Ashfaque Hossain1, Mark D Reisbig, Nancy D Hanson. 1. Center for Research in Anti-Infectives and Biotechnology, Department of Medical Microbiology and Immunology, Creighton University School of Medicine, 2500 California Plaza, Omaha, NE 68178, USA.
Abstract
OBJECTIVES: In a previous study with a Salmonella typhimurium strain containing cloned ampC-ampR from Enterobacter cloacae, it was suggested that ampC expression must be kept at low levels by AmpR to maintain normal growth and virulent phenotype. The purpose of this study was to determine whether findings obtained with a laboratory model can be extended to a virulent clinical isolate of S. typhimurium expressing the plasmid-encoded bla(CMY-7). METHODS: Disc induction assays were carried out to investigate inducibility of bla(CMY-7). Primer extension and sequence analyses were carried out to map the transcriptional start site of bla(CMY-7) and determine the relative expression. Growth and invasion potential of Salmonella strains were monitored by optical density, viable counts and cell invasion assays. RESULTS: Sequence analysis confirmed the absence of ampR upstream of bla(CMY-7) therefore confirming the negative results observed using the disc induction assay. Primer extension analysis mapped the start site of bla(CMY-7) transcription within an ISEcp1-like element. The relative expression of bla(CMY-7) was approximately 965-fold higher than the expression of a wild-type Citrobacter freundii chromosomal ampC and approximately 4.1-fold higher than ampC expression from a derepressed mutant of C. freundii. Growth and the capacity to invade mammalian cells were not compromised for either the clinical isolate or the S. typhimurium transconjugant containing bla(CMY-7). However, a Salmonella transformant containing bla(CMY-7) exhibited a compromised phenotype with respect to growth and invasion of mammalian cells. CONCLUSION: These findings indicate that the biological cost of high-level AmpC production can be compensated by plasmid-encoded factors and not by regulating ampC expression.
OBJECTIVES: In a previous study with a Salmonella typhimurium strain containing cloned ampC-ampR from Enterobacter cloacae, it was suggested that ampC expression must be kept at low levels by AmpR to maintain normal growth and virulent phenotype. The purpose of this study was to determine whether findings obtained with a laboratory model can be extended to a virulent clinical isolate of S. typhimurium expressing the plasmid-encoded bla(CMY-7). METHODS: Disc induction assays were carried out to investigate inducibility of bla(CMY-7). Primer extension and sequence analyses were carried out to map the transcriptional start site of bla(CMY-7) and determine the relative expression. Growth and invasion potential of Salmonella strains were monitored by optical density, viable counts and cell invasion assays. RESULTS: Sequence analysis confirmed the absence of ampR upstream of bla(CMY-7) therefore confirming the negative results observed using the disc induction assay. Primer extension analysis mapped the start site of bla(CMY-7) transcription within an ISEcp1-like element. The relative expression of bla(CMY-7) was approximately 965-fold higher than the expression of a wild-type Citrobacter freundii chromosomal ampC and approximately 4.1-fold higher than ampC expression from a derepressed mutant of C. freundii. Growth and the capacity to invade mammalian cells were not compromised for either the clinical isolate or the S. typhimurium transconjugant containing bla(CMY-7). However, a Salmonella transformant containing bla(CMY-7) exhibited a compromised phenotype with respect to growth and invasion of mammalian cells. CONCLUSION: These findings indicate that the biological cost of high-level AmpC production can be compensated by plasmid-encoded factors and not by regulating ampC expression.
Authors: S D Kotsakis; L S Tzouvelekis; E Lebessi; A Doudoulakakis; T Bouli; E Tzelepi; V Miriagou Journal: Antimicrob Agents Chemother Date: 2015-02-17 Impact factor: 5.191
Authors: C James Manktelow; Elitsa Penkova; Lucy Scott; Andrew C Matthews; Ben Raymond Journal: Antimicrob Agents Chemother Date: 2020-09-21 Impact factor: 5.191