Takashi Suzuki

The threat of antimicrobial resistance in aquatic environments, particularly riverine systems, is escalating, in part due to effluents discharged from healthcare facilities. This issue has been recognized not only in Japan but also in other Asian countries such as Indonesia and Nepal. Nevertheless, existing research remains limited, prompting an investigation into the prevalence of antimicrobial-resistant bacteria in the upstream and downstream sites of environmental rivers. In 2024, six samples were collected from three rivers in Hyogo Prefecture, Japan; five samples from five river sites in Indonesia; and three samples from downstream sites of rivers in Kathmandu, Nepal. These samples were subjected to selective culture–based Next Generation Sequencing and resistome analyses, based exclusively on the selective culture of bacteria propagated on CHROMagar ESBL plates. In Japan and Indonesia, Pseudomonas, Stenotrophomonas and Acinetobacter were frequently detected, whereas Klebsiella was overwhelmingly predominant in Nepal. Significant differences in the similarity of bacterial community composition among sampling sites across the three countries were observed (p < 0.001). Notably, Nepal exhibited the highest abundance level of antimicrobial resistance genes among the three countries, largely consisting of β-lactam resistance genes. In conclusion, these analyses elucidated substantial differences in bacterial community composition and degrees of environmental contamination.

Background: When the first systematic list of mosquitoes in Nepal was reported in 1990, there was no description ofAedes aegypti(L.), whileAedes albopictus(Skuse) has been included in theStegomyiasubgroup since the 1950s. The first record ofAe. aegyptiin Nepal was reported in 2009, suggesting some coincidence between the invasion of this species and the first record of dengue fever in Nepal in 2006.Results: We performed a field survey of the distribution and insecticide susceptibility ofAe. aegyptiandAe. albopictusin Nepal in 2017 and 2018. Mosquito larvae were collected from used tires located along the streets of Kathmandu, Bharatpur and Pokhara, and a simplified bioassay was used to assess the susceptibility of the larvae to pyrethroid insecticides usingd-allethrin. The presence or absence of point mutations in the voltage-gated sodium channel was also detected by direct sequencing. V1016G was detected at a high frequency and a strong correlation was observed between the frequencies of V1016G and susceptibility indices inAe. aegyptipopulations. F1534C was also detected at a relatively low frequency. InAe. albopictuspopulations, susceptibilities tod-allethrin were high and no point mutations were detected. Analysis of the cytochromecoxidase subunit 1 (cox1) gene was performed for assessing genetic diversity and the existence of two strains were identified inAe. aegyptipopulations. One consisted of 9 globally-distributed haplotypes while the other was derived from an African haplotype.Conclusions: The high pyrethroid resistance, high V1016G frequency, and relatively low quantity of insecticides used to control dengue vectors in Nepal may have resulted in only weak selection pressure favoring insecticide resistance and could support the hypothesis that this species has recently been introduced from neighboring Asian countries where pyrethroid resistance is relatively widespread.

The Anopheles gambiae sensu lato species complex consists of a number of cryptic species with different habitats and behaviours. These morphologically indistinct species are identified by chromosome banding and molecular diagnostic techniques which are still under improvement even though the current SINE method for identification between An. coluzzii and An. gambiae works reliably. This study describes a refinement of the SINE method to increase sensitivity and high throughput method for the identification of both species and An. arabiensis using amplicon dissociation characteristics. Field collected samples, laboratory reared colonies and crossed specimens of the two species were used for the design of the protocol. An. gambiae , An. coluzzii , and hybrids of the two species were provided by the insectary of Vestergaard-NMIMR Vector Labs at the Noguchi Memorial Institute for Medical Research (Ghana) and An. arabiensis from Kenya. Samples were first characterised using conventional SINE PCR method, and further assayed using SYBR green, an intercalating fluorescent dye. The three species and hybrids were clearly differentiated using the melting temperature of the dissociation curves, with derivative peaks at 72 Celsius for An. arabiensis , 75°C for An. gambiae and 86°C for An. coluzzii . The hybrids ( An. gambiae / An. coluzzii ) showed both peaks. This work is the first to describe a SYBR green real time PCR method for the characterization of An. arabiensis , An. gambiae and An. coluzzii and was purposely designed for basic melt-curve analysis (rather than high-resolution melt-curve) to allow it to be used on a wide range of real-time PCR machines.