Background: Wastewater is a major source of Antibiotic-Resistant Bacteria (ARB) and a hotspot for the exchange of Antibiotic-Resistant Genes (ARGs). The occurrence of Carbapenem-Resistant Bacteria (CRB) in wastewater samples is a major public health concern. Objectives: This study aimed to analyze Antibiotic resistance in bacteria from wastewater sources in Pakistan. Methods: We analyzed 32 bacterial isolates, including 18 Escherichia coli, 4 Klebsiella pneumoniae, and 10 other bacterial isolates using phenotypic antibiotic susceptibility assay and whole-genome sequencing. This study identified the ARGs, plasmid replicons, and integron genes cassettes in the sequenced isolates. One representative isolate was further sequenced using Illumina and Oxford nanopore sequencing technologies. Results: Our findings revealed high resistance to clinically important antibiotics: 91% of isolates were resistant to cefotaxime, 75% to ciprofloxacin, and 62.5% to imipenem, while 31% showed non-susceptibility to gentamicin. All E. coli isolates were resistant to cephalosporins, with 72% also resistant to carbapenems. Sequence analysis showed a diverse resistome, including carbapenamases (blaNDM-5, blaOXA-181), ESBLs (blaCTX-M-15, blaTEM), and AmpC-type β-lactamases (blaCMY). Key point mutations noticed in the isolates were pmrB_Y358N (colistin) and ftsI_N337NYRIN, ftsI_I336IKYRI (carbapenem). The E. coli isolates had 11 different STs, with ST410 predominating (28%). Notably, the E. coli phylogroup A isolate 45EC1, (ST10886) is reported for the first time from wastewater, carrying blaNDM-5, blaCMY-16, and pmrB_Y358N with class 1 integron gene cassette of dfrA12-aadA2-qacEΔ1 on a plasmid-borne contig. Other carbapenamase, blaNDM-1 and blaOXA-72, were detected in K. pneumoniae 22EB1 and Acinetobacter baumannii 51AC1, respectively. The integrons with the gene cassettes encoding antibiotic resistance, and the transport and bacterial mobilization protein, were identified in the sequenced isolates. Ten plasmid replicons were identified, with IncFIB prevalent in 53% of isolates. Combined Illumina and Oxford nanopore sequencing revealed blaNDM-5 on an IncFIA/IncFIC plasmid and is identical to those reported in the USA, Myanmar, and Tanzania. Conclusions: These findings highlight the environmental prevalence of high-risk and WHO-priority pathogens with clinically important ARGs, underscoring the need for a One Health approach to mitigate ARB isolates.

Porcine reproductive and respiratory syndrome virus (PRRSV) is the most economically important swine pathogen in North America and is second globally only to African swine fever virus. PRRSV is a positive sense, single stranded RNA virus associated with reproductive disorder of sows and respiratory disease of pigs at all age. Diagnostic tests are commonly used to monitor the presence of PRRSV in swine populations including sequencing the open reading frame 5 (ORF5) gene to track the epidemiology of the virus and lateral introductions into a farm. PRRSView is a web portal created at the Iowa State University Veterinary Diagnostic Laboratory (ISU VDL) to host analytical and phylogenetic tools related to PRRSV ORF5 sequences with the goal of assisting veterinarians and producers in evaluating the genetic diversity, spatial, and temporal aspects of ORF5 sequences maintained in the ISU VDL database. PRRSView works in conjunction with the broader Swine Disease Reporting System (SDRS) project to contextualize the ever changing patterns of PRRSV diversity, and supports interactive tools for veterinarians to analyze their sequence data compared to other sequences detected throughout the United States. The PRRSView homepage provides a phylogenetic overview of the sequences generated by the ISU VDL within the previous month, indicating the current strains detected in circulation. There are currently three ORF5 analytical tools available on PRRSView: a genetic sequence BLAST tool, a vaccine identity tool, and an RFLP tool. The ORF5 BLAST tool allows the users to submit their ORF5 gene sequences and returns up to 10 closely related sequences from the ISU VDL database, with metadata that includes the state, genetic lineage, RFLP, and identity to the query sequence. The vaccine identity tool allows users to quickly calculate the percent homology of their sequence(s) to five different PRRSV vaccines: Inglevac PRRS ATP, Inglevac PRRS MLV, Prime Pac PRRS, Fostera PRRS, and Prevacent PRRS, as well as the distance to the Lelystad strain. This tool also builds a neighbor joining tree with a set of curated strains to estimate the genetic lineage of the sequence, which is rendered in the web browser for viewing. Additionally, this tool will calculate the RFLP of the sequence, and the exact positions of the cut sites are shown when hovering over the RFLP value. The last analytical tool provided is the ORF5 RFLP tool, which quickly calculates the RFLP pattern of the input sequences. These analytical tools are designed to allow veterinarians and researchers to easily analyze their PRRSV ORF5 sequences against the expansive ISU VDL database to gain valuable epidemiologic information and comparative data regarding the genetic lineages and related metadata of the PRRSV circulating in a production system, while lowering the barrier of entry for use.

Rapid and reliable identification of the hemagglutinin (HA) and neuraminidase (NA) genetic clades of an influenza A virus (IAV) sequence from swine can inform control measures and multivalent vaccine composition. Current approaches to genetically characterize HA or NA sequences are based on nucleotide similarity or phylogenetic analyses. Public databases exist to acquire IAV genetic sequences for comparison, but personnel at the diagnostic or production level have difficulty in adequately updating and maintaining relevant sequence datasets for IAV in swine. Further, phylogenetic analyses are time intensive, and inference drawn from these methods is impacted by input sequence data and associated metadata. We describe here the use of the IAV multisequence identity tool as an integrated public webpage located on the Iowa State University Veterinary Diagnostic Laboratory (ISU-VDL) FLUture website: https://influenza.cvm.iastate.edu/ . The multisequence identity tool uses sequence data derived from IAV-positive cases sequenced at the ISU-VDL, employs a BLAST algorithm that identifies sequences that are genetically similar to submitted query sequences, and presents a tabulation and visualization of the most genetically similar IAV sequence and associated metadata from the FLUture database. Our tool removes bioinformatic barriers and allows clients, veterinarians, and researchers to rapidly classify and identify IAV sequences similar to their own sequences to augment interpretation of results.

A diagnostic investigation into an outbreak of fatal respiratory disease among young goats in Iowa, USA revealed bronchitis lesions of unknown etiology and secondary bacterial bronchopneumonia. Hypothesis-free metagenomics identified a previously unreported picornavirus (USA/IA26017/2023), and further phylogenetic analysis classified USA/IA26017/2023 as an aphthovirus related to bovine rhinitis B virus. Viral nucleic acid was localized to lesions of bronchitis using in situ hybridization. This marks the first report of a picornavirus putatively causing respiratory disease in goats and highlights the potential for cross-species transmission of aphthoviruses.