Wenhui Ji

Application of a Bacteriophage Lysin To Disrupt Biofilms Formed by the Animal Pathogen Streptococcus suis

ABSTRACT Bacterial biofilms are crucial to the pathogenesis of many important infections and are difficult to eradicate. Streptococcus suis is an important pathogen of pigs, and here the biofilm-forming ability of 32 strains of this species was determined. Significant biofilms were completely formed by 10 of the strains after 60 h of incubation, with exopolysaccharide production in the biofilm significantly higher than that in the corresponding planktonic cultures. S. suis strain SS2-4 formed a dense biofilm, as revealed by scanning electron microscopy, and in this state exhibited increased resistance to a number of antibiotics (ampicillin, amoxicillin, ciprofloxacin, kanamycin, and rifampin) compared to that of planktonic cultures. A bacteriophage lysin, designated LySMP, was used to attack biofilms alone and in combination with antibiotics and bacteriophage. The results demonstrated that the biofilms formed by S. suis, especially strains SS2-4 and SS2-H, could be dispersed by LySMP and with >80% removal compared to a biofilm reduction by treatment with either antibiotics or bacteriophage alone of less than 20%; in addition to disruption of the biofilm structure, the S. suis cells themselves were inactivated by LySMP. The efficacy of LySMP was not dose dependent, and in combination with antibiotics, it acted synergistically to maximize dispersal of the S. suis biofilm and inactivate the released cells. These data suggest that bacteriophage lysin could form part of an effective strategy to treat S. suis infections and represents a new class of antibiofilm agents.

Rapid simultaneous quantification of zearalenone and fumonisin B1 in corn and wheat by lateral flow dual immunoassay.

A lateral flow dual immunoassay (LFDIA) was developed for rapid quantitative detection of zearalenone (ZEN) and fumonisin B1 (FB1) in corn and wheat samples on a single test strip. Two test lines and the control line on the nitrocellulose membrane were coated with ZEN and FB1 conjugates and goat anti-mouse IgG, respectively. Colloidal gold nanoparticles were conjugated with monoclonal antibodies against ZEN or FB1. The intensity of the test lines was analyzed by a photometric strip reader to determine the concentrations of ZEN and FB1 based on the calibration curves of known concentrations versus intensity readings. Test parameters such as types of buffers, ratio of the two gold-labeled antibodies, and dilution of the sample extracts and the gold-labeled antibodies were optimized. The detection limit was 0.35 and 5.23 ng/mL for ZEN and FB1, respectively, and the corresponding detection ranges were 0.94-7.52 and 9.34-100.45 ng/mL, respectively. Spiked and natural samples were analyzed using both LFDIA and liquid chromatography-tandem mass spectrometry. The two methods had a good correlation (R(2) = 0.96). The dual quantitative LFDIA is sensitive, rapid, and easy-to-use for on-site testing of a large number of samples.

Novel chemiluminescence immunoassay for the determination of zearalenone in food samples using gold nanoparticles labeled with streptavidin-horseradish peroxidase.

A novel highly sensitive chemiluminescence immunoassay (CLIA) was developed to detect zearalenone in food samples by using both biotinylated zearalenone conjugates and gold (Au) nanoparticles labeled with streptavidin-horseradish peroxidase for signal amplification. Biotinylated zearalenone-ovalbumin conjugates and Au nanoparticles labeled with streptavidin-horseradish peroxidase were synthesized separately. The concentrations of immunoreagents and the reaction times of these immunoreagents were optimized to improve the performances of analytical methods. For the CLIA based on biotinylated zearalenone conjugates and Au nanoparticles labeled with streptavidin-horseradish peroxidase, the limit of detection was 0.008 ng/mL and the IC50 was 0.11 ng/mL. The linear working range was 0.02-0.51 ng/mL. The cross-reactivities with the zearalenone analogues (α-zearalanol, zearalanone, α-zearalenol, β-zearalanol, and β-zearalenol) were 32, 17, 12, 0.3, and 0.1%, respectively. The recovery rates in spiked food samples were 97-117%, and the intraday and interday relative standard deviations were both <10%. Parallel analysis of natural food samples showed a good correlation between this novel CLIA and liquid chromatography-tandem mass spectrometry. This method provides a rapid, accurate, and highly sensitive method to determine levels of zearalenone in food samples.

Simultaneous quantitative determination of multiple mycotoxins in cereal and feedstuff samples by a suspension array immunoassay.

Mycotoxins produced by different species of fungi may coexist in single cereal and feedstuff samples, which could become highly toxic for humans and animals. In order to quantify four mycotoxins (zearalenone, fumonisin B1, deoxynivalenol, and aflatoxin B1) in cereal and feedstuff samples simultaneously, a new suspension array immunoassay was developed. Antimycotoxin monoclonal antibodies were conjugated to the surface of different encoding microspheres (19#, 37#, 39#, and 49#), and mycotoxin-protein conjugates were then coupled with biotin. Using streptavidin-phycoerythrin as a signal reporter protein, this direct competition multiple suspension array immunoassay was optimized. The results showed that the detection limits for zearalenone, fumonisin B1, deoxynivalenol, and aflatoxin B1 were 0.51, 6.0, 4.3, and 0.56 ng/mL, respectively, with detection ranges of 0.73-6.8, 11.6-110.3, 8.6-108.1, and 1.1-14.1 ng/mL, respectively. For the detection of the spiked samples, the recovery rates were between 92.3% and 115.5%. This method also shows a good correlation coefficient (r = 0.99, P < 0.01) with liquid chromatography-tandem mass spectrometry in the detection of toxins in commercial cereal and feedstuff samples. This suspension array immunoassay was high-throughput and accurate for the rapid quantitative detection of multiple mycotoxins in commercial cereal and feedstuff samples.

Functional analysis of c-di-AMP phosphodiesterase, GdpP, in Streptococcus suis serotype 2

Streptococcus suis serotype 2 (SS2) is an important zoonotic pathogen that causes serious diseases in pigs and humans. GdpP protein is a recently discovered specific phosphodiesterase that degrades cyclic diadenosine monophosphate (c-di-AMP). It is widely distributed among the firmicutes phylum and altered expression of GdpP is associated with several phenotypes in various bacterial strains. We investigated the role of GdpP in physiology and virulence in SS2. An in-frame mutant of gdpP was constructed using homologous recombination and bacterial growth, biofilm formation, hemolytic activity, cell adherence and invasion, expression of virulence factors, and virulence were evaluated. Disruption of gdpP increased intracellular c-di-AMP level and affected growth and increased biofilm formation of SS2. Simultaneously, the gdpP mutant strain exhibited a significant decrease in hemolytic activity and adherence to and invasion of HEp-2 cells compared with the parental strain. Quantitative reverse transcriptase polymerase chain reaction indicated significantly reduced expression of the known virulence genes cps2, sly, fpbs, mrp, ef and gdh in the gdpP mutant. In murine infection models, the gdpP mutant strain was attenuated, and impaired bacterial growth was observed in specific organs. All these findings revealed a significant contribution of gdpP and its substrate (c-di-AMP) to the biology and virulence of SS2.

Muscovy duck retinoic acid-induced gene I (MdRIG-I) functions in innate immunity against H9N2 avian influenza viruses (AIV) infections.

Retinoic acid inducible gene I (RIG-I) is a cytosolic pattern recognition receptor that senses pathogen-associated molecular patterns (PAMPs). Muscovy duck (Cairina moschata) is a large duck different from other species of ducks, and is more susceptible to some microbial pathogens. In this study, the Muscovy duck RIG-I gene (MdRIG-I) was identified. Quantitative RT-PCR showed that MdRIG-I mRNA was widely expressed in different tissues, especially in those with mucosa. RIG-I null DF-1 cells transfected with DNA constructs encoding MdRIG-I or CARDs domain can activate IRF-3 and NF-κB to up-regulated activity of IFN-β promoter. The components of the signaling pathway downstream of RIG-I in mammalian cells including IRF-3, NF-κB, IFN-β and the IFN-stimulated genes Mx-1, PKR and MDA5 were significantly up-regulated in CARDs-overexpressing-DF-1 cells. Implicating RIG-I in the antiviral response to an infection in vivo, we found that RIG-I expression in brain, spleen, lung and bursa were up-regulated in ducks challenged with H9N2 avian influenza virus (AIV), whose six internal genes were closely related to the H7N9 and H10N8 AIV. In vitro, DF-1 cells transfected with MdRIG-I plasmid can respond significantly to H9N2 AIV, evident through enhancement of IFN-β promoter activity and decreased virus titer. Altogether, these results indicated that MdRIG-I is a novel member of RLR gene family, engaging in the early stage of antiviral innate immunity.

SLPW: A Virulent Bacteriophage Targeting Methicillin-Resistant Staphylococcus aureus In vitro and In vivo.

Staphylococcus aureus (S. aureus) is a Gram-positive pathogen causing a variety of infections in humans and animals. Extensive use of antibiotics has led to the emergence of methicillin-resistant S. aureus (MRSA). As an alternative antibacterial agent against drug-resistant S. aureus, a lytic phage, designated SLPW, was isolated from fecal sewage in a pig farm. The SLPW was morphologically classified under Podoviridae and contains a double-stranded DNA genome. The genome of SLPW was 17,861 bp (29.35% G+C) containing 20 open reading frames and lacked regions encoding lysogeny-related integrase gene and cI repressor gene. Phage SLPW showed a broad host range and high efficiency of plating against various types of S. aureus. One-step growth curve showed a short latency period (10 min) and a long lytic period (120 min). Phage SLPW remained stable under a wide range of temperatures or pH and was almost unaffected in chloroform or ultraviolet light. Further, it efficiently lysed MRSA strains in vitro and in vivo. Intraperitoneal phage administration at 1 h post-infection cured the mice and reduced the bacterial expression of inflammatory cytokines in mice. Specifically, the phage SLPW displayed a wide antibacterial spectrum. It was therapeutically effective against intra-abdominal infection in mice harboring different multilocus sequence typing (MLST) types of S. aureus strains. Therefore, phage SLPW is a potential therapeutic agent against MRSA infections.

A novel endolysin disrupts Streptococcus suis with high efficiency

Streptococcus suis serotype 2 (S. suis 2) is a zoonotic pathogen that exhibits high-level resistance and multi-drug resistance to classic antibiotics and causes serious human casualties and heavy economic losses in the swine industry worldwide. Therefore, alternative therapies or novel antibacterial agents need to be developed to combat this pathogen. A novel endolysin derived from the S. suis temperate phage phi7917, termed Ly7917, was identified, which had broad lytic activity against S. suis type 1, 2, 7 and 9. Ly7917 consisted of an N-terminal cysteine, histidine-dependent amidohydrolases/peptidase catalytic domain and C-terminal SH3b cell wall binding domain. The endolysin maintained activity at high pH and its catalytic activity could be improved by addition of 10 μM 1.5 mM Ca(2+). In animal studies, 90% of BALB/c mice challenged with typical virulent strain HA9801 of S. suis 2 were protected by Ly7917 treatment. The bacterial load in the blood of HA9801-challenged mice was efficiently reduced almost 50% by Ly7917 while that of penicillin-G-treated mice kept almost unchanged. Our data suggest that Ly7917 may be an alternative therapeutic agent for infections caused by virulent S. suis strains.

Escherichia coli nfuA is essential for maintenance of Shiga toxin phage Min27 lysogeny under iron-depleted condition

It has been earlier hypothesized that lysogenic infection with Stx-encoding phages influences protein expression in the bacterial host, and therefore, some differentially expressed proteins could affect survival characteristics and pathogenicity. We compared the protein expression profiles of the host MG1655 and lysogens by 2D electrophoresis. Four different genes identified were all related to Fe/S subunit production, namely, nfuA, fdoH, sdhB and ftnA. To explore the role of nfuA in the biology of Stx prophage lysogeny, gene knockout experiments and phage lysogenic conversion were performed. The inactivation of nfuA caused the prophage to enter its lytic life cycle, especially under an iron-depleted condition. A similar activity was also detected in the Escherichia coli O157:H7 strain from which the Stx phage Min 27 was originally isolated. NfuA might be the positive regulator of genes controlling lysogenic cycle such as cI, cII and cIII since their transcriptional level was significantly reduced in nfuA deletion mutant as shown by qRT-PCR. We conclude that NfuA is essential for maintenance of Stx phage lysogeny in hosts genetic reservoir under iron-deficient condition.