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Abstract Circulating bat coronaviruses present a significant pandemic threat, yet our understanding of their genetic diversity and evolutionary dynamics remains limited. Over 3 years, we sampled 1,462 bats in Cambodia’s Steung Treng province, identifying extensive and diverse coronaviruses co-circulation. Using metatranscriptomic and amplicon sequencing, we generated 33 complete sarbecovirus genomes sequences, revealing novel lineages that cluster into four distinct groups, each associated with different Rhinolophus bat species. Our analysis highlights rapid migration and recombination of sarbecovirus lineages over short distances and timescales. Of note, the receptor-binding domains of two novel viral groups exhibit high similarity to SARS-CoV-2, and pseudovirus assays confirmed the ability of this spike protein to mediate entry into cells expressing human ACE2, suggesting a potential zoonotic risk. The observed genetic diversity underscores the urgent need for continuous surveillance to identify high-risk animal-to-human interfaces and inform pandemic preparedness.
Extensively drug-resistant gram-negative bacteria harbouring dual resistance to carbapenems and colistin represent a critical global health threat. A total of 929 population-representative Enterobacter isolates were systematically collected from 29 hospitals across four regions of Taiwan between 2010 and 2020. Forty-one isolates (4.4%) were nonsusceptible to carbapenems and underwent whole-genome sequencing, resistance gene profiling, plasmid analysis, and antimicrobial susceptibility testing (AST). Among them, 35 isolates (85.4%) exhibited dual resistance to carbapenems and colistin; however, only half (17/35) were detectable by standard phenotypic AST. Colistin resistance was primarily mediated by activation of the chromosomal arnBCADTEF operon, which was frequently inducible and often undetected by standard testing, rather than by mcr-9 or mcr-10. A conserved IncHI2 plasmid carrying blaIMP-8 and mcr-9 persisted and circulated across Enterobacter species for over a decade. Species-specific resistance patterns were observed: E. roggenkampii typically exhibited colistin resistance despite lacking carbapenemases, whereas E. hormaechei commonly carried blaIMP-8 and occasionally lacked the arn operon. Both species exhibited comparable imipenem nonsusceptibility, complicating therapeutic decision-making. The convergence of carbapenem and colistin resistance in a substantial proportion of Enterobacter isolates at the population level makes this genus an emerging priority for hospital infection control and antimicrobial resistance surveillance. These findings underscore the urgent need for improved diagnostics, strengthened antimicrobial resistance surveillance, and optimized treatment strategies.
Zoonotic neglected tropical diseases (NTDs) remain a substantial but under-recognised source of human morbidity and economic concern in the Greater Mekong Subregion, particularly in settings characterised by close human-animal interaction. This article represents a narrative synthesis of zoonotic disease research conducted in Laos and Cambodia between 2000 and 2025. The review integrates published literature with findings from long-term surveillance programmes conducted by the authors and collaborating institutions in Laos, with comparative insights from Cambodia, to examine the presence, distribution, diversity, and drivers of zoonotic pathogens at the human-animal interface. Evidence demonstrates the endemic presence of a wide range of parasitic, bacterial, and viral zoonoses, including Taenia solium, Trichinella spp., Streptococcus suis, rickettsial infections, melioidosis, hepatitis E virus, and Japanese encephalitis virus. Some of these pathogens are sustained within smallholder livestock systems, informal slaughter, farming practises, food networks, and wet market environments, where limited diagnostic capacity and fragmented surveillance obscure true disease presence. Surveillance innovations, including abattoir-based sampling, cross-sectoral serological studies, environmental surveillance approaches, and molecular diagnostic tools, have improved pathogen detection but have also highlighted persistent structural and behavioural barriers to control. Socio-cultural practices, occupational exposure, wildlife trade, and economic dependencies reinforce transmission dynamics, indicating that biomedical interventions alone are insufficient. Instead, zoonotic disease persistence reflects the interaction of livestock production systems, environmental conditions, diagnostic limitations, and entrenched human behaviours. This review emphasises the need for integrated One Health approaches that combine strengthened surveillance, improved diagnostics, behavioural interventions, and regional collaboration. Addressing zoonotic NTDs in Laos and Cambodia requires coordinated strategies that account for both biological complexity and socio-economic context to achieve sustainable disease control and improved public health outcomes.
Salmonella is the most common cause of salmonellosis, a foodborne infection that is transmitted by consuming contaminated food. It is becoming more prevalent worldwide, particularly in Cambodia. Additionally, Salmonella is recognized as one of the drug-resistant bacteria, posing serious public health problems that spread quickly due to the misuse and overuse of antibiotics in healthcare, livestock husbandry, and agriculture. Therefore, the purpose of this study was to determine the occurrence of Salmonella from different food matrices in Cambodia and to assess its profile of antibiotic resistance, including the detection of extended-spectrum beta-lactamase (ESBL) resistant genes. From January to December 2023, a total of food samples (n=1,506), categorized into twenty-two sample types, were tested for Salmonella using the standard method ISO 6579-1:2017. The positive Salmonella were subjected to antimicrobial susceptibility testing by using disc diffusion method against 22 antibiotics following the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines.. The ESBL-producing Salmonella were screened for the resistance genes via conventional PCR. The results showed that Salmonella was present in 2.7% (40/1506) of the analyzed food samples originated from seafood (10.3%), fish and fish products (13.8%), and meat and meat products (28.7%). Antimicrobial susceptibility assay revealed that 57.5% (23/40) were resistant to amoxicillin, piperacillin, and ticarcillin, and 52.5% (21/40) were resistant to nalidixic acid, while 35% (14/40) of the Salmonella isolates were susceptible to all of the antibiotics tested. It was also found that 45% (18/40) of Salmonella isolates were multidrug-resistant (MDR) and 27.5% (11/40) were ESBL-producing strains originated from all samples of meat and meat products. Molecular detections revealed that 45.5% of the ESBL-producing Salmonella isolates carried bla CTX-M group 9 genes. Our findings demonstrated the primary sources of Salmonella contamination, including seafood, fish, and meat, and meat products serving as a significant reservoir for multidrug-resistant (MDR) and ESBL-producing strains. This pathogen's emergence highlights the need for regulatory measures intended to restrict the use of antibiotics in food agriculture. As a recommendation, further study should employ other PCR primers to determine the remaining ESBL-producing strains in combination with sequencing to define the specific resistance genes, or whole genome sequencing, to comprehensively identify resistance genes and trace the spread of resistant Salmonella strains in the food production environment.
Here, we present a protocol to acquire high resolution, extended depth of field images of insect specimens by photographic focus stacking using a modular digital imaging system. The method provides a standardized workflow linking equipment assembly, calibration, image acquisition, and post processing. Using a full frame mirrorless camera (61 MP) coupled to microscope objectives and synchronized strobe illumination, the protocol achieves pixel scales from 0.76 m-0.19 m and produces artifact free composites through sub-micron focus increments (0.2 m). The procedure can capture and process approximately 20 final images per week under routine laboratory conditions. Compared with existing stacking solutions, this lowcost hybrid setup (< 30% of the cost of commercial systems) maximizes accessibility while maintaining diffraction limited image quality. Representative applications include the production of color calibrated identification plates for taxonomy, biodiversity digitization, and outreach. The protocol's standardized structure facilitates reproducibility across laboratories and field stations, supporting large scale insect imaging campaigns in both resource limited and institutional environments.
