Priority establishment of soil bacteria in rhizosphere limited the spread of tetracycline resistance genes from pig manure to soil-plant systems based on synthetic communities approach.

The spread of antibiotic resistance genes (ARGs) in agroecosystems through the application of animal manure is a global threat to human and environmental health. However, the adaptability and colonization ability of animal manure-derived bacteria determine the spread pathways of ARG in agroecosystems, which have rarely been studied. Here, we performed an invasion experiment by creating a synthetic communities (SynCom) with ten isolates from pig manure and followed its assembly during gnotobiotic cultivation of a soil-Arabidopsis thaliana (A. thaliana) system. We found that Firmicutes in the SynCom were efficiently filtered out in the rhizosphere, thereby limiting the entry of tetracycline resistance genes (TRGs) into the plant. However, Proteobacteria and Actinobacteria in the SynCom were able to establish in all compartments of the soil-plant system thereby spreading TRGs from manure to soil and plant. The presence of native soil bacteria prevented the establishment of manure-borne bacteria and effectively reduced the spread of TRGs. Achromobacter mucicolens and Pantoea septica were the main vectors for the entry of tetA into plants. Furthermore, doxycycline stress promoted the horizontal gene transfer (HGT) of the conjugative resistance plasmid RP4 within the SynCom in A. thaliana by upregulating the expression of HGT-related mRNAs. Therefore, this study provides evidence for the dissemination pathways of ARGs in agricultural systems through the invasion of manure-derived bacteria and HGT by conjugative resistance plasmids and demonstrates that the priority establishment of soil bacteria in the rhizosphere limited the spread of TRGs from pig manure to soil-plant systems.

Community coalescence and plant host filtering determine the spread of tetracycline resistance genes from pig manure into the microbiome continuum of the soil-plant system.

The spread of livestock manure-borne antibiotic resistance genes (ARGs) into agroecosystems through manure application poses a potential threat to human health. However, there is still a knowledge gap concerning ARG dissemination in coalescing manure, soil and plant microbiomes. Here, we examined the fate of tetracycline resistance genes (TRGs) originating from pig manure microbiomes and spread in the soil-A thaliana system and explored the effects of microbial functions on TRGs spread at different interfaces. Our results indicate that the TRGs abundances in all microbiome continuum of the soil-A. thaliana system were significantly increased with the application of a living manure microbiome, although the addition of manure with both an active and inactive microbiome caused a shift in the microbial community composition. This was attributed to the increasing relative abundances of tetA, tetL, tetM, tetO, tetW and tolC in the system. The application of living manure with DOX residues resulted in the highest relative abundance of total TRGs (3.30×10-3 copies/16S rRNA gene copies) in the rhizosphere soil samples. Community coalescence of the manure and soil microbiomes increased the abundance of Firmicutes in the soil and root microbiome, which directly explains the increase in TRG abundance observed in these interfaces. In contrast, the leaf microbiome differed markedly from that of the remaining samples, indicating strong plant host filtering effects on Firmicutes and TRGs from pig manure. The random forest machine learning model revealed microbial functions and their significant positive correlation with TRG abundance in the microbiome continuum of the system. Our findings revealed that community coalescence is the main driver of TRG spread from manure to the soil and root microbiomes. Plant host filtering effects play a crucial role in allowing certain microbial groups to occupy ecological niches in the leaves, thereby limiting the establishment of manure-borne TRGs in aboveground plant tissues.

Short-, long-read metagenome and virome reveal the profile of phage-mediated ARGs in anoxic-oxic processes for swine wastewater treatment.

Phages are among the most widely spread viruses, but their profiles and the antibiotic resistance genes (ARGs) they carry in swine wastewater remain underexplored. The present study investigated the distribution characteristics of phages and their ARG risk in anoxic/oxic (A/O) wastewater treatment processes of swine farms using short- and long-read metagenome and virome. The results demonstrated that the virome could extract more phage sequences than the total metagenome; thus, it was more suited for studying phages in wastewater settings. Intriguingly, phages had significantly lower abundance of ARG than ARGs harbored by total microorganisms (P < 0.01). Eleven ARGs co-occurred with phages and bacteria (R > 0.6 and P < 0.05), with Siphoviridae being the phage co-occurring with the most ARGs (5). Horizontal gene transfer (HGT) events were observed between Proteobacteria and the major phyla except for Bacteroidota. Furthermore, there were prophage sequences and ARGs on the same contig in bacterial MAGs. These data strongly demonstrate that phages promote horizontal transfer of ARG between bacterial hosts in A/O processes for swine wastewater treatment. Therefore, the risk of phage-mediated horizontal transfer of ARGs cannot be overlooked despite the low abundance of phage ARGs (pARG).

Removal of antibiotic resistance genes during swine manure composting is strongly impaired by high levels of doxycycline residues.

Antibiotic resistance genes (ARGs) are emerging pollutants that enter the farm and surrounding environment via the manure of antibiotic-treated animals. Pretreatment of livestock manure by composting decreases ARGs abundance, but how antibiotic residues affect ARGs removal efficiency remains poorly understood. Here, we explored the fate of the resistome under different doxycycline residue levels during aerobic swine manure composting. Metagenomic sequencing showed that the presence of high levels of doxycycline generally had a higher abundance of tetracycline ARGs, and their dominant host bacteria of Firmicutes, especially Clostridium and Streptococcus, also had limited elimination in composting under high levels of doxycycline stress. Moreover, high levels of doxycycline impaired the removal of the total ARGs number in finished composts, with a removal rate of 51.74 % compared to 63.70 % and 71.52 % for the control and low-level doxycycline manure, respectively. Horizontal gene transfer and strengthened correlations among the bacterial community fostered ARGs preservation at high doxycycline levels during composting. In addition, ARGs carried by both plasmids and chromosomes, such as multidrug ARGs, showed wide host characteristics and rebound during compost maturation. Compared with chromosomes, a greater variety of ARGs on plasmids suggested that the majority of ARGs were characterized by horizontal mobility during composting, and the cross-host characteristics of ARGs during composting deserve further attention. This study provided deep insight into the fate of ARGs under residual antibiotic stress during manure composting and reminded the associated risk for environmental and public health.

Intramuscular therapeutic doses of enrofloxacin affect microbial community structure but not the relative abundance of fluoroquinolones resistance genes in swine manure.

Livestock manure is a major source of veterinary antibiotics and antibiotic resistance genes (ARGs). Elucidation of the residual characteristics of ARGs in livestock manure following the administration of veterinary antibiotics is critical to assess their ecotoxicological effects and environmental contamination risks. Here, we investigated the effects of enrofloxacin (ENR), a fluoroquinolone antibiotic commonly used as a therapeutic drug in animal husbandry, on the characteristics of ARGs, mobile genetic elements, and microbial community structure in swine manure following its intramuscular administration for 3 days and a withdrawal period of 10 days. The results revealed the highest concentrations of ENR and ciprofloxacin (CIP) in swine manure at the end of the administration period, ENR concentrations in swine manure in groups L and H were 88.67 ± 45.46 and 219.75 ± 88.05 mg/kg DM, respectively. Approximately 15 fluoroquinolone resistance genes (FRGs) and 48 fluoroquinolone-related multidrug resistance genes (F-MRGs) were detected in swine manure; the relative abundance of the F-MRGs was considerably higher than that of the FRGs. On day 3, the relative abundance of qacA was significantly higher in group H than in group CK, and no significant differences in the relative abundance of other FRGs, F-MRGs, or MGEs were observed between the three groups on day 3 and day 13. The microbial community structure in swine manure was significantly altered on day 3, and the altered community structure was restored on day 13. The FRGs and F-MRGs with the highest relative abundance were qacA and adeF, respectively, and Clostridium and Lactobacillus were the dominant bacterial genera carrying these genes in swine manure. In summary, a single treatment of intramuscular ENR transiently increased antibiotic concentrations and altered the microbial community structure in swine manure; however, this treatment did not significantly affect the abundance of FRGs and F-MRGs.

Pentachlorophenol affects doxycycline and tetracycline resistance genes in soil by altering microbial structure.

Tetracycline antibiotics play a vital role in animal husbandry, primarily employed to uphold the health of livestock and poultry. Consequently, when manure is reintegrated into farmland, tetracycline antibiotics can persist in the soil. Simultaneously, to ensure optimal crop production, organochlorine pesticides (OCPs) are frequently applied to farmland. The coexistence of tetracycline antibiotics and OCPs in soil may lead to an increased risk of transmission of tetracycline resistance genes (TRGs). Nevertheless, the precise mechanism underlying the effects of OCPs on tetracycline antibiotics and TRGs remains elusive. In this study, we aimed to investigate the effects of OCPs on soil tetracycline antibiotics and TRGs using different concentrations of doxycycline (DOX) and pentachlorophenol (PCP). The findings indicate that PCP and DOX mutually impede their degradation in soil. Furthermore, our investigation identifies Sphingomonas and Bacillus as potential pivotal microorganisms influencing the reciprocal inhibition of PCP and DOX. Additionally, it is observed that the concurrent presence of PCP and DOX could impede each other's degradation by elevating soil conductivity. Furthermore, we observed that a high concentration of PCP (10.7 mg/kg) reduced the content of efflux pump tetA, ribosome protective protein tetM, tetQ, and passivating enzyme tetX. In contrast, a low PCP concentration (6.4 mg/kg) only reduced the content of ribosome protective protein tetQ. This suggests that PCP may reduce the relative abundance of TRGs by altering the soil microbial community structure and inhibiting the potential host bacteria of TRGs. These findings have significant implications in understanding the combined pollution of veterinary antibiotics and OCPs. By shedding light on the interactions between these compounds and their impact on microbial communities, this study provides a theoretical basis for developing strategies to manage and mitigate their environmental impact, and may give some information regarding the sustainable use of antibiotics and pesticides to ensure the long-term health and productivity of agricultural systems.

Pet cats may shape the antibiotic resistome of their owner's gut and living environment.

BACKGROUND: Companion animals can contribute to the physical and mental health of people and often live in very close association with their owners. However, the antibiotic resistome carried by companion animals and the impact they have on their owners and living environment remain unclear. In this study, we compared the ARG profiles of cats, humans, and their living environments using metagenomic analysis to identify the core ARGs in the cat and human gut and explore the potential impact of cats on ARGs in the human gut through the environment. RESULTS: Results showed that the abundance of ARGs in the cat gut was significantly higher than that in the human gut (P < 0.0001), with aminoglycoside and tetracycline resistance genes being the dominant ARGs in the cat gut. There was no significant difference in the abundance of total ARGs in the guts of cat owners and non-owners (P > 0.05). However, the abundance of aminoglycoside resistance genes including APH(2'')-IIa and AAC(6')-Im was significantly higher in cat owners than that in non-cat owners (P < 0.001). Also, ARG abundance was positively correlated with the frequency of cat activity in the living environment. Enterobacteriaceae was the dominant ARG host co-occurring in the cat gut, human gut, and living environment. CONCLUSIONS: Our results show that cats may shape the living environment resistome and thus the composition of some ARGs in the human gut, highlighting the importance of companion animal environment health. Video Abstract.

Chinese Herbal Extracts Mitigate Ammonia Generation in the Cecum of Laying Hens: An In Vitro Study.

The objectives of the study were to screen one or several Chinese herbal extracts with good ammonia emission reduction effects using an in vitro gas production study. The study consisted of a control (without Chinese herbal extract), and 11 experimental groups with added cinnamon extract (CE), Osmanthus extract (OE), tangerine peel extract (TPE), dandelion extract (DE), Coptis chinensis extract (CCE), honeysuckle extract (HE), Pulsatilla root extract (PRE), yucca extract (YE), licorice extract (LE), Ginkgo biloba extract (GBE), or astragalus extract (AE). The results showed that HE, PRE, YE, LE, GBE, and AE significantly reduced ammonia production (p ≤ 0.05). The most significant ammonia inhibition was achieved via AE, resulting in a 26.76% reduction. In all treatments, Chinese herbal extracts had no significant effect on pH, conductivity, or uric acid, urea, and nitrate-nitrogen concentrations (p > 0.05). However, AE significantly reduced urease activity and the relative activity of uricase (p ≤ 0.05). AE significantly increased the relative abundance of Bacteroides and decreased the relative abundance of Clostridium, Desulfovibrio, and Prevotell (p ≤ 0.05). Astragalus extract inhibited ammonia emission from laying hens by changing the gut microbial community structure, reducing the relative abundance of ammonia-producing bacteria, and reducing microorganisms' uricase and urease activities.

Nitrogen removal characteristics of heterotrophic nitrification-aerobic denitrification bacterium Acinetobacter ZQ-A1 and community characteristics analysis of its application in pig farm wastewater.

A heterotrophic nitrifying aerobic denitrifying (HN-AD) strain ZQ-A1 with excellent denitrification performance, identified as Acinetobacter, was isolated from simultaneous nitrification and denitrification (SND) craft. ZQ-A1 was capable of removing NH4+, NO2-, and NO3-; the 21-hour removal rates were 84.84%, 87.13%, and 92.63%. ZQ-A1 has the ability to treat mixed nitrogen sources. In addition, ZQ-A1 can be well applied to actual sewage. According to the analysis of microbial community characteristics, the relative abundance of Acinetobacter in the experimental group increased from 0.06% to 2.38%, which is an important reason for the removal rate of NH4+ exceeding 99% within 30 days. The results of KEGG function prediction showed that with the addition of ZQ-A1, the relative abundance of pathways related to bacterial metabolism, such as tricarboxylic acid cycle metabolism, was higher. The research expanded the thinking of HN-AD bacteria in actual production and laid a foundation for its application in sewage treatment.

The abundance and diversity of antibiotic resistance genes in layer chicken ceca is associated with farm enviroment.

Industrialized layer chicken feedlots harbor complex environmental microbial communities that affect the enrichment and exchange of gut bacteria and antibiotic resistance genes (ARGs). However, the contribution of different environmental sources to the gut ARGs of layer chickens is not clear. Here, layer chicken gut and environmental samples (air, water, feed, cage, feather, maternal hen feces, uropygial glands) were collected during the early 3 month period before the laying of eggs, and the source and characteristics of the gut microorganisms and ARGs were analyzed by performing 16S rRNA and metagenomic sequencing. The results showed that the abundances of Bacteroidetes and Actinobacteria in cecum of layer chickens gradually increased, while that of Proteobacteria decreased with age, and the number and relative abundance of ARGs decreased significantly with age. On day 5, 57% of the layer chicken cecal ARGs were from feather samples, and 30% were from cage samples. Subsequently, the contribution of cage ARGs became progressively more prominent over time. At days 30 and 57, the contribution of cage ARGs to the chick cecal ARGs reached 63.3 and 69.5%, respectively. The bacterial community composition (especially the abundances of Klebsiella pneumoniae and Escherichia coli) was the major factor impacting the ARG profile. K. pneumoniae and E. coli were mainly transmitted from feathers to the layer chicken cecum, and the contribution rates were 32 and 3.4%, respectively. In addition, we observed the transmission of ARG-carrying bacteria (Bacteroides fragilis) from the cage to the gut, with a contribution rate of 11.5%. It is noteworthy that B. fragilis is an opportunistic pathogen that may cause diarrhea in laying hens. These results can provide reference data for the healthy breeding of layer chickens and the prevention and control of ARG pollution.

The tigecycline resistance gene tetX has an expensive fitness cost based on increased outer membrane permeability and metabolic burden in Escherichia coli.

Livestock-derived tetX-positive Escherichia coli with tigecycline resistance poses a serious risk to public health. Fitness costs, antibiotic residues, and other tetracycline resistance genes (TRGs) are fundamental in determining the spread of tetX in the environment, but there is a lack of relevant studies. The results of this study showed that both tetO and tetX resulted in reduction in growth and an increased in the metabolic burden of E. coli, but the presence of doxycycline reversed this phenomenon. Moreover, the protection of E. coli growth and metabolism by tetO was superior to that of tetX in the presence of doxycycline, resulting in a much lower competitiveness of tetX-carrying E. coli than tetO-carrying E. coli. The results of RNA-seq showed that the increase in outer membrane proteins (ompC, ompF and ompT) of tetX-carrying E. coli resulted in increased membrane permeability and biofilm formation, which is an important reason for fitness costs. Overall, the increased membrane permeability and metabolic burden of E. coli is the mechanistic basis for the high fitness cost of tetX, and the spread of tetO may limit the spread of tetX. This study provides new insights into the rational use of tetracycline antibiotics to control the spread of tetX.

The addition of nano zero-valent iron during compost maturation effectively removes intracellular and extracellular antibiotic resistance genes by reducing the abundance of potential host bacteria.

Applying compost to soil may lead to the spread of antibiotic resistance genes (ARGs) in the environment. Therefore, removing ARGs from compost is critical. In this study, for the first time, nano zero-valent iron (nZVI) was added to compost during the maturation stage to remove ARGs. After adding 1 g/kg of nZVI, the abundance of total intracellular and total extracellular ARGs was decreased by 97.62% and 99.60%, and that of total intracellular and total extracellular mobile genetic elements (MGEs) was decreased by 92.39% and 99.31%, respectively. A Mantel test and network analysis indicated that the reduction in potential host bacteria and intI1 after nZVI treatment promoted the removal of intracellular and extracellular ARGs. The addition of nZVI during composting reduced the horizontal transfer of ARGs and improve the total nitrogen and germination index of compost, allowing it to meet the requirements for organic fertilizers.

Doxycycline Attenuates Pig Intestinal Microbial Interactions and Changes Microbial Metabolic Pathways.

Doxycycline is a therapeutic veterinary antibiotic commonly used in pig breeding. In this study, 27 fattening pigs of 33.5 ± 0.72 kg were divided equally into 3 groups. Doxycycline at 0, 3, and 5 mg/kg body weight was added to the feed in groups CK, L and H. The medication and withdrawal periods were set at 5 and 28 days. The results showed that the doxycycline average concentrations in groups L and H during the medication period were 117.63 ± 13.54 and 202.03 ± 24.91 mg/kg dry matter, respectively. Doxycycline levels were lower than the detection limit after 20 days. Doxycycline did not affect the diversity of the intestinal microbial community structure. The relative abundances of Streptococcus were significantly higher in treatment groups than that in group CK, and Alishewanella, Vagococcus, Cloacibacterium, and Campylobacter abundances were significantly positively correlated with doxycycline concentration. Interestingly, the microbiota cooccurrence network suggested that high doxycycline concentration weakened the interactions among bacteria until day 33. Functional prediction showed that doxycycline significantly altered metabolic pathways related to the cell membrane. The results revealed that the use of doxycycline during pig breeding can affect bacterial abundance during the withdrawal period, and it may affect interactions among bacteria and change the intestinal metabolic pathways.

Differences in peripheral and central metabolites and gut microbiome of laying hens with different feather-pecking phenotypes.

Background: Feather pecking (FP) is a maladaptive behavior in laying hens that is associated with numerous physiological traits, including those involving the central neurotransmitter system and the immune system, which have been identified in many species as being regulated by the gut microbiota via the "microbiota-gut-brain" (MGB) axis. Yet, it is unknown whether and how gut microbiota influences FP by regulating multiple central neurotransmission systems and immune system. Methods: This study was measured the prevalence of severe FP (SFP) in the commercial layer farm. The chicken flock with the highest frequency of SFP were selected for FP phenotype identification. Nontargeted metabolomics was performed to investigated the differences in the peripheral and central metabolites and 16S rDNA sequencing was performed to investigated the differences in gut microbiome of laying hens with different FP phenotypes. Correlation analysis was performed to determine the potential mechanism by which the disturbed gut microbiota may modulate host physiology and behavior. Results: The results showed that pullets (12 weeks of age) showed significantly higher SFP frequencies than chicks (6 weeks of age) and adults (22 weeks of age; p < 0.05). Compared to neutrals (N), peckers (P) exhibited the stress-induced immunosuppression with the increased plasma levels of corticosterone and norepinephrine, and the decreased plasma levels of IgA, IL-1, IL-6 and tumor necrosis factor α (p < 0.05). In the cecum, the relative abundances of Bacteroides and Gemmiger were higher in the P group, while Roseburia, Ruminococcus2, Anaerostipes, Lachnospiracea_incertae_sedis and Methanobrevibacter were more enriched in the N group. Moreover, increased plasma levels of L-tryptophan, beta-tyrosine and L-histidine were found in the P group (p < 0.05). Notably, in the P group, hippocampal levels of L-tryptophan, xanthurenic acid, L-histidine and histamine were improved and showed a positive association with L-glutamic acid levels. Plasma levels of L-tryptophan, beta-tyrosine and L-histidine were both positively correlated with Bacteroides abundance but negatively correlated with Methanobrevibacter abundance. Conclusion: Overall, these findings suggest that the development of FP may be affected by the gut microbiota, which regulates the central glutamatergic nerve system by altering the metabolism of tryptophan, histidine and tyrosine.

Reducing light exposure enhances the circadian rhythm of the biological clock through interactions with the gut microbiota.

Light mainly synergistically regulates the central biological clock system. In farming, long-term light exposure may induce metabolic disorders and increase the load on the liver in laying hens. In contrast, intermittent photoperiods can reduce light exposure and increase rest time to improve the health of laying hens. The circadian rhythms of gut microbes are essential for the health of the host. However, the circadian rhythms of gut microbes and how those microbes interact with the host under intermittent photoperiods are not clear. We used laying hens as a model to evaluate the circadian rhythms of gut microbes and biological clock genes under different intermittent photoperiods. Intermittent photoperiod 1 (IP1, 16 [3 h -L/1 h -D]: 8 D) enhanced the circadian rhythms of cBmal1, cBmal2, cCry1, and cCry2 in the hypothalamus and increased the expression of cClock, cBmal1, and cCry2 in the liver and seven clock genes in the cecal wall. The intermittent photoperiod also significantly altered the composition and metabolic function of the cecal microbiota via the melatonin pathway. The concentrations of short-chain fatty acids (SCFAs) and the abundance of SCFA-producing genera such as Odoribacter significantly increased under the IP1 treatment and might have further fed back into and strengthened the peripheral and central rhythms by activating the SCFA receptor gene pathway in cecal wall. These findings clarify the mediation mechanisms for the circadian rhythms of the central circadian clock and highlight the role of intermittent photoperiod-induced regulation of the interaction between the host clock and the cecal microbial community.

Serious Risk of Tigecycline Resistance in Escherichia coli Isolated from Swine Manure.

The emergence of the plasmid-mediated tigecycline resistance gene tetX family in pig farms has attracted worldwide attention. The use of tetracycline antibiotics in pig farms has a facilitating effect on the prevalence of the tetX family, but the relationship among its presence, expression, and resistance phenotype in resistant bacteria is unknown. In this study, the presence and expression characteristics of tetracycline resistance genes (TRGs) in 89 strains of doxycycline-resistant E. coli (DRE) isolated from pig manure samples from 20 pig farms under low concentrations of doxycycline stress (2 µg/mL) were analyzed. The detection rate of tetO was 96.63%, which is higher than those of other TRGs, such as tetA (94.38%), tetX (76.40%), tetB (73.03%), and tet(X4) (69.66%). At least three TRG types were present in DRE strains, which thus showed extensive resistance to tetracycline antibiotics, and 37% of these strains were resistant to tigecycline. In the presence of a low concentration of doxycycline, tetA played an important role, and the expression and existence ratio of TRGs indicated low expression of TRGs. Furthermore, the doxycycline resistance of DRE was jointly determined by the total absolute abundance of TRGs, and the absolute abundance of tetX and tet(X4) was significantly positively associated with tigecycline resistance in DRE (P < 0.05). Overall, DRE isolated from swine manure is an important reservoir of the tetX family, which suggests that DRE in swine manure has a high risk of tigecycline resistance, poses a potential threat to human health, and should be of public concern.

Distribution and driving factors of antibiotic resistance genes in treated wastewater from different types of livestock farms.

Treated wastewater from livestock farms is an important reservoir for antibiotic resistance genes (ARGs), and is a main source of ARGs in the environment. However, the distribution and driving factors of ARGs in treated wastewater from different types of livestock farms are rarely reported. In this study, treated wastewater from 69 large-scale livestock farms of different types, including broiler, layer, and pig farms, was collected, and 11 subtypes of ARGs, 2 mobile genetic elements (MGEs) and bacterial community structure were analyzed. The results revealed detection rates of NDM-1 and mcr-1 of 90 % and 43 %, respectively, and the detection rates of other ARGs were 100 %. The relative abundance of ARGs, such as tetA, tetX and strB, in broiler farms was significantly higher than that in layer farms, but the bacterial α diversity was significantly lower than that in other farm types. Furthermore, although the treatment process had a greater impact on the physicochemical properties of the treated wastewater than the livestock type, livestock type was the main factor affecting the bacterial community in the treated wastewater. The analysis of potential host bacteria of ARGs revealed significant differences in the host bacteria of ARGs in treated wastewater from different types of livestock farms. The host bacteria of ARGs in broiler farms mainly belonged to Actinobacteria, layer farms mainly belonged to Proteobacteria, and pig farms mainly belonged to Firmicutes. Additionally, redundancy analysis showed that the distribution of ARGs may have resulted from the combination of multiple driving factors in different types of livestock farms, among which tnpA and NH4+-N were the main influencing factors. This study revealed multiple driving factors for the distribution of typical ARGs in treated wastewater from different types of livestock farms, providing basic data for the prevention and control of ARG pollution in agricultural environments.

Recovery of the Structure and Function of the Pig Manure Bacterial Community after Enrofloxacin Exposure.

At present, growth-promoting antibiotics are banned in the pig industry in many countries, but therapeutic antibiotics can still be used normally. However, the effect of therapeutic antibiotics on the structure and function of the intestinal bacterial community and its recovery is still unclear. We analyzed the effects of enrofloxacin on the pig manure bacterial community and functional genes during dosing and without dosing. Enrofloxacin caused significant changes in community structure. The changes in the diversity and structure of the bacterial community were the most obvious on the fifth day, and most of the differentially abundant genera (19/29) belonged to Firmicutes. The structure of the manure bacterial community in the low concentration enrofloxacin group was completely reverted after 10 days of drug discontinuation. In addition, enrofloxacin had a significant impact on the abundance of bacterial functional genes. Most of the differentially abundant functional genes of the manure bacterial community were significantly enriched, especially genes related to metabolic pathways, for adaptation to the antibiotic environment. Moreover, exposure to enrofloxacin increased the abundance of functional genes related to nitrogen metabolism in the manure bacterial community, and the total nitrogen content of pig manure was significantly reduced. The functional genetic differences caused by enrofloxacin exposure were completely reverted 10 days after drug discontinuation. The results of the present study suggest that enrofloxacin induces changes in the structure and function of manure bacterial communities, which may be rapidly recovered after drug discontinuation. IMPORTANCE A stable intestinal bacterial community balance is beneficial for animal health. Enrofloxacin is widely used in animal husbandry as a therapeutic drug, but it can cause intestinal environmental imbalance. Enrofloxacin is widely present in groundwater, pork, etc., which leads to a greater risk of human exposure. The effect of enrofloxacin on the structure and function of the intestinal bacterial community and its recovery is still unclear. In this study, we found that enrofloxacin, as a therapeutic drug, can enhance nitrogen metabolism in the manure bacterial community. Moreover, the structure and function of the manure bacterial community in the low concentration enrofloxacin group may be completely reverted 10 days after drug discontinuation. This study provides a reference for the effect of enrofloxacin exposure on the intestinal bacterial community.

Metagenomic assembly reveals the circadian oscillations of the microbiome and antibiotic resistance genes in a model of laying hens.

The increasing risks of antibiotic resistance genes (ARGs) in livestock feces have attracted global attention. However, how the rhythmic activity of ARGs changes in fecal microbiota remains largely unclear. In our study, we collected 52 fresh fecal samples every 6 h over 72 h from laying hens and characterized circadian oscillations of bacteria and ARGs using an approach based on assembled metagenome shotgun sequencing. We found that 14% of commensal bacterial taxonomic units fluctuated over 24 h. A total of 33 out of 281 ARGs and 17 of 574 mobile genetic elements (MGEs) featured rhythmic patterns in feces. lnuC and ANT(6)H-lb were the two most abundant ARGs with circadian oscillation identified from feces, and they increased during the day and decreased at night. Acetate, butyrate, propionate, and 78 out of 392 MetaCyc pathways relating to short-chain fatty acid (SCFA) metabolism featured circadian oscillations. Assessment of the above ARG-other element connections revealed that 17 ARGs presented strong correlations with 7 MGEs, and 2 SCFAs (acetate and propanoate) and bacterial species in feces. Structural equation model (SEM) analysis showed that ARGs were directly affected by microbial ß-diversity and MGEs. These results showed a comprehensive map of ARGs over 24 h and revealed circadian oscillations of ARGs, which are influenced by key bacterial species, MGEs, and metabolites. Together, our findings advance comprehension of circadian oscillations of ARGs in the fecal microbiota and provide a reference for ARGs control and management.

Porcine gut microbiota in mediating host metabolic adaptation to cold stress.

The gut microbiota plays a key role in host metabolic thermogenesis by activating UCP1 and increasing the browning process of white adipose tissue (WAT), especially in cold environments. However, the crosstalk between the gut microbiota and the host, which lacks functional UCP1, making them susceptible to cold stress, has rarely been illustrated. We used male piglets as a model to evaluate the host response to cold stress via the gut microbiota (four groups: room temperature group, n = 5; cold stress group, n = 5; cold stress group with antibiotics, n = 5; room temperature group with antibiotics, n = 3). We found that host thermogenesis and insulin resistance increased the levels of serum metabolites such as glycocholic acid (GCA) and glycochenodeoxycholate acid (GCDCA) and altered the compositions and functions of the cecal microbiota under cold stress. The gut microbiota was characterized by increased levels of Ruminococcaceae, Prevotellaceae, and Muribaculaceae under cold stress. We found that piglets subjected to cold stress had increased expression of genes related to bile acid and short-chain fatty acid (SCFA) metabolism in their liver and fat lipolysis genes in their fat. In addition, the fat lipolysis genes CLPS, PNLIPRP1, CPT1B, and UCP3 were significantly increased in the fat of piglets under cold stress. However, the use of antibiotics showed a weakened or strengthened cold tolerance phenotype, indicating that the gut microbiota plays important role in host thermogenesis. Our results demonstrate that the gut microbiota-blood-liver and fat axis may regulate thermogenesis during cold acclimation in piglets.