Fast and Accurate Estimation of Selection Coefficients and Allele Histories from Ancient and Modern DNA.

We here present CLUES2, a full-likelihood method to infer natural selection from sequence data that is an extension of the method CLUES. We make several substantial improvements to the CLUES method that greatly increases both its applicability and its speed. We add the ability to use ancestral recombination graphs on ancient data as emissions to the underlying hidden Markov model, which enables CLUES2 to use both temporal and linkage information to make estimates of selection coefficients. We also fully implement the ability to estimate distinct selection coefficients in different epochs, which allows for the analysis of changes in selective pressures through time, as well as selection with dominance. In addition, we greatly increase the computational efficiency of CLUES2 over CLUES using several approximations to the forward-backward algorithms and develop a new way to reconstruct historic allele frequencies by integrating over the uncertainty in the estimation of the selection coefficients. We illustrate the accuracy of CLUES2 through extensive simulations and validate the importance sampling framework for integrating over the uncertainty in the inference of gene trees. We also show that CLUES2 is well-calibrated by showing that under the null hypothesis, the distribution of log-likelihood ratios follows a χ2 distribution with the appropriate degrees of freedom. We run CLUES2 on a set of recently published ancient human data from Western Eurasia and test for evidence of changing selection coefficients through time. We find significant evidence of changing selective pressures in several genes correlated with the introduction of agriculture to Europe and the ensuing dietary and demographic shifts of that time. In particular, our analysis supports previous hypotheses of strong selection on lactase persistence during periods of ancient famines and attenuated selection in more modern periods.

Pdif-mediated antibiotic resistance genes transfer in bacteria identified by pdifFinder.

Modules consisting of antibiotic resistance genes (ARGs) flanked by inverted repeat Xer-specific recombination sites were thought to be mobile genetic elements that promote horizontal transmission. Less frequently, the presence of mobile modules in plasmids, which facilitate a pdif-mediated ARGs transfer, has been reported. Here, numerous ARGs and toxin-antitoxin genes have been found in pdif site pairs. However, the mechanisms underlying this apparent genetic mobility is currently not understood, and the studies relating to pdif-mediated ARGs transfer onto most bacterial genera are lacking. We developed the web server pdifFinder based on an algorithm called PdifSM that allows the prediction of diverse pdif-ARGs modules in bacterial genomes. Using test set consisting of almost 32 thousand plasmids from 717 species, PdifSM identified 481 plasmids from various bacteria containing pdif sites with ARGs. We found 28-bp-long elements from different genera with clear base preferences. The data we obtained indicate that XerCD-dif site-specific recombination mechanism may have evolutionary adapted to facilitate the pdif-mediated ARGs transfer. Through multiple sequence alignment and evolutionary analyses of duplicated pdif-ARGs modules, we discovered that pdif sites allow an interspecies transfer of ARGs but also across different genera. Mutations in pdif sites generate diverse arrays of modules which mediate multidrug-resistance, as these contain variable numbers of diverse ARGs, insertion sequences and other functional genes. The identification of pdif-ARGs modules and studies focused on the mechanism of ARGs co-transfer will help us to understand and possibly allow controlling the spread of MDR bacteria in clinical settings. The pdifFinder code, standalone software package and description with tutorials are available at https://github.com/mjshao06/pdifFinder.

Exploring the resistome, virulome, and mobilome of multidrug-resistant Klebsiella pneumoniae isolates: deciphering the molecular basis of carbapenem resistance.

BACKGROUND: Klebsiella pneumoniae, a notorious pathogen for causing nosocomial infections has become a major cause of neonatal septicemia, leading to high morbidity and mortality worldwide. This opportunistic bacterium has become highly resistant to antibiotics due to the widespread acquisition of genes encoding a variety of enzymes such as extended-spectrum beta-lactamases (ESBLs) and carbapenemases. We collected Klebsiella pneumoniae isolates from a local tertiary care hospital from February 2019-February 2021. To gain molecular insight into the resistome, virulome, and genetic environment of significant genes of multidrug-resistant K. pneumoniae isolates, we performed the short-read whole-genome sequencing of 10 K. pneumoniae isolates recovered from adult patients, neonates, and hospital tap water samples. RESULTS: The draft genomes of the isolates varied in size, ranging from 5.48 to 5.96 Mbp suggesting the genome plasticity of this pathogen. Various genes conferring resistance to different classes of antibiotics e.g., aminoglycosides, quinolones, sulfonamides, tetracycline, and trimethoprim were identified in all sequenced isolates. The highest resistance was observed towards carbapenems, which has been putatively linked to the presence of both class B and class D carbapenemases, blaNDM, and blaOXA, respectively. Moreover, the biocide resistance gene qacEdelta1 was found in 6/10 of the sequenced strains. The sequenced isolates exhibited a broad range of sequence types and capsular types. The significant antibiotic resistance genes (ARGs) were bracketed by a variety of mobile genetic elements (MGEs). Various spontaneous mutations in genes other than the acquired antibiotic-resistance genes were observed, which play an indirect role in making these bugs resistant to antibiotics. Loss or deficiency of outer membrane porins, combined with ESBL production, played a significant role in carbapenem resistance in our sequenced isolates. Phylogenetic analysis revealed that the study isolates exhibited evolutionary relationships with strains from China, India, and the USA suggesting a shared evolutionary history and potential dissemination of similar genes amongst the isolates of different origins. CONCLUSIONS: This study provides valuable insight into the presence of multiple mechanisms of carbapenem resistance in K. pneumoniae strains including the acquisition of multiple antibiotic-resistance genes through mobile genetic elements. Identification of rich mobilome yielded insightful information regarding the crucial role of insertion sequences, transposons, and integrons in shaping the genome of bacteria for the transmission of various resistance-associated genes. Multi-drug resistant isolates that had the fewest resistance genes exhibited a significant number of mutations. K. pneumoniae isolate from water source displayed comparable antibiotic resistance determinants to clinical isolates and the highest number of virulence-associated genes suggesting the possible interplay of ARGs amongst bacteria from different sources.

Quantitative microbial risk assessment for ingestion of antibiotic resistance genes from private wells contaminated by human and livestock fecal sources.

We used quantitative microbial risk assessment to estimate ingestion risk for intI1, erm(B), sul1, tet(A), tet(W), and tet(X) in private wells contaminated by human and/or livestock feces. Genes were quantified with five human-specific and six bovine-specific microbial source-tracking (MST) markers in 138 well-water samples from a rural Wisconsin county. Daily ingestion risk (probability of swallowing ≥1 gene) was based on daily water consumption and a Poisson exposure model. Calculations were stratified by MST source and soil depth over the aquifer where wells were drilled. Relative ingestion risk was estimated using wells with no MST detections and >6.1 m soil depth as a referent category. Daily ingestion risk varied from 0 to 8.8 × 10-1 by gene and fecal source (i.e., human or bovine). The estimated number of residents ingesting target genes from private wells varied from 910 (tet(A)) to 1,500 (intI1 and tet(X)) per day out of 12,000 total. Relative risk of tet(A) ingestion was significantly higher in wells with MST markers detected, including wells with ≤6.1 m soil depth contaminated by bovine markers (2.2 [90% CI: 1.1-4.7]), wells with >6.1 m soil depth contaminated by bovine markers (1.8 [1.002-3.9]), and wells with ≤6.1 m soil depth contaminated by bovine and human markers simultaneously (3.1 [1.7-6.5]). Antibiotic resistance genes (ARGs) were not necessarily present in viable microorganisms, and ingestion is not directly associated with infection. However, results illustrate relative contributions of human and livestock fecal sources to ARG exposure and highlight rural groundwater as a significant point of exposure.IMPORTANCEAntibiotic resistance is a global public health challenge with well-known environmental dimensions, but quantitative analyses of the roles played by various natural environments in transmission of antibiotic resistance are lacking, particularly for drinking water. This study assesses risk of ingestion for several antibiotic resistance genes (ARGs) and the class 1 integron gene (intI1) in drinking water from private wells in a rural area of northeast Wisconsin, United States. Results allow comparison of drinking water as an exposure route for antibiotic resistance relative to other routes like food and recreational water. They also enable a comparison of the importance of human versus livestock fecal sources in the study area. Our study demonstrates the previously unrecognized importance of untreated rural drinking water as an exposure route for antibiotic resistance and identifies bovine fecal material as an important exposure factor in the study setting.

Serum ARGS-aggrecan in a phase 2 clinical trial targeting osteoarthritis.

OBJECTIVE: To monitor serum concentrations of the aggrecan alanine-arginine-glycine-serine (ARGS) neoepitope in a clinical trial of a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-5 inhibition as disease-modifying therapy of knee osteoarthritis, and to investigate relationships between reduction in ARGS and change in cartilage thickness, knee-related pain and function. DESIGN: ROCCELLA trial participants received once-daily oral S201086 75, 150 or 300 mg, or placebo, for 52 weeks. Serum was collected at baseline, 4, 12, 28 and 52 weeks, and 2 weeks post-treatment with ARGS measured by an in-house immunoassay. Change from baseline to week 52 in central medial femorotibial compartment cartilage thickness was measured by magnetic resonance imaging, function and pain by Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) subscores. Associations between cumulative change in ARGS and change in cartilage thickness or WOMAC subscores were evaluated by linear regression. RESULTS: S201086 reduced serum levels of ARGS in a dose-dependent manner throughout the treatment period. Maximal reduction was at 4 weeks with a 58.5% [95% CI 60.8%, 56.2%] reduction of ARGS compared to baseline for 300 mg S201086. Two weeks post-treatment, ARGS concentrations rebounded with a dose-dependent overshoot compared to baseline levels. Cumulative change of ARGS concentration from baseline to week 52 had no effect on change in cartilage thickness (slope -0.8×10-6 [-2.9×10-6, 1.3×10-6]) or change in WOMAC pain and function (slopes -30×10-6 [-64×10-6, 5.2×10-6] and -97×10-6 [-214×10-6, 20×10-6], respectively) at week 52. CONCLUSION: Systemic inhibition of ADAMTS-5 resulted in markedly reduced serum ARGS, but change in serum ARGS concentrations showed no association with the progression of cartilage thinning, or patient reported pain and function.

Spread and persistence of antimicrobial resistance genes in wastewater from human and animal sources in São Paulo, Brazil.

The spread of antimicrobial resistance (AMR) through multiple reservoirs is a global concern. Wastewater is a critical AMR dissemination source, so this study aimed to assess the persistence of resistance genetic markers in wastewater using a culture-independent approach. Raw and treated wastewater samples (n = 121) from a wastewater treatment plant (WWTP), a human hospital, a veterinary hospital, and a pig farm were monthly collected and concentrated by filtration. DNA was extracted directly from filter membranes, and PCR was used in the qualitative search of 32 antimicrobial resistance genes (ARGs). Selected genes (blaCTX-M, blaKPC, qnrB, and mcr-1) were enumerated by quantitative real-time PCR (qPCR). Twenty-six ARGs were detected in the qualitative ARGs search, while quantitative data showed a low variation of the ARG's relative abundance (RA) throughout the months, especially at the human hospital and the WWTP. At the WWTP, despite significantly reducing the absolute number of gene copies/L after each treatment stage (p < 0.05), slight increases (p > 0.05) in the RAs of genes blaCTX-M, qnrB, and mcr-1 were observed in reused water (tertiary treatment) when compared with secondary effluent. Although the increase is not statistically significant, it is worth noting that there was some level of ARGs concentration after the disinfection process. No significant absolute or relative after-treatment quantification reductions were observed for any ARGs at the veterinary hospital or the pig farm. The spread of ARGs through sewage needs to be continuously addressed, because their release into natural environments may pose potential risks of exposure to resistant bacteria and impact local ecosystems.

Diversity, functions, and antibiotic resistance genes of bacteria and fungi are examined in the bamboo plant phyllosphere that serve as food for the giant pandas.

The phyllosphere of bamboo is rich in microorganisms that can disrupt the intestinal microbiota of the giant pandas that consume them, potentially leading to their death. In the present study, the abundance, diversity, biological functions (e.g., KEGG and CAZyme), and antibiotic resistance genes (ARGs) of bacteria and fungi in two bamboo species phyllosphere (Chimonobambusa szechuanensis, CS; Bashania fangiana, BF) in Daxiangling Nature Reserve (an important part of the Giant Panda National Park) were investigated respectively by amplicon sequencing of the whole 16S rRNA and ITS1-ITS2 genes on PacBio Sequel and whole-metagenome shotgun sequencing on Illumina NovaSeq 6000 platform. The results suggested that there were respectively 18 bacterial and 34 fungi biomarkers between the phyllosphere of the two species of bamboo. Beta diversity of bacteria and fungi communities exited between the two bamboos according to the (un)weighted UniFrac distance matrix. Moreover, the functional analysis showed that the largest relative abundance was found in the genes related to metabolism and global and overview maps. Glycoside hydrolases (GHs) and glycosyl transferases (GTs) have a higher abundance in two bamboo phyllospheres. Co-occurrence network modeling suggested that bacteria and fungi communities in CS phyllosphere employed a much more complex metabolic network than that in BF, and the abundance of multidrug, tetracycline, and glycopeptide resistance genes was higher and closely correlated with other ARGs. This study references the basis for protecting bamboo resources foraged by wild giant pandas and predicts the risk of antibiotic resistance in bamboo phyllosphere bacterial and fungal microbiota in the Giant Panda National Park, China.

Optimized Antibiotic Resistance Genes Monitoring Scenarios Promote Sustainability of Urban Water Cycle.

Dissemination of antibiotic resistance genes (ARGs) in urban water bodies has become a significant environmental and health concern. Many approaches based on real-time quantitative PCR (qPCR) have been developed to offer rapid and highly specific detection of ARGs in water environments, but the complicated and time-consuming procedures have hindered their widespread use. Herein, we developed a facile one-step approach for rapid detection of ARGs by leveraging the trans-cleavage activity of Cas12a and recombinase polymerase amplification (RPA). This efficient method matches the sensitivity and specificity of qPCR and requires no complex equipment. The results show a strong correlation between the prevalence of four ARG markers (ARGs: sul1, qnrA-1, mcr-1, and class 1 integrons: intl1) in tap water, human urine, farm wastewater, hospital wastewater, municipal wastewater treatment plants (WWTPs), and proximate natural aquatic ecosystems, indicating the circulation of ARGs within the urban water cycle. Through monitoring the ARG markers in 18 WWTPs in 9 cities across China during both peak and declining stages of the COVID epidemic, we found an increased detection frequency of mcr-1 and qnrA-1 in wastewater during peak periods. The ARG detection method developed in this work may offer a useful tool for promoting a sustainable urban water cycle.

Synergistic Control of Trimethoprim and the Antimicrobial Resistome in Electrogenic Microbial Communities.

Synergistic control of the risks posed by emerging antimicrobials and antibiotic resistance genes (ARGs) is crucial for ensuring ecological safety. Although electrogenic respiration can enhance the biodegradation of several antimicrobials and reduce ARGs accumulation, the association mechanisms of antimicrobial biodegradation (trimethoprim, TMP) with the fate of the antimicrobial resistome remain unclear. Here, the biotransformation pathway of TMP, microbial associations, and functional gene profiles (e.g., degradation, antimicrobial resistance, and electron transfer) were analyzed. The results showed that the microbial electrogenic respiration significantly enhanced the biodegradation of TMP, especially with a cosubstrate sodium acetate supply. Electroactive bacteria enriched in the electrode biofilm positively correlated with potential TMP degraders dominated in the planktonic communities. These cross-niche microbial associations may contribute to the accelerated catabolism of TMP and extracellular electron transfer. Importantly, the evolution and dissemination of overall ARGs and mobile genetic elements (MGEs) were significantly weakened due to the enhanced cometabolic biodegradation of TMP. This study provides a promising strategy for the synergistic control of the water ecological risks of antimicrobials and their resistome, while also highlighting new insights into the association of antimicrobial biodegradation with the evolution of the resistome in an electrically integrated biological process.

The connection between the antibiotic resistome and nitrogen-cycling microorganisms in paddy soil is enhanced by application of chemical and plant-derived organic fertilizers.

Antibiotic resistant genes (ARGs) present significant risks to environments and public health. In particular, there is increasing awareness of the role of soil nitrogen in ARG dissemination. Here, we investigated the connections between antibiotic resistome and nitrogen-cycling microbes in paddy soil by performing five-year field experiments with the treatments of no nitrogen fertilization (CK), reduced chemical nitrogen fertilization (LN), conventional chemical nitrogen fertilization (CN) and plant-derived organic nitrogen fertilization (ON). Compared with CK treatment, CN and ON treatments significantly increased soil NH4+ and TN concentrations by 25.4%-56.5% and 10.4%-20.1%, respectively. Redundancy analysis revealed significantly positive correlation of NH4+ with most ARGs, including tetA, macB and barA. Correspondingly, CN and ON treatments enhanced ARG abundances by 21.9%-23.2%. Moreover, CN and ON treatments promoted nitrate/nitrite-reducing bacteria and linked the corresponding N-cycling functional genes (narG, narH, nirK and nrfA) with most ARGs. Metagenomic binning was performed and identified Gemmatimonadaceae, Caulobacteraceae, Ilumatobacteraceae and Anaerolineaceae as hosts for both ARGs and nitrate/nitrite reduction genes that were enriched by CN and ON treatments. Soil resistome risk score analysis indicated that, although there was increased relation of ARG to nitrogen-cycling microorganisms with nitrogen fertilizer application, the environmental risk of ARGs was not increased due to the lower distribution of ARGs in pathogens. This study contributed to a deeper understanding of the role of soil nitrogen in shaping ARG profiles and controlling soil resistome risk.

Seasonal dynamics of antibiotic resistance genes and mobile genetic elements in a subtropical coastal ecosystem: Implications for environmental health risks.

Antibiotic resistance poses a considerable global public health concern, leading to heightened rates of illness and mortality. However, the impact of seasonal variations and environmental factors on the health risks associated with antibiotic resistance genes (ARGs) and their assembly mechanisms is not fully understood. Based on metagenomic sequencing, this study investigated the antibiotic resistome, mobile genetic elements (MGEs), and microbiomes in a subtropical coastal ecosystem of the Beibu Gulf, China, over autumn and winter, and explored the factors influencing seasonal changes in ARG and MGE abundance and diversity. Results indicated that ARG abundance and diversity were higher in winter than in autumn, with beta-lactam and multidrug resistance genes being the most diverse and abundant, respectively. Similarly, MGE abundance and diversity increased in winter and were strongly correlated with ARGs. In contrast, more pronounced associations between microbial communities, especially archaea, and the antibiotic resistome were observed in autumn than in winter. The co-occurrence network identified multiple interactions between MGEs and various multidrug efflux pumps in winter, suggesting a potential for ARG dissemination. Multivariate correlation analyses and path modeling indicated that environmental factors driving microbial community changes predominantly influenced antibiotic resistome assembly in autumn, while the relative importance of MGEs increased significantly in winter. These findings suggest an elevated health risk associated with antimicrobial resistance in the Beibu Gulf during winter, attributed to the dissemination of ARGs by horizontal gene transfer. The observed seasonal variations highlight the dynamic nature of antibiotic resistance dissemination in coastal ecosystems, emphasizing the need for comprehensive surveillance and management measures to address the growing threat of antimicrobial resistance in vulnerable environments.

Pollution characteristics of heavy metals, antibiotic and antibiotic resistance genes in the crested ibis and their habitat across different lifestyle and geography.

Antibacterial resistance in wild animals has been increasingly reported worldwide, even though they are usually not directly exposed to clinically relevant antibiotics. Crested ibis, one of the rarest birds in the world, usually forages in paddy fields and prefer to nest and breed near villages that is greatly influenced by anthropogenic activities. We sampled the feces of crested ibises, as well as their habitat environment samples, to explore the pollution characteristics of heavy metals, antibiotics and antibiotic resistance genes (ARGs). Results showed that the pollution characteristics of heavy metals, antibiotic, ARGs and gut microbiota of crested ibis were more related by host lifestyle and habitats. Captive ibises had higher relative abundances of the total ARGs and tetracycline concentrations compared with feralization and wild ibises, while the heavy metal contents had shown the opposite result. The Characteristics of pollutants in the corresponding environmental samples also exhibited high similarity with the results of fecal samples. The relative abundances of Proteobacteria and Actinobacteria were significantly different between captive and wild individuals, while the abundance of majority bacterial genera was generally higher in wild populations. The concentrations of heavy metals in soil (Cd, Cu and Zn) and water (Cd, Cu, Zn and Cr) were both exceeded the background soil levels or surface water quality standards, suggesting multi-element contamination in the habitat. Ecological risk assessments of soils by Igeo and Er showed that the habitats of wild ibises were heavily and moderately contaminated by Cd, which would possibly pose a threat to the health of ibises. PLS-PM analysis indicated that microbial compositions and residual antibiotics had the most substantial impact on the dynamic changes in ARGs of ibis. Overall, this work provides a comprehensive understanding of the characteristics, risks of those contaminations, and their effects on the ARGs in the habitat of crested ibis.

Metagenomics uncovers microbiome and resistome in soil and reindeer faeces from Ny-Ålesund (Svalbard, High Arctic).

Research on the microbiome and resistome in polar environments, such as the Arctic, is crucial for understanding the emergence and spread of antibiotic resistance genes (ARGs) in the environment. In this study, soil and reindeer faeces samples collected from Ny-Ålesund (Svalbard, High Arctic) were examined to analyze the microbiome, ARGs, and biocide/metal resistance genes (BMRGs). The dominant phyla in both soil and faeces were Pseudomonadota, Actinomycetota, and Bacteroidota. A total of 2618 predicted Open Reading Frames (ORFs) containing antibiotic resistance genes (ARGs) were detected. These ARGs belong to 162 different genes across 17 antibiotic classes, with rifamycin and multidrug resistance genes being the most prevalent. We focused on investigating antibiotic resistance mechanisms in the Ny-Ålesund environment by analyzing the resistance genes and their biological pathways. Procrustes analysis demonstrated a significant correlation between bacterial communities and ARG/BMRG profiles in soil and faeces samples. Correlation analysis revealed that Pseudomonadota contributed most to multidrug and triclosan resistance, while Actinomycetota were predominant contributors to rifamycin and aminoglycoside resistance. The geochemical factors, SiO42- and NH4+, were found to significantly influence the microbial composition and ARG distribution in the soil samples. Analysis of ARGs, BMRGs, virulence factors (VFs), and pathogens identified potential health risks associated with certain bacteria, such as Cryobacterium and Pseudomonas, due to the presence of different genetic elements. This study provided valuable insights into the molecular mechanisms and geochemical factors contributing to antibiotic resistance and enhanced our understanding of the evolution of antibiotic resistance genes in the environment.

Fish skin mucosal surface becomes a barrier of antibiotic resistance genes under apramycin exposure.

Antibiotic resistance genes (ARGs) are a kind of emerging environmental contamination, and are commonly found in antibiotic application situations, attracting wide attention. Fish skin mucosal surface (SMS), as the contact interface between fish and water, is the first line of defense against external pollutant invasion. Antibiotics are widely used in aquaculture, and SMS may be exposed to antibiotics. However, what happens to SMS when antibiotics are applied, and whether ARGs are enriched in SMS are not clear. In this study, Zebrafish (Danio rerio) were exposed to antibiotic and antibiotic resistant bacteria in the laboratory to simulate the aquaculture situation, and the effects of SMS on the spread of ARGs were explored. The results showed that SMS maintained the stability of the bacterial abundance and diversity under apramycin (APR) and bacterial exposure effectively. Until 11 days after stopping APR exposure, the abundance of ARGs in SMS (mean value was 3.32 × 10-3 copies/16S rRNA copies) still did not recover to the initial stage before exposure, which means that enriched ARGs in SMS were persistently remained. Moreover, non-specific immunity played an important role in resisting infection of external contamination. Besides, among antioxidant proteins, superoxide dismutase showed the highest activity. Consequently, it showed that SMS became a barrier of antibiotic resistance genes under APR exposure, and ARGs in SMS were difficult to remove once colonized. This study provided a reference for understanding the transmission, enrichment process, and ecological impact of antibiotics and ARGs in aquatic environments.

Cat and dog feces as reservoirs of diverse novel antibiotic resistance genes.

Companion animals have the potential to greatly enhance the physical and mental health of humans, thus leading to an increased focus on the interactions between humans and pets. Currently, the inappropriate and excessive utilization of antimicrobial agents has become prevalent in veterinary clinical practice for pets. This antibiotic contamination phenomenon has a profound impact on the enrichment of antibiotic resistance bacteria (ARB) and antibiotic resistance genes (ARGs) in pets. However, the pet-associated resistome, especially the novel ARGs in pets, represents a relatively neglected area. In this study, we successfully constructed a total of 12 libraries using the functional metagenomics approach to assess the diversity of ARGs in pet cats and dogs from four pet hospitals. Through the integration of functional screening and high-throughput sequencing, a total of 122 antibiotic resistance determinants were identified, of which 15 were classified as putative novel ARGs originating from five classes. Functional assessment demonstrated that 6 novel ARGs including one ß-lactam, two macrolides, two aminoglycosides, and one rifamycin (RIF), namely blaPF, ermPF, msrPF, aac(6')PF, aph(3')PF, and arrPF, exhibited functionally activity in conferring bacterial phenotypic resistance by increasing the minimum inhibitory concentrations (MICs) with a 4- to 128-fold. Genetic context analysis demonstrated that, with the exception of aac(6')PF and arrPF, the remaining four novel ARGs were found adjacent to mobile genetic elements (MGEs) including IS elements or transposases, which provided a prerequisite for horizontal transfer of these novel ARGs, thereby offering an explanation for their detection in diverse samples collected from various sampling sites. The current study has unveiled the significant role of cat and dog feces as one source of reservoirs of diverse novel ARGs, while also highlighting the potential adverse consequences of their further spread to medically significant pathogens and human commensal organisms.

Occurrence and distribution of antibiotic resistance genes in urban rivers with black-odor water of Harbin, China.

Antibiotic resistance gene contamination in polluted rivers remains a widely acknowledged environmental issue. This study focused on investigating the contamination conditions of antibiotic resistance genes (ARGs) in Harbin's urban black-odor rivers, specifically Dongfeng Ditch and Hejia Ditch. The research employed a SmartChip Real-Time PCR System to explore the types, abundance, and distribution of ARGs in diverse habitats, such as surface water and sediment. Additionally, the study examined the correlation of ARGs with mobile genetic elements (MGEs) and various environmental factors. It was found that antibiotic resistance genes were prevalent in both water and sediment within the black-odor ditches. The dominant types of ARGs identified included aminoglycoside, sulfonamide, multidrug-resistant, and ß-lactam ARGs. Notably, the top four ARGs, in terms of relative abundance, were sul1, fox5, qacEdelta1-01 and aadA1. Most categories of ARGs have significant positive connections with MGEs, indicating that the enrichment and spreading of ARGs in rivers are closely related to MGEs. Based on the correlation analysis, it is found that environmental factors such as dissolved oxygen (DO), ammonia nitrogen (NH4-N), and phosphate (PO4-P) played a substantial role in influencing the variations observed in ARGs. By employing a risk assessment framework based on the human association, host pathogenicity, and mobility of ARGs, the identification of seven high-risk ARGs was achieved. In addition, it is important to assess the environmental risk of ARGs from multiple perspectives (abundance,detection rateand mobility). This study provides a significant reference regarding the presence of ARGs contamination in urban inland black-odor rivers, essential for assessing the health risks associated with ARGs and devising strategies to mitigate the threat of antibiotic resistance.

A multidisciplinary approach to analyze the antimicrobial resistance in natural ecosystems.

Antimicrobial Resistance (AMR) poses a global threat to both human health and environmental well-being. Our study delved into Costa Rican wildlife reserves, uncovering a substantial human impact on these ecosystems and underscoring the imperative to pinpoint AMR hotspots. Embracing a One Health perspective, we advocated for a comprehensive landscape analysis that intricately intertwined geographic, climatic, forest, and human factors. This study illuminated the link between laboratory results and observed patterns of antimicrobial use, thereby paving the way for sustainable solutions. Our innovative methodology involved deploying open-ended questions to explore antimicrobial usage across livestock activities, contributing to establishing a comprehensive methodology. Non-invasive sampling in wildlife emerged as a critical aspect, shedding light on areas contaminated by AMR. Feline species, positioned at the apex of the food chain, acted as sentinels for environmental health due to heightened exposure to improperly disposed waste. Regarding laboratory findings, each sample revealed the presence of at least one antimicrobial resistance gene (ARG). Notably, genes encoding resistance to tetracyclines dominated (94.9%), followed by beta-lactams (75.6%), sulfonamides (53.8%), aminoglycosides (51.3%), quinolones (44.9%), phenicols (25.6%), and macrolides (20.5%). Genes encoding polymyxins were not detected. Moreover, 66% of samples carried a multi-resistant microbiome, with 15% exhibiting resistance to three antimicrobial families and 51% to four. The absence of a correlation between forest coverage and ARG presence underscored the profound human impact on wildlife reserves, surpassing previous estimations. This environmental pressure could potentially modify microbiomes and resistomes in unknown ways. As not all antimicrobial families encoding ARGs were utilized by farmers, our next step involved evaluating other human activities to identify the primary sources of contamination. This comprehensive study contributed crucial insights into the intricate dynamics of AMR in natural ecosystems, paving the way for targeted interventions and sustainable coexistence.

Response mechanism of a highly efficient partial nitritation-anammox (PN/A) process under antibiotic stress: Extracellular polymers, microbial community, and functional genes.

The Partial nitritation-Anammox (PN/A) process can be restricted when treating high ammonia nitrogen wastewater containing antibiotics. This study aims to explore the response mechanism of the PN/A process under antibiotic stress. Results showed the PN/A process achieved a nitrogen removal rate higher than 1.01 ± 0.03 kg N/m3/d under long-term sulfamethazine stress. The increase of extracellular polymers from 22.52 to 43.96 mg/g VSS was conducive to resisting antibiotic inhibitory. The increase of Denitratisoma and SM1A02 abundance as well as functional genes nirS and nirK indicated denitrifiers should play an important role in the stability of the PN/A system under sulfamethazine stress. In addition, antibiotic-resistant genes (ARGs) sul1 and intI1 significantly increased by 8.78 and 5.12 times of the initial values to maintain the resistance of PN/A process to sulfamethazine stress. This study uncovers the response mechanism of the PN/A process under antibiotic stress, offering a scientific basis and guidance for further application in the future.

Variations in antibiotic resistance genes during long-term operation of an upflow anaerobic sludge blanket reactor.

Antibiotic resistance genes (ARGs) have been widely detected in the environment. Anaerobic digestion (AD) has the potential ability to remove ARGs, and a comprehensive study is needed on the variations in ARGs during AD. In this study, variations in antibiotic resistance genes (ARGs) and microbial communities were investigated during the long-term operation of an upflow anaerobic sludge blanket (UASB) reactor. An antibiotic mixture of erythromycin, sulfamethoxazole and tetracycline was added to the UASB influent and the operation period was 360 days. The abundances of 11 ARGs and class 1 integron-integrase gene were detected in the UASB reactor, and the correlation between the ARGs and the microbial community was analyzed. The composition of ARGs indicated that the main ARGs in the effluent were sul1, sul2, and sul3, whereas the main ARG in the sludge was tetW. Correlation analysis indicated a negative correlation between microorganisms and ARGs in the UASB. In addition, most of ARGs showed a positive correlation with norank_f_Propionibacteriaceae and Clostridum_sensu_stricto_6, which were identified as potential hosts. These findings may help develop a feasible strategy for removing ARGs from aquatic environments during anaerobic digestion.

Combined applications of UV and chlorine on antibiotic resistance control: A critical review.

Environmental health problems caused by antibiotic-resistant bacteria (ARB) and antibiotic-resistant genes (ARGs) have become a global concern. ARB and ARGs have been continuously detected in various water environments, which pose a new challenge for water quality safety assurance. Disinfection is a key water treatment process to eliminate pathogenic microorganisms in water, and combined chlorine and UV processes (the UV/Cl2 process, the UV-Cl2 process, and the Cl2-UV process) are considered potential disinfection methods to control antibiotic resistance. This review documented the efficacy and mechanism of combined UV and chlorine processes for the control of antibiotic resistance, as well as the effects of chlorine dose, solution pH, UV wavelength, and water matrix on the effectiveness of the processes. There are knowledge gaps in research on the combined chlorine and UV processes for antibiotic resistance control, in particular the UV-Cl2 process and the Cl2-UV process. In addition, changes in the structure of microbial communities and the distribution of ARGs, which are closely related to the spread of antibiotic resistance in the water, induced by combined processes were also addressed. Whether these changes could lead to the re-transmission of antibiotic resistance and harm human health may need to be further evaluated.