Shuzhang Feng

VirA: A virulence-related gene of Streptococcus suis serotype 2

Streptococcus suis serotype 2 is an important porcine and human zoonotic pathogen. Few virulence factors have been identified and the pathogenesis of infection is not fully understood. We have identified a novel virulence-related gene, virA, of S. suis 2. To investigate its role in pathogenesis, a virA deletion mutant of S. suis 2 wild-type strain ZY458, 458Deltavir, was constructed using suicide vector pSET4s, and a functionally complemented strain 458Deltavir(pvir) was constructed using shuttle plasmid pAT18 containing the virA gene. All rabbits infected with ZY458 and 458Deltavir(pvir) exhibited body weight loss and developed severe clinical symptoms. All 5 rabbits infected with ZY458, and 4 of 5 infected with 458Deltavir(pvir), died within 6 days of infection. In contrast, all 5 rabbits inoculated with 458Deltavir gained weight normally and none developed any clinical symptoms during the entire course monitored. These results indicate that virA plays an important role in the pathogenicity of S. suis serotype 2. To investigate the virA gene further, we compared and analyzed the sequences of virA from wild-type virulent and avirulent strains. Results showed that functional virA appears to exist only in virulent strains and has been structurally mutated in avirulent strains. In conclusion, virA gene is a novel identified virulence-relate gene. It may play a key role in the evolution and pathogenicity of S. suis serotype 2.

Characterization of Antimicrobial Resistance and Extended-Spectrum β-Lactamase Genes in Escherichia coli Isolated from Chickens

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.

Towards an attenuated enterohemorrhagic Escherichia coli O157:H7 vaccine characterized by a deleted ler gene and containing apathogenic Shiga toxins

The aim of this study was to develop a candidate vaccine against enterohemorrhagic Escherichia coli O157:H7. A ler deletion mutant derived from wild-type EHEC O157:H7 86-24 was constructed by use of suicide vector pCVD442. The bacteriophage encoding Shiga toxin (Stx) was excised by serial passage to produce a ler/stx deletion mutant, F25. Stx1 and Stx2 mutants were constructed by site-directed mutagenesis within the active center and membrane-spanning region of the toxin A subunit. Mutants stx1 and stx2 were then introduced into F25 to construct live attenuated candidate vaccine F105. The cytotoxicity of F25 was inactivated and that of F105 was significantly reduced in comparison with wild-type E. coli strain EDL933. Mice injected with candidate vaccine strains F25 and F105 gained weight and showed no clinical signs of disease. F25 and F105 reduced the colonization of wild-type O157:H7 in mouse intestine. Immunized pregnant mice were able to protect their suckling newborns from intragastric challenge with wild-type O157:H7. Immunized mice were protected against infection with wild-type O157:H7 and exhibited normal weight gain. Such attenuated vaccine strains may therefore have potential use as oral vaccines against O157:H7.

Characterization and plasmid elimination of NDM-1-producing Acinetobacter calcoaceticus from China.

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.

Complete Genome Sequence of an Acinetobacter Strain Harboring the NDM-1 Gene

ABSTRACT The NDM-1 gene is a significant public health concern. Acinetobacter is one of the most prevalent opportunistic pathogens causing recent nosocomial infections with NDM-1, and drug-resistant strains pose serious threats to public health worldwide. Herein, we present the genomic sequence of Acinetobacter calcoaceticus subsp. anitratus XM1570, a multidrug-resistant isolate that carries the blaNDM-1 gene.

Investigation of Anthrax Cases in North-East China, 2010-2014

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.

Immune responses and protection induced by Brucella suis S2 bacterial ghosts in mice.

With the purpose of generating Brucella suis bacterial ghosts and investigating the immunogenicity of bacterial ghosts as a vaccine candidate, the lysis gene E and temperature-sensitive regulator cassette were cloned into a shuttle plasmid, pBBR1MCS-2, for construction of a recombinant temperature-sensitive shuttle lysis plasmid, pBBR1MCS-E. pBBR1MCS-E was then introduced into attenuated B. suis live vaccine S2 bacteria, and the resultant transformants were used for production of B. suis ghosts (BSGs) by inducing lysis gene E expression. The BSGs were characterized by observing their morphology by transmission electron microscopy. The safety and immunogenicity of BSGs were further evaluated using a murine model, the result suggested that BSG was as safe as formalin-killed B. suis. In mice, BSG demonstrated a similar capacity of inducing pathogen-specific serum IgG antibody response, spleen CD3(+) and CD4(+) T cell responses, induce secretion of gamma interferon and interleukin-4, and protection levels against Brucella melitensis 16M challenge, as the attenuated B. suis live vaccine. These data suggesting that BSG could confer protection against Brucella infection in a mouse model of disease and may be developed as a new vaccine candidate against Brucella infection.

A novel virulence-associated protein, vapE, in Streptococcus suis serotype 2

Streptococcus suis serotype 2 (SS2) is an important pathogen that affects pigs. However, neither its virulence nor its pathogenesis of infection has yet to be fully elucidated. The present study identifies a novel virulence‑associated protein E gene (vapE) of SS2. To investigate the importance of vapE in SS2 infection, a vapE knock‑out mutant based on SS2 wild‑type strain ZY458 was designated 458ΔvapE. 458ΔvapE was generated through homologous recombination, using a combined plasmid with a vapE knock‑out fragment and a pSET4s suicide vector. Additionally, the 458ΔvapE strain was transformed by a pAT18 shuttle plasmid containing the vapE gene. A functionally complemented strain for the vapE gene [termed 458ΔvapE (pvapE)] was constructed. Animal experiments demonstrated that mice infected with ZY458 and 458ΔvapE (pvapE) exhibited severe clinical symptoms, including depression, apathy, fever, anorexia, emaciation, swollen eyes and neural disorders, and died within two days of infection. All mice infected with ZY458, and 85% of mice infected with 458ΔvapE (pvapE), died within 2 days of infection. In contrast, mice inoculated with 458ΔvapE exhibited only mild clinical symptoms in the first 2 days following infection, and recovered within a week. A bacterial colonization assay demonstrated the ability of the 458ΔvapE mutant SS2 strain to colonize the heart, liver, spleen, lung and kidney of infected mice. PCR analysis of the vapE gene revealed that functional vapE was detected in virulent strains, but not in avirulent and carrier strains of S. suis SS2. These findings indicate that vapE is important for the pathogenesis of SS2.