Genomic analysis of Clostridium perfringens type D isolates from goat farms.

C. perfringens type D strains are the leading cause of enterotoxaemia in ruminants such as goats, sheep, and cattle. However, there has been no prior research on the genomic characteristics of C. perfringens type D strains from various regions in China. Here, we investigated the antibiotic resistance, genomic characteristics, and phylogenetic relationship of C. perfringens type D isolates recovered from goat farms in Shaanxi, Gansu, and Ningxia provinces. The antibiotic resistance test indicated that the isolates displayed high minimum inhibitory concentration (MIC) values to sulfafurazole, whereas the other antibiotics tested, such as penicillin, enrofloxacin, and florfenicol, worked well on them. Additionally, only tetracycline resistance genes [tetA(P) and tetB(P)] were identified from the isolates. A collective of 13 toxin genes, including etx and cpe were detected among the isolates. Sequence comparison revealed that the etx and cpe genes shared high sequence identities, and they could coexist on a pCW3-like plasmid, representing a potential risk to both animal breeding and public health. Phylogenetic analysis using core genome multi-locus sequence typing (cgMLST) and core genome single nucleotide polymorphisms (SNPs) revealed the close genetic relationship and potential regional/transregional transmission of the C. perfringens type D isolates in Shaanxi and Gansu provinces. Furthermore, pan-genomic analysis suggested the functional differences at the protein-coding gene level, although isolates from the same source shared a close genetic relationship. In conclusion, this study indicated the antibiotic resistance, virulence markers, potential transregional transmission, and genomic diversity of C. perfringens type D strains from various regions in China, which could provide references for the prevention of C. perfringens foodborne diseases and further research.

Oxidized glutathione reverts carbapenem resistance in blaNDM-1-carrying Escherichia coli.

The emergence of drug-resistant Enterobacteriaceae carrying plasmid-mediated ß-lactamase genes has become a significant threat to public health. Organisms in the Enterobacteriaceae family containing New Delhi metallo-ß-lactamase­1 (NDM-1) and its variants, which are capable of hydrolyzing nearly all ß-lactam antibacterial agents, including carbapenems, are referred to as superbugs and distributed worldwide. Despite efforts over the past decade, the discovery of an NDM-1 inhibitor that can reach the clinic remains a challenge. Here, we identified oxidized glutathione (GSSG) as a metabolic biomarker for blaNDM-1 using a non-targeted metabolomics approach and demonstrated that GSSG supplementation could restore carbapenem susceptibility in Escherichia coli carrying blaNDM-1 in vitro and in vivo. We showed that exogenous GSSG promotes the bactericidal effects of carbapenems by interfering with intracellular redox homeostasis and inhibiting the expression of NDM-1 in drug-resistant E. coli. This study establishes a metabolomics-based strategy to potentiate metabolism-dependent antibiotic efficacy for the treatment of antibiotic-resistant bacteria.

Characterization of Lung Microbiomes in Pneumonic Hu Sheep Using Culture Technique and 16S rRNA Gene Sequencing.

Hu sheep, a locally bred species in China known for its high productivity, is currently suffering from pneumonia. Here, we combine high-throughput 16SrRNA gene sequencing and bacterial culturing to examine the bacterial community in pneumonic Hu Sheep lungs (p < 0.05). The results showed that the abundance and diversity of lung bacteria in healthy sheep were significantly higher than those in pneumonia sheep (p = 0.139), while there was no significant difference between moderate and severe pneumonia. Furthermore, the composition of the lung microbiota community underwent significant alterations between different levels of pneumonia severity. The application of LEfSe analysis revealed a notable enrichment of Mannheimiae within the lungs of sheep afflicted with moderate pneumonia (p < 0.01), surpassing the levels observed in their healthy counterparts. Additionally, Fusobacterium emerged as the prevailing bacterial group within the lungs of sheep suffering from severe pneumonia. Integrating the results of bacterial isolation and identification, we conclusively determined that Mannheimia haemolytica was the primary pathogenic bacterium within the lungs of sheep afflicted with moderate pneumonia. Furthermore, the exacerbation of pneumonia may be attributed to the synergistic interplay between Fusobacterium spp. and other bacterial species. Our results provide new insights for guiding preventive and therapeutic measures for pneumonia of different severities in sheep.

Probiotics and vitamins modulate the cecal microbiota of laying hens submitted to induced molting.

Induced molting enables laying hens to relax, restore energy and prolong the laying hen cycle, resolving problems such as poor egg quality and minimizing economic losses caused by rising global feeding costs. However, traditional molting methods may disrupt gut microflora and promote potential pathogens infections. This study used a customized additive with a mixture of probiotics and vitamins to induce molting and examine the cecal microbiota post molting. A total of two hundred 377 day-of-ISA Brown laying hens were randomly assigned to four groups: non-molt with basal diet (C), 12-day feeding restriction (FR) in earlier-molting (B), feed again to 27.12% egg production in middle-molting (A) and reach second peak of egg production over 81.36% in post-molting (D). Sequencing 16S rRNA to analyze cecal microbial composition revealed that there is no significant change in bacterial community abundance post-molting. In contrast to group C, the number of potentially harmful bacteria such as E. coli and Enterococcus was not found to increase in groups B, A, or D. This additive keeps cecal microbiota diversity and community richness steady. In cecal contents, hens in group B had lower Lactobacillus, Lachnospiraceae and Prevotellaceae (vsC, A, and D), no significant differences were found between post-molting and the non-molting. Furthermore, cecal microbiota and other chemicals (antibodies, hormones, and enzymes, etc.) strongly affect immunological function and health. Most biochemical indicators are significantly positively correlated with Prevotellaceae, Ruminococcaceae and Subdoligranulum, while negatively with Phascolarctobacterium and Desulfovibrio. In conclusion, the additive of probiotics and vitamins improved the cecal microbiota composition, no increase in the associated pathogenic microbial community due to traditional molting methods, and enhances hepatic lipid metabolism and adaptive immunological function, supporting their application and induced molting technology in the poultry breeding industry.

The risks of hematological toxicities of nivolumab in cancer patients: A PRISMA-compliant meta-analysis.

BACKGROUND: Nivolumab is the human programmed cell death-1 (PD-1)-blocking antibody showing significant effect in many refractory cancers. However, little is known about its risks of hematological toxicities, rare but clinically serious and potentially life-threatening adverse events. We want to explore whether nivolumab can increase the risks of hematological toxicities compared with other immunotherapy or chemotherapy drugs. METHOD: The databases of PubMed, Embase, Web of science, and CNKI were searched. We used the medical subject heading terms "Nivolumab" plus keyword "Nivolumab" to search studies published from August 1990 to October 2021. For the included articles, we calculated the relative risks and the corresponding 95% confidence intervals (CIs) for the risks of anemia, neutropenia, and leukopenia in patients treated with nivolumab versus control drugs. RESULTS: Five original articles on the nivolumab trials were identified with 2399 patients enrolled in this meta-analysis. The relative risks of anemia, neutropenia, and leukopenia were 0.343 (95% CI: 0.177-0.663; P = .001), 0.020 (95% CI: 0.008-0.053; P = .000), and 0.054 (95% CI: 0.015-0.191; P = .000), respectively. CONCLUSION: The PD-1 inhibitor-nivolumab did not increase the risk of anemia, neutropenia and leukopenia. It may enhance awareness about lower risks of hematological toxicities when choosing nivolumab as PD-1 inhibitor among clinicians.

Evaluation of Toxicity, Bacteriostatic, Analgesic, Anti-Inflammatory, and Antipyretic Activities of Huangqin-Honghua-Pugongying-Jinyinhua Extract.

The extensive use of antibiotics has caused the global spread of multidrug-resistant bacteria and genes, seriously reducing antibiotic efficacy and threatening animal and human health. As an alternative, traditional Chinese veterinary medicine (TCVM) was used in this study for its lack of drug resistance and low toxicity. Huangqin-honghua-pugongying-jinyinhua extract (HHPJE), a novel TCVM, consists of the extracts of Huangqin (Scutellaria baicalensis), Honghua (Carthami Flos), Pugongying (Taraxacum) and Jinyinhua (Lonicerae Japonicae Flos), and was developed to treat bovine mastitis. In this study, we evaluated the toxicity, bacteriostatic, analgesic, anti-inflammatory, and antipyretic activities of HHPJE. Our results show that HHPJE did not show any acute oral toxicity and can be considered safe for oral administration. Additionally, HHPJE possessed a dose-dependent antibacterial effect on Staphylococcus aureus, Escherichia coli, Streptococcus agalactiae and Streptococcus dysgalactiae. HHPJE (60, 30 and 15 g/kg) can reduce the abdominal pain by 44.83 ± 7.69%, 43.15 ± 9.50% and 26.14 ± 4.17%, respectively. The percentages of anti-inflammation inhibition (60, 30 and 15 g/kg) were 35.34 ± 2.17%, 22.29 ± 2.74% and 12.06 ± 3.61%, respectively. The inhibition rates (60, 30 and 15 g/kg) of antipyretic activity were 82.05%, 65.71% and 52.80%, respectively. The evaluation of pharmacodynamics and toxicity indicate that HHPJE possesses significant bacteriostatic, analgesic, anti-inflammatory and antipyretic potential, and also that it is safe for acute oral toxicity, which means it has potential value for treating bovine mastitis in future and alleviating clinical symptoms with no drug resistance or side effects.

Improved Sample Preparation for Untargeted Metabolomics Profiling of Escherichia coli.

Metabolomics is a powerful tool that can systematically describe global changes in the metabolome of microbes, thus improving our understanding of the mechanisms of action of antibiotics and facilitating the development of next-generation antibacterial therapies. However, current sample preparation methods are not efficient or reliable for studying the effects of antibiotics on microbes. In the present study, we reported a novel sample preparation approach using cold methanol/ethylene glycol for quenching Escherichia coli, thus overcoming the loss of intracellular metabolites caused by cell membrane damage. After evaluating the extraction efficiency of several extraction methods, we employed the optimized workflow to profile the metabolome of E. coli exposed to cephalexin. In doing so, we proved the utility of the proposed approach and provided insights into the comprehensive metabolic alterations associated with antibiotic treatment. IMPORTANCE The emergence and global spread of multidrug-resistant bacteria and genes are a global problem. It is critical to understand the interactions between antibiotics and bacteria and find alternative treatments for infections when we are moving closer to a postantibiotic era. It has been demonstrated that the bacterial metabolic environment plays an important role in the modulation of antibiotic susceptibility and efficacy. In the present study, we proposed a novel metabolomic approach for intracellular metabolite profiling of E. coli, which can be used to investigate the metabolite alterations of bacteria caused by antibiotic treatment. Further understanding of antibiotic-induced perturbations of bacterial metabolism would facilitate the discovery of new therapeutic targets and pathways.

Determination of macrolides in animal tissues and egg by multi-walled carbon nanotube-based dispersive solid-phase extraction and ultra-high performance liquid chromatography-tandem mass spectrometry.

A simple and reliable analytical method was developed for the simultaneous determination of 11 macrolides in swine, chicken, bovine, and sheep tissues (muscle, liver, kidney, and fat), as well as eggs. Samples were extracted using a mixture of acetonitrile, ethyl acetate, and methanol; dispersive solid-phase extraction purification was then performed using multi-walled carbon nanotubes as the sorbent. The analytes were separated through ultra-high performance liquid chromatography and detected by electrospray ionization on a triple quadrupole mass spectrometer. The average recoveries ranged from 83.5% to 111.4%; the corresponding intra-day and inter-day relative standard deviations were less than 13.6% and 16.4%, respectively. The limit of detection and quantification of the eggs were 0.1-0.6 and 2.0 µg/kg, respectively. For other tissues, the limits of detection and quantification were 0.1-2.0 µg/kg and 5.0 µg/kg, respectively. The proposed method was successfully employed for the analysis of real samples to demonstrate its applicability.

Occurrence of pharmaceuticals and personal care products in bottled water and assessment of the associated risks.

The occurrence of 187 pharmaceuticals and personal care products (PPCPs) was investigated in bottled water samples (35 and 33 from Chinese and foreign brands, respectively). Forty-four compounds belonging to 14 PPCP categories were detected in 56 of the 68 bottled water samples. Further, more than 35% of water samples contained at least three PPCPs, and in one particular sample, 11 different PPCPs were detected. Macrolides constituted the most prevalent PPCP category, and salbutamol, erythromycin, and azithromycin showed the highest detection frequency (17.6%). The thermal stabilities of the 187 PPCPs were determined, and the results obtained showed that only 35 out of the 187 compounds were degraded by more than 50% after boiling for 5 min. Even though the risk quotients (RQs) of detected PPCPs showed low risk levels, the RQs of 13 compounds with RQs ≥ 0.0001 were 2-4 fold higher in infants than in other life stages. Moreover, further studies are necessary to evaluate the toxicity of PPCP mixtures, the effects of PPCPs on human intestinal microbiota, and their risk of induction of drug-resistant bacteria and drug-resistant genes.

Multi-class analysis of veterinary drugs in eggs using dispersive-solid phase extraction and ultra-high performance liquid chromatography-tandem mass spectrometry.

A multi-residue method has been developed for the identification and quantification of 78 compounds from seven different classes of veterinary drugs in eggs. This method was based on dispersive solid phase extraction where mixed-mode cation exchange sorbent was used to combine the isolation of compounds and sample purification. The analysis was performed using ultra-high performance liquid chromatography-tandem mass spectrometry, and the chromatographic run time of one injection was 9.5 min. The mean recovery ranged from 70.5% to 119.2% and inter-day relative standard deviation was less than 17.0%. The limit of quantification ranged between 0.1 and 1 µg/kg, which was sufficient to support surveillance monitoring. Lastly, the method was successfully used to detect residues of veterinary drug in real samples. The dietary exposure risk was subsequently assessed using the results of the survey, indicating that the evaluated daily intake and percentage of acceptable daily intake were at toxicologically acceptable levels.

Association of florfenicol residues with the abundance of oxazolidinone resistance genes in livestock manures.

Livestock and poultry manures are major reservoirs of antibiotic-resistant bacteria and antibiotic resistance genes (ARGs). Linezolid is a clinical medicine for humans and has never been approved for use in livestock. Interestingly, three linezolid resistance genes (cfr, optrA, and poxtA) have been detected in bacteria of animal origin, arousing public concern. This study investigated the abundance of three ARGs, cfr, optrA, and poxtA, in manures from 157 large-scale farms in China using real-time quantitative polymerase chain reaction. The residual concentrations of linezolid, florfenicol, tiamulin, and valnemulin were determined using ultra-high performance liquid chromatography-tandem mass spectrometry. A total of 140 livestock farms were tested positive for ARGs, and the positive detection rate was 89.17 %. OptrA was the most commonly detected ARG. The diversity and abundance of ARGs were significantly higher in poultry and swine manure than in bovine manure. Redundancy analysis presented a strong association between florfenicol and all the three ARGs targeted in the study, and tiamulin showed a significant correlation with optrA. Our results indicated that the residual concentration of florfenicol had a major effect on the distribution of the three ARGs in livestock manures, and extensive use of florfenicol may lead to the production of linezolid resistance genes.

Determination of cephalexin residual level using ultra-high-performance liquid chromatography-tandem mass spectrometry: Residue depletion study in swine.

An ultra-high-performance liquid chromatography-tandem mass spectrometry method was developed to analyze cephalexin in swine tissues, urine, and feces. Samples were extracted with 1% sulfuric acid, followed by purification using MCX cartridges. Mean recoveries were 95.4%-100.7% with inter-day relative standard deviations of <8.6%. The quantitation limit was 5 µg/kg for fat and urine, and 10 µg/kg for muscle, liver, kidney, and feces. Cephalexin residue depletion was determined using 32 healthy pigs, randomly divided into eight (seven treated and one control) groups. Treated groups were intramuscularly administered 10 mg/kg b.w. five times at 24-h intervals and euthanized 6 h and 1, 2, 3, 5, 7, and 10 days after the last injection. Cephalexin was eliminated rapidly in swine muscle, liver, fat, and feces. The highest concentrations among edible organs were detected in the kidney. Moreover, the longest elimination period of cephalexin in swine was determined in urine. These results indicated that kidney and urine were likely target matrices for cephalexin residue detection in swine.

Determination of Tranquilizers in Swine Urine by Ultra-High-Performance Liquid Chromatography-Tandem Mass Spectrometry.

A rapid, reliable, and sensitive method was developed for the determination of ten tranquilizers in swine urine. Sample preparation was based on solid-phase extraction, which combined isolation of the compounds and sample cleanup in a single step. Separation was performed on a reversed phase C18 column by gradient elution with a chromatographic run time of seven minutes, consisting of 0.1% formic acid in water and acetonitrile as the mobile phase. Multiple reaction monitoring in positive mode was applied for data acquisition. Matrix-matched calibration was used for quantification and good linearity was obtained with coefficients of determination higher than 0.99. The average recoveries of fortified samples at concentrations between 0.05 and 10 µg/L ranged from 85% to 106% with interday relative standard deviations of less than 13% in all cases. The limits of detection and limits of quantification obtained for tranquilizers in the urine were in the ranges of 0.03⁻0.1 µg/L and 0.05⁻0.25 µg/L, respectively. The applicability of the proposed method was demonstrated by analyzing real samples; diazepam was detected at concentrations between 0.3 and 0.6 µg/L.

Confirmatory Analysis of Nitroimidazoles and Hydroxy Metabolites in Honey by Dispersive-Solid Phase Extraction and Ultra-High Performance Liquid Chromatography-Tandem Mass Spectrometry.

An ultra-high performance liquid chromatography-tandem quadrupole mass spectrometry (UHPLC-MS/MS) method was developed and validated for confirmatory analysis of four nitroimidazoles and three hydroxy metabolites in honey. Honey samples were dissolved in 2% formic acid solution and nitroimidazoles and metabolites were isolated and enriched by dispersive-solid phase extraction using mixed-mode strong cation-exchange sorbent. The determination involves separation of analytes on an UHPLC C18 column and detection by multiple reaction monitoring in positive ionization mode. The recovery of the method was ranged from 90.2 to 105.6% with inter-day relative standard deviations of less than 11.2%. The limits of detection and limits of quantification were in the ranges of 0.02⁻0.07 µg/kg and 0.05⁻0.2 µg/kg, respectively. Honey samples from the market were analyzed to demonstrate the applicability of the proposed method.