Epimerization effects on coordination behaviours of phenanthroline-based phosphine-oxide ligands with uranyl ions.

Epimers of the (1,10-phenanthroline-2,9-diyl)bis(ethyl(phenyl)phosphine oxide) (Et-Ph-BPPhen) ligand with two chiral centers (R,R/S,S and R,S) were synthesized. The configurational effects on the coordination ability and mechanism between these epimeric ligands and uranyl ions were thoroughly investigated. This work is helpful to reveal the effects of different conformations of epimeric ligands on their coordination properties.

Antimicrobial resistance spectrum and virulence characterization of Escherichia coli, Klebsiella pneumoniae and Proteus mirabilis isolated from asymptomatic and diarrheal rhesus monkeys.

This study aims to deepen our understanding of the drug resistance and virulence characterization among gut bacteria in asymptomatic and diarrheal captive rhesus macaques (RMs). A total of 31 samples, including 8 asymptomatic RMs, 10 diarrheal RMs, and 1 dead RM, were collected from a breeding base in Sichuan, China, for bacterial isolation. As a result, Escherichia coli (n = 23), Klebsiella (n = 22), Proteus mirabilis (n = 10), Enterococcus (n = 10), Salmonella (n = 2), and Staphylococcus (n = 2) were isolated. All isolates were subjected to antimicrobial susceptibility testing and whole-genome sequencing, among which some E. coli, K. pneumoniae, and P. mirabilis were subjected to the Galleria mellonella and mice infection testing. The antimicrobial resistance rates of levofloxacin, enrofloxacin, and cefotaxime in diarrhea-associated isolates were higher than those of asymptomatic isolates. Consistent with the antimicrobial resistance phenotype, diarrheal isolates had a higher prevalence rate to qnrS1, blaTEM-1B and blaCTX-M-27 than asymptomatic isolates. Furthermore, compared with asymptomatic isolates, diarrheal isolates demonstrated a higher pathogenic potential against larvae and mice. Additionally, sequence types (STs) 14179-14181 in E. coli and ST 625 and ST 630-631 in Klebsiella aerogenes were firstly characterized. Our evidence underscores the considerable challenge posed by high rates of bacterial drug resistance in the effective treatment of diarrheal RMs.

Increased mutations in lipopolysaccharide biosynthetic genes cause time-dependent development of phage resistance in Salmonella.

Understanding how bacteria evolve resistance to phages has implications for phage-based therapies and microbial evolution. In this study, the susceptibility of 335 Salmonella isolates to the wide host range Salmonella phage BPSELC-1 was tested. Potentially significant gene sets that could confer resistance were identified using bioinformatics approaches based on phage susceptibility phenotypes; more than 90 potential antiphage defense gene sets, including those involved in lipopolysaccharide (LPS) biosynthesis, DNA replication, secretion systems, and respiratory chain, were found. The evolutionary dynamics of Salmonella resistance to phage were assessed through laboratory evolution experiments, which showed that phage-resistant mutants rapidly developed and exhibited genetic heterogeneity. Most representative Salmonella hosts (58.1% of 62) rapidly developed phage resistance within 24 h. All phage-resistant mutant clones exhibited genetic heterogeneity and observed mutations in LPS-related genes (rfaJ and rfaK) as well as other genes such as cellular respiration, transport, and cell replication-related genes. The study also identified potential trade-offs, indicating that bacteria tend to escape fitness trade-offs through multi-site mutations, all tested mutants increased sensitivity to polymyxin B, but this does not always affect their relative fitness or biofilm-forming capacity. Furthermore, complementing the rfaJ mutant gene could partially restore the phage sensitivity of phage-resistant mutants. These results provide insight into the phage resistance mechanisms of Salmonella and the complexity of bacterial evolution resulting from phage predation, which can inform future strategies for phage-based therapies and microbial evolution.

Reference gene catalog and metagenome-assembled genomes from the gut microbiome reveal the microbial composition, antibiotic resistome, and adaptability of a lignocellulose diet in the giant panda.

The giant panda, a strict herbivore that feeds on bamboo, still retains a typical carnivorous digestive system. Reference catalogs of microbial genes and genomes are lacking, largely limiting the antibiotic resistome and functional exploration of the giant panda gut microbiome. Here, we integrated 177 fecal metagenomes of captive and wild giant pandas to construct a giant panda integrated gene catalog (GPIGC) comprised of approximately 4.5 million non-redundant genes and reconstruct 393 metagenome-assembled genomes (MAGs). Taxonomic and functional characterization of genes revealed that the captivity of the giant panda significantly changed the core microbial composition and the distribution of microbial genes. Higher abundance and prevalence of antibiotic resistance genes (ARGs) were detected in the guts of captive giant pandas, and ARG distribution was influenced by geography, for both captive and wild individuals. Escherichia, as the prevalent genus in the guts of captive giant pandas, was the main carrier of ARGs, meaning there is a high risk of ARG transmission by Escherichia. We also found that multiple mcr gene variants, conferring plasmid-mediated mobile colistin resistance, were widespread in the guts of captive and wild giant pandas. There were low proportions of carbohydrate-active enzyme (CAZyme) genes in GPIGC and MAGs compared with several omnivorous and herbivorous mammals. Many members of Clostridium MAGs were significantly enriched in the guts of adult, old and wild giant pandas. The genomes of isolates and MAGs of Clostridiaceae harbored key genes or enzymes in complete pathways for degrading lignocellulose and producing short-chain fatty acids (SCFAs), indicating the potential of these bacteria to utilize the low-nutrient bamboo diet. Overall, our data presented an exhaustive reference gene catalog and MAGs in giant panda gut and provided a comprehensive understanding of the antibiotic resistome and microbial adaptability for a high-lignocellulose diet.

Antimicrobial resistance characteristics and phylogenetic relationships of pleuromutilin-resistant Enterococcus isolates from different environmental samples along a laying hen production chain.

Antimicrobial resistance in the laying hen production industry has become a serious public health problem. The antimicrobial resistance and phylogenetic relationships of the common conditional pathogen Enterococcus along the laying hen production chain have not been systematically clarified. 105 Enterococcus isolates were obtained from 115 environmental samples (air, dust, feces, flies, sewage, and soil) collected along the laying hen production chain (breeding chicken, chick, young chicken, and commercial laying hen). These Enterococcus isolates exhibited resistance to some clinically relevant antibiotics, such as tetracycline (92.4%), streptomycin (92.4%), and erythromycin (91.4%), and all strains had multidrug resistance phenotypes. Whole genome sequencing characterized 29 acquired antibiotic resistance genes (ARGs) that conferred resistance to 11 classes of antibiotics in 51 pleuromutilin-resistant Enterococcus isolates, and lsa(E), which mediates resistance to pleuromutilins, always co-occurred with lnu(B). Alignments with the Mobile Genetic Elements database identified four transposons (Tn554, Tn558, Tn6261, and Tn6674) with several ARGs (erm(A), ant(9)-la, fex(A), and optrA) that mediated resistance to many clinically important antibiotics. Moreover, we identified two new transposons that carried ARGs in the Tn554 family designated as Tn7508 and Tn7492. A complementary approach based on conventional multi-locus sequence typing and whole genome single nucleotide polymorphism analysis showed that phylogenetically related pleuromutilin-resistant Enterococcus isolates were widely distributed in various environments on different production farms. Our results indicate that environmental contamination by antimicrobial-resistant Enterococcus requires greater attention, and they highlight the risk of pleuromutilin-resistant Enterococcus and ARGs disseminating along the laying hen production chain, thereby warranting effective disinfection.

Whole genome sequence of Streptococcus pluranimalium SP21-2, a porcine strain harbouring optrA and lsa(E) with chromosomal location.

OBJECTIVES: The aim of this study was to characterise the whole genome sequence of multidrug-resistant Streptococcus pluranimalium strain SP21-2 of swine origin in China. METHODS: Illumina Miseq (200X coverage) and Nanopore PromethION platform (100X coverage) were used for genome sequencing. Rapid Annotation using Subsystem Technology (RAST) was used to annotate the genome of SP21-2. The antimicrobial resistance genes (ARGs) were identified using ResFinder-4.1. RESULTS: The assembled circular genome of S. pluranimalium SP21-2 was 1,987,058 bp in length with a GC content of 39.54%, and no plasmid sequence was detected. A total of 2086 coding sequences were predicted by RAST. Oxazolidinone-phenicol resistance gene, optrA, and pleuromutilin-lincosamide-streptogramin A resistance gene, lsa(E), are both located on chromosomes, associated with IS1216 and ISS1S, respectively. In addition, SP21-2 harbours lnu(B) (lincosamide), ant (6)-Ia and aac(6')-aph(2") (aminoglycoside), erm(B) (macrolide), and tet(O) (tetracycline). CONCLUSION: We firstly report the oxazolidinone-phenicol gene, optrA, and pleuromutilin-lincosamide-streptogramin A resistance gene, lsa(E), in S. pluranimalium. In this strain, we firstly identified ISS1S and IS1216 carrying ARGs in S. pluranimalium, which will provide a valuable reference to understanding potential transfer mechanisms of ARGs in S. pluranimalium.

Prevalence of pleuromutilin antibiotic resistance genes in different laying hen breeding stages in China.

Antimicrobial resistance is a rapidly evolving and extremely complex issue, particularly due to the use of various types of antimicrobials within human, animal, and environmental sectors. Pleuromutilin antibiotics are used to prevent and control respiratory diseases in the rearing stage of hen chicks, but the current status of pleuromutilin resistance in the laying hen breeding process is unclear. ATP-binding cassette transporters encoded by lsa(A), lsa(E), lsa(C), and vga(D) can be transferred by plasmids and transposons, thereby posing a potential dissemination risk. To investigate pleuromutilin resistance genes in the laying hen production chain in China, 95 samples from five environmental types were collected in four breeding stages to determine the abundances of the main resistance genes by qPCR, i.e. lsa(A), lsa(E), lsa(C), and vga(D). The abundance (5.16 log10GC/g) and detection rate (100%) of lsa(E) was highest in all of the samples, thereby suggesting high contamination with the lsa(E) gene across the large-scale laying hen breeding environment and feces. The lsa(A) (6.02 log10GC/g) and lsa(E) (6.18 log10GC/g) genes were most abundant in flies, and the abundance of vga(D) (4.50 log10GC/g) was highest in dust (P < .05). In addition to feces, flies and dust were important sources of contamination with pleuromutilin resistance along the laying hen production chain. In summary, we determined the abundances of four pleuromutilin resistance genes in the laying hen production chain and provided direct evidence of pleuromutilin resistance transmission and environmental contamination. In particular, the chicken breeding stage needs further attention.

Organophosphorus Extractants: A Critical Choice for Actinides/Lanthanides Separation in Nuclear Fuel Cycle.

The separation of actinides from lanthanides in spent nuclear fuel reprocessing is a vital step of nuclear fuel cycle process. As one class of mature industrial extractants, the organophosphorus extractants have been widely used for the extraction and separation of actinides and lanthanides in spent fuel reprocessing due to their strong extraction ability and low-cost acquisition. In this concept, the application scope of tributyl phosphate (TBP), bis(2-ethylhexyl) phosphate (HDEHP), octyl(phenyl)-N,N-diisobutylcarbamoylmethylphosphine oxide (CMPO), trialkyl phosphine oxide (TRPO), and purified Cyanex 301 (bis(2,4,4-trimethylpentyl) dithiophosphinic acid, HA301) are introduced, and their extraction mechanism, as well as structure-function relationships for separation of actinides over lanthanides are also discussed. Furthermore, the design criteria, extraction properties and mechanism of several typical newly developed organophosphorus extractants (CMPO-modified calixarene/pillarene, phenanthroline-derived organophosphorus extractants, and phosphate-modified carborane) based on pre-organized skeletons are briefly reviewed. Finally, the important role played by those organophosphorus extractants is emphasized and potential applications in separation of actinides over lanthanides in future advanced nuclear fuel cycle are identified.

Prevalence and Characterization of Salmonella Isolated from Chickens in Anhui, China.

Salmonella is one of the most important zoonotic pathogens that can cause both acute and chronic illnesses in poultry flocks, and can also be transmitted to humans from infected poultry. The purpose of this study was to investigate the prevalence, antimicrobial resistance, and molecular characteristics of Salmonella isolated from diseased and clinically healthy chickens in Anhui, China. In total, 108 Salmonella isolates (5.66%) were successfully recovered from chicken samples (n = 1908), including pathological tissue (57/408, 13.97%) and cloacal swabs (51/1500, 3.40%), and S. Enteritidis (43.52%), S. Typhimurium (23.15%), and S. Pullorum (10.19%) were the three most prevalent isolates. Salmonella isolates showed high rates of resistance to penicillin (61.11%), tetracyclines (47.22% to tetracycline and 45.37% to doxycycline), and sulfonamides (48.89%), and all isolates were susceptible to imipenem and polymyxin B. In total, 43.52% isolates were multidrug-resistant and had complex antimicrobial resistance patterns. The majority of isolates harbored cat1 (77.78%), blaTEM (61.11%), and blaCMY-2 (63.89%) genes, and the antimicrobial resistance genes in the isolates were significantly positively correlated with their corresponding resistance phenotype. Salmonella isolates carry high rates of virulence genes, with some of these reaching 100% (invA, mgtC, and stn). Fifty-seven isolates (52.78%) were biofilm-producing. The 108 isolates were classified into 12 sequence types (STs), whereby ST11 (43.51%) was the most prevalent, followed by ST19 (20.37%) and ST92 (13.89%). In conclusion, Salmonella infection in chicken flocks is still serious in Anhui Province, and not only causes disease in chickens but might also pose a threat to public health security.

Assembly of novel microbial genomes from gut metagenomes of rhesus macaque (Macaca mulatta).

Rhesus macaque (RM, Macaca mulatta), as an important model animal, commonly suffers from chronic diarrheal disease, challenging the breeding of RMs. Gut microbiomes play key roles in maintaining intestinal health of RMs. However, it is still unclear about more features of gut microbiome as responsible for intestinal health of RMs. In this study, we performed de novo assembly of metagenome-assembled genomes (MAGs) based on fecal metagenomes from chronic diarrheal RMs and asymptomatic individuals. In total of 731 non-redundant MAGs with at least 80% completeness were reconstructed in this study. More than 97% MAGs were novel genomes compared with more than 250,000 reference genomes. MAGs of Campylobacter and Helicobacteraceae from RM guts mainly carried flagella-associated virulence genes and chemotaxis-associated virulence genes, which might mediate motility and adhesion of bacteria. Comparing to MAGs of Campylobacter from humans, distributions and functions of these MAGs of Campylobacter from RMs exhibited significant differences. Most members of Bacteroidota, Spirochaetota, Helicobacteraceae, Lactobacillaceae and Anaerovibrio significantly decreased in guts of chronic diarrhea RMs. More than 92% MAGs in this study were not contained in 2,985 MAGs previously reported from other 22 non-human primates (NHPs), expanding the microbial diversity in guts of NHPs. The distributions and functions of gut microbiome were prominently influenced by host phylogeny of NHPs. Our results could help to more clearly understand about the diversity and function of RMs gut microbiome.

Deciphering risks of resistomes and pathogens in intensive laying hen production chain.

Antimicrobial resistance (AMR) and pathogens derived from food animals and their associated environments have emerged as challenging threats to humans from a health perspective, but our understanding of these risks and their key prevention and control points in the current intensive breeding industry remains poor. By creating an integral composition and risk profile of the resistome and microbiome through metagenomics in feces, flies, dust, sewage, and soil along the four-stage laying hen production chain, we found that the whole production chain is a hotspot for antimicrobial resistance genes (ARGs) with 374 known subtypes and pathogens, including 157 human pathogenic bacteria (HPB). Feces and flies were identified as major risk sources for these contaminations. Also, we confirmed a twin-risk of AMR and pathogenicity prevailing throughout the chain, but with different frequencies in each stage; thus, high-risk ARGs in the young chicken stage and highly prioritized HPB in the chick stage contributed 37.33 % to the total AMR risk and 36.36 % to the pathogenic risks, respectively, thus rendering the two stages to be the key prevention points. Moreover, the prevalence of 112 binned ARG supercarriers (for example, Klebsiella pneumoniae harboring 20 ARGs) was unraveled along the production chain, especially in feces, flies, and dust, and 87 potential hosts exhibited high pathogenic risk, high-risk AMR, or both, with 262 ARGs and 816 virulence factor genes. Overall, this study provides first-hand comprehensive data on high-risk ARGs and their pathogenic hosts in the intensive laying hen production chain, and thus is fundamentally important for developing new measures to help control the global AMR crisis induced through the animal-environment-human pathway.

Exploring the abundance and influencing factors of antimicrobial resistance genes in manure plasmidome from swine farms.

Plasmids play a critical role in the dissemination of antimicrobial resistance genes (ARGs), however, a systematical understanding of ARGs originated from plasmids in swine production is currently lacking. Herein, quantitative polymerase chain reaction was applied to determine the prevalence of ten ARGs and the class1 integron gene intI1 of plasmid source in swine manure from 44 farms in Sichuan, Hubei and Hebei provinces, China. All assayed ARGs were observed in plasmid DNA samples, and the average absolute abundance of aac(6')-Ib-cr, blaNDM, blaCTX-M, optrA, ermB, floR, mcr-1, qnrS, tetM, sul1 and intI1 were 7.09, 2.90, 4.67, 6.62, 7.55, 7.14, 4.08, 4.85, 7.16, 7.11 and 8.07 of 10 log copies/gram, respectively. IntI1 showed a high correlation (r > 0.8, P < 0.01) with the abundance of aac(6')-Ib-cr and sul1 in swine manure. Moreover, the farm scale (i.e., herd population) and geographical location were not found to be critical factors influencing the absolute abundance of ARGs of plasmid DNA in swine farms. However, the concentrations of florfenicol, Cu, Zn, Fe, total phosphorus (TP) and total potassium (TK) demonstrated a significant correlation with the abundance of several ARGs. Particularly, Cu and Zn had high correlations with optrA and blaCTX-M, respectively. Our results demonstrated that antibiotics, heavy metals and environmental nutrients are likely jointly contributing to the long-term persistence of ARGs in swine production. This study provides insights into the abundance and influencing factors of ARGs from swine manure, which is of significance for assessing and reducing the public health risks in livestock production.

Evaluating Salmonella pullorum dissemination and shedding patterns and antibody production in infected chickens.

BACKGROUND: Pullorum disease caused by Salmonella pullorum is one of the most important infectious diseases in the poultry industry, responsible for causing substantial economic losses globally. On farms, the traditional method to detect S. pullorum infection mainly involves the collection of feces and sera to test for antigens and antibodies, respectively, but the regularity of Salmonella pullorum dissemination in internal organs and shedding patterns and antibody production in infected chickens remains unclear. Herein we aimed to investigate the dissemination of S. pullorum to different organs and bacterial shedding patterns in the faeces as well as serum antibody production post-infection in chickens of different ages. RESULT: In this study, the liver and heart of 2-day-old chickens showed the highest copy numbers of S. pullorum at 6.4 × 106 and 1.9 × 106 copies of DNA target sequences/30 mg, respectively. In case of 10-day-old chickens, the percentage of S. pullorum fecal shedding (0%-40%) and antibody production (0%-56.6%) markedly fluctuated during the entire experiment; furthermore, in case of 42-week-old chickens, the percentage of birds showing S. pullorum shedding in the faeces showed a downward trend (from 63.33% to 6.6% in the oral inoculation group and from 43.3% to 10% in the intraperitoneal injection group), while that of birds showing serum antibody production remained at a high level (38.3% and 80% in the oral inoculation and intraperitoneal injection groups, respectively). We also performed cohabitation experiments, showed that 15% 10-day-old and 3.33% 42-week-old chickens were infected via the horizontal transmission in cohabitation with S. pullorum infected chickens, and revealed a high risk of horizontal transmission of S. pullorum. CONCLUSION: This study systematically evaluated the dissemination of S. pullorum in internal organs and bacterial fecal shedding patterns, and antibody production in infected chickens. Collectively, our findings indicate how to effectively screen S. pullorum-negative chickens on livestock farms and should also help in the development of measures to control and eradicate S. pullorum.

Insights into the fates of plasmids and antimicrobial resistance genes during swine manure treatment and related factors based on plasmidome and metagenome analyses.

Swine manure treatment plants are important reservoirs of plasmid-harboring antibiotic resistance genes (ARGs) and physicochemical contaminants, but the changes in the abundances of plasmids and ARGs, and their interactions with the physicochemical properties of manure, are still unclear. Thus, in the present study, plasmidome and metagenome analyses were conducted for samples collected at different stages in the swine manure treatment process. The results indicated that anaerobic digestion and aerobic digestion were the most efficient stages for reducing the abundances of ARGs in swine manure. However, the plasmids associated with ARGs were not effectively removed in these stages. Through the whole treatment process, the IncL/M, IncQ1, IncHI2A, IncA/C, and IncN plasmid groups had strong correlations (r > 0.8, P < 0.01) with most ARG types, thereby indicating that these plasmids play important roles in the persistence of ARGs in this environment. Furthermore, the pH, total nitrogen, total phosphorus, and four heavy metals (Cu, Zn, As, and Fe) significantly affected the abundances of seven ARG subtypes (tetB(P), ant(6)-Ia, tet44, aph(3'')-Ib, mefB, tet(L), and tet(39)). In particular, florfenicol had the most positive correlations with ARGs. Our results indicated that nutrients, heavy metals, and antibiotics all contributed to the presence and persistence of plasmid-harboring ARGs. This study provides insights into the fate of plasmids and ARGs, and related factors during the swine manure treatment process, thereby facilitating the development of a new treatment technique for removing ARGs and reducing the public health risk associated with livestock production.

The gut microbiome and antibiotic resistome of chronic diarrhea rhesus macaques (Macaca mulatta) and its similarity to the human gut microbiome.

BACKGROUND: Chronic diarrhea is a common disease causing morbidity and mortality of captive rhesus macaques (RMs, Macaca mulatta). Chronic diarrhea in RMs is typically characterized by long-term diarrhea and a weak response to antibiotic treatment. Diarrhea is also a common disease in humans and can cause death. However, the etiology of about half of diarrheal cases of humans is still unclear. Therefore, we performed shotgun metagenomic sequencing to characterize the differences in the gut microbiome and resistome of chronic diarrhea RMs and asymptomatic individuals. RESULTS: Our results showed Lactobacillus spp. (mainly L. johnsonii, L. reuteri and L. amylovorus) were significantly depleted in chronic diarrhea RM guts compared to asymptomatic individuals (5.2 vs 42.4%). Functional annotation of genes suggested these Lactobacillus spp. carried genes involved in the adhesion of intestinal epithelial cells and production of bacteriocin. Chronic diarrhea RM guts also had a significantly greater abundance of many other gut bacteria, including mucin-degrading bacteria and opportunistic pathogens. The metabolic pathways of chronic diarrhea RM gut microbiome were enriched in aerobactin biosynthesis, while the metabolic pathways of asymptomatic RM gut microbiome were enriched in the production of short-chain fatty acids (SCFAs). Chronic diarrhea RM guts had a significantly greater abundance of antibiotic resistance genes (ARGs), such as ermF, aph(3')-IIIa, ermB, and floR. The strains isolated from feces and tissue fluid of chronic diarrhea RMs had higher resistance rates to the majority of tested antibiotics, but not cephamycin and carbapenem antibiotics. Gut microbial composition comparisons showed that several captive nonhuman primate (NHP) guts were more similar to the guts of humans with a non-westernized diet than humans with a westernized diet. Chronic diarrhea RM gut microbiome was strikingly similar to rural-living humans with diarrhea and humans with a non-westernized diet than asymptomatic RMs. CONCLUSIONS: Our results suggested chronic diarrhea significantly altered the composition and metabolic pathways of the RM gut microbiome. The frequent use of antibiotics caused antibiotic resistance in chronic diarrhea RM gut microbiome with serious consequences for individual treatment and survival. The findings of this study will help us to improve the effective prevention and treatment of diarrhea in RMs. Video Abstract.

Florfenicol Enhances Colonization of a Salmonella enterica Serovar Enteritidis floR Mutant with Major Alterations to the Intestinal Microbiota and Metabolome in Neonatal Chickens.

Florfenicol is an important antibiotic commonly used in poultry production to prevent and treat Salmonella infection. However, oral administration of florfenicol may alter the animals' natural microbiota and metabolome, thereby reducing intestinal colonization resistance and increasing susceptibility to Salmonella infection. In this study, we determined the effect of florfenicol (30 mg/kg of body weight) on gut colonization of neonatal chickens challenged with Salmonella enterica subsp. enterica serovar Enteritidis. We then analyzed the microbial community structure and metabolic profiles of cecal contents using microbial 16S amplicon sequencing and liquid chromatography-mass spectrometry (LC-MS) untargeted metabolomics, respectively. We also screened the marker metabolites using a multi-omics technique and assessed the effect of these markers on intestinal colonization by S. Enteritidis. Florfenicol administration significantly increased the loads of S. Enteritidis in cecal contents, spleen, and liver and prolonged the residence of S. Enteritidis. Moreover, florfenicol significantly affected cecal colony structures, with reduced abundances of Lactobacillus and Bacteroidetes and increased levels of Clostridia, Clostridium, and Dorea. The metabolome was greatly influenced by florfenicol administration, and perturbation in metabolic pathways related to linoleic acid metabolism (linoleic acid, conjugated linoleic acid [CLA], 12,13-EpOME, and 12,13-diHOME) was most prominently detected. We screened CLA and 12,13-diHOME as marker metabolites, which were highly associated with Lactobacillus, Clostridium, and Dorea. Supplementation with CLA maintained intestinal integrity, reduced intestinal inflammation, and accelerated Salmonella clearance from the gut and remission of enteropathy, whereas treatment with 12,13-diHOME promoted intestinal inflammation and disrupted intestinal barrier function to sustain Salmonella infection. Thus, these results highlight that florfenicol alters the intestinal microbiota and metabolism of neonatal chickens and promotes Salmonella infection mainly by affecting linoleic acid metabolism. IMPORTANCE Florfenicol is a broad-spectrum fluorine derivative of chloramphenicol frequently used in poultry to prevent/treat Salmonella. However, oral administration of florfenicol may lead to alterations in the microbiota and metabolome in the chicken intestine, thereby reducing colonization resistance to Salmonella infection, and the possible mechanisms linking antibiotics and Salmonella colonization in poultry have not yet been fully elucidated. In the current study, we show that increased colonization by S. Enteritidis in chickens administered florfenicol is associated with large shifts in the gut microbiota and metabolic profiles. The most influential linoleic acid metabolism is highly associated with the abundances of Lactobacillus, Clostridium, and Dorea in the intestine. The screened target metabolites in linoleic acid metabolism affect S. Enteritidis colonization, intestinal inflammation, and intestinal barrier function. Our findings provide a better understanding of the susceptibility of animal species to Salmonella after antibiotic intervention, which may help to elucidate infection mechanisms that are important for both animal and human health.

Withdrawal of Colistin Reduces Incidence of mcr-1-Harboring IncX4-Type Plasmids but Has Limited Effects on Unrelated Antibiotic Resistance.

The global dissemination of plasmid-mediated colistin resistance gene mcr and its variants have posed a great threat to public health. Therefore, the Chinese government banned the use of colistin as a feed additive in livestock in April 2017. To explore the dynamic changes of overall antibiotic resistance genes (ARGs) and phylogenetic relationship of bacteria from a single pig farm before and after the withdrawal of colistin, fecal swab samples were collected from a large-scale pig farm before (n = 32; 2 months pre-withdrawal of colistin) and after withdrawal of colistin (n = 30; 13 months post-withdrawal of colistin). Escherichia coli and Klebsiella pneumoniae were isolated. Whole-genome sequencing (Illumina, MiSeq) was performed to examine ARGs, plasmids and the genetic relationship of the isolates. The overall SNP results indicated all isolates had high genetic diversity, and the evolutionary relationship across isolates was not influenced by the ban of colistin. However, the prevalence of mcr-1.1 (5.6%, p < 0.01) was significantly lower than before the ban (86.4%). Plasmid profiling analysis showed that 17 of 20 (85.0%) observed mcr-1.1 genes reside on IncX4-type plasmids, 16 of which (94.1%) were from isolates before the ban. On the contrary, the presence of blaCTX-M gene was significantly increased (p = 0.0215) post-withdrawal of colistin. Our results showed that withdrawal of colistin reduced the incidence of mcr-1-harboring IncX4-type plasmids, but had limited influences on unrelated ARGs.

Tracking Salmonella enterica by whole genome sequencing of isolates recovered from broiler chickens in a poultry production system.

Salmonella enterica is a major cause of foodborne diseases, and is also an important pathogenic bacterium in poultry industry. Whole genome sequencing (WGS) has become a crucial molecular typing technology used for the surveillance of the pathogenic bacteria. In the present study, we adopted WGS for tracking transmission of S. enterica in the production chain of broiler chickens. A total of 74 S. enterica strains were isolated from the different steps of breeding and slaughtering in a large production enterprise in Sichuan Province, China. The isolation rate of Salmonella was the highest in procedure of defeathering (50.0%) and evisceration (36.7%). Serotype identification showed that 74 Salmonella isolates included 7 serotypes, among which Mbandaka accounted for the highest proportions (35.1%). WGS revealed that 74 strains belonged to 7 different sequence types (STs), as well as 7 different ribosomal STs and 35 core genome STs. cgMLST-based Minimum Spanning Trees and phylogenetic tree based on the SNPs indicated that three serotypes, Mbandaka, Indiana and Kentucky, could be clonally transmitted between broiler farm and slaughterhouse. Heterogeneous resistant phenotypes and genotypes were found in two serotypes, Indiana and Kentucky. Our study indicated WGS in an accurate tool for molecular typing of S. enterica. Routine surveillance of S. enterica in the production chain of broiler chickens is needed.

On-farm soil resistome is modified after treating dairy calves with the antibiotic florfenicol.

We determined the immediate impact of exposure to antibiotic-treated animals on housing soil microbiome and resistome. Fecal (n = 36) and soil (n = 108) samples from dairy calves (n = 6) treated with and without florfenicol over 30 days were collected. There were temporary changes in the gut microbiome of antibiotic-treated calves as measured by Shannon diversity (16S rRNA gene sequencing; P = 0.03), but not in the housing soil microbiome (P > 0.05). Droplet-digital PCR demonstrated that floR gene increased by 1-log in soil exposed to treated animals (P < 0.001), but it remained relatively stable in the control soil whereby calves were not treated with antibiotic. Resistome in exposed soil was largely modified (P = 0.004) with the overall prevalence of antimicrobial resistance genes (ARGs) significantly elevated (3.8-fold increase by day 10; P = 0.01). In addition to florfenicol, enriched ARGs collectively conferring resistance to tetracyclines, aminoglycosides, sulfonamides, elfamycins, macrolides-lincosamides-streptrogramin A/B, and beta-lactams. Quantitative PCR validated that ARGs including str and tetG in soil exposed to florfenicol-treated calves had gradually increased fold-change difference relative to the control soil over time. Moreover, a greater diversity of transferrable ARGs was observed in exposed soil and these were associated with a greater diversity of bacterial species. Evaluation of on-farm effects to soil in situ after exposure to antibiotic-treated animals can help design effective managements to mitigate antibiotic resistance in food-animal production.