First report of environmental bla PAC-1-carrying Aeromonas enteropelogenes.

The bla PAC-1 has been reported in Central Asia and Europe countries like Afghanistan and France in Aeromonas caviae and Pseudomonas aeruginosa strains from animals and patients, respectively. However, there is no record of bla PAC-1-carrying strain from the natural environment, and bla PAC-1-carrying Aeromonas has not been reported in the Asia Pacific. Here, we report the first known enviromental bla PAC-1-carrying Aeromonas enteropelogenes in the world from reservoir water in Singapore. We have performed a comprehensive genetic environment alignment and comparison of bla PAC-1 between our strain and other strains from different countries and sources and found the bla PAC-1 located on a highly conserved gene cluster. We suggest that environmental Aeromonas strains may act as a hidden reservoir involved in the circulating of bla PAC-1. The finding of conserved bla PAC-1 cluster also suggested the existence of multiple transmission pathways of bla PAC-1 in the Asia-Pacific region, involving multiple sources and different species.

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.

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.

Honeys with anti-inflammatory capacity can alter the elderly gut microbiota in an ex vivo gut model.

The anti-inflammatory effect of different sourced honeys and the impact on elderly gut microbiota were studied in terms of chemical compositions, anti-inflammatory effect and gut microbiota modulating capacities. All four honeys suppressed the production of pro-inflammatory markers NO, IL-1ß and IL-6 induced by lipopolysaccharide and promoted the expression of anti-inflammatory cytokines IL-10 in RAW 264.7 cells. Moreover, in the ex vivo batch gut model using elderly fecal microbiota (referred to as microcosm), it was showed that the addition of honeys increased the abundance of beneficial lactobacilli, decreased the abundance of potentially harmful Gram negative enteric bacteria, and exerted a beneficial effect on the production of short chain fatty acids. The concentration of gallic acid in honeys was positively correlated with the expression level of IL-10 and the abundance of lactobacilli. These findings indicate honeys with anti-inflammatory capacity have great potential for regulating the elderly gut microbiota which would lead to health benefits.

Integrative multiomics analysis reveals host-microbe-metabolite interplays associated with the aging process in Singaporeans.

The age-associated alterations in microbiomes vary across populations due to the influence of genetics and lifestyles. To the best of our knowledge, the microbial changes associated with aging have not yet been investigated in Singapore adults. We conducted shotgun metagenomic sequencing of fecal and saliva samples, as well as fecal metabolomics to characterize the gut and oral microbial communities of 62 healthy adult male Singaporeans, including 32 young subjects (age, 23.1 ± 1.4 years) and 30 elderly subjects (age, 69.0 ± 3.5 years). We identified 8 gut and 13 oral species that were differentially abundant in elderly compared to young subjects. By combining the gut and oral microbiomes, 25 age-associated oral-gut species connections were identified. Moreover, oral bacteria Acidaminococcus intestine and Flavonifractor plautii were less prevalent/abundant in elderly gut samples than in young gut samples, whereas Collinsella aerofaciens and Roseburia hominis showed the opposite trends. These results indicate the varied gut-oral communications with aging. Subsequently, we expanded the association studies on microbiome, metabolome and host phenotypic parameters. In particular, Eubacterium eligens increased in elderly compared to young subjects, and was positively correlated with triglycerides, which implies that the potential role of E. eligens in lipid metabolism is altered during the aging process. Our results demonstrated aging-associated changes in the gut and oral microbiomes, as well as the connections between metabolites and host-microbe interactions, thereby deepening the understanding of alterations in the human microbiome during the aging process in a Singapore population.

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.

Characterization of Extended-Spectrum Beta-Lactamase-Producing Escherichia coli Isolates from Jurong Lake, Singapore with Whole-Genome-Sequencing.

Background: The fast-spreading of Extended-Spectrum Beta-Lactamase-Producing Escherichia coli (ESBL-producing E. coli) and ESBL genes has become a big challenge to public health. The risk of spreading ESBL genes and pathogens in the environment and community has raised public health concern. The characterizing and whole-genome sequencing studies of ESBL-producing bacteria from reservoir water in Singapore is still limited. Materials and methods: The reservoir water sample was taken from two randomly selected sampling points of the Chinese Garden (Jurong river reservoir), which is a popular reservoir park in Singapore. The bacteria of the water sample were collected with 0.45 µm filter membranes and enriched before processing with ESBL-producing E. coli screening. The collected ESBL positive isolates were further characterized by both phenotypic tests including disc diffusion and microdilution Minimum Inhibitory Concentration (MIC) test, and also genotypic test as whole-genome sequencing analysis. Besides, to investigate the transferability of the resistance gene, a conjugation test was performed with the J53 E. coli strain as the gene receptor. Result: Nine ESBL-producing E. coli isolates were collected and confirmed as ESBL-producing with both phenotypic and genotypic tests. A potential pathogen as ST131 clade A isolate was identified, and all isolates were determined to harbor a blaCTX-M gene. Among them, strain J1E4 was resistant to polymyxin E and confirmed to harboring a conjugatable mcr-1 gene. Further genetic environment analysis has reflected a conversed gene cluster formed by insert sequence (IS), blaCTX-M-15, and WbuC family cupin-fold metalloprotein, which may potentially jump from the plasmids to the chromosome. Conclusion: The first time we reported the whole genome sequencing (WGS) data of ESBL-producing E. coli including potential pathogen (ST131) present in reservoir water in Singapore. The ESBL-producing E. coli from reservoir water also carrying conjugatable colistin resistance genes which may become a risk to human health.

Prevalence and genomic analysis of ESBL-producing Escherichia coli in retail raw meats in Singapore.

OBJECTIVES: To determine the prevalence and genetic characteristics of ESBL-producing Escherichia coli in retail raw meats from Singapore markets. METHODS: A total of 634 raw meat (chicken, pork and beef) samples were collected from markets in Singapore during June 2017-October 2018. The samples were enriched overnight and then incubated on Brilliance™ ESBL Agar. Presumptive ESBL isolates were confirmed using the double-disc synergy test. Confirmed ESBL-producing E. coli were sent for WGS and bioinformatic analysis was performed. RESULTS: The prevalence of ESBL-producing E. coli in chicken, pork and beef meats was 51.2% (109/213), 26.9% (58/216) and 7.3% (15/205), respectively. A total of 225 ESBL-producing E. coli were isolated from 184 samples. ß-Lactam resistance genes were detected in all isolates. After ß-lactam resistance genes, the most common antimicrobial resistance genes detected were aminoglycoside resistance genes (92.4%). One hundred and seventy-two (76.4%), 102 (45.3%) and 52 (23.1%) isolates carried blaCTX-M genes, blaTEM genes and blaSHV genes, respectively. blaCTX-M-55 (57/225, 25.3%) and blaCTX-M-65 (40/225, 17.8%) were the most frequent ESBL genes. Colistin resistance genes (including mcr-1, mcr-3 and mcr-5) were found in 15.6% of all isolates. CONCLUSIONS: This study indicates that ESBL-producing E. coli are widely found in retail raw meats, especially chicken, in Singapore. Occurrence of MDR (resistance to at least three classes of antimicrobial) and colistin resistance genes in retail raw meat suggests potential food safety and public health risks.

Development of a Dairy-Free Fermented Oat-Based Beverage With Enhanced Probiotic and Bioactive Properties.

Lactobacillus fermentum PC1 with proven probiotic properties was used to ferment oats with added honey to develop a probiotic beverage with enhanced bioactive ingredients. The viable Lactobacilli were enumerated during the fermentation and storage at 4°C, as well as after exposure to simulated gastrointestinal tract conditions. Good survival was noted both during storage as well as when exposed to the in vitro digestive tract conditions. Comparative analysis of the antioxidant activity, total phenolic content, and phenolic composition indicated fermentation improved the total antioxidant capacity and phenolic acid concentration. An increase of more than 50% of gallic acid, catechin, vanillic acid, caffeic acid, p-coumaric acid, and ferulic acid was observed in the methanol extracts. Moreover, no significant decrease in the ß-glucan content was noted during fermentation and storage. In conclusion, this fermented product has a great potential as a functional food with enhanced probiotic survival and increased bioactive ingredients.

Metagenomics-Based Proficiency Test of Smoked Salmon Spiked with a Mock Community.

An inter-laboratory proficiency test was organized to assess the ability of participants to perform shotgun metagenomic sequencing of cold smoked salmon, experimentally spiked with a mock community composed of six bacteria, one parasite, one yeast, one DNA, and two RNA viruses. Each participant applied its in-house wet-lab workflow(s) to obtain the metagenomic dataset(s), which were then collected and analyzed using MG-RAST. A total of 27 datasets were analyzed. Sample pre-processing, DNA extraction protocol, library preparation kit, and sequencing platform, influenced the abundance of specific microorganisms of the mock community. Our results highlight that despite differences in wet-lab protocols, the reads corresponding to the mock community members spiked in the cold smoked salmon, were both detected and quantified in terms of relative abundance, in the metagenomic datasets, proving the suitability of shotgun metagenomic sequencing as a genomic tool to detect microorganisms belonging to different domains in the same food matrix. The implementation of standardized wet-lab protocols would highly facilitate the comparability of shotgun metagenomic sequencing dataset across laboratories and sectors. Moreover, there is a need for clearly defining a sequencing reads threshold, to consider pathogens as detected or undetected in a food sample.

Proficiency Testing of Metagenomics-Based Detection of Food-Borne Pathogens Using a Complex Artificial Sequencing Dataset.

Metagenomics-based high-throughput sequencing (HTS) enables comprehensive detection of all species comprised in a sample with a single assay and is becoming a standard method for outbreak investigation. However, unlike real-time PCR or serological assays, HTS datasets generated for pathogen detection do not easily provide yes/no answers. Rather, results of the taxonomic read assignment need to be assessed by trained personnel to gain information thereof. Proficiency tests are important instruments of validation, harmonization, and standardization. Within the European Union funded project COMPARE [COllaborative Management Platform for detection and Analyses of (Re-) emerging and foodborne outbreaks in Europe], we conducted a proficiency test to scrutinize the ability to assess diagnostic metagenomics data. An artificial dataset resembling shotgun sequencing of RNA from a sample of contaminated trout was provided to 12 participants with the request to provide a table with per-read taxonomic assignments at species level and a report with a summary and assessment of their findings, considering different categories like pathogen, background, or contaminations. Analysis of the read assignment tables showed that the software used reliably classified the reads taxonomically overall. However, usage of incomplete reference databases or inappropriate data pre-processing caused difficulties. From the combination of the participants' reports with their read assignments, we conclude that, although most species were detected, a number of important taxa were not or not correctly categorized. This implies that knowledge of and awareness for potentially dangerous species and contaminations need to be improved, hence, capacity building for the interpretation of diagnostic metagenomics datasets is necessary.

Make science evolve into a One Health approach to improve health and security: a white paper.

The World One Health Congresses are biennial gatherings of approximately 1500 professionals from relevant international organisations, OIE, FAO, WHO, World Bank, leading scientific experts and researchers in the field of One Health, animal production and trade, food safety, animal health, human health and environmentology/ecology, government representatives in public health, human health, food safety, environmental health and global health security. The Congress is organized by the One Health Platform. This white paper summarizes highlights of the 5th International One Health Congress in Saskatoon, Canada, June 2018 and serves as a roadmap for the future, detailing several concrete action points to be carried out in the run-up to the 6th World One Health Congress in Edinburgh, Scotland, June 2020.

Distribution of Salmonella Serovars in Humans, Foods, Farm Animals and Environment, Companion and Wildlife Animals in Singapore.

We analyzed the epidemiological distribution of Salmonella serovars in humans, foods, animals and the environment as a One-Health step towards identifying risk factors for human salmonellosis. Throughout the 2012-2016 period, Salmonella ser. Enteritidis was consistently the predominating serovar attributing to >20.0% of isolates in humans. Other most common serovars in humans include Salmonella ser. Stanley, Salmonella ser. Weltevreden, Salmonella ser. Typhimurium and Salmonella ser. 4,5,12:b:-(dT+). S. Enteritidis was also the most frequent serovar found among the isolates from chicken/chicken products (28.5%) and eggs/egg products (61.5%) during the same period. In contrast, S. Typhimurium (35.2%) and Salmonella ser. Derby (18.8%) were prevalent in pork/pork products. S. Weltevreden was more frequent in seafood (19.2%) than others (≤3.0%). Most isolates (>80.0%) from farms, companion and wildlife animals belonged to serovars other than S. Enteritidis or S. Typhimurium. Findings demonstrate the significance of a One-Health investigative approach to understand the epidemiology Salmonella for more effective and integrated surveillance systems.

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.

Comparative Blood and Urine Metabolomics Analysis of Healthy Elderly and Young Male Singaporeans.

Comparative metabolomics analysis of biofluids could provide information about the metabolic alterations in aging. To investigate the signature of multiple metabolic profiles associated with aging in an Asian population, we performed a pilot study in healthy Singaporeans, including 33 elderly and 33 young males. Fasting whole bloods were analyzed by routine hematology; the serum and urine metabolome profiles were obtained using NMR-based nontargeted metabolomics analysis and targeted lipoprotein analysis. Among the 90 identified compounds in serum and urine samples, 32 were significantly different between the two groups. The most obvious age-related metabolic signatures include decreased serum levels of albumin lysyl and essential amino acids and derivatives but increased levels of N-acetyl glycoproteins and several lipids and elevated urine levels of trimethylamine N-oxide, scyllo-inositol, citrate, and ascorbic acid but decreased levels of several amino acids, acetate, etc. Among 112 lipoprotein subfractions, 65 were elevated, and 2 were lower in the elderly group. These significantly age-varying metabolites, especially in the amino acid and fatty acid metabolism pathways, suggest that the regulation of these pathways contributes to the aging process in Chinese Singaporeans. Further multiomics studies including the gut microbiome and intervention studies in a larger cohort are needed to elucidate the possible mechanisms in the aging process.

Draft Genome Sequences of 12 Leuconostoc carnosum Strains Isolated from Cooked Ham Packaged in a Modified Atmosphere and from Fresh Sausages.

Leuconostoc carnosum is a lactic acid bacterium that preferentially colonizes meat. In this work, we present the draft genome sequences of 12 Leuconostoc carnosum strains isolated from modified-atmosphere-packaged cooked ham and fresh sausages. Three strains harbor bacteriocin genes.

Comparative Genomics of Leuconostoc carnosum.

Leuconostoc carnosum is a known colonizer of meat-related food matrices. It reaches remarkably high loads during the shelf life in packaged meat products and plays a role in spoilage, although preservative effects have been proposed for some strains. In this study, the draft genomes of 17 strains of L. carnosum (i.e., all the strains that have been sequenced so far) were compared to decipher their metabolic and functional potential and to determine their role in food transformations. Genome comparison and pathway reconstruction indicated that L. carnosum is a compact group of closely related heterofermentative bacteria sharing most of the metabolic features. Adaptation to a nitrogen-rich environment, such as meat, is evidenced by 23 peptidase genes identified in the core genome and by the autotrophy for nitrogen compounds including several amino acids, vitamins, and cofactors. Genes encoding the decarboxylases yielding biogenic amines were not present. All the strains harbored 1-4 of 32 different plasmids, bearing functions associated to proteins hydrolysis, transport of amino acids and oligopeptides, exopolysaccharides, and various resistances (e.g., to environmental stresses, bacteriophages, and heavy metals). Functions associated to bacteriocin synthesis, secretion, and immunity were also found in plasmids. While genes for lactococcin were found in most plasmids, only three harbored the genes for leucocin B, a class IIa antilisterial bacteriocin. Determinants of antibiotic resistances were absent in both plasmids and chromosomes.

Extended-spectrum ß-lactamase-producing Proteus mirabilis with multidrug resistance isolated from raw chicken in Singapore: Genotypic and phenotypic analysis.

OBJECTIVES: Proteus mirabilis is ubiquitous in soil and water. It is an important catheter-associated urinary tract pathogen and has reportedly been associated with antimicrobial-resistant infections. This study reports the draft genome of a multidrug-resistant P. mirabilis isolated from raw retail chicken meat in Singapore. METHODS: The P. mirabilis strain was isolated on BrillianceTM ESBL Agar and was screened for antimicrobial susceptibility against 29 antimicrobial agents using a MicroScan® Neg MIC Panel Type 44. The double-disk synergy test (DDST) was used for confirmation of extended-spectrum ß-lactamase (ESBL) production. Genomic DNA from the pure culture isolate was extracted and was sent for sequencing based on Illumina HiSeq 2500 technology. Further bioinformatics analysis was performed using online tools available at the Center for Genomic Epidemiology. RESULTS: Species identification of the isolate was performed by KmerFinder. Antimicrobial susceptibility testing of the isolate showed multidrug resistance to broad-spectrum ß-lactams, fluoroquinolones and aminoglycosides, among others. ESBL production was confirmed by the DDST. A total of 29 antimicrobial resistance genes were detected by ResFinder. CONCLUSION: To the best of our knowledge, this is the first report of the whole-genome sequence of a multidrug-resistant P. mirabilis producing an ESBL from raw chicken meat in Singapore. This indicates that raw meat in Singapore can be a reservoir for drug-resistant pathogens.