miR-146a-5p promotes replication of infectious bronchitis virus by targeting IRAK2 and TNFRSF18.

Avian infectious bronchitis virus (IBV) is a coronavirus which infects chickens (Gallus gallus) of all ages and causes significant economic losses to the poultry industry worldwide. The present study aims to analyze the miRNAs related to pathogenicity of nephropathogenic IBVs. It was found that four miRNAs (miR-1454, miR-3538, miR-146a-5p and miR-215-5p) were related to the infection of virulent nephropathogenic IBV with transcript per million (TPM) > 500 and more than a 2-fold alteration. In vitro study results showed that the alterations of these four miRNAs were consistent with in vivo data. In vitro, we found that high levels of miR-146a-5p could enhance the replication of IBV at the early stage of infection, and its down regulated level could slow down the replication of IBV. Finally, high levels of exogenous miR-146a-5p in HD11 cells led to down regulation of IL-1 receptor associated kinase-2 (IRAK2) and Tumor necrosis factor receptor superfamily member 18 (TNFRSF18) genes. Luciferase reporter assays revealed that miR-146a-5p could bind to the 3'-UTRs of IRAK2 and TNFRSF18. This is the first study demonstrating that IBV induced miR-146a-5p is related to virus pathogenesis by down regulating IRAK2 and TNFRSF18, which may serve as a therapeutic strategy for the prevention of IBV infections.

Co-occurrence of biofilm formation and quinolone resistance in Salmonella enterica serotype typhimurium carrying an IncHI2-type oqxAB-positive plasmid.

The objective of this study was to investigate the co-occurrence of biofilms and quinolone resistance in Salmonella enterica serotype Typhimurium mediated by IncHI2-type oqxAB-positive plasmids. Among the 40 Salmonella strains, we found that 27 isolates formed biofilms and displayed identical multidrug-resistance profiles to ciprofloxacin, doxycycline, sulfamethoxazole-trimethoprim, ampicillin and streptomycin, based on biofilm formation assays and antimicrobial susceptibility testing. In particular, a single S. Typhimurium isolate named SC523 produced the thickest biofilms and exhibited the highest-level resistance (MIC = 8 µg/mL) to ciprofloxacin compared to those of the other isolates. The detection of known plasmid-mediated quinolone resistance (PMQR) genes and point mutations in the quinolone resistance-determining region (QRDR) by PCR assay showed that oqxAB genes were present in 27 biofilm-positive isolates. Conjugation experiments, S1-pulse-field gel electrophoresis and biofilm formation assays demonstrated that the conjugative plasmid that encoded biofilms and quinolone resistance in Salmonella SC523 could be transferred to a recipient with a frequency of 4.7 × 10-3 per recipient cell. The results of PCR-based replicon typing (PBRT) showed that the IncHI2-type plasmids accounted for 100% of the biofilm-oqxAB-positive isolates and transconjugants. The sequence analysis of Salmonella SC523 confirmed that the oqxAB cassette and fourteen DNA transfer genes in the IncHI2-type oqxAB-positive conjugative plasmid were genetically responsible for the phenotypic quinolone resistance and biofilm formation. The conclusion is that the IncHI2-type plasmid in S. Typhimurium isolate from chicken farm was identified and sequenced, which contained oqxAB and tra/trh and encoded quinolone resistance and biofilms, and could be transferred to recipients through conjugation. Notably, the prevalence of IncHI2-type biofilm-oqxAB-positive plasmids in animal-origin Salmonella poses a threat to public health, as these Salmonella from poultry farms show a decreased susceptibility to quinolones and could spread to humans.

Frequency of antimicrobial resistance and integron gene cassettes in Escherichia coli isolated from giant pandas (Ailuropoda melanoleuca) in China.

Escherichia coli (E. coli) is considered as a common opportunistic pathogen, which causes seriously intestinal infections to giant pandas (Ailuropoda melanoleuca) and other animals. The aim of this investigation was to characterize the antimicrobial resistance and integron gene cassettes in E. coli isolated from the faeces of giant pandas in China. A total of 89 E. coli were isolated, after diagnosis of isolates and genomes were extracted. All the isolates were screened for the presence of related drug-resistance genes and integron gene cassettes through the Polymerase Chain Reaction (PCR) and sequencing. In addition, antimicrobial resistance testing was performed according to the standard disk diffusion method (CLSI 2013). The results demonstrated that all the isolates were multi-drug resistance (MDR). High resistance proportions of the E. coli isolates were to streptomycin (93%), cefazolin (90%), amikacin (75%), tetracycline (65%), ampicillin (62%), cefotaxime and trimethoprim-sulfamethoxazole (54%, each). With respect to the various resistance genes; blaCTX-M, sul1, ant (3')-Ia, tetA, qnrB, tetE, floR, aac (6')-Ib, sul2, rmtA, cmlA, rmtB and tetC were identified with the respective frequencies of 44%, 45%, 38%, 37%, 35%, 27%, 26%, 20%, 18%, 15%, 10%, 7% and 4%. None of the isolates was positive for qnrA and cfr genes. Moreover, a further investigation of integron revealed that the emergence of class 1 and 2 integrons were in 47% and 8% isolates, respectively. While class 3 integron was not screened. Six types of containing in class 1 integron specific gene cassettes (dfrA12-orfF-aadA2, dfrA17-aadA5, aadA1, aadA5, dfrA1 and dfrA7) were identified. However, only one gene cassette (dfrA1-sat2-aadA1) was detected in class 2 integron. These finding emphasize that a high level of E. coli isolates harbored antibiotics resistance and integron gene cassettes, which may bring so many potential threats to the health of giant pandas.

Comparative transcriptome analysis reveals induction of apoptosis in chicken kidney cells associated with the virulence of nephropathogenic infectious bronchitis virus.

Avian infectious bronchitis virus (IBV) that causes respiratory and nephritic diseases in chicken is a major poultry pathogen leading to serious economic loss worldwide. The nephropathogenic IBV strains cause nephritis and kidney lesions intrinsically and the pathogenic mechanism is still unclear. In the present study, SPF chicks were infected with three nephropathogenic IBVs of different virulence and their gene expression profiles in chicken kidney were compared at transcriptome level. As a result, 1279 differentially expressed (DE) genes were found in very virulent SCDY2 inoculated group, 145 in virulent SCK2 group and 74 in non-virulent LDT3-A group when compared to mock infected group. Gene Ontology (GO) and KEGG pathway enrichment analysis on SCDY2 group displayed that the up-regulated DE genes were mainly involved in cell apoptosis, and the down-regulated genes were involved in metabolic processes and DNA replication. Protein-Protein Interaction (PPI) analysis showed that DE genes in SCDY2 group formed a network, and the core of the network was composed by cell apoptosis and immune response proteins. The clustering of gene expression profile among the three virus inoculated groups indicated that the majority of up-regulated DE genes on apoptosis in very virulent SCDY2 group were up-regulated more or less in virulent SCK2 group and those down-regulated on innate immune response in SCDY2 group were also down-regulated differently in SCK2 group. In addition, the number of apoptotic cells detected experimentally in kidney tissue were very different among the three virus inoculated groups and were positively accordant with the viral titer, kidney lesions and viral virulence of each group. Taken all together, the present study revealed that virulent nephropathogenic IBV infection modified a number of gene expression and induction of apoptosis in kidney cells may be a major pathogenic determinant for virulent nephropathogenic IBV.

Prevalence of antimicrobial resistance and integron gene cassettes in Escherichia coli isolated from yaks (Poephagus grunniens) in Aba Tibetan Autonomous Prefecture, China.

BACKGROUND: Escherichia coli (E. coli) is one of the most relevant opportunistic pathogenic bacteria as it may cause severe morbidity and mortality in yaks (poephagus grunniens). In recent years, several kinds of antibiotics have been widely used in Tibetan areas to treat the bacterial diseases, resulting in serious repercussions on the bacterial antibiotic resistance in yaks. This investigation was conducted in order to determine the prevalence of antimicrobial resistance and integron gene cassettes in E. coli isolated from yaks in Aba Tibetan Autonomous Prefecture (Aba TAP), China. METHODS: A total of 278 non-duplicated fresh samples were collected from the yaks in Aba TAP for the isolation and identification of E. coli isolates. Antimicrobial susceptibility testing is performed by using the disc diffusion method according to the Clinical and Laboratory Standards Institute guidelines (CLSI, 2013). Various antibiotic resistance genes and integron gene cassettes were detected by polymerase chain reaction (PCR) and sequencing. RESULTS: Overall, a total of 228 E. coli bacteria were isolated from the fresh faeces of yaks in four different geographical regions. 58% of those isolates showed multi-drug resistance capabilities (MDR) in our study. These isolated bacteria showed a high resistance rate to streptomycin (84%), cefotaxime (79%), amikacin (61%) and trimethoprim-sulfamethoxazole (54%). The most common antimicrobial resistance genes in the isolates were blaCTX-M, sul1, aph (3')-IIa, aac (3)-IIa, aac (6')-Ib, tetB, with respective detection rates of 65%, 46%, 35%, 13%, 11%, and 10%. Furthermore, 66% and 6% of the strains carried Class 1 and 2 integrons, respectively. However, the class 3 integron was not detected. Gene cassette arrays in the class 1 integron included aadA1, aadA7, aadA5, aadA17, dfrA1, dfrA5, dfrA1-aadA1, dfrA12-aadA2 and dfrA17-aadA5. The most prevalent gene cassette was aadA1 (20%). For the class 2 integron, dfrA1-sat2-aadA1 (6%) and dfrA1-sat1-aadA1 (0.4%) were also detected as part of this research. CONCLUSION: High multi-drug resistance rates have been discovered, as well as a prevalence of antibiotic resistance genes and integron gene cassettes in the E. coli isolated from the faeces of yak. This might create a potential problem for treatment of the yaks' bacterial infections as well as food hygiene for humans. It is therefore urgently necessary to begin continuous surveillance and analysis of antibiotic resistance and integron cassettes in other bacteria from yaks.

Prevalence of Plasmid-Mediated Fosfomycin Resistance Gene fosA3 Among CTX-M-Producing Escherichia coli Isolates from Chickens in China.

The aim of this study was to investigate the prevalence of fosfomycin resistance gene fosA3 and characterize plasmids harboring fosA3 among CTX-M-producing Escherichia coli from chickens in China. A total of 234 CTX-M-producing E. coli isolates collected from chickens from 2014 to 2016 were screened for the presence of plasmid-mediated fosfomycin resistance genes (fosA, fosA3, and fosC2). Clonal relatedness of fosA3-positive isolates was determined by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). The genetic environment of fosA3 was analyzed by polymerase chain reaction (PCR) mapping. Plasmids were studied by using conjugation experiments, PCR-based replicon typing and plasmid MLST. Sixty-four (27.4%) fosA3-positive E. coli isolates were identified in this study. The gene blaCTX-M-55 (31/64) was predominant among these strains, followed by blaCTX-M-14 (18/64) and blaCTX-M-65 (14/64). Various PFGE patterns and sequence types (STs) indicated that these isolates were clonally unrelated. Seven different genetic environments of fosA3 were identified and two new combinations (ISEcp1-blaCTX-M-65-ΔIS903D-IS26-fosA3-orf1-orf2-Δorf3-IS26 and IS26-ISEcp1-blaCTX-M-3-orf477-blaTEM-1-IS26-fosA3-orf1-orf2-Δorf3-IS26) were discovered for the first time. Conjugation experiments were successful for 47 isolates and 33 transconjugants harbored a single plasmid. Plasmids carrying fosA3 belonged to incompatibility group IncFII (17/33), IncI1 (2/33), IncHI2 (3/33), and IncB/O (1/33). F33:A-:B- plasmids carrying blaCTX-M-55, IncHI2/ST3 plasmids carrying blaCTX-M-65, and F2:A-:B-plasmids carrying blaCTX-M-55 were found in E. coli isolates from different provinces. Our results revealed a considerable prevalence of fosA3 gene among CTX-M-producing E. coli with clonal diversity from chickens in China. The transmission of different kinds of plasmids is responsible for the dissemination of fosA3 in chicken farms in China.

Data-Based Optimal Switching and Control With Admissibility Guaranteed Q -Learning.

This article addresses the data-based optimal switching and control codesign for discrete-time nonlinear switched systems via a two-stage approximate dynamic programming (ADP) algorithm. Through offline policy improvement and policy evaluation, the proposed algorithm iteratively determines the optimal hybrid control policy using system input/output data. Moreover, a strict proof of the convergence is given for the two-stage ADP algorithm. Admissibility, an essential property of the hybrid control policy must be ensured for practical application. To this end, the properties of the hybrid control policies are analyzed and an admissibility criterion is obtained. To realize the proposed Q -learning algorithm, an actor-critic neural network (NN) structure that employs multiple NNs to approximate the Q -functions and control policies for different subsystems is adopted. By applying the proposed admissibility criterion, the obtained hybrid control policy is guaranteed to be admissible. Finally, two numerical simulations verify the effectiveness of the proposed algorithm.

Sampled-Data Consensus Protocols for a Class of Second-Order Switched Nonlinear Multiagent Systems.

In this study, the sampled-data consensus problem is investigated for a class of heterogeneous multiagent systems (MASs) in which each agent is described by a second-order switched nonlinear system. Owing to the heterogeneity and the occurrence of dynamic switching in the MASs, the sampled-data consensus protocol design problem is challenging. In this study, two periodic sampled-data consensus protocols and an event-triggered consensus protocol are developed. Here, we first propose a new periodic sampled-data consensus protocol that involves the local objective trajectory interaction among agents. The protocol is then improved by applying the finite-time control and sliding-mode control techniques. Notably, the improved protocol can be implemented without the transmission of constructed auxiliary dynamical variables, which is a major feature of the present study. It is shown that complete consensus of the underlying MASs can be achieved by the two proposed protocols with only sampled-data measurements. To further reduce the communication load, we introduce an event-triggered mechanism to obtain a new protocol. Finally, the effectiveness of the given schemes is demonstrated by considering a numerical example.

Finite-Time Consensus of Second-Order Switched Nonlinear Multi-Agent Systems.

In this brief, the practical finite-time consensus (FTC) problem is investigated for the second-order heterogeneous switched nonlinear multi-agent systems (MASs), where the subsystems and the switching signal for each agent are different. Mainly due to that agents' dynamics are switched and the unknown nonlinearities in the systems are more general, the practical FTC problem of the MASs is rather difficult to be solved by existing methods. As such, a new protocol design framework for the FTC problem is developed. Then, a novel adaptive protocol is proposed for the switched nonlinear MASs based on the developed design framework and the neural network method. The sufficient conditions for the practical FTC of nonlinear MASs under arbitrary switching are given. Finally, a numerical example is presented to demonstrate the effectiveness of the proposed control scheme.

Consensus Tracking Control of Switched Stochastic Nonlinear Multiagent Systems via Event-Triggered Strategy.

In this paper, the consensus tracking problem is investigated for a class of continuous switched stochastic nonlinear multiagent systems with an event-triggered control strategy. For continuous stochastic multiagent systems via event-triggered protocols, it is rather difficult to avoid the Zeno behavior by the existing methods. Thus, we propose a new protocol design framework for the underlying systems. It is proven that follower agents can almost surely track the given leader signal with bounded errors and no agent exhibits the Zeno behavior by the given control scheme. Finally, two numerical examples are given to illustrate the effectiveness and advantages of the new design techniques.

Sensitive and rapid detection of Salmonella enterica serovar Indiana by cross-priming amplification.

Salmonella enterica serovar Indiana (S. Indiana) was the most frequently reported foodborne pathogen, which has a broad host range including poultry, swine, and humans. Traditional methods used for the detection of S. Indiana from contaminated food products are time-consuming and labor-intensive. Therefore, rapid detection methods with high sensitivity and specificity are vitally important to prevent the spread of S. Indiana. In this study, we developed a nearly instrument-free, simple molecular method which incorporates cross-priming amplification (CPA) combined with a nucleic acid detection strip (NADS) for sensitive detection of S. Indiana. A set of CPA primers was designed based on S. Indiana specific nucleotide sequences and the specificity of CPA-NADS was tested against 42 bacterial strains. The results showed that this method was highly specific for detection of S. Indiana. The sensitivity of CPA-NADS was evaluated and compared with that of the serovar-specific PCR method and the real-time PCR method. The limit of detection of the CPA method was 8.997 fg/µL for genomic DNA and 6.2 × 101 CFU/mL for bacteria in pure culture. An application of the CPA assay was conducted with 90 inoculated specimens by S. Indiana. The accuracy of CPA-NADS was consistent with the results of the traditional culture-based methods in inoculated specimens. This method showed a higher sensitivity than the serovar-specific PCR method did and was more convenient to perform. In conclusion, we demonstrated that the CPA-NADS system offers high specificity, sensitivity, rapidity, and a simple detection tool for screening S. Indiana.

SEROTYPE AND ANTIMICROBIAL RESISTANCE OF ESCHERICHIA COLI ISOLATED FROM FECES OF WILD GIANT PANDAS ( AILUROPODA MELANOLEUCA) IN SICHUAN PROVINCE, CHINA.

Escherichia coli is a major pathogen leading to systemic and enteric illnesses in wild giant pandas ( Ailuropoda melanoleuca). To investigate the characteristics and distribution of E. coli in wild giant pandas across four different nature reserves in Sichuan, Republic of China, we researched serotypes, phylogenetic groups, antimicrobial resistance, and resistance genes of E. coli not previously reported for wild giant pandas. A total of 82 E. coli isolates were identified from 40 fecal samples in August 2016 to May 2017. The most-prevalent serogroups were O15 (4%, 3/82), O28 (2%, 2/82), and O44 (2%, 2/82). Antimicrobial resistance was highest for streptomycin (61%, 50/82) followed by amikacin (30%, 25/82). Among the four nature reserves, the proportion of streptomycin (86%, 12/14) and amikacin (57%, 8/14) was highest in Liziping. The frequencies of resistant genes aph(3')-IIa, ant(3″)-Ia, aac(3)-IIa, aadA1, and StrB were 28, 23, 5, 21, and 32%, respectively, while none of the strains had the tetracycline gene. In Qianfoshan, the phylogenetic group B2 was the most common, comprising the highest percentage of isolates compared with the other seven phylogenetic groups. Furthermore, many variables such as phylogenetic groups, antimicrobial susceptibility, and resistance genes differed significantly ( P<0.05) among the four nature reserves. In facilitating the safe discharge of captive giant pandas into the wild, as well as to support existing wild populations, the data from this research will prove invaluable to scientists and ecologists in their endeavors.

The normal vaginal and uterine bacterial microbiome in giant pandas (Ailuropoda melanoleuca).

While the health effects of the colonization of the reproductive tracts of mammals by bacterial communities are widely known, there is a dearth of knowledge specifically in relation to giant panda microbiomes. In order to investigate the vaginal and uterine bacterial diversity of healthy giant pandas, we used high-throughput sequence analysis of portions of the 16S rRNA gene, based on samples taken from the vaginas (GPV group) and uteri (GPU group) of these animals. Results showed that the four most abundant phyla, which contained in excess of 98% of the total sequences, were Proteobacteria (59.2% for GPV and 51.4% for GPU), Firmicutes (34.4% for GPV and 23.3% for GPU), Actinobacteria (5.2% for GPV and 14.0% for GPU) and Bacteroidetes (0.3% for GPV and 10.3% for GPU). At the genus level, Escherichia was most abundant (11.0%) in the GPV, followed by Leuconostoc (8.7%), Pseudomonas (8.0%), Acinetobacter (7.3%), Streptococcus (6.3%) and Lactococcus (6.0%). In relation to the uterine samples, Janthinobacterium had the highest prevalence rate (20.2%), followed by Corynebacterium (13.2%), Streptococcus (19.6%), Psychrobacter (9.3%), Escherichia (7.5%) and Bacteroides (6.2%). Moreover, both Chao1 and abundance-based coverage estimator (ACE) species richness indices, which were operating at the same sequencing depth for each sample, demonstrated that GPV had more species richness than GPU, while Simpson and Shannon indices of diversity indicated that GPV had the higher bacterial diversity. These findings contribute to our understanding of the potential influence abnormal reproductive tract microbial communities have on negative pregnancy outcomes in giant pandas.