rpoB mutations conferring rifampicin-resistance affect growth, stress response and motility in Vibrio vulnificus

Abstract

Rifampicin is a broad-spectrum antibiotic that binds to the bacterial RNA polymerase (RNAP), compromising DNA transcription. Rifampicin resistance is common in several microorganisms and it is typically caused by point mutations in the gene encoding the β subunit of RNA polymerase, rpoB. Different rpoB mutations are responsible for various levels of rifampicin resistance and for a range of secondary effects. rpoB mutations conferring rifampicin resistance have been shown to be responsible for severe effects on transcription, cell fitness, bacterial stress response and virulence. Such effects have never been investigated in the marine pathogen Vibrio vulnificus , even though rifampicin-resistant strains of V. vulnificus have been isolated previously. Moreover, spontaneous rifampicin-resistant strains of V. vulnificus have an important role in conjugation and mutagenesis protocols, with poor consideration of the effects of rpoB mutations. In this work, effects on growth, stress response and virulence of V. vulnificus were investigated using a set of nine spontaneous rifampicin-resistant derivatives of V. vulnificus CMCP6. Three different mutations (Q513K, S522L and H526Y) were identified with varying incidence rates. These three mutant types each showed high resistance to rifampicin [minimal inhibitory concentration (MIC) >800 µg ml−1], but different secondary effects. The strains carrying the mutation H526Y had a growth advantage in rich medium but had severely reduced salt stress tolerance in the presence of high NaCl concentrations as well as a significant reduction in ethanol stress resistance. Strains possessing the S522L mutation had reduced growth rate and overall biomass accumulation in rich medium. Furthermore, investigation of virulence characteristics demonstrated that all the rifampicin-resistant strains showed compromised motility when compared with the wild-type, but no major effects on exoenzyme production were observed. These findings reveal a wide range of secondary effects of rpoB mutations and indicate that rifampicin resistance is not an appropriate selectable marker for studies that aim to investigate phenotypic behaviour in this organism.

TheVibrio vulnificusstressosome is dispensable in nutrient-replete conditions

“…In particular, reduced motility, ethanol survival and hyperosmotic stress tolerance were observed (Figure S2). The same effects were observed, to the same extent, in the rifampicin-resistant parental strain in line with previously published work (33). This is an indication that the construction of the stressosome mutant in a Rif R strain might not allow the observation of differences caused by the VvrsbRSTX deletion, where these overlap with pleiotropic effects associated with rifampicin resistance.…”

“…To avoid the use of a rifampicin-resistant strain of V. vulnificus and pleiotropic effects previously reported associated with such mutations (33) and observed also in this work to interfere with the phenotypic characterisation of the mutant, the construction of the stressosome knock-out mutant was performed in a wild-type background. To do so, we used for the first time a conjugation protocol with V. vulnificus and a DAP (diaminopimelic acid)-auxotrophic strain of E. coli .…”

Antimicrobial Blue Light for Prevention and Treatment of Highly Invasive Vibrio vulnificus Burn Infection in Mice

“…The current standard of care for V. vulnificus infections is systemic antibiotic treatment using tetracycline associated with ceftazidime or ciprofloxacin (Tang et al, 2002 ). However, with the increasing emergence of multidrug-resistant variants, controlling V. vulnificus infections is becoming more difficult (Al-Dulaimi et al, 2019 ; Cutugno et al, 2020 ).…”

Microbial Musings – December 2020

“…cholerae and the gastrointestinal pathogen V. vulnificus [3, 4]. In the first paper from Nabendu Chatterjee’s group at the National Institute of Cholera and Enteric Diseases, Kolkata, India [3], the authors further characterize the V.…”

“…vulnificus , a nasty bug with a high fatality rate in the USA. The study by Laura Cutugno (@LauraCutugno) from the group of Aoife Boyd, with Conor O’Byrne (@cpobyrne) at the University of Galway, Ireland, looked at the responses of the well-studied clinical strain CMCP6 when this is selected for rifampicin resistance [4]. This is known to occur clinically, although fortunately these bacteria are still sensitive to most antibiotics, and from isolating independent genetic lines with this phenotype, which results in mutations in rpoB , they were able to show a wide range of pleiotropic phenotypes, which varied considerably with the nature of the allele of rpoB , with only reduced motility being a common phenotype.…”