The antimicrobial resistance (AMR) crisis urgently requires countermeasures for reducing the dissemination of plasmid-borne resistance genes. Of particular concern are opportunistic pathogens of Enterobacteriaceae. One innovative approach is the CRISPR-Cas9 system which has recently been used for plasmid curing in defined strains of Escherichia coli. Here we exploited this system further under challenging conditions: by targeting the bla_TEM–1 AMR gene located on a high-copy plasmid (i.e., 100–300 copies/cell) and by directly tackling bla_TEM–1-positive clinical isolates. Upon CRISPR-Cas9 insertion into a model strain of E. coli harboring bla_TEM–1 on the plasmid pSB1A2, the plasmid number and, accordingly, the bla_TEM–1 gene expression decreased but did not become extinct in a subpopulation of CRISPR-Cas9 treated bacteria. Sequence alterations in bla_TEM–1 were observed, likely resulting in a dysfunction of the gene product. As a consequence, a full reversal to an antibiotic sensitive phenotype was achieved, despite plasmid maintenance. In a clinical isolate of E. coli, plasmid clearance and simultaneous re-sensitization to five beta-lactams was possible. Reusability of antibiotics could be confirmed by rescuing larvae of Galleria mellonella infected with CRISPR-Cas9-treated E. coli, as opposed to infection with the unmodified clinical isolate. The drug sensitivity levels could also be increased in a clinical isolate of Enterobacter hormaechei and to a lesser extent in Klebsiella variicola, both of which harbored additional resistance genes affecting beta-lactams. The data show that targeting drug resistance genes is encouraging even when facing high-copy plasmids. In clinical isolates, the simultaneous interference with multiple genes mediating overlapping drug resistance might be the clue for successful phenotype reversal.

Despite the considerable morbidity and mortality of yellow fever virus (YFV) infections in Brazil, our understanding of disease outbreaks is hampered by limited viral genomic data. Here, through a combination of phylogenetic and epidemiological models, we reconstructed the recent transmission history of YFV within different epidemic seasons in Brazil. A suitability index based on the highly domesticated Aedes aegypti was able to capture the seasonality of reported human infections. Spatial modeling revealed spatial hotspots with both past reporting and low vaccination coverage, which coincided with many of the largest urban centers in the Southeast. Phylodynamic analysis unraveled the circulation of three distinct lineages and provided proof of the directionality of a known spatial corridor that connects the endemic North with the extra-Amazonian basin. This study illustrates that genomics linked with eco-epidemiology can provide new insights into the landscape of YFV transmission, augmenting traditional approaches to infectious disease surveillance and control.

Oropouche orthobunyavirus (OROV) is an emerging public health concern due to its expanding geographic range and increasing case numbers. In Brazil, 13,785 cases were confirmed in 2024, with an additional 3680 reported by January 2025, according to the Ministry of Health. Initially restricted to the Amazon region, OROV has recently been detected in new areas, highlighting the need for enhanced surveillance and vector control strategies. While Culicoides paraensis is the primary vector, the potential role of other species in transmitting the currently circulating OROV strain in Brazil remains unclear. Here, we experimentally assessed the infectivity and dissemination of a recently isolated Oropouche orthobunyavirus (OROV) strain in two widespread mosquito species, Aedes aegypti and Culex quinquefasciatus, collected from diverse regions of Brazil. Our results demonstrated that both mosquito species were refractory to oral infection, suggesting that natural transmission through these vectors is unlikely. However, in artificial systemic infection, Ae. aegypti showed viral replication and immune system activation, indicating its potential to support OROV replication under specific conditions. Additionally, to assess the potential impact of coinfection, we investigated whether Chikungunya virus (CHIKV), an arbovirus that naturally infects Ae. aegypti, could facilitate OROV infection dynamics in this mosquito species. Our results suggest that coinfection does not promote OROV oral infection. Furthermore, we examined whether OROV systemic infection induced an immune response in Ae aegypti. We analyzed the major immune response pathways—RNAi, Toll, IMD, and JAK-STAT—and observed that the RNAi pathway was the most strongly activated in response to OROV infection in Ae. aegypti. These findings highlight the importance of ongoing surveillance and further studies on OROV evolution, vector adaptation, and transmission dynamics, particularly in urban settings where vector populations and viral interactions may facilitate new epidemiological scenarios.

This report provides a detailed overview of the resurgence of DENV-3 in the state of Minas Gerais, Brazil, which is a concerning scenario in the context of dengue, a mosquito-borne viral disease. Historically, Brazil has grappled with dengue epidemics caused primarily by the DENV-1 and DENV-2 serotypes. However, in 2023, a significant shift in this pattern was observed as DENV-3 made a notable resurgence. This resurgence was characterized by the increase in DENV-3 cases within the country and the region of the Americas. Given the absence of sustained DENV-3 circulation in Brazil in previous years, this situation poses a significant risk, making the population highly susceptible to a potential novel epidemic. In November 2023, a 31-year-old male patient in Belo Horizonte exhibited symptoms of acute febrile syndrome. Multiplex RT-qPCR using the Kit Molecular ZC D-Tipagem confirmed DENV-3 infection, suggesting a likely autochthonous case, as the patient reported no travel history. To promptly assess this resurgence, we applied the nanopore sequencing technology. This allowed for the rapid characterization of the initial DENV-3 case isolated in Minas Gerais in 2023, representing a 13-year interval since the serotype’s previous documented circulation in that state. This case report underscores the critical importance of proactive monitoring and the swift implementation of targeted control strategies to address the evolving dynamics of dengue, with a specific emphasis on the resurgence of DENV-3 in the state.

The Chikungunya virus (CHIKV) presents global health challenges, with Brazil experiencing outbreaks since its introduction in 2014. In 2023, following a CHIKV outbreak in Minas Gerais (MG), social media was used to optimize an entomological survey aimed at identifying vectors and viral lineages and assessing insecticide resistance. Following Instagram posts, residents with suspected CHIKV infection were able to schedule mosquito aspirations. In total, 421 mosquitoes (165 Aedes aegypti and 256 Culex quinquefasciatus) were captured from 40 households in Salinas city (MG) and tested for the Dengue, Zika, and Chikungunya viruses through RT-qPCR. Twelve of 57 pools (10 Ae. aegypti and two Cx. quinquefasciatus) tested positive for CHIKV RNA. Viral RNA was also detected in the heads of nine Ae. aegypti, indicating viral dissemination but not in Cx. quinquefasciatus. Genome sequencing yielded the first near-complete genome from the 2023 outbreak, unveiling that the CHIKV strain belonged to the East/Central/South African (ECSA) genotype. Additionally, genetic analyses revealed high frequencies of kdr alleles, including in CHIKV-infected mosquitoes, suggesting resistance to pyrethroid insecticides in this Ae. aegypti population. Social media was important for guiding mosquito-capture efforts in CHIKV transmission hotspots, thus optimizing the opportunity for viral detection. These findings emphasize the urgent need for innovative vector studies and control strategies, as well as interdisciplinary approaches in public health interventions.