et al. (2020). Co-infections of SARS-CoV-2 with multiple common respiratory pathogens in infected patients. Sci China Life Sci 63, 606-609. https://doi.

19The ongoing SARS-CoV-2 outbreak has killed over twenty-one thousand and sickened over four 20 hundred thousand people worldwide, posing a great challenge to global public health. A sensitive and 21 accurate diagnosis method will substantially help to control disease expansion. Here, we developed a 22 chemiluminescence-immunoassay method based on the recombinant nucleocapsid antigen and the 23 magnetic beads for diagnosis of SARS-CoV-2 infections and surveillance of antibody changing pattern. : medRxiv preprint Serums from 29 healthy individuals, 51 tuberculosis patients, and 79 SARS-CoV-2 confirmed patients 25 were employed to evaluate the performance of this approach. Compared to the IgM testing, the IgG 26 testing was more reliable in which it identified 65 SARS-CoV-2 infections from the 79 confirmed 27 patients and only two false-positive cases from the 80 control group with a sensitivity and specificity 28 reaching 82.28% and 97.5%, respectively. However, only a slight difference (not statistically 29 significant) in the detected cases of SARS-CoV-2 infections was observed between the IgM and IgG 30 testing manner in patients at a different time of onset of disease. A performance comparison 31 between an ELISA kit using the same nucleocapsid antigen and our chemiluminescence method was 32 undertaken. The same false-positive cases were seen in both methods from the paired control group, 33 while ELISA kit can only detect half of the SARS-CoV-2 infections from paired SARS-CoV-2 confirmed 34 patients group than that of the chemiluminescence method, indicating a higher performance for the 35 chemiluminescence-immunoassay approach. Together, our studies provide a useful and valuable 36 serological testing tool for the diagnosis of SARS-CoV-2 infections in the community.37

We developed a chemiluminescence immunoassay method based on the recombinant nucleocapsid antigen and assessed its performance for the clinical diagnosis of severe acute respiratory syndrome coronavirus (SARS-CoV)-2 infections by detecting SARS-CoV-2-specific IgM and IgG antibodies in patients. Full-length recombinant nucleocapsid antigen and tosyl magnetic beads were used to develop the chemiluminescence immunoassay approach. Plasmas from 29 healthy cohorts, 51 tuberculosis patients, and 79 confirmed SARS-CoV-2 patients were employed to evaluate the chemiluminescence immunoassay method performance for the clinical diagnosis of SARS-CoV-2 infections. A commercial ELISA kit (Darui Biotech, China) using the same nucleocapsid antigen was used for the in-parallel comparison with our chemiluminescence immunoassay method. The IgM and IgG manner of testing in the chemiluminescence immunoassay method showed a sensitivity and specificity of 60.76% (95% CI 49.1 to 71.6) and 92.25% (95% CI 83.4 to 97.2) and 82.28% (95% CI 72.1 to 90.0) and 97.5% (95% CI 91.3 to 99.7), respectively. Higher sensitivity and specificity were observed in the chemiluminescence immunoassay method compared with the Darui Biotech ELISA kit. The developed high sensitivity and specificity chemiluminescence immunoassay IgG testing method combined with the RT-PCR approach can improve the clinical diagnosis for SARS-CoV-2 infections and thus contribute to the control of COVID-19 expansion.

Spectinomycin is an aminocyclitol antibiotic used clinically to treat a variety of infections in animals. Here, we characterized drug resistance prevalence in clinical Streptococcus suis isolates and discovered a novel resistance mechanism in which the s5 mutation (Gly26Asp) results in high spectinomycin resistance. Additionally, a novel integrative and conjugative element encompassing a multidrug resistance spw_like-aadE-lnu(B)-lsa(E) cluster and a cadmium resistance operon were identified, suggesting a possible cause for the wide dissemination of spectinomycin resistance in S. suis. Streptococcus suis, the leading agent for human meningitis in several Asian countries, is a significant zoonotic pathogen worldwide. Recently, it has received growing attention from the global academic community not only for its increased incidence of infections in humans but also for its important implied role in drug resistance transmission (1). The aminocyclitol antibiotic spectinomycin, often combined with lincomycin, is widely used for the treatment of pathogen infections in farm animals, including swine (2). However, few studies have examined spectinomycin resistance in S. suis globally. In this study, 191 clinical S. suis isolates, collected from different provinces in China over the period from 2006 to 2012, were initially subjected to spectinomycin susceptibility analysis by Etest method. Based on the spectinomycin MIC breakpoints to cattle respiratory pathogens (3), 20 isolates (10.4%) were found to be resistant (MIC of Ն256 g/ml) (see Table S1 in the supplemental material). To investigate resistance mechanisms within them, multi-PCR experiments were conducted to detect an array of adenyltransferase genes, including spc, aad9, spw, spd, and various aadA genes (4-8). In addition, the complete 16S RNA gene and the s5 gene were amplified and sequenced for mutation analysis in all resistant strains as well as certain susceptible strains (see Table S3 in the supplemental material). Unexpectedly, none of the widely reported resistance genes could be detected among these strains with the exception of the spw gene and its variant spw_like, which demonstrated approximately 94% amino acid identity corresponding to 15 site mutations (see Fig. S1 in the supplemental material). The spw gene and its variant spw_like were present in 13 isolates and 6 isolates, respectively (Table 1). In contrast, there were a variety of mutations within the 16S RNA and s5 genes in the resistant isolates in comparison with the susceptible strains, and, among these, two mutations were present in the 16S rRNA regions of all four operons corresponding to sites 1069(C1069T) and 1188(A1188G) in Escherichia coli 16S rRNA and one site within the 26 amino acids (aa) of the s5 protein (GGT ¡ GAT, Gly26Asp). Interestingly, in the isolate with the s5 gene mutation, the spw or spw_like genes were also not detected, suggesting that spectinomycin resistance was likely caused by this mutation.To verify these potential mechanisms, we cloned the complete spw_like genes a...

Most Salmonella serovars cause disease in many host species, while a few serovars have evolved to be host specific. Very little is known about the mechanisms that contribute to Salmonella host specificity. We compared the interactions between chicken primary macrophages (CDPM) and host-generalist serovar Salmonella enterica serovar Typhimurium, host-adapted Salmonella enterica serovar Dublin, and avian host-specific Salmonella enterica serovar Gallinarum. S. Gallinarum was taken up in lower numbers by CDPM than S. Typhimurium and S. Dublin; however, a higher survival rate was observed for this serovar. In addition, S. Typhimurium and S. Dublin caused substantially higher levels of cell death to the CDPM, while significantly higher concentrations of NO were produced by S. Gallinarum-infected cells. Global transcriptome analysis performed 2 h postinfection showed that S. Gallinarum infection triggered a more comprehensive response in CDPM with 1,114 differentially expressed genes (DEGs) compared to the responses of S. Typhimurium (625 DEGs) and S. Dublin (656 DEGs). Comparable levels of proinflammation responses were observed in CDPM infected by these three different serovars at the initial infection phase, but a substantially quicker reduction in levels of interleukin-1β (IL-1β), CXCLi1, and CXCLi2 gene expression was detected in the S. Gallinarum-infected macrophages than that of two other groups as infections proceeded. KEGG cluster analysis for unique DEGs after S. Gallinarum infection showed that the JAK-STAT signaling pathway was top enriched, indicating a specific role for this pathway in response to S. Gallinarum infection of CDPM. Together, these findings provide new insights into the interaction between Salmonella and the host and increase our understanding of S. Gallinarum host specificity.

Salmonella Gallinarum only infects avian species, where it causes a severe systemic infection in birds of all ages. It is generally accepted that interaction with phagocytic cells plays an important role in the development of systemic, host-specific Salmonella infections. The current study detailed the interaction of S. Gallinarum with macrophages derived from chicken (HD11) and cattle (Bomac) compared to interaction of the broad host range serovar, Salmonella Typhimurium and the cattle adapted serovar Salmonella Dublin. Results showed a weaker invading ability of S. Gallinarum in both kinds of macrophages, regardless whether the bacteria were opsonized or not before infections. However, opsonization of S. Gallinarum by chicken serum increased its intracellular survival rate in chicken macrophages. No significant induction of nitrogen oxide was observed in the infected HD11 cells within the first 6 h, and levels of reactive oxygen species (ROS) were similar among the three serovars. S. Gallinarum infection was associated with low cell deaths in both chicken and cattle macrophages, whereas S. Dublin only induced a comparable high level of cell death in chicken macrophages, but not in macrophages of its preferred host species (Bomac) compared to host generalist S. Typhimurium. S. Gallinarum-infected HD11 macrophages exhibited low induction of pro-inflammation genes [interleukin (IL)1β, CXCLi1, and CXCLi2] compared to the two other serovars, and contrary to the other serovars, it did not induce significant downregulation of Toll-like receptor (TLR)2, TLR4, and TLR5. In in vivo infection of 1-week-old chicken, a significant upregulation of the TLR4 and TLR5 genes in the spleen was observed in S. Gallinarum-infected chickens, but not in S. Typhimurium-infected chicken at 5 days post-infections. Taken together, results show that S. Gallinarum infection of macrophages was characterized by low uptake and low cytotoxicity, possibly allowing long-term persistence in the intracellular environment, and it caused a low induction of pro-inflammatory responses.

Salmonella Pathogenicity Islands 19 (SPI19) encodes a type VI secretion system (T6SS). SPI19 is only present in few serovars of S. enterica, including the host-adapted serovar S. Dublin and the host-specific serovar S. Gallinarum. The role of the SPI19 encoded T6SS in virulence in these serovar is not fully understood. Here we show that during infection of mice, a SPI19/T6SS deleted strain of S. Dublin 2229 was less virulent than the wild type strain after oral challenge, but not after IP challenge. The mutant strain also competed significantly poorer than the wild type strain when co-cultured with strains of E. coli, suggesting that this T6SS plays a role in pathogenicity by killing competing bacteria in the intestine. No significant difference was found between wild type S.Gallinarum G9 and its SPI19/T6SS mutant in infection, whether chicken were challenged orally or by the IP route, and the S. Gallinarum G9 SPI19/T6SS strain competed equally well as the wild type strain against strains of E. coli. However, contrary to what was observed with S. Dublin, the wild type G9 strains was significantly more cytotoxic to monocyte derived primary macrophages from hens than the mutant, suggesting that SPI19/T6SS in S. Gallinarum mediates killing of eukaryotic cells. The lack of significant importance of SPI19/T6SS after oral and systemic challenge of chicken was confirmed by knocking out SPI19 in a second strain, J91. Together the results suggest that the T6SS encoded from SPI19 have different roles in the two serovars and that it is a virulence-factor after oral challenge of mice in S. Dublin, while we cannot confirm previous results that SPI19/T6SS influence virulence significantly in S. Gallinarum.

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