Whole genome sequencing of SARS-CoV-2 isolated in Guangdong Province and factors influencing the sequencing / 中华微生物学和免疫学杂志

Objective:To obtain the genome sequences of SARS-CoV-2 in respiratory specimens in Guangdong Province with next-generation sequencing (NGS) and analyze the factors influencing sequencing.Methods:Eight upper and lower respiratory tract specimens were collected from patients with SARS-CoV-2 infection in Guangdong Province in January 2020. RNA library construction was used to obtain the genome sequences of SARS-CoV-2. A bio-informatics software package (CLC Genomics Workbench 12.0) was used to analyze and compare the genomic sequences.Results:Five SARS-CoV-2 genome sequences were obtained from the eight specimens and two were obtained from lower respiratory tract specimens. The nucleotide homology to SARS-CoV-2 was 97.74%-99.90%. The Ct values were lower, while the sequencing depth, coverage, relative abundance and genome integrity were higher in sequencing the SARS-CoV-2 in lower respiratory tract specimens.Conclusions:The low Ct value of SARS-CoV-2 in the samples was good for sequencing.

Circulation and etiological characterization of Salmonella enterica serotype in human in Guangdong province, 2007-2016 / 中华流行病学杂志

Objective To understand the circulation,drug resistance and molecular characteristics of Salmonella 1,4,[5],12:i:-in human in Guangdong province.Methods Salmonella 1,4,[5],12:i:-isolated from diarrhea patients in Guangdong during 2007-2016 were detected for drug resistance,genes and PFGE characteristics.Results A total of 2 960 strains Salmonella 1,4,[5],12:i:-were isolated from human diarrhea cases during this period.The positive rates of the isolation increased year by year.The male to female ratio of the infection cases was 1.58 ∶ 1,and the infection mainly occurred in infants and young children.Except imipenem,Salmonella 1,4,[5],12:i:-was resistant to other 17 antibiotics to some extent.The drug resistant rates to ceftazidime,cefotaxime and ciprofloxacin increased from 2011 to 2016.Multi-drug resistance was serious,for example,the multi-drug resistant strains with ASSuT accounted for 70.62％ (435/616) and the multi-drug resistant strains with ACSuGSTTm accounted for 27.11％ (167/616).The lack offljA,fljB and hin genes,as well as the retaining of iroB,STM2740,STM2757 genes,resulted in the unable expression of FljBenx gene with 8 different defection profiles.There were 934 different PFGE patterns observed in 2 347 strains,which displayed a relatively large fingerprint polymorphism.The major PFGE pattern was JPXX01.GD0226,which was found in 97 strains,accounting for 4.13％ (97/2 347).The PFGE patterns in 168 Salmonella 1,4,[5],12:i:-strains were consistent with that of Salmonella typhimurium.Conclusions Salmonella 1,4,[5],12:i:-strains has become the major serotype of Salmonella that cause diarrhea in human in Guangdong.The multi-drug resistance of Salmonella 1,4,[5],12:i:-was serious,and since the defection offljA,fljB and hin genes,the expression of FljBenx protein failed.The PFGE results were diverse,which displayed polymorphism in inheritance.

Circulation and etiological characterization of Salmonella enterica serotype in human in Guangdong province, 2007-2016 / 中华流行病学杂志

Objective To understand the circulation,drug resistance and molecular characteristics of Salmonella 1,4,[5],12:i:-in human in Guangdong province.Methods Salmonella 1,4,[5],12:i:-isolated from diarrhea patients in Guangdong during 2007-2016 were detected for drug resistance,genes and PFGE characteristics.Results A total of 2 960 strains Salmonella 1,4,[5],12:i:-were isolated from human diarrhea cases during this period.The positive rates of the isolation increased year by year.The male to female ratio of the infection cases was 1.58 ∶ 1,and the infection mainly occurred in infants and young children.Except imipenem,Salmonella 1,4,[5],12:i:-was resistant to other 17 antibiotics to some extent.The drug resistant rates to ceftazidime,cefotaxime and ciprofloxacin increased from 2011 to 2016.Multi-drug resistance was serious,for example,the multi-drug resistant strains with ASSuT accounted for 70.62％ (435/616) and the multi-drug resistant strains with ACSuGSTTm accounted for 27.11％ (167/616).The lack offljA,fljB and hin genes,as well as the retaining of iroB,STM2740,STM2757 genes,resulted in the unable expression of FljBenx gene with 8 different defection profiles.There were 934 different PFGE patterns observed in 2 347 strains,which displayed a relatively large fingerprint polymorphism.The major PFGE pattern was JPXX01.GD0226,which was found in 97 strains,accounting for 4.13％ (97/2 347).The PFGE patterns in 168 Salmonella 1,4,[5],12:i:-strains were consistent with that of Salmonella typhimurium.Conclusions Salmonella 1,4,[5],12:i:-strains has become the major serotype of Salmonella that cause diarrhea in human in Guangdong.The multi-drug resistance of Salmonella 1,4,[5],12:i:-was serious,and since the defection offljA,fljB and hin genes,the expression of FljBenx protein failed.The PFGE results were diverse,which displayed polymorphism in inheritance.

Hand, foot, and mouth disease in Guangzhou, 2016-2017: an epidemiological study and genotype analysis of non-enterovirus group A 71 non-coxsackievirus group A 16 enterovirus / 中华传染病杂志

Objective To study the epidemiology of hand,foot,and mouth disease (HFMD) and the spectrum of serotypes in the other enterovirus (EV) (non-EV-A71 and non-Coxsaekievirus group A 16,CV-A 16) from 2016 to 2017 in Guangzhou,to provide the basis for its treatment,prevention and control.Methods Enteroviruses universal type,EV-A71 and CV-A16 were detected by real time reverse transeription-polymerase chain reaction in the specimens from HFMD suspected patients from 2016 to 2017.The positive specimens of non-EV-A71 and non-CV-A16 were amplified and sequenced based on 5'-untranslated region (UTR) region.The spectrum of serotypes was analyzed with BLAST in NCBI on the basis of 5'-UTR region.Results A total of 25779 specimens from HFMD patients were collected during 2016-2017,16 300 (63.23 ％) of which were positive.The positive rates of EV-A71,CV-A16,non-EV-A71 and non-CV-A16 were 4.57％ (1 178/25 779),12.70％ (3 274/25 779) and 45.96％ (11 848/25779),respectively.The average positive rate of non-EV-A71 and non-CV-A16 in 2017 was 55.68％,which was higher than that in 2016.Sequence analysis showed that there were 16 genotypes in 95 non-EV-A71 and non-CV-A16 positive specimen,including CV-A6,CV-A10,CV-A4,CV-A2,CV-A8,CV-A12,CV-A9,Coxsakievirus B5 (CV-B5),CV-B2,CV-B4,CV-B3,Echovirus 1 (E1),E16,E30,E2 and E18.CV-A6 (26.32％),and CV-A10 (15.79％) were the most common genotypes,followed by CV-A4 (6.32％)、CV-A8(4.21％),and CV-A2 (4.21％).Conclusions The infection rate of EV-A71 is very low during 2016-2017.From April to July 2016,there is a small peak of CV-A16 infection.The non-EV-A71 and non-CV-A16 enterovirus becomes the main causative agent of HFMD during 2016 to 2017.CV-A6 and CV-A10 are the most prevalent pathogens of non-EV-A71 and non-CV-A16 enterovirus.Research and monitoring of CV-A6,CV-A10 as the main non-EV-A71and non-CV-A16 virus should be strengthened.

Surveillance and drug resistance analysis of Salmonella in Guangdong province in 2015 / 中华微生物学和免疫学杂志

Objective To analyze the serotype distribution and antibiotic resistance characteristics of Salmonella strains isolated in Guangdong province for better understanding the condition of Salmonella infection in patients with diarrhea.Methods Fecal samples collected from patients with diarrhea in Guangdong province were used to isolate Salmonella strains.Biochemical analysis was performed to identify these isolated strains.Serotyping and antimicrobial susceptibility testing were carried out for further analysis of the isolated Salmonella strains.Results The rate of Salmonella infection was 7.64％in 2015, and the male to female patient ratio was 1.52∶1.A total of 2 377 patients of all age groups were positive for Salmonella infection and the patients aged 0-6 years accounted for 81.74％.The isolation rate of Salmonella strains in the summer and autumn was higher than that in the winter and spring (10.73％ vs 4.24％;X2=463.77, P<0.01).The Salmonella isolation rates in different areas were as follows: 16.82％ in Zhuhai, 15.85％ in Heyuan, 11.81％ in Yangjiang, 10.68％ in Jiangmen, 8.49％ in Zhongshan, 8.07％ in Maoming, 8.05％ in Jieyang, 7.35％ in Shaoguan, 6.97％ in Foshan, 6.03％ in Dongguan, 5.48％ in Guangzhou and 0.00％ in Zhanjiang.And the differences between different regions were statistically significant (X2=367.67, P<0.01).The 2 377 isolated Salmonella strains were classified into 108 serotypes except for oneSalmonella strain that could not be classified.The top four predominant serotypes were 4,5,12:i:-, Salmonella enteritidis,Salmonella stanley and Salmonella typhimurium.Most Salmonella strains were sensitive to imipenem, azithromycin, ceftazidime, cefotaxime and trimethoprim/sulfamethoxazole, but multidrug resistance was common among those strains.Conclusion Salmonella serotypes of 4,5,12:i:-and Salmonella enteritidis are the predominant pathogens causing human Salmonella infections in Guangdong province.Ceftazidime and cefotaximeare are preferred in the treatment of Salmonella infections.Surveillance for drug resistance in Salmonella should be strengthened as multidrug resistant strains have become a serious problem in Guangdong province.

Preparation of simulated stool specimens for proficiency testing scheme on the detection of Salmonella and Shigella / 中华微生物学和免疫学杂志

Objective To prepare simulated stool specimens for proficiency testing ( PT) by mix-ing lentils with Salmonella, Shigella and Escherichia coli strains and to establish an assessment scheme for the detection of Salmonella and Shigella in clinical samples. Methods Salmonella, Shigella and Escherich-ia coli strains were respectively spiked to lentils in Cary-Blair transport medium to create simulated stool specimens. Various ratios of Escherichia coli to Salmonella strains were spiked to lentils to prepare mixed simulated stool specimens. The accuracy and stability of prepared stool samples for PT were tested in-house. Results of sample detection were collected from participating laboratories for further external quality assess-ment. Results The Escherichia coli and Salmonella strains mixed at ratios of 100 ∶ 1 to 106 ∶ 1 could be ef-ficiently isolated from the media. Enrichment was needed in order to effectively isolate Salmonella strains from the media when the ratios of Escherichia coli to Salmonella strains were 104 ∶ 1 to 106 ∶ 1. Of the16 participating laboratories, 14 laboratories (87. 5%) received a grade of“satisfactory” and the other 2 labo-ratories (12. 5%) received a grade of “mainly satisfactory”. Conclusion The simulated stool specimens and the PT procedures designed in this study were suitable for proficiency testing program on the detection of Salmonella, Shigella and other similar microbes.

Study on Salmonella serotyping by use of Microsphere-based Liquid Array method / 中华流行病学杂志

Objective To understand the effect ofserotyping on Salmonella isolates,by use of Microsphere-based Liquid Array method,among diarrhea patients,in Guangdong.Methods Salmonella isolated from humans in Guangdong province were serotyped on the Microsphere-based Liquid Array platform with SSA kit.Results A total of 4 942 Salmonella strains with 189 serotypes,were identified in Guangdong province in 2010-2014.The top 100 serotypes accounted for 98.08％ (4 847/4 942) of all the strains.98％ of the top 100 species serotypes could completely be serotyped with SSA kit.In order to detect O antigen among 198 isolates with SSA kit,181 strains were carrying the O antigen,with the coincidence rate as 100％.However,under the SSA,98.32％ (528/537) of the H antigen could be detected and were consistent with the traditional serum agglutination test.The coincidence rate of fljB gene was 93.09％ (175/188),with false negative rate and false positive rate of fljB gene as 7.35％ (9/134) and 7.41％ (4/54) respectively.The coincidence rate of sdf gene and Vi gene were 100％.11 out of the 12 Salmonella strains could not be serotyped under the traditional methods but were successfully serotyped by the molecular serotyping method.Conclusions Using the SSA kit,more than 96％ of the anthropogenic Salmonella strains could be serotyped in Guangdong province.Comparing with the traditional methods,the coincidence rate of serotyping appeared over 98％.Under the Microsphere-based Liquid Array techniques,the molecular serotyping method appeared faster and more accurate on Salmonella serotyping than those traditional methods.

Study on Salmonella serotyping by use of Microsphere-based Liquid Array method / 中华流行病学杂志

Objective To understand the effect ofserotyping on Salmonella isolates,by use of Microsphere-based Liquid Array method,among diarrhea patients,in Guangdong.Methods Salmonella isolated from humans in Guangdong province were serotyped on the Microsphere-based Liquid Array platform with SSA kit.Results A total of 4 942 Salmonella strains with 189 serotypes,were identified in Guangdong province in 2010-2014.The top 100 serotypes accounted for 98.08％ (4 847/4 942) of all the strains.98％ of the top 100 species serotypes could completely be serotyped with SSA kit.In order to detect O antigen among 198 isolates with SSA kit,181 strains were carrying the O antigen,with the coincidence rate as 100％.However,under the SSA,98.32％ (528/537) of the H antigen could be detected and were consistent with the traditional serum agglutination test.The coincidence rate of fljB gene was 93.09％ (175/188),with false negative rate and false positive rate of fljB gene as 7.35％ (9/134) and 7.41％ (4/54) respectively.The coincidence rate of sdf gene and Vi gene were 100％.11 out of the 12 Salmonella strains could not be serotyped under the traditional methods but were successfully serotyped by the molecular serotyping method.Conclusions Using the SSA kit,more than 96％ of the anthropogenic Salmonella strains could be serotyped in Guangdong province.Comparing with the traditional methods,the coincidence rate of serotyping appeared over 98％.Under the Microsphere-based Liquid Array techniques,the molecular serotyping method appeared faster and more accurate on Salmonella serotyping than those traditional methods.

Application and evaluation of PFGE and MLVA subtyping methods on Brucella genotype in Guangdong Province,China / 中国人兽共患病学报

To compare and evaluate the discriminatory ability and potential value of pulsed field gel electrophoresis (PF-GE) and multiple locus VNTRs analysis (MLVA) on the genotyping of Brucella ,a total of 60 strains of Brucella and three standards (16M ,544A ,1330S) were genotyped simultaneously by PFGE and MLVA .The result indicated that the similarity coefficient among the 63 isolates was from 72 .1-100 .0% by PFGE ,and could distinguish three species of B .melitensis ,B .su-is and B .abortus at the similarity level of 94 .4% .There were 14 clusters and 29 PFGE types identified by PFGE with discrim-inatory index (DI) of 0 .957 5 at the similarity level of 100% ;the similarity coefficient among the 63 isolates was from 16 .9-100 .0% by MLVA ,and could distinguish three species of Brucella at the similarity level of 52 .3% .There were 8 clusters and 47 MLVA types identified by MLVA with discriminatory index (DI) of 0 .985 2 at the similarity level of 100% .It's suggested that PFGE and MLVA could be used to distinguish three species of Brucella in the similarity coefficient of certain ,but could not effectively distinguish the type in the same species .Both of these two methods could be used for Brucella molecular typing , but MLVA is better than PFGE for its relatively higher discriminating ability .

Study on polymorphism of Brucella melitensis biovar 3 strains isolated from Guangdong province / 中华微生物学和免疫学杂志

Objective To investigate the polymorphism of Brucella melitensis biovar 3 ( B.melitensis biovar 3) strains isolated from Guangdong province .Methods PCR assays followed by agar-ose gel electrophoresis and capillary electrophoresis based on the multiple locus variable number of tandem repeats (VNTR) analysis (MLVA) were performed to analyze 43 clinic isolates of B.melitensis biovar 3 strains isolated from clinical patients in Guangdong province .Results MLVA typing showed that the simi-larities of the analyzed locus among 43 strains of Brucella ranged from 68.2%to 100%.32 genotypes identi-fied among the isolates were identical (100%similarity).27 out of 43 strains (62.8%) were single geno-types, while the other 16 strains (37.2%) belonged to 5 other genotypes with 2 to 5 strains in each of them . Conclusion B.melitensis biovar 3 isolates showed polymorphism distribution in Guangdong province as in-dicated by MLVA typing analysis .Single-genotype isolates accounted for 62.8% of all studied strains.No predominant genotype was found among all isolates .

Development and evaluation of a DNA microarray for Listeria monocytogenes detection / 中华微生物学和免疫学杂志

Objective To develop a rapid and sensitive DNA microarray for Listeria monocytogenes detection.Methods A DNA microarray was developed using gyrB,ISR,16S rRNA,23S rRNA,hlyA,iap and prfA as the target genes and tested against 18 different species of known reference for repeatability,sensitivity,and specificity to verify the effectiveness of the chip.Results After testing of samples by the LM array,results show that the 70 mer Oligos synthesized by IDT are superior to the Oligos synthesized by Sagon with respect to both probe spotting or samples detection.The comparison of 3 spotting probe concentrations of 10 μmol/L,40 μmol/L and 80 μmol/L demonstrated that the 10 pmol/L probes result in good detection signals equivalent to the 40 μmol/L and 80 μmol/L probes.The repeatability and sensitivity evaluated by sample testing on the LM array revealed that the chips developed in this study have good repeatability and the lower limit of sample detection is 0.9 ng DNA.The LM array can distinguish clearly and definitively between Listeria and non-Listeria bacteria in the sample.Conclusion The microarray is able to rapidly detect and identify Listeria monocytogenes.

Molecular characteristic of dominant serotypes of Vibrio parahaemolyticus isolated from foodborne disease outbreaks in Guangdong province / 中华微生物学和免疫学杂志

ObjectiveTo study the toxin genes and pandemic group distribution as well as the genetic correlation between the major serotypes( O3:K6,O1:Kut,O4:K8 ) of Vibrio parahaemolyticus (VP) isolated from the outbreaks of Guangdong province.MethodsThe tdh and trh genes,GS-PCR and orf8 gene were detected on the 62 isolates sourced from patient and seafood occurred in the 23 outbreaks during 2008-2010.44 isolates of which were analyzed on PFGE digested by Not Ⅰ enzyme.ResultsToxin genes distributions suggested that 96.8% (60/62)isolates were tdh+,trh-.Three tdh+ isolates sourced from seafood were found.Pandemic group distribution suggested that 97.2% (35/36) O3:K6,5.88% (1/17) O4:K8,66.7% (8/9)O1:Kut serotype was GS-PCR+ and/or orf8+,respectively.PFGE analysis suggested that 44 isolates were separated into 3 clusters,of which the similarity of PFGE profile was 80.5% in the pandemic group cluster constructed by 28 isolates,the similarity between pandemic group and non-pandemic group was 59.5%.Pandemic group of O3:K6,O1:Kut as well as 04:K8 isolated on some outbreaks were processing the same PFGE profiles.ConclusionThe characteristic of toxin genes of major serotypes VP isolated in the outbreaks of Guangdong province form 2008-2010 was tdh- present and trh- absent.Within the pandemic group,O3:K6 and O1:Kut were the major serotypes.In single outbreak,isolates belongs to pandemic group but with different serotype seem to be close correlations.

Analysis of the fatty acid components of Brucella strains in Guangdong province / 中国人兽共患病学报

To explore the possibility to type the Brucella strains isolated in Guangdong province with analytical method to detect the fatty acid components and to collect the basic data of fatty acid components of Brucella strains, 29 strains of Brucella were selected for analysis on the bacterial fatty acid components and the cluster analysis on the collected data was performed with Sherlock analysis soft-ware (MIDI). It was demonstrated that the main fatty acid components of Brucella strains isolated in Guangdong province were 19∶0 cycloω8c acid, 16∶0 acid and 18∶0 acid. The content of 19∶0cycloω8c acid was highest in B.abortus, followed by B.melitensis and lowest in B.suis.-In addition, the content differences of 19∶0cycloω8c and 18∶0 acid between B. melitensis and Brucella suis were statistically significant; and that of 19∶0cycloω8c and 18∶0 acid between strains isolated in 1965 and those isolated in recent 3 years was statistically significant. It was also shown that the fatty acid components of Brucella strains were stable, but the contents of fatty acid components were different in different species.-It is evident that at certain euclidean distance, 3 species of Brucella can be differentiated in species level.