A broad host phage, CP6, for combating multidrug-resistant Campylobacter prevalent in poultry meat.

Campylobacter is a major cause of bacterial foodborne diarrhea worldwide. Consumption of raw or undercooked chicken meat contaminated with Campylobacter is the most common causative agent of human infections. Given the high prevalence of contamination in poultry meat and the recent rise of multi-drug-resistant (MDR) Campylobacter strains, an effective intervention method of reducing bird colonization is needed. In this study, the Campylobacter-specific lytic phage CP6 was isolated from chicken feces. Phage CP6 exhibited a broad host range against different MDR Campylobacter isolates (97.4% of strains were infected). Some biological characteristics were observed, such as a good pH (3-9) stability and moderate temperature tolerance (<50 ℃). The complete genome sequence revealed a linear double-stranded DNA (178,350 bp, group II Campylobacter phage) with 27.51% GC content, including 209 predicted open reading frames, among which only 54 were annotated with known functions. Phylogenetic analysis of the phage major capsid protein demonstrated that phage CP6 was closely related to Campylobacter phage CPt10, CP21, CP20, IBB35, and CP220. CP6 phage exerted good antimicrobial effects on MDR Campylobacter in vitro culture and reduced CFUs of the host cells by up to 1-log compared with the control in artificially contaminated chicken breast meat. Our findings suggested the potential of CP6 phage as a promising antimicrobial agent for combating MDR Campylobacter in food processing.

Identification of metabolite biomarkers in Salmonella enteritidis-contaminated chickens using UHPLC-QTRAP-MS-based targeted metabolomics.

This study aimed to characterize the metabolic profile of Salmonella enteritidis (S. enteritidis) in chicken matrix and to identify metabolic biomarkers of S. enteritidis in chicken. The UHPLC-QTRAP-MS high-throughput targeted metabolomics approach was employed to analyze the metabolic profiles of contaminated and control group chickens. A total of 348 metabolites were quantified, and the application of deep learning least absolute shrinkage and selection operator (LASSO) modelling analysis obtained eight potential metabolite biomarkers for S. enteritidis. Metabolic abundance change analysis revealed significantly enriched abundances of anthranilic acid, l-pyroglutamic acid, 5-hydroxylysine, n,n-dimethylarginine, 4-hydroxybenzoic acid, and menatetrenone in contaminated chicken samples. The receiver operating characteristic (ROC) curve analysis demonstrated the strong ability of these six metabolites as biomarkers to distinguish S. enteritidis contaminated and fresh chicken samples. The findings presented in this study offer a theoretical foundation for developing an innovative approach to identify and detect foodborne contamination caused by S. enteritidis.

NamiRNA-enhancer network of miR-492 activates the NR2C1-TGF-ß/Smad3 pathway to promote epithelial-mesenchymal transition of pancreatic cancer.

Pancreatic cancer (PaCa) is one of the most fatal malignancies of the digestive system, and most patients are diagnosed at advanced stages due to the lack of specific and effective tumor-related biomarkers for the early detection of PaCa. miR-492 has been found to be upregulated in PaCa tumor tissue and may serve as a potential therapeutic target. However, the molecular mechanisms by which miR-492 promotes PaCa tumor growth and progression are unclear. In this study, we first found that miR-492 in enhancer loci activated neighboring genes (NR2C1/NDUFA12/TMCC3) and promoted PaCa cell proliferation, migration, and invasion in vitro. We also observed that miR-492-activating genes significantly enriched the TGF-ß/Smad3 signaling pathway in PaCa to promote epithelial-mesenchymal transition (EMT) during tumorigenesis and development. Using CRISPR-Cas9 and ChIP assays, we further observed that miR-492 acted as an enhancer trigger, and that antagomiR-492 repressed PaCa tumorigenesis in vivo, decreased the expression levels of serum TGF-ß, and suppressed the EMT process by downregulating the expression of NR2C1. Our results demonstrate that miR-492, as an enhancer trigger, facilitates PaCa progression via the NR2C1-TGF-ß/Smad3 pathway.

Untargeted and targeted metabolomics identify metabolite biomarkers for Salmonella enteritidis in chicken meat.

Salmonella Enteritidis easily contaminate chicken during slaughtering, processing, transportation, and sales, which seriously endangers human health. This study aimed to identify metabolite biomarkers for Salmonella Enteritidis contamination in chicken meat. UPLC-Q-Orbitrap MS untargeted metabolomics analysis identified 441 and 240 confidently metabolites in positive and negative ion mode, respectively. Thirty metabolites were defined as potential biomarkers for Salmonella enteritidis contamination in chicken meat. UPLC-QQQ-MS based targeted metabolomics was used to quantitatively analyze candidate metabolite biomarkers in Salmonella enteritidis contaminated and fresh chicken samples. A total of 10 candidate metabolite biomarkers were confirmed in the validation set, among which acetylcholine, l-Methionine, l-Proline, l-Valine, and l-Norleucine were identified as biomarkers for Salmonella Enteritidis contamination in chicken. The combined receiver operating characteristic curve analysis of the five biomarkers achieved an AUC of 0.956, indicating their high sensitivity and specificity in predicting Salmonella Enteritidis in raw chicken. In conclusion, the present study identified five metabolite biomarkers for Salmonella enteritidis in raw chicken. These results provide a potential theoretical basis for developing Salmonella Enteritidis detection methods in raw chicken.

Review on treatment and utilization of barium slag in China.

Barium slag (BS) is generated as a by-product waste during the production of barium salts from barite. A large amount of BS is discharged annually threating the ecological environment and restricting the development of the barium salts industry. In China, BS is classified as hazardous waste due to its corrosivity, and more importantly because of its extraction toxicity of barium. Soluble barium is toxic and can result in barium poisoning for environment and human beings. The current review presents a detailed summary on general characteristics, discharge and disposal status, harmless treatment pathways and comprehensive utilization of BS in China. BaO, SiO2, CaO, and SO3 occur as main chemical compositions in BS, especially BaO accounting approximately for 35-40%. The mineral compositions include unreacted barite, quartz, clay minerals, newly-formed phases from the side reactions such as BaCO3, BaSiO3 and BaSO3, and residual carbon. A special attention is given to the assessment of the harmless treatment methods for BS from hazardous waste to general waste, which will decrease its management costs. Precipitation and solidification of soluble barium is the common pathway for harmless treatment of BS, and the using of other industrial waste can realize cost-saving. Methods for comprehensive utilization of BS include recovery of barium and carbon, application in building materials, and using as adsorbents for wastewater treatment. In particular, we analyzed and discussed the advantages and disadvantages of these existing process routes, intending to promote potentials for comprehensive utilization of BS in the future.

Detection of AmpC ß-lactamases in gram-negative bacteria.

AmpC ß-lactamase genes are clinically important because they often confer resistance to most ß-lactams other than 4th-generation cephalosporins and carbapenems. However, traditional and existing detection methods are expensive, labor-intensive and range-limited. We established an efficient multiplex PCR method to simultaneously identify six families of ampC ß-lactamase genes, ACC, EBC, CIT, DHA, MOX and FOX, and evaluated the sensitivity and specificity of this assay. The multiplex method could accurately identify ACC, EBC, CIT, DHA, MOX and FOX variants among a total of 175 ampC ß-lactamase genes. The minimum concentration of genomic DNA that could be detected was 1.0×103 copies/µL. We subsequently used this method to analyze 2 Salmonella spp. with carrying CMY-2 and DHA-1, and 167 Enterobacteriaceae isolates in blinded PCR testing. Positive isolates produced bright bands that corresponded with their genotype. Results were in concordance with those of the traditional method but showed increased sensitivity and accuracy. This indicates that the newly developed multiplex PCR system could be used as a diagnostic tool to accurately distinguish the six families of ampC ß-lactamase genes with high efficiency, wide range, easy operation and good discrimination.

Protective effects of fowl-origin cadmium-tolerant lactobacillus against sub-chronic cadmium-induced toxicity in chickens.

Cadmium (Cd) directly endangers poultry health and indirectly causes harm to human health by food chain. Numerous studies have focused on removing Cd using lactic acid bacteria (LAB). However, there is still a lack of in vivo studies to validate whether Cd can be absorbed successfully by LAB to alleviate Cd toxicity. Here, we aimed to isolated and screened poultry-derived Cd-tolerant LAB with the strongest adsorption capacity in vitro and investigate the protective effect of which on sub-chronic Cd toxicity in chickens. First, nine Cd-tolerant LAB strains were selected preliminarily by isolating, screening, and identifying from poultry farms. Next, four strains with the strongest adsorption capacity were used to explore the influence of different physical and chemical factors on the ability of LAB to adsorb Cd as well as its probiotic properties in terms of acid tolerance, bile salt tolerance, drug resistance, and antibacterial effects. Resultantly, the CLF9-1 strain with the best comprehensive ability was selected for further animal protection test. The Cd-tolerant LAB treatment promoted the growth performance of chickens and reduced the Cd-elevated liver and kidney coefficients. Moreover, Cd-induced liver, kidney, and duodenum injuries were alleviated significantly by high-dose LAB treatment. Furthermore, LAB treatment also increased the elimination of Cd in feces and markedly reduced the Cd buildup in the liver and kidney. In summary, these findings determine that screened Cd-tolerant LAB strain exerts a protective effect on chickens against sub-chronic cadmium poisoning, thus providing an essential guideline for the public health and safety of livestock and poultry.

Characterization and phylogenetic evolution of mitochondrial genome in Tibetan chicken.

The objective of this study was to characterize mitochondrial genome and investigate phylogenetic evolution in Tibetan chicken. In this study, four haplotypes were identified based on D-loop sequencing in Tibetan chicken (n = 40), and each representative of four haplotypes was selected for total mitochondrial genome sequencing and analyzed together with published mitochondrial genome data of red jungle fowl. Four haplotypes belonged to three previously published clades, i.e., Clade A, clade B and clade E. Based on D-loop sequencing data, the average haplotype diversity and nucleotide diversity were 0.658 ± 0.065 and 0.00442 ± 0.00094, respectively. The mitochondrial genome of Tibetan chicken is 16,785 bp in size, consisting of 22 transfer RNA (tRNA) genes, two ribosomal RNA (rRNA) genes, 13 protein-coding genes and one non-coding control region (CR). Compared with the mitochondrial genome, a phylogenetic tree based on the D-loop sequence had a messy distribution, and no breed cluster pattern was observed in Tibetan chicken. The results indicate that Tibetan chicken populations in our study have relatively low nucleotide and haplotype diversity and likely share multiple maternal lineages. The D-loop sequence has limited power for the resolution of phylogenetic relationships in comparison with the complete mitochondrial genome.

Aminoglycoside Resistance and Possible Mechanisms in Campylobacter Spp. Isolated From Chicken and Swine in Jiangsu, China.

Campylobacter is a major food-borne pathogen in humans, and previous studies reported a high prevalence of gentamicin-resistant Campylobacter isolates from food-producing animals in China. This study aimed to investigate the aminoglycoside resistance of Campylobacter isolated from chicken and swine in Jiangsu province, China and understand the possible mechanisms responsible for aminoglycoside resistance. One hundred and eighty-five Campylobacter isolates of chicken and swine origins in 2017 and 2018 were analyzed for gentamicin and kanamycin resistance. Some aminoglycoside resistance genes were selected for PCR detection in all strains. The genomic DNAs of two strains with high resistance to gentamicin were used as donors to subject C. jejuni NCTC11168 to natural transformation. The transformants were investigated by whole-genome sequencing and analyzed comparatively with C. jejuni NCTC11168. In total, 30.5% (29/95) of C. jejuni isolates and 42.2% (38/90) of C. coli isolates were resistant to gentamicin and kanamycin. The prevalence of the aph(2")-If gene and aac(6')-Ie/aph(2")-Ia gene was 65.4% (121/185) and 36.2% (67/185) in Campylobacter isolates, respectively. The aadE-sat4-aphA-3 cluster was identified in 8.7% (8/92) and 20.4% (19/93) of all Campylobacter isolates in each year. With each donor DNA, aminoglycoside-resistant transformants were obtained. The transformants showed ≥128-fold increases in the MICs of gentamicin, kanamycin, and tobramycin. A 5200-bp segment was found to be inserted between the highly conserved genes Cj0299 and panB of Campylobacter. A total of 9.7% (18/185) strains showing high resistance to aminoglycosides had this segment by PCR detection. The genetic diversity of the insertion-fragment positive strains was determined by MLST, and seven sequence types were identified for these strains.

Role of mitochondrial dysfunction and PINK1/Parkin-mediated mitophagy in Cd-induced hepatic lipid accumulation in chicken embryos.

The present study was performed to investigate the effects of Cd exposure on lipid metabolism and mitochondrial dysfunction and to explore the role of mitophagy in Cd-induced dysregulation of lipid metabolism in chicken embryo liver tissues and hepatocytes. To this end, seven-day-old chicken embryos were exposed to different concentrations of Cd for 7 days, and primary chicken embryo hepatocytes were treated with Cd at four different concentrations for 6 h. Furthermore, the mitophagy inhibitor cyclosporine A (CsA) was used to investigate the role of mitophagy in Cd-induced disruption of lipid metabolism. Lipid accumulation, the expression levels of genes involved in lipid metabolism, mitochondrial dysfunction, and mitophagy were measured. The results demonstrated that Cd exposure increases hepatic triglyceride (TG) accumulation and the expression levels of lipogenic genes while decreasing those of lipolytic genes. Furthermore, Cd exposure was observed to alter mitochondrial morphology in terms of reduced size, excessive mitochondrial damage, and the formation of mitophagosomes. The co-localization of lysosome-associated membrane glycoprotein 2 and LC3 puncta was significantly increased in primary chicken embryo hepatocytes after Cd exposure. Moreover, Cd exposure increased LC3, PINK1, and Parkin protein expression levels. CsA effectively alleviated Cd-induced mitochondrial dysfunction, blocked mitochondrial membrane potential collapse, and suppressed PINK1/Parkin-mediated mitophagy. Furthermore, CsA treatment reversed the Cd-induced TG accumulation in liver tissues but further increased it in hepatocytes. Taken together, our findings demonstrate (for the first time) the importance of mitochondrial dysfunction and mitophagy via the PINK1/Parkin pathway in Cd-induced disruption of lipid metabolism.

A critical review on approaches for electrolytic manganese residue treatment and disposal technology: Reduction, pretreatment, and reuse.

Electrolytic manganese residue (EMR) has become a barrier to the sustainable development of the electrolytic metallic manganese (EMM) industry. EMR has a great potential to harm local ecosystems and human health, due to it contains high concentrations of soluble pollutant, especially NH4+ and Mn2+, and also the possible dam break risk because of its huge storage. There seems to be not a mature and stable industrial solution for EMR, though a lot of researches have been done in this area. Hence, by fully considering the EMM ecosystem, we analyzed the characteristics and eco-environmental impact of EMR, highlighted state-of-the-art technologies for EMR reduction, pretreatment, and reuse; indicated the factors that block EMR treatment and disposal; and proposed plausible and feasible suggestions to solve this problem. We hope that the results of this review could help solve the problem of EMR and thus promote the sustainable development of EMM industry.

Antibiotic Resistance Profiles and Molecular Mechanisms of Campylobacter From Chicken and Pig in China.

The purpose of this research was to characterize the antibiotic resistance profiles of Campylobacter spp. derived from chicken and pig feces collected from farms in Jiangsu Province, China, and to analyze the relevant resistance mechanisms among antimicrobial-resistant Campylobacter spp. isolates. Antibiotic susceptibility to nine antibiotic agents was tested with the microdilution method in 93 Campylobacter spp. (45 C. jejuni and 25 C. coli from chickens; 23 C. coli from pigs). High rates of resistance were observed to nalidixic acid (79.6%), erythromycin (75.3%), tetracycline (68.8%), azithromycin (66.7%), ciprofloxacin (64.5%), and gentamicin (35.5%), with a lower resistance rate to florfenicol (8.6%). The prevalence of the tested antibiotic resistance in C. coli was higher than in C. jejuni from chickens. The rate of antimicrobial resistance to ciprofloxacin in C. coli isolates from chickens was 100.0%, and the C. coli isolates from pigs were all resistant to erythromycin (100%). Most of C. jejuni (64.4%) and C. coli (64.5%) isolates displayed multi-drug resistance. All the Campylobacter spp. isolates resistant to fluoroquinolones had the C257T mutation in the gyrA gene. All 64 tetracycline-resistant Campylobacter spp. isolates were positive for the tetO gene. The tetA gene was also amplified in 6.5% of Campylobacter spp. isolates, whereas tetB was not detected among the isolates. The A2075G point mutation in the 23S rRNA gene occurred in 86.1% (62/72) of the macrolides-resistant Campylobacter spp. isolates, and the ermB gene was identified in 49 Campylobacter spp. isolates (30 C. jejuni and 19 C. coli). Amino acid insertions or mutations in the L4 and L22 ribosomal proteins were not linked to macrolide resistance. These results highlight the high prevalence of resistance to multiple antibiotics, particular macrolides, among Campylobacter spp. from chickens and pigs in Jiangsu Province, China, which is probably attributable to the overuse of antimicrobials in chicken and pig production. These findings recommend the more cautious use of critical antimicrobial agents in swine and poultry production. Stringent and continuous surveillance is required to reduce the drug-resistant campylobacteriosis in food animals and humans.

A new electrochemical method for simultaneous removal of Mn2+and NH4+-N in wastewater with Cu plate as cathode.

In this study, a new electrochemical method was used to simultaneously efficient removal of Mn2+ and NH4+-N in wastewater with Cu plate as cathode. The effects of various reaction parameters on the concentrations of Mn2+, NH4+-N and by-products (NO3--N and NO2--N, free chlorine and residual chlorine), as well as the removal mechanism were investigated. The results showed that the removal efficiencies of Mn2+ and NH4+-N were 99.1% and 92.9%, and the concentrations of NO3--N, NO2--N, free chlorine and residue chlorine were 0.73 mg/L, 0.15 mg/L, 0.13 mg/L and 0.63 mg/L reacting for 3 h at room temperature, respectively, when the current density was 10 mA/cm2, the mass ratio of ClO- and Cl- was 1:1, the initial pH was 9. The concentrations of Mn2+, NH4+-N and by-products in wastewater met the integrated wastewater discharge standard (GB8978-1996). In addition, spherical manganese oxide was deposited on the anode plate, and spherical manganese oxide collapsed over electrolysis time. Manganese was mainly removed in the form of MnO, Mn(OH)2 and MnO2. NH4+-N was mainly oxidized to N2. Economic evalution revealed that the treatment cost was 2.93 $/m3.

Expression of the Campylobacter jejuni FliD protein and its reaction to chicken sera.

Campylobacter is a leading causative pathogen of acute bacterial gastroenteritis among humans. Contaminated chicken products are regarded as major sources of human infection. The flagellar capping protein (FliD), which plays important roles in colonization and adhesion to the mucosal surface of chicken ceca, is conserved among Campylobacter jejuni strains. In this study, the recombinant C. jejuni FliD protein was expressed, purified and used as a coated protein to examine the prevalence of C. jejuni antibodies in chickens. The anti-FliD antibody was prevalent among chicken serum samples taken from different farms in the diverse regions of Jiangsu province by using enzyme-linked immunosorbent assay. The Campylobacter antibody was present in culture-negative chickens. No strong dose-response relationships were observed between serum FliD antibody levels and Campylobacter cultural status. These results provide a basis for further evaluating FliD as a vaccine candidate for broiler chickens or for examining host-C. jejuni interactions, with implications for improving food safety.

Prevalence and Characteristics of Campylobacter Throughout the Slaughter Process of Different Broiler Batches.

Handling and consumption of chicken meat are risk factors for human campylobacteriosis. This study was performed to describe the Campylobacter population in broiler carcasses and environmental samples throughout the slaughter process. Moreover, the genetic diversity and antimicrobial resistance of the Campylobacter strains were evaluated. Cloacal swabs, samples from carcasses at different stages, and environmental samples were collected thrice from the different flocks at the same abattoir located in Central Jiangsu, China. Campylobacter isolated from the three batches (n = 348) were identified as Campylobacter jejuni (n = 117) and Campylobacter coli (n = 151) by multiplex PCR. Characterization by multilocus sequence typing revealed a specific genotype of Campylobacter for each batch. Antimicrobial sensitivity to 18 antibiotics were analyzed for all selected strains according to the agar diffusion method recommended by the Clinical and Laboratory Standards Institute. Antibiotic susceptibility tests indicated that the majority of the tested isolates were resistant to quinolones (>89.7%). Less resistance to macrolide (59.8%), gentamicin (42.7%), amikacin (36.8%) was observed. Results showed that 94.0% of the tested strains demonstrated multidrug resistance.

The genetic diversity of chicken breeds from Jiangxi, assessed with BCDO2 and the complete mitochondrial DNA D-loop region.

The Jiangxi Province of China has numerous native domestic chicken breeds, including some black skin breeds. The genetic diversity of Jiangxi native chickens is largely unknown, and specifically, the genetic contribution of the grey junglefowl to black skin chickens is not well understood. To address these questions, the complete D-loop region of the mitochondrial DNA (mtDNA) and beta-carotene dioxygenase 2(BCDO2)gene was sequenced in a total of 209 chickens representing seven Jiangxi native breeds. Thirty-one polymorphic sites were identified across the complete mtDNA D-loop region sequence. Twenty-three haplotypes were observed in the seven breeds, which belonged to four distinct mitochondrial clades (A, B, C and E). Clade A and B were dominant in the chickens with a frequency of approximately 67.9%. There were five SNPs that defined two haplotypes, W and Y in BCDO2. Four breeds had one haplotype and three breeds had two. We conclude that Jiangxi native chicken breeds have relatively low genetic diversity and likely share four common maternal lineages from two different maternal ancestors of junglefowl. Furthermore, some Jiangxi chicken populations may have been mixed with chickens with exotic lineage. Further research should be established to protect these domestic chicken resources.

The investigation of genetic diversity and evolution of Daweishan Mini chicken based on the complete mitochondrial (mt)DNA D-loop region sequence.

This study evaluated the genetic diversity and origin of Daweishan Mini chickens using mtDNA sequence polymorphism. Blood samples from 30 Daweishan Mini chickens were collected. The complete D-loop was PCR amplified, sequenced and compared with the DNA data of five Red Junglefowl (Gallus gallus) subspecies. Eighteen variable sites that defined six haplotypes were observed. The six haplotypes were clustered into four clades (A, B, D and E), of which clade A and B were dominant. Clades Aand B were clustered with G.g. spadiceus, indicating these two clades may have originated from this subspecies. These results show there is diversity in the middle of the mtDNA D-loop, and indicate there are multiple maternal origins for Daweishan Mini chickens. It appears that G.g. spadiceus contributed more to the evolution of the Daweishan Mini chickens breed than the other four subspecies tested here.