TAL effector-dependent Bax gene expression in transgenic rice confers disease resistance to Xanthomonas oryzae pv. oryzae.

The hypersensitive response (HR) is a form of programmed cell death of plant cells occurring in the local region surrounding pathogen infection site to prevent the spread of infection by pathogens. Bax, a mammalian pro-apoptotic member of Bcl-2 family, triggers HR-like cell death when expressed in plants. However, constitutive expression of the Bax gene negatively affects plant growth and development. The Xa10 gene in rice (Oryza sativa) is an executor resistance (R) gene that confers race-specific disease resistance to Xanthomonas oryzae pv. oryzae strains harboring TAL effector gene AvrXa10. In this study, the Xa10 promoter was used to regulate heterologous expression of the Bax gene from mouse (Mus musculus) in Nicotiana benthamiana and rice. Cell death was induced in N. benthamiana after co-infiltration with the PXa10:Bax:TXa10 gene and the PPR1:AvrXa10:TNos gene. Transgenic rice plants carrying the PXa10:Bax:TXa10 gene conferred specific disease resistance to Xa10-incompatible X. oryzae pv. oryzae strain PXO99A(pHM1AvrXa10), but not to the Xa10-compatible strain PXO99A(pHM1). The resistance specificity was confirmed by the AvrXa10-dependent induction of the PXa10:Bax:TXa10 gene in transgenic rice. Our results demonstrated that the inducible expression of the Bax gene in transgenic rice was achieved through the control of the executor R gene promoter and the heterologous expression of the pro-apoptosis regulator gene in rice conferred disease resistance to X. oryzae pv. oryzae.

Occurrence and Patterns of Enterotoxin Genes, spa Types and Antimicrobial Resistance Patterns in Staphylococcus aureus in Food and Food Contact Surfaces in Singapore.

Staphylococcus aureus contamination of food and food contact surfaces is a public health concern given its virulent and antimicrobial-resistant properties worldwide. In this study, a total of 181 MSSA isolates were analyzed for SE genes, antimicrobial resistance patterns, and spa types. Overall, 24.9% of isolates were positive for SE gene detection, with sea being the most prevalent classical SE (18.8%). The most predominant sample sources for SE gene contamination were hand swabs for sea (6/48), meat dishes for seb (3/14) and seafood dishes for sec (2/24). Antimicrobial resistance was also observed at relatively high frequencies for the clinically important antibiotics penicillin G and ampicillin (both 54.7%), followed by tetracycline (14.9%) and azithromycin (8.8%). In addition, characterization of spa types revealed spa type t5078 to be the most predominant (40.3%), with significant associations between spa types t127 and t5521 and the sea gene. This study offers insights into the enterotoxin gene and antimicrobial resistance profiles of S. aureus in cooked or ready-to-eat food to inform future surveillance and epidemiological studies.

Quantitative Risk Evaluation of Antimicrobial-Resistant Vibrio parahaemolyticus Isolated from Farmed Grey Mullets in Singapore.

Vibrio parahaemolyticus is a causative pathogen for gastroenteritis involving the consumption of undercooked or raw seafood. However, there is a paucity of data regarding the quantitative detection of this pathogen in finfish, while no study reported the enumeration of haemolytic antimicrobial-resistant (AMR) V. parahaemolyticus. In this study, ampicillin-, penicillin G- and tetracycline-resistant and non-AMR haemolytic V. parahaemolyticus isolates were monitored and quantified in grey mullet samples reared locally from different premises within the food chain (farm and retail). Occurrence data for haemolytic V. parahaemolyticus were 13/45 (29%) in farm fish samples, 2/6 (one third) from farm water samples and 27/45 (60%) from retail fish samples. Microbial loads for haemolytic V. parahaemolyticus microbial loads ranged from 1.9 to 4.1 Log CFU/g in fish samples and 2.0 to 3.0 Log CFU/g in farm water samples. AMR risk assessments (ARRAs) for both the full farm-to-home and partial retail-to-home chains in the risk modelling framework were conducted, specifically for ampicillin, penicillin G, tetracycline and haemolytic (non-AMR) scenarios. The haemolytic ARRA predicted an average probability of illness of 2.9 × 10-4 and 4.5 × 10-5 per serving for the farm-to-home and retail-to-home chains, respectively, translating to 57 and 148 cases annually. The ratios of the average probability of illness per year for the three ARRAs to the haemolytic ARRA were 1.1 × 10-2 and 3.0 × 10-4 (ampicillin and penicillin G, respectively) for the farm-to-home chain and 1.3, 1.6 and 0.4 (ampicillin, penicillin G and tetracycline, respectively) for the retail-to-home chain. Sensitivity analysis showed that the initial concentrations of haemolytic V. parahaemolyticus in the gills and intestines of the fish and the cooking and washing of the fish cavity were the major variables influencing risk outputs in all modelled ARRAs. The findings of this study are useful for relevant stakeholders to make informed decisions regarding risk management to improve overall food safety.

Antimicrobial Resistance Risk Assessment of Vibrio parahaemolyticus Isolated from Farmed Green Mussels in Singapore.

Vibrio parahaemolyticus, commonly found in seafood products, is responsible for gastroenteritis resulting from the consumption of undercooked seafood. Hence, there is a need to characterize and quantify the risk involved from this pathogen. However, there has been no study reporting the quantification of hemolytic antimicrobial-resistant (AMR) Vibrio parahaemolyticus in locally farmed shellfish in Singapore. In this study, ampicillin, penicillin G, tetracycline resistant, and non-AMR hemolytic V. parahaemolyticus were surveyed and quantified in green mussel samples from different premises in the food chain (farm and retail). The occurrence data showed that 31/45 (68.9%) of farmed green mussel samples, 6/6 (100%) farm water samples, and 41/45 (91.1%) retail shellfish samples detected the presence of hemolytic V. parahaemolyticus. V. parahaemolyticus counts ranged from 1.6-5.9 Log CFU/g in the retail shellfish samples and 1.0-2.9 Log CFU/g in the farm water samples. AMR risk assessments (ARRA), specifically for ampicillin, penicillin G, tetracycline, and hemolytic (non-AMR) scenarios were conducted for the full farm-to-home and partial retail-to-home chains. The hemolytic ARRA scenario estimated an average probability of illness of 5.7 × 10-3 and 1.2 × 10-2 per serving for the full and partial chains, respectively, translating to 165 and 355 annual cases per total population or 2.9 and 6.2 cases per 100,000 population, respectively. The average probability of illness per year ratios for the three ARRAs to the hemolytic ARRA were 0.82, 0.81, and 0.47 (ampicillin, penicillin G, and tetracycline, respectively) for the full chain and 0.54, 0.39, and 0.09 (ampicillin, penicillin G, and tetracycline, respectively) for the partial chain. The sensitivity analysis showed that the overall cooking effect, initial concentrations of pathogenic V. parahaemolyticus, and harvest duration and harvest temperature were key variables influencing the risk estimates in all of the modelled ARRAs. The study findings can be used by relevant stakeholders to make informed decisions for risk management that improve food safety.

Characterisation of Salmonella Enteritidis ST11 and ST1925 Associated with Human Intestinal and Extra-Intestinal Infections in Singapore.

Salmonella Enteritidis is a major foodborne pathogen worldwide. In this study, a total of 276 S. enteritidis isolates, collected between 2016 and 2017 from human, food and farm/slaughterhouse samples, were studied to enhance the understanding of the epidemiology of human salmonellosis in Singapore. Results showed all 276 isolates belonged either to ST1925 (70.3%) or ST11 (29.7%), with ST11 being significantly more frequent in extra-intestinal isolates and chicken isolates. Food isolates, most of which were from poultry, showed the highest prevalence of resistance (33-37%) against beta-lactams or beta-lactams/beta-lactamase inhibitor combination (ampicillin, piperacillin and ampicillin/sulbactam). The analysis showed the detection of genes associated with resistance to aminoglycoside genes (99.6%), tetracycline (55.1%), and beta-lactams (14.9%) of all isolates. Nine types of plasmids were found in 266 isolates; the most common incompatibility group profiles were IncFIB(S)-IncFII(S)-IncX1 (72.2%) and IncFIB(S)-IncFII(S) (15.8%). Most plasmid harbouring isolates from chicken (63.6%, 14/22) and from human (73.8%, 175/237) shared the same plasmid profile (IncFIB(S)-IncFII(S)-IncX1). SNP analysis showed clustering of several isolates from poultry food products and human isolates, suggesting phylogenetic relatedness among these isolates. Lastly, this study provides important epidemiological insights on the application of phenotypic and next-generation sequencing (NGS) tools for improved food safety and public health surveillance and outbreak investigation of S.enteritidis.

Whole-Genome Sequencing Analysis of Salmonella Isolates from Poultry Farms, a Slaughterhouse, and Retail Stalls in Thailand.

The draft genome sequences of 21 Salmonella isolates obtained from poultry production chains in Hat Yai City, Songkhla Province, Thailand, are reported in this study. Our study revealed that there was Salmonella environmental contamination along poultry production chains and cross-contamination among poultry through inanimate surfaces in the environment.

Inactivation of retrotransposon Tos17 Chr.7 in rice cultivar Nipponbare through CRISPR/Cas9-mediated gene editing.

Retrotransposons are mobile genetic elements capable of transposition via reverse transcription of RNA intermediates. Rice cultivar Nipponbare contains two nearly identical genomic copies of Tos17, an endogenous copia-like LTR retrotransposon, on chromosomes 7 (Tos17 Chr.7) and 10 (Tos17 Chr.10), respectively. Previous studies demonstrated that only Tos17 Chr.7 is active in transposition during tissue culture. Tos17 Chr.7 has been extensively used for insertional mutagenesis as a tool for functional analysis of rice genes. However, Tos17 Chr.7 transposition might generate somaclonal mutagenesis with undesirable traits during rice transformation, which would affect the evaluation or application of transgenes. In this study, we generated a Tos17 Chr.7 knockout mutant D873 by using CRISPR/Cas9 gene editing system. The gene-edited allele of Tos17 Chr.7 in D873, designated as Tos17 D873, has an 873-bp DNA deletion in the pol gene of Tos17 Chr.7, which caused the deletion of the GAG-pre-integrase domain and the integrase core domain. Although the transcription of Tos17 D873 was activated in D873 calli, no transposition of Tos17 D873 was detected in the regenerated D873 plants. The results demonstrate that the GAG-pre-integrase domain and the integrase core domain are essential for Tos17 Chr.7 transposition and the deletion of the two domains could be not complemented by other LTR retrotransposons in rice genome. As the Tos17 Chr.7-derived somaclonal mutagenesis is blocked in the D873 plants, the generation of the Tos17 D873 allele will be helpful in production of transgenic rice plants for gene function study and genetic engineering. Similar approach can be used to inactivate other retrotransposons in crop breeding.

Occurrence and Antimicrobial Resistance Traits of Escherichia coli from Wild Birds and Rodents in Singapore.

Antimicrobial resistance (AMR) in Escherichia coli (E. coli) poses a public health concern worldwide. Wild birds and rodents, due to their mobility, are potential vehicles for transmission of AMR bacteria to humans. Ninety-six wild birds' faecal samples and 135 rodents' droppings samples were collected and analysed in 2017. Forty-six E. coli isolates from wild birds and rodents were subjected to AMR phenotypic and genotypic characterisation. The proportion of E. coli isolates resistant to at least one of the antimicrobials tested from wild birds (80.8%) was significantly higher than that of isolates from rodents (40.0%). The proportion of E. coli isolates resistant to each antimicrobial class for wild birds was 3.8% to 73.1% and that for rodents was 5.0% to 35.0%. Six out of 26 E. coli isolates from wild birds (23.1%) and two out of 20 (10.0%) isolates from rodents were multi-drug resistant (MDR) strains. These MDR E. coli isolates were detected with various antimicrobial resistance genes such as blaTEM-1B and qnrS1 and could be considered as part of the environmental resistome. Findings in this study suggested that wild birds and rodents could play a role in disseminating antimicrobial resistant E. coli, and this underscores the necessity of environment management and close monitoring on AMR bacteria in wild birds and rodents to prevent spreading of resistant organisms to other wildlife animals and humans.

Marker-assisted breeding of the rice restorer line Wanhui 6725 for disease resistance, submergence tolerance and aromatic fragrance.

BACKGROUND: Rice is a staple food crop in the world. With the increase in world population and economic development, farmers need to produce more rice in limited field. However, the rice production is frequently affected by biotic and abiotic stresses. The use of natural disease resistance and stress tolerance through genetic breeding is the most efficient and economical way to combat or acclimate to these stresses. In addition, rice with aromatic fragrance can significantly increase market value for its good grain quality. Mianhui 725 (MH725) is an elite restorer line that has been widely used to produce three-line hybrid rice in China. We previously introduced rice bacterial blight resistance genes Xa4 and Xa21 into MH725 and obtained an introgression rice line Wanhui 421 (WH421), which theoretically possesses 96.9% genetic background of MH725. RESULTS: Here we report the introduction and pyramiding of disease resistance genes Xa27 and Pi9, submergence tolerance gene Sub1A and aromatic fragrance gene badh2.1 in WH421 through backcrossing and marker-assisted selection. The newly developed introgression rice line was designated as Wanhui 6725 (WH6725), which theoretically possesses 95.0% genetic background of MH725. WH6725 and its hybrid rice conferred disease resistance to both blast and bacterial blight diseases and showed tolerance to submergence for over 14 days without significant loss of viability. Compared with non-aromatic rice MH725, WH6725 has strong aromatic fragrance. The major important agronomic traits and grain quality of WH6725 and its hybrid rice obtained in field trials were similar to those of MH725 and the control hybrid rice, indicating that WH6725 is as good as MH725 when it is used as a restorer line for three-line hybrid rice production. CONCLUSION: We have successfully developed a new restorer line WH6725 with disease resistance to rice blast and bacterial blight, tolerance to submergence and aromatic fragrance, which can be used to replace MH725 for hybrid rice production.