Characterization of an International High-Risk Escherichia coli ST410 Clone Coproducing NDM-5 and OXA-181 in a Food Market in China.

During a 2020 routine epidemiological investigation of carbapenem-resistant Enterobacterales at a local food market in Guangzhou, China, two Escherichia coli ST410 isolates coproducing NDM-5 and OXA-181 were obtained from environmental samples. Antimicrobial susceptibility testing, whole-genome sequencing, and conjugation assays were applied to identify their resistance phenotypes, phylogenetic relatedness, and genetic characteristics. Phylogenetic analysis showed that the two isolates were clonally related with only one core-genome single-nucleotide polymorphism (SNP) difference and clustered into a branch with 87 E. coli ST410 isolates deposited in GenBank. These 89 ST410 isolates were closely related (≤51 SNPs), and most were from humans in Southeast Asian countries (n = 47). A Vietnamese clinical isolate collected in 2017 showed the strongest epidemiological link (seven SNPs) to the two ST410 isolates detected in this study. Complete-genome analysis revealed that the carbapenem resistance determinants blaNDM-5 and blaOXA-181 were located on an IncF1:A1:B49-IncQ1 plasmid and IncX3 plasmid, respectively. Conjugation experiments confirmed that the IncX3 plasmid was self-transmissible while the IncF1:A1:B49-IncQ1 plasmid was nonconjugative. BLASTn analysis indicated that the two plasmids showed high similarity to other blaNDM-5-bearing IncF1:A1:B49-IncQ1 and blaOXA-181-bearing IncX3 plasmids from other countries. Altogether, the high similarity of the core genomes and plasmids between the ST410 isolates found in this study and those human source isolates from foreign countries suggested the clonal spread of E. coli ST410 strains and horizontal transmission of blaOXA-181-bearing IncX3 plasmids across Southeast Asian countries. Stringent sanitary management of food markets is important to prevent the dissemination of high-risk clones to the public. IMPORTANCE This is the first report of an Escherichia coli ST410 clone that coproduces NDM-5 and OXA-181 in China. The high similarity of the core genomes and plasmids between the ST410 isolates characterized in this study and human source isolates from foreign countries strongly suggests that this ST410 lineage is an international high-risk clone, highlighting the need for continuous global surveillance of ST410 clones.

Efficacy and safety of salvianolate and enoxaparin in the prevention of perioperative deep venous thrombosis in gastrointestinal surgery.

OBJECTIVE: In this study, the efficacy and safety of salvianolate were compared with enoxaparin in the prevention of perioperative deep vein thrombosis in gastrointestinal surgery. METHODS: From October 2017 to September 2019, 563 patients who underwent gastrointestinal surgery were collected. Based on the inclusion and exclusion criteria, 119 patients were divided into two groups: enoxaparin group (n = 65) and salvianolate group (n = 54). Comparisons were made regarding the outcomes: prothrombin time (PT), prothrombin activity (PTA), international normalized ratio (INR), activated partial thromboplastin time (APTT), fibrinogen (FIB), thrombin time (TT), D-dimer level (D-D), platelet count (PLT), hematokrit (HCT), and incidence of deep vein thrombosis (DVT). RESULTS: The main outcomes showed no significance between enoxaparin group and salvianolate group (p > .05). The incidence of DVT in salvianolate group was 1.85%, significantly lower than that in enoxaparin group (12.3%) (p < .05). No serious adverse reactions occurred in the two groups during treatment. CONCLUSION: Compared with enoxaparin, salvianolate has an advantage in the prevention of perioperative thrombosis in gastrointestinal surgery with a lower incidence of DVT.

Characterization of NDM-5-producing Enterobacteriaceae isolates from retail grass carp ( Ctenopharyngodon idella) and evidence of bla NDM-5-bearing IncHI2 plasmid transfer between ducks and fish.

We aimed to characterize NDM-5-producing Enterobacteriaceae from aquatic products in Guangzhou, China. A total of 196 intestinal samples of grass carp collected in 2019 were screened for carbapenemase genes. Characterization of bla NDM-5 positive isolates and plasmids was determined by antimicrobial susceptibility testing, conjugation experiments, Illumina HiSeq, and Nanopore sequencing. One Citrobacter freundii and six Escherichia coli strains recovered from seven intestinal samples were verified as bla NDM-5 carriers (3.57%, 7/196). The bla NDM-5 genes were located on the IncX3 ( n=5), IncHI2 ( n=1), or IncHI2-IncF ( n=1) plasmids. All bla NDM-5-bearing plasmids were transferred by conjugation at frequencies of ~10 -4-10 -6. Based on sequence analysis, the IncHI2 plasmid pHNBYF33-1 was similar to other bla NDM-5-carrying IncHI2 plasmids deposited in GenBank from Guangdong ducks. In all IncHI2 plasmids, bla NDM-5 was embedded in a novel transposon, Tn 7051 (IS 3000-ΔIS Aba125-IS 5-ΔIS Aba125- bla NDM-5- ble MBL- trpF- tat-∆ dct-IS 26-∆ umuD-∆IS Kox3-IS 3000), which was identical to the genetic structure surrounding bla NDM-5 found in some IncX3 plasmids. The IncHI2-IncF hybrid plasmid pHNTH9F11-1 was formed by homologous recombination of the bla NDM-5-carrying IncHI2 plasmid and a heavy-metal-resistant IncF plasmid through ∆Tn 1721. To the best of our knowledge, this is the first report on the characterization of bla NDM-5-bearing plasmids in fish in China. The IncHI2 plasmid pHNBYF33-1 may be transmitted from ducks, considering the common duck-fish freshwater aquaculture system in Guangdong. Tn 7051 is likely responsible for the transfer of bla NDM-5 from IncX3 to IncHI2 plasmids in Enterobacteriaceae, resulting in the expansion of transmission vectors of bla NDM-5.

Genome-wide identification and characterization of long non-coding RNAs in Tribolium castaneum.

Long non-coding RNAs (lncRNAs) are poorly understood in insects. In this study, we performed genome-wide analysis of lncRNAs in Tribolium castaneum by RNA-seq. In total, 4516 lncRNA transcripts corresponding to 3917 genes were identified from late embryos, early larvae, late larvae, early pupae, late pupae and early adults of T. castaneum, including 3152 novel lncRNAs and 1364 known lncRNAs. These lncRNAs have few exons and transcripts, and are short in length. During development, they exhibited nine different expression patterns. Functionally, they can act either by targeting messenger RNAs (1813 lncRNAs) and lncRNAs (45 lncRNAs) or as micro RNA (miRNA) precursors (46 lncRNAs). LncRNAs were observed to target the metabolic enzymes of glycolysis, TCA cycle and amino acids, demonstrating that lncRNAs control metabolism by regulating metabolic enzymes. Moreover, lncRNAs were shown to participate in cell differentiation and development via their targets. As miRNA precursors, lncRNAs could participate in the ecdysone signaling pathway. This study provides comprehensive information for lncRNAs of T. castaneum, and will promote functional analysis and target identification of lncRNAs in the insect.

Low prevalence of antibodies against Toxoplasma gondii in dairy cattle from China's central region.

BACKGROUND: Toxoplasma gondii is an intracellular protozoan that can infect humans and other animals, including cattle. Cattle are one of the world's main sources of meat, and people who consume raw or undercooked meat and milk of cattle infected with T. gondii can become infected. In this study, a total of 5292 dairy cattle serum samples, collected from 17 cities (Henan Province, China) from January 2015 to September 2017, were screened for antibodies against T. gondii. RESULTS: Antibodies to T. gondii were found in 1.93% (102/5292) (95% CI, 1.56-2.30) of dairy cattle using a modified agglutination test (cut-off 1:100). The results showed that geographic location and season may be risk factors for T. gondii infection of cattle (P < 0.05), and the seroprevalence of T. gondii in cattle along the Yellow River is higher than other areas. CONCLUSIONS: This is the first large-scale investigation on the seroprevalence of T. gondii infection in cattle from Central China. This survey shows that the T. gondii infection rate of dairy cattle is low; however, these findings provide additional information on the epidemiology of Chinese T. gondii. The possibility of dairy cattle exposure to T. gondii in Central China can not be ignored, and the consumption of raw or undercooked beef or milk may pose a risk to human health.

Cyclooxygenase-2 up-regulates hepatic somatostatin receptor 2 expression.

Somatostatin and its analogues, which function by binding to somatostatin receptors (SSTRs) 1-5, play a protective role in liver cirrhosis. Hepatic SSTR-2 expression is up-regulated in subjects with liver cirrhosis. However, little is known about the mechanisms underlying this process. In the present study, we observed the up-regulation of hepatic SSTR-2 expression in thioacetamide (TAA)-induced cirrhotic rats and further showed that cyclooxygenase-2 (COX-2) might play a role in this process via the protein kinase C (PKC)-cAMP response element binding protein (CREB) signaling pathway. In vivo, the up-regulated SSTR-2 in liver cirrhosis was inhibited by the addition of a selective COX-2 inhibitor, such as celecoxib. In vitro, the up-regulation of COX-2 by either transfection with COX-2 plasmids or treatment with TAA increased levels of SSTR-2 and phosphorylated CREB (p-CREB) in the human hepatocyte cell line L02. Furthermore, the increase in SSTR-2 expression was inhibited by the addition of celecoxib and a PKC inhibitor. Moreover, for comparable DNA methylation levels in the region upstream of the hepatic SSTR-2 gene in normal and cirrhotic livers, DNA methylation may not contribute to the up-regulation of SSTR-2 expression in cirrhotic livers. In conclusion, the up-regulation of hepatic SSTR-2 might be induced by COX-2 via the PKC-CREB signaling pathway but is probably not induced by DNA methylation.

Avirulence and lysozyme secretion in Paneth cells after infection of BALB/c mice with oocysts of Toxoplasma gondii strains TgCatCHn2 (ToxoDB#17) and TgCatCHn4 (ToxoDB#9).

Toxoplasma gondii has a complex life cycle and pathogenic mechanisms. Acute T. gondii infections in mice often result in death, whereas in chronic infections, the parasites may persist in the host tissues as intraneuronal or intramuscular cysts. However, the virulence of T. gondii strains in mice varies with its genetic background. The present study investigated the pathogenicity and pathological lesions of two T. gondii isolates from China: namely, TgCatCHn2 (ToxoDB#17) and TgCatCHn4 (ToxoDB#9). The virulent (ToxoDB#216) and avirulent (VEG) strains were employed as controls. Toxoplasmosis was induced by inoculating BALB/c mice with oocysts of the strains of T. gondii VEG, ToxoDB#216, TgCatCHn2 (ToxoDB#17), and TgCatCHn4 (ToxoDB#9), respectively. As a result, one oocyst of ToxoDB#216 could kill a mouse within 10 days post inoculation (DPI). The survival time of the mice for T. gondii TgCatCHn2 and TgCatCHn4 was >60 DPI for 106/mL oocysts, but this concentration (106/mL oocysts) of VEG strain could kill mice within 11 DPI. Compared with the strains of T. gondii ToxoDB#216 and VEG, the lesions in the small intestines of the strains of TgCatCHn2- and TgCatCHn4-infected mice were significantly smaller (P < 0.01). The positive area of T. gondii antigen in the ileum of mice infected with the strains of T. gondii VEG, TgCatCHn2 and TgCatCHn4 were significantly lower than that T. gondii ToxoDB#216 at 8 DPI (P < 0.01). Paneth cells (PCs) in the small intestines was eliminated by ToxoDB#216 (5-6 DPI) (P < 0.05). The strains of T. gondii TgCatCHn2-, TgCatCHn4- and VEG-infected mice, the number of PCs and granules decreased in the intestines, compared to T. gondii free mice, but the difference was not significant (8 DPI, P > 0.05). However, the granules in the PCs showed negative lysozyme expression in the intestines of mice infected with T. gondii TgCatCHn2 and TgCatCHn4. Thus, T. gondii strains of TgCatCHn2 (ToxoDB#17) and TgCatCHn4 (ToxoDB#9) were avirulent strains, they triggered an inhibition of lysozyme expression in the granules of PCs of the mouse intestine. These effects may in turn lead to intestinal dysbiosis, which may be related to further parasitic invasion of the intestines. The findings of the present study further expand the spectrum of the pathogenic features of various Chinese isolates of T. gondii.

Antibody Detection, Isolation, Genotyping, and Virulence of Toxoplasma gondii in Captive Felids from China.

The felids are the only definitive hosts of Toxoplasma gondii, which could excrete oocysts into the environment and provide an infection source for toxoplasmosis in various warm-blooded animal species, particularly the captive felids that live close to human communities. The infection rate of the captive felids is a perfect standard in detecting the presence of Toxoplasma gondii oocysts in the environment. In this study, sera or tissue samples from zoo (1 young tiger, 2 adult tigers, 6 young lions), farm (10 masked palm civets), and pet hospital (28 cats) from Henan Province (China) were collected. The sera (n = 47) were tested for immunoglobulin G (IgG) antibodies against T. gondii by using modified agglutination test (MAT), whereas the hearts tissue (n = 40) were bioassayed in mice to isolate T. gondii strains. The genotype was distinguished by using PCR-RFLP of 10 loci (SAG1, SAG2, SAG3, GRA6, BTUB, L358, c22-8, PK1, c29-2, and Apico). The detection rate for the T. gondii antibody in captive felids was 21.3% (10/47). One viable T. gondii strain (TgCatCHn4) was obtained from a cat heart tissue, and its genotype was ToxoDB#9. The oocysts of ToxoDB#9 were collected from a T. gondii-free cat. The virulence of TgCatCHn4 was low and no cysts were detected in the brain of mice at 60 days post-inoculation. The finding of the present study suggested a widespread exposure of T. gondii for felids in Henan Province of central China, particularly those from the zoological gardens and homes. ToxoDB#9 was the predominant strain in China. Preventive measures against T. gondii oocyst contamination of various components of the environment should thus be implemented, including providing pre-frozen meat, well-cooked cat food, cleaned fruits and vegetables, monitoring birds and rodents, inactive T. gondii oocysts in felids feces, and proper hygiene.

Celecoxib and octreotide synergistically ameliorate portal hypertension via inhibition of angiogenesis in cirrhotic rats.

Abnormal angiogenesis is critical for portal hypertension in cirrhosis. Except for etiological treatment, no efficient medication or regime has been explored to treat the early stage of cirrhosis when angiogenesis is initiated or overwhelming. In this study, we explored an anti-angiogenesis effort through non-cytotoxic drugs octreotide and celecoxib to treat early stage of cirrhotic portal hypertension in an animal model. Peritoneal injection of thioacetamide (TAA) was employed to induce liver cirrhosis in rats. A combination treatment of celecoxib and octreotide was found to relieve liver fibrosis, portal venous pressure, micro-hepatic arterioportal fistulas, intrahepatic and splanchnic angiogenesis. Celecoxib and octreotide exerted their anti-angiogenesis effect via an axis of cyclooxygenase-2/prostaglandin E2/EP-2/somatostatin receptor-2, which consequently down-regulated phosphorylation of extracellular signal-regulated kinase (p-ERK)-hypoxia-inducible factor-1α (HIF-1α)-vascular endothelial growth factor (VEGF) integrated signaling pathways. In conclusions, combination of celecoxib and octreotide synergistically ameliorated liver fibrosis and portal hypertension of the cirrhotic rats induced by TAA via the inhibition of intrahepatic and extrahepatic angiogenesis. The potential mechanisms behind the regimen may due to the inactivation of p-ERK-HIF-1α-VEGF signaling pathway.

Celecoxib attenuates hepatic cirrhosis through inhibition of epithelial-to-mesenchymal transition of hepatocytes.

BACKGROUND AND AIM: The epithelial-mesenchymal transition (EMT) of hepatocytes is a key step for hepatic fibrosis and cirrhosis. Long-term administration of celecoxib, a selective cyclooxygenase-2 (COX-2) inhibitor, can ameliorate hepatic fibrosis. This research aimed to examine the effect of celecoxib on the EMT of hepatocytes during the development of liver cirrhosis. METHODS: Cirrhotic liver model of rat was established by peritoneal injection of thiacetamide (TAA). Thirty-six rats were randomly assigned to control, TAA, and TAA + celecoxib groups. Hepatic expressions of tumor necrosis factor-α (TNF-α), interleukin 6 (IL-6), COX-2, prostaglandin E2 (PGE2 ), matrix metalloproteinase (MMP)-2 and -9, transforming growth factor-ß1 (TGF-ß1), Phospho-Smad2/3, Snail1, α-smooth muscle actin (α-SMA), vimentin, collagen I, fibroblast-specific protein (FSP-1), E-cadherin and N-cadherin were quantitated. Hepatic fibrosis was assessed by the visible hepatic fibrotic areas and Ishak's scoring system. RESULTS: Exposed to TAA treatment, hepatocytes underwent the process of EMT during hepatic fibrosis. Compared with those in TAA group, celecoxib significantly downregulated the hepatic expressions of TNF-α, IL-6, COX-2, PGE2 , MMP-2, MMP-9, TGF-ß1, Phospho-Smad2/3, Snail1, α-SMA, FSP-1, and vimentin while greatly restoring the levels of E-cadherin. The fibrotic areas and collagen I levels of TAA + celecoxib group were much lower than those in TAA group. CONCLUSIONS: Celecoxib could ameliorate hepatic fibrosis and cirrhosis in TAA-rat model through suppression of the mesenchymal biomarkers in the hepatocytes while restoring the levels of their epithelial biomarkers. The inhibitory effect of celecoxib on the EMT of hepatocytes is associated with reduction of intrahepatic inflammation, preservation of normal basement matrix, and inhibition of TGF-ß1/Smad pathway.

Celecoxib ameliorates portal hypertension of the cirrhotic rats through the dual inhibitory effects on the intrahepatic fibrosis and angiogenesis.

BACKGROUND: Increased intra-hepatic resistance to portal blood flow is the primary factor leading to portal hypertension in cirrhosis. Up-regulated expression of cyclooxygenase-2 (COX-2) in the cirrhotic liver might be a potential target to ameliorate portal hypertension. OBJECTIVE: To verify the effect of celecoxib, a selective inhibitor of COX-2, on portal hypertension and the mechanisms behind it. METHODS: Cirrhotic liver model of rat was established by peritoneal injection of thiacetamide (TAA). 36 rats were randomly assigned to control, TAA and TAA+celecoxib groups. Portal pressures were measured by introduction of catheters into portal vein. Hepatic fibrosis was assessed by the visible hepatic fibrotic areas and mRNAs for collagen III and α-SMA. The neovasculature was determined by hepatic vascular areas, vascular casts and CD31 expression. Expressions of COX-2, vascular endothelial growth factor (VEGF), VEGF receptor-2 (VEGFR-2) and related signal molecules were quantitated. RESULTS: Compared with TAA group, the portal pressure in TAA+celecoxib group was significantly decreased by 17.8%, p<0.01. Celecoxib treatment greatly reduced the tortuous hepatic portal venules. The data of fibrotic areas, CD31expression, mRNA levels of α-SMA and collagen III in TAA+celecoxib group were much lower than those in TAA group, p<0.01. Furthermore, the up-regulation of hepatic mRNA and protein levels of VEGF, VEGFR-2 and COX-2 induced by TAA was significantly inhibited after celecoxib treatment. The expressions of prostaglandin E2 (PGE2), phosphorylated extracellular signal-regulated kinase (p-ERK), hypoxia-inducible factor-1α (HIF-1α), and c-fos were also down-regulated after celecoxib treatment. CONCLUSIONS: Long term administration of celecoxib can efficiently ameliorate portal hypertension in TAA rat model by its dual inhibitory effects on the intrahepatic fibrosis and angiogenesis. The anti-angiogenesis effect afforded by celecoxib may attribute to its modulation on VEGF/VEGFR-2 through the down-regulation of integrated signal pathways involving PGE2- HIF-1α- VEGF and p-ERK- c-fos- VEGFR-2.