Broad adaptations of plasmid-carrying Escherichia coli and Klebsiella pneumoniae to water as a reservoir

Broad adaptations of plasmid-carrying Escherichia coli and Klebsiella pneumoniae to water as a reservoir | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Broad adaptations of plasmid-carrying Escherichia coli and Klebsiella pneumoniae to water as a reservoir Phillip Lübcke, Sascha Knauf, Elias Eger, Sylvia Dreyer, Timo Homeier-Bachmann, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9425840/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 8 You are reading this latest preprint version 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. Biological sciences/Genetics Biological sciences/Microbiology high-risk clones in water One Health transcriptomics Enterobacterales plasmid-cured variants Figures Figure 1 Figure 2 Figure 3 1 Introduction The global spread of antimicrobial resistance (AMR) is one of the most critical health challenges, threatening human, animal, and environmental health 1 . While healthcare settings are particularly affected by AMR, aquatic environments are increasingly recognized as reservoirs and potential hotspots 2 – 5 , as they serve as crucial interfaces between different ecological compartments 6 , 7 . Among the most clinically relevant pathogens in these environments are Enterobacterales , particularly Escherichia ( E .) coli and Klebsiella ( K .) pneumoniae , driving significant AMR-related mortality worldwide 1 . Of particular concern is the emergence and environmental spread of high-risk clonal lineages, such as E. coli sequence type (ST)648 and K. pneumoniae ST307 8,9 . Known for their successful global dissemination, high virulence potential, and extensive AMR profiles 10 , their ability to persist outside clinical settings raises important questions regarding how they survive and thrive in nutrient-depleted and potentially stressful aquatic conditions. A key driver of this adaptability seems to be the high genomic variability, often mediated by the accessory genome through extrachromosomal elements, such as plasmids 11 , 12 . While plasmids are well documented for their role in conferring AMR and virulence 13 , they often also impose a metabolic burden or, conversely, provide fitness advantages depending on the host’s phylogenetic background and environmental context 14 . Although the contribution of plasmids to clinical success is well established, their influence on bacterial adaptation to natural aquatic environments remains poorly understood. Despite the importance of these pathogens, there is still a clear lack of comprehensive data on how high-risk E. coli and K. pneumoniae lineages adjust their gene expression to survive and persist in water. Previous studies of environmental adaptation have often been limited in scope, such as relying on microarray technologies that analyze only a fraction of the genome 15 . As a result, the global gene expression changes that enable long-term survival in water, and specifically the role of plasmids in modulating these transcriptomic responses, remain largely unexplored. To address these gaps, this study investigates the survival and transcriptomic adaptation of two high-risk E. coli strains (ST648) and one K. pneumoniae strain (ST307) in river and tap water microcosms over a 14-day period. The decision to examine a 14-day interval was based on a previous study 15 . Using RNA sequencing, we provide a comprehensive analysis of the metabolic and stress-response pathways affected during this transition. We also compared wild-type isolates with their respective plasmid-cured variants (PCVs) to explicitly examine the contribution of extrachromosomal elements to environmental adaptability. This approach offers new insights into the molecular mechanisms contributing to the persistence of AMR pathogens in aquatic habitats. 2 Results 2.1 Survival of high-risk E. coli and K. pneumoniae in different water sources We investigated the environmental persistence of high-risk E. coli and K. pneumoniae lineages in nutrient-limited aquatic environments. The tested strains included three wild-type strains: E. coli ST648 isolated from surface water (Ec1 2 ) and a blackbird (Ec2 16 ), and K. pneumoniae ST307 from a hospital outbreak (Kp 17 ). To assess the specific impact of plasmid carriage, we included plasmid-cured variants (PCVs), namely PCV-Ec2 for Ec2, which lost an extended-spectrum β-lactamase (ESBL)-encoding plasmid. For Kp, we included PCV-Kp (PCV1935), a PCV of PBIO1935. PBIO1935 originated from the same hospital outbreak as Kp and belonged to the same clonal lineage (15 SNPs). PCV-Kp had lost parts of a carbapenemase-encoding plasmid alongside a second smaller plasmid ( Supplementary Table S1 ). We would hypothesize that different origins of the bacterial strains could result in different survival in aquatic habitats. Survival of bacterial strains in water was tested in autoclaved water from the local river Ryck (RW) and tap water (TW) over a 14-day period. RW represents an environmental habitat, while TW is subject to strict regulations. Comparison of both water types should clarify, whether bacterial survival and adaptation reflects more general or specific adaptations to different aquatic compartments. Viable cells of all five strains were recovered in both water types at every time point (day 2, day 7, and day 14; Fig. 1 , Supplementary Table S3 – S5 ). Despite nutrient-depleted conditions, bacterial loads remained stable or increased depending on the initial inoculation density. In setups with lower starting concentrations (OD 600 0.1 and 0.01; Supplementary Figure S1 ), we observed that colony-forming units (CFUs) increased significantly in RW by day 14 from approximately 4.72 × 10 7 to 1.18 × 10 8 (OD 600 0.1 p = 0.0134), whereas a decrease from 3.54 × 10 6 to 8.2 × 10 4 was noted in TW for OD 600 0.01 ( p = 0.0134). In contrast, setups with a higher starting concentration (OD 600 0.2) showed no significant changes in CFU counts, indicating successful persistence at high densities. Notably, the loss of plasmids did not impair survival as there were no significant differences in survival rates between the wild-type strains and their respective PCVs in either water type. 2.2 Transcriptomic profiles reveal strain-specific and plasmid-dependent adaptations To understand the genetic basis of this survival, we isolated RNA from OD 600 0.1 cultures (triplicates) directly after inoculation (t 0 ), after 2 days (t 2 ), and after 14 days (t 14 ), followed by a differential gene expression analysis. Principal component analysis (PCA) indicated that the most significant transcriptional shifts occurred when comparing t 0 with t 14 with relatively minor changes between t 2 and t 14 ( Supplementary Figures S2 – S6 ). Our analysis focused primarily on the long-term adaptation observed between t 0 and t 14 to investigate adaptation processes of high-risk clonal lineages after introduction into aquatic habitats. Notably, the number of annotated genes varied between the genomes, particularly between wild-type and PCVs, which was considered when interpreting individual effects ( Supplementary Table S2 ). The magnitude of the transcriptional response varied substantially across species and environmental conditions (Table 1 ). Kp exhibited a profound transcriptomic shift in RW, with 1,005 differentially expressed genes (DEGs). In contrast, Ec2 showed a much more limited response in the same condition, with only 76 DEGs, using the same thresholds (p |2|). However, this low reactivity was condition-specific, as Ec2 showed a considerably stronger response in TW with 288 DEGs. Similar to Ec2, PCV-Ec2 and PCV-Kp displayed a greater number of DEGs under TW conditions, whereas Ec1 and Kp showed substantially higher DEG counts following exposure to RW. Table 1 Differentially expressed genes (DEGs) detected in the bacterial strains following incubation in river water or tap water. The total number of DEGs is shown alongside their localization, distinguishing between chromosomal and plasmid-encoded genes. Strains include the wildtype isolates for Escherichia coli (Ec1, Ec2) and Klebsiella pneumoniae (Kp) and the corresponding plasmid-cured variants (PCV-Ec2, PCV-Kp). “NA” indicates missing information because hybrid sequencing was not performed for the respective strain. Strain Differentially expressed genes River water Tap water Total Chromosome Plasmid Total Chromosome Plasmid Ec1 528 NA NA 416 NA NA Ec2 76 73 3 288 276 12 PCV-Ec2 115 115 0 168 168 0 Kp 1005 891 114 370 343 27 PCV-Kp 294 268 20 497 434 63 In addition to differences in the total number of DEGs, there were also differences in the direction of regulation (Fig. 2 a). Overall, the number of downregulated DEGs ranged from 48 (Ec2 RW ) to 497 (Kp RW ), while upregulated DEGs ranged from 28 (Ec2 RW ) to 508 (Kp RW ). In most conditions, the number of downregulated genes exceeded that of upregulated ones, with notable exceptions in PCV-Ec2 RW , Kp RW , and PCV-Kp RW , where the proportions were more evenly distributed. In RW, Kp exhibited more than a threefold higher number of both up- and downregulated genes relative to PCV-Kp (upregulated: 508 vs. 150, 9.44% vs. 2.93%; downregulated: 497 vs. 144, 9.24% vs. 2.81%). This indicates that plasmid loss reduced the global transcriptional response to environmental changes in K. pneumoniae . Conversely, this pattern was reversed in E. coli ; PCV-Ec2 exhibited more DEGs than its wild-type strain in both RW and TW. To distinguish shared from water-specific (RW, TW) transcriptional responses, we analyzed the overlap of DEGs identified under RW and TW conditions in the t 0 -t 14 interval (Fig. 2 b). The comparison of downregulated genes overlapping between Ec1 RW and Ec1 TW , along with the number of genes exclusively differentially expressed in both conditions, revealed that there were more differentially expressed genes shared between both conditions than genes exclusively downregulated in Ec1 TW (145 vs. 137; 51.42%). Conversely, the lowest proportions were detected in upregulated (10 vs. 109; 8.4%) and downregulated (15 vs. 150; 9.09%) genes in Ec2 TW and upregulated genes in Kp RW (55 vs. 453; 10.83%), reflecting the lower total DEG counts or distinct responses in those specific conditions. Finally, a comparison of time intervals using Upset plots revealed substantial differences between early (t 0 –t 2 ) and later (t 2 –t 14 ) phases of incubation ( Supplementary Figures S7 – S11 ). Generally, more DEGs were observed between t 0 and t 2 . Especially for Kp and PCV-Kp, comparatively few DEGs were detected between t 2 and t 14 . However, exceptions were noted in the E. coli strains. Ec1 had more upregulated genes in RW during the later phase (89 in t 2 –t 14 vs. 76 in t 0 –t 2 ), and Ec2 similarly showed increased upregulation in RW during t 2 –t 14 (47 vs. 15). This trend was reinforced in PCV-Ec2, where both upregulated and downregulated genes (75 vs. 12 up; 29 vs. 18 down) were higher during t 2 –t 14 in RW. In summary, our analysis showed substantial gene expression changes, with the magnitude of the response being highly species- and condition-specific. Plasmid carriage significantly influenced global regulation, substantially increasing the number of DEGs in K. pneumoniae but paradoxically reducing them in E. coli , suggesting a strain- and species-dependent role in environmental adaptation. 2.3 Metabolic reprogramming drives adaptation to aquatic environments To contextualize the transcriptomic responses across water matrices, DEGs were assigned to cluster of orthologous groups (COG) functional categories (Fig. 3 ; Supplementary Data 1 ) and mapped to Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways ( Supplementary Data 2 ). Overall, functional shifts were dominated by metabolic and regulatory functions, indicating broad physiological reprogramming during persistence in water. Across the E. coli isolates, DEGs were primarily enriched in COG categories linked to amino acid transport and metabolism (E) and inorganic ion transport and metabolism (P), accompanied by pronounced changes in transcription (K) and, depending on strain and matrix, energy production and conversion (C). In Ec1, RW triggered broader functional changes than TW, whereas Ec2 showed fewer DEGs in RW and a stronger response in TW, including functions related to environmental interaction (e.g., biofilm-associated pathways). In both Ec2 backgrounds, KEGG mapping repeatedly highlighted ABC transporters and two-component systems, consistent with an increased emphasis on nutrient scavenging and environmental sensing in oligotrophic water matrices. Plasmid curing did not fundamentally change the dominant functional signature but modulated the magnitude and balance of affected categories, particularly transport- and signaling-related functions ( Supplementary Table S7 ). In K. pneumoniae , the response was broader, encompassing additional metabolic modules compared with E. coli , with prominent representation of DEGs assigned to the KEGG pathways associated with porphyrin metabolism and biosynthesis of cofactors, alongside central carbon metabolism, particularly in RW. At the species level, E and P consistently accounted for the largest DEG fractions, while defense mechanisms (V) contributed the smallest proportion ( Supplementary Table S8 ). Collectively, these profiles suggest that persistence in drinking- and surface-water matrices is accompanied by coordinated changes in gene regulation, with metabolism, transport and signal transduction systems as recurring functional hallmarks relevant to environmental survival and potential regrowth in water systems. 2.4 Modulation of expression of resistance determinants and virulence-associated factors The differential gene expression analysis revealed time-dependent remodeling of AMR- and virulence-associated transcription (Table 2 ). Overall, AMR genes tended to be downregulated by day 14, whereas a smaller set of stress- and environment-associated functions was induced in a condition-specific manner. Table 2 Differentially expressed genes associated with AMR and virulence in wild-type Escherichia coli (Ec1, Ec2) , Klebsiella pneumoniae (Kp) and their respective plasmid-cured variants (PCV-Ec2 and PCV-Kp) incubated in river water (RW) and tap water (TW). The table is divided according to the direction of gene regulation (↑...upregulation, ↓...downregulation) and the respective intervals, in which the differential gene expression took place (t 0 -t 2 ...between inoculation and day 2, t 2 -t 14 ...between day 2 and 14, t 0 -t 14 ...between inoculation and day 14). Only genes with significant differential expression are depicted (p |2|). Numbers in superscript display whether genes were chromosomally or plasmid-encoded ( 1 =chromosomal, 2 =plasmid-encoded). Gene localization in Ec1 was predicted with Plasmer. Strain Database t 0 -t 14 ↓ t 0 -t 2 ↓ t 2 -t 14 ↓ t 0 -t 14 ↑ t 0 -t 2 ↑ t 2 -t 14 ↑ Ec1 RW AMRFinderPlus ariR 1 , mdtM 1 , mph (A) 2 , catA1 2 , bla EC 1 ariR 1 , mdtM 1 , mph (A) 2 , catA1 2 VFDB entF 1 , fepB 1 , ompA 1 , fyuA 1 , iutA 2 iucABCD 2 , fes 1 , entF 1 , irp2 1 , fyuA 1 , iutA 2 allB 1 cgsF 1 , galF 1 , rpoS 1 galF 1 , allB 1 , tssG 1 iucABC 2 , ybtA 1 Ec1 TW AMRFinderPlus ariR 1 , mdtM 1 , mph (A) 2 ariR 1 , mdtM 1 , mph (A) 2 parE 1 , bla OXA−1 2 parE 1 , bla OXA−1 2 VFDB entBF 1 , irp2 1 , fyuA 1 entBF 1 , irp2 1 , fyuA 1 csgB 1 , ibeB 1 tssFGL 1 , ibeB 1 allB 1 Ec2 RW AMRFinderPlus ariR 1 ariR 1 VFDB fimADF 1 fimACDFGI 1 , csgA 1 kpsS 1 Ec2 TW AMRFinderPlus ariR 1 , mph (A) 2 ariR 1 , tet (A) 2 VFDB aslA 1 , kpsEF 1 aslA 1 , fepG 1 allB 1 ibeB 1 , chuS 1 , csgBFG 1 csgBF 1 PCV-Ec2 RW AMRFinderPlus ariR 1 ariR 1 VFDB fimA 1 gspF 1 , kpsS 1 , fepE 1 gspF 1 , kpsS 1 , fepE 1 PCV-Ec2 TW AMRFinderPlus ariR 1 ariR 1 VFDB aslA 1 , kpsDFM 1 allB 1 ibeB 1 , csgBF 1 ibeB 1 , csgABF 1 , allB 1 Kp RW AMRFinderPlus emrD 1 , terB 2 , ble 2 , aph(3’’)-Ib 2 , aph(6)-Id 2 armA 2 , aph(3’’)-Ib 2 , aph(6)-Id 2 silP 2 , arsC 2 VFDB fimCDFGHI 1 , mrkABCDI 1 , tssG 1 , rmpA 2 fimCDFGH 1 , mrkABCDI 1 , yagXZ 1 iucA 2 , ybtA 1 , entC 1 , fepB 1 iucAB 2 , ybtA 1 , entC 1 Kp TW AMRFinderPlus emrD 1 , aph(3’)-Ia 2 emrD 1 , aph(3’)-Ia 2 oqxB19 1 , oqxA 1 , pcoBCD 2 pcoBCD 2 VFDB fimFGH 1 , mrkABCDHIJ 1 , rfbD 1 fimFGH 1 , mrkABCDHI 1 iucABCD 2 , rfbD 1 PCV-Kp RW AMRFinderPlus aph(6)-Id 2 VFDB mrkABCDHI 1 ybtPX 1 PCV-Kp TW AMR emrD 1 , bla OXA−48 2 oqxB19 1 , oqxA 1 , pcoABCD 2 , silAB 2 pcoABCD 2 , silAB 2 VFDB fimC 1 , mrkABCDHI 1 , entF 1 mrkABCD 1 In Ec1, AMR determinants were strongly affected after prolonged incubation. In Ec1 RW , the chromosomally-encoded ariR , mdtM , and catA1 and the plasmid-encoded mph (A) and catA1 were significantly downregulated at t 14 relative to t 0 . Ec1 TW showed a similar reduction of ariR , mdtM and mph (A), but additionally displayed significant upregulation of parE and the plasmid-encoded bla OXA−1 . While the downregulated AMR genes already showed differential expression during early incubation (t 0 –t 2 ), parE and bla OXA−1 remained upregulated beyond t 2 compared to t 0 , indicating sustained expression in TW. In Ec2, AMR-associated differential expression was more limited to ariR . In Ec2 RW , only ariR was downregulated after 14 days and also in t 0 –t 2 . In Ec2 TW , ariR and mph (A) were downregulated over 14 days, and the plasmid-encoded tetracycline efflux pump gene tet (A) was significantly downregulated in t 0 –t 2 . In the plasmid-cured Ec2 (PCV-Ec2), differential expression of AMR genes was also limited. In both PCV-Ec2 RW and PCV-Ec2 TW , only ariR was downregulated, consistent with deletion of plasmid-borne AMR genes such as mph (A) and tet (A). In Kp, AMR transcription changed markedly and differed between matrices and plasmid backgrounds. In Kp RW , terB , emrD , ble , and the aminoglycoside phosphotransferases aph(3'')-Ib and aph(6)-Id were downregulated at t 14 versus t 0 . In early incubation (t 0 –t 2 ), aph(3'')-Ib , aph(6)-Id and armA were also downregulated, while the heavy-metal tolerance genes silP and arsC were upregulated. In Kp TW , emrD was likewise downregulated, but the remaining DEGs differed: aph(3')-Ia was downregulated in both t 0 –t 2 and t 0 –t 14 , whereas copper resistance genes ( pcoBCD ) and the efflux genes oqxA and oqxB19 were upregulated in t 0 –t 14 . In PCV-Kp RW , AMR-associated differential expression was reduced to the downregulation of the plasmid-encoded aph(6)-Id in t 0 –t 14 . In PCV-Kp TW , emrD and bla OXA−48 were downregulated and oqxA , oqxB19 , pcoABCD , and silAB were upregulated from t 0 to t 14 . Virulence-associated genes also exhibited condition-dependent shifts. In Ec1 RW , iron utilization genes ( entF, fepB, fyuA, iutA ) and ompA were downregulated, whereas csgF , galF and rpoS were upregulated from t 0 to t 14 . The aerobactin locus iucABCD was downregulated in t 0 –t 2 but subsequently upregulated in t 2 –t 14 . In addition, fes and irp2 were downregulated in t 0 –t 2 . In Ec1 TW , entBF , irp2 and fyuA were downregulated in both t 0 –t 2 and t 0 –t 14 , while csgB and ibeB showed increased expression. In Ec2 RW , fimADF were downregulated at t 14 , and csgA was downregulated in t 0 –t 2 . In Ec2 TW , aslA and kpsEF were downregulated in t 0 –t 14 , while ibeB , chuS and csgBFG were upregulated over the same period. In PCV-Ec2 RW , only fimA was downregulated in t 0 –t 14 ; gspF , kpsS and fepE were upregulated in t 2 –t 14 and t 0 –t 14 , and no virulence-associated genes were differentially expressed in t 0 –t 2 . PCV-Ec2 TW resembled Ec2 TW , with downregulation of aslA and kpsDFM and upregulation of ibeB and csgBF in t 0 –t 14 . In K. pneumoniae , both wild-type and PCV showed downregulation of mrk operon genes in t 0 –t 14 , and several conditions additionally showed downregulation of fim operon genes. In Kp RW , tssG and plasmid-encoded rmpA were downregulated and iron acquisition genes ( iucA, ybtA, entC, fepB ) were upregulated in t 0 –t 14 ; in Kp TW , iucABCD and rfbD were upregulated at t 14 . In PCV-Kp RW , only mrkABCDHI were downregulated in t 0 –t 14 , while PCV-Kp TW contained downregulated entF and fimC . Based on the differential expression of AMR- and virulence-associated genes, we also examined master regulators known to control stress responses and pathogenicity (Table 3 ). Table 3 Differentially expressed master regulators in wild-type Escherichia coli (Ec1, Ec2) , Klebsiella pneumoniae (Kp), and the associated plasmid-cured variants (PCV-Ec2 and PCV-Kp), which were incubated in either river water (RW) or tap water (TW). Genes are grouped based on the direction of gene expression (↓...downregulation, ↑...upregulation) and whether expression changes occurred early (t 0 -t 2 ...between inoculation and day 2), later (t 2 -t 14 ...between day 2 and day 14) or over the whole period (t 0 -t 14 ...between inoculation and day 14) of their regulation. Only genes with significant differential expression are shown (p |2|). The p-values are rounded to 4 decimal digits, with smaller values displayed as 0.0000. Strain t0-t14↓ t0-t2↓ t0-t14↑ t0-t2↑ t2-t14↑ Gene l2fc p-value Gene l2fc p-value Gene l2fc p-value Gene l2fc p-value Gene l2fc p-value Ec1 RW rpoS flhC flhD narP lexA 2.08 3.52 4.09 2.51 2.28 0.0355 0.0141 0.0052 0.0345 0.0084 Ec1 TW arcA -2.09 0.0269 arcA -2.18 0.0136 flhC flhD 4.91 3.42 0.0025 0.0482 Ec2 RW flhC 4.89 0.0002 flhC flhD 4.65 4.26 0.0014 0.0154 Ec2 TW lrp flhC flhD 2.43 3.76 3.36 0.0000 0.0329 0.0425 flhC flhD 4.11 4.07 0.0250 0.0067 PCV-Ec2 RW flhC flhD 4.46 3.87 0.0089 0.0420 flhC flhD 4.50 4.15 0.0137 0.0285 PCV-Ec2 TW flhC flhD 3.65 3.04 0.0000 0.0060 Kp RW fis narL -2.20 -2.84 0.0453 0.0004 fis -2.92 0.0004 csrA arcA lexA marA 2.87 4.32 5.15 5.32 0.0019 0.0000 0.0000 0.0000 csrA hns arcA marA 3.09 3.17 3.25 5.64 0.0006 0.0066 0.0000 0.0000 PCV-Kp RW arcA lexA 2.70 4.28 0.0133 0.0003 PCV-Kp TW soxS 2.77 0.0104 First, a marked upregulation of the flagellar master regulators flhC and flhD was observed in E. coli strains. In Ec1 RW , Ec1 TW , and Ec2 RW , flhCD upregulation was sustained or increased over the 14-day period. This suggests an active investment in motility, potentially to escape nutrient-poor conditions. Second, the general stress response regulator rpoS and the SOS response repressor lexA were upregulated in Ec1 RW . Similarly, Kp in RW upregulated lexA and arcA (anoxic redox control), along with the carbon storage regulator csrA . Finally, the PCVs maintained similar regulatory patterns to their wild-type counterparts in some contexts; for example, PCV-Kp in RW also upregulated arcA and lexA . However, PCV-Kp TW showed unique behavior, with only the superoxide response regulator soxS significantly upregulated. 3 Discussion This study examined the survival and gene expression of E. coli ST648 and K. pneumoniae ST307 strains in RW and TW over 14 days. All strains, including their PCVs, were able to survive in both water types. Bacterial survival in water compartments has been reported to range from a few days to several hundred days, depending on the specific (experimental) conditions 18 , 19 . Our results are consistent with published data, suggesting these strains could maintain a constant CFU count well beyond our 14-day observation period. Lower cell concentrations may reduce survival by limiting cell-to-cell communication, such as quorum sensing 20 , which could partly explain the CFU decrease in TW OD 600 0.01. In contrast, the lack of a similar reduction in RW at OD 600 0.01 could reflect differences in water composition, such as nutrient availability 21 . Additionally, E. coli and K. pneumoniae can also enter a viable but non-culturable (VBNC) state in water, which can influence the results of colony-based methods 22 , 23 . Initially, we hypothesized that the distinct origins of the bacterial strains would lead to different survival rates due to prior habitat or host adaptation 24 , 25 . However, our data suggest that strain origin did not influence survival or growth in water. It is important to note that our use of sterilized water likely supported survival. The absence of microbial competition is known to reduce bacterial decline compared to multi-species co-cultures 26 , 27 and prevents interspecies interactions that otherwise influence gene regulation and adaptive responses 28 . Transcriptomic alteration is a key driver of bacterial adaptation to environmental changes. While previous studies have shown rapid, extensive gene expression changes within minutes or hours of water exposure 29 , 30 , our extended timeframe focused on long-term adaptation. Nevertheless, our data also suggested that transcriptomic adaptations occurred rather quickly after introduction of the strains into water. Although data on aquatic bacterial adaptation remain limited, existing literature suggests broad regulatory changes. For example, Legionella pneumophila exhibits widespread downregulation of genes across various COG categories after 24 hours in artificial freshwater, with its composition modeled after North American freshwater 31 . In contrast, our strains showed a more balanced distribution between up- and downregulated genes. Similarly, while Duffitt et al. 15 found that E. coli O157:H7 exhibited far fewer DEGs in sterile stream water compared to soil, our findings demonstrate that high-risk E. coli and K. pneumoniae clones are capable of mounting extensive adaptive responses to aquatic habitats. The complex metabolism of E. coli and K. pneumoniae is strongly influenced by their high genetic variability 32 , 33 . Transitioning from nutrient-rich LB medium to nutrient-depleted RW or TW may represent a severe nutritional shift 34 . Nutrient limitations, particularly of nitrogen and carbohydrates, are known triggers for extensive adaptive processes 35 , 36 . This likely explains the high prevalence of metabolism-associated DEGs and transcriptional regulators across our data set 15 , 31 , 37 . The concurrent up- and downregulation of metabolic genes suggests active metabolic readjustment rather than a uniform shutdown 38 . Virulence-associated genes and resistance determinants also play nuanced roles in environmental survival. While biofilm formation can protect bacteria from suboptimal conditions 39 , motility allows them to escape stress and seek nutrients 29 , 40 – 42 , . We observed upregulation of the master flagellar regulators flhCD , alongside general downregulation of adhesion- and biofilm-associated genes. These observations are reasonable within the context of our experimental setup. The continuous shaking of the flasks likely limited the possibility of biofilm formation compared to setups involving low flow rate or minimal turbulence. Additionally, enhancing motility would represent a strategy favoring the movement toward nutrients rather than remaining stationary 43 , 44 . Interestingly, general stress pathways were largely unprovoked. The alternative sigma factor RpoS ( rpoS ) regulates the general stress response in Enterobacterales during starvation or osmotic shock 45 – 48 , . However, we observed little to no differential expression of rpoS , suggesting our experimental conditions were not stressful enough to trigger its activation. Similarly, soxS , a key mediator of oxidative stress 49 , was rarely differentially expressed, indicating low oxidative stress levels in these microcosms. While plasmids are well known for mediating AMR and virulence 50 , 51 , their influence on bacterial survival in aquatic environments is less clear. In our study, plasmid carriage did not affect survival rates of the wild-type strains compared to their PCVs, consistent with findings in extraintestinal pathogenic E. coli 52 . However, plasmids can impose fitness costs that are later mitigated by adaptive evolution and compensation 53 , 54 . Although survival remained steady, the cured genetic segments could have affected other fitness parameters, such as growth rates or competitive ability. This is supported by the differential expression patterns of iron utilization-associated genes observed in Kp and PCV-Kp. Upregulation of siderophore synthesis likely indicates active iron scavenging in depleted water 55 , 56 , whereas downregulation could minimize biosynthesis costs when ferric iron concentrations are sufficient 57 . Notably, the absence of the plasmid-encoded iutA and iucABCD genes in PCV-Kp, in contrast to their upregulation in Kp, did not translate into differences in survival. Simultaneously, the contrasting downregulation of additional siderophore-associated genes in PCV-Kp and their upregulation in Kp suggests that the cured plasmid regions influenced iron acquisition-related responses beyond the deleted loci. Importantly, plasmids substantially influenced global gene regulation, though its influence was strain-dependent. Assuming all other conditions were identical, the pronounced transcriptomic differences between wild-type strains and PCVs reflect the loss of these extrachromosomal elements. Plasmid acquisition is known to alter host gene expression, particularly in metabolic pathways 58 , 59 . We primarily detected variations in metabolism-linked DEGs between the wild-type K. pneumoniae and its PCV, confirming that plasmids modulate metabolic transcription in response to environmental changes. While previous studies noted plasmid-induced changes in ABC transporters 58 and two-component systems 60 , our data showed these changes were highly strain- and water-dependent, precluding a uniform trend. While our study provides valuable insights into the transcriptomic adaptation of high-risk Enterobacterales lineages in aquatic environments, several limitations should be noted. First, the use of sterilized water microcosms eliminated microbial competition, predation, and complex interspecies interactions, which likely enhanced bacterial survival and altered transcriptomic responses compared to natural ecosystems. Follow-up co-culture experiments would help to assess how such biotic interactions influence survival and gene regulation under more ecologically realistic conditions. Second, our investigation was limited to a 14-day observation period under static laboratory conditions; thus, it may not fully capture the long-term evolutionary dynamics or the impact of environmental stressors present in natural aquatic reservoirs, such as UV radiation and temperature fluctuations. Finally, the study evaluated a small number of strains from specific high-risk lineages (ST648 and ST307) and their respective PCVs. Therefore, broader generalizations regarding the adaptive capacities of diverse E. coli and K. pneumoniae clones, or the universal influence of distinct plasmid types, require further research involving larger strain panels in non-sterile settings. To conclude, strains belonging to high-risk clonal lineages of E. coli and K. pneumoniae , along with their respective PCVs, survived in TW and RW over 14 days with no observable differences in CFU counts. However, their transcriptomic responses varied considerably, revealing highly strain-specific regulatory and metabolic adaptations to aquatic conditions. These findings underscore the capacity of clinically relevant, high-risk Enterobacterales to persist in natural environments, emphasizing the role of aquatic compartments as critical reservoirs within the One Health framework. Future investigations incorporating a broader panel of strains with diverse plasmid backgrounds are essential to further disentangle the complex dynamics of strain-specific and plasmid-associated environmental adaptation. 4 Methods 4.1 Bacterial strains All bacterial strains used in this study are listed in Table 4 and were stored at − 80°C in LB broth (Carl Roth, Karlsruhe, Germany) supplemented with 20% (v/v) glycerol (anhydrous; Merck, Darmstadt, Germany). Routine cultivation was performed at 37°C in LB broth or on Luria agar (LA) plates (Carl Roth). Table 4 Summary of bacterial strains used in this study. PCV1935 is a plasmid-cured variant (PCV) of PBIO1935, which belongs to the same clonal lineage as PBIO1953 (15 SNPs). ST...sequence type. Strain Original designation Species ST Source Year of isolation Reference Ec1 PBIO3806 E. coli 648 Surface water (Kumasi, Ghana) 2021 Eger et al. (2024) 2 Ec2 PBIO730/ IMT16316 E. coli 648 Eurasian blackbird feces (Giessen, Germany) 2008 Guenther et al. (2010) 16 PCV-Ec2 PCV730 E. coli 648 Plasmid-cured variant of Ec2 2013 Schaufler et al. (2013) 69 Kp PBIO1953/ va20750 K. pneumoniae 307 Tracheal secretion from hospital patient (Greifswald, Germany) 2019 Heiden et al. (2020) 17 PCV-Kp PCV1935 K. pneumoniae 307 Throat from hospital patient (Greifswald, Germany) 2019 Heiden et al. (2020) 17 Environmental water samples were collected on March 21, 2022, using sterile 1000 mL blue-cap bottles. Tap water was sourced directly from the laboratory supply (Greifswald, Germany), while river water was collected from the river Ryck at coordinates 54.09879° N, 13.40529° E. To ensure a sterile matrix for subsequent experiments, both samples were autoclaved (121°C, 15 min) and stored at 4°C until further use. The previously assembled genomes of the strains were annotated with Bakta (v1.11.3; database version 6.0) to provide a consistent and comparable annotation. Additional functional annotation was obtained using the eggNOG-mapper 61 , 62 web application (emapper-2.1.12; accessed July 23, 2025), and COG assignments as well as KEGG 63 Orthology (KO) identifiers were extracted from the output. 4.2 Survival in water Biological triplicates of overnight cultures were diluted 1:100 in 5 mL of fresh LB and incubated at 37°C with shaking at 180 rpm until visible turbidity was reached. The cells were harvested by centrifugation at 7,500 x g for 5 min at room temperature, and the resulting pellets were resuspended in either the prepared tap water or river water. For the survival assay, 30 mL of the respective water samples were transferred into sterile 100 mL Erlenmeyer flasks. Each flask was inoculated in triplicates with the bacterial suspension to achieve an initial OD 600 of 0.01, 0.1, or 0.2. The cultures were then incubated at room temperature (22 ± 2°C) with continuous agitation at 150 rpm. CFUs were performed by plating serial dilutions of the cultures onto LA plates, followed by overnight incubation at 37°C. Significance testing was performed using the two-sided Kruskal-Wallis statistical test followed by Dunn’s multiple-comparison post-hoc test. 4.3 RNA isolation, sequencing and analysis At the time points t 0 , t 2 and t 14 , 1 mL aliquots of the cultures (initial OD 600 = 0.1) were harvested and immediately snap-cooled in liquid nitrogen for 5 s to arrest transcription. The samples were then centrifuged at 16,000 x g for 3 min at 4°C. Supernatants were completely discarded, and the resulting pellets were snap-frozen in liquid nitrogen for 2 s and stored at − 70°C for a maximum of 6 h prior to extraction. Total RNA was extracted using the RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. RNA quantity was determined with a Qubit 4 fluorometer (Thermo Fisher Scientific, Waltham, MA, USA) and RNA integrity was assessed using Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Total RNA was shipped on dry ice to the Competence Centre for Genomic Analysis (CCGA, Kiel, Germany). Stranded total RNA-seq libraries were prepared using the Illumina Stranded Total RNA Prep with Ribo-Zero Plus kit (Illumina, San Diego, CA, USA) according to the manufacturer’s instructions. Libraries were sequenced on an Illumina NovaSeq 6000 platform to generate 2 × 100-bp paired-end reads. 4.4 Read processing and mapping Raw reads were processed with fastp (v0.23.4) 64 to remove adapter sequences and low-quality bases. Filtered reads were aligned against the corresponding strain-specific genome assembly using Bowtie 2 (v2.5.1) 65 in local alignment mode (--local). Gene-level read counts were generated with featureCounts (v2.0.1) 66 using the corresponding strain-specific genome annotation. Unless otherwise stated, software was run with default parameters. Putative plasmid-derived sequences in strain Ec1 were identified using Plasmer v0.1 (20220816) 67 with the database version dated 2025-01-07. 4.5 Differential gene expression analysis Gene count tables were imported into R and analyzed with DESeq2 (v1.48.1) 68 . Differential gene expression analysis was performed separately for each strain and water type, i.e. RW and TW, with the respective time point serving as the experimental factor. DESeq2-based size-factor normalization, dispersion estimation and model fitting were performed following the standard workflow. To assess sample clustering, PCA was performed in DESeq2 using the plotPCA function (default parameters) on variance-stabilized transformed counts. Pairwise contrasts were calculated for t 2 versus t 0 , t 14 versus t 0 , and t 14 versus t 2 . Statistical significance for each contrast was assessed using Wald tests, and P values were adjusted for multiple testing using the Benjamini-Hochberg procedure. For each comparison l2fcs were extracted. A gene was considered differential expressed if it met the following criteria: |l2fcs|>=2 and an adjusted p-value < 0.05. Declarations Potential conflicts of interest. All authors declare no potential conflicts. Funding. This work was supported by two grants from the Federal Ministry of Research, Technology and Space (BMFTR, Germany) to K.S. entitled “Standardized One Health surveillance of antibiotic residues and antibiotic and heavy metal resistance in Baltic water environments and wild birds” (01KI2402A) and “Disarming pathogens as a different strategy to fight antimicrobial-resistant Gram-negatives” (01KI2410). Author Contribution Conceptualization, K.S.; methodology, E.E., M.S. and P.L.; software, M.S. and S.E.H.; validation, E.E., M.S. and P.L.; formal analysis, E.E., K.S., M.S., P.L., S.D., S.E.H., S.K. and T.H.-B.; investigation, E.E., P.L. and M.S..; resources, K.S. and S.K.; data curation, M.S., P.L. and S.E.H.; writing—original draft preparation, P.L.; writing—review and editing, E.E., K.S. and M.S.; visualization, M.S. and P.L.; supervision, K.S.; project administration, K.S.; funding acquisition, K.S. and S.K. All authors have read and agreed to the published version of the manuscript. Acknowledgement We thank Sara-Lucia Skwara for her excellent technical assistance. Data Availability The data for this study have been deposited in the European Nucleotide Archive (ENA) at EMBL-EBI under accession number PRJEB110490 (https://www.ebi.ac.uk/ena/browser/view/PRJEB110490). References Antimicrobial Resistance Collaborators. 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Heiden","email":"","orcid":"","institution":"Helmholtz Institute for One Health, Helmholtz Centre for Infection Research HZI","correspondingAuthor":false,"prefix":"","firstName":"Stefan","middleName":"E.","lastName":"Heiden","suffix":""},{"id":625627840,"identity":"9b6e58f7-ab8c-458d-9ba3-2767948879cc","order_by":6,"name":"Michael Schwabe","email":"","orcid":"","institution":"Helmholtz Institute for One Health, Helmholtz Centre for Infection Research HZI","correspondingAuthor":false,"prefix":"","firstName":"Michael","middleName":"","lastName":"Schwabe","suffix":""},{"id":625627843,"identity":"b26dab5e-d7b2-45a4-baa3-4b2b8ba30722","order_by":7,"name":"Katharina Schaufler","email":"data:image/png;base64,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","orcid":"","institution":"Helmholtz Institute for One Health, Helmholtz Centre for Infection Research HZI","correspondingAuthor":true,"prefix":"","firstName":"Katharina","middleName":"","lastName":"Schaufler","suffix":""}],"badges":[],"createdAt":"2026-04-15 10:53:45","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9425840/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9425840/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":107749269,"identity":"89e4d416-0bf4-4b87-a469-26d3281261f8","added_by":"auto","created_at":"2026-04-24 16:46:32","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":245438,"visible":true,"origin":"","legend":"\u003cp\u003eSurvival of \u003cem\u003eEscherichia\u0026nbsp;coli\u003c/em\u003e (Ec1, Ec2) and \u003cem\u003eKlebsiella\u0026nbsp;pneumoniae\u003c/em\u003e (Kp), along with their respective plasmid-cured variants (PCV-Ec2, PCV-Kp), in river water and tap water adjusted to a starting concentration of OD\u003csub\u003e600\u003c/sub\u003e 0.2 (approximately 1.19\u0026nbsp;×\u0026nbsp;10\u003csup\u003e8\u003c/sup\u003e cells/mL). The results represent mean values and standard errors across replicates (n = 3) and are displayed as time course diagrams illustrating overall survival trends. Lines between data points are shown to aid visual orientation.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9425840/v1/8d0df22722424316dfde8e70.jpeg"},{"id":107749272,"identity":"7b271de1-a20d-4589-9946-efa90cb7b168","added_by":"auto","created_at":"2026-04-24 16:46:32","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":182101,"visible":true,"origin":"","legend":"\u003cp\u003eOverview of the general results of the transcriptomic analysis of wild-type and plasmid-cured variants (PCV) of \u003cem\u003eEscherichia coli\u003c/em\u003e(Ec1, Ec2, and PCV-Ec2) and \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e (Kp and PCV-Kp) strains. a number of differentially expressed genes (DEGs) detected after 14 days of incubation in river water (RW) and tap water (TW). Blue bars depict upregulated genes, while red bars portray downregulated genes. b Absolute DEG counts for all five strains with respect to their direction of regulation and water source, depicted as a Venn diagram. Areas without labels represent combinations of water sources and regulations for which no DEGs occurred.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9425840/v1/e29f86dd9b06d99f7c67c61b.jpeg"},{"id":107869855,"identity":"86b76ef6-e9bc-44de-8483-e7a1d935b34e","added_by":"auto","created_at":"2026-04-27 07:38:19","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":287804,"visible":true,"origin":"","legend":"\u003cp\u003eResults of the differential gene expression analysis of wild-type and plasmid-cured variants (PCVs) of \u003cem\u003eEscherichia coli\u003c/em\u003e (Ec1, Ec2, and PCV-Ec2) and \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e(Kp and PCV-Kp) after incubation in river water (RW) and tap water (TW). The functional classification was based on the Clusters of Orthologous Genes (COG) categories split into upregulated and downregulated genes. For reasons of clarity, genes linked to the COG categories R (general function prediction only) and S (function unknown) are not shown. The absolute numbers of DEGs for every category can be found in the supplements (Supplementary Data 1).\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9425840/v1/8bdffe07acf50934c71b7b89.jpeg"},{"id":109206809,"identity":"b410e09a-109c-494b-a280-61846981c57d","added_by":"auto","created_at":"2026-05-13 15:16:04","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1399186,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9425840/v1/54d3c637-7594-4553-b6c5-a096c53049e6.pdf"},{"id":109204170,"identity":"23f244bd-7030-4650-a844-4e8f295aed09","added_by":"auto","created_at":"2026-05-13 14:54:46","extension":"xlsx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":23812,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryData2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-9425840/v1/182709bd037480baed3ea305.xlsx"},{"id":108181361,"identity":"b96e44ce-4eae-40d9-a605-caaa85ab30ef","added_by":"auto","created_at":"2026-04-30 08:58:34","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":13870,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryData1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-9425840/v1/5d14c59ba18989155313d54b.xlsx"},{"id":107749273,"identity":"79fdb115-f709-4888-85ba-e062dfd3a844","added_by":"auto","created_at":"2026-04-24 16:46:32","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":616645,"visible":true,"origin":"","legend":"","description":"","filename":"20260220SormasSupplementsFigures.docx","url":"https://assets-eu.researchsquare.com/files/rs-9425840/v1/5c6f55aff48b518b54b4aee1.docx"},{"id":107868936,"identity":"465ebe40-dfd9-4a18-b1f7-2d199cd76994","added_by":"auto","created_at":"2026-04-27 07:35:08","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":50293,"visible":true,"origin":"","legend":"","description":"","filename":"20260220SormasSupplementsTables.docx","url":"https://assets-eu.researchsquare.com/files/rs-9425840/v1/b9097436576bfcd809d069a6.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eBroad adaptations of plasmid-carrying \u003cem\u003eEscherichia coli\u003c/em\u003e and Klebsiella pneumoniae to water as a reservoir\u003c/p\u003e","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003eThe global spread of antimicrobial resistance (AMR) is one of the most critical health challenges, threatening human, animal, and environmental health\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. While healthcare settings are particularly affected by AMR, aquatic environments are increasingly recognized as reservoirs and potential hotspots\u003csup\u003e\u003cspan additionalcitationids=\"CR3 CR4\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e, as they serve as crucial interfaces between different ecological compartments\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Among the most clinically relevant pathogens in these environments are \u003cem\u003eEnterobacterales\u003c/em\u003e, particularly \u003cem\u003eEscherichia\u003c/em\u003e (\u003cem\u003eE\u003c/em\u003e.) \u003cem\u003ecoli\u003c/em\u003e and \u003cem\u003eKlebsiella\u003c/em\u003e (\u003cem\u003eK\u003c/em\u003e.) \u003cem\u003epneumoniae\u003c/em\u003e, driving significant AMR-related mortality worldwide\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Of particular concern is the emergence and environmental spread of high-risk clonal lineages, such as \u003cem\u003eE. coli\u003c/em\u003e sequence type (ST)648 and \u003cem\u003eK. pneumoniae\u003c/em\u003e ST307\u003csup\u003e8,9\u003c/sup\u003e. Known for their successful global dissemination, high virulence potential, and extensive AMR profiles\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e, their ability to persist outside clinical settings raises important questions regarding how they survive and thrive in nutrient-depleted and potentially stressful aquatic conditions.\u003c/p\u003e \u003cp\u003eA key driver of this adaptability seems to be the high genomic variability, often mediated by the accessory genome through extrachromosomal elements, such as plasmids\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. While plasmids are well documented for their role in conferring AMR and virulence\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e, they often also impose a metabolic burden or, conversely, provide fitness advantages depending on the host\u0026rsquo;s phylogenetic background and environmental context\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Although the contribution of plasmids to clinical success is well established, their influence on bacterial adaptation to natural aquatic environments remains poorly understood.\u003c/p\u003e \u003cp\u003eDespite the importance of these pathogens, there is still a clear lack of comprehensive data on how high-risk \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eK. pneumoniae\u003c/em\u003e lineages adjust their gene expression to survive and persist in water. Previous studies of environmental adaptation have often been limited in scope, such as relying on microarray technologies that analyze only a fraction of the genome\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. As a result, the global gene expression changes that enable long-term survival in water, and specifically the role of plasmids in modulating these transcriptomic responses, remain largely unexplored.\u003c/p\u003e \u003cp\u003eTo address these gaps, this study investigates the survival and transcriptomic adaptation of two high-risk \u003cem\u003eE. coli\u003c/em\u003e strains (ST648) and one \u003cem\u003eK. pneumoniae\u003c/em\u003e strain (ST307) in river and tap water microcosms over a 14-day period. The decision to examine a 14-day interval was based on a previous study\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Using RNA sequencing, we provide a comprehensive analysis of the metabolic and stress-response pathways affected during this transition. We also compared wild-type isolates with their respective plasmid-cured variants (PCVs) to explicitly examine the contribution of extrachromosomal elements to environmental adaptability. This approach offers new insights into the molecular mechanisms contributing to the persistence of AMR pathogens in aquatic habitats.\u003c/p\u003e"},{"header":"2 Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Survival of high-risk \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eK. pneumoniae\u003c/em\u003e in different water sources\u003c/h2\u003e \u003cp\u003eWe investigated the environmental persistence of high-risk \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eK. pneumoniae\u003c/em\u003e lineages in nutrient-limited aquatic environments. The tested strains included three wild-type strains: \u003cem\u003eE. coli\u003c/em\u003e ST648 isolated from surface water (Ec1\u003csup\u003e2\u003c/sup\u003e) and a blackbird (Ec2\u003csup\u003e16\u003c/sup\u003e), and \u003cem\u003eK. pneumoniae\u003c/em\u003e ST307 from a hospital outbreak (Kp\u003csup\u003e17\u003c/sup\u003e). To assess the specific impact of plasmid carriage, we included plasmid-cured variants (PCVs), namely PCV-Ec2 for Ec2, which lost an extended-spectrum β-lactamase (ESBL)-encoding plasmid. For Kp, we included PCV-Kp (PCV1935), a PCV of PBIO1935. PBIO1935 originated from the same hospital outbreak as Kp and belonged to the same clonal lineage (15 SNPs). PCV-Kp had lost parts of a carbapenemase-encoding plasmid alongside a second smaller plasmid (\u003cb\u003eSupplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e). We would hypothesize that different origins of the bacterial strains could result in different survival in aquatic habitats.\u003c/p\u003e \u003cp\u003eSurvival of bacterial strains in water was tested in autoclaved water from the local river Ryck (RW) and tap water (TW) over a 14-day period. RW represents an environmental habitat, while TW is subject to strict regulations. Comparison of both water types should clarify, whether bacterial survival and adaptation reflects more general or specific adaptations to different aquatic compartments. Viable cells of all five strains were recovered in both water types at every time point (day 2, day 7, and day 14; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, \u003cb\u003eSupplementary Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e \u0026ndash; S5\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eDespite nutrient-depleted conditions, bacterial loads remained stable or increased depending on the initial inoculation density. In setups with lower starting concentrations (OD\u003csub\u003e600\u003c/sub\u003e 0.1 and 0.01; \u003cb\u003eSupplementary Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e), we observed that colony-forming units (CFUs) increased significantly in RW by day 14 from approximately 4.72 \u0026times; 10\u003csup\u003e7\u003c/sup\u003e to 1.18 \u0026times; 10\u003csup\u003e8\u003c/sup\u003e (OD\u003csub\u003e600\u003c/sub\u003e 0.1 \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0134), whereas a decrease from 3.54 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e to 8.2 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e was noted in TW for OD\u003csub\u003e600\u003c/sub\u003e 0.01 (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0134). In contrast, setups with a higher starting concentration (OD\u003csub\u003e600\u003c/sub\u003e 0.2) showed no significant changes in CFU counts, indicating successful persistence at high densities. Notably, the loss of plasmids did not impair survival as there were no significant differences in survival rates between the wild-type strains and their respective PCVs in either water type.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Transcriptomic profiles reveal strain-specific and plasmid-dependent adaptations\u003c/h2\u003e \u003cp\u003eTo understand the genetic basis of this survival, we isolated RNA from OD\u003csub\u003e600\u003c/sub\u003e 0.1 cultures (triplicates) directly after inoculation (t\u003csub\u003e0\u003c/sub\u003e), after 2 days (t\u003csub\u003e2\u003c/sub\u003e), and after 14 days (t\u003csub\u003e14\u003c/sub\u003e), followed by a differential gene expression analysis. Principal component analysis (PCA) indicated that the most significant transcriptional shifts occurred when comparing t\u003csub\u003e0\u003c/sub\u003e with t\u003csub\u003e14\u003c/sub\u003e with relatively minor changes between t\u003csub\u003e2\u003c/sub\u003e and t\u003csub\u003e14\u003c/sub\u003e (\u003cb\u003eSupplementary Figures \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e \u0026ndash; S6\u003c/b\u003e). Our analysis focused primarily on the long-term adaptation observed between t\u003csub\u003e0\u003c/sub\u003e and t\u003csub\u003e14\u003c/sub\u003e to investigate adaptation processes of high-risk clonal lineages after introduction into aquatic habitats. Notably, the number of annotated genes varied between the genomes, particularly between wild-type and PCVs, which was considered when interpreting individual effects (\u003cb\u003eSupplementary Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eThe magnitude of the transcriptional response varied substantially across species and environmental conditions (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Kp exhibited a profound transcriptomic shift in RW, with 1,005 differentially expressed genes (DEGs). In contrast, Ec2 showed a much more limited response in the same condition, with only 76 DEGs, using the same thresholds (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, log2 fold-change (l2fc) \u0026gt; |2|). However, this low reactivity was condition-specific, as Ec2 showed a considerably stronger response in TW with 288 DEGs. Similar to Ec2, PCV-Ec2 and PCV-Kp displayed a greater number of DEGs under TW conditions, whereas Ec1 and Kp showed substantially higher DEG counts following exposure to RW.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u003cb\u003eDifferentially expressed genes (DEGs) detected in the bacterial strains following incubation in river water or tap water.\u003c/b\u003e The total number of DEGs is shown alongside their localization, distinguishing between chromosomal and plasmid-encoded genes. Strains include the wildtype isolates for \u003cem\u003eEscherichia coli\u003c/em\u003e (Ec1, Ec2) and \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e (Kp) and the corresponding plasmid-cured variants (PCV-Ec2, PCV-Kp). \u0026ldquo;NA\u0026rdquo; indicates missing information because hybrid sequencing was not performed for the respective strain.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eStrain\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"6\" nameend=\"c7\" namest=\"c2\"\u003e \u003cp\u003eDifferentially expressed genes\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cp\u003eRiver water\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c7\" namest=\"c5\"\u003e \u003cp\u003eTap water\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTotal\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eChromosome\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePlasmid\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eTotal\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eChromosome\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003ePlasmid\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEc1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e528\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e416\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEc2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e288\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e276\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePCV-Ec2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e115\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e115\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e168\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e168\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eKp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1005\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e891\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e114\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e370\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e343\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e27\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePCV-Kp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e294\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e268\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e497\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e434\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e63\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eIn addition to differences in the total number of DEGs, there were also differences in the direction of regulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). Overall, the number of downregulated DEGs ranged from 48 (Ec2\u003csub\u003eRW\u003c/sub\u003e) to 497 (Kp\u003csub\u003eRW\u003c/sub\u003e), while upregulated DEGs ranged from 28 (Ec2\u003csub\u003eRW\u003c/sub\u003e) to 508 (Kp\u003csub\u003eRW\u003c/sub\u003e). In most conditions, the number of downregulated genes exceeded that of upregulated ones, with notable exceptions in PCV-Ec2\u003csub\u003eRW\u003c/sub\u003e, Kp\u003csub\u003eRW\u003c/sub\u003e, and PCV-Kp\u003csub\u003eRW\u003c/sub\u003e, where the proportions were more evenly distributed.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn RW, Kp exhibited more than a threefold higher number of both up- and downregulated genes relative to PCV-Kp (upregulated: 508 vs. 150, 9.44% vs. 2.93%; downregulated: 497 vs. 144, 9.24% vs. 2.81%). This indicates that plasmid loss reduced the global transcriptional response to environmental changes in \u003cem\u003eK. pneumoniae\u003c/em\u003e. Conversely, this pattern was reversed in \u003cem\u003eE. coli\u003c/em\u003e; PCV-Ec2 exhibited more DEGs than its wild-type strain in both RW and TW.\u003c/p\u003e \u003cp\u003eTo distinguish shared from water-specific (RW, TW) transcriptional responses, we analyzed the overlap of DEGs identified under RW and TW conditions in the t\u003csub\u003e0\u003c/sub\u003e-t\u003csub\u003e14\u003c/sub\u003e interval (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). The comparison of downregulated genes overlapping between Ec1\u003csub\u003eRW\u003c/sub\u003e and Ec1\u003csub\u003eTW\u003c/sub\u003e, along with the number of genes exclusively differentially expressed in both conditions, revealed that there were more differentially expressed genes shared between both conditions than genes exclusively downregulated in Ec1\u003csub\u003eTW\u003c/sub\u003e (145 vs. 137; 51.42%). Conversely, the lowest proportions were detected in upregulated (10 vs. 109; 8.4%) and downregulated (15 vs. 150; 9.09%) genes in Ec2\u003csub\u003eTW\u003c/sub\u003e and upregulated genes in Kp\u003csub\u003eRW\u003c/sub\u003e (55 vs. 453; 10.83%), reflecting the lower total DEG counts or distinct responses in those specific conditions.\u003c/p\u003e \u003cp\u003eFinally, a comparison of time intervals using Upset plots revealed substantial differences between early (t\u003csub\u003e0\u003c/sub\u003e\u0026ndash;t\u003csub\u003e2\u003c/sub\u003e) and later (t\u003csub\u003e2\u003c/sub\u003e\u0026ndash;t\u003csub\u003e14\u003c/sub\u003e) phases of incubation (\u003cb\u003eSupplementary Figures S7 \u0026ndash; S11\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eGenerally, more DEGs were observed between t\u003csub\u003e0\u003c/sub\u003e and t\u003csub\u003e2\u003c/sub\u003e. Especially for Kp and PCV-Kp, comparatively few DEGs were detected between t\u003csub\u003e2\u003c/sub\u003e and t\u003csub\u003e14\u003c/sub\u003e. However, exceptions were noted in the \u003cem\u003eE. coli\u003c/em\u003e strains. Ec1 had more upregulated genes in RW during the later phase (89 in t\u003csub\u003e2\u003c/sub\u003e\u0026ndash;t\u003csub\u003e14\u003c/sub\u003e vs. 76 in t\u003csub\u003e0\u003c/sub\u003e\u0026ndash;t\u003csub\u003e2\u003c/sub\u003e), and Ec2 similarly showed increased upregulation in RW during t\u003csub\u003e2\u003c/sub\u003e\u0026ndash;t\u003csub\u003e14\u003c/sub\u003e (47 vs. 15). This trend was reinforced in PCV-Ec2, where both upregulated and downregulated genes (75 vs. 12 up; 29 vs. 18 down) were higher during t\u003csub\u003e2\u003c/sub\u003e\u0026ndash;t\u003csub\u003e14\u003c/sub\u003e in RW.\u003c/p\u003e \u003cp\u003eIn summary, our analysis showed substantial gene expression changes, with the magnitude of the response being highly species- and condition-specific. Plasmid carriage significantly influenced global regulation, substantially increasing the number of DEGs in \u003cem\u003eK. pneumoniae\u003c/em\u003e but paradoxically reducing them in \u003cem\u003eE. coli\u003c/em\u003e, suggesting a strain- and species-dependent role in environmental adaptation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Metabolic reprogramming drives adaptation to aquatic environments\u003c/h2\u003e \u003cp\u003eTo contextualize the transcriptomic responses across water matrices, DEGs were assigned to cluster of orthologous groups (COG) functional categories (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e; \u003cb\u003eSupplementary Data 1\u003c/b\u003e) and mapped to Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways (\u003cb\u003eSupplementary Data 2\u003c/b\u003e). Overall, functional shifts were dominated by metabolic and regulatory functions, indicating broad physiological reprogramming during persistence in water.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAcross the \u003cem\u003eE. coli\u003c/em\u003e isolates, DEGs were primarily enriched in COG categories linked to amino acid transport and metabolism (E) and inorganic ion transport and metabolism (P), accompanied by pronounced changes in transcription (K) and, depending on strain and matrix, energy production and conversion (C). In Ec1, RW triggered broader functional changes than TW, whereas Ec2 showed fewer DEGs in RW and a stronger response in TW, including functions related to environmental interaction (e.g., biofilm-associated pathways). In both Ec2 backgrounds, KEGG mapping repeatedly highlighted ABC transporters and two-component systems, consistent with an increased emphasis on nutrient scavenging and environmental sensing in oligotrophic water matrices.\u003c/p\u003e \u003cp\u003ePlasmid curing did not fundamentally change the dominant functional signature but modulated the magnitude and balance of affected categories, particularly transport- and signaling-related functions (\u003cb\u003eSupplementary Table S7\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eIn \u003cem\u003eK. pneumoniae\u003c/em\u003e, the response was broader, encompassing additional metabolic modules compared with \u003cem\u003eE. coli\u003c/em\u003e, with prominent representation of DEGs assigned to the KEGG pathways associated with porphyrin metabolism and biosynthesis of cofactors, alongside central carbon metabolism, particularly in RW. At the species level, E and P consistently accounted for the largest DEG fractions, while defense mechanisms (V) contributed the smallest proportion (\u003cb\u003eSupplementary Table S8\u003c/b\u003e). Collectively, these profiles suggest that persistence in drinking- and surface-water matrices is accompanied by coordinated changes in gene regulation, with metabolism, transport and signal transduction systems as recurring functional hallmarks relevant to environmental survival and potential regrowth in water systems.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Modulation of expression of resistance determinants and virulence-associated factors\u003c/h2\u003e \u003cp\u003eThe differential gene expression analysis revealed time-dependent remodeling of AMR- and virulence-associated transcription (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Overall, AMR genes tended to be downregulated by day 14, whereas a smaller set of stress- and environment-associated functions was induced in a condition-specific manner.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u003cb\u003eDifferentially expressed genes associated with AMR and virulence in wild-type\u003c/b\u003e \u003cb\u003eEscherichia coli\u003c/b\u003e \u003cb\u003e(Ec1, Ec2)\u003c/b\u003e, \u003cb\u003eKlebsiella pneumoniae\u003c/b\u003e \u003cb\u003e(Kp) and their respective plasmid-cured variants (PCV-Ec2 and PCV-Kp) incubated in river water (RW) and tap water (TW).\u003c/b\u003e The table is divided according to the direction of gene regulation (\u0026uarr;...upregulation, \u0026darr;...downregulation) and the respective intervals, in which the differential gene expression took place (t\u003csub\u003e0\u003c/sub\u003e-t\u003csub\u003e2\u003c/sub\u003e...between inoculation and day 2, t\u003csub\u003e2\u003c/sub\u003e-t\u003csub\u003e14\u003c/sub\u003e...between day 2 and 14, t\u003csub\u003e0\u003c/sub\u003e-t\u003csub\u003e14\u003c/sub\u003e...between inoculation and day 14). Only genes with significant differential expression are depicted (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, log2 fold-change (l2fc) \u0026gt; |2|). Numbers in superscript display whether genes were chromosomally or plasmid-encoded (\u003csup\u003e1\u003c/sup\u003e=chromosomal, \u003csup\u003e2\u003c/sup\u003e=plasmid-encoded). Gene localization in Ec1 was predicted with Plasmer.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStrain\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDatabase\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003et\u003csub\u003e0\u003c/sub\u003e-t\u003csub\u003e14\u003c/sub\u003e\u0026darr;\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003et\u003csub\u003e0\u003c/sub\u003e-t\u003csub\u003e2\u003c/sub\u003e\u0026darr;\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003et\u003csub\u003e2\u003c/sub\u003e-t\u003csub\u003e14\u003c/sub\u003e\u0026darr;\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003et\u003csub\u003e0\u003c/sub\u003e-t\u003csub\u003e14\u003c/sub\u003e\u0026uarr;\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003et\u003csub\u003e0\u003c/sub\u003e-t\u003csub\u003e2\u003c/sub\u003e\u0026uarr;\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e 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class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eiucABCD\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e, \u003cem\u003erfbD\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003ePCV-Kp\u003csub\u003eRW\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAMRFinderPlus\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eaph(6)-Id\u003c/em\u003e\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eVFDB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003emrkABCDHI\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eybtPX\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003ePCV-Kp\u003csub\u003eTW\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAMR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eemrD\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e, \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eOXA\u0026minus;48\u003c/sub\u003e\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eoqxB19\u003c/em\u003e\u003csup\u003e1\u003c/sup\u003e, \u003cem\u003eoqxA\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e, \u003cem\u003epcoABCD\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e, \u003cem\u003esilAB\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cem\u003epcoABCD\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e, \u003cem\u003esilAB\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eVFDB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003efimC\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e, \u003cem\u003emrkABCDHI\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e, \u003cem\u003eentF\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003emrkABCD\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eIn Ec1, AMR determinants were strongly affected after prolonged incubation. In Ec1\u003csub\u003eRW\u003c/sub\u003e, the chromosomally-encoded \u003cem\u003eariR\u003c/em\u003e, \u003cem\u003emdtM\u003c/em\u003e, and \u003cem\u003ecatA1\u003c/em\u003e and the plasmid-encoded \u003cem\u003emph\u003c/em\u003e(A) and \u003cem\u003ecatA1\u003c/em\u003e were significantly downregulated at t\u003csub\u003e14\u003c/sub\u003e relative to t\u003csub\u003e0\u003c/sub\u003e. Ec1\u003csub\u003eTW\u003c/sub\u003e showed a similar reduction of \u003cem\u003eariR\u003c/em\u003e, \u003cem\u003emdtM\u003c/em\u003e and \u003cem\u003emph\u003c/em\u003e(A), but additionally displayed significant upregulation of \u003cem\u003eparE\u003c/em\u003e and the plasmid-encoded \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eOXA\u0026minus;1\u003c/sub\u003e. While the downregulated AMR genes already showed differential expression during early incubation (t\u003csub\u003e0\u003c/sub\u003e\u0026ndash;t\u003csub\u003e2\u003c/sub\u003e), \u003cem\u003eparE\u003c/em\u003e and \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eOXA\u0026minus;1\u003c/sub\u003e remained upregulated beyond t\u003csub\u003e2\u003c/sub\u003e compared to t\u003csub\u003e0\u003c/sub\u003e, indicating sustained expression in TW.\u003c/p\u003e \u003cp\u003eIn Ec2, AMR-associated differential expression was more limited to \u003cem\u003eariR\u003c/em\u003e. In Ec2\u003csub\u003eRW\u003c/sub\u003e, only \u003cem\u003eariR\u003c/em\u003e was downregulated after 14 days and also in t\u003csub\u003e0\u003c/sub\u003e\u0026ndash;t\u003csub\u003e2\u003c/sub\u003e. In Ec2\u003csub\u003eTW\u003c/sub\u003e, \u003cem\u003eariR\u003c/em\u003e and \u003cem\u003emph\u003c/em\u003e(A) were downregulated over 14 days, and the plasmid-encoded tetracycline efflux pump gene \u003cem\u003etet\u003c/em\u003e(A) was significantly downregulated in t\u003csub\u003e0\u003c/sub\u003e\u0026ndash;t\u003csub\u003e2\u003c/sub\u003e. In the plasmid-cured Ec2 (PCV-Ec2), differential expression of AMR genes was also limited. In both PCV-Ec2\u003csub\u003eRW\u003c/sub\u003e and PCV-Ec2\u003csub\u003eTW\u003c/sub\u003e, only \u003cem\u003eariR\u003c/em\u003e was downregulated, consistent with deletion of plasmid-borne AMR genes such as \u003cem\u003emph\u003c/em\u003e(A) and \u003cem\u003etet\u003c/em\u003e(A).\u003c/p\u003e \u003cp\u003eIn Kp, AMR transcription changed markedly and differed between matrices and plasmid backgrounds. In Kp\u003csub\u003eRW\u003c/sub\u003e, \u003cem\u003eterB\u003c/em\u003e, \u003cem\u003eemrD\u003c/em\u003e, \u003cem\u003eble\u003c/em\u003e, and the aminoglycoside phosphotransferases \u003cem\u003eaph(3'')-Ib\u003c/em\u003e and \u003cem\u003eaph(6)-Id\u003c/em\u003e were downregulated at t\u003csub\u003e14\u003c/sub\u003e versus t\u003csub\u003e0\u003c/sub\u003e. In early incubation (t\u003csub\u003e0\u003c/sub\u003e\u0026ndash;t\u003csub\u003e2\u003c/sub\u003e), \u003cem\u003eaph(3'')-Ib\u003c/em\u003e, \u003cem\u003eaph(6)-Id\u003c/em\u003e and \u003cem\u003earmA\u003c/em\u003e were also downregulated, while the heavy-metal tolerance genes \u003cem\u003esilP\u003c/em\u003e and \u003cem\u003earsC\u003c/em\u003e were upregulated. In Kp\u003csub\u003eTW\u003c/sub\u003e, \u003cem\u003eemrD\u003c/em\u003e was likewise downregulated, but the remaining DEGs differed: \u003cem\u003eaph(3')-Ia\u003c/em\u003e was downregulated in both t\u003csub\u003e0\u003c/sub\u003e\u0026ndash;t\u003csub\u003e2\u003c/sub\u003e and t\u003csub\u003e0\u003c/sub\u003e\u0026ndash;t\u003csub\u003e14\u003c/sub\u003e, whereas copper resistance genes (\u003cem\u003epcoBCD\u003c/em\u003e) and the efflux genes \u003cem\u003eoqxA\u003c/em\u003e and \u003cem\u003eoqxB19\u003c/em\u003e were upregulated in t\u003csub\u003e0\u003c/sub\u003e\u0026ndash;t\u003csub\u003e14\u003c/sub\u003e. In PCV-Kp\u003csub\u003eRW\u003c/sub\u003e, AMR-associated differential expression was reduced to the downregulation of the plasmid-encoded \u003cem\u003eaph(6)-Id\u003c/em\u003e in t\u003csub\u003e0\u003c/sub\u003e\u0026ndash;t\u003csub\u003e14\u003c/sub\u003e. In PCV-Kp\u003csub\u003eTW\u003c/sub\u003e, \u003cem\u003eemrD\u003c/em\u003e and \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eOXA\u0026minus;48\u003c/sub\u003e were downregulated and \u003cem\u003eoqxA\u003c/em\u003e, \u003cem\u003eoqxB19\u003c/em\u003e, \u003cem\u003epcoABCD\u003c/em\u003e, and \u003cem\u003esilAB\u003c/em\u003e were upregulated from t\u003csub\u003e0\u003c/sub\u003e to t\u003csub\u003e14\u003c/sub\u003e.\u003c/p\u003e \u003cp\u003eVirulence-associated genes also exhibited condition-dependent shifts. In Ec1\u003csub\u003eRW\u003c/sub\u003e, iron utilization genes (\u003cem\u003eentF, fepB, fyuA, iutA\u003c/em\u003e) and \u003cem\u003eompA\u003c/em\u003e were downregulated, whereas \u003cem\u003ecsgF\u003c/em\u003e, \u003cem\u003egalF\u003c/em\u003e and \u003cem\u003erpoS\u003c/em\u003e were upregulated from t\u003csub\u003e0\u003c/sub\u003e to t\u003csub\u003e14\u003c/sub\u003e. The aerobactin locus \u003cem\u003eiucABCD\u003c/em\u003e was downregulated in t\u003csub\u003e0\u003c/sub\u003e\u0026ndash;t\u003csub\u003e2\u003c/sub\u003e but subsequently upregulated in t\u003csub\u003e2\u003c/sub\u003e\u0026ndash;t\u003csub\u003e14\u003c/sub\u003e. In addition, \u003cem\u003efes\u003c/em\u003e and \u003cem\u003eirp2\u003c/em\u003e were downregulated in t\u003csub\u003e0\u003c/sub\u003e\u0026ndash;t\u003csub\u003e2\u003c/sub\u003e. In Ec1\u003csub\u003eTW\u003c/sub\u003e, \u003cem\u003eentBF\u003c/em\u003e, \u003cem\u003eirp2\u003c/em\u003e and \u003cem\u003efyuA\u003c/em\u003e were downregulated in both t\u003csub\u003e0\u003c/sub\u003e\u0026ndash;t\u003csub\u003e2\u003c/sub\u003e and t\u003csub\u003e0\u003c/sub\u003e\u0026ndash;t\u003csub\u003e14\u003c/sub\u003e, while \u003cem\u003ecsgB\u003c/em\u003e and \u003cem\u003eibeB\u003c/em\u003e showed increased expression. In Ec2\u003csub\u003eRW\u003c/sub\u003e, \u003cem\u003efimADF\u003c/em\u003e were downregulated at t\u003csub\u003e14\u003c/sub\u003e, and \u003cem\u003ecsgA\u003c/em\u003e was downregulated in t\u003csub\u003e0\u003c/sub\u003e\u0026ndash;t\u003csub\u003e2\u003c/sub\u003e. In Ec2\u003csub\u003eTW\u003c/sub\u003e, \u003cem\u003easlA\u003c/em\u003e and \u003cem\u003ekpsEF\u003c/em\u003e were downregulated in t\u003csub\u003e0\u003c/sub\u003e\u0026ndash;t\u003csub\u003e14\u003c/sub\u003e, while \u003cem\u003eibeB\u003c/em\u003e, \u003cem\u003echuS\u003c/em\u003e and \u003cem\u003ecsgBFG\u003c/em\u003e were upregulated over the same period. In PCV-Ec2\u003csub\u003eRW\u003c/sub\u003e, only \u003cem\u003efimA\u003c/em\u003e was downregulated in t\u003csub\u003e0\u003c/sub\u003e\u0026ndash;t\u003csub\u003e14\u003c/sub\u003e; \u003cem\u003egspF\u003c/em\u003e, \u003cem\u003ekpsS\u003c/em\u003e and \u003cem\u003efepE\u003c/em\u003e were upregulated in t\u003csub\u003e2\u003c/sub\u003e\u0026ndash;t\u003csub\u003e14\u003c/sub\u003e and t\u003csub\u003e0\u003c/sub\u003e\u0026ndash;t\u003csub\u003e14\u003c/sub\u003e, and no virulence-associated genes were differentially expressed in t\u003csub\u003e0\u003c/sub\u003e\u0026ndash;t\u003csub\u003e2\u003c/sub\u003e. PCV-Ec2\u003csub\u003eTW\u003c/sub\u003e resembled Ec2\u003csub\u003eTW\u003c/sub\u003e, with downregulation of \u003cem\u003easlA\u003c/em\u003e and \u003cem\u003ekpsDFM\u003c/em\u003e and upregulation of \u003cem\u003eibeB\u003c/em\u003e and \u003cem\u003ecsgBF\u003c/em\u003e in t\u003csub\u003e0\u003c/sub\u003e\u0026ndash;t\u003csub\u003e14\u003c/sub\u003e.\u003c/p\u003e \u003cp\u003eIn \u003cem\u003eK. pneumoniae\u003c/em\u003e, both wild-type and PCV showed downregulation of \u003cem\u003emrk\u003c/em\u003e operon genes in t\u003csub\u003e0\u003c/sub\u003e\u0026ndash;t\u003csub\u003e14\u003c/sub\u003e, and several conditions additionally showed downregulation of \u003cem\u003efim\u003c/em\u003e operon genes. In Kp\u003csub\u003eRW\u003c/sub\u003e, \u003cem\u003etssG\u003c/em\u003e and plasmid-encoded \u003cem\u003ermpA\u003c/em\u003e were downregulated and iron acquisition genes (\u003cem\u003eiucA, ybtA, entC, fepB\u003c/em\u003e) were upregulated in t\u003csub\u003e0\u003c/sub\u003e\u0026ndash;t\u003csub\u003e14\u003c/sub\u003e; in Kp\u003csub\u003eTW\u003c/sub\u003e, \u003cem\u003eiucABCD\u003c/em\u003e and \u003cem\u003erfbD\u003c/em\u003e were upregulated at t\u003csub\u003e14\u003c/sub\u003e. In PCV-Kp\u003csub\u003eRW\u003c/sub\u003e, only \u003cem\u003emrkABCDHI\u003c/em\u003e were downregulated in t\u003csub\u003e0\u003c/sub\u003e\u0026ndash;t\u003csub\u003e14\u003c/sub\u003e, while PCV-Kp\u003csub\u003eTW\u003c/sub\u003e contained downregulated \u003cem\u003eentF\u003c/em\u003e and \u003cem\u003efimC\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eBased on the differential expression of AMR- and virulence-associated genes, we also examined master regulators known to control stress responses and pathogenicity (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u003cb\u003eDifferentially expressed master regulators in wild-type\u003c/b\u003e \u003cb\u003eEscherichia coli\u003c/b\u003e \u003cb\u003e(Ec1, Ec2)\u003c/b\u003e, \u003cb\u003eKlebsiella pneumoniae\u003c/b\u003e \u003cb\u003e(Kp), and the associated plasmid-cured variants (PCV-Ec2 and PCV-Kp), which were incubated in either river water (RW) or tap water (TW).\u003c/b\u003e Genes are grouped based on the direction of gene expression (\u0026darr;...downregulation, \u0026uarr;...upregulation) and whether expression changes occurred early (t\u003csub\u003e0\u003c/sub\u003e-t\u003csub\u003e2\u003c/sub\u003e...between inoculation and day 2), later (t\u003csub\u003e2\u003c/sub\u003e-t\u003csub\u003e14\u003c/sub\u003e...between day 2 and day 14) or over the whole period (t\u003csub\u003e0\u003c/sub\u003e-t\u003csub\u003e14\u003c/sub\u003e...between inoculation and day 14) of their regulation. Only genes with significant differential expression are shown (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, log2 fold-change (l2fc) \u0026gt; |2|). The p-values are rounded to 4 decimal digits, with smaller values displayed as 0.0000.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"16\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c12\" colnum=\"12\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c13\" colnum=\"13\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c14\" colnum=\"14\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c15\" colnum=\"15\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c16\" colnum=\"16\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eStrain\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cp\u003et0-t14\u0026darr;\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c7\" namest=\"c5\"\u003e \u003cp\u003et0-t2\u0026darr;\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c10\" namest=\"c8\"\u003e \u003cp\u003et0-t14\u0026uarr;\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c13\" namest=\"c11\"\u003e \u003cp\u003et0-t2\u0026uarr;\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c16\" namest=\"c14\"\u003e \u003cp\u003et2-t14\u0026uarr;\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGene\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003el2fc\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003ep-value\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eGene\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003el2fc\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003ep-value\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eGene\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003el2fc\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003ep-value\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c11\"\u003e \u003cp\u003eGene\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c12\"\u003e \u003cp\u003el2fc\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c13\"\u003e \u003cp\u003ep-value\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c14\"\u003e \u003cp\u003eGene\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c15\"\u003e \u003cp\u003el2fc\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c16\"\u003e \u003cp\u003ep-value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEc1\u003csub\u003eRW\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cem\u003erpoS\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eflhC\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eflhD\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003enarP\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003elexA\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e2.08\u003c/p\u003e \u003cp\u003e3.52\u003c/p\u003e \u003cp\u003e4.09\u003c/p\u003e \u003cp\u003e2.51\u003c/p\u003e \u003cp\u003e2.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0.0355\u003c/p\u003e \u003cp\u003e0.0141\u003c/p\u003e \u003cp\u003e0.0052\u003c/p\u003e \u003cp\u003e0.0345\u003c/p\u003e \u003cp\u003e0.0084\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c16\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEc1\u003csub\u003eTW\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003earcA\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e-2.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.0269\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003earcA\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e-2.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.0136\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cem\u003eflhC\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eflhD\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e4.91\u003c/p\u003e \u003cp\u003e3.42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0.0025\u003c/p\u003e \u003cp\u003e0.0482\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c16\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEc2\u003csub\u003eRW\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cem\u003eflhC\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e4.89\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0.0002\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e \u003cp\u003e\u003cem\u003eflhC\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eflhD\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e \u003cp\u003e4.65\u003c/p\u003e \u003cp\u003e4.26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c16\"\u003e \u003cp\u003e0.0014\u003c/p\u003e \u003cp\u003e0.0154\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEc2\u003csub\u003eTW\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cem\u003elrp\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eflhC\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eflhD\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e2.43\u003c/p\u003e \u003cp\u003e3.76\u003c/p\u003e \u003cp\u003e3.36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0.0000\u003c/p\u003e \u003cp\u003e0.0329\u003c/p\u003e \u003cp\u003e0.0425\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e \u003cp\u003e\u003cem\u003eflhC\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eflhD\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e \u003cp\u003e4.11\u003c/p\u003e \u003cp\u003e4.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c16\"\u003e \u003cp\u003e0.0250\u003c/p\u003e \u003cp\u003e0.0067\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePCV-Ec2\u003csub\u003eRW\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cem\u003eflhC\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eflhD\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e4.46\u003c/p\u003e \u003cp\u003e3.87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0.0089\u003c/p\u003e \u003cp\u003e0.0420\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e \u003cp\u003e\u003cem\u003eflhC\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eflhD\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e \u003cp\u003e4.50\u003c/p\u003e \u003cp\u003e4.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c16\"\u003e \u003cp\u003e0.0137\u003c/p\u003e \u003cp\u003e0.0285\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePCV-Ec2\u003csub\u003eTW\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cem\u003eflhC\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eflhD\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e3.65\u003c/p\u003e \u003cp\u003e3.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0.0000\u003c/p\u003e \u003cp\u003e0.0060\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c16\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eKp\u003csub\u003eRW\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003efis\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003enarL\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e-2.20\u003c/p\u003e \u003cp\u003e-2.84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.0453\u003c/p\u003e \u003cp\u003e0.0004\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003efis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e-2.92\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.0004\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cem\u003ecsrA\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003earcA\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003elexA\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003emarA\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e2.87\u003c/p\u003e \u003cp\u003e4.32\u003c/p\u003e \u003cp\u003e5.15\u003c/p\u003e \u003cp\u003e5.32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0.0019\u003c/p\u003e \u003cp\u003e0.0000\u003c/p\u003e \u003cp\u003e0.0000\u003c/p\u003e \u003cp\u003e0.0000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e\u003cem\u003ecsrA\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003ehns\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003earcA\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003emarA\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e3.09\u003c/p\u003e \u003cp\u003e3.17\u003c/p\u003e \u003cp\u003e3.25\u003c/p\u003e \u003cp\u003e5.64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e0.0006\u003c/p\u003e \u003cp\u003e0.0066\u003c/p\u003e \u003cp\u003e0.0000\u003c/p\u003e \u003cp\u003e0.0000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c16\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePCV-Kp\u003csub\u003eRW\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cem\u003earcA\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003elexA\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e2.70\u003c/p\u003e \u003cp\u003e4.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0.0133\u003c/p\u003e \u003cp\u003e0.0003\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c16\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePCV-Kp\u003csub\u003eTW\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cem\u003esoxS\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e2.77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0.0104\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c16\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eFirst, a marked upregulation of the flagellar master regulators \u003cem\u003eflhC\u003c/em\u003e and \u003cem\u003eflhD\u003c/em\u003e was observed in \u003cem\u003eE. coli\u003c/em\u003e strains. In Ec1\u003csub\u003eRW\u003c/sub\u003e, Ec1\u003csub\u003eTW\u003c/sub\u003e, and Ec2\u003csub\u003eRW\u003c/sub\u003e, \u003cem\u003eflhCD\u003c/em\u003e upregulation was sustained or increased over the 14-day period. This suggests an active investment in motility, potentially to escape nutrient-poor conditions. Second, the general stress response regulator \u003cem\u003erpoS\u003c/em\u003e and the SOS response repressor \u003cem\u003elexA\u003c/em\u003e were upregulated in Ec1\u003csub\u003eRW\u003c/sub\u003e. Similarly, Kp in RW upregulated \u003cem\u003elexA\u003c/em\u003e and \u003cem\u003earcA\u003c/em\u003e (anoxic redox control), along with the carbon storage regulator \u003cem\u003ecsrA\u003c/em\u003e. Finally, the PCVs maintained similar regulatory patterns to their wild-type counterparts in some contexts; for example, PCV-Kp in RW also upregulated \u003cem\u003earcA\u003c/em\u003e and \u003cem\u003elexA\u003c/em\u003e. However, PCV-Kp\u003csub\u003eTW\u003c/sub\u003e showed unique behavior, with only the superoxide response regulator \u003cem\u003esoxS\u003c/em\u003e significantly upregulated.\u003c/p\u003e \u003c/div\u003e"},{"header":"3 Discussion","content":"\u003cp\u003eThis study examined the survival and gene expression of \u003cem\u003eE. coli\u003c/em\u003e ST648 and \u003cem\u003eK. pneumoniae\u003c/em\u003e ST307 strains in RW and TW over 14 days. All strains, including their PCVs, were able to survive in both water types. Bacterial survival in water compartments has been reported to range from a few days to several hundred days, depending on the specific (experimental) conditions\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Our results are consistent with published data, suggesting these strains could maintain a constant CFU count well beyond our 14-day observation period. Lower cell concentrations may reduce survival by limiting cell-to-cell communication, such as quorum sensing\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e, which could partly explain the CFU decrease in TW OD\u003csub\u003e600\u003c/sub\u003e 0.01. In contrast, the lack of a similar reduction in RW at OD\u003csub\u003e600\u003c/sub\u003e 0.01 could reflect differences in water composition, such as nutrient availability\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Additionally, \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eK. pneumoniae\u003c/em\u003e can also enter a viable but non-culturable (VBNC) state in water, which can influence the results of colony-based methods\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eInitially, we hypothesized that the distinct origins of the bacterial strains would lead to different survival rates due to prior habitat or host adaptation\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. However, our data suggest that strain origin did not influence survival or growth in water. It is important to note that our use of sterilized water likely supported survival. The absence of microbial competition is known to reduce bacterial decline compared to multi-species co-cultures\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e and prevents interspecies interactions that otherwise influence gene regulation and adaptive responses\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eTranscriptomic alteration is a key driver of bacterial adaptation to environmental changes. While previous studies have shown rapid, extensive gene expression changes within minutes or hours of water exposure\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e, our extended timeframe focused on long-term adaptation. Nevertheless, our data also suggested that transcriptomic adaptations occurred rather quickly after introduction of the strains into water.\u003c/p\u003e \u003cp\u003eAlthough data on aquatic bacterial adaptation remain limited, existing literature suggests broad regulatory changes. For example, \u003cem\u003eLegionella pneumophila\u003c/em\u003e exhibits widespread downregulation of genes across various COG categories after 24 hours in artificial freshwater, with its composition modeled after North American freshwater\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. In contrast, our strains showed a more balanced distribution between up- and downregulated genes. Similarly, while Duffitt \u003cem\u003eet al.\u003c/em\u003e\u003csup\u003e15\u003c/sup\u003e found that \u003cem\u003eE. coli\u003c/em\u003e O157:H7 exhibited far fewer DEGs in sterile stream water compared to soil, our findings demonstrate that high-risk \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eK. pneumoniae\u003c/em\u003e clones are capable of mounting extensive adaptive responses to aquatic habitats.\u003c/p\u003e \u003cp\u003eThe complex metabolism of \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eK. pneumoniae\u003c/em\u003e is strongly influenced by their high genetic variability\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Transitioning from nutrient-rich LB medium to nutrient-depleted RW or TW may represent a severe nutritional shift\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. Nutrient limitations, particularly of nitrogen and carbohydrates, are known triggers for extensive adaptive processes\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e,\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. This likely explains the high prevalence of metabolism-associated DEGs and transcriptional regulators across our data set\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e,\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. The concurrent up- and downregulation of metabolic genes suggests active metabolic readjustment rather than a uniform shutdown\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eVirulence-associated genes and resistance determinants also play nuanced roles in environmental survival. While biofilm formation can protect bacteria from suboptimal conditions\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e, motility allows them to escape stress and seek nutrients\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan additionalcitationids=\"CR41\" citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e,\u003c/sup\u003e. We observed upregulation of the master flagellar regulators \u003cem\u003eflhCD\u003c/em\u003e, alongside general downregulation of adhesion- and biofilm-associated genes. These observations are reasonable within the context of our experimental setup. The continuous shaking of the flasks likely limited the possibility of biofilm formation compared to setups involving low flow rate or minimal turbulence. Additionally, enhancing motility would represent a strategy favoring the movement toward nutrients rather than remaining stationary\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e,\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eInterestingly, general stress pathways were largely unprovoked. The alternative sigma factor RpoS (\u003cem\u003erpoS\u003c/em\u003e) regulates the general stress response in \u003cem\u003eEnterobacterales\u003c/em\u003e during starvation or osmotic shock\u003csup\u003e\u003cspan additionalcitationids=\"CR46 CR47\" citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e,\u003c/sup\u003e. However, we observed little to no differential expression of \u003cem\u003erpoS\u003c/em\u003e, suggesting our experimental conditions were not stressful enough to trigger its activation. Similarly, \u003cem\u003esoxS\u003c/em\u003e, a key mediator of oxidative stress\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e, was rarely differentially expressed, indicating low oxidative stress levels in these microcosms.\u003c/p\u003e \u003cp\u003eWhile plasmids are well known for mediating AMR and virulence\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e,\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e, their influence on bacterial survival in aquatic environments is less clear. In our study, plasmid carriage did not affect survival rates of the wild-type strains compared to their PCVs, consistent with findings in extraintestinal pathogenic \u003cem\u003eE. coli\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. However, plasmids can impose fitness costs that are later mitigated by adaptive evolution and compensation\u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e,\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e. Although survival remained steady, the cured genetic segments could have affected other fitness parameters, such as growth rates or competitive ability. This is supported by the differential expression patterns of iron utilization-associated genes observed in Kp and PCV-Kp. Upregulation of siderophore synthesis likely indicates active iron scavenging in depleted water\u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e,\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e, whereas downregulation could minimize biosynthesis costs when ferric iron concentrations are sufficient\u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e. Notably, the absence of the plasmid-encoded \u003cem\u003eiutA\u003c/em\u003e and \u003cem\u003eiucABCD\u003c/em\u003e genes in PCV-Kp, in contrast to their upregulation in Kp, did not translate into differences in survival. Simultaneously, the contrasting downregulation of additional siderophore-associated genes in PCV-Kp and their upregulation in Kp suggests that the cured plasmid regions influenced iron acquisition-related responses beyond the deleted loci.\u003c/p\u003e \u003cp\u003eImportantly, plasmids substantially influenced global gene regulation, though its influence was strain-dependent. Assuming all other conditions were identical, the pronounced transcriptomic differences between wild-type strains and PCVs reflect the loss of these extrachromosomal elements. Plasmid acquisition is known to alter host gene expression, particularly in metabolic pathways\u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e,\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e. We primarily detected variations in metabolism-linked DEGs between the wild-type \u003cem\u003eK. pneumoniae\u003c/em\u003e and its PCV, confirming that plasmids modulate metabolic transcription in response to environmental changes. While previous studies noted plasmid-induced changes in ABC transporters\u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e and two-component systems\u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e, our data showed these changes were highly strain- and water-dependent, precluding a uniform trend.\u003c/p\u003e \u003cp\u003eWhile our study provides valuable insights into the transcriptomic adaptation of high-risk \u003cem\u003eEnterobacterales\u003c/em\u003e lineages in aquatic environments, several limitations should be noted. First, the use of sterilized water microcosms eliminated microbial competition, predation, and complex interspecies interactions, which likely enhanced bacterial survival and altered transcriptomic responses compared to natural ecosystems. Follow-up co-culture experiments would help to assess how such biotic interactions influence survival and gene regulation under more ecologically realistic conditions. Second, our investigation was limited to a 14-day observation period under static laboratory conditions; thus, it may not fully capture the long-term evolutionary dynamics or the impact of environmental stressors present in natural aquatic reservoirs, such as UV radiation and temperature fluctuations. Finally, the study evaluated a small number of strains from specific high-risk lineages (ST648 and ST307) and their respective PCVs. Therefore, broader generalizations regarding the adaptive capacities of diverse \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eK. pneumoniae\u003c/em\u003e clones, or the universal influence of distinct plasmid types, require further research involving larger strain panels in non-sterile settings.\u003c/p\u003e \u003cp\u003eTo conclude, strains belonging to high-risk clonal lineages of \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eK. pneumoniae\u003c/em\u003e, along with their respective PCVs, survived in TW and RW over 14 days with no observable differences in CFU counts. However, their transcriptomic responses varied considerably, revealing highly strain-specific regulatory and metabolic adaptations to aquatic conditions. These findings underscore the capacity of clinically relevant, high-risk \u003cem\u003eEnterobacterales\u003c/em\u003e to persist in natural environments, emphasizing the role of aquatic compartments as critical reservoirs within the One Health framework. Future investigations incorporating a broader panel of strains with diverse plasmid backgrounds are essential to further disentangle the complex dynamics of strain-specific and plasmid-associated environmental adaptation.\u003c/p\u003e"},{"header":"4 Methods","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e4.1 Bacterial strains\u003c/h2\u003e \u003cp\u003eAll bacterial strains used in this study are listed in Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e and were stored at \u0026minus;\u0026thinsp;80\u0026deg;C in LB broth (Carl Roth, Karlsruhe, Germany) supplemented with 20% (v/v) glycerol (anhydrous; Merck, Darmstadt, Germany). Routine cultivation was performed at 37\u0026deg;C in LB broth or on Luria agar (LA) plates (Carl Roth).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u003cb\u003eSummary of bacterial strains used in this study.\u003c/b\u003e PCV1935 is a plasmid-cured variant (PCV) of PBIO1935, which belongs to the same clonal lineage as PBIO1953 (15 SNPs). ST...sequence type.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStrain\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOriginal designation\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSpecies\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eST\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSource\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eYear of isolation\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eReference\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEc1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePBIO3806\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eE.\u0026nbsp;coli\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e648\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSurface water (Kumasi, Ghana)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e2021\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eEger \u003cem\u003eet\u0026nbsp;al.\u003c/em\u003e (2024)\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEc2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePBIO730/\u003c/p\u003e \u003cp\u003eIMT16316\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eE.\u0026nbsp;coli\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e648\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eEurasian blackbird feces (Giessen, Germany)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e2008\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eGuenther \u003cem\u003eet\u0026nbsp;al.\u003c/em\u003e (2010)\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePCV-Ec2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePCV730\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eE.\u0026nbsp;coli\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e648\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePlasmid-cured variant of Ec2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e2013\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eSchaufler \u003cem\u003eet\u0026nbsp;al.\u003c/em\u003e (2013)\u003csup\u003e\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eKp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePBIO1953/\u003c/p\u003e \u003cp\u003eva20750\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eK.\u0026nbsp;pneumoniae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e307\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eTracheal secretion from hospital patient (Greifswald, Germany)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e2019\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eHeiden \u003cem\u003eet\u0026nbsp;al.\u003c/em\u003e (2020)\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePCV-Kp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePCV1935\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eK.\u0026nbsp;pneumoniae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e307\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eThroat from hospital patient (Greifswald, Germany)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e2019\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eHeiden \u003cem\u003eet\u0026nbsp;al.\u003c/em\u003e (2020)\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eEnvironmental water samples were collected on March 21, 2022, using sterile 1000 mL blue-cap bottles. Tap water was sourced directly from the laboratory supply (Greifswald, Germany), while river water was collected from the river Ryck at coordinates 54.09879\u0026deg; N, 13.40529\u0026deg; E. To ensure a sterile matrix for subsequent experiments, both samples were autoclaved (121\u0026deg;C, 15 min) and stored at 4\u0026deg;C until further use.\u003c/p\u003e \u003cp\u003eThe previously assembled genomes of the strains were annotated with Bakta (v1.11.3; database version 6.0) to provide a consistent and comparable annotation. Additional functional annotation was obtained using the eggNOG-mapper\u003csup\u003e\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e,\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e web application (emapper-2.1.12; accessed July 23, 2025), and COG assignments as well as KEGG\u003csup\u003e\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u003c/sup\u003e Orthology (KO) identifiers were extracted from the output.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e4.2 Survival in water\u003c/h2\u003e \u003cp\u003eBiological triplicates of overnight cultures were diluted 1:100 in 5 mL of fresh LB and incubated at 37\u0026deg;C with shaking at 180 rpm until visible turbidity was reached. The cells were harvested by centrifugation at 7,500 x \u003cem\u003eg\u003c/em\u003e for 5 min at room temperature, and the resulting pellets were resuspended in either the prepared tap water or river water. For the survival assay, 30 mL of the respective water samples were transferred into sterile 100 mL Erlenmeyer flasks. Each flask was inoculated in triplicates with the bacterial suspension to achieve an initial OD\u003csub\u003e600\u003c/sub\u003e of 0.01, 0.1, or 0.2. The cultures were then incubated at room temperature (22\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C) with continuous agitation at 150 rpm. CFUs were performed by plating serial dilutions of the cultures onto LA plates, followed by overnight incubation at 37\u0026deg;C. Significance testing was performed using the two-sided Kruskal-Wallis statistical test followed by Dunn\u0026rsquo;s multiple-comparison post-hoc test.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e4.3 RNA isolation, sequencing and analysis\u003c/h2\u003e \u003cp\u003eAt the time points t\u003csub\u003e0\u003c/sub\u003e, t\u003csub\u003e2\u003c/sub\u003e and t\u003csub\u003e14\u003c/sub\u003e, 1 mL aliquots of the cultures (initial OD\u003csub\u003e600\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.1) were harvested and immediately snap-cooled in liquid nitrogen for 5 s to arrest transcription. The samples were then centrifuged at 16,000 x \u003cem\u003eg\u003c/em\u003e for 3 min at 4\u0026deg;C. Supernatants were completely discarded, and the resulting pellets were snap-frozen in liquid nitrogen for 2 s and stored at \u0026minus;\u0026thinsp;70\u0026deg;C for a maximum of 6 h prior to extraction.\u003c/p\u003e \u003cp\u003eTotal RNA was extracted using the RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer\u0026rsquo;s instructions. RNA quantity was determined with a Qubit 4 fluorometer (Thermo Fisher Scientific, Waltham, MA, USA) and RNA integrity was assessed using Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Total RNA was shipped on dry ice to the Competence Centre for Genomic Analysis (CCGA, Kiel, Germany). Stranded total RNA-seq libraries were prepared using the Illumina Stranded Total RNA Prep with Ribo-Zero Plus kit (Illumina, San Diego, CA, USA) according to the manufacturer\u0026rsquo;s instructions. Libraries were sequenced on an Illumina NovaSeq 6000 platform to generate 2 \u0026times; 100-bp paired-end reads.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e4.4 Read processing and mapping\u003c/h2\u003e \u003cp\u003eRaw reads were processed with fastp (v0.23.4)\u003csup\u003e64\u003c/sup\u003e to remove adapter sequences and low-quality bases. Filtered reads were aligned against the corresponding strain-specific genome assembly using Bowtie 2 (v2.5.1)\u003csup\u003e65\u003c/sup\u003e in local alignment mode (--local). Gene-level read counts were generated with featureCounts (v2.0.1)\u003csup\u003e66\u003c/sup\u003e using the corresponding strain-specific genome annotation. Unless otherwise stated, software was run with default parameters. Putative plasmid-derived sequences in strain Ec1 were identified using Plasmer v0.1 (20220816)\u003csup\u003e\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e\u003c/sup\u003e with the database version dated 2025-01-07.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e4.5 Differential gene expression analysis\u003c/h2\u003e \u003cp\u003eGene count tables were imported into R and analyzed with DESeq2 (v1.48.1)\u003csup\u003e68\u003c/sup\u003e. Differential gene expression analysis was performed separately for each strain and water type, i.e. RW and TW, with the respective time point serving as the experimental factor. DESeq2-based size-factor normalization, dispersion estimation and model fitting were performed following the standard workflow. To assess sample clustering, PCA was performed in DESeq2 using the plotPCA function (default parameters) on variance-stabilized transformed counts. Pairwise contrasts were calculated for t\u003csub\u003e2\u003c/sub\u003e versus t\u003csub\u003e0\u003c/sub\u003e, t\u003csub\u003e14\u003c/sub\u003e versus t\u003csub\u003e0\u003c/sub\u003e, and t\u003csub\u003e14\u003c/sub\u003e versus t\u003csub\u003e2\u003c/sub\u003e. Statistical significance for each contrast was assessed using Wald tests, and P values were adjusted for multiple testing using the Benjamini-Hochberg procedure. For each comparison l2fcs were extracted. A gene was considered differential expressed if it met the following criteria: |l2fcs|\u0026gt;=2 and an adjusted p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003ePotential conflicts of interest.\u003c/h2\u003e \u003cp\u003e All authors declare no potential conflicts.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding.\u003c/h2\u003e \u003cp\u003eThis work was supported by two grants from the Federal Ministry of Research, Technology and Space (BMFTR, Germany) to K.S. entitled \u0026ldquo;Standardized One Health surveillance of antibiotic residues and antibiotic and heavy metal resistance in Baltic water environments and wild birds\u0026rdquo; (01KI2402A) and \u0026ldquo;Disarming pathogens as a different strategy to fight antimicrobial-resistant Gram-negatives\u0026rdquo; (01KI2410).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eConceptualization, K.S.; methodology, E.E., M.S. and P.L.; software, M.S. and S.E.H.; validation, E.E., M.S. and P.L.; formal analysis, E.E., K.S., M.S., P.L., S.D., S.E.H., S.K. and T.H.-B.; investigation, E.E., P.L. and M.S..; resources, K.S. and S.K.; data curation, M.S., P.L. and S.E.H.; writing\u0026mdash;original draft preparation, P.L.; writing\u0026mdash;review and editing, E.E., K.S. and M.S.; visualization, M.S. and P.L.; supervision, K.S.; project administration, K.S.; funding acquisition, K.S. and S.K. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe thank Sara-Lucia Skwara for her excellent technical assistance.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe data for this study have been deposited in the European Nucleotide Archive (ENA) at EMBL-EBI under accession number PRJEB110490 (https://www.ebi.ac.uk/ena/browser/view/PRJEB110490).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAntimicrobial Resistance Collaborators. 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ESBL-plasmids carrying toxin-antitoxin systems can be \"cured\" of wild-type \u003cem\u003eEscherichia coli\u003c/em\u003e using a heat technique. \u003cem\u003eGut Pathog\u003c/em\u003e 5, 34 (2013). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/1757-4749-5-34\u003c/span\u003e\u003cspan address=\"10.1186/1757-4749-5-34\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"npj-emerging-contaminants","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [npj Emerging Contaminants](https://www.nature.com/npjemergcontam/)","snPcode":"44454","submissionUrl":"https://submission.springernature.com/new-submission/44454/3","title":"npj Emerging Contaminants","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Unsupported Journal","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"high-risk clones in water, One Health, transcriptomics, Enterobacterales, plasmid-cured variants","lastPublishedDoi":"10.21203/rs.3.rs-9425840/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9425840/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe spread of antimicrobial-resistant \u003cem\u003eEnterobacterales\u003c/em\u003e 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 \u003cem\u003eEscherichia coli\u003c/em\u003e and \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e, 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 \u003cem\u003eK. pneumoniae\u003c/em\u003e, 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.\u003c/p\u003e","manuscriptTitle":"Broad adaptations of plasmid-carrying Escherichia coli and Klebsiella pneumoniae to water as a reservoir","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-24 16:46:28","doi":"10.21203/rs.3.rs-9425840/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2026-05-18T04:47:54+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"241772126754471074695805306557744930333","date":"2026-05-08T23:52:27+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"291630600003799803937481248373864655069","date":"2026-05-08T13:05:12+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"179787907896131742489402203348531562416","date":"2026-05-08T09:00:37+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-17T05:47:43+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-16T12:44:33+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-16T12:04:02+00:00","index":"","fulltext":""},{"type":"submitted","content":"npj Emerging Contaminants","date":"2026-04-15T10:49:13+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"npj-emerging-contaminants","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [npj Emerging Contaminants](https://www.nature.com/npjemergcontam/)","snPcode":"44454","submissionUrl":"https://submission.springernature.com/new-submission/44454/3","title":"npj Emerging Contaminants","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Unsupported Journal","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"e1034114-b722-47e5-8359-db564af08984","owner":[],"postedDate":"April 24th, 2026","published":true,"recentEditorialEvents":[{"type":"editorInvitedReview","content":"","date":"2026-05-18T04:47:54+00:00","index":45,"fulltext":""},{"type":"reviewerAgreed","content":"241772126754471074695805306557744930333","date":"2026-05-08T23:52:27+00:00","index":44,"fulltext":""},{"type":"reviewerAgreed","content":"291630600003799803937481248373864655069","date":"2026-05-08T13:05:12+00:00","index":42,"fulltext":""},{"type":"reviewerAgreed","content":"179787907896131742489402203348531562416","date":"2026-05-08T09:00:37+00:00","index":39,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":66969272,"name":"Biological sciences/Genetics"},{"id":66969273,"name":"Biological sciences/Microbiology"}],"tags":[],"updatedAt":"2026-04-24T16:46:28+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-24 16:46:28","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9425840","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9425840","identity":"rs-9425840","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}