Adaptive laboratory evolution of Salmonella enterica in acid stress

Abstract

IntroductionAdaptive laboratory evolution (ALE) studies play a crucial role in understanding the adaptation and evolution of different bacterial species. In this study, we have investigated the adaptation and evolution of Salmonella enterica serovar Enteritidis to acetic acid using ALE.Materials and methodsAcetic acid concentrations below the minimum inhibitory concentration (sub-MIC) were used. Four evolutionary lineages (EL), namely, EL1, EL2, EL3, and EL4, of S. Enteritidis were developed, each demonstrating varying levels of resistance to acetic acid.ResultsThe acetic acid MIC of EL1 remained constant at 27 mM throughout 70 days, while the MIC of EL2, EL3, and EL4 increased throughout the 70 days. EL4 was adapted to the highest concentration of acetic acid (30 mM) and demonstrated the highest increase in its MIC against acetic acid throughout the study, reaching an MIC of 35 mM on day 70. The growth rates of the evolved lineages increased over time and were dependent on the concentration of acetic acid used during the evolutionary process. EL4 had the greatest increase in growth rate, reaching 0.33 (h−1) after 70 days in the presence of 30 mM acetic acid as compared to EL1, which had a growth rate of 0.2 (h−1) after 70 days with no exposure to acetic acid. Long-term exposure to acetic acid led to an increased MIC of human antibiotics such as ciprofloxacin and meropenem against the S. enterica evolutionary lineages. The MIC of ciprofloxacin for EL1 stayed constant at 0.016 throughout the 70 days while that of EL4 increased to 0.047. Bacterial whole genome sequencing revealed single-nucleotide polymorphisms in the ELs in various genes known to be involved in S. enterica virulence, pathogenesis, and stress response including phoP, phoQ, and fhuA. We also observed genome deletions in some of the ELs as compared to the wild-type S. Enteritidis which may have contributed to the bacterial acid adaptation.DiscussionThis study highlights the potential for bacterial adaptation and evolution under environmental stress and underscores the importance of understanding the development of cross resistance to antibiotics in S. enterica populations. This study serves to enhance our understanding of the pathogenicity and survival strategies of S. enterica under acetic acid stress.

Transcriptomic analysis using RNA sequencing and phenotypic analysis of Salmonella enterica after acid exposure for different time durations using adaptive laboratory evolution

“…Adaptive laboratory evolution of S . Enteritidis in acid stress was performed by continuing the method described in our previous study with some additions (Ghoshal et al, 2023 ). Briefly, at the beginning of the ALE study, MIC of WT (wild type) S .…”

“…The MIC of the antibiotics meropenem, ciprofloxacin, gentamycin, and streptomycin were determined using MTS strips (MIC test strips, Liofilchem) (Matuschek et al, 2018 ; van den Bijllaardt et al, 2018 ). The test strips for each antibiotic had a concentration range of 0.016–256 μg/mL, and MIC was determined using the method previously described (Ghoshal et al, 2023 ). Briefly, overnight cultures of the various evolutionary lineages were cultivated using their respective acetic acid concentrations.…”

“…Genomic analysis of EL5 from ALE day 90 was performed using the methods from our previous study (Ghoshal et al, 2023 ). The genomes of EL5 on ALE day 90 and EL4 on ALE day 70 were mapped to that of the WT genome.…”

“…Enteritidis during prolonged exposure to acetic acid. ALE led to the generation of four acid-evolved lineages (EL) in our previous study, which were named EL1-EL4 (Ghoshal et al, 2023 ). We studied the changes in the growth rates of the ELs after prolonged and continuous exposure to acetic acid.…”

“…This study applies a multi-pronged approach, where we analyzed the stability of the phenotypic changes observed in the previous study (Ghoshal et al, 2023 ) after the removal and subsequent reintroduction of the acid stress. The ALE process was continued in EL4, and we worked on the development of another evolutionary lineage, EL5.…”