Objective To avoid false positive detection ofVibrio cholerae and improve the detection correct rate.Methods 1~7 months of 2013 were randomly selected,the national various provinces and cities CDC to China cholera CDC positive screening 14 strains.LB nutrient agar 12 hours,take single colony to Vibrio cholera serum agglutination,extraction of strain DNA at the same time boiled template method.For Vibrio 16SrDNA sequence and design primers for PCR detection of Vib-rio,16SrDNA,electrophoresis were used to observe the 16SrDNA products,16SrDNA positive products sent to sequencing company sequencing,sequencing results were Blast comparison on the NCBI website for the analysis and comparison of ser-um agglutination and Blast alignment.Results 12 strains was positive for agglutination and 2 strains of non agglutination in 14 strains.The Vibrio 16SrDNA amplification,electrophoresis were used to observe the 14 isolates that were amplified frag-ment corresponding,that the selected strains were vibrio.The 16SrDNA positive products were 14 strains,and the sequen-cing the Blast results:2 strains of bacteria were Vibrio harveyi for non agglutination,in 12 positive strains of serum aggluti-nation;1 strains was Vibrio natriegen and 11 strains wereVibrio cholerae .Conclusion Detection ofVibrio cholerae cholerae combined with serum agglutination and gene sequencing can avoid false positive result ofVibrio cholerae ,and improve cor-rect rate of the detection.

Objective To compare and analyze the phylogenetic tree and sequence variant characteristics of Klebsiella species between 16S rDNA and rpoB. Methods Eighteen isolates identified as genus Klebsiella (with 15 of K. Pneumoniae and 3 of K. Oxytoca) by automated biochemical tests were selected. DNA were extracted, 16S rDNA and rpoB genes were amplified and sequenced with Klebsiella 16S rDNA and rpoB primers. Together with already published 8 species of Klebsiella and 9 species of Enterobacteriaceae 16S rDNA and rpoB sequences from GenBank, totally 35 sequences of 16S rDNA and rpoB respectively, phylogenetic trees were constructed with MEGA 4.0 to the analysis of groups. DNAStar/MegAlign was used for comparison of variable regions of 16S rDNA and rpoB, with analysis of degree of divergent at the same time. Results As for all 35 sequences, both 16S rDNA and rpoB phylogenetic trees divided Klebsiella species into three groups, 15 of K. Pneumoniae in this study and 6 of K. Pneumoniae from GenBank (except for K. Oxytoca and K. Mobilis) cluster to group Ⅰ, K. Oxytoca and K. Mobilis were cluster to group Ⅱ and Ⅲ, respectively. In rpoB phylogenetic tree, no matter group Ⅰ and group Ⅱ, or subgroup within group Ⅰ, the bootstrap values in each node of rpoB phylogenetic tree is obviously higher than that of 16S rDNA. Moreover, as for cluster to K. Oxytoca, rpoB is better than 16S rDNA. Analysis nucleic acid sequences of Klebsiella species, with 41 variable regions and 4 most significant regions were found within the Klebsiella 16S rDNA, while rpoB with 63 variable regions, and 1 most significant region. The similarity of 16S rDNA and rpoB within Klebsiella were 95.9%-100% and 90.2%-100% respectively. Further analysis divergent degree of 16S rDNA and rpoB within Klebsiella, the divergent value of rpoB (0-10.6) is higher than that of the 16S rDNA(0-4.0). Conclusion As for molecular classification and identification within KlebsieUa species, rpoB has more advantages than 16S rDNA.

Objective To identity the distribution of enterotoxin and hemolysin,as well as the clonal complexes and drug resistance of the strains of methicillin-resistant Staphylococcus aureus (MRSA) in Maanshan region.Methods Automatic enzyme-linked fluorescent assay system and PCR technology were used to identify the distribution of enterotoxin and hemolysin genes.Seven Staphylococcus aureus hourskeeping genes were choosed as the target genes for multilocus sequence typing (MLST) on 34 strains of MRSA and 3 strains of methicillin-sensitive Staphylococcus aureus (MSSA),comparing the data with the online database and obtaining the sequence typing (ST),conducting affinity analysis on its ST based on eBURST,testing in agar dilution method the drug resistance of MRSA against 12 antibiotics.Results 50.9％ of the 210 Staphylococcus aureus strains were enterotoxin positive,and 97.1％ of them carried hemolysin genes as all 51 strains of MRSA carried hemolsin genes.The 34 MRSA strains were divided into 10 STs,ranging in sequence ST239 (47.1％,16/34),ST5 (17.6％,6/34).Three MSSA strains belonged to ST188,ST1281 and ST7,respectively.Seventeen strains from the patients were divided into 6 STs,ranging in sequence ST239 (35.3％,6/17) and ST5 (29.4％,5/17).Twenty strains from food sources were divided into 9 STs,ranging in sequence ST239 (45.0％,9/20) and ST7 (15.0％,3/20).STs of ST585,ST630 and ST239 were close in affinity,while the rest were distant in affinity.Except for vancomycin,all the strains were found with drug resistance to varying extent to the 10 antibiotics tested.Conclusion Existence of Staphylococcus aureus hemotoxin was universal; ST239 was the main predominant MRSA in Maanshan region,with distant affinity among the STs.

Objective To develop methodology of both multiple PCR and real-time SYBR green PCR for the detection of Vibrio cholerae (V.cholerae) serogroups non-O1 and non-O139.Methods The outer membrane protein gene (ompW) specific for V.cholerae,as well as O antigen rfb genes specific for both O1 and O139,were used for the design of the PCR primers.Both multiple PCR and real-time SYBR green PCR systems were used to detect both O1 and O139.Specific rfb genes and ompW were developed to evaluate their specificity,limit of detection,reproducibility and consistency.Results We established multiple PCR and real-time SYBR green PCR methods.According to the specific electrophoretic bands (multiple PCR) and the specific melt curve temperature (real-time SYBR green PCR),both methods could specifically detect the non-O1,non-O139 V.cholerae,and to differentiate them from O1,O139 V.cholerae,other five Vibrios and 3 intestinal bacteria.The detection limits were 7 × 104 cfu/ml (multiple PCR) and 7 × 102 cfu/ml (real-time SYBR green PCR),with statistically significant difference seen (P＜0.05).For the reproducibility of real-time SYBR green PCR,the external coefficient variation ranging from 0.22％ to 0.92％ while the internal coefficient variation ranging from 0.27％ to 1.41％.370 strains of non-O1,non-O139 V.cholerae,were detected,with both consistency rates as 100％.Conclusion Both multiple PCR and real-time SYBR green PCR could detect non-O1,non-O139 V.cholerae,rapidly,specifically,and reproducibly,that could all be used for the detection and identification of non-O 1,non-O 139 under different conditions.

OBJECTIVETo analyze the etiologic characteristics of O1/O139 Vibrio cholerae in Guangdong province in 2009-2013.

METHODSIsolates from cholera cases and from the environment surveillance points were investigated by serological typing, antibiotic susceptibility testings, toxic genes detection and molecular typing to analyze the similarities and differences of the identified species.

RESULTSTotally, 190 isolations of O1/O139 V. cholerae were obtained from cholera cases (16 strains) and environmental samples (174 strains) in Guangdong province in 2009-2013. The sero-types would include Inaba (3 isolates), Ogawa (7 isolates) and O139 (6 isolates) in all the isolates from the cholera cases. Ten strains from the ctxA positive cases were detected by PCR. Two Ogawa strains carried incomplete CTXΦ phage. Results from the antibiotic susceptibility test indicated that 5 strains were absolutely sensitive to 11 antibiotic discs in vitro, while another 3 strains were simultaneously resistant to 4 antibiotic discs. Except for 2 stains, all the O139 strains from the environment were ctxA negative, detected by PCR. Incomplete CTXΦ phage was found in the Inaba (53 isolates), Ogawa (22 isolates) and O139 (2 isolates), respectively. Results from the antibiotic susceptibility test exhibited that 25 strains were resistant simultaneously to 4 and/or more antibiotic discs, especially the Inaba strains from the seafoods(13 isolates). 2 Inaba strains from seafood were simultaneously resistant to 7 antibiotic discs. Results from PFGE molecular typing indicated that the PFGE types digested by Not I expressed significant diversity. Inaba and O139 strains from cases were gathered in the same clusters, while the Ogawa strains from cases scattered in different clusters but no significant correlation among these strains were found. Our results suggested that a distant genetic relationship might exist between these two different sources strains.

CONCLUSIONComplex and diverse as the virulence genes and genetic characteristics and with the grim situation of multi-drug resistant strains, all seemed important to strengthen the surveillance programs on the variation of strain types and antibiotics resistance of O1/O139 V. cholerae in Guangdong province.

Bacterial Typing Techniques ; China ; epidemiology ; Cholera ; epidemiology ; microbiology ; Drug Resistance, Multiple, Bacterial ; Genes, Bacterial ; Humans ; Microbial Sensitivity Tests ; Vibrio cholerae ; classification ; drug effects ; isolation & purification ; Virulence

Background China is a big country in the production and use of antibiotics. The abuse of antibiotics enables bacteria in water environment to acquire resistance, and promotes the generation and spread of antibiotics resistance genes (ARGs). The problem of antibiotic-resistant bacteria is increasingly serious and has become a public security issue of global concern. Water environment is a huge reservoir of antibiotics and ARGs. It is of great significance to study the pollution of antibiotics and ARGs in water to protect water sources and optimize the biosecurity of drinking water. Objective To evaluate the detection of antibiotics and ARGs in typical water sources, and to explore the relationship between antibiotics and ARGs. Methods Water samples were collected in Heilongjiang, Liaoning, and Hubei provinces during the wet season (from August to October) in 2020. Ten water samples were collected from each of the three places, and a total of 30 water samples were collected in this study. Five kinds of antibiotics, including macrolides, quinolones, sulfonamides, tetracycline, and β-lactam, were detected by ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The integron (Intl1), 16S rRNA, and 6 kinds of ARGs were detected by quantitative real-time PCR (qPCR). The ARGs include one macrolide ARGs (ermB), one β-lactam ARGs (bla_TEM), two tetracycline ARGs (tetC, tetQ), and two sulfonamide ARGs (sul1, sul2). Results The types of detected antibiotics varied by the three regions, and the concentration ranges of the same antibiotics varied by the three regions (P<0.05). The concentration ranges of selected five kinds of antibiotics were 0.11-418.80 ng·L⁻¹ in region A, 0.12-23.23 ng·L⁻¹ in region B, and 4.69-285.75 ng·L⁻¹ in region C, respectively. The detection rates of all six ARGs were 100%. The absolute abundance of ARGs in region A ranged from 22.56 to 94355.91 copies·mL⁻¹, that in region B ranged from 27.99 to 80584.32 copies·mL⁻¹, and that in region C ranged from 41.99 to 111068.19 copies·mL⁻¹. The absolute abundance of bla_TEM was higher among the ARGs, followed by sul1 and sul2. In addition, the absolute abundance of Intl1 was also at a high level. The results of correlation analysis showed that the abundance of ARGs was positively correlated with each other. There was no correlation between specific antibiotics and corresponding ARGs. There was a positive correlation between Intl1 and sul1 or sul2 (P<0.05). Conclusion The types and concentrations of antibiotics and the abundance of ARGs in source water vary greatly in the study areas. The association between antibiotics and ARGs is uncertain. Intl1 may play an important role in the horizontal transfer of sulfonamide resistance genes.

OBJECTIVETo investigate the infection status and virulent genes of Aeromonas in patients with acute diarrhea in Pudong New Area, Shanghai.

METHODSIn 2012, stool samples were collected from diarrhea patients in 12 sentinel hospitals in Pudong for the detections of 13 pathogens causing diarrhea, and the detections of 5 diarrhea related virulent genes were conducted for Aeromonas isolates.

RESULTSA total of 101 patients were infected with Aeromonas in 2533 patients (4.0%). A total of 101 Aeromonas strains were isolated, including 17 Aeromonas hydrophila strains (18.8%), 44 Aeromonas veronii biovar sobria strains (52.5%) and 12 Aeromonas caviae strains (29.7%). And 44 coinfections with other pathogens were detected. Aeromonas infection mainly occurred in summer and in people aged ≥20 years. Among the patients infected with Aeromonas, 71 (70.3%) had watery diarrhea, 20 (19.8%) had vomiting and 11 (10.9%) had fever. Virulent genes detection showed that 95.0% of the Aeromonas. strains carried virulent genes, and the detection rates of hlyA, aerA, act, alt, and ast genes were 5.9%, 6.9%, 67.3%, 42.6% and 13.9%, respectively.

CONCLUSIONSHigh incidence of Aeromonas infection was found in the patients with acute diarrhea in Pudong, and a high proportion of coinfections with other pathogens was detected too. Most Aeromonas strains carried virulent genes, and the distribution varied.

Aeromonas ; genetics ; Aeromonas hydrophila ; genetics ; China ; epidemiology ; Diarrhea ; microbiology ; Gram-Negative Bacterial Infections ; epidemiology ; microbiology ; Humans ; Seasons ; Virulence ; genetics ; Young Adult