Human angiotensin converting enzyme 2 specific antisense oligonucleotides reduce infection with SARS-CoV-2 variants.

BACKGROUND: The Spike protein mutation of SARS-CoV-2 led to decreased protective effect of various vaccines and monoclonal antibodies, suggesting that blocking SARS-CoV-2 infection by targeting host factors would make the therapy more resilient against virus mutations. Angiotensin converting enzyme 2 (ACE2) is the host receptor of SARS-CoV-2 and its variants, as well as many other coronaviruses. Down-regulation of ACE2 expression in the respiratory tract may prevent viral infection. Antisense oligonucleotides (ASOs) can be rationally designed based on sequence data, require no delivery system, and can be administered locally. OBJECTIVE: We sought to design ASOs that can block SARS-CoV-2 by down-regulating ACE2 in human airway. METHODS: ACE2-targeting ASOs were designed using a bioinformatic method and screened in cell lines. Human primary nasal epithelial cells cultured at the air-liquid interface and humanized ACE2 mice were used to detect the ACE2 reduction levels and the safety of ASOs. ASOs pretreated nasal epithelial cells and mice were infected and then used to detect the viral infection levels. RESULTS: ASOs reduced ACE2 expression on mRNA and protein level in cell lines and in human nasal epithelial cells. Furthermore they efficiently suppressed virus replication of three different SARS-CoV-2 variants in human nasal epithelial cells. In vivo, ASOs also down-regulated human ACE2 in humanized ACE2 mice and thereby reduced viral load, histopathological changes in lungs, and they increased survival of mice. CONCLUSION: ACE2-targeting ASOs can effectively block SARS-COV-2 infection. Our study provides a new approach for blocking SARS-CoV-2 and other ACE2-targeting virus in high-risk populations.

Dynamic changes in polioviruses identified by environmental surveillance in Guangzhou, 2009-2021.

Polio cases can be missed by acute flaccid paralysis (AFP) case surveillance alone, emphasizing the importance of environmental surveillance (ES). In this study, to investigate the serotype distribution and epidemiological trends of poliovirus (PV), we characterized PV isolated from domestic sewage in Guangzhou City, Guangdong Province, China from 2009 to 2021. A total of 624 sewage samples were collected from the Liede Sewage Treatment Plant, and the positive rates of PV and non-polio enteroviruses were 66.67% (416/624) and 78.37% (489/624), respectively. After sewage sample treatment, each sewage sample was inoculated in six replicate tubes of three cell lines, and 3370 viruses were isolated during the 13-year surveillance period. Among these, 1086 isolates were identified as PV, including type 1 PV (21.36%), type 2 PV (29.19%), and type 3 PV (49.48%). Based on VP1 sequences, 1057 strains were identified as Sabin-like, 21 strains were high-mutant vaccines, and eight strains were vaccine-derived poliovirus (VDPV). The numbers and serotypes of PV isolates in sewage were influenced by the vaccine switch strategy. After type 2 OPV was removed from the trivalent oral PV (OPV) vaccine and a bivalent OPV (bOPV) was adopted in May 2016, the last type 2 PV strain was isolated from sewage, with no detection thereafter. Type 3 PV isolates increased significantly and became the dominant serotype. Before and after the second vaccine switch in January 2020, that is, from the first dose of IPV and second-fourth doses of bOPV to the first two doses of IPV and third-fourth doses of bOPV, there was also a statistical difference in PV positivity rates in sewage samples. Seven type 2 VDPVs and one type 3 VDPV were identified in sewage samples in 2009-2021, and phylogenetic analysis indicated that all VDPVs isolated from ES in Guangdong are newly discovered VDPVs, different from VDPV previously discovered in China, and were classified as ambiguous VDPV. It is noteworthy that no VDPV cases were reported in AFP case surveillance in the same period. In conclusion, continued PV ES in Guangzhou since April 2008 has been a useful supplement to AFP case surveillance, providing an important basis for evaluating the effectiveness of vaccine immunization strategies. ES improves early detection, prevention, and control; accordingly, this strategy can curb the circulation of VDPVs and provide a strong laboratory basis for maintaining a polio-free status.

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.

Comparison of model-specific histopathology in mouse models of COVID-19.

A novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has been identified as the causative agent of the current coronavirus disease 2019 pandemic. Development of animal models that parallel the clinical and pathologic features of disease are highly essential to understanding the pathogenesis of SARS-CoV-2 infection and the development of therapeutics and prophylactics. Several mouse models that express the human angiotensin converting enzyme 2 (hACE2) have been created, including transgenic and knock-in strains, and viral vector-mediated delivery of hACE2. However, the comparative pathology of these mouse models infected with SARS-CoV-2 are unknown. Here, we perform systematic comparisons of the mouse models including K18-hACE2 mice, KI-hACE2 mice, Ad5-hACE2 mice and CAG-hACE2 mice, which revealed differences in the distribution of lesions and the characteristics of pneumonia induced. Based on these observations, the hACE2 mouse models meet different needs of SARS-CoV-2 researches. The similarities or differences among the model-specific pathologies may help in better understanding the pathogenic process of SARS-CoV-2 infection and aiding in the development of effective medications and prophylactic treatments for SARS-CoV-2.

Single-dose rAAV5-based vaccine provides long-term protective immunity against SARS-CoV-2 and its variants.

The COVID-19 pandemic, caused by the SARS-CoV-2 virus and its variants, has posed unprecedented challenges worldwide. Existing vaccines have limited effectiveness against SARS-CoV-2 variants. Therefore, novel vaccines to match mutated viral lineages by providing long-term protective immunity are urgently needed. We designed a recombinant adeno-associated virus 5 (rAAV5)-based vaccine (rAAV-COVID-19) by using the SARS-CoV-2 spike protein receptor binding domain (RBD-plus) sequence with both single-stranded (ssAAV5) and self-complementary (scAAV5) delivery vectors and found that it provides excellent protection from SARS-CoV-2 infection. A single-dose vaccination in mice induced a robust immune response; induced neutralizing antibody (NA) titers were maintained at a peak level of over 1:1024 more than a year post-injection and were accompanied by functional T-cell responses. Importantly, both ssAAV- and scAAV-based RBD-plus vaccines produced high levels of serum NAs against the circulating SARS-CoV-2 variants, including Alpha, Beta, Gamma and Delta. A SARS-CoV-2 virus challenge showed that the ssAAV5-RBD-plus vaccine protected both young and old mice from SARS-CoV-2 infection in the upper and lower respiratory tracts. Whole genome sequencing demonstrated that AAV vector DNA sequences were not found in the genomes of vaccinated mice one year after vaccination, demonstrating vaccine safety. These results suggest that the rAAV5-based vaccine is safe and effective against SARS-CoV-2 and several variants as it provides long-term protective immunity. This novel vaccine has a significant potential for development into a human prophylactic vaccination to help end the global pandemic.

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.

No evidence of a role for mitochondrial complex I in Helicobacter pylori pathogenesis.

BACKGROUND: Complex I is the first enzyme complex in the mitochondrial respiratory chain, responsible for generating a large fraction of energy during oxidative phosphorylation. Recently, it has been identified that complex I deficiency can result in increased inflammation due to the generation of reactive oxygen species by innate immune cells. As a reduction in complex I activity has been demonstrated in human stomachs with atrophic gastritis, we investigated whether complex I deficiency could influence Helicobacter pylori pathogenesis. MATERIALS AND METHODS: Ndufs6gt/gt mice have a partial complex I deficiency. Complex I activity was quantified in the stomachs and immune cells of Ndufs6gt/gt mice by spectrophotometric assays. Ndufs6gt/gt mice were infected with H. pylori and bacterial colonization assessed by colony-forming assay, gastritis assessed histologically, and H. pylori -specific humoral response quantified by ELISA. RESULTS: The immune cells and stomachs of Ndufs6gt/gt mice were found to have significantly decreased complex I activity, validating the model for assessing the effects of complex I deficiency in H. pylori infection. However, there was no observable effect of complex I deficiency on either H. pylori colonization, the resulting gastritis, or the humoral response. CONCLUSIONS: Although complex I activity is described to suppress innate immune responses and is decreased during atrophic gastritis in humans, our data suggest it does not affect H. pylori pathogenesis.

[Taq Man probe-based quadruple real-time PCR for detection of Salmonella paratyphi A/B/C and Salmonella typhi].

OBJECTIVE: Establishment and application of Taq Man probe-based quadruple real-time PCR for detection of Salmonella paratyphi A/B/C and Salmonella typhi. Primers specific to Salmonella paratyphi A( SPAP), Salmonella paratyphi B( SPBP), Salmonella paratyphi C( SPCP), and Salmonella typhi( STP) were designed. METHODS: A method of Taq Man probe-based quadruple real-time PCR was established according to the distinction of the 5'end of the probe mark of TET, ROX, FAM and HEX. 5 strains of SPA, 4 strains of SPB, 7 strains of SPC and 11 strains of ST were identified by amplification from SPAP, SPBP, SPCP and STP. RESULTS: While other serotypes of salmonella and17 strains of non-salmonella got negative results of amplification. Amplification rate of SPAP, SPBP, SPCP, and STP were 84. 5%, 101. 8%, 92. 4% and 90. 9%, respectively. The linear correlation coefficient( R~2) were 0. 996, 0. 975, 0. 996 and 0. 984, respectively. CONCLUSION: The PCR system is specific and sensitive for the identification of SPA, SPB, SPC and ST.

Serotypes, seasonal trends, and antibiotic resistance of non-typhoidal Salmonella from human patients in Guangdong Province, China, 2009-2012.

BACKGROUND: Non-typhoidal Salmonella is a common cause of infectious diarrhea in humans. Antimicrobial-resistant Salmonella has become a global concern. METHODS: Using laboratory-based surveillance system for Salmonella from September 2009 to December 2012 in Guangdong Province of China. The clinical information and samples of diarrhea patients were collected, according to the surveillance case definition. The lab tests were followed by standardized protocols, including sample isolation, isolates confirmation, serotyping, and antimicrobial susceptibility testing (AST). RESULTS: A total of 1,826 Salmonella isolates were identified from40,572 patients in 28 hospitals in11 prefectures. The isolates ratio was highest in autumn (38.8%, 708/1826) and lowest in winter (6.4%, 117/1826). Children aged <5 years were the group most affected by Salmonella in Guangdong Province accounting for 73% (1,329/1,826), of whom the infants (<1 year) were 81.5% (1084/1329) especially. A total of 108 serotypes were identified among the isolates. S. Typhimurium represented the most common serotype followed by serotype 4,5,12:i:-. S. Typhimurium was also the common serotype followed by S. Enteritidis among infants and children aged 1-3 years old. However, S. Enteritidis became the common serotype followed by S. Typhimurium among children aged 3-5 and >5 years. Resistance to at least one antimicrobial was found in 72% (1321/1,826) of the isolates. Resistance to at least three antimicrobials was found in 46% (850/1,826) of the isolates. Resistance to all 12 antimicrobials screened was observed in 8 isolates (0.44%, 8/1,826). The resistant prevalence to quinolones including nalidixic acid and ciprofloxacin was 61.9% (1131/1826), of which ciprofloxacin resistance rate was 8.05% (147/1826). The prevalence resistance to all three cephalosporin antimicrobials (cefepime, cefotaxime, and caftazidime) in <5 yr age group was accounted for 90% (89/99). CONCLUSIONS: Additional data and more refined methods can improve future surveillance. The invasive Salmonella isolates should also be included to the antibiotic resistance surveillance for clinical care or public health.

Tissue-specific splicing of an Ndufs6 gene-trap insertion generates a mitochondrial complex I deficiency-specific cardiomyopathy.

Mitochondrial complex I (CI) deficiency is the most common mitochondrial enzyme defect in humans. Treatment of mitochondrial disorders is currently inadequate, emphasizing the need for experimental models. In humans, mutations in the NDUFS6 gene, encoding a CI subunit, cause severe CI deficiency and neonatal death. In this study, we generated a CI-deficient mouse model by knockdown of the Ndufs6 gene using a gene-trap embryonic stem cell line. Ndufs6(gt/gt) mice have essentially complete knockout of the Ndufs6 subunit in heart, resulting in marked CI deficiency. Small amounts of wild-type Ndufs6 mRNA are present in other tissues, apparently due to tissue-specific mRNA splicing, resulting in milder CI defects. Ndufs6(gt/gt) mice are born healthy, attain normal weight and maturity, and are fertile. However, after 4 mo in males and 8 mo in females, Ndufs6(gt/gt) mice are at increased risk of cardiac failure and death. Before overt heart failure, Ndufs6(gt/gt) hearts show decreased ATP synthesis, accumulation of hydroxyacylcarnitine, but not reactive oxygen species (ROS). Ndufs6(gt/gt) mice develop biventricular enlargement by 1 mo, most pronounced in males, with scattered fibrosis and abnormal mitochondrial but normal myofibrillar ultrastructure. Ndufs6(gt/gt) isolated working heart preparations show markedly reduced left ventricular systolic function, cardiac output, and functional work capacity. This reduced energetic and functional capacity is consistent with a known susceptibility of individuals with mitochondrial cardiomyopathy to metabolic crises precipitated by stresses. This model of CI deficiency will facilitate studies of pathogenesis, modifier genes, and testing of therapeutic approaches.

Serovar distribution, antimicrobial resistance profiles, and PFGE typing of Salmonella enterica strains isolated from 2007-2012 in Guangdong, China.

BACKGROUND: Salmonella enterica includes the major serovars associated with human salmonellosis. In this study, 1764 clinical Salmonella enterica isolates from diarrhea outpatients were collected from fifteen cities in Guangdong province, China, between 2007 and 2012. These isolates represent all of the Salmonella isolates collected from the province during that period. METHODS: The isolates were characterized by serovar determination, antimicrobial susceptibility tests and PFGE fingerprint typing. RESULTS: The serovar distribution results demonstrated that Salmonella Typhimurium (n=523, 29.65%) and Salmonella 4,5,12:i:- (n=244, 13.83%) are the most common serovars causing infant salmonellosis, whereas Salmonella Enteritidis (n=257, 14.57%) mainly causes human salmonellosis in adults. The serovar shift from Salmonella Enteritidis to Salmonella Typhimurium occurred in 2008. Antimicrobial susceptibility data showed a high burden of multidrug resistance (MDR) (n=1128, 56.58%), and a 20%-30% increase in the number of isolates resistant to ciprofloxacin (n=142, 8.05%) and third-generation cephalosporins (n=88, 4.99%) from 2007-2012. Only 9.97% of isolates (n=176) were fully susceptible to all agents tested. A high burden of MDR was observed in Salmonella Typhimurium and Salmonella 4,5,12:i:- for all age groups, and a reduced susceptibility to third-generation cephalosporins and quinolones occurred particularly in infants (≤ 6 years). The dominant PFGE patterns were JPXX01.GD0004, JEGX01.GD0006-7 and JNGX01.GD0006-7. ACSSuT was the predominant MDR profile in the Salmonella Typhimurium & 4,5,12:i:- complexes, while ASSuT-Nal and ASSu-Nal were the major MDR profiles in Salmonella Enteritidis. The predominant PFGE patterns of the Salmonella Typhimurium & 4,5,12:i:- complexes and Salmonella Stanley were most prevalent in infants (≤ 6 years). However, no obvious relationship was observed between these PFGE profiles and geographic location. CONCLUSIONS: These data reveal the serovar distribution of isolates recovered from diarrhea patients, the characteristics of resistant strains and fingerprint typing in Guangdong from 2007 to 2012. These results highlight a serovar shift and a worrying percentage of MDR strains with increasing resistance to quinolones and third-generation cephalosporins. Thus, continued surveillance of Salmonella and their MDR profiles using combined molecular tools and efforts to control the rapid increase in antimicrobial resistance among Salmonella in Guangdong are needed.

Epidemiology and etiology characteristics of foodborne outbreaks caused by Vibrio parahaemolyticus during 2008-2010 in Guangdong province, China.

Vibrio parahaemolyticus infection has been considered the leading cause of bacterial illnesses mainly associated with seafood consumption in Guangdong province in China. In this study, epidemiological and etiological characteristics of 36 V. parahaemolyticus outbreaks that occurred from 2008 to 2010 in Guangdong province were analyzed; 284 strains involved were characterized by serotyping; virulence genes and 66 strains from four outbreaks therein were subtyped by pulsed-field gel electrophoresis (PFGE). Epidemiological evidence showed that 36% (13/36) of outbreaks were caused by food contamination, of which 84.6 % (11/13) were related to salted food, including viscera of pigs and cattle, meat, and vegetable salad. It was also indicated that 88.9% (32/36) of V. parahaemolyticus outbreaks appeared from June to September, 44.4% (16/36) of which occurred in canteens as well as 41.7% (15/36) in restaurants. As for the etiology, 31% (11/36) of outbreaks were caused by single serovar of strains, while 69% (25/36) were caused by multiserovars; O3:K6, O4:K8, O1:Kut, and O2:K3 were the dominant serovars. Among the 284 strains, 98.8% (254/257) of strains from patients were tdh-present and trh-absent, whereas 37.0% (10/27) from food were tdh-present. Cluster analysis of PFGE patterns demonstrated that strains in the same outbreak with identical serovar seemed to be diversified, whereas strains with various serovars could be closely related genetically. Moreover, cross-contamination between salted food and seafood was first confirmed by molecular subtyping in Guangdong, revealing that salted food might be a vital risk factor associated with V. parahaemolyticus outbreaks.

Antimicrobial resistance and population genomics of emerging multidrug-resistant Salmonella 4,[5],12:i:- in Guangdong, China.

Salmonella 4,[5],12:i:-, a monophasic variant of Salmonella Typhimurium, has emerged as a global cause of multidrug-resistant salmonellosis and has become endemic in many developing and developed countries, especially in China. Here, we have sequenced 352 clinical isolates in Guangdong, China, during 2009-2019 and performed a large-scale collection of Salmonella 4,[5],12:i:- with whole genome sequencing (WGS) data across the globe, to better understand the population structure, antimicrobial resistance (AMR) genomic characterization, and transmission routes of Salmonella 4,[5],12:i:- across Guangdong. Salmonella 4,[5],12:i:- strains showed broad genetic diversity; Guangdong isolates were found to be widely distributed among the global lineages. Of note, we identified the formation of a novel Guangdong clade (Bayesian analysis of population structure lineage 1 [BAPS1]) genetically diversified from the global isolates and likely emerged around 1990s. BAPS1 exhibits unique genomic features, including large pan-genome, decreased ciprofloxacin susceptibility due to mutation in gyrA and carriage of plasmid-mediated quinolone resistance (PMQR) genes, and the multidrug-resistant IncHI2 plasmid. Furthermore, high genetic similarity was found between strains collected from Guangdong, Europe, and North America, indicating the association with multiple introductions from overseas. These results suggested that global dissemination and local clonal expansion simultaneously occurred in Guangdong, China, and horizontally acquired resistance to first-line and last-line antimicrobials at local level, underlying emergences of extensive drug and pan-drug resistance. Our findings have increased the knowledge of global and local epidemics of Salmonella 4,[5],12:i:- in Guangdong, China, and provided a comprehensive baseline data set essential for future molecular surveillance.IMPORTANCESalmonella 4,[5],12:i:- has been regarded as the predominant pandemic serotype causing diarrheal diseases globally, while multidrug resistance (MDR) constitutes great public health concerns. This study provided a detailed and comprehensive genome-scale analysis of this important Salmonella serovar in the past decade in Guangdong, China. Our results revealed the complexity of two distinct transmission modes, namely global transmission and local expansion, circulating in Guangdong over a decade. Using phylogeography models, the origin of Salmonella 4,[5],12:i:- was predicted from two aspects, year and country, that is, Salmonella 4,[5],12:i:- emerged in 1983, and was introduced from the UK, and subsequently differentiated into the local endemic lineage circa 1991. Additionally, based on the pan-genome analysis, it was found that the gene accumulation rate in local endemic BAPS 1 lineage was higher than in other lineages, and the horizontal transmission of MDR IncHI2 plasmid associated with high resistance played a major role, which showed the potential threat to public health.

Design, synthesis and immunological evaluation of monophosphoryl lipid A derivatives as adjuvants for a RBD-hFc based SARS-CoV-2 vaccine.

Toll-like receptor 4 (TLR4) is a reliable target for the development of vaccine adjuvants. To identify novel TLR4 ligands with improved immunological properties for use as adjuvants for a RBD-hFc based SARS-CoV-2 vaccine, herein, natural E. coli monophosphoryl lipid A (MPLA) and nine of its derivatives were designed and synthesized. Immunological evaluation showed that compounds 1, 3, 5 and 7 exhibited comparative or better adjuvant activity than clinically used Al adjuvants, and are expected to be a promising platform for the development of new adjuvants used for a RBD-hFc based SARS-CoV-2 vaccine. Preliminary structure-activity relationship analysis of the MPLA derivatives showed that the replacement of the functional groups at the C-1, C-4' or C-6' position of E. coli MPLA has an effect on its biological activity. In addition, we found that the combination of MPLA and Al was feasible for immunotherapy and could further enhance immune responses, providing a new direction toward the immunological enhancement of RBD-hFc based SARS-CoV-2 vaccines.

Metagenomic profiles of planktonic bacteria and resistome along a salinity gradient in the Pearl River Estuary, South China.

Estuarine ecosystems undergo pronounced and intricate changes due to the mixing of freshwater and saltwater. Additionally, urbanization and population growth in estuarine regions result in shifts in the planktonic bacterial community and the accumulation of antibiotic resistance genes (ARGs). The dynamic changes in bacterial communities, environmental factors, and carriage of ARGs from freshwater to seawater, as well as the complex interrelationships among these factors, have yet to be fully elucidated. Here, we conducted a comprehensive study based on metagenomic sequencing and full-length 16S rRNA sequencing, covering the entire Pearl River Estuary (PRE) in Guangdong, China. The abundance and distribution of the bacterial community, ARGs, mobile genetic elements (MGEs), and bacterial virulence factors (VFs) were analyzed on a site-by-site basis through sampling along the salinity gradient in PRE, from upstream to downstream. The structure of the planktonic bacterial community undergoes continuous changes in response to variations in estuarine salinity, with the phyla Proteobacteria and Cyanobacteria being dominant bacterial throughout the entire region. The diversity and abundance of ARGs and MGEs gradually decreased with the direction of water flow. A large number of ARGs were carried by potentially pathogenic bacteria, especially in Alpha-proteobacteria and Beta-proteobacteria. Multi-drug resistance genes have the highest abundance and subtypes in PRE. In addition, ARGs are more linked to some MGEs than to specific bacterial taxa and disseminate mainly by HGT and not by vertical transfer in the bacterial communities. Various environmental factors, such as salinity and nutrient concentrations, have a significantly impact on the community structure and distribution of bacteria. In conclusion, our results represent a valuable resource for further investigating the intricate interplay between environmental factors and anthropogenic disturbances on bacterial community dynamics. Moreover, they contribute to a better understanding of the relative impact of these factors on the dissemination of ARGs.

Carriage and Transmission of mcr-1 in Salmonella Typhimurium and Its Monophasic 1,4,[5],12:i:- Variants from Diarrheal Outpatients: a 10-Year Genomic Epidemiology in Guangdong, Southern China.

The banning of colistin as a feed additive for food-producing animals in mainland China in 2017 caused the decline in the prevalence of Escherichia coli-mobilized colistin resistance (mcr-1) in China. Salmonella Typhimurium and its monophasic 1,4,[5],12:i:- variants are also the main species associated with the spread of mcr-1; however, the evidence of the prevalence and transmission of mcr-1 among Salmonella is lacking. Herein, the 5,354 Salmonella isolates recovered from fecal samples of diarrheal patients in Guangdong, Southern China, from 2009 to 2019 were screened for colistin resistance and mcr-1, and mcr-1-positive isolates were characterized based on whole-genome sequencing (WGS) data. Relatively high prevalence rates of colistin resistance and mcr-1 (4.05%/4.50%) were identified, and more importantly, the prevalence trends of colistin-resistant and mcr-1-positive Salmonella isolates had a similar dynamic profile, i.e., both were first detected in 2012 and rapidly increased during 2013 to 2016, followed by a sharp decrease since 2017. WGS and phylogenetic analysis indicate that, whether before or after the ban, the persistence and cross-hospital transmission of mcr-1 are primarily determined by IncHI2 plasmids with similar backbones and sequence type 34 (ST34) Salmonella in specific clades that are associated with a high prevalence of IncHI2 plasmids and clinically important antimicrobial resistance genes, including blaCTX-M-14-fosA3-oqxAB-floR genotypes. Our work reveals the difference in the prevalence rate of mcr-1 in clinical Salmonella before and after the Chinese colistin ban, whereas mcr-1 transmission was closely linked to multidrug-resistant IncHI2 plasmid and ST34 Salmonella across diverse hospitals over 10 years. Continued surveillance is required to explore the factors related to a sharp decrease in mcr-1 after the recent ban and determine whether the ban has affected the carriage of mcr-1 in Salmonella circulating in the health care system. IMPORTANCE Colistin is one of the last-line antibiotics for the clinical treatment of Enterobacteriaceae. However, the emergence of the mobilized colistin resistance (mcr-1) gene has spread throughout the entire human health system and largely threatens the usage of colistin in the clinical setting. In this study, we investigated the existence of mcr-1 in clinical Salmonella from a 10-year continuous surveillance and genomic study. Overall, the colistin resistance rate and mcr-1 carriage of Salmonella in tertiary hospitals in Guangdong (2009 to 2019) were relatively high and, importantly, rapidly increased from 2013 to 2016 and significantly decreased after the Chinese colistin withdrawal. However, before or after the ban, the MDR IncHI2 plasmid with a similar backbone and ST34 Salmonella were the main vectors involved in the spread of mcr-1. Interestingly, these Chinese mcr-1-carrying Salmonella obtain phylogenetically and phylogeographically distinct patterns compared with those from other continents and are frequently associated with clinically important ARGs including the extended-spectrum ß-lactamases. Our data confirmed that the national stewardship intervention seems to be successful in blocking antibiotic resistance determinants and that continued surveillance of colistin resistance in clinical settings, farm animals, and related products is necessary.

Prevalence of Non-Polio Enteroviruses in the Sewage of Guangzhou City, China, from 2013 to 2021.

Continuous surveillance of enteroviruses (EVs) in urban domestic sewage can timely reflect the circulation of EVs in the environment and crowds, and play a predictive and early warning role in EV-related diseases. To better understand the long-term epidemiological trends of circulating EVs and EV-related diseases, we conducted a 9-year (2013 to 2021) surveillance study of non-polio EVs (NPEVs) in urban sewage in Guangzhou city, China. After concentrating and isolating the viruses from the sewage samples, NPEVs were detected and molecular typing was performed. Twenty-one different NPEV serotypes were identified. The most isolated EVs were echovirus 11 (E11), followed by coxsackievirus (CV) B5, E6, and CVB3. EV species B prevailed in sewage samples, but variations in the annual frequency of different serotypes were also observed in different seasons, due to spatial and temporal factors. E11 and E6 were detected continuously before 2017, and the number of isolates was relatively stable during the surveillance period. However, after their explosive growth in 2018 and 2019, their numbers suddenly decreased significantly. CVB3 and CVB5 had alternating trends; CVB5 was most frequently detected in 2013 to 2014 and 2017 to 2018, while CVB3 was most frequently detected in 2015 to 2016 and 2020 to 2021. Phylogenetic analysis showed that at least two different transmission chains of CVB3 and CVB5 were prevalent in Guangzhou City. Our results show that in the absence of a comprehensive and systematic EV-related disease surveillance system in China, environmental surveillance is a powerful and effective tool to strengthen and further investigate the invisible transmission of EVs in the population. IMPORTANCE This study surveilled urban sewage samples from north China for 9 years to monitor enteroviruses. Samples were collected, processed, and viral identification and molecular typing were performed. We detected 21 different non-polio enteroviruses (NPEVs) with yearly variations in prevalence and peak seasons. In addition, this study is very important for understanding the epidemiology of EVs during the COVID-19 pandemic, as the detection frequency and serotypes of EVs in sewage changed considerably around 2020. We believe that our study makes a significant contribution to the literature because our results strongly suggest that environmental surveillance is an exceptionally important tool, which can be employed to detect and monitor organisms of public health concern, which would otherwise be missed and under-reported by case-based surveillance systems alone.

Somatically hypermutated antibodies isolated from SARS-CoV-2 Delta infected patients cross-neutralize heterologous variants.

SARS-CoV-2 Omicron variants feature highly mutated spike proteins with extraordinary abilities in evading antibodies isolated earlier in the pandemic. Investigation of memory B cells from patients primarily with breakthrough infections with the Delta variant enables isolation of a number of neutralizing antibodies cross-reactive to heterologous variants of concern (VOCs) including Omicron variants (BA.1-BA.4). Structural studies identify altered complementarity determining region (CDR) amino acids and highly unusual heavy chain CDR2 insertions respectively in two representative cross-neutralizing antibodies-YB9-258 and YB13-292. These features are putatively introduced by somatic hypermutation and they are heavily involved in epitope recognition to broaden neutralization breadth. Previously, insertions/deletions were rarely reported for antiviral antibodies except for those induced by HIV-1 chronic infections. These data provide molecular mechanisms for cross-neutralization of heterologous SARS-CoV-2 variants by antibodies isolated from Delta variant infected patients with implications for future vaccination strategy.

Survey of physician diagnostic and treatment practices for patients with acute diarrhea in Guangdong province, China.

Although international clinical guidelines generally recommend performing bacterial stool culture in patients with acute diarrhea and fever and discourage routine antibiotic prescribing, clinical practice varies. Understanding practice patterns can help health officials assess the sensitivity of laboratory-based enteric infection surveillance systems and the need to improve antibiotic prescribing practices. We surveyed physicians in Guangdong province, China, to measure their practices for patients with acute diarrhea. A standardized questionnaire was used to interview physicians working in hospitals participating in a Salmonella surveillance system in Guangdong, China. The questionnaire asked physicians about their routine practice for patients with diarrhea, including how they managed the last patient they had seen with acute diarrhea. We calculated the odds ratio and 95% confidence interval for factors associated with ordering a stool culture and for prescribing antibiotics. We received surveys from 237 physicians across 22 hospitals in Guangdong. For the last patient with diarrhea whom they had evaluated, 134 (57%) reported ordering a stool culture. The most common reasons for not ordering a stool culture included that it takes too long to receive the result, that the patient is not willing to pay for the test, and that the patient's illness was too mild to warrant testing. Most physicians prescribed at least one medication for the last patient with diarrhea whom they had evaluated. Of the 237 physicians surveyed, 153 (65%) prescribed antibiotics, 135 (57%) probiotics, and 115 (49%), a gastric mucosal protective drug. In conclusion, physicians in Guangdong, China, reported high rates of ordering bacterial stool cultures from patients with diarrhea, possibly associated with their hospital's participation in a special surveillance project. The high rate of antibiotic prescribing suggests that efforts to promote judicious antibiotic use, such as physician education, are needed.

Laboratory-based surveillance of non-typhoidal Salmonella infections in Guangdong Province, China.

Salmonella is one of the most common foodborne pathogens in humans. Laboratory-based surveillance for non-typhoidal Salmonella infection was conducted in Guangdong Province, China to improve understanding about the disease burden and detection of dispersed outbreaks. Salmonella isolated from patients with diarrhea were sent from 16 sentinel hospitals to local public health laboratories for confirmation, serotyping, antimicrobial susceptibility testing, and pulsed-field gel electrophoresis (PFGE). PFGE patterns were analyzed to identify clusters representing potential outbreaks. Between September 2009 and October 2010, 352 (4%) Salmonella isolates were obtained from 9167 stool specimens. Salmonella enterica serotype Typhimurium (45%) and Salmonella enterica serotype Enteritidis (13%) were the most common serotypes, and multidrug resistance was high, especially in Salmonella Typhimurium isolates. PFGE patterns of obtained Salmonella isolates were found to be diverse, but a unique PFGE pattern comprising 53 Salmonella Typhimurium isolates were found to occur almost exclusively in infants. Epidemiologic studies are ongoing to determine whether a common exposure is the source of the Salmonella Typhimurium strain frequently isolated from infants.