Findings demonstrate strain-specific adaptive responses to aquatic environments and underline the context-dependent influence of plasmids in shaping adaptation.
Abstract
The spread of antimicrobial-resistant Enterobacterales is a major One Health issue, with aquatic environments increasingly recognized as potential reservoirs. However, data on bacterial adaptation to water is limited. We examined the survival and transcriptomic adaptation of Escherichia coli and Klebsiella pneumoniae, including plasmid-cured variants (PCVs), during 14 days in sterilized tap and river water. The strains belonged to sequence types (ST)648 and ST307, representing international high-risk clonal lineages. Viable cell counts of wild-type strains and PCVs remained stable in both water types. We used RNA sequencing, followed by functional analysis of the differentially expressed genes. Considerable transcriptomic changes occurred, especially in K. pneumoniae, with extensive regulation of genes related to inorganic ion transport, and coenzyme and nutrient transport and metabolism. Adaptational differences between wild-type strains and PCVs highlighted plasmid-associated effects. These findings demonstrate strain-specific adaptive responses to aquatic environments and underline the context-dependent influence of plasmids in shaping adaptation.
Klebsiella aerogenes is an opportunistic pathogen increasingly associated with healthcare-associated infections and severe respiratory complications. However, it's in vivo adaptation during host colonization remains poorly understood. We characterized three clonal K. aerogenes ST93 isolates sequentially recovered on days 4, 14, and 19 of hospitalization from a patient with fatal viral pneumonia and concurrent abdominal sepsis. While initially identified generically as Klebsiella spp. by automated clinical systems, Whole-Genome Sequencing and Average Nucleotide Identity confirmed their identity at the species level. Genomic analysis revealed an in vivo plasmid curing event characterized by the loss of a 6.1 kb blaOXA-232-carrying plasmid in the final isolate (18281). Despite this plasmid loss, isolate 18281 sustained persistent carbapenem resistance due to a conserved ompK36 chromosomal mutation. Phenotypically, 18281 exhibited significantly higher lethality in Galleria mellonella larvae and induced progressive body weight loss in dexamethasone-treated mice compared to the other isolates. This enhanced virulence strongly correlated with a tissue-specific, compartmentalized transcriptional reprogramming, characterized by a marked 15.66-fold up-regulation of the yersiniabactin gene irp1 within hepatic tissues, whereas the colibactin gene clbA was consistently downregulated in vivo across both isolates and organs. Our findings demonstrate that intra-host microevolution of K. aerogenes involves structural plasmid instability and targeted transcriptional remodeling during host adaptation. This localized adaptive behavior underscores the clinical threat posed by transitional clonal variants in critically ill patients undergoing viral-mediated immune dysregulation.
Luis Duarte-Zambrano, Neli Nava-Domínguez, J. P. Ramírez-Hinojosa et al.· International Microbiology· 0 citations
Wastewater treatment plants (WWTPs) are increasingly recognized as environmental reservoirs for antimicrobial resistance (AMR), yet genome-level data on non-clinical Gram-negative bacteria in wastewater remain scarce in many low- and middle-income settings. This study characterized the resistome, plasmid content, and genetic diversity of environmentally associated Gram-negative bacteria recovered from wastewater systems to elucidate their contribution to environmental AMR persistence. Influent and effluent samples (n = 30) were collected from three wastewater treatment plants. Sixteen Gram-negative isolates were recovered and identified using the VITEK system and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Antimicrobial susceptibility testing was performed phenotypically, followed by whole-genome sequencing and bioinformatic analyses to identify antimicrobial resistance genes (ARGs), plasmid replicons, virulence-associated genes, and sequence types.The isolates comprised of
Enterobacter cloacae
(n = 5),
Raoultella planticola
(n = 3),
Raoultella ornithinolytica
(n = 3),
Citrobacter freundii
(n = 3), and
Aeromonas spp
. (n = 2). Multidrug resistance was observed phenotypically across influent and effluent samples. Genomic analysis identified clinically relevant ARGs, including
bla
ACT
,
bla
CTX-M-15
,
bla
CMY
,
qnr
variants,
fosA
,
aadA
,
sul1
, and
tet
genes, frequently associated with IncF, IncQ, IncL/M, and Col-type plasmids and virulent determinants including
yersiniabactin
and
enterobactin
. Resistance and plasmid profiles persisted in effluent-derived isolates, suggesting environmental persistence and potential transmission.This study provides genome-level baseline evidence that wastewater-associated, non-clinical Gram-negative bacteria in Namibia harbor clinically relevant AMR determinants, reinforcing the value of wastewater-based genomic surveillance for understanding environmental AMR dissemination, and emphasizing the public health implications of wastewater reuse.
Graça K. Kandanda, L. M. Mwapagha· PLOS Water· 0 citations
OBJECTIVES
Carbapenem-resistant Enterobacteriaceae (CRE) pose a major public health threat due to extensive drug resistance and nosocomial spread. This study characterized the genome sequence of a multidrug-resistant E. coli strain EC6030 carrying blaNDM-5 recovered from a 10-month-old patient, and investigated its relationship to global blaNDM-5-positive E. coli.
METHODS
Whole-genome sequencing was performed to analyze antimicrobial resistance genes, virulence determinants, plasmid content, and the genetic context of blaNDM-5 in EC6030. A total of 7,606 blaNDM-5-carrying E. coli genomes were retrieved from the NCBI RefSeq database. Core-genome multilocus sequence typing, minimum-spanning tree analysis, and SNP-based phylogenetic analysis were used to assess global distribution and genetic relatedness.
RESULTS
EC6030 was resistant to multiple antimicrobial classes, including β-lactams, carbapenems, fluoroquinolones, trimethoprim-sulfamethoxazole, chloramphenicol, and tetracycline, but remained susceptible to amikacin and gentamicin. Genomic analysis assigned EC6030 to ST410 and phylogroup C. The blaNDM-5 gene was located on a 46,161 bp IncX3 plasmid flanked by multiple insertion sequences. This plasmid showed high sequence similarity to previously reported blaNDM-carrying plasmids among E. coli and K. pneumoniae isolates in the public database. Globally, blaNDM-5-carrying E. coli strains are most prevalent in Asia, primarily from clinical specimens, and occur sporadically. The closest relative to EC6030 is a ST410 strain P24_WM1_05.20, recovered from the United Kingdom in 2020 which differing by 61 SNPs.
CONCLUSION
These findings highlight the clinical significance and potential clonal and plasmid-mediated dissemination of blaNDM-5-carrying E. coli ST410 lineage and support strengthened genomic surveillance, antimicrobial stewardship, and infection control strategies globally.
Guoli Li, Shuangshuang Li, Menglu Zhang et al.· Journal of Global Antimicrob...· 0 citations
Antimicrobial resistance (AMR) is a major global health threat, and environmental reservoirs such as wastewater (WWTPs) and drinking water treatment plants (DWTPs) may facilitate its persistence and spread despite reducing bacterial loads. We investigated 152 antibiotic-resistant Escherichia coli strains collected across treatment stages from two WWTPs and one DWTP in Barcelona, Spain. Strains were characterized through antimicrobial susceptibility testing, whole-genome sequencing, multilocus sequence typing, biofilm assays, and screening of antimicrobial resistance genes (ARGs), virulence factors (VFGs), biocide and heavy-metal tolerance genes (HMTGs). Although E. coli bacterial loads decreased along treatment, AMR remained highly prevalent: 85.5% of strains were multidrug-resistant (MDR), 5.3% extensively drug-resistant, and 11.2% carbapenemase-producers. Strains harboring integrase genes were 2.4 to 11.8 times more likely to harbor ARGs for sulfonamide, aminoglycoside, phenicol, trimethoprim, mercury and quaternary-ammonium compounds. Strains carrying blaCTX-M genes were 3.0 to 20.3 times more likely to carry VFGs, while high-risk clones were 3.2 to 7.0 times more associated with VFGs. Some MDR and high-risk E. coli clones persisted in reclaimed water, and one MDR strain was detected at the DWTP inlet. These findings highlight environmental AMR reservoirs as a public health concern and support a One Health approach integrating antibiotic stewardship and environmental monitoring.
Victoria Ballén, Enmanuel Cornielle, Anna Pinar-Méndez et al.· PLoS ONE· 0 citations
AIMS
Precise characterization of Escherichia coli isolates plays a crucial role in the treatment and prevention of diseases in animal production. The classical diagnostic approach to define pathotypes of E. coli relies on detection of virulence genes. However, by targeting a limited set of genetic markers, routine PCR-based approaches may hinder the detection of atypical pathogenic isolates, especially in bacteria with high genomic plasticity.
METHODS AND RESULTS
Genomic approaches were used to characterize a hybrid ETEC/ExPEC E. coli strain isolated from lambs submitted for necropsy over the course of six months, during an investigation of persistent neonatal mortality. Most lambs submitted for necropsy showed lesions suggestive of a septicemia. Routine PCR analysis detected genes encoding two ETEC-associated toxins but none of the ExPEC-associated virulence genes commonly targeted in routine diagnostics. Whole-genome sequencing revealed a diverse set of virulence genes, consistent with the hybrid nature of the strain. Genes encoding toxins were located on plasmids, while ExPEC-associated virulence factors were found to be chromosomally encoded. Genomic analyses revealed rapid antimicrobial resistance evolution, driven by plasmid acquisition.
CONCLUSIONS
This case highlights how horizontal gene transfer promotes the development of hybrid pathotype and facilitates resistance genes acquisition, compromising traditional diagnostic approaches and treatment. ExPEC strains are particularly difficult to identify due to their diverse and ill-defined virulence markers, which are not always targeted by standard genotyping tests. Emergence of atypical E. coli strains such as those with hybrid pathotypes reinforces the need for more comprehensive methods such as whole-genome sequencing in veterinary diagnostics.
Maud de Lagarde, G. Vanier, J. Fairbrother et al.· Journal of Applied Microbiol...· 1 citation
Uropathogenic Escherichia coli (UPEC) strains are the primary cause of urinary tract infections and pose a serious clinical problem due to their remarkable genetic variability, diverse virulence traits, and rapidly increasing antibiotic resistance. In this study, five clinical UPEC isolates (EC3, EC149, EC164, EC179, and EC256) were deeply investigated to better understand their pathogenic potential. The strains differed substantially in genome architecture, mobile genetic elements, plasmid composition, virulence factors composition, and antibiotic resistance profiles, illustrating the highly dynamic nature of UPEC populations. Of particular concern was the detection of plasmids linked to resistance against “last-resort” antibiotics, emphasizing the growing epidemiological risk associated with these pathogens. To address the limitations of conventional therapy, five lytic bacteriophages active against the analyzed UPEC strains were isolated and thoroughly characterized. Comparative genomic and phylogenetic analyses revealed that all phages represent previously undescribed species belonging to the genera Hanrivervirus, Warwickvirus, Nonavirus, Kagunavirus, and Vectrevirus. Functional assays demonstrated pronounced differences in host range and infectivity. Among them, phage vB_EcoS-149_4M emerged as the most promising candidate, combining broad antibacterial activity with high stability and a favourable safety profile, including the absence of toxin or antibiotic resistance genes and no detectable cytotoxicity toward human cell lines. Taken together, these findings highlight both the complexity and clinical threat posed by UPEC strains and the substantial potential of carefully selected bacteriophages as effective and precise antimicrobial tools. In effect, this multi-faceted approach provides a strong foundation for the development of precision phage therapy against multidrug-resistant UPEC infections.
Wojciech Wesołowski, Grzegorz Czerwonka, Katarzyna Zegadło et al.· Scientific Reports· 0 citations
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