Epidemiological and Whole-Genome Sequencing Analysis of a Gastroenteritis Outbreak Caused by a New Emerging Serotype of Vibrio parahaemolyticus in China.

Vibrio parahaemolyticus is an important foodborne pathogen with diverse serotypes. In May 2021, we investigated a gastroenteritis outbreak that occurred in China, caused by V. parahaemolyticus O10:K4 infection. Based on the epidemiological curve, this outbreak was identified as a homologous exposure event. A case-control study demonstrated that emperor crab with mashed garlic (odds ratio [OR] = 4.60, p = 0.030; 95% confidence interval [95% CI]: 1.11-19.14), goose liver geoduck (OR = 4.50, p = 0.029; 95% CI: 1.12-18.13), shrimp (OR = 4.89, p = 0.021; 95% CI: 1.22-19.65), and sea cucumber (OR = 7.36, p = 0.005; 95% CI: 1.68-32.26) were the potential sources of the food poisoning. V. parahaemolyticus isolates from 18 laboratory-confirmed cases were all serotyped O10:K4, and determined to be sequence type ST3 via multilocus sequence typing. Pulsed field gel electrophoresis and whole-genome sequencing analysis revealed the identical pattern and 0-2 single nucleotide variation among these isolates. tdh was positive in all isolates, while trh and Orf8 were absent. Seven essential base positions in toxRS for pandemic clone identification were identical between the O10:K4 and O3:K6 pandemic clones. Phylogenetic analysis with 45 additional genomes of 13 different serotypes showed the closest genetic relationship between O10:K4 and O1: KUT. O10:K4 was thought to evolve from the O3:K6 pandemic clone. The new serovariant of O3:K6 poses a challenge for the prevention and control of V. parahaemolyticus disease outbreaks, or even epidemics, in the future.

Prevalence and Population Analysis of Vibrio parahaemolyticus Isolated from Freshwater Fish in Zhejiang Province, China.

Objectives: The previous researches revealed that Vibrio parahaemolyticus has been detected in freshwater fish samples. However, the molecular characteristics of V. parahaemolyticus isolated from freshwater fish, including pathogenic and pandemic strains, are still unknown. This study aims to characterize and identify molecular properties of the bacterium. In addition, it identifies the source of V. parahaemolyticus from freshwater fish samples in Zhejiang Province, China. Methods: Four hundred and twenty-one freshwater fish samples (from fishing farms, retail markets, and restaurants) and 212 seafood samples (from retail markets) were collected in 10 cities of Zhejiang Province. V. parahaemolyticus strains were isolated from these samples and comparatively analyzed by multilocus sequence typing, serotyping, antimicrobial susceptibility test, and polymerase chain reaction, targeting common toxin genes (tdh, trh) and markers for pandemic strains (orf8, toxRS/new). Results: Sixty-eight V. parahaemolyticus strains were isolated from the 421 freshwater fish samples, and 89 V. parahaemolyticus isolates were identified out of 212 seafood samples. The detection rate of V. parahaemolyticus was significantly different (p < 0.05) between the fishing farms, the retail markets, and the restaurants. The isolates from freshwater fish samples were divided into eight O serotypes with three O3:K6 isolates, which contain three pandemic complexes (tdh+, orf8+, toxRS/new+). A total of 53 different sequence types (STs) were identified among the 68 isolates, including 28 novel STs. Antimicrobial susceptibility results indicated that 76.5% of the strains were resistant to ampicillin. A third (3/9) of the isolates from fishing farm sources shared the same STs with their counterparts from retail markets. Compared with the isolates from the seafood samples collected in the same sampling sites, 13.2% (9/68) freshwater fish isolates overlapped with seafood isolates. Conclusions: Our study showed that V. parahaemolyticus population in freshwater fish is genetically diverse. The V. parahaemolyticus contaminates might have come from both fishing farm sources and cross-contamination from seafood in the closed area at the markets. Freshwater fish may work as a reservoir of pathogenic and pandemic V. parahaemolyticus isolates, indicating potential public health and food safety risks associated with the consumption of freshwater fish.

Comparative genomic analyses of IncpA1763-KPC plasmids.

Multi-replicon plasmids harboring the IncpA1763-KPC replicon together with other replicons are being increasingly reported among Enterobacteriaceae species. However, plasmids with single IncpA1763-KPC replicons are poorly studied as a different incompatibility (Inc) group, despite their rise in appearance in some strains. IncpA1763-KPC plasmids, pA1763-KPC, and p427113-2, from two clinical Klebsiella pneumoniae isolates were fully sequenced by high-throughput genome sequencing. Linear structural comparisons of IncpA1763-KPC backbone region were made between these two plasmids and six arbitrarily selected representative IncpA1763-KPC plasmids sequenced previously. A further detailed genomic comparison was carried out between plasmids pA1763-KPC, p427113-2, and pFB2.2, which show high homology across the backbone sequence to one another. Among all sequenced IncpA1763-KPC plasmids considered in this study, plasmids pA1763-KPC and p427113-2 showed the most complete IncpA1763-KPC backbones. These were composed of the IncpA1763-KPC replicon (repAIncpA1763-KPC and its iterons), the 5.6-kb IncpA1763-KPC -type maintenance region, the 27.7-kb IncFIIK -type maintenance region, and the 36.6-kb IncFIIK -type conjugal transfer regions. Compared with pA1763-KPC or p427113-2, the backbone regions of the other analyzed IncpA1763-KPC plasmids had gradually undergone different deletions or truncations, but shared small and core IncpA1763-KPC backbones including the IncpA1763-KPC replicon, IncpA1763-KPC -type maintenance region, and residual IncFIIK -type maintenance region. Accessory modules integrated into IncpA1763-KPC backbones included the multidrug-resistant module blaKPC-2 region in pA1763-KPC, the metal-resistance modules ars region together with ncr region in pFB2.2 and sil in pKPN-9a0d, the ISKpn14-to-IS26 region in p427113-2, and other non-resistance region in the respective plasmids. This detailed comparative genomics analysis of IncpA1763-KPC plasmids provides a deep insight into their diversification and evolution.

Prevalence and genetic characteristics of Cronobacter spp. from food and human clinical stool samples in Wenzhou, China 2008-2018.

Pathogenic Cronobacter species are responsible for life-threatening illness in neonates. A ten-year comprehensive survey was conducted to examine the population structure and antimicrobial resistant patterns of Cronobacter isolates from food (n = 78) and clinical (n = 12) sources in Wenzhou, China. A total of 90 (4.4%) isolates were recovered from 2051 collected samples. The occurrence of Cronobacter spp. was highest in spices with a rate of 22% (26/119), whereas the lowest contamination rate of 1% was found in powered infant and toddler formula (7/494), special medical infant formula (1/95) and human stool samples (12/1024). Cronobacter strains revealed a high degree of genetic diversity among the isolates tested. Pulsed-field gel electrophoresis (PFGE) distinguished 75 clonal groups, and the biggest cluster consisted of four strains. Multilocus sequence typing (MLST) method displayed 43 sequence types (STs), of which ST1, ST4, ST8, ST64, ST148 and ST201 were most frequently identified. Meanwhile, two new sequence types were discovered and added to the PubMLST international database. Resistance to ceftriaxone, cefotaxiv, amoxicillin, ampicillin, cefoxitin, tetracycline, streptomycin, azithromycin, chloramphenicol, as well as multidrug resistance, was noted. Taken together, this large-scale surveillance study highlights the wide dissemination and diverse molecular features of Cronobacter spp. in Wenzhou China.

Epidemiological and Whole Genomic Sequencing Analysis of a Campylobacter jejuni Outbreak in Zhejiang Province, China, May 2019.

Campylobacter is well recognized as the leading cause of bacterial foodborne diarrheal disease worldwide with a very low outbreak reported in China. In May 2019, we investigated an outbreak of Campylobacter jejuni infections among students in a junior high school in Eastern China. Cases were interviewed to identify a common source of contamination. As cases were identified in the same school during a period of time, menus were reviewed and food items included in the questionnaire. Rectal swabs from school kitchen staff and suspected food items (raw chicken) from a local market from where the school food came were examined for C. jejuni. Pulsed-field gel electrophoresis and whole genome sequencing were performed to determine the relatedness of the isolates. To identify the source of the contamination, a case-control study was conducted. Forty-five cases were reported with diarrhea among 1696 students and staff. Stool samples for 10 of the 45 and 5 tested positive for C. jejuni. WGS analysis revealed a 0-4 single nucleotide variation in case-patient isolates. Although we were unable to identify the specific food item, a specific menu was identified as the potential source of the contamination (odds ratios = 20.82; 95% confidence interval = 6.472-66.957). In this menu, chicken was served. A food isolate collected from chicken in Zhejiang province in 2018 was positive for the same identical strain (5-7 single nucleotide polymorphisms). This is one of the few reports in China about outbreak caused by C. jejuni. This investigation illustrates the potential risk of outbreaks caused by Chinese cold dishes of chicken.

Multilocus sequence type profiles of Bacillus cereus isolates from infant formula in China.

Bacillus cereus sensu stricto is an opportunistic foodborne pathogen. The multilocus sequence type (MLST) of 74 B. cereus isolated from 513 non-random infant formula in China was analyzed. Of 64 sequence types (STs) detected, 50 STs and 6 alleles were newly found in PubMLST database. All isolates except for one singleton (ST-1049), were classified into 7 clonal complexes (CC) by BURST (n-4), in which CC1 with core ancestral clone ST-26 was the largest group including 86% isolates, and CC2, 3, 9, 10 and 13 were first reported in China. MLST profiles of the isolates from 8 infant formula brands were compared. It was found the brands might be potentially tracked by the variety of STs, such as ST-1049 of singleton and ST-1062 of isolate from goat milk source, though they could not be easily tracked just by clonal complex types of the isolates.

Quantitative Prevalence, Phenotypic and Genotypic Characteristics of Bacillus cereus Isolated from Retail Infant Foods in China.

Bacillus cereus is an important foodborne pathogen, which can cause severe food poisoning. The aim of this study was (i) to evaluate the quantitative prevalence of B. cereus in retail prepackaged infant formula and ready-to-eat rice flour in China and (ii) to gain the basic information on pheno- and genotypic characteristics of B. cereus isolates. We found that 40 out of the 587 samples were positive for B. cereus. B. cereus in 3.5% of infant formula samples and 1.0% of rice flour samples outnumbered 100 Colony-Forming Units (CFU)/g. B. cereus level even attained 103-104 CFU/g in four infant formula samples and one rice flour sample. Furthermore, we identified the distribution patterns of toxin genes in B. cereus isolates. The results showed that 97.5% of B. cereus isolates harbored at least one enterotoxin gene. Antibiotic susceptibility tests revealed that all isolated B. cereus strains were resistant to penicillin and 50% of them were multidrug resistant. Thirteen new sequence types (STs) and four new alleles were identified via multilocus sequence typing. Clonal Complex (CC) ST-205 and CC ST-142 were predominant clonal complexes. Interestingly, we revealed the special relationship between STs of B. cereus isolates and the geographical distributions of infant food manufacturers for the first time. The data implied that B. cereus of different STs might have a distinct ecological niche in China. In view of relatively high contamination level of enterotoxin- producing B. cereus in a proportion of infant foods, especially in those suitable for the ≤6-month-old infant group, appropriate safety criteria and hygienic control measures for infant foods should be drafted in China to prevent B. cereus infection.

[Antimicrobial resistance profiles and genetic diversity of bovine staphylococcus aureus isolated in 5 provinces of China in 2013].

OBJECTIVE: To investigate antimicrobial resistance profiles and genetic diversity of staphylococcus aureus isolated from lactating cows of 5 provinces in China, 2013. METHODS: A total of 680 samples were collected from 15 herds (12 farms, 3 artels) in 5 provinces of China in 2013, including swabs of extramammary sites (bovine teat skin and milking machine liners) and quarter milk samples from lactating cows diagnosed with subclinical mastitis. The antimicrobial resistance of the isolates were tested by broth microdilution method and the genotypes were determined by PFGE (pulsed-field gel electrophoresis) method. RESULTS: A total of 111 isolates were isolated and identified as staphylococcus aureus. Resistance to penicillin (90.1% (100/111)), erythromycin (48.6% (54/111)), ciprofloxacin (36.9% (41/111)), clindamycin (27.9% (31/111)), gentamycin (18.9% (21/111)), chloramphenicol (9.0% (10/111)), tetracycline (7.2% (8/111)) of these strains were observed. All isolates were sensitive to oxacillin, vancomycin and selectrin. 92.8% (103/111) staphylococcus aureus isolates were resistant to at least one antimicrobial. 38.7% (43/111) strains were multi-drug resistant isolates. The resistance rate of isolates in artels (100% (48/48)) was higher than it in farms (87% (55/63)) and the difference was statistically significant (χ(2) = 4.80, P < 0.05). The multi-resistance rate of isolates in artels (54% (26/48)) was higher than it in farms (27% (17/63)) and the difference was also statistically significant (χ(2) = 8.48, P < 0.05). The 111 strains were clustered into 8 types, 6 out of which were consisted of 98% isolates (109/111), and were prevalent in 2 to 9 herds. Every herd had 1 to 4 types, and tend to be comprised by one major type. Most swab isolates were indistinguishable from isolates infecting the mammary gland. There were no relationship between antimicrobial resistance profiles and genotypes of these isolates. CONCLUSION: The drug resistance of staphylococcus aureus isolates associated with lactating cows of 5 provinces in China is serious, especially the isolates collected from artels. A few specialized clones were responsible for most of the cases of bovine mastitis in a single herd and some clones might have a broad geographic distribution.

[Analysis of molecular features of clinical Vibrio parahaemolyticus strains in China].

OBJECTIVE: To explore the phenetic and genetic features of clinical Vibrio parahaemolyticus strains from 2007-2009 in China. METHODS: A total of 135 clinical Vibrio parahaemolyticus strains, isolated from Zhejiang, Jiangsu, Sichuan, Guangxi, Liaoning Provinces during 2007 to 2009, were selected for the research. The occurrence of virulence genes thermostable direct hemolysin (tdh) and TDH-related hemolysin (trh), species-specific genes thermolabile hemolysin (tlh), toxR, VPM and gyrB, the pandemic clone gene markers(GS-PCR, PGS-PCR, orf8 and HU-α) in 135 Vibrio parahaemolyticus strains was detected by PCR. The antimicrobial susceptibilities to eight antimicrobial agents of the experimental strains were determined by the broth microdilution method. All strains were serotyped and underwent the cluster analysis with pulsed-field gel electrophoreses. RESULTS: The results of PCR methods claim that all experiment strains carry species-specific genes such as tlh, toxR, gyrB, VPM. Among clinical strains, 85.9% (116/135) carry tdh and/or trh. 85.2% (115/135) were positive for tdh, and 3.0% (4/135) were positive for trh; while 3 strains carried both.66.7% (90/135) , 80.7% (109/135) , 65.2% (88/135) , 66.7% (90/135) clinical strains carried the genes of GS-PCR, PGS-PCR, orf8, HU-α, respectively. The results of antibiotics susceptibility test showed that 8.1% (11/135) strains were resistant to at least one agent, including 9 strains were resistant to ampicillin, 2 strains were resistant to trimethoprim and sulphamethoxazole, and 1 strain were resistant to tetracycline. All clinical strains were sensitive to cefotaxime, ceftazidime, gentamicin, ciprofloxacin and chloromycetin.Serological analysis of the O and K antigens claimed that a total of 29 serotypes were identified for clinical strains, predominantly O3, O4 and O1 groups, accounting for 89.6% (121/135). O3: K6 was dominant serotype, accounting for 56.3% (76/135). The pandemic flora in China included O3: K6, O4: K68, O1: K36, O1: K25, O1: K5 and O3: K29 serotypes.Genomic DNAs of 135 clinical strains were digested with SfiI and NotI, the molecular size of PFGE restriction fragments used for analysis mainly ranged from 30-700 kb.When subjected to UPGMA clustering, 6 and 9 clusters were grouped by SfiI and NotI, and the minimal similarity was 52.6% and 58.7%, and pandemic flora were located in C groups and D group, respectively. CONCLUSION: Most of Vibrio parahaemolyticus strains isolated from clinical sources in China were pathogenic. The pandemic clone, especially O3: K6 was prevalent. The GS-PCR and HU-α genes were reliable markers to identify the pandemic flora. The serotype by PFGE was reliable to distinguish the pandemic flora and the sporadic strains.

Molecular analysis of non-O1/non-O139 Vibrio cholerae isolated from hospitalised patients in China.

BACKGROUND: Cholera is still a significant public health issue in developing countries. The aetiological agent is Vibrio cholerae and only two serogroups, O1 and O139, are known to cause pandemic or epidemic cholera. In contrast, non-O1/non-O139 V. cholerae has only been reported to cause sporadic cholera-like illness and localised outbreaks. The aim of this study was to determine the genetic diversity of non-O1/non-O139 V. cholerae isolates from hospitalised diarrhoeal patients in Zhejiang Province, China. RESULTS: In an active surveillance of enteric pathogens in hospitalised diarrhoeal patients, nine non-O1/non-O139 V. cholerae isolates were identified from 746 diarrhoeal stool samples at a rate of 1.2%. These isolates and an additional 31 isolates from sporadic cases and three outbreaks were analysed using pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). PFGE divided the isolates into 25 PFGE types while MLST divided them into 15 sequence types (STs). A single ST, ST80, was predominant which persisted over several years in different cities and caused two outbreaks in recent years. Antibiotic resistance varied with the majority of the isolates resistant to sulphamethoxazole/trimethoprim and nearly all isolates either resistant or intermediate to erythromycin and rifampicin. None of the isolates carried the cholera toxin genes or toxin co-regulated pilus genes but the majority carried a type III secretion system as the key virulence factor. CONCLUSIONS: Non-O1/non-O139 V. cholerae is an important contributor to diarrhoeal infections in China. Resistance to commonly used antibiotics limits treatment options. Continuous surveillance of non-O1/non-O139 V. cholerae is important for control and prevention of diarrhoeal infections.

[Sequence-based typing for 61 Legionella pneumophila serogroup 1 isolates in Zhejiang Province].

OBJECTIVE: To analyze the sequence characteristics of the serogroup 1 Legionella pneumophila (Lp1) strains isolated from the cooling tower water in Zhejiang province by sequence-based typing (SBT). METHODS: 61 strains of Lp1 isolated from cooling tower water in ten cities of Zhejiang Province from 2005 to 2011 were genotyped by SBT method. The nucleotide polymorphism of the sequencing results was analyzed by DnaSP 5.0, and the SBT results were cluster analyzed using SplitsTree and eBURST software. RESULTS: All 61 isolates of Lp1 were resolved into 11 STs, and 5 new STs were found in this study. The ST with the largest number of isolates was ST1 (n=50), and these ST-1 strains were found in 10 cities. The nucleotide polymorphism (Pi) of these seven housekeeping genes ranged from 0.01095 (mip) to 0.05355 (pilE). The STs of the Lpl isolates were clustered to four clonal complexes (CC), and ST-1, ST-149, ST-154 were the three main clonal complexes (CC). CONCLUSION: Genetic diversity was shown in the serogroup 1 Legionella pneumophila strains isolated from cooling tower water in Zhejiang province.

A Pseudovirus-Based Entry Assay to Evaluate Neutralizing Activity against Respiratory Syncytial Virus.

Respiratory syncytial virus (RSV) infection can cause life-threatening pneumonia and bronchiolitis, posing a significant threat to human health worldwide, especially to children and the elderly. Currently, there is no specific treatment for RSV infection. The most effective measures for preventing RSV infection are vaccines and prophylactic medications. However, not all population groups are eligible for the approved vaccines or antibody-based preventive medications. Therefore, there is an urgent need to develop novel vaccines and prophylactic drugs available for people of all ages. High-throughput assays that evaluate the efficacy of viral entry inhibitors or vaccine-induced neutralizing antibodies in blocking RSV entry are crucial for evaluating vaccine and prophylactic drug candidates. We developed an efficient entry assay using a lentiviral pseudovirus carrying the fusion (F) protein of type A or B RSV. In addition, the essential parameters were systematically optimized, including the number of transfected plasmids, storage conditions of the pseudovirus, cell types, cell numbers, virus inoculum, and time point of detection. Furthermore, the convalescent sera exhibited comparable inhibitory activity in this assay as in the authentic RSV virus neutralization assay. We established a robust pseudovirus-based entry assay for RSV, which holds excellent promise for studying entry mechanisms, evaluating viral entry inhibitors, and assessing vaccine-elicited neutralizing antibodies against RSV.

A new intracellular targeting motif in the cytoplasmic tail of the spike protein may act as a target to inhibit SARS-CoV-2 assembly.

Infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses a threat to global public health, underscoring the urgent need for the development of preventive and therapeutic measures. The spike (S) protein of SARS-CoV-2, which mediates receptor binding and subsequent membrane fusion to promote viral entry, is a major target for current drug development and vaccine design. The S protein comprises a large N-terminal extracellular domain, a transmembrane domain, and a short cytoplasmic tail (CT) at the C-terminus. CT truncation of the S protein has been previously reported to promote the infectivity of SARS-CoV and SARS-CoV-2 pseudoviruses. However, the underlying molecular mechanism has not been precisely elucidated. In addition, the CT of various viral membrane glycoproteins play an essential role in the assembly of virions, yet the role of the S protein CT in SARS-CoV-2 infection remains unclear. In this study, through constructing a series of mutations of the CT of the S protein and analyzing their impact on the packaging of the SARS-CoV-2 pseudovirus and live SARS-CoV-2 virus, we identified V1264L1265 as a new intracellular targeting motif in the CT of the S protein, that regulates the transport and subcellular localization of the spike protein through the interactions with cytoskeleton and vesicular transport-related proteins, ARPC3, SCAMP3, and TUBB8, thereby modulating SARS-CoV-2 pseudovirus and live SARS-CoV-2 virion assembly. Either disrupting the V1264L1265 motif or reducing the expression of ARPC3, SCAMP3, and TUBB8 significantly repressed the assembly of the live SARS-CoV-2 virion, raising the possibility that the V1264L1265 motif and the host responsive pathways involved could be new drug targets for the treatment of SARS-CoV-2 infection. Our results extend the understanding of the role played by the S protein CT in the assembly of pseudoviruses and live SARS-CoV-2 virions, which will facilitate the application of pseudoviruses to the study of SARS-CoV-2 and provide potential strategies for the treatment of SARS-CoV-2 infection.

The E484K Substitution in a SARS-CoV-2 Spike Protein Subunit Vaccine Resulted in Limited Cross-Reactive Neutralizing Antibody Responses in Mice.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), especially emerging variants, poses an increased threat to global public health. The significant reduction in neutralization activity against the variants such as B.1.351 in the serum of convalescent patients and vaccinated people calls for the design of new potent vaccines targeting the emerging variant. However, since most vaccines approved and in clinical trials are based on the sequence of the original SARS-CoV-2 strain, the immunogenicity and protective efficacy of vaccines based on the B.1.351 variant remain largely unknown. In this study, we evaluated the immunogenicity, induced neutralization activity, and protective efficacy of wild-type spike protein nanoparticle (S-2P) and mutant spike protein nanoparticle (S-4M-2P) carrying characteristic mutations of B.1.351 variant in mice. Although there was no significant difference in the induction of spike-specific IgG responses in S-2P- and S-4M-2P-immunized mice, neutralizing antibodies elicited by S-4M-2P exhibited noteworthy, narrower breadth of reactivity with SARS-CoV-2 variants compared with neutralizing antibodies elicited by S-2P. Furthermore, the decrease of induced neutralizing antibody breadth at least partly resulted from the amino acid substitution at position 484. Moreover, S-4M-2P vaccination conferred insufficient protection against live SARS-CoV-2 virus infection, while S-2P vaccination gave definite protection against SARS-CoV-2 challenge in mice. Together, our study provides direct evidence that the E484K substitution in a SARS-CoV-2 subunit protein vaccine limited the cross-reactive neutralizing antibody breadth in mice and, more importantly, draws attention to the unfavorable impact of this mutation in spike protein of SARS-CoV-2 variants on the induction of potent neutralizing antibody responses.

AluScan: a method for genome-wide scanning of sequence and structure variations in the human genome.

BACKGROUND: To complement next-generation sequencing technologies, there is a pressing need for efficient pre-sequencing capture methods with reduced costs and DNA requirement. The Alu family of short interspersed nucleotide elements is the most abundant type of transposable elements in the human genome and a recognized source of genome instability. With over one million Alu elements distributed throughout the genome, they are well positioned to facilitate genome-wide sequence amplification and capture of regions likely to harbor genetic variation hotspots of biological relevance. RESULTS: Here we report on the use of inter-Alu PCR with an enhanced range of amplicons in conjunction with next-generation sequencing to generate an Alu-anchored scan, or 'AluScan', of DNA sequences between Alu transposons, where Alu consensus sequence-based 'H-type' PCR primers that elongate outward from the head of an Alu element are combined with 'T-type' primers elongating from the poly-A containing tail to achieve huge amplicon range. To illustrate the method, glioma DNA was compared with white blood cell control DNA of the same patient by means of AluScan. The over 10 Mb sequences obtained, derived from more than 8,000 genes spread over all the chromosomes, revealed a highly reproducible capture of genomic sequences enriched in genic sequences and cancer candidate gene regions. Requiring only sub-micrograms of sample DNA, the power of AluScan as a discovery tool for genetic variations was demonstrated by the identification of 357 instances of loss of heterozygosity, 341 somatic indels, 274 somatic SNVs, and seven potential somatic SNV hotspots between control and glioma DNA. CONCLUSIONS: AluScan, implemented with just a small number of H-type and T-type inter-Alu PCR primers, provides an effective capture of a diversity of genome-wide sequences for analysis. The method, by enabling an examination of gene-enriched regions containing exons, introns, and intergenic sequences with modest capture and sequencing costs, computation workload and DNA sample requirement is particularly well suited for accelerating the discovery of somatic mutations, as well as analysis of disease-predisposing germline polymorphisms, by making possible the comparative genome-wide scanning of DNA sequences from large human cohorts.

Salmonella enterica subsp. diarizonae Harboring ST233, ST1263, and ST1845 in Children.

Objective: This study aims to analyze the molecular epidemiology, resistance, and pathogenicity of Salmonella enterica subsp. diarizonae isolated from children. Methods: Whole genome sequencing was carried out, and molecular serotypes, sequence types, resistance genes, and virulence genes of S. enterica subsp. diarizonae isolates were analyzed. Antimicrobial susceptibility test was determined by commercialized microdilution method. Results: A total of three isolates of S. enterica subsp. diarizonae were isolated during 2015 to 2020. The molecular serotypes of the three strains were 61:c:z35, 61:l,v:1,5,7:[z57], and 65:k:z, respectively, and the sequence types were ST1845, ST233, and ST1263. All the three isolates were susceptible to ceftriaxone, ceftazidime, cefepime, amoxycillin/clavulanic acid, piperacillin/tazobactam, ertapenem, imipenem, levofloxacin, and trimethoprim/sulfamethoxazole. No other resistant gene was detected except aac(6')-Iaa. There were no resistant plasmids detected in all the three isolates. A total of 76 genes were present in all isolates, containing 49 genes of Type III Secretion System (T3SS) mediated by SPI-1and SPI-2, 13 genes of adherence (type 1 fimbriae, Agf, and MisL-related genes), 11 genes of iron uptake (Yersiniabactin), two genes of magnesium uptake, and one gene of typhoid toxin(cdtB). Conclusion: The serotypes and sequence types of S. enterica subsp. diarizonae isolates were rarely reported in children; all the S. enterica subsp. diarizonae isolates were susceptible to detected antibiotics; T3SS, adherence, iron uptake, magnesium uptake, and typhoid toxin were responsible for pathogenicity of the S. enterica subsp. diarizonae isolates in children.

The Epidemiology of Meningococcal Disease and Carriage, Genotypic Characteristics and Antibiotic Resistance of Neisseria meningitidis Isolates in Zhejiang Province, China, 2011-2021.

Neisseria meningitidis (Nm) remains a worldwide leading cause of epidemic meningitis. During 2011-July 2021, 55 meningococcal disease (MD) cases were reported with a case fatality rate of 5.45% in Zhejiang Province, China. The median age was 7 years. The annual incidence was 0.0017-0.0183 per 100,000 population. The highest age-specific incidence was observed in the group younger than 1 year. Serogroup was identified in 30 laboratory-confirmed MD cases, and MenB was most predominant. MenB was mainly observed in two age groups: younger than 5 and older than 35 years. MenB incidence was significantly increasing from 0.0018 per 100,000 in 2013 to 0.0070 per 100,000 in 2019. During 2015-2020, 17 positive samples were detected from 2,827 throat swabs from healthy population, of which 70.59% was MenB. Twenty multilocus sequence typing sequence types (STs) containing eight newly assigned STs (ST15881-ST15888) were determined in all Nm isolates. Either in MD cases or in healthy population, MenB CC ST-4821 was the predominant ST. It was worth noting that two MenY CC ST-23 cases occurred in 2019 and 2021, respectively. MenY CC ST-23 MD cases increased gradually in China. Phylogeny results based on genome sequencing indicated that Chinese MenW CC ST-11 isolates were genetically linked and grouped together with Japanese isolates, separated from MenW CC ST-11 isolates from Saudi Arabia Hajj outbreak, Europe, South Africa, South America, North America, and Oceania. MenW CC ST-11 isolates from East Asia might have evolved locally. Antibiotic susceptibility tests revealed a relatively high resistance rate (22.86%) of Nm isolates to penicillin. This study provided valuable data for Chinese public health authorities to grasp the temporal epidemiological characteristics of MD and healthy carriage.

Foodborne pathogens in retail oysters in south China.

OBJECTIVE: To investigate the occurrence of important foodborne pathogens in shellstock Pacific oysters in the food markets in South China. METHODS: From July 2007 to June 2008, retail oysters were collected in different seasons from South China and analyzed for the prevalence and levels of Listeria monocytogenes, Vibrio vulnificus and Vibrio parahaemolyticus. RESULTS: None of L. monocytogenes could be detected in any of the 202 oyster samples tested, while E vulnificus and V. parahaemolyticus could be detected in 67 (54.9%) and 109 (89.3%) of the 122 oyster samples analyzed, respectively, with an MPN (most probable number) value greater than or equal to 3. V. vulnificus and V. parahaemolyticus with a more than 102 MPN/g were found in 36 (29.5%) and 59 (48.4%) of the 122 oyster samples, respectively. The tdh and trh genes were detected in 4 (0.3%) and 8 (0.6%) of the 1 349 V parahaemolyticus isolates, respectively. Of the 122 samples, 4 (3.3%) was positive for either tdh or trh. The levels of V. vulnificus and total V. parahaemolyticus in oysters in South China varied in different seasons. CONCLUSION: V. vulnificus and pathogenic V. parahaemolyticus are frequently found in oysters in south China, which may pose a potential threat to public health. Data presented here will be useful for the microbiological risk assessment in oysters in China.

Site-directed modification of adenoviral vector with combined DNA assembly and restriction-ligation cloning.

Commonly used and well accepted approaches are lacking for site-directed modification of adenoviral vectors. Here, we attempt to introduce an easy-to-implement strategy for such purpose with an example of establishing a replication competent adenoviral vector system from pKAd5 plasmid, an infectious clone of human adenovirus 5 (HAdV-5). PCR products of GFP expression cassette and plasmid backbone were fused with the EcoRI/NdeI-digested fragment of pKAd5 to generate a modified intermediate plasmid pMDXE3GA by DNA assembly. NdeI-digested fragment of pMDXE3GA was brought back to pKAd5 to form the adenoviral plasmid pKAd5XE3GA by restriction-ligation cloning. Recombinant adenovirus HAdV5-XE3GA was rescued, amplified and purified. The expression of GFP and the propagation of virus in adherent HEp-2 and suspension K562 cells were investigated. Expression of target gene was significantly enhanced in both cell lines infected with HAdV5-XE3GA due to virus replication. However, propagation of virus could not sustain in culture of K562 cells. Shuttle plasmid pSh5RC-GFP was constructed to facilitate exchange of transgene. In summary, the strategy of combined DNA assembly and restriction-ligation cloning is functional, cost-effective and suitable for genetic modification of adenovirus.

Molecular Characterization and Antibiotic Resistant Profiles of Campylobacter Species Isolated From Poultry and Diarrheal Patients in Southeastern China 2017-2019.

Campylobacter is a zoonotic pathogen that causes foodborne diarrheal illness globally. To better understand health risks in Southeastern China, Campylobacter spp. were surveyed in humans and representative poultry products over 3 years. One hundred and ninety-five representative isolates (n = 148, Campylobacter jejuni; n = 45, Campylobacter coli; n = 2 Campylobacter hyointestinalis) were examined for genetic relatedness and antimicrobial susceptibility. Nearly all Campylobacter isolates (99.0%, 193/195) were resistant to at least one class of antimicrobials, and 45.6% (89/195) of the isolates exhibited multidrug resistance. Genotypic analysis revealed high diversity among tested strains. Multilocus sequence typing (MLST) displayed 120 sequence types (STs) including 42 novel STs being added to the PubMLST international database. Sixty-two STs belonged to 16 previously characterized clonal complexes (CCs), of which CC-21, CC-45, CC-464, CC-574, CC-353, and CC-828 were most frequently identified. In addition, pulsed-field gel electrophoresis (PFGE) fingerprinting resulted in 66 PFGE SmaI patterns among the 125 isolates, with eight patterns shared between human and poultry sources. Subtyping data did not correlate with antimicrobial resistance phenotypes. Taken together, this large-scale surveillance study highlights high antimicrobial resistance and molecular features of Campylobacter isolates in Southeastern China.