Molecular Epidemiology of Plasmid-Mediated Types 1 and 3 Fimbriae Associated with Biofilm Formation in Multidrug Resistant Escherichia coli from Diseased Food Animals in Guangdong, China.

Types 1 and 3 fimbriae in Enterobacteriaceae play versatile roles in bacterial physiology including attachment, invasion, cell motility as well as with biofilm formation and urinary tract infections. Herein, we investigated the prevalence and transmission of plasmid-mediated types 1 and 3 fimbriae from 1753 non-duplicate Enterobacteriaceae from diseased food Animals. We identified 123 (7.01%) strong biofilm producers and all was identified as E. coli. WGS analysis of 43 selected strong biofilm producers revealed that they harbored multiple ARGs, including ESBLs, PMQR and mcr-1. The gene clusters mrkABCDF and fimACDH encoding types 1 and 3 fimbriae, respectively, were identified among 43 (34.96%) and 7 (5.7%) of 123 strong biofilm isolates, respectively. These two operons were able to confer strong biofilm-forming ability to an E. coli weak-biofilm forming laboratory strain. Plasmid analysis revealed that mrk and fim operons were found to co-exist with ARGs and were primarily located on IncX1 and IncFII plasmids with similar backbones, respectively. mrkABCDF operons was present in all of 9457 Klebsiella pneumoniae using archived WGS data, and shared high homology to those on plasmids of 8 replicon types and chromosomes from 6 Enterobacteriaceae species from various origins and countries. In contrast, fimACDH operons was present in most of Enterobacter cloacae (62.15%), and shared high homology to those with only a small group of plasmids and Enterobacteriaceae species. This is the first comprehensive report of the prevalence, transmission and homology of plasmid-encoded type 1 and 3 fimbriae among the Enterobacteriaceae. Our findings indicated that plasmid-encoded mrkABCDF and fimACDH were major contributors to enhanced biofilm formation among E. coli and these two operons, in particular mrk could be as a potential anti-biofilm target. IMPORTANCE Biofilms allow bacteria to tolerate disinfectants and antimicrobials, as well as mammalian host defenses, and are therefore difficult to treat clinically. Most research concerning biofilm-related infections is typically focused on chromosomal biofilm-associated factors, including types 1 and 3 fimbriae of biofilm-forming Enterobacterium. However, the transmission and homology of the mobile types 1 and 3 fimbriae among Enterobacteriaceae is largely unknown. The findings revealed that the plasmid-encoded type 3 fimbriae encoded by mrkABCDF and type 1 fimbriae encoded by fimACDH were major contributors to enhancing biofilm formation among strong biofilm E. coli from diseased food producing animals. Additionally, mrk operon with high homology at an amino acid sequence was present both on plasmids of various replicon types and on chromosomes from diverse Enterobacteriaceae species from numerous origins and countries. These findings provide important information on the transmission of the mobile types 1 and 3 fimbriae among Enterobacteriaceae, indicating a potential antibiofilm target.

Phylogenomic analysis of Salmonella Indiana ST17, an emerging MDR clonal group in China.

OBJECTIVES: To reconstruct the genomic epidemiology and evolution of MDR Salmonella Indiana in China. METHODS: A total of 108 Salmonella Indiana strains were collected from humans and livestock in China. All isolates were subjected to WGS and antimicrobial susceptibility testing. Phylogenetic relationships and evolutionary analyses were conducted using WGS data from this study and the NCBI database. RESULTS: Almost all 108 Salmonella Indiana strains displayed the MDR phenotype. Importantly, 84 isolates possessed concurrent resistance to ciprofloxacin and cefotaxime. WGS analysis revealed that class 1 integrons on the chromosome and IncHI2 plasmids were the key vectors responsible for multiple antibiotic resistance gene (ARG) [including ESBL and plasmid-mediated quinolone resistance (PMQR) genes] transmission among Salmonella Indiana. The 108 Salmonella Indiana dataset displayed a relatively large core genome and ST17 was the predominant ST. Moreover, the global ST17 Salmonella Indiana strains could be divided into five distinct lineages, each of which was significantly associated with a geographical distribution. Genomic analysis revealed multiple antimicrobial resistance determinants and QRDR mutations in Chinese lineages, which almost did not occur in other global lineages. Using molecular clock analysis, we hypothesized that ST17 isolates have existed since 1956 and underwent a major population expansion from the 1980s to the 2000s and the genetic diversity started to decrease around 2011, probably due to geographical barriers, antimicrobial selective pressure and MDR, favouring the establishment of this prevalent multiple antibiotic-resistant lineage and local epidemics. CONCLUSIONS: This study revealed that adaptation to antimicrobial pressure was possibly pivotal in the recent evolutionary trajectory for the clonal spread of ST17 Salmonella Indiana in China.

Occurrence and Transmission of blaNDM-Carrying Enterobacteriaceae from Geese and the Surrounding Environment on a Commercial Goose Farm.

We investigated the prevalence and transmission of NDM-producing Enterobacteriaceae in fecal samples of geese and environmental samples from a goose farm in southern China. The samples were cultivated on MacConkey agar plates supplemented with meropenem. Individual colonies were examined for blaNDM, and blaNDM-positive bacteria were characterized based on whole-genome sequencing (WGS) data from the Illumina and Oxford Nanopore Technologies (ONT) platforms. Of 117 samples analyzed, the carriage rates for New Delhi metallo-ß-lactamase (NDM)-positive Enterobacteriaceae were 47.1, 18, and 50% in geese, inanimate environments (sewage, soil, fodder, and dust), and mouse samples, respectively. Two variants (blaNDM-1 and blaNDM-5, in 4 and 40 isolates, respectively) were found among 44 blaNDM-positive Enterobacteriaceae; these variants belonged to eight species, and Escherichia coli was the most prevalent (50%). WGS analysis revealed that blaNDM coexisted with diverse antibiotic resistance genes (ARGs). Population structure analysis showed that most E. coli and Enterobacter sp. isolates were highly heterogeneous, while most Citrobacter sp. and P. stuartii isolates possessed extremely high genetic similarities. In addition, blaNDM-5-positive ST4358/ST48 E. coli isolates were found to be clonally spread between geese and the environment and were highly genetically similar to those reported from ducks, farm environments, and humans in China. Plasmid analysis indicated that IncX3 pHNYX644-1-like (n = 40) and untypeable pM2-1-like plasmids (n = 4) mediated blaNDM spread. pM2-1-like plasmids possessed diverse ARGs, including blaNDM-1, the arsenical and mercury resistance operons, and the maltose operon. Our findings revealed that the goose farm is a reservoir for NDM-positive Enterobacteriaceae The blaNDM contamination of wild mice and the novel pM2-1-like plasmid described here likely adds to the risk for dissemination of blaNDM and associated resistance genes.IMPORTANCE Carbapenem-resistant bacteria, in particular NDM-producing Enterobacteriaceae, have become a great threat to global public. These bacteria have been found not only in hospital and community environments but also among food animal production chains, which are recognized as reservoirs for NDM-producing Enterobacteriaceae However, the dissemination of NDM-producing bacteria in waterfowl farms has been less well explored. Our study demonstrates that the horizontal spread of blaNDM-carrying plasmids and the partial clonal spread of blaNDM-positive Enterobacteriaceae contribute to the widespread contamination of blaNDM in the goose farm ecosystem, including mice. Furthermore, we found a novel and transferable blaNDM-1-carrying multidrug resistance (MDR) plasmid that possessed multiple environmental adaptation-related genes. The outcomes of this study contribute to a better understanding of the prevalence and transmission of blaNDM-carrying Enterobacteriaceae among diverse niches in the farm ecosystem.

Global clonal spread of mcr-3-carrying MDR ST34 Salmonella enterica serotype Typhimurium and monophasic 1,4,[5],12:i:- variants from clinical isolates.

OBJECTIVES: To investigate the prevalence and transmission of mcr-3 among Salmonella enterica serotype Typhimurium and 1,4,[5],12:i:-. METHODS: A total of 4724 clinical Salmonella isolates were screened for the presence of mcr-3 in China during 2014-19. The clonal relationship of the mcr-3-positive isolates and their plasmid contents and complete sequence were also characterized based on WGS data from the Illumina and MinION platforms. RESULTS: We identified 10 mcr-3-positive isolates, and all were MDR, mostly resistant to colistin, cefotaxime, ciprofloxacin, doxycycline and florfenicol. mcr-3 was co-present with blaCTX-M-55-qnrS1 on hybrid ST3-IncC-FII conjugatable plasmids (n = 6) and an ST3-IncC non-conjugatable plasmid (n = 1) and embedded into a pCHL5009T-like IncFII plasmid on the Salmonella chromosome (n = 3). Four distinctive genetic contexts surrounded mcr-3 and all but one were closely related to each other and to the corresponding region of IncFII plasmid pCHL5009T. IS15DI was most likely the vehicle for integration of mcr-3-carrying IncFII plasmids into ST3-IncC plasmids and the chromosome and for shaping the MDR regions. In addition, a phylogenetic tree based on the core genome revealed a unique Salmonella lineage (≤665 SNPs) that contained these 10 mcr-3-positive isolates and another 38 (33 from patients) mcr-3-positive Salmonella from five countries. In particular, most of the 51 mcr-3-positive isolates belonged to ST34 and harboured diverse antibiotic resistance genes (ARGs), including mcr-3-blaCTX-M-55-qnrS1, and possessed similar ARG profiles. CONCLUSIONS: Our findings revealed global clonal spread of MDR ST34 Salmonella from clinical isolates co-harbouring mcr-3 with blaCTX-M-55 and qnrS1 and a flexibility of mcr-3 co-transmittance with other ARGs mediated by mobile genetic elements.

Co-occurrence of Plasmid-Mediated Tigecycline and Carbapenem Resistance in Acinetobacter spp. from Waterfowls and Their Neighboring Environment.

Tigecycline serves as one of the antibiotics of last resort to treat multidrug-resistant (including carbapenem-resistant) pathogens. However, the recently emerged plasmid-mediated tigecycline resistance mechanism, Tet(X), challenges the clinical efficacy of this class of antibiotics. In this study, we detected 180 tet(X)-harboring Acinetobacter isolates (8.9%, n = 180) from 2,018 samples collected from avian farms and adjacent environments in China. Eighteen tet(X)-harboring isolates (10.0%) were found to cocarry the carbapenemase gene blaNDM-1, mostly from waterfowl samples (94.4%, 17/18). Interestingly, among six Acinetobacter strains, tet(X) and blaNDM-1 were found to colocalize on the same plasmids. Moreover, whole-genome sequencing (WGS) revealed a novel orthologue of tet(X) in the six isolates coharboring tet(X) and blaNDM-1 Inverse PCR suggested that the two tet(X) genes form a single transposable unit and may be cotransferred. Sequence comparison between six tet(X)- and blaNDM-1-coharboring plasmids showed that they shared a highly homologous plasmid backbone even though they were isolated from different Acinetobacter species (three from Acinetobacter indicus, two from Acinetobacter schindleri, and one from Acinetobacter lwoffii) from various sources and from different geological regions, suggesting the horizontal genetic transfer of a common tet(X)- and blaNDM-1-coharboring plasmid among Acinetobacter species in China. Emergence and spread of such plasmids and strains are of great clinical concern, and measures must be implemented to avoid their dissemination.

Diverse and Flexible Transmission of fosA3 Associated with Heterogeneous Multidrug Resistance Regions in Salmonella enterica Serovar Typhimurium and Indiana Isolates.

We identified fosA3 at a rate of 2.6% in 310 Salmonella isolates from food animals in Guangdong province, China. The fosA3 gene was genetically linked to diverse antibiotic resistance genes (ARGs), including mcr-1, blaCTX-M-14/55, oqxAB, and rmtB These gene combinations were embedded in heterogeneous fosA3-containing multidrug resistance regions on the transferable ST3-IncHI2 and F33:A-:B- plasmids and the chromosome. This indicated a great flexibility of fosA3 cotransmission with multiple important ARGs among Salmonella species.

Structural characterization of two endopolysaccharides from Phellinus sp. and their immunologic effects by intragastric administration in a healthy mammalian model.

Two purified endopolysaccharides derived from cultured Phellinus sp., individually named SHIP-1 and 2, were structurally characterized, along with an evaluation of their in vivo influential immunomodulatory activity in a healthy mammalian model. The structure of SHIP-1 was mainly composed of →4)-α-d-Fucp-(1→, →3,6)-α-d-Araf-(1→ and →2,4)-ß-d-Galp-(→, with four residuals of α-d-Manp-(1→ and one α-d-Glcp-(1→ as sidegroups, while the planar structure and the heteronuclear multiple-bond correlation of SHIP-2 were not able to be analyzed. Biochemical analysis in the healthy mice model demonstrated that SHIP-1 increased the concentrations of the detected cytokines in a dosage-dependent manner but not in a time-dependent way. SHIP-2 exerted a positive effect in a dose-dependent manner over time for interferon gamma (IFN-γ) and interleukin (IL)-2 cytokine production at elevated dosages of 200, or 350 mg kg-1 d-1, while IFN-alpha(α) and IL-4 production was observed only in a dosage-dependent manner even at high dosages. Thus, SHIP-1 and 2 significantly improved the immune response through the intragastric administration of the tested high dosages by increasing the production of cytokines in the healthy mice, and these polysaccharides could possibly be used as an immunopotentiator in health foods or dietary supplements.

[Biorecovery of Palladium from Simulated Wastewaters and Its Catalytic Property for Methylene Blue].

By using Enterococcus faecalis Z5 strain (CCTCC M2012445) as a microbial resource, this study explored the possibility of recovering palladium (Pd) in the form of nanoparticles by adding an electron donor; investigated the Pd biorecovery efficiency of three kinds of simulated wastewaters including industrial waste processing leachates (IW) , printed circuit board scrap (PCBS) , and spent automotive catalyst (SAC); and analyzed the effect of other metal ions contained in simulated wastewater on Pd biorecovery efficiency. The results showed that the E. faecalis Z5 could recover Pd(Ⅱ) as palladium nanoparticles from the three simulated wastewaters. X-ray diffraction and transmission electron microscopy analysis indicated that the recovered product was Pd nanoparticles that were about 10 nm in size and mainly distributed in the periplasm of the cells. The order of Pd(Ⅱ) biorecovery efficiency from the three kinds of wastewaters was IW> SAC> PCBS. The biosorption efficiencies for IW, SAC, and PCBS were 99.8% (6 h), 99.7% (8 h), and 90.3% (12 h), respectively, and the bioreduction efficiencies were 99.9% (4 h), 99.9% (6 h), and 80.4% (36 h). Other metal ions contained in the simulated wastewaters such as Pt(Ⅳ), Au(Ⅲ), Ag(Ⅰ), Cu(Ⅱ), and Fe(Ⅱ) affected both the biosorption and bioreduction processes. The degree of matrix effects on the Pd(Ⅱ) bioreduction efficiency were in the order Au(Ⅲ)> Pt(Ⅳ)> Cu(Ⅱ)> Ag(Ⅰ)> Fe(Ⅱ). Further doping the recovered Pd nanoparticles with ferriferous oxide enabled the products to catalyze the degradation of methylene blue in heterogeneous Fenton reactions, which showed 96.7% degradation rate of methyl blue within 80 min.

[Detection of interferon-induced transmembrane-1 gene expression for clinical diagnosis of colorectal cancer].

OBJECTIVE: To investigate the expression of the interferon-induced transmembrane-1 (IFITM1) gene in colorectal cancer (CRC) tissue and the serum anti-IFITM1 antibody responses of the patients and assess their value in clinical diagnosis of CRC. METHODS: Semi-quantitative RT-PCR was performed to detect IFITM1 mRNA expression in the specimens of normal colonic mucosa, CRC tissue, inflammatory polyps, adenomatous polyps, gastric cancer, esophageal carcinoma and liver cancer tissues. Serum samples were collected from the patients to detect anti-IFITM1 antibody responses using Western blotting. The clinicopathological features of the carcinoma expressing IFITM1 gene were analyzed. RESULTS: IFITM1 mRNA was expressed in 47.4 % (18/38) of the CRC specimens, a rate significantly higher than that in adenomatous polyps [15% (3/20)] and gastric cancer [4.8% (1/21)]; no obvious IFITM1 expression was found in normal colonic mucosa, inflammatory polyp, esophageal carcinoma or liver cancer tissues (P<0.001 or P<0.05). IFITM1 mRNA was strongly expressed in CRC at the expression level of 0.8048-/+0.2273, which was significantly higher than that in adenomatous polyps (0.4447-/+0.0989, P<0.001). No anti-IFITM1 antibody response was detected in healthy human sera, but in the CRC patients, the serum antibody response was detected at the rate of 36.8% (14/38), significantly higher than the rate of 9.5% (2/21) in gastric cancer (P<0.05). No antibody response was detected in esophageal carcinoma, liver cancer, inflammatory polyp or adenomatous polyps. Most of the IFITM1-expressing CRC had a diameter exceeding 5 cm, often invading the serous membrane with metastasis to the lymph nodes and the distant organs; these tumors were identified mostly as well-differentiated adenocarcinoma in Dukes stage C or D. CONCLUSION: IFITM1 gene may play an important role in the pathogenesis, development and metastasis of CRC, and may serve as a potential biomarker for clinical diagnosis of CRC.

[Studies on sensitization for electrochemiluminescence of luminol with platinum-gold bimetallic nanoparticles modified electrode].

The present paper reports the researches on sensitization of nano-bimetalic material for the electrochemiluminescence (ECL) of luminol. The platinum-gold bimetallic nanoparticles with different component ratio and size were prepared by chemical reduction. The analytical methods such as UV-Vis spectra, TEM and XRD were applied to characterize the properties ofthe nanoparticles. The information obtained from these methods revealed that the prepared bimetallic nanoparticles were truly of alloy structure and absolutely not the mixture of two kinds of metallic nanoparticles. The components of the nanoparticles could be regulated for a series of Pt/Au ratios and the diameters determined by laser-granulometer. In alkaline medium of pH 12.0, the bimetallic Pt-Au nanoparticle modified electrode sensitized the electrochemiluminescence of luminol. The sensitization efficiency reached as high as one order of magnitude with the 6:1 of Pt/Au ratio. The specific surface area of nanoparticles would be larger as the size diminishes, resulting in a higher surface activity, so the smallest size of nanoparticles led to the highest efficiency.

[Effect of potassium level on output and diosgenin content of Dioscorea zingiberensisg].

OBJECTIVE: To study the effect of potassium level on physiological characteristics and diosgenin content of Dioscorea zingiberensisg and provide experimental basis for proper use of potassium fertilizer. METHODS: Field experiment including four potassium levels was carried out. The physiological characteristics of leaves were determined at different growth stages and the diosgenin content in bulk root was assayed after harvest. RESULTS: Within the arrange of 0-180 kg K2SO4/hm2, the content of chlorophyll and amino acid, Fv/Fo, Fv/Fm, and phiPs II were increased with the increase in potassium level. Meanwhile, the ability to defense the damage caused by active oxygen was obviously enhanced. Further increase in potassium level resulted in the decrease in efficiency of potassium fertilizer. CONCLUSION: It indicats that higher yield and diosgenin content can be obtained when the potassium level is 180 kg K2/hm2.

[Screening and preliminary analysis of colorectal carcinoma-associated antigen genes].

OBJECTIVE: To screen and identify the genes coding for colorectal carcinoma-associated antigen and analyze the bioinformation of their cDNA sequences. METHODS: Immunoscreening of the cDNA phage-display library derived from human colorectal carcinoma was performed with autologous or allogeneic serum antibody from patients with colorectal cancer through SEREX approach. After amplification of the positive phage clones, the phage DNA was extracted and purified with Qiagen kit, and the fragment sizes of the cDNA of positive clones were identified by PCR and EcoR I and Hind III restriction endonucleases. The cDNAs of the positive clones were ligated into pUCm-T vector and sequenced. The bioinformation of cDNA sequences were analyzed against GenBank+EMBL+DDBJ+PDB Sequences Database. RESULTS AND CONCLUSION: Eleven positive clones were obtained after immunoscreening, and the sizes of the cDNA fragments were 1100, 1300, 1000, 2000, 1200, 1200, 700, 900, 600, 1200 and 1000 bp, respectively, representing 9 antigen genes, including 7 with homology with the known genes. Among the 11 obtained positive clones, 3 were the same cDNA having homology with interferon-induced transmembrance protein-1 and possessing anti-proliferation effect; another 6 represented different genes, namely human BAC clone RP11-453E17 whose function have not been cleared, human cartilage-hair hypoplasia region gene responsible for cartilage-hair hypoplasia, human chromosome 5 clone CTD-2030B15 with insertion mutation, human gene similar to anti tumor necrosis factor-alpha antibody light-chain Fab fragment associated with tumor growth, mRNA of human beta-2-microglobulin in relation to tumor cell proliferation, and human aldolase A gene promoting tumor cell proliferation. The other two cDNA sequences were not identified for homology with currently known genes in GenBank, and their functions awaits further investigation.

[PCR amplification, cloning and protein expression of interferon-inducible transmembrane protein-1 gene].

OBJECTIVE: To study the PCR amplification, cloning and protein expression of interferon-inducible transmembrane protein-1 (IFITMP-1) gene. METHODS: With the cDNA fragment containing IFITMP-1 gene as template, IFITMP-1 gene was amplified using Pfu enzyme by means of PCR. After EcoRI and HindIII digestion, the target gene fragment was linked to pUCm-T plasmid and sequenced. The IFITMP-1 gene was cloned into pET-Trx protein expression plasmid, and the condition for protein expression was optimized. RESULTS: The length of the PCR product of IFITMP-1 gene-containing cDNA fragment was about 1000 bp. The IFITMP-1 gene was successfully inserted into pUCm-T plasmid with correct sequence and cloning of the IFITMP-1 gene into the pET-Trx protein expression plasmid was achieved. Expression of the fusion protein of pUCm-T plasmid and IFITMP-1 gene was detected after IPTG induction. CONCLUSION: Successful amplification and cloning of the IFITMP-1 gene and its protein expression may facilitate further study of the role of IFITMP-1 gene in colorectal cancer.

[The study on electrochemiluminescence of luminol on ITO glass].

The indium tin oxide(ITO) glass was applied as the electrode to study the electrochemilumuinescent behavior of luminol on its surface. The experimental results indicated that it was an excellent electrode material for ECL of luminol with very high sensitivity. The detection limit of the luminol is as low as 10(-15) mol x L(-1) order of magnitude. Compared with the voltammetric behavior of luminol, it was revealed that both the two oxidation processes of luminol led to the emission of ECL on the surface of ITO glass,which was different from the ECL behavior of luminol on platinum electrode. Although the ECL intensity of first step oxidation of luminol was relative weak than that of the second step oxidation, it was profitable because it was a reversible process and more stable. It might be useful to fabricate the flow-cell of ECL based on its ECL activity and transparence for light. The influences of the electric parameters and the dissolved oxygen on luminol's ECL on the ITO glass, and the mechanism were also studied in the present paper.