Bixia Ke

Laboratory-Based Surveillance of Non-typhoidal Salmonella Infections in Guangdong Province, China

Salmonella is one of the most common foodborne pathogens in humans. Laboratory-based surveillance for non-typhoidal Salmonella infection was conducted in Guangdong Province, China to improve understanding about the disease burden and detection of dispersed outbreaks. Salmonella isolated from patients with diarrhea were sent from 16 sentinel hospitals to local public health laboratories for confirmation, serotyping, antimicrobial susceptibility testing, and pulsed-field gel electrophoresis (PFGE). PFGE patterns were analyzed to identify clusters representing potential outbreaks. Between September 2009 and October 2010, 352 (4%) Salmonella isolates were obtained from 9167 stool specimens. Salmonella enterica serotype Typhimurium (45%) and Salmonella enterica serotype Enteritidis (13%) were the most common serotypes, and multidrug resistance was high, especially in Salmonella Typhimurium isolates. PFGE patterns of obtained Salmonella isolates were found to be diverse, but a unique PFGE pattern comprising 53 Salmonella Typhimurium isolates were found to occur almost exclusively in infants. Epidemiologic studies are ongoing to determine whether a common exposure is the source of the Salmonella Typhimurium strain frequently isolated from infants.

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

ABSTRACT 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 103 CFU/ml for detection of V. cholerae O1 and O139 strains in a suspension containing 10 different species of enterobacterial strains (total, 105 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.

Brucellosis in Guangdong Province, People’s Republic of China, 2005–2010

To the Editor: Brucellosis is one of the most prevalent zoonotic diseases in the world. It is principally an animal disease, but globally, >500,000 human cases are reported each year (1). Transmission to humans occurs primarily through contact with infected animals and consumption of contaminated food (2,3). Persons with occupational exposure are at highest risk for brucellosis, in particular those performing husbandry activities, butchering, and livestock trading (4,5). Although brucellosis has been eradicated from many industrialized countries, new foci of disease continually appear, particularly in parts of Asia (6–8). In China, 160,214 brucellosis cases were reported during 2005–2010; 90% of them occurred in 6 northern agricultural provinces: Neimenggu, Shanxi, Heilongjiang, Hebei, Jilin, and Shaanxi. Livestock, such as goats, cattle, and pigs, are the main infectious source. However, factors such as the rapid movement of people from northern to southern China, increased livestock trading, and lack of livestock quarantine mean that infected livestock or their products readily traverse provincial borders and transmit disease to persons who have no direct contact with livestock. With an illness rate of 400). The male:female ratio among these patients was 66:46. The age ranges were similar by sex; male patients were 18–71 (median 47) years of age, and female patients were 20–70 (median 43) years of age. The first 3 cases of brucellosis in 2005 were reported in Shenzhen in Guangdong Province. One case was culture confirmed by clinical laboratory, and the isolate was identified as Brucella melitensis biovar 3 by SAT and phage biotyping. The other 2 cases were in dairy farm workers; their infections were laboratory confirmed by SAT but could not be identified by biovar. Since 2005, more cities in Guangdong have reported brucellosis cases (Figure, Appendix). The Pearl River Delta region reported 100 cases: 48 in Guangzhou, 27 in Shenzhen, 7 in Zhongshan, 6 in Foshan, 6 in Jiangmen, 4 in Zhuhai, and 2 in Dongguan. Only 12 cases were reported from undeveloped rural areas in Guangdong: 5 in Zhaoqing, 2 in Yangjiang, and 1 each in Huizhou, Qingyuan, Meizhou, Maoming, and Yunfu. Figure Reported cases of brucellosis, by city, Guandong Province, China, 2005–2010. A total of 42 Brucella isolates were cultured during 2005–2009, and all were identified as B. melitensis biovar 3. However, of 19 Brucella isolates cultured during 2010, a total of 13 were identified as B. melitensis biovar 3, 4 as B. melitensis biovar 1, and 2 as B. suis biovar 3. These results indicate a shift in species and biovar for Brucella spp. circulating in China. We conducted a retrospective epidemiologic investigation of the 112 brucellosis cases reported during 2005–2010 to identify the vehicles and sources of infection. Among the cases identified, 33 (29.46%) patients had occupational exposure history: 13 were pig or goat butchers, 12 dairy farmers, 5 animal market workers in charge of leading the animals to and from transportation, and 3 mutton and pork sellers in wet markets. The remaining 79 (70.54%) cases were in patients who denied having contact with living animals. Among these patients were retired persons, housekeeping matrons, teachers, doctors, white collar workers, and the unemployed. However, 17 of these patients recalled having purchased or handled goat placenta to be prepared for home consumption or having eaten goat products through barbequing or hot pot. The other 62 could not remember if they had contacted with livestock or their products. These findings indicate that nonoccupational exposure may pose a risk for brucellosis infection among persons who handle fresh meat and meat products for home cooking. In conclusion, Guangdong Province has become an emerging foci for brucellosis in China. The species and biovars of Brucella spp. circulating in this region are changing, and many persons are infected by nonoccupational exposure. Measures need to be taken by central and provincial governments to address these issues and prevent epidemics of brucellosis in humans.

Inapparent Streptococcus agalactiae infection in adult/commercial tilapia

We report on inapparent infections in adult/commercial tilapia in major tilapia fish farms in Guangdong. A total of 146 suspected isolates were confirmed to be S. agalactiae using an API 20 Strep system and specific PCR amplification. All isolates were identified as serotype Ia using multiplex serotyping PCR. An MLST assay showed single alleles of adhP (10), atr (2), glcK (2), glnA (1), pheS (1), sdhA (3) and tkt (2), and this profile was designated ‘unique ST 7’. The analysis of virulence genes resulted in 10 clusters, of which dltr-bca-sodA-spb1-cfb-bac (62, 42.47%) was the predominant virulence gene profile. The PFGE analysis of S. agalactiae yielded 6 distinct PFGE types (A, B, C, D, F and G), of which Pattern C (103) was the predominant type, accounting for approximately 70.55% (103/146) of the total S. agalactiae strains. Therefore, unlike what has been found in juvenile tilapia, in which PFGE pattern D/F is the major prevalent pattern, we found that pattern C was the major prevalent pattern in inapparent infected adult/commercial tilapia in Guangdong, China. In conclusion, we close a gap in the current understanding of S. agalactiae epidemiology and propose that researchers should be alert for inapparent S. agalactiae infections in adult/commercial tilapia to prevent a potential threat to food safety.

A rapid pulsed-field gel electrophoresis method of genotyping Haemophilus parasuis isolates.

Aims:  To develop a modified pulsed‐field gel electrophoresis (PFGE) method for characterizing Haemophilus parasuis isolates.

Phenotypic and phylogenetic analysis of Vibrio parahaemolyticus isolates recovered from diarrhea cases in Guangdong Province, China

Vibrio parahaemolyticus has emerged as a common foodborne pathogen of global concern. In this study, 108 V. parahaemolyticus isolates that recovered from diarrhea cases (n=96) and seafood products (n=12) in Guangdong Province from 2007 to 2011 were characterized by serotyping, tdh and trh toxin gene detection and multilocus sequence typing (MLST). The dominant serotypes from the cases were O3:K6, O4:K8 and O1: KUT (untyped). However, most isolates recovered from seafood products belonged to other serotypes. None of the isolates carried the trh gene, while the major isolates from the cases were tdh positive. MLST analysis revealed 31 sequence types (STs); 17 STs were unique in this study. eBURST analysis revealed four clonal complexes (CC), The majority of the isolates (n=58, all from cases and tdh+) were grouped into the CC3, which included O3:K6, O4:K68 and O1:KUT isolates. The CC3 was the most prevalent clonal complex, and all of the CC3 isolates were recovered from clinical cases of geographically diverse origin. As to the CC345, which was completely constituted by O4:K8, was another important clonal complex affecting Guangdong Province. Ongoing surveillance of V. parahaemolyticus in diarrhea patients and seafood products remains a public health priority for Guangdong Province, China.

Survey of Physician Diagnostic and Treatment Practices for Patients with Acute Diarrhea in Guangdong Province, China

Although international clinical guidelines generally recommend performing bacterial stool culture in patients with acute diarrhea and fever and discourage routine antibiotic prescribing, clinical practice varies. Understanding practice patterns can help health officials assess the sensitivity of laboratory-based enteric infection surveillance systems and the need to improve antibiotic prescribing practices. We surveyed physicians in Guangdong province, China, to measure their practices for patients with acute diarrhea. A standardized questionnaire was used to interview physicians working in hospitals participating in a Salmonella surveillance system in Guangdong, China. The questionnaire asked physicians about their routine practice for patients with diarrhea, including how they managed the last patient they had seen with acute diarrhea. We calculated the odds ratio and 95% confidence interval for factors associated with ordering a stool culture and for prescribing antibiotics. We received surveys from 237 physicians across 22 hospitals in Guangdong. For the last patient with diarrhea whom they had evaluated, 134 (57%) reported ordering a stool culture. The most common reasons for not ordering a stool culture included that it takes too long to receive the result, that the patient is not willing to pay for the test, and that the patients illness was too mild to warrant testing. Most physicians prescribed at least one medication for the last patient with diarrhea whom they had evaluated. Of the 237 physicians surveyed, 153 (65%) prescribed antibiotics, 135 (57%) probiotics, and 115 (49%), a gastric mucosal protective drug. In conclusion, physicians in Guangdong, China, reported high rates of ordering bacterial stool cultures from patients with diarrhea, possibly associated with their hospitals participation in a special surveillance project. The high rate of antibiotic prescribing suggests that efforts to promote judicious antibiotic use, such as physician education, are needed.

Vibrio parahaemolyticus O4:K8 forms a potential predominant clone in southern China as detected by whole-genome sequence analysis

Vibrio parahaemolyticus has been the most common food-borne pathogen in southern China, especially the O3:K6 pandemic clone and its serovariants. Recently, the serotype O4:K8 became more and more prevalent in southern China, which was different from the O3:K6 pandemic clone. Thus, the aim of the present work was to elucidate the molecular characteristics of the O4:K8. Some O3:K6 pandemic clone and its serovariants isolated in the same period were selected for comparative analysis, which were still dominant clone locally. The whole genome sequencing (WGS) was applied to characterize 20 strains of V. parahaemolyticus isolated from food-borne diarrheal cases and belonging to the serotype O4:K8, O3:K6 and O1:KUT (untypable), prevalent serotypes in recent southern China. The results showed that all these isolates were positive for the thermostable direct hemolysin gene (tdh), while negative for the TDH-related hemolysin gene (trh). We compared the V. parahaemolyticus strains to those of 31 strains isolated overseas and were available from NCBI genome database. A WGS-SNPs phylogenetic analysis of all the genomes revealed that the strains formed an important genetic lineage, which was genetically distinct from the O3:K6, O1:KUT and other internationals strains. Comparative genome analysis also revealed that all the O4:K8 strains carried the entire T3SS-1 and VpaI-7 (T3SS-2) regions, the most important virulent elements of the O3:K6 pandemic clone. However, all the O4:K8 strains lacked the entire VpaI-1 and VpaI-4 regions and carried only few ORFs of the VpaI-5 and VpaI-6, which were considered to be unique among post-1995 strains belonging to the O3:K6 pandemic clone. Our data showed that the O4:K8 strains possessed the virulence factors similar to the O3:K6 pandemic clone, which may have enabled them to become prevalent in southern China. Our study also revealed that WGS-bases analysis may help improve understanding epidemiology of this bacterium in food-borne disease surveillance.

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.

Distribution and molecular characteristics of Vibrio cholerae O1 El Tor isolates recovered in Guangdong Province, China, 1961-2013.

Chinas Guangdong Province is located along the same latitude as Kolkata, India and Dhaka, Bangladesh, and is also considered a source of epidemic cholera. However, molecular description and the genetic relationships between Vibrio cholerae O1 El Tor isolates in Guangdong remain unclear. In this study, 381 clinical V. cholerae O1 isolates recovered from cholera cases presenting in Guangdong between 1961 and 2013 were investigated by PCR, amplicon sequencing and pulsed-field gel electrophoresis (PFGE). During this time frame, four distinct epidemic periods (1-4) were observed based on the different dominant serotype leading its epidemic, correspond to years; or time periods from/to 1961-1969, 1978-1989, 1990-2000, 2001-2013, respectively. Molecular analysis of representative isolates indicated that a single dominating clone was associated with each epidemic stage. All isolates from periods 1 and 2 carried the typical El Tor ctxB; this allele was displaced by classical ctxB beginning in 1993. However all isolates carried the El Tor-specific toxin-coregulated pili subunit A (tcpA). Isolates were grouped into five clusters on the basis of Not I enzyme digested PFGE, and the first four clusters were associated with specific periods, cluster I (period 1), II (period 3), III (period 2) and IV (period 4), respectively. While cluster V consisted of isolates from all four epidemic periods, but was most heterogeneous in appearance. Our data indicate genetic variations that shape the relationship among emerging isolates of V. cholerae O1 in Guangdong Province contribute to the 7th global pandemic.