High-resolution mapping of age- and gender-specific risk of Clonorchis sinensis infection risk in Guangdong, China: a geostatistical modeling study.

BACKGROUND: The latest national survey on the distribution of human parasites in China demonstrated that Guangdong was among the endemic provinces with the highest Clonorchis sinensis infection rates. High-resolution, age- and gender-specific risk maps of the temporal and spatial distributions are essential for the targeted control work. METHODS: Disease data on the prevalence of C. sinensis infection from 1990 onwards, either age- and gender-specific or aggregated across age and gender, were collected through systematic review and four large-scale surveys in Guangdong Province. Environmental and socioeconomic variables were obtained from open-access databases and employed as potential predictors. A Bayesian geostatistical model was developed to estimate the C. sinensis infection risk at high spatial resolution. RESULTS: The final dataset included 606 surveys at 463 unique locations for C. sinensis infection. Our findings suggested that following an initial increase and stabilization, the overall population-adjusted prevalence had declined to 2.2% (95% Bayesian credible interval: 1.7-3.0%) in the period of 2015 onwards. From 2015 onwards, moderate and high infection risks were found in the northern regions (e.g. Heyuan and Shaoguan cities) and the southern Pearl River Delta (e.g. Foshan, Zhongshan, Zhuhai and Jiangmen cities), respectively. Age- and gender-specific risk maps revealed that males had a higher infection risk than females, and the infection risk was higher in adults compared to children. CONCLUSIONS: Our high-resolution risk maps of C. sinensis infection in Guangdong Province identified the spatial, temporal, age and gender heterogeneities, which can provide useful information assisting tailored control strategies.

The adenosine analog prodrug ATV006 is orally bioavailable and has preclinical efficacy against parental SARS-CoV-2 and variants.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus driving the ongoing coronavirus disease 2019 (COVID-19) pandemic, continues to rapidly evolve. Because of the limited efficacy of vaccination in prevention of SARS-CoV-2 transmission and continuous emergence of variants of concern (VOCs), orally bioavailable and broadly efficacious antiviral drugs are urgently needed. Previously, we showed that the parent nucleoside of remdesivir, GS-441524, has potent anti-SARS-CoV-2 activity. Here, we report that esterification of the 5'-hydroxyl moieties of GS-441524 markedly improved antiviral potency. This 5'-hydroxyl-isobutyryl prodrug, ATV006, demonstrated excellent oral bioavailability in rats and cynomolgus monkeys and exhibited potent antiviral efficacy against different SARS-CoV-2 VOCs in vitro and in three mouse models. Oral administration of ATV006 reduced viral loads and alleviated lung damage when administered prophylactically and therapeutically to K18-hACE2 mice challenged with the Delta variant of SARS-CoV-2. These data indicate that ATV006 represents a promising oral antiviral drug candidate for SARS-CoV-2.

Detection of Major SARS-CoV-2 Variants of Concern in Clinical Samples via CRISPR-Cas12a-Mediated Mutation-Specific Assay.

Objectives: Emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants pose a great threat and burden to global public health. Here, we evaluated a clustered regularly interspaced short palindromic repeat-associated enzyme 12a (CRISPR-Cas12a)-based method for detecting major SARS-CoV-2 variants of concern (VOCs) in SARS-CoV-2 positive clinical samples. Methods: Allele-specific CRISPR RNAs (crRNAs) targeting the signature mutations in the spike protein of SARS-CoV-2 are designed. A total of 59 SARS-CoV-2 positive oropharyngeal swab specimens were used to evaluate the performance of the CRISPR-Cas12a-mediated assay to identify major SARS-CoV-2 VOCs. Results: Compared with Sanger sequencing, the eight allele-specific crRNAs analyzed can specifically identify the corresponding mutations with a positive predictive value of 83.3-100% and a negative predictive value of 85.7-100%. Our CRISPR-Cas12a-mediated assay distinguished wild-type and four major VOCs (Alpha, Beta, Delta, and Omicron) of SARS-CoV-2 with a sensitivity of 93.8-100.0% and a specificity of 100.0%. The two methods showed a concordance of 98.3% (58/59) with a κ value of 0.956-1.000, while seven (11.9%) samples were found to be positive for extra mutations by the CRISPR-based assay. Furthermore, neither virus titers nor the sequences adjacent to the signature mutations were associated with the variation of fluorescence intensity detected or the false-positive reaction observed when testing clinical samples. In addition, there was no cross-reaction observed when detecting 33 SARS-CoV-2 negative clinical samples infected with common respiratory pathogens. Conclusions: The CRISPR-Cas12a-based genotyping assay is highly sensitive and specific when detecting both the SARS-CoV-2 wild-type strain and major VOCs. It is a simple and rapid assay that can monitor and track the circulating SARS-CoV-2 variants and the dynamics of the coronavirus disease 2019 (COVID-19) pandemic and can be easily implemented in resource-limited settings.

Comparison of model-specific histopathology in mouse models of COVID-19.

A novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has been identified as the causative agent of the current coronavirus disease 2019 pandemic. Development of animal models that parallel the clinical and pathologic features of disease are highly essential to understanding the pathogenesis of SARS-CoV-2 infection and the development of therapeutics and prophylactics. Several mouse models that express the human angiotensin converting enzyme 2 (hACE2) have been created, including transgenic and knock-in strains, and viral vector-mediated delivery of hACE2. However, the comparative pathology of these mouse models infected with SARS-CoV-2 are unknown. Here, we perform systematic comparisons of the mouse models including K18-hACE2 mice, KI-hACE2 mice, Ad5-hACE2 mice and CAG-hACE2 mice, which revealed differences in the distribution of lesions and the characteristics of pneumonia induced. Based on these observations, the hACE2 mouse models meet different needs of SARS-CoV-2 researches. The similarities or differences among the model-specific pathologies may help in better understanding the pathogenic process of SARS-CoV-2 infection and aiding in the development of effective medications and prophylactic treatments for SARS-CoV-2.

Viral infection and transmission in a large, well-traced outbreak caused by the SARS-CoV-2 Delta variant.

The SARS-CoV-2 Delta variant has spread rapidly worldwide. To provide data on its virological profile, we here report the first local transmission of Delta in mainland China. All 167 infections could be traced back to the first index case. Daily sequential PCR testing of quarantined individuals indicated that the viral loads of Delta infections, when they first become PCR-positive, were on average ~1000 times greater compared to lineage A/B infections during the first epidemic wave in China in early 2020, suggesting potentially faster viral replication and greater infectiousness of Delta during early infection. The estimated transmission bottleneck size of the Delta variant was generally narrow, with 1-3 virions in 29 donor-recipient transmission pairs. However, the transmission of minor iSNVs resulted in at least 3 of the 34 substitutions that were identified in the outbreak, highlighting the contribution of intra-host variants to population-level viral diversity during rapid spread.

[Etiologic characteristics of Vibrio cholerae in Guangdong province in 2009-2013].

OBJECTIVE: To analyze the etiologic characteristics of O1/O139 Vibrio cholerae in Guangdong province in 2009-2013. METHODS: Isolates 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. RESULTS: Totally, 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. CONCLUSION: Complex 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.

High prevalence and diversity of pre-CTXΦ alleles in the environmental Vibrio cholerae†O1 and O139 strains in the Zhujiang River estuary.

Toxigenic conversion of environmental Vibrio cholerae strains through lysogenic infection by the phage CTXΦ is an important step in the emergence of new pathogenic clones. The precursor form of the CTXΦ phage, pre-CTXΦ, does not carry the cholera toxin gene. During our investigation, we frequently found pre-CTXΦ prophages in non-toxigenic isolates in the serogroups of O1 and O139 strains in the Zhujiang estuary. We observed high amounts of sequence variation of rstR and gIII(CTX) in the pre-CTXΦ alleles as well as in the tcpA sequences within the strains. In addition, a new pre-CTXΦ allele, with a novel rstR sequence type and hybrid RS2, was identified. Our findings show that active, complicated gene recombination and horizontal transfer of pre-CTXΦs occurs within V. cholerae environmental strains, which creates a complex intermediate pool for the generation of toxigenic clones in the estuarine environment.

[Surveillance and analysis on the pathogenic features of Salmonella in Guangdong province in 2010].

OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

MLVA genotyping of Chinese human Brucella melitensis biovar 1, 2 and 3 isolates.

BACKGROUND: Since 1950, Brucella melitensis has been the predominant strain associated with human brucellosis in China. In this study we investigated the genotypic characteristics of B. melitensis isolates from China using a multiple-locus variable-number tandem-repeat analysis (MLVA) and evaluated the utility of MLVA with regards to epidemiological trace-back investigation. RESULTS: A total of 105 B. melitensis strains isolated from throughout China were divided into 69 MLVA types using MLVA-16. Nei's genetic diversity indices for the various loci ranged between 0.00 - 0.84. 12 out 16 loci were the low diversity with values < 0.2 and the most discriminatory markers were bruce16 and bruce30 with a diversity index of > 0.75 and containing 8 and 7 alleles, respectively. Many isolates were single-locus or double-locus variants of closely related B. melitensis isolates from different regions, including the north and south of China. Using panel 1, the majority of strains (84/105) were genotype 42 clustering to the 'East Mediterranean' B. melitensis group. Chinese B. melitensis are classified in limited number of closely related genotypes showing variation mainly at the panel 2B loci. CONCLUSION: The MLVA-16 assay can be useful to reveal the predominant genotypes and strain relatedness in endemic or non-endemic regions of brucellosis. However it is not suitable for biovar differentiation of B. melitensis. Genotype 42 is widely distributed throughout China during a long time. Bruce 16 and bruce 30 in panel 2B markers are most useful for typing Chinese isolates.

[Surveillance on Salmonella infection in Guangdong province, 2008 - 2009].

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 quinolone and 59.3% of them were multiresistant to the antibiotics. CONCLUSION: S. enteritidis and S. typhimurium were the most common serotypes that caused infectious diarrhoea and food poisoning in Guangdong province.

Phenotypic and genotypic characterization Vibrio cholerae O139 of clinical and aquatic isolates in China.

To enhance the understanding of epidemiological impact of environmental Vibrio cholerae O139 strains, we characterized 10 clinical and 20 environmental isolates collected from human clinical samples and Pear River estuary during 2006 to 2008. Isolates were tested by PCR for eight virulence genes: cholera toxin (ctxA), zonula occludens toxin (zot), accessory cholera enterotoxin (ace), hemolysin (hlyA), NAG-specific heat-stable toxin (st), toxin-coregulated pilus (tcpA), outer membrane protein (ompU), and regulatory protein genes (tcpI). Genetic relatedness was assessed by pulsed-field gel electrophoresis (PFGE), and antibiotic susceptibility was determined using disk diffusion. Seven of eight virulence markers were detected in six clinical isolates and one environmental isolate. One clinical and one environmental isolate were positive for six virulence markers. 60% clinical isolates showed multi-drug resistance to tetracycline (TET), Nalidixic acid (NAL), chloramphenicol (CHL), and ampicillin (AMP), 70% were resistant to Trimethoprim + Sulfamethoxazole (SXT), while only 35% environmental strains were resistant to SXT. PFGE analysis revealed that the isolates in this study were formed three clusters. Cluster III was more related to strains from diarrheal patients than the strains in other clusters. Different from the clinical strains, most environmental strains lacked CTX and TCP gene clusters. Most environmental strains possess a single resistance profile, while most clinical isolates show multidrug resistant. PFGE analysis indicated the cluster III has more possibility to become a potential pathogenic clonal cluster.

[Etiologic characteristics of Vibrio parahaemolyticus strains causing outbreaks and sporadic cases in Guangdong, 2009].

OBJECTIVE: To study the serotypes, virulence features and molecular characteristics of Vibrio parahaemolyticus 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].

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 non-epidemic 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].

OBJECTIVE: To clarify the diagnosis of one suspected case of diphtheria in Guangdong province by epidemiological analysis and etiologic detection. METHODS: 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 ß-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. RESULTS: 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 ß-Corynebacteriophage, Corynebacterium diphtheriae was negative according to the PCR test. CONCLUSION: 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.

[Development of a DNA microarray for detecting 8 common species of food-borne bacterial pathogens in south China].

OBJECTIVE: To prepare a DNA Microarray that can detect 8 common species of food borne bacterial pathogens in south China. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Detection of Vibrio cholerae O1 and O139 in environmental water samples by an immunofluorescent-aggregation assay.

Environmental waters are an important reservoir for Vibrio cholerae, and effective surveillance of the pathogen can help to warn of and prevent infection with this potentially fatal pathogen. An immunofluorescent-aggregation (IFAG) assay to detect V. cholerae O1 and O139 was established and evaluated with estuarine water samples. The practical application of this assay was compared with the conventional culture method and real-time PCR. The IFAG method had a sensitivity of 10(3) CFU/ml for detection of V. cholerae O1 and O139 strains in a suspension containing 10 different species of enterobacterial strains (total, 10(5) CFU/ml). Ten fluorescent bacterial aggregate colonies were randomly picked and tested positive in serum agglutination tests for the V. cholerae O1 and O139 strains, showing a high specificity. The enrichment broths of 146 samples of estuarine water were tested, and the percentage positive by the IFAG assay was 19.9% (29/146), which was significantly higher than that of the conventional culture method (10.3%, 15/146; P < 0.01) but lower than that of real-time PCR (29.5%, 43/146; P < 0.01). The coincidence rates of real-time PCR and IFAG detection were decreased with the reduction of the V. cholerae concentration. The IFAG method, with a high specificity and a relatively high sensitivity, may be used for detection and isolation of V. cholerae in environmental water samples.

[Identification and characterization of the Francisella sp. strain 08HL01032 isolated in air condition systems].

OBJECTIVE: To identify and characterize the strain 08H101032 was isolated from air condition systems in the routine investigations of Legionella in Guangzhou, China, in 2008. METHODS: We adopted several phenotypic and genotypical methods, such as the growth status on various media, morphological, physical and biochemical characteristics, animal test, antibiotic susceptivities, PCR identification, sequence analysis of 16S RNA and RNA polymerase beta-subunit (ropB) gene etc, to determinate the phylogenetic position and outline the basic biological characteristics. RESULTS: Strain 08H101032 was Gram-negative with polymorphic short rods or coccobacillus; with no flagella; devoid of spores; well growth on buffered charcoal yeast extraction (BCYE) agar and BCYE supplemented with glycine (3 g/L), polymyxin B sulfate (80000 iu/L), vancomycin (1 mg/L) and cycloheximide (80 mg/L) (GVPC medium) within 2 days, but delayed growth on ordinary sheep blood agar untill 5 - 7 days; catalase positive; oxidase negative; no reduction of nitrate; no hydrolysis of urea; delayed fermention of glucose to produce acid; which was primarily considered as Legionella. It was lastly identified to the genus Fransicella, characterized by a variety of biochemical and molecular phylogenetic tests, which shared the highest similarities to F. Philomiragia with 95.3% to 16S rRNA gene of 1377 oligo nucleotides and 87.3% to ropB gene of 367 oligo nucleotides (GenBank accession number: FJ591095, FJ939309). Growth were observed after a treatment for 10 minutes with the KCl-HCl buffer of pH 2.2, 20 degrees C, and at 25 degrees C, 37 degrees C (optimum 25 degrees C - 28 degrees C), but not at 42 degrees C. The cells had capsule-like construction by transmission electron microscopy, however no virulence found to mice. CONCLUSIONS: Strain 08H101032 was a potential new species of the genus Fransicella with a typical characteristic of L-cysteine growth stimulating activity, distinguishingly to Legionella with L-cysteine growth dependent activity.

[MLST typing of Streptococcus suis isolated from clinical patients in Guangdong Province in 2005].

OBJECTIVE: 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.