Abstract
Characterizing the physiological response of bacterial cells to antibiotics is crucial for designing diagnostic techniques, treatment choices, and drug development. While bacterial cells at sublethal doses of antibiotics are commonly characterized, the impact of exposure to high concentrations of antibiotics on bacteria after long-term serial exposure and their effect on withdrawal need attention for further characterization. This study investigated the effect of increasing imipenem concentrations on carbapenem-susceptible (S) and carbapenem-resistant (R) E. coli on their growth adaptation and cell surface structure. We exposed the bacterial population to increasing imipenem concentrations through 30 exposure cycles. Cell morphology was observed using a 3D laser scanning confocal microscope (LSCM) and transmission electron microscope (TEM). Results showed that the exposure resulted in significant morphological changes in E. coli (S) cells, while minor changes were seen in E. coli (R) cells. The rod-shaped E. coli (S) gradually transformed into round shapes. Further, the exposed E. coli (S) cells’ surface area-to-volume ratio (SA/V) was also significantly different from the control, which is non-exposed E. coli (S). Then, the exposed E. coli (S) cells were re-grown in antibiotic-free environment for 100 growth cycles to determine if the changes in cells were reversible. The results showed that their cell morphology remained round, showing that the cell morphology was not reversible. The morphological response of these cells to imipenem can assist in understanding the resistance mechanism in the context of diagnostics and antibacterial therapies.
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References
Wellington EMH, Boxall ABA, Cross P, Feil EJ, Gaze WH, Hawkey PM, et al. The role of the natural environment in the emergence of antibiotic resistance in Gram-negative bacteria. Lancet Infect Dis. 2013;13:155–65.
Caliskan-Aydogan O, Alocilja EC. A review of carbapenem resistance in enterobacterales and its detection techniques. Microorganism. 2023;11:1491. https://www.mdpi.com/2076-2607/11/6/1491.
Gullberg E, Cao S, Berg OG, Ilbäck C, Sandegren L, Hughes D, et al. Selection of resistant bacteria at very low antibiotic concentrations. PLoS Pathog. 2011;7:1–9.
Serwecińska L. Antimicrobials and antibiotic-resistant bacteria: a risk to the environment and to public health. Water. 2020;12:3313. https://www.mdpi.com/2073-4441/12/12/3313.
Sandegren L. Selection of antibiotic resistance at very low antibiotic concentrations. Ups J Med Sci. 2014;119:103–7.
Hughes D, Andersson DI. Environmental and genetic modulation of the phenotypic expression of antibiotic resistance. FEMS Microbiol Rev. 2017;41:374–91.
Andersson DI, Hughes D. Evolution of antibiotic resistance at non-lethal drug concentrations. Drug Resist Updat. 2012;15:162–72.
Ter Kuile BH, Kraupner N, Brul S. The risk of low concentrations of antibiotics in agriculture for resistance in human health care. FEMS Microbiol Lett. 2016;363. Available from: https://academic.oup.com/femsle/article-abstract/363/19/fnw210/2236218.
Olofsson SK, Cars O. Optimizing drug exposure to minimize selection of antibiotic resistance. Clin Infect Dis. 2007;45:129–36.
Capita R, Alonso-Calleja C. Antibiotic-resistant bacteria: a challenge for the food industry. Crit Rev Food Sci Nutr. 2013;53:11–48.
Munita JM, Arias CA. Mechanisms of antibiotic resistance. Virulence Mech Bact Pathog. 2016;4:481–511.
Taggar G, Rheman MA, Boerlin P, Diarra MS. Molecular epidemiology of carbapenemases in enterobacteriales from humans, animals, food and the environment. Antibiotics. 2020;9:1–22.
Rabaan AA, Eljaaly K, Alhumaid S, Albayat H, Al-Adsani W, Sabour AA, et al. An overview on phenotypic and genotypic characterisation of carbapenem-resistant enterobacterales. Medicina. 2022;58:1675. https://www.mdpi.com/1648-9144/58/11/1675.
Band VI, Weiss DS. Heteroresistance: a cause of unexplained antibiotic treatment failure? Coers J, editor. PLOS Pathog. 2019;15:e1007726. Available from: https://dx.plos.org/10.1371/journal.ppat.1007726.
Abdeta A, Bitew A, Fentaw S, Tsige E, Assefa D, Lejisa T, et al. Phenotypic characterization of carbapenem non-susceptible gram-negative bacilli isolated from clinical specimens. PLoS One. 2021;16:1–18. https://doi.org/10.1371/journal.pone.0256556.
https://www.who.int/news/item/27-02-2017-who-publishes-list-of-bacteria-for-which-new-antibiotics-are-urgently-needed World Health Organization (WHO). WHO publishes list of bacteria for which new antibiotics are urgently needed [Internet]. WHO.
https://www.cdc.gov/drugresistance/pdf/threats-report/2019-ar-threats-report-508.pdf Antibiotic resistance threats in the United States [Internet]. 2019.
Dankittipong N, Fischer EAJ, Swanenburg M, Wagenaar JA, Stegeman AJ, de Vos CJ. Quantitative risk assessment for the introduction of carbapenem-resistant Enterobacteriaceae (CPE) into Dutch Livestock Farms. Antibiotics. 2022;11:281. https://www.mdpi.com/2079-6382/11/2/281.
Lutgring JD, Limbago BM. The Problem of Carbapenemase-Producing-Carbapenem-Resistant-Enterobacteriaceae Detection. J Clin Microbiol. 2016;54:529–34. Available from: https://journals.asm.org/doi/10.1128/JCM.02771-15.
Woodford N, Wareham DW, Guerra B, Teale C. Carbapenemase-producing Enterobacteriaceae and non-Enterobacteriaceae from animals and the environment: an emerging public health risk of our own making? J Antimicrob Chemother. 2014;69:287–91. https://academic.oup.com/jac/article-lookup/doi/10.1093/jac/dkt392.
Fernández J, Guerra B, Rodicio MR. Resistance to carbapenems in non-typhoidal Salmonella enterica serovars from humans, animals and food. Veterinary Sci. 2018;5:40.
Morrison BJ, Rubin JE. Carbapenemase producing bacteria in the food supply esca** detection. Plos One. 2015;10:e0126717.
Fischer J, Schmoger S, Jahn S, Helmuth R, Guerra B. NDM-1 carbapenemase-producing Salmonella enterica subsp. enterica serovar Corvallis isolated from a wild bird in Germany. J Antimicrob Chemother. 2013;68:2954–6. https://academic.oup.com/jac/article-lookup/doi/10.1093/jac/dkt260.
Mills MC, Lee J. The threat of carbapenem-resistant bacteria in the environment: evidence of widespread contamination of reservoirs at a global scale. Environ Pollut. 2019;255:113143 https://doi.org/10.1016/j.envpol.2019.113143.
Codjoe F, Donkor E. Carbapenem resistance: a review. Med Sci. 2017;6:1 http://www.mdpi.com/2076-3271/6/1/1.
Smith HZ, Kendall B. Carbapenem Resistant Enterobacteriaceae. StatPearls Publishing: Tampa, FL, USA; 2021.
Papp-Wallace KM, Endimiani A, Taracila MA, Bonomo RA. Carbapenems: past, present, and future. Antimicrob Agents Chemother. 2011;55:4943–60.
Capita R, Riesco-Peláez F, Alonso-Hernando A, Alonso-Calleja C. Exposure of Escherichia coli ATCC 12806 to sublethal concentrations of food-grade biocides influences its ability to form biofilm, resistance to antimicrobials, and ultrastructure. Appl Environ Microbiol. 2014;80:1268–80.
Lorian V. Low concentrations of antibiotics. J Antimicrob Chemother. 1985;15:15–26.
Cushnie TPT, O’Driscoll NH, Lamb AJ. Morphological and ultrastructural changes in bacterial cells as an indicator of antibacterial mechanism of action [Internet]. Cell Mol Life Sci. 2016;73:4471–92. https://springer.longhoe.net/article/10.1007/s00018-016-2302-2.
O’Driscoll NH, Cushnie TPT, Matthews KH, Lamb AJ. Colistin causes profound morphological alteration but minimal cytoplasmic membrane perforation in populations of Escherichia coli and Pseudomonas aeruginosa. Arch Microbiol. 2018;200:793–802. https://doi.org/10.1007/s00203-018-1485-3.
Nishino M, Matsuzaki I, Musangil FY, Takahashi Y, Iwahashi Y, Warigaya K, et al. Measurement and visualization of cell membrane surface charge in fixed cultured cells related with cell morphology. Plos One. 2020;15:e0236373.
Furchtgott L, Wingreen NS, Huang KC. Mechanisms for maintaining cell shape in rod‐shaped Gram‐negative bacteria. Mol Microbiol. 2011;81:340–53.
Chang T-W, Weinstein L. Morphological changes in gram-negative bacilli exposed to cephalothin. J Bacteriol. 1964;88:1790–7.
Murtha AN, Kazi MI, Schargel RD, Cross T, Fihn C, Cattoir V, et al. High-level carbapenem tolerance requires antibiotic-induced outer membrane modifications. PLOS Pathog. 2022;18:e1010307. Available from: https://dx.plos.org/10.1371/journal.ppat.1010307.
Wang S, Ding Q, Zhang Y, Zhang A, Wang Q, Wang R, et al. Evolution of Virulence, Fitness, and Carbapenem Resistance Transmission in ST23 Hypervirulent Klebsiella pneumoniae with the Capsular Polysaccharide Synthesis. Am Soc Microbiol. 2022; Available from: https://journals.asm.org/doi/abs/10.1128/spectrum.02400-22.
Horii T, Kobayashi M, Sato K, Ichiyama S, Ohta M. An in-vitro study of carbapenem-induced morphological changes and endotoxin release in clinical isolates of gram-negative bacilli. J Antimicrob Chemother. 1998;41:435–42.
Cylke C, Si F, Banerjee S. Effects of antibiotics on bacterial cell morphology and their physiological origins. Biochem Soc Trans. 2022;50:1269–79. https://portlandpress.com/biochemsoctrans/article/50/5/1269/231752/Effects-of-antibiotics-on-bacterial-cell.
Toprak E, Veres A, Michel J-B, Chait R, Hartl DL, Kishony R. Evolutionary paths to antibiotic resistance under dynamically sustained drug selection. Nat Genet. 2012;44:101–5.
Cusack TP, Ashley EA, Ling CL, Rattanavong S, Roberts T, Turner P, et al. Impact of CLSI and EUCAST breakpoint discrepancies on reporting of antimicrobial susceptibility and AMR surveillance. Clin Microbiol Infect. 2019;25:910–1. https://linkinghub.elsevier.com/retrieve/pii/S1198743X19301090.
Andrews JM. Determination of minimum inhibitory concentrations. J Antimicrob Chemother. 2001;48:5–16. http://academic.oup.com/jac/article/48/suppl_1/5/2473513/Determination-of-minimum-inhibitory-concentrations.
Shah PM. Parenteral carbapenems. Clin Microbiol Infect. 2008;14:175–80. https://linkinghub.elsevier.com/retrieve/pii/S1198743X14604900.
Reynoso EC, Laschi S, Palchetti I, Torres E. Advances in antimicrobial resistance monitoring using sensors and biosensors: a review. Chemosensors. 2021;9:232.
Spagnolo F, Rinaldi C, Sajorda DR, Dykhuizen DE. Evolution of resistance to continuously increasing streptomycin concentrations in populations of Escherichia coli. Antimicrob Agents Chemother. 2016;60:1336–42.
Schmid M, Steiner O, Fasshold L, Goessler W, Holl A-M, Kühn K-D. The stability of carbapenems before and after admixture to PMMA-cement used for replacement surgery caused by Gram-negative bacteria. Eur J Med Res. 2020;25:34. https://eurjmedres.biomedcentral.com/articles/10.1186/s40001-020-00428-z.
George AM, Levy SB. Amplifiable resistance to tetracycline, chloramphenicol, and other antibiotics in Escherichia coli: involvement of a non-plasmid-determined efflux of tetracycline. J Bacteriol. 1983;155:531–40.
Adam M, Murali B, Glenn NO, Potter SS. Epigenetic inheritance based evolution of antibiotic resistance in bacteria. BMC Evol Biol. 2008;8:52. https://bmcevolbiol.biomedcentral.com/articles/10.1186/1471-2148-8-52.
Nhung NT, Thuy CT, Trung NV, Campbell J, Baker S, Thwaites G, et al. Induction of antimicrobial resistance in Escherichia coli and non-typhoidal Salmonella strains after adaptation to disinfectant commonly used on farms in Vietnam. Antibiotics. 2015;4:480–94.
Nozaki U, Kawashima F, Imada A. C-19393 S2 AND H2, New carbapenem antibiotics. J Antibiot. 1981;34:206–11.
Bernabeu-Wittel M, García-Curiel A, Pichardo C, Pachon-Ibanez ME, Jimenez-Mejias ME, Pachón J. Morphological changes induced by imipenem and meropenem at sub-inhibitory concentrations in Acinetobacter baumannii. Clin Microbiol Infect. 2004;10:931–4.
Cross T, Ransegnola B, Shin JH, Weaver A, Fauntleroy K, VanNieuwenhze MS, Westblade LF, Dörr T. Spheroplast-mediated carbapenem tolerance in Gram-negative pathogens. Antimicrob Agents Chemother. 2019;63:e00756–19.
Toyofuku M, Nomura N, Eberl L. Types and origins of bacterial membrane vesicles. Nat Rev Microbiol. 2019;17:13–24.
Wojnicz D, Kłak M, Adamski R, Jankowski S. Influence of subinhibitory concentrations of amikacin and ciprofloxacin on morphology and adherence ability of uropathogenic strains. Folia Microbiol. 2007;52:429–36.
Shen JP, Chou CF. Morphological plasticity of bacteria-Open questions. Biomicrofluidics. 2016;10:031501 http://aip.scitation.org/doi/10.1063/1.4953660.
Yang DC, Blair KM, Salama NR. Staying in shape: the impact of cell shape on bacterial survival in diverse environments. Microbiol Mol Biol Rev. 2016;80:187–203.
Young KD. Bacterial morphology: why have different shapes? Curr Opin Microbiol. 2007;10:596–600.
Sandoval‐Motta S, Aldana M. Adaptive resistance to antibiotics in bacteria: a systems biology perspective. Wiley Interdiscip Rev Syst Biol Med. 2016;8:253–67.
Schaechter M, Maaløe O, Kjeldgaard NO. Dependency on medium and temperature of cell size and chemical composition during balanced growth of Salmonella Typhimurium. Microbiology. 1958;19:592–606.
Young KD. Bacterial shape: two-dimensional questions and possibilities. Annu Rev Microbiol. 2010;64:223–40.
Amir A. Cell size regulation in bacteria. Phys Rev Lett. 2014;112:208102. https://journals.aps.org/prl/abstract/10.1103/PhysRevLett.112.208102.
Harris LK, Theriot JA. Surface area to volume ratio: a natural variable for bacterial morphogenesis. Trends Microbiol. 2018;26:815–32. https://linkinghub.elsevier.com/retrieve/pii/S0966842X18301021.
Ojkic N, Serbanescu D, Banerjee S. Surface-to-volume scaling and aspect ratio preservation in rod-shaped bacteria. Elife [Internet]. 2019;8:e47033. https://elifesciences.org/articles/47033.
Si F, Li D, Cox SE, Sauls JT, Azizi O, Sou C, et al. Invariance of initiation mass and predictability of cell size in Escherichia coli. Curr Biol. 2017;27:1278–87.
Otero F, Santiso R, Tamayo M, Ferna L, Lepe A, Mcconnell MJ, et al. Rapid detection of antibiotic resistance in gram-negative bacteria through assessment. Micro Drug Resist. 2017;23:157–62.
Longo G, Trampuz MarquesL, Dietler A, Bizzini G, Kasas A. S. Antibiotic-induced modi fi cations of the stiffness of bacterial membranes. J Microbiol Methods. 2013;93:80–4. https://doi.org/10.1016/j.mimet.2013.01.022.
Syal K, Mo M, Yu H, Iriya R, **g W, Guodong S, et al. Current and emerging techniques for antibiotic susceptibility tests. Theranostics. 2017;7:1795–805.
Khan ZA, Siddiqui MF, Park S. Current and emerging methods of antibiotic susceptibility testing. Diagnostics. 2019;9:49. https://www.mdpi.com/2075-4418/9/2/49.
Soon RL, Nation RL, Cockram S, Moffatt JH, Harper M, Adler B, et al. Different surface charge of colistin-susceptible and -resistant Acinetobacter baumannii cells measured with zeta potential as a function of growth phase and colistin treatment. J Antimicrob Chemother. 2011;66:126–33.
Domingues MM, Silva PM, Franquelim HG, Carvalho FA, Castanho MARB, Santos NC. Antimicrobial protein rBPI21-induced surface changes on Gram-negative and Gram-positive bacteria. Nanomed Nanotechnol, Biol Med. 2014;10:543–51. https://doi.org/10.1016/j.nano.2013.11.002.
Gan SK-E, Phua S-X, Yeo JY. Sagacious epitope selection for vaccines, and both antibody-based therapeutics and diagnostics: tips from virology and oncology. Antib Ther. 2022;5:63–72. https://academic.oup.com/abt/article/5/1/63/6533537.
Alkhudhairy MK, Alshadeedi SMJ, Mahmood SS, Al-Bustan SA, Ghasemian A. Comparison of adhesin genes expression among Klebsiella oxytoca ESBL-non-producers in planktonic and biofilm mode of growth, and imipenem sublethal exposure. Micro Pathog. 2019;134:103558. https://linkinghub.elsevier.com/retrieve/pii/S0882401018304844.
Dhabaan GN, AbuBakar S, Cerqueira GM, Al-Haroni M, Pang SP, Hassan H. Imipenem treatment induces expression of important genes and phenotypes in a resistant Acinetobacter baumannii Isolate. Antimicrob Agents Chemother. 2016;60:1370–6. https://journals.asm.org/doi/10.1128/AAC.01696-15.
Fajardo-Lubián A, Ben Zakour NL, Agyekum A, Qi Q, Iredell JR. Host adaptation and convergent evolution increases antibiotic resistance without loss of virulence in a major human pathogen. PLOS Pathog. 2019;15:e1007218. Available from: https://dx.plos.org/10.1371/journal.ppat.1007218.
Pajerski W, Ochonska D, Brzychczy-Wloch M, Indyka P, Jarosz M, Golda-Cepa M, et al. Attachment efficiency of gold nanoparticles by Gram-positive and Gram-negative bacterial strains governed by surface charges. J Nanoparticle Res. 2019;21:1–12.
Dester E, Alocilja E. Current methods for extraction and concentration of foodborne bacteria with glycan-coated magnetic nanoparticles: a review. Biosensing. 2022;12:112. https://www.mdpi.com/2079-6374/12/2/112.
Bohara RA, Pawar SH. Innovative developments in bacterial detection with magnetic nanoparticles. Appl Biochem Biotechnol. 2015;176:1044–58.
Caliskan-Aydogan O, Sharief SA, Alocilja EC. Rapid isolation of low-level carbapenem-resistant. E coli Water Foods Using Glycan-Coat Magn Nanopart Biosens. 2023;13:902. https://www.mdpi.com/2079-6374/13/10/902.
Acknowledgements
Research related to this article was supported by the Targeted Support Grant for Technology Development (TSGTD), Michigan State University Foundation; the USDA Hatch MICL 02782; USDA Hatch Multistate NC1194 MICL 04233 (RA101064) and the USDA-NIFA project 2022-67017-36982. Further, the Turkish Ministry of Education sponsored O. Caliskan‐Aydogan’s Ph.D. program at Michigan State University. Furthermore, we want to thank Dr. Alicia Withrow for assisting in taking TEM images and Dr. Shannon Manning and Dr. Saad A. Sharief for many helpful discussions and communication of the research results.
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Oznur Caliskan-Aydogan: Conceptualization, Methodology, Data curation, Investigation, Roles/Writing - original draft, and Writing - review & editing. Chloe Zaborney Kline: Investigation, Visualization, Methodology, Evangelyn C. Alocilja: Conceptualization, Methodology Resources, Funding acquisition, Project administration, Writing - review & editing, and Supervision.
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Caliskan-Aydogan, O., Zaborney Kline, C. & Alocilja, E.C. Cell morphology as biomarker of carbapenem exposure. J Antibiot (2024). https://doi.org/10.1038/s41429-024-00749-9
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DOI: https://doi.org/10.1038/s41429-024-00749-9
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