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.

Breed differences in the expression levels of gga-miR-222a in laying hens influenced H2S production by regulating methionine synthase genes in gut bacteria.

BACKGROUND: The microbiota in the cecum of laying hens is crucial for host digestion, metabolism, and odor gas production. The results of recent studies have suggested that host microRNAs (miRNAs) can regulate gene expression of the gut microbiota. In the present study, the expression profiles of host-derived miRNAs in the cecal content of two laying hen breeds; Hy-line Gray and Lohmann Pink, which have dissimilar H2S production, were characterized; and their effects on H2S production by regulating the expression of gut microbiota-associated genes were demonstrated. RESULTS: The differential expression of microbial serine O-acetyltransferase, methionine synthase, aspartate aminotransferase, methionine-gamma-lyase, and adenylylsulfate kinase between the two hen breeds resulted in lower H2S production in the Hy-line hens. The results also revealed the presence of miRNA exosomes in the cecal content of laying hens, and an analysis of potential miRNA-target relationships between 9 differentially expressed miRNAs and 9 differentially expressed microbial genes related to H2S production identified two methionine synthase genes, Odosp_3416 and BF9343_2953, that are targeted by gga-miR-222a. Interestingly, in vitro fermentation results showed that gga-miR-222a upregulates the expression of these genes, which increased methionine concentrations but decreased H2S production and soluble sulfide concentrations, indicating the potential of host-derived gga-miR-222a to reduce H2S emission in laying hens. CONCLUSION: The findings of the present study reveal both a physiological role by which miRNAs shape the cecal microbiota of laying hens and a strategy to use host miRNAs to manipulate the microbiome and actively express key microbial genes to reduce H2S emissions and breed environmentally friendly laying hens. Video Abstract.

Microbial Diversity and Community Variation in the Intestines of Layer Chickens.

The intestinal microbiota is increasingly recognized as an important component of host health, metabolism and immunity. Early gut colonizers are pivotal in the establishment of microbial community structures affecting the health and growth performance of chickens. White Lohmann layer is a common commercial breed. Therefore, this breed was selected to study the pattern of changes of microbiota with age. In this study, the duodenum, caecum and colorectum contents of white Lohmann layer chickens from same environment control farm were collected and analyzed using 16S rRNA sequencing to explore the spatial and temporal variations in intestinal microbiota. The results showed that the diversity of the microbial community structure in the duodenum, caecum and colorectum increased with age and tended to be stable when the layer chickens reached 50 days of age and the distinct succession patterns of the intestinal microbiota between the duodenum and large intestine (caecum and colorectum). On day 0, the diversity of microbes in the duodenum was higher than that in the caecum and colorectum, but the compositions of intestinal microbes were relatively similar, with facultative anaerobic Proteobacteria as the main microbes. However, the relative abundance of facultative anaerobic bacteria (Escherichia) gradually decreased and was replaced by anaerobic bacteria (Bacteroides and Ruminococcaceae). By day 50, the structure of intestinal microbes had gradually become stable, and Lactobacillus was the dominant bacteria in the duodenum (41.1%). The compositions of dominant microbes in the caecum and colorectum were more complex, but there were certain similarities. Bacteroides, Odoribacter and Clostridiales vadin BB60 group were dominant. The results of this study provide evidence that time and spatial factors are important factors affecting the intestinal microbiota composition. This study provides new knowledge of the intestinal microbiota colonization pattern of layer chickens in early life to improve the intestinal health of layer chickens.

The microbiota structure in the cecum of laying hens contributes to dissimilar H2S production.

BACKGROUND: Host genotype plays a crucial role in microbial composition of laying hens, which may lead to dissimilar odor gas production. The objective of this study was to investigate the relationship among layer breed, microbial structure and odor production. RESULTS: Thirty Hy-Line Gray and thirty Lohmann Pink laying hens were used in this study to determine the impact of cecal microbial structure on odor production of laying hens. The hens were managed under the same husbandry and dietary regimes. Results of in vivo experiments showed a lower hydrogen sulfide (H2S) production from Hy-Line hens and a lower concentration of soluble sulfide (S2-) but a higher concentration of butyrate in the cecal content of the Hy-Line hens compared to Lohmann Pink hens (P < 0.05), which was consistent with the in vitro experiments (P < 0.05). However, ammonia (NH3) production was not different between genotypes (P > 0.05). Significant microbial structural differences existed between the two breed groups. The relative abundance of some butyrate producers (including Butyricicoccus, Butyricimonas and Roseburia) and sulfate-reducing bacteria (including Mailhella and Lawsonia) were found to be significantly correlated with odor production and were shown to be different in the 16S rRNA and PCR data between two breed groups. Furthermore, some bacterial metabolism pathways associated with energy extraction and carbohydrate utilization (oxidative phosphorylation, pyruvate metabolism, energy metabolism, two component system and secretion system) were overrepresented in the Hy-Line hens, while several amino acid metabolism-associated pathways (amino acid related enzymes, arginine and proline metabolism, and alanine-aspartate and glutamate metabolism) were more prevalent in the Lohmann hens. CONCLUSION: The results of this study suggest that genotype of laying hens influence cecal microbiota, which in turn modulates their odor production. Our study provides references for breeding and enteric manipulation for defined microbiota to reduce odor gas emission.

The metabolism and morphology mutation response of probiotic Bacillus coagulans for lead stress.

Lead is among the most common toxic heavy metals and its contamination is of great public concern. Bacillus coagulans is the probiotic which can be considered as the lead absorption sorbent to apply in the lead contaminant water directly or indirectly. A better understanding of the lead resistance and tolerance mechanisms of B. coagulans would help further its development and utilization. Wild-type Bacillus coagulans strain R11 isolated from a lead mine, was acclimated to lead-containing culture media over 85 passages, producing two lead-adapted strains, and the two strains shown higher lead intracellular accumulation ability (38.56-fold and 19.36-fold) and reducing ability (6.94-fold and 7.44-fold) than that of wild type. Whole genome sequencing, genome resequencing, and comparative transcriptomics identified lead resistance and tolerance process significantly involved in these genes which regulated glutathione and sulfur metabolism, flagellar formation and metal ion transport pathways in the lead-adapted strains, elucidating the relationships among the mechanisms regulating lead deposition, deoxidation, and motility and the evolved tolerance to lead. In addition, the B. coagulans mutants tended to form flagellar and chemotaxis systems to avoid lead ions rather than export it, suggesting a new resistance strategy. Based on the present results, the optimum lead concentration in environment should be considered when employed B. coagulans as the lead sorbent, due to the bacteria growth ability decreased in high lead concentration and physiology morphology changed could reduce the lead removal effectiveness. The identified deoxidization and compound secretion genes and pathways in B. coagulans R11 also are potential genetic engineering candidates for synthesizing glutathione, cysteine, methionine, and selenocompounds.

Bacillus coagulans R11 maintained intestinal villus health and decreased intestinal injury in lead-exposed mice by regulating the intestinal microbiota and influenced the function of faecal microRNAs.

Lead contamination is an environmental problem, especially in developing countries; due to the nondegradable characteristics of lead, it is easily deposited in human and animal bodies by the food chain. Probiotics are regarded as a good tool to remove lead ions in the intestine and maintain gut health conditions, but previous studies failed to elucidate the relationship among probiotics, the host and the gut microbiota. In the present study, B. coagulans R11 was employed as the "lead removal tool" in lead-exposed mouse, and the effects of B. coagulans R11 on intestinal cells, the microbiota and faecal microRNAs were tested. The results indicated that B. coagulans R11 had no negative effects on mouse intestine model cells and helped keep cells in a normal proliferation ratio and reduce the reactive oxygen species and apoptosis ratios under lead exposure conditions. An in vivo mouse experiment also showed that B. coagulans R11 feeding could reduce the intestinal villi damage caused by lead through adjusting the microbiota structure and function, such as increasing the genus abundance of Akkermansia and Alistipes, decreasing the genus abundance of Alloprevotella, Lachnospiraceae, Parabacteroides and Ruminiclostridium, and keeping the protein dltD existing. Host faecal microRNAs may be influenced by lead and B. coagulans R11, which may change the microbiota structure. Thus, B. coagulans R11 has the potential to be developed and considered as the probiotic that protects the host gut against villi damage and gut microbiota structure and function disorders during lead exposure.

Effect of different sulfadimidine addition methods on its degradation behaviour in swine manure.

Sulfadimidine (SM2) is commonly used in the swine industry and enters the environment via faeces. In recent years, advances in the ecotoxicology of SM2 have become a popular research interest with two common research methods including swine manure collection from swine fed with a diet containing SM2 and directly adding SM2. The purpose of this experiment was to compare SM2 degradation behaviour in pig manure with two different SM2 addition methods. The results showed that the degradation half-lives of SM2 in manure from SM2-fed swine treatment were 33.2 and 32.0 days at the initial addition level of SM2 at 32.1 and 64.3 mg/kg, respectively. This was significantly longer than that in manure directly adding SM2 treatment with the half-lives of 21.4 and 14.8 days. The metabolite of SM2 N4-acetyl-sulfamethazine occurred in manure from SM2-fed swine treatment but was not detected in directly adding SM2 treatment. The pH in manure from SM2-fed swine treatment was significantly lower than that in directly adding SM2 treatment, but the values of organic carbon, total nitrogen, and electrical conductivity in manure from SM2-fed swine treatment were significantly higher than those in manure directly adding SM2 treatment. Meanwhile, although the copy number of bacteria had no significant difference between two treatments, there was a significant difference in bacteria diversity. Results of the present study demonstrated that the presence of the metabolites, chemical property, and microbial diversity might be the reason for different SM2 degradation behaviours on different addition methods. Thus, the method using manure with SM2 collected from swine could obtain more accurate results for the ecotoxicological study of SM2.

In vitro fermentative capacity of swine large intestine: comparison between native Lantang and commercial Duroc breeds.

Native Lantang and commercial Duroc pigs were used as animal models to evaluate the differences existing in dietary fiber utilization ability between breeds. Animals were fed the same diet from weaning (4 weeks) to 4 months of age. Neutral detergent fiber (NDF) from wheat bran (as substrate) and fecal samples from the two breeds (as inoculum) were used in an in vitro gas production trial. Results showed that cumulative and maximum gas productions were higher in inocula from Lantang than those from the Duroc breed (P < 0.05). The degradation capacity of NDF for microbiome from Lantang fecal samples were significantly higher compared to Duroc (P < 0.01). The total quantity of short-chain fatty acids and its constituents from the fermentation liquors were different between breeds, suggesting that the dynamic characteristics of fermentation differed between the two breeds. The PCR denaturing gradient gel electrophoresis fingerprint and cluster analysis demonstrated that microbial communities of the two breeds were separated into two clusters and the bacterial community structure of large intestine among the two breed of pigs was different. Our results concluded that Lantang had higher dietary fiber degradation capacity than Duroc pigs, and the higher degradation capacity for the former breed was due to differences in the inherent microbial community in their respective large intestines.

The excretion and environmental effects of amoxicillin, ciprofloxacin, and doxycycline residues in layer chicken manure.

The excretion rates and ecological risk to the environment of three commonly used veterinary antibiotics (VAs), amoxicillin, ciprofloxacin, and doxycycline, in layer hen manure during the application and withdrawal periods were investigated in a study consisting of a control group fed with VA-free basal diet and nine treatment groups consisted of three levels (200 mg/kg, 100 mg/kg, and 50 mg/kg) of amoxicillin (AMX), ciprofloxacin (CIP), or doxycycline (DOC). Each treatment group was replicated seven times with three layer hens per replication. Results of the study showed that the average excretion rates of AMX in the 200, 100, and 50 mg/kg groups were 67.88, 55.82, and 66.15%, respectively, while those for CIP and DOC were 47.84, 51.85, and 44.87% and 82.67, 94.39, and 95.72%, respectively. The concentrations of the above veterinary drugs in manure decreased sharply in the withdrawal period (7, 28, and 10 d, respectively), for AMX, DOC, and CIP. Neither AMX nor DOC was detected in the manure after the withdrawal period. In contrast to AMX and DOC, the excretion rate of CIP was significantly lower and thus had a longer residence time. Ecological risk study, estimated using hazard quotient values, showed that AMX in the 100 and 50 mg/kg groups posed no risk to the environment after d 1 of withdrawal, while CIP in the 50 mg/kg group posed no risk to the environment from d 5 of withdrawal. CIP in the 200 and 100 mg/kg groups required 10 d withdrawal in order to pose no risk to the environment. In contrast, DOC residue during withdrawal in the manure posed no risk to the environment, thus making it more environmentally safe.

Co-addition of manure increases the dissipation rates of tylosin A and the numbers of resistance genes in laboratory incubation experiments.

The behavior of veterinary antibiotics in the soil is commonly studied using the following methods to add antibiotics to the soil: (A) adding manure collected from animals fed a diet that includes antibiotics; (B) adding antibiotic-free animal manure spiked with antibiotics; and (C) the direct addition of antibiotics. However, most studies have only used methods (B) and (C) in their research, and few studies have simultaneously compared the different antibiotic addition methods. This study used tylosin A (TYLA) as a model antibiotic to compare the effects of these three commonly used antibiotic addition methods on the dissipation rates of TYLA and the numbers of resistance genes in laboratory incubation experiments. The results showed that the three treatment methods produced similar TYLA degradation trends; however, there were significant differences (P<0.05) in the TYLA degradation half-life (t1/2) among the three methods. The half-life of TYLA degradation in treatments A, B and C was 2.44 ± 0.04, 1.21 ± 0.03 and 5.13 ± 0.11 days, respectively. The presence of manure resulted in a higher electrical conductivity (EC), higher relative abundance of Citrobacter amalonaticus, higher macrolide resistant gene (ermB, ermF and ermT) count and lower ecological toxicity in the soil, which could partially explain the higher TYLA degradation rate in the treatments containing manure. The higher degradation rate of TYLA in treatment B when compared to treatment A could be due to the lower concentrations of tylosin B (TYLB) and tylosin D (TYLD). The main route for veterinary antibiotics to enter the soil is via the manure of animals that have been administered antibiotics. Therefore, the more appropriate method to study the degradation and ecotoxicity of antibiotic residues in the soil is by using manure from animals fed/administered the particular antibiotic rather than by adding the antibiotic directly to the soil.

Effect of different oxytetracycline addition methods on its degradation behavior in soil.

The degradation behavior of veterinary antibiotics in soil is commonly studied using the following methods of adding antibiotics to the soil: (i) adding manure collected from animals fed with a diet containing antibiotics, (ii) adding antibiotic-free animal manure spiked with antibiotics and (iii) directly adding antibiotics. No research simultaneously comparing different antibiotic addition methods was found. Oxytetracycline (OTC) was used as a model antibiotic to compare the effect of the three commonly used antibiotic addition methods on OTC degradation behavior in soil. The three treatment methods have similar trends, though OTC degradation half-lives show the following significant differences (P<0.05): manure from swine fed OTC (treatment A)

[Development of SNP multiplexes utilizing universal reporter primers for forensic purposes].

OBJECTIVE: To establish a simple, fast and economical technique for multiplex-typing SNPs and to explore its potential forensic application. METHODS: Five Y-SNP loci (IMS-JST164520, IMS-JST021354, IMS-JST003305, M119 and M134) were selected and the allele specific primers of each locus were designed with the universal reporter primers tailed at their 5' end. Alleles of these loci were amplified first by allele specific primers, then amplified by universal reporter primers tagged by fluorescent dye. RESULTS: A fluorescent-multiplex PCR system of the five Y-SNP loci was established. The typing results showed that two different colors of product peaks denoted two different alleles of a SNP locus, and the fragment sizes of alleles among different SNP loci were different. The haplotype diversity of these five loci was estimated to be 0.8655 in Wuhan Han population. CONCLUSION: The multiplex-typing SNPs based on the universal reporter primers is a simple, fast, and economical technique, and may have good application value in forensic medicine.