Isolation and Genetic Characterization of Japanese Encephalitis Virus Two Decades after Its Elimination in Singapore.

Japanese encephalitis virus (JEV) is an important arbovirus in Asia that can cause serious neurological disease. JEV is transmitted by mosquitoes in an enzootic cycle involving porcine and avian reservoirs, in which humans are accidental, dead-end hosts. JEV is currently not endemic in Singapore, after pig farming was abolished in 1992; the last known human case was reported in 2005. However, due to its location along the East-Asian Australasian Flyway (EAAF), Singapore is vulnerable to JEV re-introduction from the endemic regions. Serological and genetic evidence in the last decade suggests JEV's presence in the local fauna. In the present study, we report the genetic characterization and the first isolation of JEV from 3214 mosquito pools consisting of 41,843 Culex mosquitoes, which were trapped from April 2014 to May 2021. The findings demonstrated the presence of genotype I of JEV (n = 10), in contrast to the previous reports of the presence of genotype II of JEV in Singapore. The genetic analyses also suggested that JEV has entered Singapore on several occasions and has potentially established an enzootic cycle in the local fauna. These observations have important implications in the risk assessment and the control of Japanese encephalitis in non-endemic countries, such as Singapore, that are at risk for JEV transmission.

The use of anticoagulants for rodent control in a mixed-use urban environmefent in Singapore: A controlled interrupted time series analysis.

Vector control remains an important strategy in preventing rodent-borne diseases. Studies quantifying the impact of anticoagulant bait use on rodent populations are scarce in tropical settings. This study examined the impact of anticoagulant bait use on three measures of rodent activity in Singapore to inform rodent-borne disease control strategies. Using a controlled interrupted time-series analytical design with negative binomial and linear regression models, the average rodent activity levels were compared in the pre- and post-intervention periods. There was a 62.7% (Incidence Rate Ratio (IRR): 0.373, 95% CI: [0.224, 0.620]) reduction in the number of rodents caught, a 25.8-unit (coefficient = -25.829, 95% CI: [-29.855, -21.804]) reduction in the number of 30 g/unit baits consumed and a 61.9% (IRR: 0.381, 95% CI: [0.218, 0.665]) reduction in the number of marred bait stations relative to the pre-intervention period. There was a rise in all three outcome measures within four months after the post-intervention period. This study provided strong evidence that anticoagulant baits substantially reduces rodent activity. The population resurgence after the post-intervention period reinforces the importance of timing the resumption of control measures aimed at reducing rodent-borne disease transmission.

VKORC1 mutations in rodent populations of a tropical city-state as an indicator of anticoagulant rodenticide resistance.

Anticoagulant rodenticides are commonly used in rodent control because they are economical and have great deployment versatility. However, rodents with Single Nucleotide Polymorphism (SNP) mutations within the Vkorc1 gene are resistant to the effects of anticoagulant rodenticide use and this influences the effectiveness of control strategies that rely on such rodenticides. This study examined the prevalence of rat SNP mutations in Singapore to inform the effectiveness of anticoagulant rodenticide use. A total of 130 rat tail samples, comprising 83 Rattus norvegicus (63.8%) and 47 Rattus rattus complex (36.2%) were conveniently sampled from November 2016 to December 2019 from urban settings and sequenced at exon 3 of Vkorc1. Sequencing analysis revealed 4 synonymous and 1 non-synonymous mutations in Rattus rattus complex samples. A novel synonymous mutation of L108L was identified and not previously reported in other studies. Non-synonymous SNPs were not detected in the notable codons of 120, 128 and 139 in R. norvegicus, where these regions are internationally recognised to be associated with resistance from prior studies. Our findings suggest that the prevalence of anticoagulant rodenticide resistance in Singapore is low. Continued monitoring of rodenticide resistance is important for informing rodent control strategies aimed at reducing rodent-borne disease transmission.

Adaptation to an Amoeba Host Leads to Pseudomonas aeruginosa Isolates with Attenuated Virulence.

The opportunistic pathogen Pseudomonas aeruginosa is ubiquitous in the environment, and in humans, it is capable of causing acute or chronic infections. In the natural environment, predation by bacterivorous protozoa represents a primary threat to bacteria. Here, we determined the impact of long-term exposure of P. aeruginosa to predation pressure. P. aeruginosa persisted when coincubated with the bacterivorous Acanthamoeba castellanii for extended periods and produced genetic and phenotypic variants. Sequencing of late-stage amoeba-adapted P. aeruginosa isolates demonstrated single nucleotide polymorphisms within genes that encode known virulence factors, and this correlated with a reduction in expression of virulence traits. Virulence for the nematode Caenorhabditis elegans was attenuated in late-stage amoeba-adapted P. aeruginosa compared to early-stage amoeba-adapted and nonadapted counterparts. Further, late-stage amoeba-adapted P. aeruginosa showed increased competitive fitness and enhanced survival in amoebae as well as in macrophage and neutrophils. Interestingly, our findings indicate that the selection imposed by amoebae resulted in P. aeruginosa isolates with reduced virulence and enhanced fitness, similar to those recovered from chronic cystic fibrosis infections. Thus, predation by protozoa and long-term colonization of the human host may represent similar environments that select for similar losses of gene function. IMPORTANCE Pseudomonas aeruginosa is an opportunistic pathogen that causes both acute infections in plants and animals, including humans, and chronic infections in immunocompromised and cystic fibrosis patients. This bacterium is commonly found in soils and water, where bacteria are constantly under threat of being consumed by bacterial predators, e.g., protozoa. To escape being killed, bacteria have evolved a suite of mechanisms that protect them from being consumed or digested. Here, we examined the effect of long-term predation on the genotypes and phenotypes expressed by P. aeruginosa. We show that long-term coincubation with protozoa gave rise to mutations that resulted in P. aeruginosa becoming less pathogenic. This is particularly interesting as similar mutations arise in bacteria associated with chronic infections. Importantly, the genetic and phenotypic traits possessed by late-stage amoeba-adapted P. aeruginosa are similar to those observed in isolates obtained from chronic cystic fibrosis infections. This notable overlap in adaptation to different host types suggests similar selection pressures among host cell types as well as similar adaptation strategies.

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.

Salmonella in Retail Food and Wild Birds in Singapore-Prevalence, Antimicrobial Resistance, and Sequence Types.

Non-typhoidal salmonellosis is a leading cause of foodborne zoonosis. To better understand the epidemiology of human salmonellosis, this study aimed to determine the prevalence, antimicrobial resistance and sequence types of Salmonella in retail food and wild birds (proximity to humans) in Singapore. We analyzed 21,428 cooked and ready-to-eat food and 1,510 residual faecal samples of wild birds collected during 2010-2015. Thirty-two Salmonella isolates from food and wild birds were subjected to disc diffusion and multi-locus sequence typing (MLST). Salmonella was isolated from 0.08% (17/21,428) of food and 0.99% (15/1510) of wild birds. None of the isolates from wild birds (n = 15) exhibited phenotypic resistance, while the isolates from food (47.1%, 8/17) showed a high prevalence of phenotypic resistance to, at least, one antimicrobial. These findings suggested that the avian Salmonella isolates had been subjected to less antimicrobial selection pressure than those from food samples. MLST revealed specific sequence types found in both food and wild birds. The study can guide future studies with whole-genome analysis on a larger number of isolates from various sectors for public health measures.