RAA Enzyme Is a New Family of Class A Extended-Spectrum ß-Lactamase from Riemerella anatipestifer Strain RCAD0122.

Whole-genome sequencing of Riemerella anatipestifer isolate RCAD0122 revealed a chromosomally located ß-lactamase gene, blaRAA-1, which encoded a novel class A extended-spectrum ß-lactamase (ESBL), RAA-1. RAA-1 shared ≤65% amino acid sequence identity with other characterized ß-lactamases. The kinetic assay of native purified RAA-1 revealed ESBL-like hydrolysis activity. Furthermore, blaRAA-1 could be transferred to a homologous strain by natural transformation. However, an epidemiological study showed that the blaRAA-1 gene is not prevalent currently.

A Long Noncoding RNA Regulates Hepatitis C Virus Infection Through Interferon Alpha-Inducible Protein 6.

Long noncoding RNAs (lncRNAs) play a critical role in the regulation of many important cellular processes. However, the mechanisms by which lncRNAs regulate viral infection and host immune responses are not well understood. We sought to explore lncRNA regulation of hepatitis C virus (HCV) infection and interferon response. We performed RNA sequencing (RNAseq) in Huh7.5.1 cells with or without interferon alpha (IFNα) treatment. Clustered regularly interspaced short palindromic repeats/Cas9 guide RNA (gRNA) was used to knock out selected genes. The promoter clones were constructed, and the activity of related interferon-stimulated genes (ISGs) were detected by the secrete-pair dual luminescence assay. We constructed the full-length and four deletion mutants of an interferon-induced lncRNA RP11-288L9.4 (lncRNA-IFI6) based on predicted secondary structure. Selected gene mRNAs and their proteins, together with HCV infection, in Huh7.5.1 cells and primary human hepatocytes (PHHs) were monitored by quantitative real-time PCR (qRT-PCR) and western blot. We obtained 7,901 lncRNAs from RNAseq. A total of 1,062 host-encoded lncRNAs were significantly differentially regulated by IFNα treatment. We found that lncRNA-IFI6 gRNA significantly inhibited HCV infection compared with negative gRNA control. The expression of the antiviral ISG IFI6 was significantly increased following lncRNA-IFI6 gRNA editing compared with negative gRNA control in Japanese fulminant hepatitis 1 (JFH1)-infected Huh7.5.1 cells and PHHs. We observed that lncRNA-IFI6 regulation of HCV was independent of Janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling. lncRNA-IFI6 negatively regulated IFI6 promoter function through histone modification. Overexpression of the truncated spatial domain or full-length lncRNA-IFI6 inhibited IFI6 expression and increased HCV replication. Conclusion: A lncRNA, lncRNA-IFI6, regulates antiviral innate immunity in the JFH1 HCV infection model. lncRNA-IFI6 regulates HCV infection independently of the JAK-STAT pathway. lncRNA-IFI6 exerts its regulatory function via promoter activation and histone modification of IFI6 through its spatial domain.

Development of an indirect ELISA for detecting humoral immunodominant proteins of Mycoplasma hyopneumoniae which can discriminate between inactivated bacterin-induced hyperimmune sera and convalescent sera.

BACKGROUND: Mycoplasma hyopneumoniae (M. hyopneumoniae) is the primary pathogen of porcine enzootic pneumonia, which has been associated with economic losses due to reduced daily weight gain and feed efficiency. Although it has a small genome and no more than 1000 genes, M. hyopneumoniae can be cultured in cell free media. However, some proteins were not expressed or were only expressed in negligible amounts under culture conditions. Nevertheless, some of these proteins can be expressed at a high level and induce a strong and rapid immune response after M. hyopneumoniae infection. The unexpressed or less expressed proteins may play critical roles in pathogenesis and/or immune response. In order to find the differentially expressed proteins of M. hyopneumoniae between culture condition and infected animals, we established an indirect ELISA for the detection of humoral immunodominant proteins which can discriminate between inactivated bacterin-induced hyperimmune sera and convalescent sera by using Mhp366 protein which did not react with sera from bacterin-immunized pigs, but revealed a strong immunoreaction with porcine convalescent sera. RESULTS: The checkerboard titration method was done by using porcine convalescent sera as positive sera and inactivated bacterin-induced hyperimmune sera as negative sera. The bacterial lysates of fusion proteins and free GST protein without dilution were the optimal coating antigens. The optimal blocking buffer was PBS with 10% FBS and 2.5% skimmed milk. In the checkboard ELISAs, when the sera were diluted at 1:500 and the HRP-labeled rabbit anti-pig IgG were diluted at 1:20000, most positive result was obtained for the assay. CONCLUSIONS: This established indirect ELISA can be used as a tool for the detection of humoral immunodominant proteins of M. hyopneumoniae which can discriminate between inactivated bacterin-induced hyperimmune sera and convalescent sera.

Prevalence, antimicrobial resistance, and staphylococcal toxin genes of blaTEM-1a -producing Staphylococcus aureus isolated from animals in Chongqing, China.

BACKGROUND: Staphylococcus aureus infection of livestock animals and humans is a major public health issue. There are reports of antimicrobial resistance and multiple staphylococcal superantigen genes in many countries and several provinces of China, but the status in Chongqing, China is uncertain. OBJECTIVES: The aim of this study was to determine the prevalence, antimicrobial susceptibility, and other molecular characteristics of S. aureus isolates from livestock animals in Chongqing. METHODS: Staphylococcus aureus was isolated and identified by selective enrichment and amplification of the nuc gene from 1371 samples collected at farms in Chongqing. The agar dilution method was used to determine the resistant phenotype, and extended spectrum ß-lactamase genes were amplified by PCR. Methicillin-resistant S. aureus was verified by the presence of the mecA gene, and the presence or absence of SE, SEl, and TSST-1 genes was detected in the isolates. RESULTS: We cultured 89 S. aureus isolates from 1371 samples between March 2014 and December 2017. These isolates were from pigs, cattle, goats, rabbits, and chickens. There were four methicillin-resistant S. aureus strains (three from pigs and one from a chicken). The 89 isolates had high resistance to penicillin (93.3%) and ampicillin (92.1%), but most were susceptible to amikacin and ofloxacin, with resistance rates below 10%. A total of 62.9% of the isolates had varying degrees of multidrug resistance. Almost all strains, except for three isolates from chickens, were positive for blaTEM-1a . There were 19 of 20 tested staphylococcal SE/SEl/TSST-1 genes present (all except for seq), and the predominant genes were sei (58.4%), tst-1 (56.2%), and seg (51.7%). CONCLUSIONS: The high antimicrobial resistance and prevalence of blaTEM-1a reinforce the need to reduce the usage of antimicrobials in livestock. The universal existence of staphylococcal toxin genes implies a potential threat to public health by animal-to-human transmission via the food chain.

BlaTEM-positive Salmonella enterica serovars Agona and Derby are prevalent among food-producing animals in Chongqing, China.

Salmonella is one of the most important foodborne zoonotic pathogens, causing global morbidity and mortality in both humans and animals. Due to the extensive use of antimicrobials in food-producing animals, the antimicrobial resistance of Salmonella has attracted increasing attention globally. There have been many reports concerning the antimicrobial resistance of Salmonella from food-producing animals, meats and the environment. However, few studies on Salmonella from food-producing animals have been reported in Chongqing municipality, China. The aim of the present study was to determine the prevalence, serovar diversity, sequence types, and antimicrobial resistance of Salmonella isolated from livestock and poultry in Chongqing. Meanwhile, we also want to know the presence of ß-lactamase genes, plasmid-mediated quinolone resistance (PMQR) genes and quinolone resistance-determining region (QRDR) mutations of Salmonella isolates. A total of 129 Salmonella strains were recovered from 2,500 fecal samples at 41 farms from pigs, goats, beef cattle, rabbits, chickens, and ducks. Fourteen serovars were identified, with S. Agona and S. Derby being the dominant serovars. The 129 isolates had high resistance to doxycycline (87.6%), ampicillin (80.6%), tetracycline (79.8%), trimethoprim (77.5%), florfenicol (76.7%) chloramphenicol (72.9%), and trimethoprim-sulfamethoxazole (71.3%), but were susceptible to cefepime. A total of 114 (88.4%) isolates showed multidrug resistant phenotypes. The prevalence of ß-lactamase genes in Salmonella isolates was 89.9% (116/129), and among these isolates, 107 (82.9%) harbored blaTEM, followed by blaOXA (26, 20.2%), blaCTX-M (8, 6.2%), and blaCMY (3, 2.3%). In addition, qnrB, qnrD, qnrS, oqxA, oqxB, and aac(6')-Ib-cr were detected in 11, 2, 34, 34, 43, and 72 PMQR-producing isolates, respectively. Moreover, QRDR mutations were very common in PMQR-positive Salmonella isolates (97.2%, 70/72) with mutation(s) in parC or combinative mutations in gyrA and parC. More significantly, 32 extended spectrum beta-lactamase (ESBL)-producing isolates were identified, and 62.5% of them were found to harbor one to four PMQR genes. Furthermore, 11 sequence types were identified from the isolates, and most of ESBL-producing isolates were attributed to ST34 (15.6%) and ST40 (62.5%). The coexistence of PMQR genes with ß-lactamase genes and the extensive mutations in QRDR present in Salmonella isolates from food-producing animals suggest a potential threat to public health. Reasonable utilization and strict control strategies for antimicrobials in animal husbandry and animal treatment are necessary to reduce the emergence and dissemination of drug-resistant Salmonella isolates.

The absence of MyD88 has no effect on the induction of alternatively activated macrophage during Fasciola hepatica infection.

UNLABELLED: Alternatively activated macrophages (AAMφ) play important roles in allergies and responses toparasitic infections. However, whether signaling through toll-like receptors (TLRs) plays any role in AAMφ induction when young Fasciola hepatica penetrates the liver capsule and migrates through the liver tissue is still unclear. RESULTS: The data show that the lack of myeloid differentiation factor 88 (MyD88) has no effect on the AAMφ derived from the bone marrow (BMMφ) in vitro and does not impair the mRNA expression of arginase-1, resistin-like molecule (RELMα), and Ym1 in BMMφs. The Th2 cytokine production bias in splenocytes was not significantly altered in F. hepatica-infected mice in the absence of MyD88 in vitro and in the pleural cavity lavage in vivo. In addition, MyD88-deficiency has no effect on the arginase production of the F. hepatica elicited macrophages (Fe Mφs), production of RELMα and Ym1 proteins and mRNA expression of Ym1 and RELMα of macrophages in the peritoneal cavity 6 weeks post F. hepatica infection. CONCLUSIONS: The absence of MyD88 has no effect on presence of AAMφ 6 weeks post F. hepatica infection.

[Advances in bacterial type VI secretion system--a review].

Type VI secretion system (T6SS) is a newly found secretion system, which distributes widely in Gram-negative bacteria. It consists of structural proteins (constitute secretion system), translocated proteins (form transmembrane pipe structure), secretory proteins and a few auxiliary function proteins for secretion systems. T6SS can enhance the adaptability of bacteria on the environment,mediate pathogenicity to host cells and has some other functions.

Immunization with outer membrane vesicles of avian pathogenic Escherichia coli O78 induces protective immunity in chickens.

Avian pathogenic Escherichia coli (APEC) typically causes colibacillosis and is a major concern for the poultry industry and public health. As a vaccine platform, the outer membrane vesicles (OMVs) derived from various gram-negative bacteria and even some gram-positive bacteria have been reported to be immunogenic in laboratories or upon commercial usage worldwide. Here, we purified OMVs from APEC serotype O78 strain by ultracentrifugation and gradient isolation. By SDS-PAGE and LC-MS/MS analysis, the 20 most abundant proteins located on OMVs were identified and analyzed; the lipopolysaccharide (LPS) profiles of OMVs were not different from those of the bacteria. Moreover, three groups of chickens were immunized with OMV-, outer membrane protein (OMP)- and PBS, with the latter two serving as positive and negative controls, respectively. By analyzing the anti-OMP and anti-LPS IgG titers stimulated by the tested vaccine candidates, the macrophage opsonophagocytic activity and the bactericidal activity mediated by serum antibodies in vaccinated chickens, we found that the OMV-vaccinated chicken group was superior to the two other groups. These findings were confirmed by additional chicken challenge tests, in which all OMV-vaccinated group chickens obtained complete protection but those of the other two groups were barely protected. Our data demonstrate that native APEC O78 OMVs can induce protective immunity in chickens and therefore be used as a candidate vaccine for APEC serotype O78 strain infections.

Genetically engineered Salmonella Typhimurium: Recent advances in cancer therapy.

Bacteria have been investigated as anti-tumor therapeutic agents for more than a century, since Coley first observed successful curing of a patient with inoperable cancer by injection of streptococcal organisms. Previous studies have demonstrated that some obligate or facultative anaerobes can selectively accumulate and proliferate within tumors and suppress their growth. Developments in molecular biology as well as the complete genome sequencing of many bacterial species have increased the applicability of bacterial organisms for cancer treatment. In particular, the facultative anaerobe Salmonella Typhimurium has been widely studied and genetically engineered to improve its tumor-targeting ability as well as to reduce bacterial virulence. Moreover, the effectiveness of engineered attenuated S. Typhimurium strains employed as live delivery vectors of various anti-tumor therapeutic agents or combined with other therapies has been evaluated in a large number of animal experiments. The well-known S. Typhimurium mutant VNP20009 and its derivative strain TAPET-CD have even been applied in human clinical trials. However, Salmonella-mediated cancer therapies have not achieved the expected success, except in animal experiments. Many problems remain to be solved to exploit more promising strategies for combatting cancer with Salmonella bacteria. Here, we summarize the promising studies regarding cancer therapy mediated by Salmonella bacteria and highlight the main mechanisms of Salmonella anti-tumor activities.

[Role of bacterial minicells in cancer therapy].

Cancer is one of the most important diseases threatening human health. Frequently-used traditional cancer treatment methods, like radiotherapy, chemotherapy and surgery, have serious toxic side effects and limitations. The widely-used drug delivery carriers (liposomes, nanoparticles, etc.) have also possessed many issues such as drug leakage and incomplete loading in the late clinical stage. Currently, using tumor-targeting vectors to deliver anti-tumor drugs or small molecules is one of the promising strategies for mediating safe and effective tumor therapy. In recent years, bacterial-derived non-replicating minicells, which are nanoscale non-nucleated cells produced during abnormal bacterial division, have got more and more attention. With a diameter of 200-400 nm, minicells have a large drug loading capacity. Meanwhile, the surface of minicells are able to be modified to load the assembly of antibodies/ligands that bind to tumor cell surface specific antigens or receptors, which can significantly improve tumor targeting of minicells. This tumor-targeting nanomaterials of minicells not only are used to deliver anti-tumor chemotherapeutic drugs, functional nucleic acids or plasmids encoding functional small molecules to mammalian cells, but also greatly increase drug loading and reduce drug penetration. Thus, the use of minicells combining with chemical therapy could help reduce the toxicity and maximize the effectiveness of the drug in the body. This paper summarizes the research and development of production purification, drug loading, tumor cells targeting, and internalization process of minicells, as well as its use in the delivery of anti-tumor drugs, to provide some information for the development and utilization of minicell carriers.

Epidemiological survey on Escherichia coli O157 in Chongqing and Three-Gorge Reservoir Areas of China.

Prevalence, presence of virulence and adherence associated genes, genetic diversity, biochemical characteristics, and antibiotic susceptibility were determined for Escherichia coli O157 isolated over 4 months in Chongqing city and Three-Gorge Reservoir Areas. 11 isolates of E. coli O157 were isolated from 1504 samples and 7 of them are O157:H7 and 4 are O157:H? All O157:H7 isolates had eaeA, ehxA, EspA and Tccp genes, but did not have stx1 and stx2. All O157:H? isolates did not have stx1, stx2, eaeA, ehxA, EspA and Tccp genes except for the isolate obtained from Yunyang county which had stx1. When eaeA and ehxA presented in isolates were digested by restriction enzymes, the numbers and the sizes of the segments were the same as the control E. coli O157 strains. This suggests that eaeA and ehxA exhibit poor polymorphism. Most E. coli O157 isolates showed identical biochemical activities to the standard strains for sorbitol and rhamnose, and all E. coli O157:H7 obtained from feces at the same dairy cattle farm had similar biochemical characteristics. Antibiotic susceptibility demonstrated resistance of the isolates to penicillin, ampicillin, bacitracin, cefuroxime, erythromycin, gentamycin and tetracycline, indicating the isolates obtained in this study had a multi-drug resistance.

[Development of an ELISA for identification of immunodominant protein antigens of Mycoplasma hyopneumoniae].

We developed a method to identify serological humoral immunodominant proteinic antigen of Mycoplasma hyopneumoniae (Mhp). After constructing the recombinant plasmid pGEX-6P-1-mhp366 and transforming it into Escherichia coli BL21(DE3), the recombinant GST-Mhp366 protein was expressed successfully. The lysates of the recombinant GST-Mhp366 and genetic engineering GST were added into glutathione coated plates and reacted with 17 positive sera or 13 negative sera. Meanwhile, the optimization of experimental conditions, including coated antigen, blocking buffer, dilutions of sera and second antibody were determined. The optimal concentration of the coated antigen was the original bacteria lysates without dilution, and the optimal blocking buffer contained 10% FBS and 2.5% skim milk in PBS. Besides, the working concentration of serum samples and the HRP-tagged rabbit anti-pig IgG secondary antibody were 1:500 and 1:40 000, respectively. Thus, an indirect ELISA was established for identification of immunodominant protein antigens of Mhp. Meanwhile, this method was confirmed by the identified serological humoral immunodominant proteinic antigen Mhp156 and Mhp364. This method can be used for identification of the candidate vaccine antigens on a genome-wide scale. Furthermore, it can lay the foundation for identifying the candidate vaccine antigens through colostra and the nasal mucosal secretions.

[Techniques of separation and identification of enterohemorrhagic Escherichia coli O157 in laboratory].

Escherichia coli O157 is an important food-borne human pathogen. Separation and identification of this bacterium and further research in molecular level could have important significance. The techniques of separation and identification of E. coli O157 in laboratory, such as plate separation, immunomagnetic separation, and polymerase chain reaction were reviewed, and made a comparision of various methods.