Abstract
Uropathogenic Escherichia coli (UPECs) are the predominant cause of asymptomatic bacteriuria (ABU) and symptomatic UTI. In this study, multidrug-resistant (MDR) ABU-UPECs from hospitalized patients of Kolkata, India, were characterized with respect to their ESBL phenotype, acquisition of β-lactamase genes, mobile genetic elements (MGEs), phylotype property, ERIC-PCR profile, sequence types (STs), clonal complexes (CCs) and evolutionary and quantitative relationships and compared to the symptomatic ones to understand their epidemiology and evolutionary origin. Statistically significant incidence of ESBL producers, β-lactamase genes, MGEs and novel phylotype property (NPP) among ABU-UPECs similar to the symptomatic ones indicated the probable incidence of chromosomal plasticity on resistance gene acquisition through MGEs due to indiscriminate drug usage. ERIC-PCR typing and MLST analysis showed clonal heterogeneity and predominance of ST940 (CC448) among asymptomatic isolates akin to symptomatic ones along with the evidence of zoonotic transmissions. Minimum spanning tree analysis showed a close association between ABU-UPEC with known and unidentified STs having NPPs with isolates that belonged to phylogroups clade I, D, and B2. This is the first study that reported the occurrence of MGEs and NPPs among ABU-UPECs with the predominance of ESBL production which displayed the deleterious effect of MDR among this pathogen demanding alternative therapeutic interventions. Moreover, this study for the first time attempted to introduce a new approach to ascertain the phylotype property of unassigned UPECs. Withal, increased recognition, proper understanding and characterization of ABU-UPECs with the implementation of appropriate therapeutic measures against them when necessary are the need of the era which otherwise might lead to serious complications in the vulnerable population.
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Acknowledgements
The authors would like to express their sincere gratitude to Director and Head of Department of Tropical Medicine, School of Tropical Medicine, Kolkata West Bengal, India, for their kind support. The authors would also like to thank Mr. Snehashis Koley, for his assistance in acquiring access to the BioNumerics version 7.6 software, Applied Maths, Sint-Martens-Latem, Belgium.
Funding
Consumable support to carry out the research was provided from an extramural grant from the Science and Engineering Research Board (SERB), Department of Science and Technology, Government of India (Grant No. EMR/2016/001670 dated August 22, 2017). Arunita Ghosh was the recipient of the INSPIRE fellowship grant from the Department of Science and Technology, New Delhi, Government of India, No.-DST/INSPIRE Fellowship/2016/ IF160069 dated October 31, 2018, and October 10, 2019.
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This work was performed as collaboration among all the authors. MM and AG conceived the study. BG participated in the study design. AG performed all the experiments, participated in statistical and bioinformatics analysis, and also wrote the first draft of the manuscript. BG participated only in the PCR-based phylogenetic assays. MM supervised the entire study and also reviewed and finalized the manuscript. All of the authors had read and approved the final manuscript.
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The present study was approved by the Clinical Research Ethics Committee, School of Tropical Medicine, Kolkata (CREC-STM), Ref No. CREC-STM/317 dated March 29, 2016.
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Supplementary Fig. 1 Cluster analysis performed on Heat maps generated using R software package (version 3.2.5)., based on the presence and absence three β-lactamase genes in the plasmid DNA of each of the individual isolate from (a) Asymptomatic UPEC group and (b) Symptomatic UPEC group. Numbers in the text box provided on the righthand side represented sample ID of the E. coli isolates considered in each group. Colour key represented the variation in colours from red to white illustrating the complete absence of a particular gene to its complete presence respectively. Supplementary Fig. 2 Cluster analysis performed on Heat maps generated using R software package (version 3.2.5)., based on the presence and absence three β-lactamase genes in the genomic DNA of each of the individual isolate from (a) Asymptomatic UPEC group and (b) Symptomatic UPEC group. Numbers in the text box provided on the righthand side represented sample ID of the E. coli isolates considered in each group. Colour key represented the variation in colours from red to white illustrating the complete absence of a particular gene to its complete presence respectively. Supplementary Fig. 3 Graphical representation of correlation coefficient values computed with confidence level of 95% (p values ≤ 0.05) using GraphPad Prism version 9 (Prism software package) based on the correlation of three different β-lactamase genes in the genomic DNA of each of individual (a) asymptomatic and (b) symptomatic UPECs. Different β-lactamases were represented by interleaved symbols with varied colours. Dotted lines were used to differentiate correlation of each of the β-lactamase gene with two others. Supplementary Fig. 4 Dendogram generated on ERIC-PCR profiles of (a) asymptomatic (n=20) and (b) symptomatic (n=20) UPECs. Dice similarity coefficient values were used to generate the dendogram by UPGMA method of clustering using SPSS version 21.0 software. Isolates were distinctly unrelated at a coefficient of similarity value ≥ 96% (indicated by a solid line). ERIC-banding pattern of 20 each of ABU and Symptomatic UPECs respectively were represented as grouped individual lanes that contained the amplicons of each isolate. Five and six clonal groups were identified based on the cluster analysis of the individual ERIC profiles of asymptomatic and symptomatic isolates respectively. The extreme left column represented respective ESBL /BLIR phenotype of the isolates. Immediately adjacent to that was the phylogroup of the isolates.
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Ghosh, A., Ghosh, B. & Mukherjee, M. Epidemiologic and molecular characterization of β-lactamase-producing multidrug-resistant uropathogenic Escherichia coli isolated from asymptomatic hospitalized patients. Int Microbiol 25, 27–45 (2022). https://doi.org/10.1007/s10123-021-00187-9
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DOI: https://doi.org/10.1007/s10123-021-00187-9