Identification of knowledge gaps in whole-genome sequence analysis of multi-resistant thermotolerant Campylobacter spp.

BACKGROUND: Campylobacter spp. is the most frequent cause of bacterial food-borne gastroenteritis and a high priority antibiotic resistant bacterium according to the World Health Organization (WHO). European monitoring of thermotolerant Campylobacter spp. does not reflect the global burden of resistances already circulating within the bacterial population worldwide. METHODS: We systematically compared whole genome sequencing with comprehensive phenotypic antimicrobial susceptibility, analyzing 494 thermotolerant Campylobacter poultry isolates from Vietnam and Germany. Any discrepancy was checked by repeating the wet lab and improving the dry lab part. Selected isolates were additionally analyzed via long-read Oxford Nanopore technology, leading to closed chromosomes and plasmids. RESULTS: Overall, 22 different resistance genes and gene variants (e. g. erm(B), aph(3')-IIIa, aph(2'')-If, catA, lnu(C), blaOXA, sat4) and point mutations in three distinct genes (gyrA, 23S rRNA, rpsL) associated with AMR were present in the Campylobacter isolates. Two AMR genes were missing in the database and one falsely associated with resistance. Bioinformatic analysis based on short-read data partly failed to identify tet(O) and aadE, when the genes were present as duplicate or homologous gene variants. Intriguingly, isolates also contained different determinants, redundantly conferring resistance to chloramphenicol, gentamicin, kanamycin, lincomycin and streptomycin. We found a novel tet(W) in tetracycline sensitive strains, harboring point mutations. Furthermore, analysis based on assemblies from short-read data was impaired to identify full length phase variable aad9, due to variations of the poly-C tract within the gene. The genetic determinant responsible for gentamicin resistance of one isolate from Germany could not be identified. GyrT86I, presenting the main determinant for (fluoro-)quinolone resistance led to a rare atypical phenotype of ciprofloxacin resistance but nalidixic acid sensitivity. Long-read sequencing predicted AMR genes were mainly located on the chromosome, and rarely on plasmids. Predictions from long- and short-read sequencing, respectively, often differed. AMR genes were often organized in multidrug resistance islands (MDRI) and partially located in proximity to transposase genes, suggesting main mobilization of resistance determinants is via natural transformation and transposition in Campylobacter. CONCLUSIONS: The results of this study suggest that there is frequent resistance gene duplication, mosaicism, and mutation leading to gene variation and truncation in Campylobacter strains that have not been reported in previous studies and are missing from databases. Furthermore, there is a need for deciphering yet unknown resistance mechanisms and resistance spread in thermotolerant Campylobacter spp. that may pose a challenge to global food safety.

Long-Term Epidemiology and Evolution of Swine Influenza Viruses, Vietnam.

Influenza A viruses are a One Health threat because they can spill over between host populations, including among humans, swine, and birds. Surveillance of swine influenza virus in Hanoi, Vietnam, during 2013-2019 revealed gene pool enrichment from imported swine from Asia and North America and showed long-term maintenance, persistence, and reassortment of virus lineages. Genome sequencing showed continuous enrichment of H1 and H3 diversity through repeat introduction of human virus variants and swine influenza viruses endemic in other countries. In particular, the North American H1-δ1a strain, which has a triple-reassortant backbone that potentially results in increased human adaptation, emerged as a virus that could pose a zoonotic threat. Co-circulation of H1-δ1a viruses with other swine influenza virus genotypes raises concerns for both human and animal health.

Brunner's gland hyperplasia associated with lipomatous pseudohypertrophy of the pancreas presenting with gastrointestinal bleeding: A case report.

BACKGROUND: Brunner's gland hyperplasia (BGH) is a rare benign lesion of the duodenum. Lipomatous pseudohypertrophy (LiPH) of the pancreas is an extremely rare disease. Because each condition is rare, the probability of purely coincidental coexistence of both conditions is extremely low. CASE SUMMARY: We report a 26-year-old man presenting to our hospital with symptoms of recurrent upper gastrointestinal bleeding. Upper gastrointestinal endoscopy showed a huge pedunculated polypoid lesion in the duodenum with bleeding at the base of the lesion. Histopathological examination of the duodenal biopsy specimens showed BGH. Besides, abdominal computed tomography and magnetic resonance imaging revealed marked fat replacement over the entire pancreas, confirmed by histopathological evaluation on percutaneous pancreatic biopsies. Based on the radiological and histological findings, LiPH of the pancreas and BGH were diagnosed. The patient refused any surgical intervention. Therefore, he was managed with supportive treatment. The patient's symptoms improved and there was no further bleeding. CONCLUSION: This is the first well-documented case showing the coexistence of LiPH of the pancreas and BGH.

Prospective One Health genetic surveillance in Vietnam identifies distinct blaCTX-M-harbouring Escherichia coli in food-chain and human-derived samples.

OBJECTIVES: We performed a One Health surveillance in Hanoi-a region with a high-density human population and livestock production, and a recognized hotspot of animal-associated antimicrobial resistance (AMR)-to study the contribution of blaCTX-M-carrying Escherichia coli and plasmids from food-animal sources in causing human community-acquired urinary tract infections (CA-UTIs). METHODS: During 2014-2015, 9090 samples were collected from CA-UTI patients (urine, n = 8564), pigs/chickens from farms and slaughterhouses (faeces, carcasses, n = 448), and from the slaughterhouse environment (surface swabs, water, n = 78). E. coli was identified in 2084 samples. Extended-spectrum ß-lactamase (ESBL) production was confirmed in 235 and blaCTX-M in 198 strains by PCR with short-read plasmid sequencing. Fourteen strains were long-read sequenced to enable plasmid reconstruction. RESULTS: The majority of the ESBL-producing E. coli strains harboured blaCTX-M (n = 198/235, 84%). High clonal diversity (48 sequence types, STs) and distinct, dominant STs in human sources (ST1193, n = 38/137; ST131, n = 30/137) and non-human sources (ST155, n = 25/61) indicated lack of clonal transmission between habitats. Eight blaCTX-M variants were identified; five were present in at least two sample sources. Human and food-animal strains did not show similar plasmids carrying shared blaCTX-M genes. However, IS6 elements flanking ISEcp1-blaCTX-M-orf477/IS903B structures were common across habitats. CONCLUSIONS: In this study, animal-associated blaCTX-ME. coli strains or blaCTX-M plasmids were not direct sources of CA-UTIs or ESBL resistance in humans, respectively, suggesting evolutionary bottlenecks to their adaptation to a new host species. Presence of common IS6 elements flanking blaCTX-M variants in different plasmid backbones, however, highlighted the potential of these transposable elements for AMR transmission either within or across habitats.

Dataset on the effects of spacing and fruit truss limitation on the growth, yield and quality of open-field tomato plants.

This article presents data on the effects of spacing and fruit truss limitation on tomato plant growth, yield and fruit quality. Plants with two, three, and four fruit trusses (T1-T3) were grown in four different spaces (S1-S4) to create 12 treatments. The experiment was conducted on an open field with a randomized complete block design and three replications. Data on fruit quantity, weight, and yield were collected to assess the effects of plant density and fruit truss limitation on tomato fruit produced and marketable fruit produced. This data could help develop a strategy for breeding new tomato cultivars for high density planting on the rice-based rotational crop systems in the Red River Delta of Vietnam and other similar sub-tropical regions.

Unexpected cases in field diagnosis of African swine fever virus in Vietnam: The needs consideration when performing molecular diagnostic tests.

Background: The first confirmed case of African swine fever (ASF) in Vietnam was reported officially in February 2019. To date, ASF virus (ASFV) have been detected in 63/63 provinces in Vietnam. Currently, real-time polymerase chain reaction (PCR) is considered to be a powerful tool for viral detection in field samples, including ASFV. However, some recent reports have suggested that mismatches in primer and probe binding regions may directly affect real-time PCR qualification, leading a false-negative result. Aim: This study aims to further examine a conflicting result obtained from two OIE recommended methods, conventional PCR and real-time PCR, for ASFV detection. Methods: Two ASF suspected pigs from different provinces in the north of Vietnam were selected for this study based on clinical signs and postmortem lesions. The different results obtained by OIE-recommended conventional PCR and real-time PCR were further analyzed by the Sanger sequencing method and virus isolation in combination with hemadsorption (HAD) test using porcine alveolar macrophages cells. Results: The results showed that when the primer sequence matched perfectly with the sequences of field isolates, a mutation in probe binding region was found, indicating that a single mismatch in the probe binding site may cause a false-negative result by real-time PCR in detecting ASFV in clinical samples in Vietnam. An agreement between conventional PCR, using PPA1/PPA2 primers and two golden standard methods, virus isolation in combination with HAD assay, and sequencing method was observed in this study. Conclusion: A single mismatch in the probe binding site caused a failse-negative result by realtime PCR method in field diagnosis of ASFV. The needs consideration when selecting the appropriate molecular diagnostic methods is based on the current databases of ASFV sequences, particularly for epidemiological surveillance of ASF.

Genetic Characterisation of African Swine Fever Virus in Outbreaks in Ha Nam Province, Red River Delta Region of Vietnam, and Activity of Antimicrobial Products Against Virus Infection in Contaminated Feed.

INTRODUCTION: African swine fever (ASF) was officially reported in Vietnam in February 2019 and spread across the whole country, affecting all 63 provinces and cities. MATERIAL AND METHODS: In this study, ASF virus (ASFV) VN/Pig/HaNam/2019 (VN/Pig/HN/19) strain was isolated in primary porcine alveolar macrophage (PAM) cells from a sample originating from an outbreak farm in Vietnam's Red River Delta region. The isolate was characterised using the haemadsorption (HAD) test, real-time PCR, and sequencing. The activity of antimicrobial feed products was evaluated via a contaminated ASFV feed assay. RESULTS: Phylogenetic analysis of the viral p72 and EP402R genes placed VN/Pig/HN/19 in genotype II and serogroup 8 and related it closely to Eastern European and Chinese strains. Infectious titres of the virus propagated in primary PAMs were 106 HAD50/ml. Our study reports the activity against ASFV VN/Pig/HN/19 strain of antimicrobial Sal CURB RM E Liquid, F2 Dry and K2 Liquid. Our feed assay findings suggest that the antimicrobial RM E Liquid has a strong effect against ASFV replication. These results suggest that among the Sal CURB products, the antimicrobial RM E Liquid may have the most potential as a mitigant feed additive for ASFV infection. Therefore, further studies on the use of antimicrobial Sal CURB RM E Liquid in vivo are required. CONCLUSIONS: Our study demonstrates the threat of ASFV and emphasises the need to control and eradicate it in Vietnam by multiple measures.

An improvement of real-time polymerase chain reaction system based on probe modification is required for accurate detection of African swine fever virus in clinical samples in Vietnam.

OBJECTIVE: The rapid and reliable detection of the African swine fever virus (ASFV) plays an important role in emergency control and preventive measures of ASF. Some methods have been recommended by FAO/OIE to detect ASFV in clinical samples, including realtime polymerase chain reaction (PCR). However, mismatches in primer and probe binding regions may cause a false-negative result. Here, a slight modification in probe sequence has been conducted to improve the qualification of real-time PCR based on World Organization for Animal Health (OIE) protocol for accurate detection of ASFV in field samples in Vietnam. METHODS: Seven positive confirmed samples (four samples have no mismatch, and three samples contained one mutation in probe binding sites) were used to establish novel real-time PCR with slightly modified probe (Y = C or T) in comparison with original probe recommended by OIE. RESULTS: Both real-time PCRs using the OIE-recommended probe and novel modified probe can detect ASFV in clinical samples without mismatch in probe binding site. A high correlation of cycle quantification (Cq) values was observed in which Cq values obtained from both probes arranged from 22 to 25, suggesting that modified probe sequence does not impede the qualification of real-time PCR to detect ASFV in clinical samples. However, the samples with one mutation in probe binding sites were ASFV negative with OIE recommended probe but positive with our modified probe (Cq value ranked between 33.12-35.78). CONCLUSION: We demonstrated for the first time that a mismatch in probe binding regions caused a false negative result by OIE recommended real-time PCR, and a slightly modified probe is required to enhance the sensitivity and obtain an ASF accurate diagnosis in field samples in Vietnam.

Impaired expression of excitatory amino acid transporter 2 (EAAT2) and glutamate homeostasis in canine necrotizing meningoencephalitis.

To clarify the involvement of excitatory and inhibitory amino acids in canine necrotizing meningoencephalitis (NME), glutamate, aspartate, taurine and gamma-aminobutylic acid (GABA) were determined in the cerebrospinal fluids (CSF) from eight NME cases and ten healthy controls. NME dogs exhibited significantly higher concentrations of glutamate and aspartate than those in controls (p<0.001 and p<0.001, respectively), while there was no difference in taurine or GABA between the two groups. When fetal canine astrocytes were cultured for 24 hr in the presence of NME-CSF, supernatant concentrations of glutamate, aspartate and taurine were significantly elevated. Simultaneously, expression of excitatory amino acid transporter 2 (EAAT2) mRNA was significantly reduced in the astrocytes without change in EAAT1 mRNA. Hence, reduced expression of EAAT2 and impaired glutamate homeostasis may contribute to the pathogenesis of NME.