Identification and analysis of 2 Corynebacterium diphtheria strains in Guangdong Province / 中华预防医学杂志

Objective: To identify and analyze two strains of C. diphtheriae in Guangdong Province by combining whole genome sequencing with traditional detection methods. Methods: The C. diphtheriae was isolated from Guangzhou in 2010 and Zhuhai in 2020 respectively. Isolates were identified by API Coryne strips and MALDI-TOF-MS. Genomic DNA was sequenced by using Illumina. The assembly was performed for each strain using CLC software. J Species WS online tool was used for average nucleoside homology identification, then narKGHIJ and tox gene were detected by NCBI online analysis tool BLSATN. MEGA-X was used to build a wgSNP phylogenetic tree. Results: GD-Guangzhou-2010 was Belfanti and GD-Zuhai-2020 was Gravis. ANIb between GD-Guangzhou-2010 and C. belfantii was 99.61%. ANI between GD-Zhuhai-2020 and C. diphtheriae was 97.64%. BLASTN results showed that the nitrate reduction gene narKGHIJ and tox gene of GD-Guangzhou-2010 was negative, while GD-Zhuhai-2020 nitrate reduction gene narKGHIJ was positive. There were two obvious clades in wgSNP phylogenetic tree. The first clades included all Mitis and Gravis types strains as well as GD-Zhuhai-2020. The second clades contained all isolates of C.belfantii, C.diphtheriae subsp. lausannense and GD-guangzhou-2010. Conclusion: Two non-toxic C. diphtheriae strains are successfully isolated and identified. The phylogenetic tree suggests that GD-Guangzhou-2010 and GD-Zhuhai-2020 are located in two different evolutionary branches.

Analysis of the brucella pathogen and its molecular genotype in Guangdong province / 中国地方病学杂志

Objective To analysis the etiology and molecular classification of brucella strains isolated in Guangdong province in 2010.Methods The strains of 19 brucella were verified and identified by some methods including traditional biology phenotype confirmation,PCR amplification and pulsed field gel electrophoresis (PFGE).Results On phenotype level,4 strains were brucella melitensis biovar 1,2 strains were brucella suis biovar 3,and the rest were brucella melitensis biovar 3,which were specific B genes positive strains,and the PFGE typing similar values ranging from 67.9％ to 100％.In addition to the four strains from Zhuhai for the outbreak,the homology was 100％,and the rest were sporadic cases.Conclusions Brucella cases,in Guangdong province,are highly sporadic and dispersed outbreaks.Compared with a few years ago,it shows species diversification,and brucella melitensis biovar 3 is still the dominant serotype.PFGE can be used to distinguish the three species of brucella,but it can't effectively distinguish the allotypes.

Surveillance and analysis on the pathogenic features of Salmonella in Guangdong province in 2010 / 中华预防医学杂志

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

Surveillance on Salmonella infection in Guangdong province, 2008-2009 / 中华流行病学杂志

Objective To understand the infection of Salmonella (S.) in patients with diarrhea and outbreaks caused by Salmonella to identify the serotypes, resistance to antibiotics and PFGE types of the strains from the surveillance program in Guangdong province. Methods S. strains from patients with diarrhea were detected, and all the positive strains collected in routine and outbreak surveillance programs, were tested by serum agglutination, antibiotic susceptibility and PFGE.Results 71 S. strains were isolated from 1922 stool samples in 2008, with positive rate as 3.7%.85 S. strains were isolated from 2110 stool samples in 2009, with positive rate as 4.0%. All the 156 strains were divided into 37 serotypes, with S. serotype typhimurium and enteritidis as the most common serotypes. 10 incidents of food poisoning were detected, of which 4 were caused by enteritidis and 3 by typhimurium. A suspected outbreak by enteritidis was discovered and under epidemiological investigation. The findings indicated that 2 of the 4 patients from this outbreak were infected with identical enteritidis isolates. 80% of the 229 isolates were found susceptible to cephalosporins and quinoione and 59.3% of them were muitiresistant to the antibiotics. Conclusion S. enteritidis and S. typhimurium were the most common serotypes that caused infectious diarrhoea and food poisoning in Guangdong province.

Etiologic characteristics of Vibrio parahaemolyticus strains causing outbreaks and sporadic cases in Guangdong,2009 / 中华流行病学杂志

Objective To study the serotypes,virulence features and molecular characteristics of Vibrio parahaemaolyticus isolated in food poisoning cases and surveillance program on diarrhea patients in Guangdong,2009.Methods 95 Vibrio parahaemolyticus strains from food poisoning cases and 15 strains from surveillance program on diarrhea patients were serotyped and detected for tdh(thermostable direct hemolysin,tdh)and trh(tdh-related hemolysin gene,trh)by PCR.81 sero-variant Vibrio parahaemolyticus strains were selected through PFGE subtyping.Results There were 15 Vibrio parahaemolyticus strains isolated from surveillance program on diarrhea patients and 95 strains were isolated from 11 Vibrio parahaemolyticus-caused food poisoning cases in 2009.Among these strains,O3:K6(46.67％ and 44.21％)and O4:K8(33.33％ and 28.42％)were the dominant serotypes,but not the 7 food-borne strains.There were 93(84.54％)tdh +trh-,13(11.81％)tdh-trh-,and 3(3.65％)tdh+ trh + strains.The similarity value was between 57.7％ to 100.0％ of the 81 strains after PFGE sub-typing method and 36 PFGE subtypes were identified.PFGE001 and PFGE029 appeared to be the dominant subtypes.Conclusion O3:K6 and O4:K8 were the most dominant serotypes in Vibrio parahaemolyticus-caused diarrhea and food poisoning cases in Guangdong and tdh were detected in most of the strains.Dominant PFGE subtypes were causing both sporadic and outbreak cases in different areas in Guangdong province.

Surveillance program on and the distribution related to the virulence-associated genes of Vibrio cholerae in estuary of Pearl River / 中华流行病学杂志

Objective To understand the distribution,molecular characteristics and virulence genes of the O1 and O139 Vibrio cholerae isolates from the Pearl River Estuary water.Methods Vibrio cholerae isolates collected from the Pearl River estuary waters from January 2009 to December 2010,were tested by PCR for eight virulence-related genes,including cholera toxin(ctxA),zonula occludens toxin(zot),accessory cholera enterotoxin(ace),hemolysin(hlyA),toxin-coregulated pilus (tcpA),outer membrane protein(ompU),and the regulatory protein genes(tcpⅠ,toxR).Genetic relation was assessed by pulsed-field gel electrophoresis(PFGE)and the patterns were clustered by BioNumerics.Results From 1152 aquatic samples,69 isolates were identified,including 41 Inaba,18 Ogawa and 10 O139.All the isolates showed ctxA negative,while the hlyA and toxR genes were positive in all the isolates.34.15％(14/41)of the Inaba strains were hlyA + toxR + ompU + ace + zot + tcpI+,while 66.67％(12/18)belonged to Ogawa strains and 70％(7/10)of the O139 strains were hlyA + toxR+.Through PFGE analysis,the O1 isolates formed three clusters in this study.The patterns of O1 isolates differed widely,with the similarity as 72.8％-100.0％,while the patterns of O139 isolates having the similarity of 69.9％-95.5％.Conclusion The non-toxigenic O1 and O139 V.cholerae had a wide distribution in the environment of Pearl River estuary water during the nonepidemic period of cholera.All the aquatic isolates presented diversities on the related virulent genes.

Etiologic detection and epidemiological analysis of one suspected case of diphtheria in Guangdong province / 中华预防医学杂志

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

Development of a DNA microarray for detecting 8 common species of food-borne bacterial pathogens in south China / 南方医科大学学报

<p><b>OBJECTIVE</b>To prepare a DNA Microarray that can detect 8 common species of food borne bacterial pathogens in south China.</p><p><b>METHODS</b>All the 70mer oligo probes were designed on the characteristic genome loci of the 8 species of food borne bacterial pathogens. Eight subarrays corresponding to the 8 food borne bacterial pathogens were spotted onto the slide and integrated into a pan-array on the chip. A number of identified and known bacterial samples from the storage bank were selected for the validation test.</p><p><b>RESULTS</b>Based on the PPR ranking, for LM sub-array, the PPR of the 3 Listeria bacteria LM, Lin and Liv was 68.8%, 51.8% and 59.6%, respectively, while that of the non-Listeria bacterial samples was all below 43%. For VC sub-array, the PPR of VC sample was 54.1% and that of the non-VC bacterial samples was lower than 17.2%. For VP sub-array, the PPR was 66.7% for VP sample and below 24.2% for non-VP bacterial samples. For Sal sub-array, the PPR was 55.9% for Sal sample and below 50.5% for non-Sal bacterial samples. For Shi sub-array, the PPR of Shi sample and the non-Shi bacterial samples was 53.8% and below 36.6%, respectively. For SA sub-array, the PPR of SA sample and non-SA bacterial samples was 65.2% and below 22.7%, respectively. For CJ sub-array, the PPR of the 2 Campylobacter bacteria CJ and CC were 88.2% and 58.8%, respectively, and that of the non-Campylobacter bacterial samples was lower than 35.3%. For EC sub-array, the PPR of EC sample was 47.9%, and that of the non-EC bacterial samples was lower than 41.6%. Evaluation of the Biosafood-8 chip developed in this study by 18 biological samples from different origins demonstrated its good specificity and accuracy in the identification of the pathogens.</p><p><b>CONCLUSION</b>The chip we developed can clearly differentiate the target food borne pathogenic bacteria and non-target bacteria and allows specific and accurate identification of the species of the tested bacteria isolates.</p>

MLST typing of Streptococcus suis isolated from clinical patients in Guangdong Province in 2005 / 南方医科大学学报

Intensive surveillance of human S.suis infection was carried out in July and August of 2005 in Guangdong Province, which coincided with the Sichuan outbreak. Five isolated cases of human infections were identified during this period, from which 5 S. suis serotype 2 isolates were recovered. MLST analysis showed that these 5 isolates shared identical sequences of 6 MLST housekeeping genes except for one point mutation found within the thrA gene fragment, a neutral mutation (TTA to TTG) in the third nucleotide (360 nt) of the codon for leucine. MLST analysis identified 2 sequence types in the Guangdong sporadic infection. Three Guangdong isolates L-SS002, L-SS003 and L-SS005 belonged to ST7, while the other two isolates L-SS004 and L-SS006 belonged to ST1, but they all belonged to ST1 clonal complex. This finding represents a striking feature that differs from the Sichuan outbreak caused by a single ST7 SS2 clone. The 3 isolates of ST7 were probably imported from Sichuan Province, while the origin of the other 2 isolates of ST1 still remain to be clarified.

Distribution and molecular characteristics of Vibrio cholerae serogroups O1 and O139 isolates in estuary of Pearl River / 中华流行病学杂志

<p><b>OBJECTIVE</b>Through systematic monitoring of the number and strain types of O1 and O139 Vibrio cholerae in the Pearl River estuary waters to analyze it's relevance with the temperature of environment, and the relevance between strains in water and isolates during outbreaks and epidemics as well as to estimate the methods used for environmental water detection and the potential role in cholera surveillance program.</p><p><b>METHODS</b>Twenty-four stations along the Pearl River were selected and the water samples were collected monthly from March 2006 to February 2007. V. cholerae O1 and O139 strains were isolated from the samples. Real-time PCR established in our laboratory was used to detect V. cholerae O1 and O139. Air temperature and water temperature were collected during sampling. Pulsed field gel electrophoresis (PFGE) was applied in molecular typing of the isolates.</p><p><b>RESULTS</b>862 water samples were collected during the study period. A total number of 77 O1 and O139 V. cholerae were isolated in 67 water samples and the positive rates were 7.77% for isolation and 26.33% for real-time PCR. Seasonal trend of positive rates by month were approximately coincident with the change of water temperature. The positive rates in the stations in urban area were higher than those in other areas. Toxigenic O139 strains were found in one station located in downstream of a marine market. Most of the O1 and O139 isolates were non-toxigenic. No trend of seasonal variation of the strains was noticed. Within these 75 isolates, 49 PFGE patterns were identified and the patterns differed widely with the similarity of 57.4% - 100%.</p><p><b>CONCLUSION</b>V. cholerae existed as the natural habitat in estuary water of the Pearl River and showed obvious genetic diversity. Data from monitoring waters might show the separation of strains with certain seasonal association. But the crowd did not show the relationship between the infections. Results from water surveillance program might provide indicators on the appearance of cholera pathogen which might be used in assessing the environmental risk of cholera epidemics as well as the alert of cholera.</p>

Development and application of real-time polymerase chain reaction to detect Vibrio cholerae O1 and O139 in river water / 中华流行病学杂志

<p><b>OBJECTIVE</b>To develop a real-time SYBR Green polymerase chain reaction (PCR) for detection of Vibrio cholerae serogroups O1 and O139, and to evaluate its reliability through detection of estuary water samples.</p><p><b>METHODS</b>O antigen rfb genes specific for O1 and O139 were used for the design of PCR primers. The real-time SYBR Green PCR system in detecting O1 and O139 specific rfb genes in one tube was developed, and its sensitivity, specificity and reproducibility were evaluated. The ability of the real-time PCR in detection of estuary water samples was compared with the routine PCR and bacteria isolation.</p><p><b>RESULTS</b>The amplification of O1 or O139 specific target gene could be detected according to the melt curve temperature of amplicons. No amplification was observed in the templates of other 10 non-cholerae vibrios. When comparing to the real-time PCR to bacteria isolation in detection of 524 estuary water samples, it showed high sensitivity, plus also positive in real-time PCR detection among all the samples in which bacteria of O1 or O139 were isolated.</p><p><b>CONCLUSION</b>The real-time SYBR Green PCR could be used as the first step of rapid environment screen of V. cholerae in water samples thus might enhance the efficiency of isolation in screening of large amount of water samples.</p>