Prevalence, genomic characterization and antimicrobial resistance of Campylobacter spp. isolates in pets in Shenzhen, China.

The prevalence of Campylobacter spp.in pets is a potential concern for human health. However, little is known about the pet-related Campylobacter spp. in China. A total of 325 fecal samples were collected from dogs, cats, and pet foxes. Campylobacter spp. were isolated by culture, and MALDI-TOF MS was used to identify 110 Campylobacter spp. isolates in total. C. upsaliensis (30.2%, 98/325), C. helveticus (2.5%, 8/325), and C. jejuni (1.2%, 4/325) were the three found species. In dogs and cats, the prevalence of Campylobacter spp. was 35.0% and 30.1%, respectively. A panel of 11 antimicrobials was used to evaluate the antimicrobial susceptibility by the agar dilution method. Among C. upsaliensis isolates, ciprofloxacin had the highest rate of resistance (94.9%), followed by nalidixic acid (77.6%) and streptomycin (60.2%). Multidrug resistance (MDR) was found in 55.1% (54/98) of the C. upsaliensis isolates. Moreover, 100 isolates, including 88 C. upsaliensis, 8 C. helveticus, and 4 C. jejuni, had their whole genomes sequenced. By blasting the sequence against the VFDB database, virulence factors were identified. In total, 100% of C. upsaliensis isolates carried the cadF, porA, pebA, cdtA, cdtB, and cdtC genes. The flaA gene was present in only 13.6% (12/88) of the isolates, while the flaB gene was absent. By analyzing the sequence against the CARD database, we found that 89.8% (79/88) of C. upsaliensis isolates had antibiotic target alteration in the gyrA gene conferring resistance to fluoroquinolone, 36.4% (32/88) had the aminoglycoside resistance gene, and 19.3% (17/88) had the tetracycline resistance gene. The phylogenetic analysis using the K-mer tree method obtained two major clades among the C. upsaliensis isolates. All eight isolates in subclade 1 possessed the gyrA gene mutation, the aminoglycoside and tetracycline resistance genes, and were phenotypically resistant to six classes of antimicrobials. It has been established that pets are a significant source of Campylobacter spp. strains and a reservoir for them. This study is the first to have documented the presence of Campylobacter spp. in pets in Shenzhen, China. In this study, C. upsaliensis of subclade 1 required additional attention due to its broad MDR phenotype and relatively high flaA gene prevalence.

Genetic characteristics, antimicrobial resistance, and prevalence of Arcobacter spp. isolated from various sources in Shenzhen, China.

Arcobacter spp. is a globally emerging zoonotic and foodborne pathogen. However, little is known about its prevalence and antimicrobial resistance in China. To investigate the prevalence of Arcobacter spp. isolated from various sources, 396 samples were collected from human feces, chicken cecum, and food specimens including chicken meat, beef, pork, lettuce, and seafood. Arcobacter spp. was isolated by the membrane filtration method. For 92 strains, the agar dilution method and next-generation sequencing were used to investigate their antimicrobial resistance and to obtain whole genome data, respectively. The virulence factor database (VFDB) was queried to identify virulence genes. ResFinder and the Comprehensive Antibiotic Resistance Database (CARD) were used to predict resistance genes. A phylogenetic tree was constructed using the maximum likelihood (ML) method with core single-nucleotide polymorphisms (SNPs). We found that 27.5% of the samples (n = 109) were positive for Arcobacter spp., comprising Arcobacter butzleri (53.0%), Arcobacter cryaerophilus (39.6%), and Arcobacter skirrowii (7.4%). Chicken meat had the highest prevalence (81.2%), followed by seafood (51.9%), pork (43.3%), beef (36.7%), lettuce (35.5%), chicken cecum (8%), and human fecal samples (0%, 0/159). Antimicrobial susceptibility tests revealed that 51 A. butzleri and 40 A. cryaerophilus strains were resistant to streptomycin (98.1, 70%), clindamycin (94.1, 90%), tetracycline (64.7, 52.5%), azithromycin (43.1%, 15%), nalidixic acid (33.4, 35%), and ciprofloxacin (31.3, 35%) but were susceptible to erythromycin, gentamicin, chloramphenicol, telithromycin, and clindamycin (≤10%). A. skirrowii was sensitive to all experimental antibiotics. The virulence factors tlyA, mviN, cj1349, ciaB, and pldA were carried by all Arcobacter spp. strains at 100%, and the following percentages were cadF (95.7%), iroE (23.9%), hecB (2.2%), hecA, and irgA (1.1%). Only one A. butzleri strain (F061-2G) carried a macrolide resistance gene (ereA). One A. butzleri and one A. cryaerophilus harbored resistance island gene clusters, which were isolated from pork and chicken. Phylogenetic tree analysis revealed that A. butzleri, A. cryaerophilus, and A. skirrowii were separated from each other. To our knowledge, this is the first report of the isolation of Arcobacter spp. from vegetables and seafood in China. The resistance island gene cluster found in pork and chicken meat and the presence of virulence factors could be a potential risk to human health.

FilmArray GI-panel performance for the rapid and multiple detection of gastrointestinal microorganisms in foodborne illness outbreaks in Shenzhen during 2018-2019.

Foodborne illness outbreaks can be caused by a great many of gastrointestinal microorganisms including bacteria, viruses and parasites. Acute gastroenteritis is most commonly found in such patients infected with at least one pathogen through food intake. The stool culture has been conventionally used to guide a single diagnosis and therapy. However, traditional methods for identification of a pathogen are time-consuming and have limited sensitivity, leading to false negatives and co-infection omission. The aim of this study was to characterize the multiple etiology of each foodborne illness outbreak in Shenzhen during 2018-2019 by the FilmArray GI panel, and to reveal the seasonality of each causative organism incurring outbreaks. All patients included had a FilmArray GI panel performance and the seasonal characteristics were recorded. A total of 173 patients suffered from foodborne illnesses in 32 outbreaks in Nanshan District of Shenzhen. In total, 365 microorganisms were detected of which 83.8% (306/365) corresponded to bacteria and 16.2% (59/365) to viruses. Co-infections with more than one microorganism were detected in 81.3% (26/32) of the outbreaks. In 153 (88.4%) of 173 patients at least two pathogens were identified. The most common diarrheal pathogen related to outbreaks was EPEC (56%), followed by ETEC (38%), Norovirus (34%), EAEC (28%), Vibrio (25%), Salmonella (22%), P. shigelloides (22%), C. difficile (16%), STEC (3%) and Sapovirus (3%). Bacterial outbreaks occurred with a seasonal distribution with the exception of C. difficile whereas Norovirus outbreaks predominated during the autumn-winter months. The use of the FilmArray GI panel has given us worthy information regarding the epidemiology of pathogens detected in patients with acute diarrhea. It also highlights the importance of multi-pathogen infections and the frequency of diarrheogenic E. coli in foodborne disease outbreaks. More significantly, the rapid and multiple findings may help quickly taking an appropriate precaution, control and treatment.

Comparison of the Filtration Culture and Multiple Real-Time PCR Examination for Campylobacter spp. From Stool Specimens in Diarrheal Patients.

Campylobacter is one of the most common pathogens leading to the bacterial diarrheal illness. In order to set up one effective culture independent assay for the screen of the Campylobacter infection in the diarrheal patients, the quadruple real-time PCR method comparing to the culture based on the enriched filtration method which was recognized as the most effective isolation method was assessed for 190 stool samples from the diarrheal patients collected during the Foodborne Diseases Active Surveillance Network in Beijing. This multiple real-time PCR was designed to identify the Campylobacter genus, C. jejuni, C. coli, and C. lari simultaneously. With the enrichment culture method, 23 (12.1%, 23/190) Campylobacter isolates were obtained (20 C. jejuni and 3 C. coli), however, 31 samples (16.3%, 31/190) were detected positively with the real-time PCR (21 C. jejuni, 8 C. coli, and 2 Campylobacter genus only). With the comparison, the real-time-PCR method is more sensitive than the enrichment filtration method (16.3 vs. 12.1%, p = 0.021). Among the culture-positive samples, 95.7% (22/23) were detected positively by PCR which indicate the specificity of this method was higher. These two methods were consistent well (Kappa = 0.785, p < 0.05). Comparing to the culture methods, the result of the multiple real-time PCR method is sensitive, reliable and rapid. The present study indicated this multiple real-time PCR can be used both for the surveillance network and the preceding screen for bacteria isolation. This is first comparative study between the culture and multiple real-time PCR method for Campylobacter identification in stool specimens from the diarrheal patients.

[Genetic characterization of Vibrio parahaemolyticus O3: K6 serovariant isolated in Shenzhen].

OBJECTIVE: To characterize the O3: K6 serovariant of Vibrio parahaemolyticus on virulence gene and molecular typing, and analyze the genetic relationship between O3: K6 and O3: K6 serovariants. METHODS: PFGE was performed on 115 strains of V.parahaemolyticus which were collected from the anal swab of cases of foodbrone diseases in Shenzhen during 2006-2012. According to isolation times and locations, 7 strains of O3: K6 were selected as control strains. Tdh gene, trh gene, orf8 gene were detected, GS-PCR, multi-locus sequence typing (MLST) were used to chracterize 7 strains of O3: K6 and O3: K6 serovariants. RESULTS: PFGE indicated that 58.3% (67/115) of V. parahaemolyticus strains shared a high similarity of band pattern (similarity > 80%) , which comprised of O3: K6 (44/67), O1: KUT(4/67), and O3: K6 serovariants(19/67). Among the O3: K6 serovariants, O1: K25 accounted for 7% (5/67), O4: K68 accounted for 10% (7 /67), O11: K36 accounted for 10% (7 /67). They all carried both tdh and trh gene, and 53% (10/19) was GS-PCR positive and carried orf8 gene, 26% (5/19) was both GS-PCR and orf8 gene negative, 21% (4/19) was GS-PCR negative, orf8 gene positive, 89% (17/19) was assigned to ST-3, 11% (2/19) was assigned to ST-305. Seven strains of O3: K6 was GS-PCR positive, carried orf8 gene, assigned to ST-3. ST-305 and ST-3 had differences in 2 housekeeping genes, which was dtdS gene and pntA gene. In the 305th base of dtdS gene, ST-305(147 allele profile) was T, while ST-3(4 allele profile) was C. In the 33th base of pntA gene, ST-305(93 allele profile) was T, while ST-3(29 allele profile ) was C. CONCLUSION: O4: K68,O1: K25 and O11: K36 were highly similar in virulencec gene carriage, MLST type of O3: K6, and aslo shared a close genetic relationship with O3: K6, thus were considered as O3: K6 serovirants.

[Characterization of Vibrio parahaemolyticus obtained from environment and cases of foodborne disease in Shenzhen, during 2006 - 2008].

OBJECTIVE: To analyze the serological and genetic divergence in the Vibrio parahaemolyticus from environment and cases of foodborne disease, and to compare these two groups in terms of virulence and other biological traits. METHODS: Serotyping, multi-PCR, and pulse-field gel electrophoresis (PFGE) were carried out. RESULTS: The main serotypes of cases isolates were O3:K6 (40.8%), O1: KUT (7.1%), O4:K8 (7.1%), and the main serotypes of environmental isolates were O3:KUT (14.2%), O1: KUT (11.4%) and O2:K3 (11.4%). No O3:K6 strain was isolated from environment. Most cases isolates were tdh positive and trh negative, which account for 83.7%, while most environmental isolates were tdh negative and trh negative, which account for 88.6%. PFGE indicated that Clone P1 was the dominant clone cluster, including serotype O3: K6 (29 isolates), O4: K68 (4 isolates), O11: K36 (3 isolates), O1: K25 (2 isolates), and these strains were all obtained from cases of foodborne disease. No dominant clone cluster was found in environmental isolates. CONCLUSIONS: V. parahaemolyticus from cases of foodborne disease in Shenzhen mostly were hemolytic, tdh positive and trh negative, there were dominant PFGE type in cases isolates, the main serotype was O3:K6, while environmental isolates mostly were non-hemolytic, tdh negative and trh negative, no dominant PFGE type was found.