This report presents the complete genome sequence of an atypical Bacillus cereus group strain carrying anthrax virulence genes isolated in China. Based on a comparison of the reference genome sequences of each species from the B. cereus group, the strain was determined to be Bacillus tropicus.

BackgroundWohlfahrtiimonas chitiniclastica is an emerging fly-borne zoonotic pathogen, which causes infections in immunocompromised patients and some animals. Herein, we reported a W. chitiniclastica BM-Y from a dead zebra in China.MethodsThe complete genome sequencing of BM-Y showed that this isolate carried one chromosome and one novel type of blaVEB–1-carrying plasmid. Detailed genetic dissection was applied to this plasmid to display the genetic environment of blaVEB–1.ResultsThree novel insertion sequence (IS) elements, namely ISWoch1, ISWoch2, and ISWoch3, were found in this plasmid. aadB, aacA1, and gcuG were located downstream of blaVEB–1, composing a gene cassette array blaVEB–1–aadB–aacA1–gcuG bracketed by an intact ISWoch1 and a truncated one, which was named the blaVEB–1 region. The 5′-RACE experiments revealed that the transcription start site of the blaVEB–1 region was located in the intact ISWoch1 and this IS provided a strong promoter for the blaVEB–1 region.ConclusionThe spread of the blaVEB–1-carrying plasmid might enhance the ability of W. chitiniclastica to survive under drug selection pressure and aggravate the difficulty in treating infections caused by blaVEB–1-carrying W. chitiniclastica. To the best of our knowledge, this is the first report of the genetic characterization of a novel blaVEB–1-carrying plasmid with new ISs from W. chitiniclastica.

Objectives: To characterize the antimicrobial resistance and virulence of pathogenic Escherichia coli isolated from diseased captive giant pandas.Methods: Antimicrobial susceptibility and minimum inhibitory concentration (MIC) were determined by the broth dilution method. Whole-genome sequencing was used to characterize the phylogeny, serotype, virulence, resistome, plasmids, and genetic structures of the cefotaxime (CTX)-M genes.Results: Four extended-spectrum beta-lactamase (ESBL)-producing E. coli strains were identified and the MICs against 11 antibiotics in vitro were determined. All ESBL-producing E. coli strains were resistant to more than eight antibiotics and carried the bla(CTX-M-55) or bla(CTX-M-105) gene in different sizes of replicon-type plasmids (pAMSH1-IncHI2, 257 kb; pAMPD2-IncFII, 89 kb; pAMPD02-IncFIB, 129 kb; and pAMSC4-IncN, 47 kb). Distinct insertional sequences and transposases were identified up-/downstream of bla(CTX-Ms), including IS26, ISEcp1, ISKpn72, IS903B, and Tn2. These strains also possessed at least three virulence genes of pathogenic E. coli and originated in four different evolutionary branches. One strain carried the complete locus of the enterocyte effacement pathogenicity island, but lacked the virulence genes stx and bfpA, indicating atypical enteropathogenic E. coli, whereas the other strains were considered to be extraintestinal pathogenic E. coli.Conclusions: The emergence of ESBL-producing pathogenic E. coli strains from diseased captive giant pandas warrants greater attention. The findings of this study will help to prevent the spread of these strains among captive giant pandas as well as from wild animals to humans.

Multi-drug-resistant Escherichia coli, Pseudomonas aeruginosa, and Enterococcus faecalis were isolated from the tissue samples of a giant panda. The E. coli strain SH-YH-DH that we isolated carried a self-transferable plasmid associated with multi-drug-resistant genes (bla(CTX-M-55), bla(TEM-1), sul1, floR, strB, aac(6')-Ib, and tetA/R). This strain exhibited phenotypic resistance to gentamicin, cefotaxime, ampicillin, chloramphenicol, trimethoprim-sulfamethoxazole, and tetracycline. We report the participation of multi-drug-resistant E. coli in a extraintestinal polyinfection in giant pandas and that further research is warranted to pay attention to the threat of multi-drug-resistant bacteria in the panda population.

半胱氨酸是一种食品添加剂,被应用于食品的增色、增香以及抗氧化.单细胞生物传感器可以用于氨基酸(如苯丙氨酸,缬氨酸等)的快速检测,但有关半胱氨酸的单细胞生物传感器还没有被报道.本研究基于转录调控因子CcdR,构建了荧光信号与半胱氨酸浓度相偶联的单细胞生物传感器,并对其线性范围进行了检测.在0 mmol/L至32 mmol/L的半胱氨酸浓度下,荧光信号强度与半胱氨酸浓度之间具有良好的线性关系.利用构建的半胱氨酸单细胞传感器,结合等离子诱变和流式细胞分选方法,从约5万株突变菌中筛选到两株半胱氨酸高产菌,其产量分别是野生型菌株的2.1倍和3.2倍.实验结果表明构建的单细胞生物传感器可以应用于半胱氨酸的快速检测及高产菌株的筛选.

Numerous environmental reservoirs contribute to the problem of acquired antibiotic resistance of human pathogens. One environmental reservoir of particular importance is the intestinal bacteria of poultry. Antibiotics are often used to prevent sickness and improve production in chicken farms. Hence, the intestinal microbial community of chickens as a reservoir for antibiotic resistance genes (ARGs) has received increasing attention. Therefore, the aim of this study was to investigate the occurrence, diversity, and abundance of ARGs and mobile genetic elements (MGEs), and the distribution of plasmids in layer and broiler feces using Illumina high-throughput sequencing. Metagenomic analysis showed that Proteobacteria, Firmicutes, Bacteroidetes, and Actinobacteria were predominate in chicken feces. At the genus level, Escherichia coli may be predominant in chicken feces with a relative abundance of plasmids. Sequencing reads revealed differences in the distribution of plasmids in microbial communities between layer and broiler feces. Genes coding for antibiotic resistance were identified in both communities. BLAST analysis against the Antibiotic Resistance Genes Database (ARDB) further revealed that layer and broiler feces contained various ARGs with high abundances, among which vancomycin (van) was the most abundant, accounting for >36% of all reads of ARGs. Beta-lactam resistance genes (bl) were highly enriched in the broiler feces, among which bl2e_cbla was the most abundant, but was absent in layer feces. Over 20 types of insertion sequences were detected in each fecal sample. These results highlight the prevalence of ARGs, plasmids, and MGEs in chicken feces, which warrants further studies to better elucidate the risk to public health.

Dozens of wild bharals died suddenly in Tibet. Necropsy showed severe congestion and hemorrhage in multiple organs, with large numbers of Gram-positive bacilli. Strains of Clostridium perfringens type A were isolated from the different organs and the intestinal contents. The other possible pathogens were ruled out by PCR.

多杀性巴氏杆菌（Pasteurella multocida）是巴氏杆菌属的成员之一,能感染鸡、鸭、兔、猪和牛等多种家养动物和部分野生动物,不同动物感染后的疾病命名不同,如禽霍乱（Eveleth et al.,1949）、猪肺疫和牛出血性败血症（陆承平,2001）。该菌为革兰氏阴性球杆菌或短杆菌,新分离菌株瑞氏染色两极着色明显,有荚膜,人工培养

A comprehensive study of cross-species transmission and inter-regional migration would provide insights into the global ecology of influenza A viruses (IAVs). To this end, we assembled 17,241 non-redundant IAV whole-genome sequences with complete epidemiological information. We hierarchically divided the movements of IAVs into the cross-species transmission in each region and the inter-regional migration driven by each host species. We then systematically identified the potential cross-species transmission and inter-regional migration events. Cross-species transmission networks were obtained for each gene segment of the IAVs. Waterfowl, domestic birds and swine showed higher degrees of connection than did other species in all of the transmission networks. East Asia and Southeast Asia were hot regions for avian-mammal transmissions. Swine and migratory birds were the dominant species for global virus delivery. The importance of swine was reemphasized because it has not only provided an environment for adaptive evolution during the avian-human transmission of IAVs (as incubators) but also served as a key species for the global dissemination of the viruses (as carriers). Therefore, monitoring the global live trade of swine and survey of migratory birds along flyways would be beneficial for the prevention and control of IAVs.

ABSTRACT Here, a complete genome sequence of Wohlfahrtiimonas chitiniclastica strain BM-Y is presented. The whole genome is 2.18-Mb and contains a bla VEB-1 gene cassette which endows it with resistance to ceftazidime, ampicillin, tetracycline, etc. To our knowledge, this is the first time that an extended spectrum beta-lactamase (ESBL) type W. chitiniclastica strain has been found.

We determined the genotypes of seven Bacillus anthracis strains that were recovered from nine anthrax outbreaks in North-East China from 2010 to 2014, and two approved vaccine strains that are currently in use in China. The causes of these cases were partly due to local farmers being unaware of the presence of anthrax, and butchers with open wounds having direct contact with anthrax-contaminated meat products. The genotype of five of the seven recovered strains was A.Br.001/002 sub-lineage, which was concordant with previously published research. The remaining two cases belongs to the A.Br.Ames sub-lineage. Both of these strains displayed an identical SNR pattern, which was the first time that this genotype was identified in North-East China. Strengthening education in remote villages of rural China is an important activity aimed at fostering attempts to prevent and control anthrax. The genotype of the vaccine strain Anthrax Spore Vaccine No. II was A.Br.008/009 and A.Br.001/002 for the vaccine strain Anthrax Spore Vaccine Non-capsulated. Further studies of their characteristics are clearly warranted.

OBJECTIVETo generate polyclonal antibodies against Shigella flexneri 5a M90T heat shock protein 70 (DnaK) and identify the specificity and titer of antibodies.METHODSThe sequence of Shigella flexneri 5a M90T dnaK gene was amplified by PCR and inserted into expression vector pET-24a to obtain recombinant plasmid pET24a-dnaK. Then the recombinant plasmid pET24a-dnaK was transformed into E.coli BL21 (DE3) and expressed under IPTG induction. The recombinant DnaK proteins with His-tag (DnaK-His) were then purified by affinity purification. The polyclonal antibodies were prepared by immunizing the mice with the purified recombinant proteins and then harvesting mouse sera. The specificity and titer of the polyclonal antibodies were identified by Western blotting, ELISA and immunofluorescent staining.RESULTSThe recombinant plasmid pET24a-dnaK was expressed efficiently in E.coli BL21 (DE3). The polyclonal antibodies harvested from the mice immunized with DnaK-His were demonstrated being specific to Shigella flexneri 5a M90T DnaK proteins, and could be efficient in Western blotting, ELISA and immunofluorescent staining.CONCLUSIONThe polyclonal antibodies against Shigella flexneri 5a M90T DnaK protein have been successfully prepared.

Extended-spectrum β-lactamase (ESBL)-producing Escherichia coli have been frequently isolated from food-producing animals and pose a serious threat to human health. This study collected 195 ESBL-producing E. coli isolates from 20 chicken farms and 3 live-bird markets located in Northeast China (Heilongjiang, Liaoning, Jilin) and Jiangsu province from February 2011 to October 2013. ESBL genes, including blaCTX-M, blaTEM, and blaSHV, were detected and characterized, and the susceptibilities of these strains to various antimicrobial agents were determined. One hundred ninety-one of these isolates carried 1 or more bla genes. blaCTX-M, blaTEM-1, and blaSHV-5 were identified in 183, 121, and 2 isolates, respectively. The most common blaCTX-M genes were blaCTX-M-15 (68 strains), blaCTX-M-65 (41 strains), blaCTX-M-55 (35 strains), blaCTX-M-14 (32 strains), followed by blaCTX-M-3, blaCTX-M-13, blaCTX-M-79, and blaCTX-M-101, as well as the chimeric genes blaCTX-M-64, blaCTX-M-123, and blaCTX-M-132. Fifteen strains (7.7%) co-harboring CTX-M-1 group and CTX-M-9 group genes were detected in 195 ESBL-producing strains. Pulsed-field gel electrophoresis of 45 strains showed that these CTX-M-producing isolates belonged to 34 different types. To our knowledge, this is the first study to report the blaSHV-5 gene in E. coli isolated from chickens in China. Conjugation experiments demonstrated that the blaCTX-M and blaTEM genes could be transferred to E. coli strain J53, while conjugative transfer of the blaSHV-5 gene from two isolates was not detectable. blaCTX-M genes are carried by many kinds of transferable and untypable plasmids. Our findings demonstrate that the CTX-M enzymes are predominant in both type and quantity.

An entry from the Cambridge Structural Database, the world’s repository for small molecule crystal structures. The entry contains experimental data from a crystal diffraction study. The deposited dataset for this entry is freely available from the CCDC and typically includes 3D coordinates, cell parameters, space group, experimental conditions and quality measures.

VirG is outer membrane protein of Shigella and affects the spread of Shigella. Recently it has been reported that apyrase influences the location of VirG, although the underlying mechanism remains poorly understood. The site of interaction between apyrase and VirG is the focus of our research. First we constructed recombinant plasmid pHIS-phoN2 and pS-(v1–1102, v53–758, v759–1102, v53–319, v320–507, v507–758) by denaturation-renaturation, the phoN2:kan mutant of Shigella flexneri 5a M90T by a modified version of the lambda red recombination protocol originally described by Datsenko and Wanner and the complemented strain M90TΔphoN2/pET24a(PhisphoN2). Second, the recombinant plasmid pHIS-phoN2 and the pS-(v1–1102, v53–758, v759–1102, v53–319, v320–507, v507–758) were transformed into E. coli BL21 (DE3) and induced to express the fusion proteins. Third, the fusion proteins were purified and the interaction of VirG and apyrase was identified by pull-down. Fourth, VirG was divided and the interaction site of apyrase and VirG was determined. Finally, how apyrase affects the function of VirG was analyzed by immunofluorescence. Accordingly, the results provided the data supporting the fact that apyrase combines with the α-domain of VirG to influence the function of VirG.

OBJECTIVE:To prepare mouse polyclonal antibodies against Shigella flexneri M90T glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and verify its titer and specificity.METHODS:The gapA gene of Shigella flexneri M90T was amplified by PCR from extracted genomic DNA and inserted into expression vector pET24a. The plasmid pET24a-gapA was transformed into E.coli BL21(DE3) and induced to express the tagged protein GAPDH-His. After condition optimization, the protein was purified with Ni Sepharose(TM); 6 Fast Flow. The polyclonal antibodies were prepared by immunizing the mice with the purified recombinant protein and then harvesting mouse sera. The specificity and efficiency of antibodies in sera were analyzed by Western blotting and ELISA against whole cell proteins of Shigella flexneri M90T.RESULTS:The recombinant plasmid pET24a-gapA was successfully constructed. The soluble expression of the tagged protein GAPDH-His was achieved with 2-hour induction of 1 mmol/L IPTG at 30°C. Western blotting and ELISA demonstrated that the polyclonal antibody harvested from the mice immunized with GAPDH-His was specific and efficient against Shigella flexneri M90T GAPDH. Conclusion The mouse polyclonal antibodies against the Shigella flexneri M90T GAPDH protein were successfully prepared.

The presence of multidrug-resistant bacterial pathogens in the environment poses a serious threat to public health. The opportunistic Acinetobacter spp. are among the most prevalent causes of nosocomial infections. Here, we performed complete genome sequencing of the Acinetobacter calcoaceticus strain XM1570, which was originally cultivated from the sputum of a patient diagnosed with pneumonia in Xiamen in 2010. We identified carbapenem resistance associated gene bla(NDM-1) located on a 47.3-kb plasmid. Three methods--natural reproduction, sodium dodecyl sulfate treatment and nalidixic acid treatment--were used to eliminate the bla(NDM-1)-encoding plasmid, which achieved elimination rates of 3.32% (10/301), 83.78% (278/332), and 84.17% (298/354), respectively. Plasmid elimination dramatically increased antibiotic sensitivity, reducing the minimum bacteriostatic concentration of meropenem from 256 µg/ml in the clinical strain to 0.125 µg/ml in the plasmid-eliminated strain. Conjugation transfer assays showed that the bla(NDM-1)-containing plasmid could be transferred into Escherichia coli DH5α:pBR322 in vitro as well as in vivo in mice. The bla(NDM-1) genetic environment was in accordance with that of other bla(NDM-1) genes identified from India, Japan, and Hong-Kong. The multilocus sequence type of the isolate was identified as ST-70. Two novel genes encoding intrinsic OXA and ADC were identified and named as OXA-417 and ADC-72. The finding of bla(NDM-1) in species like A. calcoaceticus demonstrates the wide spread of this gene in gram-negative bacteria which is possible by conjugative plasmid transfer. The results of this study may help in the development of a treatment strategy for controlling NDM-1 bacterial infection and transmission.