Persistence phenotype of adherent-invasive Escherichia coli in response to ciprofloxacin, revealing high-persistence strains

Abstract

Persister cells correspond to a bacteria subpopulation able to survive antibiotic treatments and are thought to contribute to chronicity and symptom relapse of chronic diseases. Crohns disease (CD) is a multifactorial chronic inflammatory condition of the gastrointestinal tract, and adherent-invasive Escherichia coli (AIEC) has emerged as a key contributor to its pathogenesis. AIEC can survive, replicate, and produce persister cells within macrophages; however, out of the LF82 reference strain, a lack of knowledge of the persistence phenotype and its variability among AIEC strains is lacking. Here, two AIEC reference strains were analyzed after ciprofloxacin challenge, a fluoroquinolone antibiotic common in CD treatments. Four clinical isolates with an AIEC phenotype and five different E. coli pathotypes were also analyzed. We investigated the roles of the resident antibiotic resistance plasmid and the stress response protein HtrA, along with macrophage-induced persister formation. We observed a broad variability of persister cell formation capability among AIEC strains. Remarkably, the reference NRG857c strain showed a threatening high-persistence phenotype, with persistence levels higher than LF82 (65-fold) and some AIEC clinical isolates. The resident antibiotic resistance plasmid and the stress protein HtrA were dispensable for the NRG857c persistence phenotype. Among E. coli pathotypes, only EPEC showed persistence levels comparable to NRG857c, while other pathotypes resembled the lower persistence levels of LF82. Macrophage internalization did not increase NRG857c persister fraction, opposite to the LF82 behavior. Overall, our findings show that variable persistence phenotypes are found among AIEC. Opposite to LF82, NRG857c is a high-persistence strain, and this characteristic is shared with one AIEC clinical isolated and the EPEC reference strain. Undoubtedly, bacterial persistence should be considered in CD antibiotic treatments, especially with the feasible presence of AIEC high-persistence strains. Author SummaryAdherent-invasive Escherichia coli (AIEC) are common bacteria found in Crohns disease patients and recognized to survive within macrophages. The reference strain LF82 is known to form persistent cells, an enigmatic subpopulation of cells able to survive antibiotic treatments and thought to contribute to chronicity and symptoms relapses of chronic diseases. However, out of LF82, there is a lack of knowledge on AIEC persister cell formation, as well as the genetic factors contributing to this phenotype. In this study, it was observed a that the persistence phenotype was variable among both AIEC strains and E. coli pathotypes. Unlike LF82, the reference strain NRG857c displayed inherently high-persistence levels, regardless of whether it carries its native antibiotic resistance plasmid or the htrA and hipA genes. Notably, stress conditions within macrophages did not trigger increased persister cell formation in NRG857c. This study highlights the importance to decipher the role of AIEC persisters in ongoing Crohns disease, symptom relapses, and responses to antibiotic treatment, especially given the potential existence of high-persistence AIEC strains.