Clinically relevant sequence types of carbapenemase-producing Escherichia coli and Klebsiella pneumoniae detected in Finnish wastewater in 2021-2022.

BACKGROUND: Antimicrobial resistance (AMR) is a critical threat to human health. Escherichia coli and Klebsiella pneumoniae are clinically the most important species associated with AMR and are the most common carbapenemase-producing (CP) Enterobacterales detected in human specimens in Finland. Wastewater surveillance has emerged as a potential approach for population-level surveillance of AMR, as wastewater could offer a reflection from a larger population with one sample and minimal recognized ethical issues. In this study, we investigated the potential of wastewater surveillance to detect CP E. coli and K. pneumoniae strains similar to those detected in human specimens. METHODS: Altogether, 89 composite samples of untreated community wastewater were collected from 10 wastewater treatment plants across Finland in 2021-2022. CP E. coli and K. pneumoniae were isolated using selective culture media and identified using MALDI-TOF MS. Antimicrobial susceptibility testing was performed using disk diffusion test and broth microdilution method, and a subset of isolates was characterized using whole-genome sequencing. RESULTS: CP E. coli was detected in 26 (29.2%) and K. pneumoniae in 25 (28.1%) samples. Among E. coli, the most common sequence type (ST) was ST410 (n = 7/26, 26.9%), while ST359 (n = 4/25, 16.0%) predominated among K. pneumoniae. Globally successful STs were detected in both E. coli (ST410, ST1284, ST167, and ST405) and K. pneumoniae (ST512, ST101, and ST307). K. pneumoniae carbapenemases (KPC) were the most common carbapenemases in both E. coli (n = 11/26, 42.3%) and K. pneumoniae (n = 13/25, 52.0%), yet also other carbapenemases, such as blaNDM-5, blaOXA-48, and blaOXA-181, were detected. We detected isolates harboring similar ST and enzyme type combinations previously linked to clusters in Finland, such as E. coli ST410 with blaKPC-2 and K. pneumoniae ST512 with blaKPC-3. CONCLUSIONS: Our study highlights the presence of clinically relevant strains of CP E. coli and K. pneumoniae in community wastewater. The results indicate that wastewater surveillance could serve as a monitoring tool for CP Enterobacterales. However, the specificity and sensitivity of the methods should be improved, and technologies, like advanced sequencing methods, should be utilized to distinguish data with public health relevance, harness the full potential of wastewater surveillance, and implement the data in public health surveillance.

Insights into the Phylogeny and Evolution of Cold Shock Proteins: From Enteropathogenic Yersinia and Escherichia coli to Eubacteria.

Psychrotrophic foodborne pathogens, such as enteropathogenic Yersinia, which are able to survive and multiply at low temperatures, require cold shock proteins (Csps). The Csp superfamily consists of a diverse group of homologous proteins, which have been found throughout the eubacteria. They are related to cold shock tolerance and other cellular processes. Csps are mainly named following the convention of those in Escherichia coli. However, the nomenclature of certain Csps reflects neither their sequences nor functions, which can be confusing. Here, we performed phylogenetic analyses on Csp sequences in psychrotrophic enteropathogenic Yersinia and E. coli. We found that representative Csps in enteropathogenic Yersinia and E. coli can be clustered into six phylogenetic groups. When we extended the analysis to cover Enterobacteriales, the same major groups formed. Moreover, we investigated the evolutionary and structural relationships and the origin time of Csp superfamily members in eubacteria using nucleotide-level comparisons. Csps in eubacteria were classified into five clades and 12 subclades. The most recent common ancestor of Csp genes was estimated to have existed 3585 million years ago, indicating that Csps have been important since the beginning of evolution and have enabled bacterial growth in unfavorable conditions.

Changes in Transcriptome of Yersinia pseudotuberculosis IP32953 Grown at 3 and 28°C Detected by RNA Sequencing Shed Light on Cold Adaptation.

Yersinia pseudotuberculosis is a bacterium that not only survives, but also thrives, proliferates, and remains infective at cold-storage temperatures, making it an adept foodborne pathogen. We analyzed the differences in gene expression between Y. pseudotuberculosis IP32953 grown at 3 and 28°C to investigate which genes were significantly more expressed at low temperature at different phases of growth. We isolated and sequenced the RNA from six distinct corresponding growth points at both temperatures to also outline the expression patterns of the differentially expressed genes. Genes involved in motility, chemotaxis, phosphotransferase systems (PTS), and ATP-binding cassette (ABC) transporters of different nutrients such as fructose and mannose showed higher levels of transcripts at 3°C. At the beginning of growth, especially genes involved in securing nutrients, glycolysis, transcription, and translation were upregulated at 3°C. To thrive as well as it does at low temperature, Y. pseudotuberculosis seems to require certain cold shock proteins, especially those encoded by yptb3585, yptb3586, yptb2414, yptb2950, and yptb1423, and transcription factors, like Rho, IF-1, and RbfA, to maintain its protein synthesis. We also found that genes encoding RNA-helicases CsdA (yptb0468), RhlE (yptb1214), and DbpA (yptb1652), which unwind frozen secondary structures of nucleic acids with cold shock proteins, were significantly more expressed at 3°C, indicating that these RNA-helicases are important or even necessary during cold. Genes involved in excreting poisonous spermidine and acquiring compatible solute glycine betaine, by either uptake or biosynthesis, showed higher levels of transcripts at low temperatures. This is the first finding of a strong connection between the aforementioned genes and the cold adaptation of Y. pseudotuberculosis. Understanding the mechanisms behind the cold adaptation of Y. pseudotuberculosis is crucial for controlling its growth during cold storage of food, and will also shed light on microbial cold adaptation in general.