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Salmonella, one of the major causes of foodborne infections, can cause bacterial foodborne illness. We investigated the serotype distribution, multidrug resistance (MDR), and β-lactamase resistance genes of human Salmonella isolates collected from clinical specimens in Guizhou, China, between 2013 and 2018. A total of 363 Salmonella isolates were collected from clinical specimens at 17 surveillance hospitals. Twenty-four serotypes were identified by sliding agglutination test. S. Enteritidis (33.9%), Salmonella 4,[5],12:i:- (24.0%), S. Typhimurium (16.3%), S. London (6.3%), and S. Derby (3.9%) were the top five serotypes. In 2018, the most common serotype changed from S. Enteritidis to S. Typhimurium. Among the 363 Salmonella isolates, 97.5% of isolates were resistant to at least one class of antimicrobial agents. For cephalosporins, ceftriaxone had the highest resistance rate of 10.5%, and cefepime and cefoxitin were 8.0% and 2.2%, respectively. Three hundred and one (82.9%) Salmonella isolates showed MDR. Salmonella 4,[5],12:i:- had the highest MDR rate with 94.2%, followed by S. London (91.3%) and S. Typhimurium (88.1%). Multidrug resistance rates of Salmonella isolates in Guizhou from 2013 to 2017 increased from 75.8% to 86.7%. Sixteen isolates (4.4%) showed extensive drug resistance. One hundred thirty-four antimicrobial resistance patterns were found. Two hundred and forty-one (66.4%) isolates carried at least one β-lactamase resistance gene. The blaTEM gene (61.2%) was the most prevalent resistant gene in all Salmonella isolates, followed by the blaCTX-M gene (6.1%) and blaOXA-1 gene (4.1%). Our findings showed that the MDR rate of Salmonella isolates from Guizhou province increased year by year. Therefore, systematic and long-term surveillance on MDR Salmonella isolates from clinical patients should be further strengthened.

Evidence shows that chaperone-mediated autophagy (CMA) is involved in cancer cell pathogenesis and progression. However, the potential role of CMA in breast cancer angiogenesis remains unknown. We first manipulated CMA activity by knockdown and overexpressing of lysosome-associated membrane protein type 2A (LAMP2A) in MDA-MB-231, MDA-MB-436, T47D and MCF7 cells. We found that the tube formation, migration and proliferation abilities of human umbilical vein endothelial cells (HUVECs) were inhibited after cocultured with tumor-conditioned medium from breast cancer cells of LAMP2A knockdown. While the above changes were promoted after cocultured with tumor-conditioned medium from breast cancer cells of LAMP2A overexpression. Moreover, we found that CMA could promote VEGFA expression in breast cancer cells and in xenograft model through upregulating lactate production. Finally, we found that lactate regulation in breast cancer cells is hexokinase 2 (HK2) dependent, and knockdown of HK2 can significantly reduce the ability of CMA-mediated tube formation capacity of HUVECs. Collectively, these results indicate that CMA could promote breast cancer angiogenesis via regulation of HK2-dependent aerobic glycolysis, which may serve as an attractive target for breast cancer therapies.

Nontyphoidal Salmonella is a bacterial and foodborne pathogen that poses a severe public health threat. However, the genetic diversity of different serogroups of nontyphoidal Salmonella in Guizhou is unknown. This study aimed to obtain the RNA secondary structure of the typical direct repeat sequences, the characteristics of clustered regularly interspaced short palindromic repeats (CRISPR) genotypes, and the genetic diversity of different serogroups of nontyphoidal Salmonella strains. The 342 nontyphoidal Salmonella strains were collected from nine cities (prefectures) of Guizhou province during 2013–2018, serotyped by slide agglutination, and examined the molecular genotypes by CRISPR method. The strains were divided into five serogroups. The dominant serogroup was group B (47.08%), followed by group D1 (36.55%). One hundred and thirty-five CRISPR genotypes were detected with 108 novel spacer sequences amongst 981 unique spacer sequences. The diversity of nontyphoidal Salmonella CRISPR loci was not only the deletion, duplication, or point mutation of spacer sequences but also the acquisition of new spacer sequences to form novel genotypes. The CRISPR genotyping was an effective typing method that could reveal the genetic diversity of different nontyphoidal Salmonella serotypes except for S. Enteritidis.

BackgroundEchinococcus multilocularis causes alveolar echinococcosis (AE), a rising zoonotic disease in the northern hemisphere. Treatment of this fatal disease is limited to chemotherapy using benzimidazoles and surgical intervention, with frequent disease recurrence in cases without radical surgery. Elucidating the molecular mechanisms underlying E. multilocularis infections and host-parasite interactions ultimately aids developing novel therapeutic options. This study explored an involvement of unfolded protein response (UPR) and endoplasmic reticulum-stress (ERS) during E. multilocularis infection in mice.MethodsE. multilocularis- and mock-infected C57BL/6 mice were subdivided into vehicle, albendazole (ABZ) and anti-programmed death ligand 1 (αPD-L1) treated groups. To mimic a chronic infection, treatments of mice started six weeks post i.p. infection and continued for another eight weeks. Liver tissue was then collected to examine inflammatory cytokines and the expression of UPR- and ERS-related genes.ResultsE. multilocularis infection led to an upregulation of UPR- and ERS-related proteins in the liver, including ATF6, CHOP, GRP78, ERp72, H6PD and calreticulin, whilst PERK and its target eIF2α were not affected, and IRE1α and ATF4 were downregulated. ABZ treatment in E. multilocularis infected mice reversed, or at least tended to reverse, these protein expression changes to levels seen in mock-infected mice. Furthermore, ABZ treatment reversed the elevated levels of interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α and interferon (IFN)-γ in the liver of infected mice. Similar to ABZ, αPD-L1 immune-treatment tended to reverse the increased CHOP and decreased ATF4 and IRE1α expression levels.Conclusions and significanceAE caused chronic inflammation, UPR activation and ERS in mice. The E. multilocularis-induced inflammation and consecutive ERS was ameliorated by ABZ and αPD-L1 treatment, indicating their effectiveness to inhibit parasite proliferation and downregulate its activity status. Neither ABZ nor αPD-L1 themselves affected UPR in control mice. Further research is needed to elucidate the link between inflammation, UPR and ERS, and if these pathways offer potential for improved therapies of patients with AE.

Salmonella enterica serovar 4,[5],12:i:-, a monophasic variant of Salmonella Typhimurium lacking the phase 2 flagellin, is one of the common serotypes causing Salmonellosis worldwide. However, information on Salmonella serovar 4,[5],12:i:- from Guizhou Province has lacked so far. This study aimed to investigate the antimicrobial resistance, the presence of antimicrobial resistance genes and virulence genes, and characterize the MLST genotypes of Salmonella serovar 4,[5],12:i:- isolates from Guizhou province, China. We collected 363 non-typhoid Salmonella (NTS) isolates of Guizhou from 2013 to 2018. Biochemical identification, serogroups testing, and specific multiplex polymerase chain reaction (mPCR) assay were conducted to identify Salmonella 4,[5],12:i:- isolates. Isolates were determined the antimicrobial resistance by the micro broth dilution method, detected the presence of antimicrobial resistance genes and virulence genes by PCR, and examined the molecular genotyping by Multilocus sequence typing (MLST). Eighty-seven Salmonella 4,[5],12:i:- isolates were detected, accounting for 23.9% (87/363) of the total NTS isolates. All Salmonella 4,[5],12:i:- isolates showed highly resistant to sulfaoxazole (93.1%), streptomycin (90.8%), ampicillin (88.5%), tetracycline (86.2%) and doxycycline (86.2%). A high proportion (94.2%) of multi-drug resistance (MDR) isolates were found. Most (83.9%) Salmonella 4,[5],12:i:- isolates carried four antimicrobial resistance genes, especially blaTEM-1, strA-strB, sul2, and tetB genes. Salmonella 4,[5],12:i:- isolates showed a high rate of invA, sseL, mgtC, siiE, sopB, gipA, gtgB, sspH1, and sspH2 (72.4%~98.9%). On the contrary, none of the isolates were detected the spvC and pefA genes. MLST analysis revealed three sequence types (STs), and ST34 (97.7%) was the dominant sequence type. This study is the first report of Salmonella 4,[5],12:i:- in humans from Guizhou province, China. The data might be useful for rational antimicrobial usage against Salmonella 4,[5],12:i:- infections, risk management, and public health strategies in Guizhou.

We present a comprehensive analysis of the hepatic miRNA transcriptome at one month post-infection of experimental primary alveolar echinococcosis (AE), a parasitic infection caused upon ingestion of E. multilocularis eggs. Liver tissues were collected from infected and non-infected C57BL/6 mice, then small RNA libraries were prepared for next-generation sequencing (NGS). We conducted a Stem-loop RT-qPCR for validation of most dysregulated miRNAs. In infected mice, the expression levels of 28 miRNAs were significantly altered. Of these, 9 were up-regulated (fold change (FC) ≥ 1.5) and 19 were down-regulated (FC ≤ 0.66) as compared to the non-infected controls. In infected livers, mmu-miR-148a-3p and mmu-miR-101b-3p were 8- and 6-fold down-regulated, respectively, and the expression of mmu-miR-22-3p was reduced by 50%, compared to non-infected liver tissue. Conversely, significantly higher hepatic levels were noted for Mus musculus (mmu)-miR-21a-5p (FC = 2.3) and mmu-miR-122-5p (FC = 1.8). In addition, the relative mRNA expression levels of five genes (vegfa, mtor, hif1-α, fasn and acsl1) that were identified as targets of down-regulated miRNAs were significantly enhanced. All the five genes exhibited a higher expression level in livers of E. multilocularis infected mice compared to non-infected mice. Finally, we studied the issue related to functionally mature arm selection preference (5p and/or 3p) from the miRNA precursor and showed that 9 pre-miRNAs exhibited different arm selection preferences in normal versus infected liver tissues. In conclusion, this study provides first evidence that miRNAs are regulated early in primary murine AE. Our findings raise intriguing questions such as (i) how E. multilocularis affects hepatic miRNA expression;(ii) what are the alterations in miRNA expression patterns in more advanced AE-stages; and (iii) which hepatic cellular, metabolic and/or immunologic processes are modulated through altered miRNAs in AE. Thus, further research on the regulation of miRNAs during AE is needed, since miRNAs constitute an attractive potential option for development of novel therapeutic approaches against AE.

This paper presents the use of the Aimsun microsimulation program to simulate vehicle violating behaviors and observe their impact on road traffic crash risk. Plugins for violations of speeding, slow driving, and abrupt stopping were developed using Aimsun's API and SDK module. A safety analysis plugin for investigating probability of rear-end collisions was developed, and a method for analyzing collision risk is proposed. A Fuzzy C-mean Clustering algorithm was developed to identify high risk states in different road segments over time. Results of a simulation experiment based on the G15 Expressway in Shanghai showed that abrupt stopping had the greatest impact on increasing collision risk, and the impact of violations increased with traffic volume. The methodology allows for the evaluation and monitoring of risks, alerting of road hazards, and identification of hotspots, and could be applied to the operations of existing facilities or planning of future ones.

BACKGROUND:The growth potential of the tumor-like Echinococcus multilocularis metacestode (causing alveolar echinococcosis, AE) is directly linked to the nature/function of the periparasitic host immune-mediated processes. We previously showed that Fibrinogen-like-protein 2 (FGL2), a novel CD4+CD25+ Treg effector molecule, was over-expressed in the liver of mice experimentally infected with E. multilocularis. However, little is known about its contribution to the control of this chronic helminth infection. METHODS/FINDINGS:Key parameters for infection outcome in E. multilocularis-infected fgl2-/- (AE-fgl2-/-) and wild type (AE-WT) mice at 1 and 4 month(s) post-infection were (i) parasite load (i. e. wet weight of parasitic metacestode tissue), and (ii) parasite cell proliferation as assessed by determining E. multilocularis 14-3-3 gene expression levels. Serum FGL2 levels were measured by ELISA. Spleen cells cultured with ConA for 48h or with E. multilocularis Vesicle Fluid (VF) for 96h were analyzed ex-vivo and in-vitro. In addition, spleen cells from non-infected WT mice were cultured with rFGL2/anti-FGL2 or rIL-17A/anti-IL-17A for further functional studies. For Treg-immune-suppression-assays, purified CD4+CD25+ Treg suspensions were incubated with CD4+ effector T cells in the presence of ConA and irradiated spleen cells as APCs. Flow cytometry and qRT-PCR were used to assess Treg, Th17-, Th1-, Th2-type immune responses and maturation of dendritic cells. We showed that AE-fgl2-/- mice exhibited (as compared to AE-WT-animals) (a) a significantly lower parasite load with reduced proliferation activity, (b) an increased T cell proliferative response to ConA, (c) reduced Treg numbers and function, and (d) a persistent capacity of Th1 polarization and DC maturation. CONCLUSIONS:FGL2 appears as one of the key players in immune regulatory processes favoring metacestode survival by promoting Treg cell activity and IL-17A production that contributes to FGL2-regulation. Prospectively, targeting FGL2 could be an option to develop an immunotherapy against AE and other chronic parasitic diseases.

Pathogenesis of chronically developing alveolar echinococcosis (AE) is characterized by a continuous, granulomatous, periparasitic infiltration of immune cells surrounding the metacestode of Echinococcus multilocularis (E.multilocularis) in the affected liver. A detailed cytokine and chemokine profile analysis of the periparasitic infiltrate in the liver has, however, not yet been carried out in a comprehensive way all along the whole course of infection in E. multilocularis intermediate hosts. We thus assessed the hepatic gene expression profiles of 18 selected cytokine and chemokine genes using qRT-PCR in the periparasitic immune reaction and the subsequent adjacent, not directly affected, liver tissue of mice from day 2 to day 360 post intra-hepatic injection of metacestode. DNA microarray analysis was also used to get a more complete picture of the transcriptional changes occurring in the liver surrounding the parasitic lesions. Profiles of mRNA expression levels in the hepatic parasitic lesions showed that a mixed Th1/Th2 immune response, characterized by the concomitant presence of IL-12α, IFN-γ and IL-4, was established very early in the development of E. multilocularis. Subsequently, the profile extended to a combined tolerogenic profile associating IL-5, IL-10 and TGF-β. IL-17 was permanently expressed in the liver, mostly in the periparasitic infiltrate; this was confirmed by the increased mRNA expression of both IL-17A and IL-17F from a very early stage, with a subsequent decrease of IL-17A after this first initial rise. All measured chemokines were significantly expressed at a given stage of infection; their expression paralleled that of the corresponding Th1, Th2 or Th17 cytokines. In addition to giving a comprehensive insight in the time course of cytokines and chemokines in E. multilocularis lesion, this study contributes to identify new targets for possible immune therapy to minimize E. multilocularis-related pathology and to complement the only parasitostatic effect of benzimidazoles in AE.

Alveolar echinococcosis (AE) is characterized by the development of irreversible fibrosis and of immune tolerance towards Echinococcus multilocularis (E. multilocularis). Very little is known on the presence of transforming growth factor-β (TGF-β) and other components of TGF-β/Smad pathway in the liver, and on their possible influence on fibrosis, over the various stages of infection. Using Western Blot, qRT-PCR and immunohistochemistry, we measured the levels of TGF-β1, TGF-β receptors, and down-stream Smads activation, as well as fibrosis marker expression in both a murine AE model from day 2 to 360 post-infection (p.i.) and in AE patients. TGF-β1, its receptors, and down-stream Smads were markedly expressed in the periparasitic infiltrate and also in the hepatocytes, close to and distant from AE lesions. Fibrosis was significant at 180 days p.i. in the periparasitic infiltrate and was also present in the liver parenchyma, even distant from the lesions. Over the time course after infection TGF-β1 expression was correlated with CD4/CD8 T-cell ratio long described as a hallmark of AE severity. The time course of the various actors of the TGF-β/Smad system in the in vivo mouse model as well as down-regulation of Smad7 in liver areas close to the lesions in human cases highly suggest that TGF-β plays an important role in AE both in immune tolerance against the parasite and in liver fibrosis.

BACKGROUND: Alveolar echinococcosis (AE) is characterized by the tumor-like growth of Echinococcus (E.) multilocularis. Very little is known on the influence of helminth parasites which develop in the liver on the proliferation/growth arrest metabolic pathways in the hepatocytes of the infected liver over the various stages of infection. METHODOLOGY/PRINCIPAL FINDINGS: Using Western blot analysis, qPCR and immunohistochemistry, we measured the levels of MAPKs activation, Cyclins, PCNA, Gadd45β, Gadd45γ, p53 and p21 expression in the murine AE model, from day 2 to 360 post-infection. Within the early (day 2-60) and middle (day60-180) stages, CyclinB1 and CyclinD1 gene expression increased up to day30 and then returned to control level after day60; Gadd45β, CyclinA and PCNA increased all over the period; ERK1/2 was permanently activated. Meanwhile, p53, p21 and Gadd45γ gene expression, and caspase 3 activation, gradually increased in a time-dependent manner. In the late stage (day180-360), p53, p21 and Gadd45γ gene expression were significantly higher in infected mice; JNK and caspase 3 were activated. TUNEL analysis showed apoptosis of hepatocytes. No significant change in CyclinE, p53 mRNA and p-p38 expression were observed at any time. CONCLUSIONS: Our data support the concept of a sequential activation of metabolic pathways which 1) would first favor parasitic, liver and immune cell proliferation and survival, and thus promote metacestode fertility and tolerance by the host, and 2) would then favor liver damage/apoptosis, impairment in protein synthesis and xenobiotic metabolism, as well as promote immune deficiency, and thus contribute to the dissemination of the protoscoleces after metacestode fertility has been acquired. These findings give a rational explanation to the clinical observations of hepatomegaly and of unexpected survival of AE patients after major hepatic resections, and of chronic liver injury, necrosis and of hepatic failure at an advanced stage and in experimental animals.

BACKGROUND: Alveolar echinococcosis (AE) is a severe chronic parasitic disease which behaves like a slow-growing liver cancer. Clinical observations suggest that the parasite, Echinococcus multilocularis (E. multilocularis) influences liver homeostasis and hepatic cell metabolism. However, this has never been analyzed during the time course of infection in the common model of secondary echinococcosis in experimental mice. METHODOLOGY/PRINCIPAL FINDINGS: Gene expression profiles were assessed using DNA microarray analysis, 1, 2, 3 and 6 months after injection of E. multilocularis metacestode in the liver of susceptible mice. Data were collected at different time points to monitor the dynamic behavior of gene expression. 557 differentially expressed genes were identified at one or more time points, including 351 up-regulated and 228 down-regulated genes. Time-course analysis indicated, at the initial stage of E. multilocularis infection (month 1-2), that most of up-regulated pathways were related to immune processes and cell trafficking such as chemokine-, mitogen-activated protein kinase (MAPK) signaling, and down-regulated pathways were related to xenobiotic metabolism; at the middle stage (month 3), MAPK signaling pathway was maintained and peroxisome proliferator-activated receptor (PPAR) signaling pathway emerged; at the late stage (month 6), most of up-regulated pathways were related to PPAR signaling pathway, complement and coagulation cascades, while down-regulated pathways were related to metabolism of xenobiotics by cytochrome P450. Quantitative RT-PCR analysis of a random selection of 19 genes confirmed the reliability of the microarray data. Immunohistochemistry analysis showed that proliferating cell nuclear antigen (PCNA) was increased in the liver of E. multilocularis infected mice from 2 months to 6 months. CONCLUSIONS: E. multilocularis metacestode definitely exerts a deep influence on liver homeostasis, by modifying a number of gene expression and metabolic pathways. It especially promotes hepatic cell proliferation, as evidenced by the increased PCNA constantly found in all the experimental time-points we studied and by an increased gene expression of key metabolic pathways.