Genomic typing and virulence gene profile analysis of Salmonella Derby from different sources.

Salmonella enterica serovar Derby (S. Derby) is one of the most common Salmonella serovars which can infect poultry, swine, and humans. With the reduction of the sequencing cost and the improvement of sequencing technology, whole genome sequencing (WGS) has become an important method for bacterial determination, molecular investigation, and pathogenic tracing analysis. In this study, we investigated S. Derby isolates from different sources in China using in-silico multilocus sequence typing (MLST), core genome MLST (cgMLST) and whole genome MLST (wgMLST) analysis based on WGS. The results showed that 21 S. Derby strains were divided into 3 STs using MLST analysis, including ST40 (n = 19, accounting for 90.48%), ST71 (n = 1, accounting for 4.76%) and ST8016 (n = 1, accounting for 4.76%). cgMLST and wgMLST analysis categorized the tested strains into 13 cgSTs and 21 wgSTs, respectively. The minimum spanning trees of cgMLST and wgMLST both divided these strains into 3 clusters and 4 singletons. In addition, virulence gene profiles of S. Derby isolates were also analyzed, and a total of 174 virulence genes belonged to 8 categories were identified. In summary, we studied genomic typing, phylogenetic relationship and virulence gene profiles of S. Derby strains from different sources in China. These findings were beneficial for the epidemiology and pathogenesis of Salmonella.

Biofilm-forming ability of Salmonella enterica strains of different serotypes isolated from multiple sources in China.

Salmonella is an important zoonotic and foodborne pathogen that can infect humans and animals, causing severe concerns about food safety and a heavy financial burden worldwide. The pathogen can adhere to living and abiotic surfaces by forming biofilms, which increases the risk of transmission and infection. In this study, we investigated the biofilm-forming ability of 243 Salmonella strains of 36 serotypes from different sources in China using microplate crystal violet staining method. The results showed that 99.6% tested strains, with the exception of one strain of S. Thompson, were capable of forming biofilms. The strains with the biofilm-forming ability of strong, medium and weak accounted for 2.88%, 24.28% and 72.43%, respectively. The strains of S. Havana and S. Hvittingfoss had the strongest biofilm-forming ability, with the OD570 of 0.81 ± 0.02 and 0.81 ± 0.38, respectively, while the strains of S. Agona and S. Bovismorbificans had the weakest biofilm-forming ability, with the OD570 of 0.16 ± 0.02 and 0.15 ± 0.00, respectively. Furthermore, statistical analysis results demonstrated that isolation of source had no effect on the biofilm formation ability, while the detection rates of pefABCD and ddhC were positively correlated with the biofilm formation ability of Salmonella. In particular, the detection rate of ddhC gene was more than 60% in the biofilm forming strains. These findings have important guiding significance for the investigation of pathogenesis, as well as the prevention and control of salmonellosis.

Optimization of fermentation, purification, and properties of blue pigment produced from Quambalaria cyanescens QY229.

AIM: Blue pigments have broad applications in foods, cosmetics, and clothing. However, natural blue pigments are rare. At present, the majority of blue pigments for sale are chemically synthetic. Owing to the safety risks of chemical pigments, it is an urgent demand to develop novel natural blue pigments. METHODS AND RESULTS: The fermentation medium and culture conditions of blue pigment produced by Quambalaria cyanescens QY229 were optimized by Plackett-Burman (PB) experimental design and response surface methodology (RSM) for the first time. The stability, bioactivity, and toxicity of the obtained blue pigment were studied after isolation and purification. CONCLUSION: The results showed that the optimal fermentation parameters were 34.61 g·L-1 of peptone concentration, 31.67°C of growing temperature, and 72.33 mL of medium volume in a 250-mL flask, and the yield of blue pigment reached 348.2 ± 7.1 U·mL-1. QY229 blue pigment is stable to light, heat, pH, most metal ions, and additives, and has certain antioxidant and inhibitory activity of α-glucosidase in vitro. QY229 blue pigment at concentrations of 0-1.25 mg·mL-1 was nontoxic to Caenorhabditis elegans in an acute toxicity trial.

Genomic virulence genes profile analysis of Salmonella enterica isolates from animal and human in China from 2004 to 2019.

Salmonella is a momentously zoonotic and food-borne pathogen that seriously threats human and animal health around the world. Salmonella pathogenicity is closely related to its virulence genes profile. However, conventional virulence gene analysis methods cannot truly reveal whole virulence genes carried by Salmonella. In this study, whole genome sequencing in combination with Virulence Factor Database were applied to investigate whole virulence gene profiles of 243 Salmonella isolates from animals and humans in China from 2004 to 2019. The results showed that a total of 670 virulence genes were identified in Salmonella, among them, 319 virulence genes were found in all the Salmonella tested isolates, and 9 virulence genes were unique to Salmonella. The 670 virulence genes were classified into 14 categories according to their functions, and the genes related to adherence, effector delivery system, immune modulation, motility and nutritional/metabolic factors accounted for 84.63%. Relationships between virulence genes and serovars, sequence types indicated that strains belonged to the same serovar or sequence type had similar virulence genes profiles, however, isolates from different sources, years and locations of isolation had variable virulence gene profiles. In addition, copy number of virulence genes and homologous virulence genes shared with other pathogens were also analyzed in this study. In summary, we investigated pan-genomic virulence gene profiles and molecular epidemiology of Salmonella isolates from humans and animals in China from 2004 to 2019. These findings are beneficial for pathogenic monitoring, investigation of virulence evolution as well as prevention and control of Salmonella.

Detection of Antibiotic Resistance and Analysis of Resistance Genes and Virulence Genes of Salmonella Isolated from Human.

Salmonella is a food-borne pathogen that can cause zoonoses. The emergence of drug-resistant strains of Salmonella is of great concern. It is necessary to understand the prevalence of antibiotic resistance, antibiotic resistance genes and virulence genes in human Salmonella. In this study, drug susceptibility test was used to detect and analyze the drug resistance of 24 Salmonella strains collected from human. A multi-drug resistant strain QLUF123 was selected for whole genome sequencing, and its drug resistance genes and virulence genes were analyzed. The results showed that 24 Salmonella strains were resistant to the tested antibiotics, 87.50% of the strains had multi-drug resistance, the resistance rate to ceftazidime, sulfamethoxazole and tilmicosin reached more than 80%. The alignment results based on the whole genome sequence showed that there were multiple types of drug-resistant genes in QLUF123, among which efflux pump system genes were the most abundant, including sdiA, mdtK, baeR and other multidrug-resistant efflux pump system genes. QLUF123 carried 46 kinds of virulence factors and 249 related virulence genes, among which the three functions of secretory system, adhesion and motility accounted for the most virulence genes, accounting for 93.57%. In this study, antibiotic resistance of human Salmonella was detected by drug sensitivity test, and drug resistance and virulence genes in Salmonella were analyzed by whole genome sequencing technology, which is of great significance for scientific treatment and rational drug use of related diseases caused by Salmonella infection.

Genomes-based MLST, cgMLST, wgMLST and SNP analysis of Salmonella Typhimurium from animals and humans.

Salmonella Typhimurium (S. Typhimurium) is an important food-borne and zoonotic pathogen that causes salmonellosis. With the development of whole genome sequencing (WGS), genome-based typing has been widely applied to bacteriology. In this study, we investigated genotyping and phylogenetic clusters of S. Typhimurium isolates from humans and animals in different provinces (including Beijing, Shandong, Guangxi, Shaanxi, Henan, and Shanghai) of China during 2009-2018 using multi locus sequence typing (MLST), core genome MLST (cgMLST), whole genome MLST (wgMLST) and single nucleotide polymorphism (SNP) based on WGS. 29 S. Typhimurium isolates from chicken (n = 22), sick pigeon (n = 2), patients (n = 4) and sick swine (n = 1) were tested. MLST analysis showed S. Typhimurium strains were divided into four STs, namely ST19 (n = 14), ST34 (n = 12), ST128 (n = 2) and ST1544 (n = 1). cgMLST and wgMLST divided 29 strains into 27 cgSTs and 29 wgST, respectively. Phylogenetic clustering showed that all isolates were divided into 4 clusters and 4 singletons. SNP analysis was used to examine MLST, cgMLST, wgMLST analysis. Finally, comparisons of MLST, cgMLST, wgMLST, and SNP were analyzed and the results showed their precision increased in order. In summary, genomic typing and phylogenetic relationships of 29 S. Typhimurium strains from different sources in China were analyzed. These findings were beneficial to investigate molecular pathogenesis, bacterial diversity, and traceability analysis of Salmonella.

Whole genome sequencing analysis of avian pathogenic Escherichia coli from China.

Avian pathogenic Escherichia coli (APEC) can cause localized or systemic infection in poultry herds, i.e., colibacillosis, which is an economically devastating bacterial disease of the poultry industry worldwide. Additionally, some APEC may have zoonotic potential. In this study, we sequenced 125 APEC isolates from chickens and ducks with obvious clinical symptoms in poultry farms in China and performed genomic epidemiological analysis along with 16 APEC reference genomes downloaded from NCBI. The phylogenetic analysis indicated a great diversity of APEC isolates, and a total of 35 different O types, 22 H types, and 29 ST types were identified. Several virulence-associated genes (VAGs), such as ompT (96.45 %), iss (97.87 %), and hlyF (90.78 %), as well as four complete siderophore gene clusters, including the Sit transport system (86.52 %), aerobactin (89.36 %), salmochelin (79.43 %), and yersiniabactin (54.61 %), were detected in APEC isolates with high prevalence, which could serve as virulence markers of APEC. Several virulence-associated gene clusters, including the two T6SS systems and the K1 capsule biosynthesis gene clusters, were significantly associated with APEC of phylogroups B2, D, and F but very rarely encoded by the APEC from phylogroups C and E. In addition, several virulence-associated genes, which have been reported in other E. coli pathotypes but have not been reported in APEC, were identified in this study. Our findings in this study have implications for a better understanding of APEC evolution and pathogenesis and may lead to the development of new diagnostic tools for APEC.

Serotyping, MLST, and Core Genome MLST Analysis of Salmonella enterica From Different Sources in China During 2004-2019.

Salmonella enterica (S. enterica) is an important foodborne pathogen, causing food poisoning and human infection, and critically threatening food safety and public health. Salmonella typing is essential for bacterial identification, tracing, epidemiological investigation, and monitoring. Serotyping and multilocus sequence typing (MLST) analysis are standard bacterial typing methods despite the low resolution. Core genome MLST (cgMLST) is a high-resolution molecular typing method based on whole genomic sequencing for accurate bacterial tracing. We investigated 250 S. enterica isolates from poultry, livestock, food, and human sources in nine provinces of China from 2004 to 2019 using serotyping, MLST, and cgMLST analysis. All S. enterica isolates were divided into 36 serovars using slide agglutination. The major serovars in order were Enteritidis (31 isolates), Typhimurium (29 isolates), Mbandaka (23 isolates), and Indiana (22 isolates). All strains were assigned into 43 sequence types (STs) by MLST. Among them, ST11 (31 isolates) was the primary ST. Besides this, a novel ST, ST8016, was identified, and it was different from ST40 by position 317 C → T in dnaN. Furthermore, these 250 isolates were grouped into 185 cgMLST sequence types (cgSTs) by cgMLST. The major cgST was cgST235530 (11 isolates), and only three cgSTs contained isolates from human and other sources, indicating a possibility of cross-species infection. Phylogenetic analysis indicated that most of the same serovar strains were putatively homologous except Saintpaul and Derby due to their multilineage characteristics. In addition, serovar I 4,[5],12:i:- and Typhimurium isolates have similar genomic relatedness on the phylogenetic tree. In conclusion, we sorted out the phenotyping and genotyping diversity of S. enterica isolates in China during 2004-2019 and clarified the temporal and spatial distribution characteristics of Salmonella from different hosts in China in the recent 16 years. These results greatly supplement Salmonella strain resources, genetic information, and traceability typing data; facilitate the typing, traceability, identification, and genetic evolution analysis of Salmonella; and therefore, improve the level of analysis, monitoring, and controlling of foodborne microorganisms in China.

Probing the pH sensitivity of R-phycoerythrin: investigations of active conformational and functional variation.

Crystal structures of phycobiliproteins have provided valuable information regarding the conformations and amino acid organizations of peptides and chromophores, and enable us to investigate their structural and functional relationships with respect to environmental variations. In this work, we explored the pH-induced conformational and functional dynamics of R-phycoerythrin (R-PE) by means of absorption, fluorescence and circular dichroism spectra, together with analysis of its crystal structure. R-PE presents stronger functional stability in the pH range of 3.5-10 compared to the structural stability. Beyond this range, pronounced functional and structural changes occur. Crystal structure analysis shows that the tertiary structure of R-PE is fixed by several key anchoring points of the protein. With this specific association, the fundamental structure of R-PE is stabilized to present physiological spectroscopic properties, while local variations in protein peptides are also allowed in response to environmental disturbances. The functional stability and relative structural sensitivity of R-PE allow environmental adaptation.

Tip30-induced apoptosis requires translocation of Bax and involves mitochondrial release of cytochrome c and Smac/DIABLO in hepatocellular carcinoma cells.

TIP30 (Tat-interacting protein 30), a newly found proapoptotic factor, appears to be involved in multiple functions including metabolic suppression, apoptosis induction, and diminishing angiogenic properties. In the present study, we reported that mitochondrial events were required for apoptosis induced by TIP30 in hepatocellular carcinoma cells (HCC cells). Translocation of Bax was essential for TIP30-induced apoptosis, whereas overexpression of the anti-apoptotic protein Bcl-xL delayed both second mitochondria-derived activator of caspases (Smac/DIABLO) release and onset of apoptosis. Furthermore, TIP30-induced apoptosis was dependent on caspase activity because the pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp (OMe)-fluoromethyl ketone (z-VAD-fmk) blocked DNA fragmentation. Release of Smac/DIABLO from the mitochondria through the TIP30-P53-Bax cascade was required to remove the inhibitory effect of XIAP (X-linked Inhibitor of Apoptosis) and allowed apoptosis to proceed. Our results showed for the first time that Bax-dependent release of Smac/DIABLO, cytochrome c and AIF from the mitochondria mediated the contribution of the mitochondrial pathway to TIP30-mediated apoptosis. Our data suggested that adenovirus-mediated overexpression of TIP30 was capable of inducing therapeutic programmed cell death in vitro by activating the mitochondrial pathway of apoptosis. On the basis of these studies, elucidating the mechanism by which TIP30 induces cell death might establish it as an anticancer approach.

[Spectral changes of C-phycocyanin with different molar ratios of SPDP].

Pure C-phycocyanin was prepared from Spirulina platensis using one-step anion-exchange chromatography. The C-PC obtained was with an absorption maximum at 620 nm and a fluorescence emission maximum at 640 nm when excited by 580 nm. SPDP is an excellent heterobifunctional crosslinker for thiolating amines. Different molar ratios of SPDP have remarkable influence on the absorption and fluorescence spectra of C-phycocyanin. The absorption maximum and fluorescence emission maximum both decreased and blue-shifted from 640 nm to 630 nm as the molar ratios of SPDP increased. It was found that the molar ratios of SPDP to C-phycocyanin was not more than 100 was appropriate to being conjugated with other biomolecules from the absorption and fluorescence spectra of C-phycocyanin.

Transcriptional Control of Dual Transporters Involved in α-Ketoglutarate Utilization Reveals Their Distinct Roles in Uropathogenic Escherichia coli.

Uropathogenic Escherichia coli (UPEC) are the primary causative agents of urinary tract infections. Some UPEC isolates are able to infect renal proximal tubule cells, and can potentially cause pyelonephritis. We have previously shown that to fulfill their physiological roles renal proximal tubule cells accumulate high concentrations of α-ketoglutarate (KG) and that gene cluster c5032-c5039 contribute to anaerobic utilization of KG by UPEC str. CFT073, thereby promoting its in vivo fitness. Given the importance of utilizing KG for UPEC, this study is designed to investigate the roles of two transporters KgtP and C5038 in KG utilization, their transcriptional regulation, and their contributions to UPEC fitness in vivo. Our phylogenetic analyses support that kgtP is a widely conserved locus in commensal and pathogenic E. coli, while UPEC-associated c5038 was acquired through horizontal gene transfer. Global anaerobic transcriptional regulators Fumarate and nitrate reduction (FNR) and ArcA induced c5038 expression in anaerobiosis, and C5038 played a major role in anaerobic growth on KG. KgtP was required for aerobic growth on KG, and its expression was repressed by FNR and ArcA under anaerobic conditions. Analyses of FNR and ArcA binding sites and results of EMS assays suggest that FNR and ArcA likely inhibit kgtP expression through binding to the -35 region of kgtP promoter and occluding the occupancy of RNA polymerases. Gene c5038 can be specifically induced by KG, whereas the expression of kgtP does not respond to KG, yet can be stimulated during growth on glycerol. In addition, c5038 and kgtP expression were further shown to be controlled by different alternative sigma factors RpoN and RpoS, respectively. Furthermore, dual-strain competition assays in a murine model showed that c5038 mutant but not kgtP mutant was outcompeted by the wild-type strain during the colonization of murine bladders and kidneys, highlighting the importance of C5038 under in vivo conditions. Therefore, different transcriptional regulation led to distinct roles played by C5038 and KgtP in KG utilization and fitness in vivo. This study thus potentially expanded our understanding of UPEC pathobiology.

mcr-1 identified in Avian Pathogenic Escherichia coli (APEC).

Antimicrobial resistance associated with colistin has emerged as a significant concern worldwide threatening the use of one of the most important antimicrobials for treating human disease. Here, we examined a collection (n = 980) of Avian Pathogenic Escherichia coli (APEC) isolated from poultry with colibacillosis from the US and internationally for the presence of mcr-1 and mcr-2, genes known to encode colistin resistance. Included in the analysis was an additional set of avian fecal E. coli (AFEC) (n = 220) isolates from healthy birds for comparative analysis. The mcr-1 gene was detected in a total of 12 isolates recovered from diseased production birds from China and Egypt. No mcr genes were detected in the healthy fecal isolates. The full mcr-1 gene from positive isolates was sequenced using specifically designed primers and were compared with sequences currently described in NCBI. mcr-1 positive isolates were also assessed for phenotypic colistin resistance and extended spectrum beta lactam phenotypes and genotypes. This study has identified mcr-1 in APEC isolates dating back to at least 2010 and suggests that animal husbandry practices could result in a potential source of resistance to the human food chain in countries where application of colistin in animal health is practiced.

[Biological characteristics of three Newcastle disease virus isolates and entire genome sequences analysis].

Three Newcastle disease virus (NDV) strains recovered from ND outbreaks in chickens and duck flocks in north china during 2009 to 2011 were completely sequenced and biologically characterized. All the strains were velogenic and had the velogenic motif 112R-R-Q-K-R-F117 which was consistent with the results of biological tests. Analysis of the variable region (nucleotide 47 to 420) of the F gene indicated that the three isolates belonged to genotype VII d. Cross hemagglutination inhibition test indicated that the antigen homology between three isolates and LaSota were 82.5%-89.4%, the homology between the two isolates from chicken was 90%. A cross-protection experiment in which specific-pathogen-free chickens vaccinated with LaSota were challenged by SDLY01 isolate showed that LaSota vaccine could provide complete protection against SDLY01, however virus discharge could be detected on fifth day. Challenge experiment in which Cherry Valley duck of 30 day old challenged with SD03 strain indicated that cherry valley duck had no disease in experiment period, but virus discharge could be detected from Larynx and cloaca until fifth day. Genome length of three NDV isolates was 15192bp and belonged to genotype VII d. Sequence analysis clarified that the whole genomic sequence of these three isolates shared high homology with NDV virus strains isolated from goose and duck over the same period, which elucidated that NDV isolated from goose, duck or chicken had close genetics and epidemiological relationship.

Avian influenza virus and Newcastle disease virus (NDV) surveillance in commercial breeding farm in China and the characterization of Class I NDV isolates.

In order to determine the actual prevalence of avian influenza virus (AIV) and Newcastle disease virus (NDV) in ducks in Shandong province of China, extensive surveillance studies were carried out in the breeding ducks of an intensive farm from July 2007 to September 2008. Each month cloacal and tracheal swabs were taken from 30 randomly selected birds that appeared healthy. All of the swabs were negative for influenza A virus recovery, whereas 87.5% of tracheal swabs and 100% cloacal swabs collected in September 2007, were positive for Newcastle disease virus isolation. Several NDV isolates were recovered from tracheal and cloacal swabs of apparently healthy ducks. All of the isolates were apathogenic as determined by the MDT and ICPI. The HN gene and the variable region of F gene (nt 47-420) of four isolates selected at random were sequenced. A 374 bp region of F gene and the full length of HN gene were used for phylogenetic analysis. Four isolates were identified as the same isolate based on nucleotide sequences identities of 99.2-100%, displaying a closer phylogenetic relationship to lentogenic Class I viruses. There were 1.9-9.9% nucleotide differences between the isolates and other Class I virus in the variable region of F gene (nt 47-420), whereas there were 38.5-41.2% nucleotide difference between the isolates and Class II viruses. The amino acid sequences of the F protein cleavage sites in these isolates were 112-ERQERL-117. The full length of HN gene of these isolates was 1851 bp, coding 585 amino acids. The homology analysis of the nucleotide sequence of HN gene indicated that there were 2.0-4.2% nucleotide differences between the isolates and other Class I viruses, whereas there were 29.5-40.9% differences between the isolates and Class II viruses. The results shows that these isolates are not phylogenetically related to the vaccine strain (LaSota). This study adds to the understanding of the ecology of influenza viruses and Newcastle disease viruses in ducks and emphasizes the need for constant surveillance in times of an ongoing and expanding epidemic of AIV and NDV.