Antimicrobial Resistance Analysis and Whole-Genome Sequencing of Salmonella Isolates from Environmental Sewage - Guangzhou City, Guangdong Province, China, 2022-2023.

What is already known about this topic?: S.1,4,[5],12:i:- and S. Rissen are emerging serotypes of Salmonella that require close monitoring for antimicrobial resistance and containment of their spread. What is added by this report?: The study aimed to identify antimicrobial resistance genes (ARGs) in S.1,4,[5],12:i:- and S. Rissen strains isolated from environmental sewage in Guangzhou City, Guangdong Province, China. A phylogenetic tree was constructed using single nucleotide polymorphism data to assess genetic relatedness among strains, offering insights for Salmonella infection outbreak investigations in the future. What are the implications for public health practice?: It is crucial to implement strategies, such as integrating different networks, to control the spread of drug-resistant Salmonella. Novel technologies must be utilized to disinfect sewage and eliminate ARGs. Ensuring food safety and proper sewage disinfection are essential to curb the dissemination of Salmonella.

Seroprevalence of Diphtheria and Tetanus Immunoglobulin G among the General Health Population in Guangzhou, China.

A seroepidemiological study was conducted in 2018 to assess diphtheria and tetanus antibodies in Guangzhou, China. Diphtheria and tetanus antibody concentrations were measured with an enzyme-linked immunosorbent assay. A total of 715 subjects were enrolled in the study. The overall diphtheria and tetanus toxoid IgG-specific antibody levels were 0.126 IU/mL (95% CI: 0.115, 0.137) and 0.210 IU/mL (95% CI: 0.185, 0.240), respectively; the overall positivity rate was 61.82% (95% CI: 58.14, 65.39) and 71.61% (95% CI: 68.3, 74.92), respectively. The diphtheria and tetanus antibody concentration was decreased by age and increased by doses. The geometric mean concentrations and positivity rate of diphtheria and tetanus antibodies were lowest and below the essential protection level in people over 14 years of age. Compared to children and adolescents, middle-aged people and the aged are at much higher risk of infection with Corynebacterium diphtheriae and Clostridium tetani. The current diphtheria and tetanus immunization schedule does not provide persistent protection after childhood. There is an urgent need to adjust the current immunization schedule.

Role of myeloid-derived suppressor cells in chronic brucellosis.

Introduction: Human brucellosis, a Brucella infection caused most common zoonosis in the world, remains a serious public health burden in China. Brucella chronic infection always causes immunosuppressive status and results in severe organ or tissue damages. The aim of this work was to study the role of the myeloid-derived suppressor cells (MDSCs) in human chronic brucellosis. Methods: Fifty cases of chronic brucellosis and 40 healthy individual controls were enrolled in this study. We analyzed the frequency and subsets of MDSCs in PBMC between the chronic brucellosis and healthy control groups by flow cytometry. Furthermore, we also measured the inflammatory-related cytokines in serum samples and the MDSCs inhibition ability to the proliferation of T cells in vitro. Results: We found that the frequency of MDSCs in peripheral blood and the level of IL-6 and IL-10 Th2 cytokines and Arginase-1 were significantly increased in chronic brucellosis patients. In addition, we also found that the T cell function was suppressed in vitro by co-culturing with MDSCs from brucellosis patients. Conclusion: Our study described an increase of immunosuppressive MDSCs in peripheral blood of chronic brucellosis patients. These results contribute to the understanding of Brucella persistent infection, which may provide an insight for effective treatment of chronic brucellosis patients in clinical practice.

Novel Vertical Flow Immunoassay with Au@PtNPs for Rapid, Ultrasensitive, and On-Site Diagnosis of Human Brucellosis.

Brucellosis is an infectious zoonosis caused by Brucella with clinical symptoms of wavy fever, fatigue, and even invasion of tissues and organs in the whole body, posing a serious threat to public health around the world. Herein, a novel vertical flow immunoassay based on Au@Pt nanoparticles (Au@PtNPs-VFIA) was established for detection of Brucella IgG antibody in clinical serum samples. The testing card of Au@PtNPs-VFIA was manufactured by printing the purified Brucella LPS and goat antimouse IgG on the nitrocellulose membrane as the test-spot or control-spot, respectively. Au@PtNPs labeled with protein G (Au@PtNPs-prG) were concurrently employed as detection probes presenting visible spots and catalysts mimicking catalytic enzymes to catalyze the DAB substrate (H2O2 plus O-phenylenediamine) for deepening color development. The testing procedure of Au@PtNPs-VFIA takes 2-3 min, and the limit of detection (LOD) for Brucella antibody is 0.1 IU/mL, which is faster and more sensitive than that of Au@PtNP-based lateral flow immunoassay (Au@PtNPs-LFIA: 15 min and 1.56 IU/mL, respectively). By comparing with vertical flow immunoassay based on classic Au nanoparticles (AuNPs-VFIA), the Au@PtNPs-VFIA is 32 times or 16 times more sensitive with or without further development of DAB substrate catalysis. Au@PtNPs-VFIA did not react with the serum samples of Gram-negative bacterium infections but only weakly cross-reacted with diagnostic serum of Y. enterocolitica O9 infection. In detection of clinical samples, Au@PtNPs-VFIA was validated for possessing 98.33% sensitivity, 100% specificity, and 99.17% accuracy, which were comparable with or even better than those obtained by the Rose-Bengal plate agglutination test, serological agglutination test, AuNPs-VFIA, and Au@PtNPs-LFIA. Therefore, this newly developed Au@PtNPs-VFIA has potential for rapid, ultrasensitive, and on-site diagnosis of human Brucellosis in clinics.

Development of a smartphone-based quantum dot lateral flow immunoassay strip for ultrasensitive detection of anti-SARS-CoV-2 IgG and neutralizing antibodies.

BACKGROUND: As several vaccines for SARS-CoV-2 have been developed, a large proportion of individuals have been vaccinated worldwide so far. The rapid and accurate immunoassays are urgently needed for detecting the specific virus-neutralizing antibody (NAb), which reflect the protective effect of the vaccines among different populations. METHODS: In this study, we designed a quantum dot lateral flow immunoassay strip (QD-LFIA) for smartphones for the detection of specific IgG or neutralizing antibodies in SARS-CoV-2 in human serum or whole blood samples. The recombinant receptor binding domain of the SARS-CoV-2 spike protein was used as the antigen to combine with NAb or angiotensin-converting enzyme 2. RESULTS: Among 81 patients who recovered from COVID-19 who were diagnosed using the nucleic acid test initially, 98.8% (80/81) were positive for IgG and 88.9% (72/81) were positive for NAb by QD-LFIA. Among 64 individuals inoculated with inactivated vaccines and six subunit vaccines, 90% (63/70) were positive for IgG and 82.9% (58/70) were positive for NAb by QD-LFIA, whereas no cross-reaction was found in 150 healthy blood donors, two patients with influenza B, and three patients with common cold. CONCLUSION: The established platform could achieve a rapid and accurate detection of NAb specific to SARS-CoV-2, which could be used for detecting the protective effect of the vaccines in areas of world that currently affected by the pandemic.

Development of a Smartphone-Based Nanozyme-Linked Immunosorbent Assay for Quantitative Detection of SARS-CoV-2 Nucleocapsid Phosphoprotein in Blood.

Since December 2019, a novel coronavirus (SARS-CoV-2) has resulted in a global pandemic of coronavirus disease (COVID-19). Although viral nucleic acid test (NAT) has been applied predominantly to detect SARS-CoV-2 RNA for confirmation diagnosis of COVID-19, an urgent need for alternative, rapid, and sensitive immunoassays is required for primary screening of virus. In this study, we developed a smartphone-based nanozyme-linked immunosorbent assay (SP-NLISA) for detecting the specific nucleocapsid phosphoprotein (NP) of SARS-CoV-2 in 37 serum samples from 20 COVID-19 patients who were diagnosed by NAT previously. By using SP-NLISA, 28/37 (75.7%) serum samples were detected for NP antigens and no cross-reactivity with blood donors' control samples collected from different areas of China. In a control assay using the conventional enzyme-linked immunosorbent assay (ELISA), only 7/37 (18.91%) serum samples were detected for NP antigens and no cross-reactivity with control samples. SP-NLISA could be used for rapid detection of SARS-CoV-2 NP antigen in primary screening of SARS-CoV-2 infected individuals.

Prime-boost vaccination of mice and rhesus macaques with two novel adenovirus vectored COVID-19 vaccine candidates.

ABSTRACTCOVID-19 vaccines are being developed urgently worldwide. Here, we constructed two adenovirus vectored COVID-19 vaccine candidates of Sad23L-nCoV-S and Ad49L-nCoV-S carrying the full-length gene of SARS-CoV-2 spike protein. The immunogenicity of two vaccines was individually evaluated in mice. Specific immune responses were observed by priming in a dose-dependent manner, and stronger responses were obtained by boosting. Furthermore, five rhesus macaques were primed with 5 × 109 PFU Sad23L-nCoV-S, followed by boosting with 5 × 109 PFU Ad49L-nCoV-S at 4-week interval. Both mice and macaques well tolerated the vaccine inoculations without detectable clinical or pathologic changes. In macaques, prime-boost regimen induced high titers of 103.16 anti-S, 102.75 anti-RBD binding antibody and 102.38 pseudovirus neutralizing antibody (pNAb) at 2 months, while pNAb decreased gradually to 101.45 at 7 months post-priming. Robust T-cell response of IFN-γ (712.6 SFCs/106 cells), IL-2 (334 SFCs/106 cells) and intracellular IFN-γ in CD4+/CD8+ T cell (0.39%/0.55%) to S peptides were detected in vaccinated macaques. It was concluded that prime-boost immunization with Sad23L-nCoV-S and Ad49L-nCoV-S can safely elicit strong immunity in animals in preparation of clinical phase 1/2 trials.

Microbiological Contamination of Strawberries from U-Pick Farms in Guangzhou, China.

This study quantified the association of rodent fruit damage and the microbiological quality of irrigation water on the risk of microbiological contamination of strawberries collected from 18 U-pick farms across five different districts in the Guangzhou metropolitan region of southern China. Fifty-four composite strawberries samples, with or without evidence of rodent or avian foraging damage (i.e., bitten), along with 16 irrigation water samples, were collected during the spring of 2014 and winter of 2015 from our cohort of 18 farms. Composite strawberry samples and irrigation water were analyzed for total coliforms, E. coli, Salmonella, E. coli O157, Giardia, and Cryptosporidium. Total coliforms and E. coli were detected in 100% and ~90% of irrigation water samples, respectively. In contrast, Cryptosporidium was detected in only two water samples, while Salmonella, E. coli O157, and Giardia were not detected in any water samples. Strawberries with signs of being bitten by wildlife had significantly higher concentrations of total coliforms and E. coli, compared to strawberries with no physical evidence of rodent damage (p < 0.001). Similarly, Cryptosporidium was detected in 7/18 (39%) of bitten, 4/18 (22%) of edge, and 5/18 (28%) of central strawberry samples, respectively. Concentration of E. coli on strawberries (p < 0.001), air temperature (p = 0.025), and presence of Cryptosporidium in irrigation water (p < 0.001) were all associated with the risk of Cryptosporidium contamination on strawberries. Salmonella and Giardia were detected in <4% strawberry samples and E. coli O157 was not detected in any samples. These results indicate the potential food safety and public health risks of consuming unwashed strawberries from U-pick farms, and the need for improved rodent biosecurity of U-pick strawberry fields and enhanced microbiological quality of irrigation water used at these facilities.

Use of syringe filters to isolate Campylobacter species from stool samples.

We investigated the efficacy of syringe filters and membrane filters with different pore sizes for recovering Campylobacter. A syringe filter with a 0.45 µm pore size achieved the highest recovery rate (0.29%), while polycarbonate membrane filters with pore sizes of 0.6 µm and 0.4 µm recovered less Campylobacter, at 0.01% and 1.3 × 10-3%, respectively. We also tested 601 diarrheic stool samples using membrane and syringe filtration methods. A total of 23 Campylobacter jejuni/coli were isolated from both syringe and membrane filtration; nine and one were isolated from only syringe or only membrane filtration, respectively (p < .05). The syringe filtration technique was better than membrane filtration for the isolation of Campylobacter.

Assessment of Microbiological Safety of Water in Public Swimming Pools in Guangzhou, China.

This study assessed microbiological safety of water from public swimming pools in Guangzhou, China. Water samples from 39 outdoor municipal swimming pools were collected from late June to early September, 2013 and subjected to detection of protozoa (Giardia and Cryptosporidium) and bacteria (Pseudomonas aeruginos, total coliforms, E. coli, E. coli O157, Shigella, and Salmonella). Cryptosporidium and Giardia were both detected in 5 (12.8%) swimming pools. Total coliforms were detected in 4 (10.3%) samples with concentrations ranging from 1.3 to 154.0 MPN/100 mL while E. coli was detected in 4 (10.3%) samples with concentrations ranging from 0.5 to 5.3 MPN/100 mL. P. aeruginosa was detected in 27 (69.2%) samples but E. coli O157, Shigella and Salmonella were not detected. Among these swimming pools, 9 (23%) met the Chinese National Standard of residual chlorine levels and 24 (62%) were tested free of residual chlorine at least once. The multi-locus sequence typing (MLST) analysis showed that all P. aeruginosa isolates belonged to new sequence types (STs) with dominant ST-1764 and ST-D distributed in different locations within the area. Some P. aeruginosa strains were resistant to medically important antibiotics. Results indicate potential public health risks due to the presence of microbiological pathogens in public swimming pools in this area.

Complete Genome Sequence of Campylobacter fetus subsp. testudinum Strain 772, Isolated from Ascites of a Patient with Chronic Kidney Disease.

Campylobacter fetus subsp. testudinum originating in reptiles can cause invasive infections in humans. Here, we present the whole-genome sequence of C. fetus subsp. testudinum strain 772, isolated from a human patient in China.

Molecular typing of Legionella pneumophila from air-conditioning cooling waters using mip gene, SBT, and FAFLP methods.

Legionella spp. are important waterborne pathogens. Molecular typing has become an important method for outbreaks investigations and source tracking of Legionnaires. In a survey program conducted by the Guangzhou Center for Disease Control and Prevention, multiple serotypes Legionella pneumophila (L. pneumophila) were isolated from waters in air-conditioning cooling towers in urban Guangzhou region, China between 2008 and 2011. Three genotyping methods, mip (macrophage infectivity potentiator) genotyping, SBT (sequence-based typing), and FAFLP (fluorescent amplified fragment length polymorphism analysis) were used to type these waterborne L. pneumophila isolates. The three methods were capable of typing all the 134 isolates and a reference strain of L. pneumophila (ATCC33153), with discriminatory indices of 0.7034, 0.9218, and 0.9376, for the mip, SBT, and FAFLP methods respectively. Among the 9 serotypes of the 134 isolates, 10, 50, and 34 molecular types were detected by the mip, SBT, and FAFLP methods respectively. The mip genotyping and SBT typing are more feasible for inter-laboratory results sharing and comparison of different types of L. pneumophila. The SBT and FAFLP typing methods were rapid with higher discriminatory abilities. Combinations of two or more of the typing methods enables more accurate typing of Legionella isolates for outbreak investigations and source tracking of Legionnaires.

Characterization of Francisella species isolated from the cooling water of an air conditioning system.

Strains of Francisella spp. were isolated from cooling water from an air conditioning system in Guangzhou, China. These strains are Gram negative, coccobacilli, non-motile, oxidase negative, catalase negative, esterase and lipid esterase positive. In addition, these bacteria grow on cysteine-supplemented media at 20 °C to 40 °C with an optimal growth temperature of 30 °C. Analysis of 16S rRNA gene sequences revealed that these strains belong to the genus Francisella. Biochemical tests and phylogenetic and BLAST analyses of 16S rRNA, rpoB and sdhA genes indicated that one strain was very similar to Francisella philomiragia and that the other strains were identical or highly similar to the Francisella guangzhouensis sp. nov. strain 08HL01032 we previously described. Biochemical and molecular characteristics of these strains demonstrated that multiple Francisella species exist in air conditioning systems.

Characterization of Francisella species isolated from the cooling water of an air conditioning system

Strains of Francisella spp. were isolated from cooling water from an air conditioning system in Guangzhou, China. These strains are Gram negative, coccobacilli, non-motile, oxidase negative, catalase negative, esterase and lipid esterase positive. In addition, these bacteria grow on cysteine-supplemented media at 20 °C to 40 °C with an optimal growth temperature of 30 °C. Analysis of 16S rRNA gene sequences revealed that these strains belong to the genus Francisella. Biochemical tests and phylogenetic and BLAST analyses of 16S rRNA, rpoB and sdhA genes indicated that one strain was very similar to Francisella philomiragia and that the other strains were identical or highly similar to the Francisella guangzhouensis sp. nov. strain 08HL01032 we previously described. Biochemical and molecular characteristics of these strains demonstrated that multiple Francisella species exist in air conditioning systems.

Potential risk of blood transfusion-transmitted brucellosis in an endemic area of China.

BACKGROUND: Brucellosis is a severe zoonotic disease that is increasingly prevalent in north China. A study evaluating Brucella infection in blood donors was conducted at Kashi central blood station, Xinjiang, China. STUDY DESIGN AND METHODS: Four serologic and two molecular methods of detection of Brucella infection were used in plasma samples from blood donations collected from Kashi in northwest China, considered a brucellosis-endemic area. Blood donor samples collected in Shenzhen, southern China, a brucellosis-nonendemic area, were tested as a negative control group. RESULTS: In 3896 plasma samples collected from Kashi central blood station, 135 (3.5%) plasma samples were reactive by the Rose Bengal plate test (RBPT), and 120 (3.1%) of the 135 RBPT-reactive sample were also reactive with the standard tube agglutination test (SAT), respectively. All samples of the control group of 399 blood samples from Shenzhen blood center tested negative with RBPT and SAT. Of 135 seroreactive plasma samples, 39 (1.0%) reacted with B. melitensis membrane protein extracts by enzyme-linked immunosorbent assay and 25 were reactive to either rBP26 or rOMP31 by Western blot. Thirteen plasma samples and two follow-up blood samples were identified as carrying Brucella DNA by quantitative polymerase chain reaction (PCR) and nested PCR. Overall 15 (1:300) Kashi blood donations were found positive by nucleic acid testing, confirmed specific by DNA sequencing. CONCLUSIONS: The data indicate a probable high rate of Brucella bacteremia, suggesting a potential risk of transfusion-transmitted brucellosis. Blood donation screening for Brucella infection may be considered in the high Brucella-endemic areas of China.

Increasing threat of brucellosis to low-risk persons in urban settings, China.

Cases of brucellosis were diagnosed in 3-month-old twins and their mother. An epidemiologic survey suggested that raw sheep or goat meat might be the source of Brucella melitensis infection. This finding implies that the increasing threat of brucellosis might affect low-risk persons in urban settings in China.

Establishment of loop-mediated isothermal amplification (LAMP) for rapid detection of Brucella spp. and application to milk and blood samples.

Brucella spp. are facultative intracellular bacteria that infect humans and animals. In this study, the loop-mediated isothermal amplification (LAMP) was used to detect the Brucella-specific gene omp25. Reaction conditions were optimized as temperature 65°C, reaction time 60 min, Mg(2+) concentration 8.0 mmol/L, polymerase content Bst DNA, 0.5 µL, deoxyribonucleotide concentration 1.6 mmol/L, and inner/outer primer ratio 1:8. The LAMP method was evaluated with 4 Brucella species and 29 non-Brucella bacteria species. Positive reactions were observed on all the 4 Brucella species but not on any non-Brucella species. The limit of detection of the LAMP method was 3.81 CFU Brucella spp. Using the LAMP method, 7 of 110 raw milk samples and 5 of 59 sheep blood samples were detected positive of Brucella spp. Results indicated that LAMP is a fast, specific, sensitive, inexpensive, and suitable method for diagnosis of Brucella spp. infection.