Co-Localization of Sampling and Sequencing for Zoonotic Pathogen Identification in the Field Monitoring Using Mobile Laboratories.

Introduction: Accurate etiological detection is needed to evaluate the risk of zoonotic diseases. Metagenomic next-generation sequencing (mNGS) can be used to monitor pathogens in animal species and identify potential zoonotic threats. The current sampling model for zoonotic pathogen monitoring in wild animals requires samples to be transferred from the field to a laboratory for further detection. Methods: We constructed a zoonotic pathogen survey model using a set of mobile laboratories. Results: The monitoring in this study was preplanned to detect Yersinia pestis, but the mNGS unexpectedly identified Bartonella spp. in the rodent samples, thus exposing the threat of bartonellosis to humans in this region. The co-localization of sampling and sequencing (CLOSS) model we tested required no long-distance transferring of samples and expands the regional coverage of zoonotic surveys by using a mobile laboratory. Discussion: Using this mNGS technique will enable detection of more zoonotic pathogens beyond the preplanned monitoring targets. This may increase the surveillance efficiency compared with that of the previous workflow and expand the application of the mobile laboratories for infectious diseases identification and surveillance in the field.

Prevalence of 16S rRNA Methylation Enzyme Gene armA in Salmonella From Outpatients and Food.

Salmonella is the primary cause of community-acquired foodborne infections, so its resistance to antimicrobials, such as aminoglycosides, is a public health issue. Of concern, aminoglycoside resistance in Salmonella is increasing rapidly. Here, we performed a retrospective study evaluating the prevalence of Salmonella harboring armA-mediated aminoglycoside resistance in community-acquired infections and in food or environmental sources. The prevalence rates of armA-harboring Salmonella strains were 1.1/1,000 (13/12,095) and 8.7/1,000 (32/3,687) in outpatient and food/environmental isolates, respectively. All the armA-harboring Salmonella strains were resistant to multiple drugs, including fluoroquinolone and/or extended-spectrum cephalosporins, and most (34/45) belonged to serovar Indiana. The armA gene of these strains were all carried on plasmids, which spanned five replicon types with IncHI2 being the dominant plasmid type. All the armA-carrying plasmids were transferable into Escherichia coli and Acinetobacter baumannii recipients. The conjugation experiment results revealed that the armA-harboring S. Indiana strains had a relatively higher ability to acquire armA-carrying plasmids. The low similarity of their pulsed field gel electrophoresis patterns indicates that the armA-harboring Salmonella strains were unlikely to have originated from a single epidemic clone, suggesting broad armA spread. Furthermore, the genetic backgrounds of armA-harboring Salmonella strains isolated from outpatients exhibited higher similarity to those isolated from poultry than to those isolated from swine, suggesting that poultry consumption maybe an infection source. These findings highlight an urgent need to monitor the prevalence and transmission of armA-harboring Salmonella, especially S. Indiana, to better understand the potential public health threat and prevent the further spread of these strains.

Rapid Identification of Plasmid Replicon Type and Coexisting Plasmid-Borne Antimicrobial Resistance Genes by S1-Pulsed-Field Gel Electrophoresis-Droplet Digital Polymerase Chain Reaction.

Bacterial drug resistance is a significant food safety problem and public health threat. Plasmids carrying drug resistance genes may result in the rapid spread of resistance among different bacteria, hosts, and environments; therefore, antibiotic resistance monitoring and continuing research into the mechanisms of drug resistance are urgently needed. Southern blotting with probes for antibiotic resistance genes and even next-generation sequencing have been used previously to detect plasmid-borne resistance genes, but these approaches are complex and time-consuming. The next-generation sequencing requires strict laboratory conditions and bioinformatics analysis ability. In this study, we developed a simplified and sensitive method to detect plasmid-borne antimicrobial resistance genes and plasmid replicon types. Salmonella strains carrying plasmids of three different replicon types that contained mcr-1 and two ESBL-producing genes were used to verify the new method. The plasmids harbored by the Salmonella strains were separated by S1 nuclease treatment and pulsed-field gel electrophoresis (PFGE), then recovered and used as the templates for droplet digital polymerase chain reaction (ddPCR) to identify target genes. The target genes were present in significantly higher copy numbers on the plasmids than the background noise. These results were consistent with the plasmid sequencing results. This S1-PFGE-ddPCR method was less time-consuming to perform than Southern blot and complete plasmid sequencing. Therefore, this method represents a time-saving alternative for detecting plasmid-borne genes, and is likely to be a valuable tool for detecting coexisting plasmid-borne drug resistance genes.

Development of a Rapid and Fully Automated Multiplex Real-Time PCR Assay for Identification and Differentiation of Vibrio cholerae and Vibrio parahaemolyticus on the BD MAX Platform.

Vibrio cholerae and Vibrio parahaemolyticus are common diarrheal pathogens of great public health concern. A multiplex TaqMan-based real-time PCR assay was developed on the BD MAX platform; this assay can simultaneously detect and differentiate V. cholerae and V. parahaemolyticus directly from human fecal specimens. The assay includes two reactions. One reaction, BDM-VC, targets the gene ompW, the cholera toxin (CT) coding gene ctxA, the O1 serogroup specific gene rfbN, and the O139 serogroup specific gene wbfR of V. cholerae. The other, BDM-VP, targets the gene toxR and the toxin coding genes tdh and trh of V. parahaemolyticus. In addition, each reaction contains a sample process control. When evaluated with spiked stool samples, the detection limit of the BD MAX assay was 195-780 CFU/ml for V. cholerae and 46-184 CFU/ml for V. parahaemolyticus, and the amplification efficiency of all genes was between 95 and 115%. The assay showed 100% analytical specificity, using 63 isolates. The BD MAX assay was evaluated for its performance compared with conventional real-time PCR after automated DNA extraction steps, using 164 retrospective stool samples. The overall percent agreement between the BD MAX assay and real-time PCR was ≥ 98.8%; the positive percent agreement was 85.7% for ompW, 100% for toxR/tdh, and lower (66.7%) for trh because of a false negative. This is the first report to evaluate the usage of the BD MAX open system in detection and differentiation of V. cholerae and V. parahaemolyticus directly from human samples.

A multiplex PCR assay for the detection of five human pathogenic Vibrio species and Plesiomonas.

A multiplex PCR (mPCR) assay was established to detect five pathogenic Vibrio species and Plesiomonas shigelloides. Twelve genes were included: ompW, ctxA, rfbN, and wbfR from V. cholerae; tl, tdh, and trh from V. parahaemolyticus; toxR and vmhA from V. mimicus; toxR from V. fluvialis; vvhA from V. vulnificus; and the 23S rRNA gene from P. shigelloides. The specificity of the mPCR assay was 100% for the detection of 136 strains and the limits of detection (LoD) were 12.5-50 pg/reaction. The assay exhibited higher sensitivity than cultivation methods in the detection of APW cultures of 113 diarrhea samples. In the analysis of 369 suspected Vibrio populations from estuarine water samples, the specificity of the mPCR for V. cholerae and V. parahaemolyticus was 100% for both, while the sensitivities were 100% and 96.1%, respectively. The assay can be applied to screen enrichment cultures and suspected colonies from environmental and clinical samples.

Fur Represses Vibrio cholerae Biofilm Formation via Direct Regulation of vieSAB, cdgD, vpsU, and vpsA-K Transcription.

Attached Vibrio cholerae biofilms are essential for environmental persistence and infectivity. The vps loci (vpsU, vpsA-K, and vpsL-Q) are required for mature biofilm formation and are responsible for the synthesis of exopolysaccharide. Transcription of vps genes is activated by the signaling molecule bis-(3'-5')-cyclic di-GMP (c-di-GMP), whose metabolism is controlled by the proteins containing the GGDEF and/or EAL domains. The ferric uptake regulator (Fur) plays key roles in the transcription of many genes involved in iron metabolism and non-iron functions. However, roles for Fur in Vibrio biofilm production have not been documented. In this study, phenotypic assays demonstrated that Fur, independent of iron, decreases in vivo c-di-GMP levels and inhibits in vitro biofilm formation by Vibrio cholerae. The Fur box-like sequences were detected within the promoter-proximal DNA regions of vpsU, vpsA-K, vieSAB, and cdgD, suggesting that transcription of these genes may be under the direct control of Fur. Indeed, the results of luminescence, quantitative PCR (qPCR), electrophoretic mobility shift assay (EMSA), and DNase I footprinting assays demonstrated Fur to bind to the promoter-proximal DNA regions of vpsU, vpsA-K, and cdgD to repress their transcription. In contrast, Fur activates the transcription of vieSAB in a direct manner. The cdgD and vieSAB encode proteins with GGDEF and EAL domains, respectively. Thus, data presented here highlight a new physiological role for Fur wherein it acts as a repressor of V. cholerae biofilm formation mediated by decreasing the production of exopolysaccharide and the intracellular levels of c-di-GMP.

Comparison of BioFire FilmArray gastrointestinal panel versus Luminex xTAG Gastrointestinal Pathogen Panel (xTAG GPP) for diarrheal pathogen detection in China.

OBJECTIVES: To compare the performance of two syndromic panels: Luminex xTAG Gastrointestinal Pathogen Panel (GPP) and FilmArray Gastrointestinal (GI) panel. METHODS: A total of 243 diarrhea specimens were detected by two panels in parallel, and the inconsistent results were analyzed by real-time PCR or reverse transcription PCR (RT-PCR). The target concentration in specimens was examined by comparing the crossing point values of FilmArray, the median fluorescence intensity of xTAG and the cycle threshold values in any discrepancies. RESULTS: For pathogens detected by both panels, the positive rates of FilmArray GI and xTAG GPP were 65.0% and 48.6%, respectively. The two panels showed high consistency (kappa ≥0.74) in detecting norovirus, rotavirus and Campylobacter, while there was low consistency (kappa ≤0.40) in detecting Cryptosporidium, Salmonella, Shiga toxin-producing Escherichia coli (STEC) and enterotoxigenic Escherichia coli (ETEC). Samples with low concentration targets were more often detected by FilmArray than with xTAG GPP. The xTAG GPP was more likely to be affected by amplification inhibitors. Several defects of xTAG GPP were found in detecting ETEC. CONCLUSIONS: FilmArray was more sensitive. For specimens with low target concentrations or containing ETEC heat stable enterotoxin, the false negatives of xTAG GPP need to be considered.

Quorum sensing regulation confronts the development of a viable but non-culturable state in Vibrio cholerae.

Vibrio cholerae can enter a viable but non-culturable (VBNC) state when it encounters unfavourable environments; VBNC cells serve as important reservoirs and still pose threats to public health. The genetic regulation of V. cholerae entering its VBNC state is not well understood. Here, we show a confrontation strategy adapted by V. cholerae O1 in which it utilizes a quorum sensing (QS) system to prevent transition into a VBNC state under low nutrition and temperature conditions. The upregulation of hapR resulted in a prolonged culturable state of V. cholerae in artificial sea water at 4°C, whereas the mutation of hapR led to fast entry into the VBNC state. We also observed that different V. cholerae O1 natural isolates with distinct QS functions present a variety of abilities to maintain culturability during the transition to a VBNC state. The strain groups with higher or constitutive expression of QS genes exhibit a greater tendency to maintain the culturable state during VBNC induction than those lacking QS functional groups. In summary, HapR-mediated QS regulation is associated with the transition to the VBNC state in V. cholerae. HapR expression causes V. cholerae to resist VBNC induction and become dominant over competitors in changing environments.

Co-existence of multiple distinct lineages in Vibrio parahaemolyticus serotype O4:K12.

Vibrio parahaemolyticus is an important cause of foodborne gastroenteritis globally. Thermostable direct haemolysin (TDH) and the TDH-related haemolysin are the two key virulence factors in V. parahaemolyticus. Vibrio pathogenicity islands harbour the genes encoding these two haemolysins. The serotyping of V. parahaemolyticus is based on the combination of O and K antigens. Frequent recombination has been observed in V. parahaemolyticus, including in the genomic regions encoding the O and K antigens. V. parahaemolyticus serotype O4:K12 has caused gastroenteritis outbreaks in the USA and Spain. Recently, outbreaks caused by this serotype of V. parahaemolyticus have been reported in China. However, the relationships among this serotype of V. parahaemolyticus strains isolated in different regions have not been addressed. Here, we investigated the genome variation of the V. parahaemolyticus serotype O4:K12 using the whole-genome sequences of 29 isolates. We determined five distinct lineages in this strain collection. We observed frequent recombination among different lineages. In contrast, little recombination was observed within each individual lineage. We showed that the lineage of this serotype of V. parahaemolyticus isolated in America was different from those isolated in Asia and identified genes that exclusively existed in the strains isolated in America. Pan-genome analysis showed that strain-specific and cluster-specific genes were mostly located in the genomic islands. Pan-genome analysis also showed that the vast majority of the accessory genes in the O4:K12 serotype of V. parahaemolyticus were acquired from within the genus Vibrio. Hence, we have shown that multiple distinct lineages exist in V. parahaemolyticus serotype O4:K12 and have provided more evidence about the gene segregation found in V. parahaemolyticus isolated in different continents.

Application of digital PCR and next generation sequencing in the etiology investigation of a foodborne disease outbreak caused by Vibrio parahaemolyticus.

Globally, V. parahaemolyticus infection is a leading cause of bacterial diarrheal diseases. Pathogenic V. parahaemolyticus strains that produce hemolysins are responsible for these diseases. The composition of pathogenic and non-pathogenic V. parahaemolyticus and the change of the bacterial composition before and after traditional selective enrichment in a single sample associated with disease outbreak remain unclear. We investigated an outbreak by using next generation sequencing and digital PCR to address those questions. NGS showed that the V. parahaemolyticus caused the outbreak belonged to s single clone. In contrast, among the seven non-pathogenic V. parahaemolyticus isolated from the suspected food sample, 4 serotypes and 6 PFGE patterns were identified. And nearly 70,000 SNPs were identified among the non-pathogenic strains. This result confirmed that the outbreak was caused by V. parahaemolyticus. Furthermore, NGS results clearly showed the diversity of non-pathogenic V. parahaemolyticus in a single contaminated food sample. The ratios of non-pathogenic and pathogenic V. parahaemolyticus were 31.41 and 620.11 in the original and enriched food samples respectively showed by digital PCR. Meta-genomic data indicated the top 3 species were Weissella cibaria, Weissella confusa, and Enterobacter cloacae in the original food sample, and Vibrio sp Ex25, Vibrio sp 712i, and V. parahaemolyticus in the enriched sample. Therefore, the combing of NGS and digital PCR results showed that traditional Vibrio selective enrichment media could facilitate the growth of Vibrios, however, it provided no advantages to pathogenic V. parahaemolyticus. Hence, our results indicated that the traditional culture methods alone may lead to wrong conclusions and so improvements in culture methods are needed.

Expression of Hemolysin Is Regulated Under the Collective Actions of HapR, Fur, and HlyU in Vibrio cholerae El Tor Serogroup O1.

The biotype El Tor of serogroup O1 and most of the non-O1/non-O139 strains of Vibrio cholerae can produce an extracellular pore-forming toxin known as cholera hemolysin (HlyA). Expression of HlyA has been previously reported to be regulated by the quorum sensing (QS) and the regulatory proteins HlyU and Fur, but lacks the direct evidence for their binding to the promoter of hlyA. In the present work, we showed that the QS regulator HapR, along with Fur and HlyU, regulates the transcription of hlyA in V. cholerae El Tor biotype. At the late mid-logarithmic growth phase, HapR binds to the three promoters of fur, hlyU, and hlyA to repress their transcription. At the early mid-logarithmic growth phase, Fur binds to the promoters of hlyU and hlyA to repress their transcription; meanwhile, HlyU binds to the promoter of hlyA to activate its transcription, but it manifests direct inhibition of its own gene. The highest transcriptional level of hlyA occurs at an OD600 value of around 0.6-0.7, which may be due to the subtle regulation of HapR, Fur, and HlyU. The complex regulation of HapR, Fur, and HlyU on hlyA would be beneficial to the invasion and pathogenesis of V. cholerae during the different infection stages.

Chromosomal rearrangement features of Yersinia pestis strains from natural plague foci in China.

The Yersinia pestis chromosome contains a large variety and number of insert sequences that have resulted in frequent chromosome rearrangement events. To identify the chromosomal rearrangement features of Y. pestis strains from five typical plague foci in China and study spontaneous DNA rearrangements potentially stabilized in certain lineages of Y. pestis genomes, we examined the linking mode of locally collinear blocks (LCBs) in 30 Y. pestis strains by a polymerase chain reaction-based method. Our results suggest most strains have relatively stable chromosomal arrangement patterns, and these rearrangement characteristics also have a very close relationship with the geographical origin. In addition, some LCB linking modes are only present in specific strains. We conclude Y. pestis chromosome rearrangement patterns may reflect the genetic features of specific geographical areas and can be applied to distinguish Y. pestis isolates; furthermore, most of the rearrangement events are stable in certain lineages of Y. pestis genomes.