Reservoir Of Antibiotic Resistance Genes Research Articles

Free-floating extracellular DNA (exDNA) in different wastewaters: Status quo on exDNA-associated antimicrobial resistance genes

Wastewater treatment plants (WWTPs) have been reported as major anthropogenic reservoirs for the spread of antibiotic-resistant bacteria (ARB) and antibiotic resistance genes (ARGs) into the environment, worldwide. While most studies mainly focus on the intracellular DNA (iDNA), extracellular DNA (exDNA) accounting for a significant proportion of the total DNA in wastewater, was usually neglected. Following the One Health approach, this study focuses on wastewaters of municipal, clinical, and livestock origins (n = 45) that undergo different treatment processes (i.e., conventional activated sludge, ultrafiltration, and ozonation). Water samples were analysed for 12 ARGs as indicators of the different compartments associated with iDNA and exDNA by quantitative real-time PCR (qPCR). Taxonomic profiling of exDNA-fractions, obtained using nucleic acid adsorption particles, was conducted by sequencing the V3–V4 hypervariable regions of the 16S rRNA gene. Notified exDNA concentrations varied between on-site WWTPs and treatment stages, and ranged from 314.0 ± 70.2 ng/mL in untreated livestock wastewater down to 0.7 ± 0.1 ng/mL in effluents after ultrafiltration. In general, influents exhibited higher concentrations compared to effluents, while wastewater treated by advanced treatment processes (i.e., ultrafiltration and ozonation) showed the lowest exDNA concentrations. Despite the lower concentrations, free-floating exDNA accounted for up to 80.0 ± 5.8% of the total DNA in effluents. Target ARGs were more common in the iDNA (100%, n = 45/45), compared to the exDNA-fractions (51.1%, n = 23/45), whereas exDNA-ARGs were mostly detected in clinical and slaughterhouse wastewaters as well as in the municipal influents. Compared to the iDNA-ARGs, the concentrations of exDNA-ARGs were in general lower. Nevertheless, significant higher concentrations for exDNA-associated genes were measured in clinical wastewaters for blaNDM (4.07 ± 0.15 log gene copies (GC)/L) and blaVIM-2 (6.0 ± 0.2 log GC/L). Overall, our results suggest that depending on the origin of wastewater and its treatment methods, exDNA represents an important reservoir for ARGs, particularly in clinical wastewater.

Marine bacteria harbor the sulfonamide resistance gene sul4 without mobile genetic elements.

Marine bacteria are possible reservoirs of antibiotic-resistance genes (ARGs) originating not only from clinical and terrestrial hot spots but also from the marine environment. We report here for the first time a higher rate of the sulfonamide-resistance gene sul4 in marine bacterial isolates compared with other sul genes. Among four sulfonamide-resistance genes (sul1, sul2, sul3, and sul4), sul4 was most abundant (45%) in 74 sulfonamide-resistant marine isolates by PCR screening. The order of abundance was sul4 (33 isolates) >sul2 (6 isolates) >sul3 (5 isolates) >sul1 (1 isolate). Whole-genome sequencing of 23 isolates of sul4-expressing α- and γ-proteobacteria and bacilli revealed that sul4 was not accompanied by known mobile genetic elements. This suggests that sul4 in these marine isolates is clonally transferred and not horizontally transferable. Folate metabolism genes formed a cluster with sul4, suggesting that the cluster area plays a role in folate metabolism, at which sul4 functions as a dihydropteroate synthase. Thus, sul4 might be expressed in marine species and function in folate synthesis, but it is not a transferable ARG.

Integrative conjugative elements mediate the high prevalence of tmexCD3-toprJ1b in Proteus spp. of animal source.

Integrative conjugative elements (ICEs) are important mobile elements that are associated with the dissemination of antibiotic resistance genes (ARGs) in Proteus. Recent studies demonstrated that the tigecycline resistance gene cluster tmexCD-toprJ has emerged in ICEs of Proteus. However, the prevalence of tmexCD-toprJ positive Proteus from animal sources is unclear. To cover the gap, a total of 762 Proteus spp. were isolated from animal source from six provinces of China to identify the tmexCD-toprJ positive isolates. Eight tmexCD-toprJ positive isolates were identified, with the tmexCD-toprJ positive rate of 1.05% against all Proteus spp. and 2.79% against ICE-bearing Proteus spp. The tmexCD-toprJ gene cluster in these Proteus spp. were tmexCD3-toprJ1b and all of them were carried by ICEs. Genetic structure analysis showed that tmexCD3-toprJ1b-bearing ICEs were complicated and plastic, but the tmexCD3-toprJ1b was specifically integrated into variable region III (VRIII) of ICEs with the help of integrases. Furthermore, we found that the umuC gene, which is presented in VRIII of ICEs and other genetic structures of many other bacterial genomes, was a hotspot for the integration of tmexCD-toprJ-bearing potential mobile elements. In summary, our results suggested that Proteus were important reservoirs for tmexCD-toprJ due to the high prevalence of ICEs. Therefore, continuous surveillance of ICEs-associated ARGs in Proteus is necessary and significant for controlling their future transmission. IMPORTANCE The emergence and spread of tmexCD-toprJ have greatly weakened the function of tigecycline. Although studies have demonstrated the significance of Proteus as carriers for tmexCD-toprJ, the epidemic mechanism and characteristics of tmexCD-toprJ in Proteus remain unclear. Herein, we deciphered that the umuC gene in VRIII of SXT/R391 ICEs was a hotspot for the integration of tmexCD3-toprJ1b-bearing mobile genetic elements by genomic analysis. The mobilization and dissemination of tmexCD3-toprJ1b in Proteus were mediated by highly prevalent ICEs. Furthermore, the co-occurrence of tmexCD3-toprJ1b-bearing ICEs with other chromosomally encoded multidrug resistance gene islands warned that the chromosomes of Proteus are significant reservoirs of ARGs. Overall, our results provide significant insights for the prevention and control of tmexCD3-toprJ1b in Proteus.

Fate of high-risk antibiotic resistance genes in large-scale aquaculture sediments: Geographical differentiation and corresponding drivers

Antibiotic resistance genes (ARGs), emerging environmental contaminants, have become challenges of public health security. However, the distribution and drivers of ARGs, especially high-risk ARGs, in large-scale aquaculture sediments remain unknown. Here, we collected sediment samples from 40 crayfish ponds in seven main crayfish culture provinces in China and then investigated the distribution and risk of ARGs based on high-throughput sequencing and quantitative PCR techniques. Our results suggested that aquaculture sediment was potential reservoir of ARGs and the abundance of aadA-02 was the highest. High-risk ARG (floR) was also prevalent in the sediment and was the most abundant in Jiangsu Province, where opportunistic pathogens were also enriched. The abundance of floR was positively correlated with different environmental factors, such as total phosphorus in water and total carbon in sediment. In addition, Mycobacterium sp., opportunistic pathogenic bacteria, might be potential host for floR. Furthermore, the potential propagation pathway of ARGs was from sediment to crayfish gut, and Bacteroidetes and Proteobacteria might be the main bacterial groups responsible for the proliferation of ARGs. Generally, our results illustrate that pond sediment may be an ARG reservoir of aquatic animals. Meanwhile, our study helps develop valuable strategies for accessing risks and managing ARGs.

Antibiotic resistance indicator genes in biofilm and planktonic microbial communities after wastewater discharge.

The spread of bacteria with antibiotic resistance genes (ARGs) in aquatic ecosystems is of growing concern as this can pose a risk of transmission to humans and animals. While the impact of wastewater treatment plant (WWTP) effluent on ARG abundance in surface waters has been studied extensively, less is known about the fate of ARGs in biofilms. The proximity and dense growth of microorganisms in combination with the accumulation of higher antibiotic concentrations in biofilms might render biofilms a reservoir for ARGs. Seasonal parameters such as water temperature, precipitation, and antibiotic concentrations should be considered as well, as they may further influence the fate of ARGs in aquatic ecosystems. Here we investigated the effect of WWTP effluent on the abundance of the sulfonamide resistance genes sul1 and sul2, and the integrase gene intI1 in biofilm and surface water compartments of a river in Germany with a gradient of anthropogenic impact using quantitative PCR. Furthermore, we analyzed the bacterial community structure in both compartments via 16S rRNA gene amplicon sequencing, following the river downstream. Additionally, conventional water parameters and sulfonamide concentrations were measured, and seasonal aspects were considered by comparing the fate of ARGs and bacterial community diversity in the surface water compartment between the summer and winter season. Our results show that biofilm compartments near the WWTP had a higher relative abundance of ARGs (up to 4.7%) than surface waters (<2.8%). Sulfonamide resistance genes were more persistent further downstream (>10 km) of the WWTP in the hot and dry summer season than in winter. This finding is likely a consequence of the higher proportion of wastewater and thus wastewater-derived microorganisms in the river during summer periods. We observed distinct bacterial communities and ARG abundance between the biofilm and surface water compartment, but even greater variations when considering seasonal and spatiotemporal parameters. This underscores the need to consider seasonal aspects when studying the fate of ARGs in aquatic ecosystems.

Detection of tetracycline resistance genes and their diversity in Escherichia coli isolated from pig farm waste in Banten province, Indonesia.

Livestock waste in the form of feces and liquid represents an important reservoir of antibiotic resistance genes (ARGs). Because many ARGs can be horizontally transferred to other pathogens, livestock waste plays an essential role in the emergence and transmission of various ARGs in the environment. Therefore, this study aimed to detect and assess the diversity of tet genes in Escherichia coli isolated from pig farm waste in Banten province, Indonesia. Solid waste (feces) and wastewater were collected from 44 pig farms in Banten province. The isolation and identification of E. coli referred to the Global Tricycle Surveillance extended-spectrum beta-lactamase E. coli World Health Organization (2021) guidelines. tet genes were detected using quantitative real-time polymerase chain reaction after dividing pig farms in the province into four clusters based on their adjacent areas and characteristics. tetA, tetB, tetC, tetM, tetO, and tetX were detected in solid waste and wastewater from pig farms, whereas tetE was not detected in either sample type. tetX (100%) and tetO (75%) were the most dominant genes in solid waste, whereas wastewater samples were dominated by tetA, tetM, tetO, and tetX (prevalence of 50% each). Furthermore, eight tet gene patterns were found in pig farm waste (prevalence of 12.5% each). The results showed a high prevalence of tetO and tetX in solid waste and wastewater from pig farms in Banten province. This significant prevalence and diversity indicated the transmission of tet genes from pigs to the environment, posing a serious threat to public health.

Identification of antibiotic resistant bacteria communities and a GeoChip based study of resistome in Fusarium wilt diseased and healthy soil

Soil plays a crucial role as a significant reservoir for antibiotic resistance genes (ARGs). Despite the extensive research conducted in this area, there remains a need for further investigation, particularly concerning diverse types of agricultural soil. This comprehensive study examined Fusarium wilt diseased and healthy soil samples to investigate the spectrum of ARGs and identify bacteria potentially harboring these genes. The level of antibiotic resistance (AR) in the two soil types was detected by using culture plate counts (PC). We quantified and identified antibiotic resistant bacteria (ARB) against nine antibiotics. From the PC results, the relative number of ARB (number of ARB/total number culturable bacteria) resistant to erythromycin, lincomycin, sulfonamides, streptomycin sulfate, kanamycin, and enrofloxacin in the diseased soil was significantly higher than that in the healthy soil (One-way ANOVA, p < 0.01). The GeoChip (version 5.0) was employed to identify ARGs and antibiotic biosynthetic genes (ABGs). Interestingly, despite the presence of similar ARGs in both soil types, the Fusarium wilt diseased soil demonstrated a significantly elevated abundance of the tetracycline (tetX) resistance gene compared to the healthy soil. A robust correlation (r ≥ 0.8) between multidrug efflux pumps and ARGs indicates that natural antibiotics as selective pressures for transportation mechanisms. Our analysis identified diverse ARB phylotypes and multiple ARGs in both soil types. Incorporating these soil types into ARGs risk assessments and relevant monitoring is essential for a comprehensive understanding of AR dynamics in various environments.

Phenotypic and genotypic study of antibiotic-resistant Escherichia coli isolates from a wastewater treatment plant in Zulia state, Venezuela.

Antibiotic-resistance in bacteria is a global health problem, and wastewater treatment plants can play a role in their dissemination. In this work, we used PCR and plasmid transformation to characterize antibiotic-resistance and the phylogenetic groups of Escherichia coli isolated from a treatment plant in Zulia, a state in western Venezuela. Thirty-six bacteria isolates were analyzed, of which 27 resulted resistant by disc diffusion primarily to tetracycline and sulfisoxazole but also to trimethoprim, chloramphenicol, ampicillin, and cip-rofloxacin. The tetA, sul2, floR, and blaTEM resistance genes were frequently present and, in most cases, transferable. dfrA12, tetB, sul3, sul1, and aadA2genes also were detected. The integrase gene intI1 was common in multidrug-resistant isolates. These results suggest that E. coli from the treatment plant is a reservoir of antibiotic-resistance genes, which signify a potential health threat. Additionally, the phylogroup C was predominant, which is unusual and may represent an adaptation of this group to environmental conditions or per-haps the most frequent phylogroup entering from the influent.

Metagenomic analysis reveals the dissemination mechanisms and risks of resistance genes in plateau lakes

Antibiotic resistance genes (ARGs) are emerging as environmental pollutants that can persist and disseminate in aquatic environments. Lakes, as important sources of freshwater, also serve as potential natural reservoirs of ARGs. In this study, we analyzed the distribution and potential risks of resistance genes in five typical freshwater lakes on the Yunnan-Guizhou Plateau. Our findings revealed that multidrug and MLS ARGs dominated in the studied lakes. Notably, while Lugu Lake exhibited higher abundance of ARGs, mobile genetic elements (MGEs), and metal resistance genes (MRGs), a greater resistome risk was observed in the eutrophic Xingyun Lake. The dissemination processes of ARGs and MRGs are primarily driven by microbial communities and the horizontal gene transfer via MGEs. Limnohabitans, Flavobacterium, and Acinetobacter were identified as key players in the dissemination of ARGs. Our study highlights the persistence of ARGs and provides valuable baseline data and risk assessment of ARGs in plateau freshwater lakes.

Dynamics of antibiotic resistance genes and bacterial community during pig manure, kitchen waste, and sewage sludge composting

Organic solid wastes (OSWs) are important reservoirs for antibiotic resistance genes (ARGs). Aerobic composting transforms OSWs into fertilizers. In this study, we investigated ARGs dynamics and their driving mechanisms in three OSW composts: pig manure (PM), kitchen waste (KC), and sewage sludge (SG). The dominant ARGs were different in each OSW, namely tetracycline, aminoglycoside, and macrolide resistance (PM); tetracyclines and aminoglycosides (KC); and sulfonamides (SG). ARGs abundance decreased in PM (71%) but increased in KC (5.9-fold) and SG (1.3-fold). Interestingly, the ARGs abundance was generally similar in all final composts, which was contributed to the similar bacterial community in final composts. In particular, sulfonamide and β-lactam resistant genes removed (100%) in PM, while sulfonamide in KC (38-fold) and tetracycline in SG (5-fold) increased the most. Additionally, ARGs abundance rebounded during the maturation period in all treatments. Firmicutes, Proteobacteria, and Actinobacteria were the main ARGs hosts. Several persistent and high-risk genes included tetW, aadA, aadE, tetX, strB, tetA, mefA, intl1, and intl2. The structural equation models showed ARGs removal was mainly affected by physicochemical parameters and bacterial communities in PM, the ARGs enrichment in KC composting correlated with increased mobile genetic elements (MGEs). In general, thermophilic aerobic composting can inhibit the vertical gene transfer (VGT) of pig manure and horizontal gene transfer (HGT) of sludge, but it increases the HGT of kitchen waste, resulting in a dramatic increase of ARGs in KC compost. More attention should be paid to the ARGs risk of kitchen waste composting.

Unveiling the Antibiotic Susceptibility and Antimicrobial Potential of Bacteria from Human Breast Milk of Pakistani Women: An Exploratory Study.

Human life quality and expectancy have increased dramatically over the past 5 decades because of improvements in nutrition and antibiotic's usage fighting against infectious diseases. Yet, it was soon revealed that the microbes adapted to develop resistance to any of the drugs that were used. Recently, there is great concern that commensal bacteria from food and the gastrointestinal tract of humans and animals could act as a reservoir for antibiotic resistance genes. Methodology. This study was intended for evaluating the phenotypic antibiotic resistance/sensitivity profiles of probiotic bacteria from human breast milk and evaluating the inhibitory effect of the probiotic bacteria against both Gram-negative and Gram-positive bacteria. The results point out that some of the isolated bacteria were resistant to diverse antibiotics including gentamycin, imipenem, trimethoprim sulfamethoxazole, and nalidixic acid. Susceptibility profile to certain antibiotics like vancomycin, tetracycline, ofloxacin, chloramphenicol, streptomycin, rifampicin, and bacitracin was also observed. The antimicrobial qualities of cell-free supernatants of some probiotic bacteria inhibited the growth of indicator bacteria. Also, antimicrobial properties of the probiotic bacteria from the present study attributed to the production of organic acid, bacterial adhesion to hydrocarbons (BATH), salt aggregation, coaggregation with pathogens, and bacteriocin production. Some isolated bacteria from human milk displayed higher hydrophobicity in addition to intrinsic probiotic properties like Gram-positive classification, catalase-negative activity, resistance to gastric juice (pH 2), and bile salt (0.3%) concentration. This study has added to the data of the antibiotic and antimicrobial activity of some probiotic bacteria from some samples of Pakistani women breast milk. Probiotic bacteria are usually considered to decrease gastrointestinal tract diseases by adhering to the gut epithelial and reducing population of pathogens and in the case of Streptococcus lactarius MB622 and Streptococcus salivarius MB620 in terms of hydrophobicity and exclusion of indicator pathogenic strains.

Metagenomics analysis of the neonatal intestinal resistome.

The intestinal microbiome forms a major reservoir for antibiotic resistance genes (ARGs). Little is known about the neonatal intestinal resistome. The objective of this study was to investigate the intestinal resistome and factors that influence the abundance of ARGs in a large cohort of neonates. Shotgun metagenomics was used to analyse the resistome in stool samples collected at 1 week of age from 390 healthy, term-born neonates who did not receive antibiotics. Overall, 913 ARGs belonging to 27 classes were identified. The most abundant ARGs were those conferring resistance to tetracyclines, quaternary ammonium compounds, and macrolide-lincosamide-streptogramin-B. Phylogenetic composition was strongly associated with the resistome composition. Other factors that were associated with the abundance of ARGs were delivery mode, gestational age, birth weight, feeding method, and antibiotics in the last trimester of pregnancy. Sex, ethnicity, probiotic use during pregnancy, and intrapartum antibiotics had little effect on the abundance of ARGs. Even in the absence of direct antibiotic exposure, the neonatal intestine harbours a high abundance and a variety of ARGs.

Accelerated dissipation, soil microbial toxicity and dispersal of antimicrobial resistance in soils repeatedly exposed to tiamulin, tilmicosin and sulfamethoxazole

The application of manures leads to the contamination of agricultural soils with veterinary antibiotics (VAs). These might exert toxicity on the soil microbiota and threaten environmental quality, and public health. We obtained mechanistic insights about the impact of three VAs, namely, sulfamethoxazole (SMX), tiamulin (TIA) and tilmicosin (TLM), on the abundance of key soil microbial groups, antibiotic resistance genes (ARGs) and class I integron integrases (intl1). In a microcosm study, we repeatedly treated two soils (differing in pH and VA dissipation capacity) with the studied VAs, either directly or via fortified manure. This application scheme resulted in accelerated dissipation of TIA, but not of SMX, and accumulation of TLM. Potential nitrification rates (PNR), and the abundance of ammonia-oxidizing microorganism (AOM) were reduced by SMX and TIA, but not by TLM. VAs strongly impacted the total prokaryotic and AOM communities, whereas manure addition was the main determinant of the fungal and protist communities. SMX stimulated sulfonamide resistance, while manure stimulated ARGs and horizontal gene transfer. Correlations identified opportunistic pathogens like Clostridia, Burkholderia-Caballeronia-Paraburkholderia, and Nocardioides as potential ARG reservoirs in soil. Our results provide unprecedented evidence about the effects of understudied VAs on soil microbiota and highlight risks posed by VA-contaminated manures. Environmental implicationThe dispersal of veterinary antibiotics (VAs) through soil manuring enhances antimicrobial resistance (AMR) development and poses a threat to the environment and the public health. We provide insights about the impact of selected VAs on their: (i) microbially-mediated dissipation in soil; (ii) ecotoxicity on the soil microbial communities; (iii) capacity to stimulate AMR. Our results (i) demonstrate the effects of VAs and their application-mode on the bacterial, fungal, and protistan communities, and on the soil ammonia oxidizers; (ii) describe natural attenuation processes against VA dispersal, (iii) depict potential soil microbial AMR reservoirs, essential for the development of risk assessment strategies.

Escherichia ruysiae May Serve as a Reservoir of Antibiotic Resistance Genes across Multiple Settings and Regions.

Gut colonization with multidrug-resistant Enterobacterales (MDR-Ent) has reached worrisome levels worldwide. In this context, Escherichia ruysiae is a recently described species mostly found in animals. However, its spread and impact on humans is poorly understood. A stool sample from a healthy individual living in India was screened for the presence of MDR-Ent using culture-based methods. Colonies were routinely identified using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and phenotypically characterized by broth microdilution. Illumina and Nanopore whole-genome sequencing (WGS) platforms were implemented to generate a complete assembly. E. ruysiae genomes deposited in international databases were used for a core genome phylogenetic analysis. An extended-spectrum β-lactamase (ESBL)-producing E. coli strain (S1-IND-07-A) was isolated from the stool. WGS confirmed that S1-IND-07-A was indeed E. ruysiae, belonged to sequence type 5792 (ST5792), core genome (cg) ST89059, serotype O13/O129-:H56-like, clade IV phylogroup, and possessed five virulence factors. A copy of blaCTX-M-15 and five other antimicrobial resistance genes (ARGs) were detected in a conjugative IncB/O/K/Z plasmid. A database search identified 70 further E. ruysiae strains from 16 countries (44, 15, and 11 strains isolated from animals, the environment, and humans, respectively). The core genome phylogeny revealed five major STs: ST6467, ST8084, ST2371, ST9287, and ST5792. Three out of the seventy strains possessed important ARGs: OTP1704 (blaCTX-M-14; ST6467), SN1013-18 (blaCTX-M-15; ST5792), and CE1758 (blaCMY-2; ST7531). These strains were of human, environmental, and wild animal origin, respectively. E. ruysiae may acquire clinically important ARGs and transmit them to other species. Due to its zoonotic potential, further efforts are needed to improve routine detection and surveillance across One Health settings. IMPORTANCE Escherichia ruysiae is a recently described species of the cryptic clades III and IV of the genus Escherichia and is commonly found in animals and the environment. This work highlights the zoonotic potential of E. ruysiae, as it has been shown to colonize the human intestinal tract. Importantly, E. ruysiae may be associated with conjugative plasmids carrying clinically relevant antibiotic resistance genes. Therefore, it is important to closely monitor this species. Overall, this study highlights the need for improved identification of Escherichia species and continued surveillance of zoonotic pathogens in One Health settings.

Antibiotic resistance genes in bacteriophages from wastewater treatment plant and hospital wastewaters

Antibiotic resistant bacteria (ARB) are a major health risk caused particularly by anthropogenic activities. Acquisition of antibiotic resistances by bacteria is known to have happened before the discovery of antibiotics and can occur through different routes. Bacteriophages are thought to have an important contribution to the dissemination of antibiotic resistance genes (ARGs) in the environment. In this study, seven ARGs (blaTEM, blaSHV, blaCTX-M, blaCMY, mecA, vanA, and mcr-1) were investigated, in the bacteriophage fraction, in raw urban and hospital wastewaters. The genes were quantified in 58 raw wastewater samples collected at five WWTPs (n = 38) and hospitals (n = 20). All genes were detected in the phage DNA fraction, with the bla genes found in higher frequency. On the other hand, mecA and mcr-1 were the least frequently detected genes. Concentrations varied between 102 copies/L and 106 copies/L. The gene coding for the resistance to colistin (mcr-1), a last-resort antibiotic for the treatment of multidrug-resistant Gram-negative infections, was identified in raw urban and hospital wastewaters with positivity rates of 19 % and 10 %, respectively. ARGs patterns varied between hospital and raw urban wastewaters, and within hospitals and WWTP. This study suggests that phages are reservoirs of ARGs, and that ARGs (with particularly emphasis on resistance to colistin and vancomycin) in the phage fraction are already widely widespread in the environment with potential large implications for public health.

The diurnal fluctuation of colonic antibiotic resistome is correlated with nutrient substrates in a pig model

The increasing prevalence of antimicrobial resistance (AMR) poses a significant threat to public health, and the gut microbiota of livestock (e.g., pigs) are considered a crucial reservoir of antibiotic resistance genes (ARGs), contributing to the long-term persistence of AMR. However, there is still a lack of relevant research on the composition and diurnal fluctuation of ARGs, and their correlation with nutrient substrates in the gut of pigs. To address this knowledge gap, we characterized the antibiotic resistome structure, and circadian oscillations in 45 colonic metagenomically sequenced samples, covering 9-time points within 24 h, from growing pigs. We identified 227 unique types of ARGs, which belonged to 35 drug resistance classes. Tetracycline resistance and antibiotic target protection were the most enriched class and mechanism of drug resistance in colon samples, respectively. The relative abundance of ARGs fluctuated over time within 24 h, with the total abundance peaking at T21 (sampling time at 21:00 p.m.) and the total numbers reaching the peak at T15. A total of 70 core ARGs were identified, which contributed to 99 % of all ARGs. Rhythmicity analysis revealed that 50 out of 227 ARGs and 15 of 49 mobile genetic elements (MGEs) exhibited rhythmic patterns. TetW was the most abundant ARG with circadian rhythm frequently found in Limosilactobacillus reuteri. The concentration of ammonia nitrogen in the colon was significantly correlated with the host genera of rhythmic ARGs. Partial least squares path modeling (PLS-PM) analysis indicated that rhythmic ARGs were significantly correlated with bacterial community, MGEs, and colonic ammonia nitrogen. This study provides new insight into the diurnal fluctuation of ARG profiles in the colon of growing pigs, which was likely driven by the dynamic change of the availability of colonic nutrients substrates.

Resistance to Antibiotics by Enteric Bacteria Associated with the Swine Industry: in silico Exploration of the Distribution of Resistance Genes

Abstract Multidrug-resistant (MDR) bacteria are a significant contributor to the global antibiotic resistance crisis, which is predicted to kill more people than cancer by 2050. Livestock production is a contributing factor as it has been one of the fastest-growing industries in the previous century but has the most harmful effects on the environment and human health. The pig is the most widely raised and consumed food-producing animal globally, with an upward trend. The derived residues and the meat products constitute an important reservoir of antibiotic resistance genes (ARGs) that can be transmitted to humans through consumption, direct contact, the environment, or poor handling, leading to relevant zoonotic diseases, especially enteric ones. It is essential to know the diversity, abundance, and distribution of ARGs to have better control and monitoring of their dispersion. In the present study, the ARGs and Mobile Genetic Elements (MGEs) of five enteric and pathogenic species commonly present in the microbiota of both pigs and humans were examined by bioinformatic analysis. This analysis showed that 157 ARGs were distributed across 1869 genomes of five bacterial species, ranked from highest to lowest diversity of ARGs: Klebsiella pneumoniae, Escherichia coli, Enterococcus faecium, Salmonella enterica, and Enterococcus faecalis. This study contributes to better management of antibiotics, which directly impact the health of both humans and animals.

High prevalence and genomic characteristics of carbapenem-resistant Enterobacteriaceae and colistin-resistant Enterobacteriaceae from large-scale rivers in China

The widespread presence of carbapenem-resistant Enterobacteriaceae (CRE) and mcr-positive Escherichia coli (MCREC) poses a huge threat to both animal and human health. River water environments are vital reservoirs of antibiotic resistance genes, however, the prevalence and characteristics of CRE and MCREC from large-scale rivers in China have not been reported. In the current study, we sampled 86 rivers from four cities in Shandong Province, China in 2021 and analyzed the prevalence of CRE and MCREC. The blaNDM/blaKPC-2/mcr-positive isolates were characterized with methods including PCR, antimicrobial susceptibility testing, conjugation, replicon typing, whole-genome sequencing and phylogenetic analysis. We found that the prevalence of CRE and MCREC in 86 rivers was 16.3% (14/86) and 27.9% (24/86), respectively and eight rivers carried both mcr-1 and blaNDM/blaKPC-2. A total of 48 Enterobacteriaceae isolates (10 ST11 Klebsiella pneumoniae with blaKPC-2, 12 blaNDM-positive E. coli and 26 MCREC carrying only mcr-1) were obtained in this study and 47 displayed multidrug resistance (MDR). Notably, 10 of the 12 blaNDM-positive E. coli isolates also harbored the mcr-1 gene. The blaKPC-2 gene was located within mobile element ISKpn27-blaKPC-2-ISKpn6 on novel F33:A-:B- non-conjugative MDR plasmids in ST11 K. pneumoniae. The dissemination of blaNDM was mediated by transferable MDR IncB/O plasmids or IncX3 plasmids while mcr-1 was primarily disseminated by highly similar IncI2 plasmids. Notably, these waterborne IncB/O, IncX3 and IncI2 plasmids were all highly similar to previously identified plasmids from animal and human isolates. A phylogenomic analysis revealed that the CRE and MCREC isolates from water environments might be derived from animals and trigger infections in humans. The high prevalence of CRE and MCREC in large-scale environmental rivers is alarming and needs sustained surveillance due to the potential risk for transmission to humans via the food chain (irrigation) or direct contact.

Seasonal and spatial variations of antibiotic resistance genes and bacterial biodiversity in biofilms covering the equipment at successive stages of drinking water purification

The presence of ARGs (antibiotic resistance genes) in the aquatic environment is a serious threat to public health especially in environmental biofilms as natural reservoirs of ARGs in water treatment plants (WTP). It has been shown that the treatment technology and source of water have a significant impact on the abundance and type of genes determining antibiotic resistance. The following indicator genes were proposed that should absolutely be controlled in environmental biofilms: intl1, sul2, sul1, tetA, blaOXA, and blaTEM. In both studied WTPs, the highest number of copies was determined for the intI1 gene. Among the tested ARGs, the highest values were obtained for genes sul1 and tetA. The qPCR analysis also revealed that the amounts of determined ARGs decreased in the following order: sulphonamides>carbapenems >tetracyclines > β-lactams >macrolides. The dominant bacterial types in all analysed samples were Proteobacteria and Bacteroidetes. Both ARGs and bacterial biodiversity was determined rather by sampling site (spatial variation) than seasonality. The obtained results show that biofilms are reservoirs of ARGs. This may affect the microbiological quality of water entering the water system. It is therefore necessary to include their analysis in the classical studies of water quality.

Doxycycline induces the rebound of three tetracycline resistance genes during maturation of laying hen manure composting by increasing the abundance of potential host bacteria

Livestock manure is considered as an important reservoir of antibiotic resistance genes (ARGs). Antibiotics used in livestock production can stress microorganisms and lead to the spread of ARGs, yet composting is viewed as an effective means of reducing ARGs. However, it remains unclear why ARGs typically decline at high composting temperatures but rebound during compost maturation with varied concentrations of antibiotics. Two concentrations of doxycycline (5 and 20 mg/kg) were added to manure of laying hen mixed with sawdust for composting to evaluate changes in ten tetracycline resistance genes (TRGs) and two mobile genetic elements (MGEs), and to investigate the rebound of TRGs during compost maturation. The results showed that the total absolute abundances of TRGs continuously decreased in all compost treatments. Compared with the start (day 0), the relative abundance of tetA, tetG and tetX in the three compost treatments significantly decreased at thermophilic stage (day 3-day 8), but rebounded in the maturation stage (day 9-day 25) (P < 0.01). 16S rRNA sequencing showed that the relative abundance of Proteobacteria and Bacteroidetes in the three groups decreased significantly at thermophilic stage (day 3-day 8) compared with the mesophilic stage (day 0-day 2) and then increased significantly during the maturation stage (day 9-day 25) (P < 0.05), while Firmicutes showed the opposite trend. Pearson correlation analysis showed the strong correlation between the rebound TRGs and major bacteria, such as Proteobacteria and Bacteroidetes, under doxycycline stress. It suggested that the potential host bacteria caused the rebound of TRGs during compost maturation under doxycycline stress. RDA and VPA analysis showed that Marinimicrobium, Pusillimonas and Sphingobacterium contributed to the rebound of tetA, tetG and tetX. This study provides insights into the rebound of ARGs typically seen during the compost maturation stage and a reference for evaluating the risk of spreading ARGs in the process of manure composting treatment.