work we describe the occurrence of antibiotic resistant faecal coliforms and their mechanisms of antibiotic resistance in the effluent of two urban WWTPs in Ireland. Effluent samples were collected from two WWTPs in Spring and Autumn of 2015 and 2016. The bacterial susceptibility patterns to 13 antibiotics were determined. The phenotypic tests were carried out to identify AmpC or extended-spectrum {beta}-lactamase (ESBL) producers. The presence of ESBL genes were detected by PCR. Plasmids carrying ESBL genes were transformed into Escherichia coli DH5 recipient and underwent plasmid replicon typing to identify incompatibility groups. More than 90% of isolated faecal coliforms were resistant to amoxicillin and ampicillin, followed by tetracycline (up to 39.82%), ciprofloxacin (up to 31.42%) and trimethoprim (up to 37.61%). Faecal coliforms resistant to colistin and imipenem were detected in all effluent samples. Up to 53.98% of isolated faecal coliforms expressed a multi-drug resistance (MRD) phenotype. AmpC production was confirmed in 5.22% of isolates. The ESBL genes were confirmed for 11.84% of isolates (9.2% of isolates carried blaTEM, 1.4% blaSHV-12, 0.2% blaCTX-M-1 and 1% blaCTX-M-15). Plasmids extracted from 52 ESBL isolates were successfully transformed into recipient E. coli. The detected plasmid incompatibility groups included the IncF group, IncI1, IncHI1/2 and IncA/C. These results provide evidence that treated wastewater is polluted with ARB and MDR faecal coliforms and are sources of ESBL-producing, carbapenem and colistin resistant Enterobacteriaceae. ImportanceAntibiotic resistant bacteria (ARB) are an emerging environmental concern with a potential impact on human health. The results provide the evidence that treated wastewater is polluted with antibiotic resistant bacteria containing mobile resistance mechanisms of importance to clinical treatment of pathogens and multi-drug resistant (MDR) faecal coliforms. They are sources of relatively high proportions of ESBL-producing Enterobacteriaceae, and include carbapenem and colistin resistant Enterobacteriaceae. The significance of this study is the identification of the role of WWTPs as a potential control point to reduce or stop the movement of ESBL, MDR and colistin resistant bacteria into the environment from further upstream sources, such as human or animal waste.

microbiology↗

bioRxiv2019

Antibiotic resistant and extended-spectrum β-lactamase producing faecal coliforms in wastewater treatment plant effluent

Wastewater treatment plants (WWTPs) provide optimal conditions for the maintenance and spread of antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs). In this work we describe the occurrence of antibiotic resistant faecal coliforms and their mechanisms of antibiotic resistance in the effluent of two urban WWTPs in Ireland. Effluent samples were collected from two WWTPs in Spring and Autumn of 2015 and 2016. The bacterial susceptibility patterns to 13 antibiotics were determined. The phenotypic tests were carried out to identify AmpC or extended-spectrum {beta}-lactamase (ESBL) producers. The presence of ESBL genes were detected by PCR. Plasmids carrying ESBL genes were transformed into Escherichia coli DH5 recipient and underwent plasmid replicon typing to identify incompatibility groups. More than 90% of isolated faecal coliforms were resistant to amoxicillin and ampicillin, followed by tetracycline (up to 39.82%), ciprofloxacin (up to 31.42%) and trimethoprim (up to 37.61%). Faecal coliforms resistant to colistin and imipenem were detected in all effluent samples. Up to 53.98% of isolated faecal coliforms expressed a multi-drug resistance (MRD) phenotype. AmpC production was confirmed in 5.22% of isolates. The ESBL genes were confirmed for 11.84% of isolates (9.2% of isolates carried blaTEM, 1.4% blaSHV-12, 0.2% blaCTX-M-1 and 1% blaCTX-M-15). Plasmids extracted from 52 ESBL isolates were successfully transformed into recipient E. coli. The detected plasmid incompatibility groups included the IncF group, IncI1, IncHI1/2 and IncA/C. These results provide evidence that treated wastewater is polluted with ARB and MDR faecal coliforms and are sources of ESBL-producing, carbapenem and colistin resistant Enterobacteriaceae. ImportanceAntibiotic resistant bacteria (ARB) are an emerging environmental concern with a potential impact on human health. The results provide the evidence that treated wastewater is polluted with antibiotic resistant bacteria containing mobile resistance mechanisms of importance to clinical treatment of pathogens and multi-drug resistant (MDR) faecal coliforms. They are sources of relatively high proportions of ESBL-producing Enterobacteriaceae, and include carbapenem and colistin resistant Enterobacteriaceae. The significance of this study is the identification of the role of WWTPs as a potential control point to reduce or stop the movement of ESBL, MDR and colistin resistant bacteria into the environment from further upstream sources, such as human or animal waste.

microbiology↗