Effects of osmotic stress on Listeria monocytogenes ATCC 7644: persistent cells and heat resistance

Submitted: 15 September 2022
Accepted: 12 January 2023
Published: 8 March 2023
Abstract Views: 1481
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Persistent bacteria are a microbial subpopulation that, exposed to bactericidal treatment, is killed at a slower rate than the rest of the population they are part of. They can be triggered either following stress or stochastically without external signals. The hallmark of persistent bacteria is the biphasic killing curve, a sign that, within a microbial population, two subpopulations are inactivated at a different rate. Furthermore, when plated into a fresh medium and in the absence of stressors, persistent bacteria typically remain in the lag phase longer before resuming active replication. This study aims to evaluate in vitro whether the formation of persistent cells in a strain of Listeria monocytogenes can be triggered by exposure to osmotic stress and if this phenomenon can increase heat resistance in the bacterial population. In a first experiment, the lag time distribution of a L. monocytogenes strain grown in a 6% NaCl broth was evaluated using the software ScanLag. A stationary phase broth culture was inoculated on agar plates placed on an office scanner inside an incubator at 37°C. The plates were scanned every 20’ for 4 days and the acquired images were automatically elaborated with the aid of MatLab software in order to evaluate the appearance times of every single colony. The experiment was also carried out on a control culture obtained by growing the strain in the broth without salt. In a second experiment, the same broth cultures, after proper dilutions to rebalance NaCl concentration, were subjected to a heat treatment at 51°C and the death curves obtained were parameterized using the GinaFit system. Results showed that the lag phase of 31.40% of the salt culture colonies was long enough to suppose the formation of persistent bacteria. Analyses of the thermal survival curves showed that the shoulder and tail model was the one that best represented the inactivation trend of the salt culture, unlike the control culture, whose trend was essentially linear. Results of the present study show how exposure to salt could induce the formation of persistent bacteria in a L. monocytogenes strain. The last raises concerns as persistent cells may not only be undetected with common analytical techniques but they even show a greater heat resistance.



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Al-Nabulsi AA, Osaili TM, Shaker RR, Olaimat AN, Jaradat ZW, Elabedeen NAZ, Holley RA, 2015. Effects of osmotic pressure, acid, or cold stresses on antibiotic susceptibility of Listeria monocytogenes. Food Microbiol 46:154-60. DOI: https://doi.org/10.1016/j.fm.2014.07.015
Aryani DC, Den Besten HMW, Hazeleger WC, Zwietering MH, 2015. Quantifying variability on thermal resistance of Listeria monocytogenes. Int J Food Microbiol 193:130-138. DOI: https://doi.org/10.1016/j.ijfoodmicro.2014.10.021
Balaban NQ, Helaine S, Lewis K, Ackermann M, Aldridge B, Andersson DI, Brynildsen MP, Bumann D, Camilli A, Collins JJ, Dehio C, Fortune S, Ghigo J, Hardt W, Harms A, Heinemann M, Hung DT, Jenal U, Levin BR, Michiels J, Storz G, Tan M, Tenson T, Van Melderen L, Zinkernagel, A, 2019. Definitions and guidelines for research on antibiotic persistence. Nat Rev Microbiol 17:441- 8. DOI: https://doi.org/10.1038/s41579-019-0196-3
Baranyi J, Jones A, Walker C, Kaloti A, Robinson TP, Mackey BM, 1996. A combined model for growth and subsequent thermal inactivation of Brochothrix thermosphacta. Appl Environ Microbiol 62:1029-35. DOI: https://doi.org/10.1128/aem.62.3.1029-1035.1996
Blair J, Webber MA, Baylay AJ, Ogbolu DO, Piddock LJ, 2015. Molecular mechanisms of antibiotic resistance. Nat Rev Microbiol 13:42-51. DOI: https://doi.org/10.1038/nrmicro3380
Brauner A, Fridman O, Gefen O, Balaban NQ, 2016. Distinguishing between resistance, tolerance and persistence to antibiotic treatment. Nat Rev Microbiol 14:320-30. DOI: https://doi.org/10.1038/nrmicro.2016.34
Cerf O, 1977. A review. Tailing of survival curves of bacterial spores. J Appl Microbiol 42:1-19. DOI: https://doi.org/10.1111/j.1365-2672.1977.tb00665.x
Doyle ME, Mazzotta AS, Wang TIM, Wiseman DW, Scott VN, 2001. Heat resistance of Listeria monocytogenes. J Food Prot 64:410-29. DOI: https://doi.org/10.4315/0362-028X-64.3.410
Ed-Dra A, Filali FR, Presti VL, Zekkori B, Nalbone L, Bouymajane A, Giuffrida A, Giarratana F, 2020. Chemical composition, antioxidant capacity and antibacterial action of five Moroccan essential oils against Listeria monocytogenes and different serotypes of Salmonella enterica. Microb Pathog 149:104510. DOI: https://doi.org/10.1016/j.micpath.2020.104510
Ed-Dra A, Filali FR, Presti VL, Zekkori B, Nalbone L, Elsharkawy ER, Bentayeb A, Giarratana, F, 2021. Effectiveness of essential oil from the Artemisia herba-alba aerial parts against multidrug-resistant bacteria isolated from food and hospitalized patients. Biodiversitas J Biol Diver 22:2995-3005. DOI: https://doi.org/10.13057/biodiv/d220753
Fang T, Wu Y, Xie Y, Sun L, Qin X, Liu Y, Li H, Dong Q, Wang X, 2021. Inactivation and Subsequent Growth Kinetics of Listeria monocytogenes After Various Mild Bactericidal Treatments. Front Microbiol 12:646735. DOI: https://doi.org/10.3389/fmicb.2021.646735
Geeraerd AH, Herremans CH, Van Impe JF, 2000. Structural model requirements to describe microbial inactivation during a mild heat treatment. Int J Food Microbiol 59:185-209. DOI: https://doi.org/10.1016/S0168-1605(00)00362-7
Geeraerd AH, Valdramidis VP, Van Impe JF, 2005. GInaFiT, a freeware tool to assess non-log-linear microbial survivor curves. Int J Food Microbiol 102:95-105. DOI: https://doi.org/10.1016/j.ijfoodmicro.2004.11.038
Gefen O, Balaban NQ, 2009. The impor- tance of being persistent: heterogeneity of bacterial populations under antibiotic stress. FEMS Microbiol Reviews 33:704-17. DOI: https://doi.org/10.1111/j.1574-6976.2008.00156.x
Giarratana F, Nalbone L, Ziino G, Donato G, Marotta SM, Lamberta F, Giuffrida A. 2022. Temperature fluctuations along food supply chain: A dynamic and stochastic predictive approach to establish the best temperature value in challenge tests for Listeria monocytogenes. Ital J Food Safety 11:9981. DOI: https://doi.org/10.4081/ijfs.2022.9981
Guillier L, Pardon P, Augustin JC, 2005. Influence of stress on individual lag time distributions of Listeria monocytogenes. Appl Environ Microbiol 71:2940-8. DOI: https://doi.org/10.1128/AEM.71.6.2940-2948.2005
ISO, 2017. Microbiology of the food chain- Horizontal method for the detection and enumeration of Listeria monocytogenes and of Listeria spp.-Part 2: Enumeration method. ISO norm 11290-2:2017. International Standardization Organization ed., Geneva, Switzerland.
Kaplan Y, Reich S, Oster E, Maoz S, Levin-Reisman I, Ronin I, Gefen O, Agam O, Balaban NQ, 2021. Observation of universal ageing dynamics in antibiotic persistence. Nature 600:290-4. DOI: https://doi.org/10.1038/s41586-021-04114-w
Kilstrup M, Jacobsen S, Hammer K, Vogensen FK, 1997. Induction of heat shock proteins DnaK, GroEL, and GroES by salt stress in Lactococcus lactis. Appl Environ Microbiol 63:1826-37. DOI: https://doi.org/10.1128/aem.63.5.1826-1837.1997
Kowalska-Krochmal B, Dudek-Wicher R, 2021. The minimun inhibitory concentration of antibiotics: Methods, interpretation, clinical relevance. Pathogens 10:165. DOI: https://doi.org/10.3390/pathogens10020165
Levin-Reisman I, Balaban NQ, 2016. Quantitative measurements of type I and type II persisters using ScanLag. In: Michiels J, Fauvart M, eds. Bacterial Persistence. Humana Press 75-81. DOI: https://doi.org/10.1007/978-1-4939-2854-5_7
Levin-Reisman I, Gefen O, Fridman O, Ronin I, Shwa D, Sheftel H, Balaban NQ, 2010. Automated imaging with ScanLag reveals previously undetectable bacterial growth phenotypes. Nat Methods 7:737-9. DOI: https://doi.org/10.1038/nmeth.1485
Liu JF, Gefen O, Zhang ZY, Liu MM, Bar-Meir M, Balaban NQ, 2022. Interaction Tolerance Detection Test for Understanding the Killing Efficacy of Directional Antibiotic Combinations. ASM 13:e00004-22. DOI: https://doi.org/10.1128/mbio.00004-22
Mancuso G, Midiri A, Gerace E, Biondo C, 2021. Bacterial antibiotic resistance: the most critical pathogens. Pathogens 10:1310. DOI: https://doi.org/10.3390/pathogens10101310
Maria-Rosario A, Davidson I, Debra M, Verheul A, Abee T, Booth IR, 1995. The role of peptide metabolism in the growth of Listeria monocytogenes ATCC 23074 at high osmolarity. Microbiol 141:41-9. DOI: https://doi.org/10.1099/00221287-141-1-41
Reygaert WC, 2018. An overview of the antimicrobial resistance mechanisms of bacteria. AIMS Microbiol 4:482. DOI: https://doi.org/10.3934/microbiol.2018.3.482
Robinson TP, Ocio MJ, Kaloti A, Mackey BM, 1998. The effect of the growth environment on the lag phase of Listeria monocytogenes. Int J Food Microbiol 44:83-92. DOI: https://doi.org/10.1016/S0168-1605(98)00120-2
Trabelsi N, Nalbone L, Di Rosa AR, Ed-Dra A, Nait-Mohamed S, Mhamdi R, Giuffrida A, Giarratana F, 2021. Marinated anchovies (Engraulis encrasicolus) prepared with flavored olive oils (Chétoui cv.): Anisakicidal effect, microbiological, and sensory evaluation. Sustainability 13:5310. DOI: https://doi.org/10.3390/su13095310
WHO, 2019. Listeriosis– Spain, 2019. Available from: https://www. who. int/ csr/ don/ 16- september- 2019- listerio- sis- spain/ en/
Wu S, Yu PL, Flint S, 2017. Persister cell formation of Listeria monocytogenes in response to natural antimicrobial agent nisin. Food Control 77:243-50. DOI: https://doi.org/10.1016/j.foodcont.2017.02.011

How to Cite

Nalbone L, Sorrentino G, Giarratana F, Schiopu-Mariean A, Ziino G, Giuffrida A. Effects of osmotic stress on <em>Listeria monocytogenes</em> ATCC 7644: persistent cells and heat resistance. Ital J Food Safety [Internet]. 2023 Mar. 8 [cited 2024 Jul. 23];12(1). Available from: https://www.pagepressjournals.org/ijfs/article/view/10880