Wastewater is a geospatially- and temporally-linked microbial fingerprint of a given population, making it a potentially valuable tool for tracking public health across locales and time. Here, we integrate targeted and bulk RNA sequencing (N = 2238 samples) to track the viral, bacterial, and functional content over geospatially distinct areas within Miami Dade County, USA, from 2020-2022. We used targeted amplicon sequencing to track diverse SARS-CoV-2 variants across space and time, and we found a tight correspondence with positive PCR tests from University students and Miami-Dade hospital patients. Additionally, in bulk metatranscriptomic data, we demonstrate that the bacterial content of different wastewater sampling locations serving small population sizes can be used to detect putative, host-derived microorganisms that themselves have known associations with human health and diet. We also detect multiple enteric pathogens (e.g., Norovirus) and characterize viral diversity across sites. Moreover, we observed an enrichment of antimicrobial resistance genes (ARGs) in hospital wastewater; antibiotic-specific ARGs correlated to total prescriptions of those same antibiotics (e.g Ampicillin, Gentamicin). Overall, this effort lays the groundwork for systematic characterization of wastewater that can potentially influence public health decision-making.

A novel lytic bacteriophage, PSA-KC1, was isolated from wastewater. In this study, the whole genome of the bacteriophage PSA-KC1 was analyzed, and its lytic properties were assessed. PSA-KC1 has a linear double-stranded DNA genome with a total length of 43,237 base pairs and a GC content of 53.6%. In total, 65 genes were predicted, 46 of which were assigned functions as structural proteins involved in genome replication, packaging or phage lysis. PSA-KC1 belongs to the genus Septimatrevirus under the Caudoviricetes class. The aim of this study was to investigate the efficacy of the lytic bacteriophage PSA-KC1 and compare it with that of the Pyophage phage cocktail on 25 multi drug resistant (MDR) Pseudomonas aeruginosa strains isolated from sputum samples of cystic fibrosis patients. Seventeen of these strains were susceptible (68%) to the PSA-KC1 lytic phage we isolated, whereas eight clinical strains were resistant. However, 22 (88%) of the P. aeruginosa strains were susceptible to the Pyophage cocktail, and three (12%) were resistant to the Phage cocktail. At the end of our study, a new lytic phage active against multidrug-resistant P. aeruginosa strains from CF patients was isolated, and its genome was characterized. Since the PSA-KC1 phage does not contain virulence factors, toxins or integrase genes, it can be expected to be a therapeutic candidate with the potential to be used safely in phage therapy.

Background/aim: The aim of this study was to investigate the effects of PhiKZ phage therapy and meropenem alone or combined treatments in a pneumonia mouse model induced by the Pseudomonas aeruginosa PAO1 strain. The cross- talk between lungs and kidneys was also determined. Materials and methods: The systemic, lung-specific, and kidney-specific inflammation levels and the bacterial load in lung tissue and biochemical parameters were investigated after PhiKZ phage therapy and meropenem alone or combined treatments in a pneumonia mouse model induced by the P. aeruginosa PAO1 strain. The cross-talk between lungs and kidneys was also determined by measuring plasma levels of glycocalyx components. Results: The greatest reduction in lung bacterial load was obtained with the combined use of the PhiKZ phage and meropenem. The C-reactive protein level in the patient group was significantly higher than in the treatment groups and decreased after treatment. Serum interleukin IL-6 levels were statistically significantly higher than in the phage serum and phage + meropenem groups. Pulmonary infection can trigger proinflammatory cytokines such as IL-6, TNF-alpha, and IL-1 beta. Increased cytokines trigger insulin resistance in the liver. Lung infection triggers liver inflammation because there is communication between the lungs and liver. Conclusion: Elevated proinflammatory cytokines due to infection were decreased because of the reduced burden of bacterial load after treatment. This study might have proved communication between lungs and kidneys related to proinflammatory cytokines.

Background/Objectives: Klebsiella pneumoniae is a clinically important pathogen that causes respiratory tract infections, pneumonia, wound infections, urinary tract infections, and sepsis. It is on the World Health Organization (WHO) priority pathogen list as it causes antimicrobial-resistant infections. The aim of this study was to isolate bacteriophages against pan-resistant K. pneumoniae isolated from clinical wound infections. Results: One of the isolated phages, CTF-1, possesses a linear double-stranded DNA genome that is 40,841 base pairs (bp) long and contains 44 predicted genes. Functional assignments were made for 31 of the predicted gene products, which are associated with genome replication, phage packaging, structural proteins, and host lysis, leaving 13 annotated as hypothetical proteins. Based on sequencing analysis, phage CTF-1 is a new member of the genus Przondovirus within the order Autographivirales. Phage CTF-1 was effective against 22 of 25 (88%) pan-resistant K. pneumoniae isolates. The latent period and lytic cycle of the phage were approximately 40 min, with a burst size of about 92 PFU/mL. Conclusions: Our findings suggest that Klebsiella phage CTF-1 is an excellent candidate for phage therapy due to its high lytic activity against pan-resistant K. pneumoniae strains and lack of genes encoding antibiotic resistance, toxins, virulence factors, or integrases.

Probiotic quality and safety require strain-specific characterization supported by controlled production and comprehensive evaluation. This study aimed to integrate controlled fermentation and biomass production, downstream processing and lyophilization, microbiological quality control, batch reproducibility, whole-genome characterization, in vitro functional and safety-related assessment, real-time stability monitoring, and OECD-compliant in vivo safety evaluation of the human-derived Limosilactobacillus reuteri strain ATA-LTC-Lr050600. The strain was produced under controlled fermentation conditions, followed by harvesting, protective formulation, and lyophilization. Product quality was assessed by standardized microbiological testing, whole-genome sequencing, and in vitro assays evaluating acid and bile tolerance, adhesion to a fibroblast cell model, hemolytic activity, and cytotoxicity. Real-time stability was monitored during storage, and safety was further evaluated using OECD-compliant acute and repeated-dose oral toxicity studies. The production process generated a lyophilized preparation with consistent microbiological quality and reproducible manufacturing characteristics. Whole-genome sequencing confirmed strain identity and did not detect acquired antimicrobial resistance determinants. The strain tolerated simulated gastrointestinal conditions, adhered to the evaluated fibroblast cell model, and showed no detectable hemolytic activity or cytotoxicity under the experimental conditions. Product viability and microbiological quality were maintained throughout the monitored storage period. In the in vivo studies, no treatment-related adverse effects were identified, and the repeated-dose study established a no-observed-adverse-effect level of 1000 mg/kg/day under the conditions examined. Collectively, these integrated findings provide a basis for further strain-specific evaluation and support the continued development of L. reuteri ATA-LTC-Lr050600 as a well-characterized probiotic candidate under the conditions examined.