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.

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.

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.

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.

Epidemiological Surveillance: Genetic Diversity of Rotavirus Group A in the Pearl River Delta, Guangdong, China in 2019.

Objective: This study aimed to understand the epidemic status and phylogenetic relationships of rotavirus group A (RVA) in the Pearl River Delta region of Guangdong Province, China. Methods: This study included individuals aged 28 days-85 years. A total of 706 stool samples from patients with acute gastroenteritis collected between January 2019 and January 2020 were analyzed for 17 causative pathogens, including RVA, using a Gastrointestinal Pathogen Panel, followed by genotyping, virus isolation, and complete sequencing to assess the genetic diversity of RVA. Results: The overall RVA infection rate was 14.59% (103/706), with an irregular epidemiological pattern. The proportion of co-infection with RVA and other pathogens was 39.81% (41/103). Acute gastroenteritis is highly prevalent in young children aged 0-1 year, and RVA is the key pathogen circulating in patients 6-10 months of age with diarrhea. G9P[8] (58.25%, 60/103) was found to be the predominant genotype in the RVA strains, and the 41 RVA-positive strains that were successfully sequenced belonged to three different RVA genotypes in the phylogenetic analysis. Recombination analysis showed that gene reassortment events, selection pressure, codon usage bias, gene polymorphism, and post-translational modifications (PTMs) occurred in the G9P[8] and G3P[8] strains. Conclusion: This study provides molecular evidence of RVA prevalence in the Pearl River Delta region of China, further enriching the existing information on its genetics and evolutionary characteristics and suggesting the emergence of genetic diversity. Strengthening the surveillance of genotypic changes and gene reassortment in RVA strains is essential for further research and a better understanding of strain variations for further vaccine development.

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.

[Surveillance and analysis on the pathogenic features of Salmonella in Guangdong province in 2010].

OBJECTIVE: In order to better understand the nature of Salmonella infection in diarrheal patients in Guangdong province, the study analyzed the serum types, antibiotic resistance and molecular determinants of the isolated Salmonella strains. METHODS: In year 2010, 8405 diarrhea patients from 16 surveillant hospital in Guangzhou, Zhongshan, Dongguan, Zhuhai, Maoming, Yangjiang and Jiangmen cities in Guangdong province, were recruited in the study. A total of 8405 fecal specimen were collected and subjected to Salmonella isolation and culture. The isolated Salmonella strains were further analyzed via serotyping, antimicrobial susceptibility testing, and PFGE. The χ(2) test was applied to compare the differences between the isolated Salmonella strains in different seasons and districts. BioNumerics software was used to analyze the PFGE results in order to determine the correlation between different Salmonella strains. RESULTS: The positive rate of the surveillant Salmonella in Guangdong province was 3.58% (301/8405) in 2010; with the gender ratio at 1.34:1 (166/124). Salmonella infection was found in all age groups, and most in infants, accounting for 57.48% (173/301). The isolated rates of Salmonella were separately 3.48% (61/1751), 4.97% (134/2695), 3.08% (73/2370) and 2.08% (33/1589) in the four seasons; and the difference was statistically significant (χ(2) = 27.29, P < 0.01). The isolated rates of Salmonella in different regions were as follows: Zhuhai 15.43% (25/162), Maoming 7.53% (18/239), Dongguan 6.51% (39/599), Yangjiang 3.64% (14/385), Zhongshan 3.03% (70/2309), Guangzhou 2.90% (126/4349) and Jiangmen 2.49% (9/362). The difference between regions was statistically significant (χ(2) = 100.75, P < 0.01). Except one strain of the isolated Salmonella cannot be serotyped, the other 300 strains were divided into 42 serotypes, of which Salmonella typhimurium and Salmonella enteritidis were dominant, account for 45.18% (136/301) and 10.96% (33/301) respectively. Although over 85% of Salmonella were sensitive to cephalosporin, ACSSuT resistance patterns (defined as resistance to at least ampicillin, chloramphenicol, streptomycin, sulfamethoxazole and tetracycline) reached 34.88% (105/301), the highest resistant rate was found in serotype Salmonella typhimurium, as high as 65.44% (89/136). 136 strains of Salmonella typhimurium were divided into 51 PFGE types, showed great genetic diversity. 33 strains of Salmonella enteritidis were divided into 18 PFGE types. The strains with same PFGE pattern may have different drug-resistant patterns, and vice versa. CONCLUSION: Salmonella typhimurium and Salmonella enteritidis were the dominant serotypes causing infectious diarrhea in Guangdong province. Cephalosporin was the primary choice in clinical medicine. However, Salmonella typhimurium was resistant to drug most seriously in Guangdong province. There was no significant correlation between Salmonella resistance patterns and PFGE type.

Epidemiology of bla CTX-M-Positive Salmonella Typhimurium From Diarrhoeal Outpatients in Guangdong, China, 2010-2017.

Salmonella enterica can lead to intestinal diarrhea, and the emergence and spread of cephalosporin-resistant Salmonella have brought great challenges to clinical treatment. Therefore, this study investigated the prevalence and transmission of bla CTX-M genes among S. Typhimurium from diarrhoeal outpatients in Guangdong, China, from 2010 to 2017. A total of 221 bla CTX-M-positive isolates were recovered from 1,263 S. Typhimurium isolates from the facal samples of diarrhoea patients in 45 general hospitals from 11 cities. The most popular CTX-M gene was bla CTX-M-55 (39.6%, 72/182) in the CTX-M-1 group, followed by bla CTX-M-14 (22.5%, 41/182) and bla CTX-M-65 (19.2%, 35/182) in the CTX-M-9 group. The isolates that carried bla CTX-M-9G had significantly higher resistance rates to multiple antibacterials compared with bla CTX-M-1G (p < 0.01). Meanwhile, PFGE analysis not only showed the clonal transmission of bla CTX-M-55/14/65-positve isolates of diarrhoeal outpatients' origins from different hospitals in Guangdong province, but also the characteristic of bla CTX-M-55/14/65-positve isolates' bacterial persistence. Multilocus sequence typing (MLST) analysis indicated that these S. Typhimurium isolates possessed ST34 and ST19. Furthermore, genomic Beast phylogenomic analysis provided the evidence of a close relationship of bla CTX-M-positive S. Typhimurium isolates between the outpatients and pork. Most bla CTX-M-55/14/65 genes were transmitted by non-typeable or IncI1/IncFII/IncHI2 plasmids with the size of ranging from ~80 to ~280 kb. Moreover, whole-genome sequencing (WGS) analysis further revealed that bla CTX-M-55/14/65 coexisted with other 25 types of ARGs, of which 11 ARGs were highly prevalent with the detection rates >50%, and it first reported the emergence of bla TEM-141 in S. Typhimurium. This study underscores the importance of surveillance for bla CTX-M-positive microbes in diarrhea patients.

Role of glutaredoxin1 and glutathione in regulating the activity of the copper-transporting P-type ATPases, ATP7A and ATP7B.

The copper-transporting P-type ATPases (Cu-ATPases), ATP7A and ATP7B, are essential for the regulation of intracellular copper homeostasis. In this report we describe new roles for glutathione (GSH) and glutaredoxin1 (GRX1) in Cu homeostasis through their regulation of Cu-ATPase activity. GRX1 is a thiol oxidoreductase that catalyzes the reversible reduction of GSH-mixed disulfides to their respective sulfhydryls (deglutathionylation). Here, we demonstrated that glutathionylation of the Cu-ATPases and their interaction with GRX1 were affected by alterations in Cu levels. The data support our hypothesis that the Cu-ATPases serve as substrates for Cu-dependent GRX1-mediated deglutathionylation. This in turn liberates the Cu-ATPase cysteinyl thiol groups for Cu binding and transport. GSH depletion experiments led to reversible inhibition of the Cu-ATPases that correlated with effects on intracellular Cu levels and GRX1 activity. Finally, knockdown of GRX1 expression resulted in an increase in intracellular Cu accumulation. Together, these data directly implicate GSH and GRX1 with important new roles in redox regulation of the Cu-ATPases, through modulation of Cu binding by the Cu-ATPase cysteine motifs.

Phenotypic and genotypic characterization Vibrio cholerae O139 of clinical and aquatic isolates in China.

To enhance the understanding of epidemiological impact of environmental Vibrio cholerae O139 strains, we characterized 10 clinical and 20 environmental isolates collected from human clinical samples and Pear River estuary during 2006 to 2008. Isolates were tested by PCR for eight virulence genes: cholera toxin (ctxA), zonula occludens toxin (zot), accessory cholera enterotoxin (ace), hemolysin (hlyA), NAG-specific heat-stable toxin (st), toxin-coregulated pilus (tcpA), outer membrane protein (ompU), and regulatory protein genes (tcpI). Genetic relatedness was assessed by pulsed-field gel electrophoresis (PFGE), and antibiotic susceptibility was determined using disk diffusion. Seven of eight virulence markers were detected in six clinical isolates and one environmental isolate. One clinical and one environmental isolate were positive for six virulence markers. 60% clinical isolates showed multi-drug resistance to tetracycline (TET), Nalidixic acid (NAL), chloramphenicol (CHL), and ampicillin (AMP), 70% were resistant to Trimethoprim + Sulfamethoxazole (SXT), while only 35% environmental strains were resistant to SXT. PFGE analysis revealed that the isolates in this study were formed three clusters. Cluster III was more related to strains from diarrheal patients than the strains in other clusters. Different from the clinical strains, most environmental strains lacked CTX and TCP gene clusters. Most environmental strains possess a single resistance profile, while most clinical isolates show multidrug resistant. PFGE analysis indicated the cluster III has more possibility to become a potential pathogenic clonal cluster.

[Etiologic detection and epidemiological analysis of one suspected case of diphtheria in Guangdong province].

OBJECTIVE: To clarify the diagnosis of one suspected case of diphtheria in Guangdong province by epidemiological analysis and etiologic detection. METHODS: On July 6th 2010, the corynebacterium diphtheria was detected from the nasal secretions of one nasopharyngeal carcinoma patient in a college-town hospital in Guangzhou City, Guangdong Province. The patient and the close contacts were asked to participate in the epidemiological survey; and their nasopharyngeal swabs (3 samples) and the nasal secretions of the patient (1 sample) were collected. The bacteria of the samples were isolated and cultured by blood plate and agar loefflera. The smears of positive strains were dyed and identified by BioMerieux API Coryne biochemical card. Gene tox of ß-Corynebacteriophage, Corynebacterium diphtheriae was tested by PCR method, the aliphatic acid was analyzed by gas chromatography method and the Corynebacterium diphtheriae (CMCC 38009) was selected as positive control. RESULTS: The patient had not gone out, neither had been visited. The patient denied history of vaccines or the immunizations. From the survey on patient's family members and close contacts, no similar symptoms had been found. One strain of Corynebacterium diphtheriae was isolated from the patient's nasal secretions, Gram positive and shape diversified. After cultured by agar loefflera and Gram-dyed and Neisser-dyed, one end or both two ends of the strain showed typical metachromatic granule. API Coryne was identified to Corynebacterium diphtheriae mitis/belfanti (99.4%). The result of gas chromatography method also indicated Corynebacterium diphtheriae. No Corynebacterium diphtheriae was isolated from the nasopharyngeal swabs, neither of the patient nor of the close contacts. The gene tox of ß-Corynebacteriophage, Corynebacterium diphtheriae was negative according to the PCR test. CONCLUSION: The isolated Corynebacterium diphtheriae did not produce toxin as there was no biological structure gene of toxin. The patient was a health carrier of nontoxic Corynebacterium diphtheriae.

Kinetic Characteristics of Neutralizing Antibody Responses Vary among Patients with COVID-19.

OBJECTIVE: The coronavirus disease 2019 (COVID-19) pandemic continues to present a major challenge to public health. Vaccine development requires an understanding of the kinetics of neutralizing antibody (NAb) responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). METHODS: In total, 605 serum samples from 125 COVID-19 patients (from January 1 to March 14, 2020) varying in age, sex, severity of symptoms, and presence of underlying diseases were collected, and antibody titers were measured using a micro-neutralization assay with wild-type SARS-CoV-2. RESULTS: NAbs were detectable approximately 10 days post-onset (dpo) of symptoms and peaked at approximately 20 dpo. The NAb levels were slightly higher in young males and severe cases, while no significant difference was observed for the other classifications. In follow-up cases, the NAb titer had increased or stabilized in 18 cases, whereas it had decreased in 26 cases, and in one case NAbs were undetectable at the end of our observation. Although a decreasing trend in NAb titer was observed in many cases, the NAb level was generally still protective. CONCLUSION: We demonstrated that NAb levels vary among all categories of COVID-19 patients. Long-term studies are needed to determine the longevity and protective efficiency of NAbs induced by SARS-CoV-2.

The immunodominant and neutralization linear epitopes for SARS-CoV-2.

Although vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are under development, the antigen epitopes on the virus and their immunogenicity are poorly understood. Here, we simulate the 3D structures and predict the B cell epitopes on the spike (S), envelope (E), membrane (M), and nucleocapsid (N) proteins of SARS-CoV-2 using structure-based approaches and validate epitope immunogenicity by immunizing mice. Almost all 33 predicted epitopes effectively induce antibody production, six of these are immunodominant epitopes in individuals, and 23 are conserved within SARS-CoV-2, SARS-CoV, and bat coronavirus RaTG13. We find that the immunodominant epitopes of individuals with domestic (China) SARS-CoV-2 are different from those of individuals with imported (Europe) SARS-CoV-2, which may be caused by mutations on the S (G614D) and N proteins. Importantly, we find several epitopes on the S protein that elicit neutralizing antibodies against D614 and G614 SARS-CoV-2, which can contribute to vaccine design against coronaviruses.

[Molecular characters of epidemic cerebrospinal W135 Neisseria meningitidis strain firstly isolated in Guangdong province].

OBJECTIVE: To analyze the molecular characters of the W135 Neisseria meningitidis strain firstly isolated from patients in Guangdong province. METHODS: Biochemical profile by using the API NH system (bio-Merieux, France) was used for confirmation,and sero-grouping of the meningococcal isolates including one serogroup W135, one serogroup C and three serogroups of a Neisseria meningitidis isolated from patients in Guangdong province in recent two years were performed. The subtype was determined after amplifying porA and porB respectively from the genome DNA of Neisseria meningitidis. Multilocus sequence typing (MLST) was performed for determining the allele profiles and the sequence types (STs). The polygenetic tree was obtained by analyzing the allele profiles with program Splits tree online. The molecular characters of the serogroup W135 Neisseria meningitidis was analyzed by its evolution relationship and the variable regions in porA and porB which encoding the outer membranes proteins (OMPs). RESULTS: The subtype determined by porA variable regions of the serogroup W135 Neisseria meningitidis was P1.5,2, which was one of the most invasive types. The types of variable regions (VRs) I, IV, V, VII with porB were 1, 1, 1, 17, and there was no VI and VIII in porB. The allele profile of the W135 strain in this study was 2, 123, 4, 3, 8, 4, 6, and its sequence type was ST-2960, which belonged to ST-11/ET-37 clone complex. The subtypes of the serogroup C and serogroup A strains were P1.20, while their types of VR IV were all 7, and they all hadn't VR VII in porB. The strain serogroup C belonged to ST-4821 clone complex, and the 3 serogroup A strains belonged to ST-5 clone complex. CONCLUSION: The molecular character of the serogroup W135 Neisseria meningitidis should be the same with the strains isolated in foreign country, and be different from the epidemic types in the area. This serogroup W135 Neisseria meningitis isolated from patients in Guangdong for the first time was thought to be a new type appearing in the local area.

ATP7A transgenic and nontransgenic mice are resistant to high copper exposure.

The protein affected in Menkes disease, ATP7A, is a copper (Cu)-transporting P-type ATPase that plays an important role in Cu homeostasis, but the full extent of this role has not been defined at a systemic level. Transgenic mice that overexpress the human ATP7A from the chicken beta-actin composite promoter (CAG) were used to further investigate the physiological function of ATP7A. Overexpression of ATP7A in the mice caused disturbances in Cu homeostasis, with depletion of Cu in some tissues, especially the heart. To investigate the effect of overexpression of ATP7A when dietary Cu intake was markedly increased, normal and transgenic mice were exposed to drinking water containing 300 mg/L of Cu as Cu acetate for 3 mo. Cu exposure resulted in partial restoration of heart Cu concentrations in male transgenic mice. Despite the extended period of Cu exposure, Cu concentrations in the liver remained relatively unaffected, with a significant increase in male nontransgenic mice. Liver pathology was unremarkable except for small areas of fibrosis that were detected only in livers of the Cu-exposed transgenic mice. Intracellular localization of ATP7A in various tissues was not affected by Cu exposure. Plasma Cu concentration and ceruloplasmin oxidase activity were reduced in both Cu-exposed transgenic and nontransgenic mice. The expression levels of other candidate Cu homeostatic proteins, endogenous Atp7b, ceruloplasmin, Ctr1, and transgenic ATP7A were not altered significantly by Cu exposure. Overall, mice are remarkably resistant to high Cu loads and the overexpression of ATP7A has only moderate effects on the response to Cu exposure.

Correction of a mouse model of Menkes disease by the human Menkes gene.

The brindled mouse is an accurate model of the fatal human X-linked copper deficiency disorder, Menkes disease. Males carrying the mutant allele of the Menkes gene orthologue Atp7a die in the second week of life. To determine whether the genetic defect in the brindled mice could be corrected by expression of the human Menkes gene, male transgenic mice expressing ATP7A from the chicken beta-actin composite promoter (CAG) were mated with female carriers of the brindled mutation (Atp7a(Mo-br)). Mutant males carrying the transgene survived and were fertile but the copper defect was not completely corrected. Unexpectedly males corrected with one transgenic line (T25#5) were mottled and resembled carrier females, this effect appeared to be caused by mosaic expression of the transgene. In contrast, males corrected with another line (T22#2) had agouti coats. Copper concentrations in tissues of the rescued mutants also resembled those of the heterozygous females, with high levels in kidney (84.6+/-4.9 microg/g in corrected males vs. 137.0+/-44.3 microg/g in heterozygotes) and small intestine (15.6+/-2.5 microg/g in corrected males vs. 15.7+/-2.8 microg/g in heterozygotes). The results show that the Menkes defect in mice is corrected by the human Menkes gene and that adequate correction is obtained even when the transgene expression does not match that of the endogenous gene.

[Identifying group G beta-hemolytic Streptococci strains by 16S rDNA sequencing].

OBJECTIVE: To identify the DNA sequences of 26 beta-hemolytic group G streptococci strains isolated from the swabs of patients in a glomerulonephritis outbreak event. METHODS: API20 Strep system and 16S rDNA sequencing were performed on all of the 26 isolated strains respectively. The dendrogram based on the analysis of the 16S rDNA sequences of four groups of G streptococci strains was constructed by the neighbor-joining method and boot-strap test. RESULTS: The API20 Strep system failed to identify the strains with less than 62% of success rates. All of the 26 group G beta-hemolytic streptococci isolates were identified as Streptococcus anginosus by the 16S rDNA sequencing, with more than 97% of success rates. The dendrogram illustrated that the four groups of G streptococci strains were clustered at the same branch with Streptococcus anginosus. CONCLUSION: The 16S rDNA sequencing has unique advantages over the phenotypic methods. It can provide detailed information of nucleotides and identify species of beta-hemolytic G group streptococci.

Deficiency in mitochondrial complex I activity due to Ndufs6 gene trap insertion induces renal disease.

AIMS: Defects in the activity of enzyme complexes of the mitochondrial respiratory chain are thought to be responsible for several disorders, including renal impairment. Gene mutations that result in complex I deficiency are the most common oxidative phosphorylation disorders in humans. To determine whether an abnormality in mitochondrial complex I per se is associated with development of renal disease, mice with a knockdown of the complex I gene, Ndufs6 were studied. RESULTS: Ndufs6 mice had a partial renal cortical complex I deficiency; Ndufs6gt/gt, 32% activity and Ndufs6gt/+, 83% activity compared with wild-type mice. Both Ndufs6gt/+ and Ndufs6gt/gt mice exhibited hallmarks of renal disease, including albuminuria, urinary excretion of kidney injury molecule-1 (Kim-1), renal fibrosis, and changes in glomerular volume, with decreased capacity to generate mitochondrial ATP and superoxide from substrates oxidized via complex I. However, more advanced renal defects in Ndufs6gt/gt mice were observed in the context of a disruption in the inner mitochondrial electrochemical potential, 3-nitrotyrosine-modified mitochondrial proteins, increased urinary excretion of 15-isoprostane F2t, and up-regulation of antioxidant defence. Juvenile Ndufs6gt/gt mice also exhibited signs of early renal impairment with increased urinary Kim-1 excretion and elevated circulating cystatin C. INNOVATION: We have identified renal impairment in a mouse model of partial complex I deficiency, suggesting that even modest deficits in mitochondrial respiratory chain function may act as risk factors for chronic kidney disease. CONCLUSION: These studies identify for the first time that complex I deficiency as the result of interruption of Ndufs6 is an independent cause of renal impairment.