Project description:Streptococcus agalactiae is among the few pathogens that have not developed resistance to ß-lactam antibiotics despite decades of clinical use. The molecular basis of this long-lasting susceptibility has not been investigated, and it is uncertain whether specific mechanisms constraint the emergence of resistance. In this study, we first report a conserved role of the signaling nucleotide cyclic-di-AMP in the sensitivity of S. agalactiae to ß-lactam. Specifically, we demonstrate that inactivation of the phosphodiesterase GdpP confers ß-lactam tolerance. Characterizing the signaling pathway revealed an antagonistic regulation by the transcriptional factor BusR, which is activated by c-di-AMP and negatively regulates ß-lactam susceptibility. Furthermore, we show that simultaneous inhibition of osmolyte transporters activity and transcription by c-di-AMP has an additive effect, sustaining ß-lactam tolerance. Finally, transposon mutagenesis for ß-lactam reduced susceptibility reveals a convergent pattern of mutations, including in the KhpAB small RNA chaperone and the protein S immunomodulator. Overall, our findings suggest mechanisms that may foster antibiotic resistance in S. agalactiae and demonstrate that c-di-AMP acts as a turgor pressure rheostat, coordinating an integrated response to cell wall weakening due to ß-lactam activity.
Project description:P. aeruginosa isolates were grown in LB broth media. The bacterial media was then digested after incubation for 24 hours and analyzed to identify bacterial proteins related to beta-lactam drug resistance. Bottom-up proteomics analysis was performed.
Project description:Non-typeable Haemophilus influenzae (NTHi) is a common acute otitis media pathogen, with an incidence that is increased by previous antibiotic treatment. NTHi is also an emerging causative agent of other chronic infections in humans, some linked to morbidity, and all of which impose substantial treatment costs. In this study we explore the possibility that antibiotic exposure may stimulate biofilm formation by NTHi bacteria. We discovered that sub-inhibitory concentrations of beta-lactam antibiotic (i.e., amounts that partially inhibit bacterial growth) stimulated the biofilm-forming ability of NTHi strains, an effect that was strain and antibiotic dependent. When exposed to sub-inhibitory concentrations of beta-lactam antibiotics NTHi strains produced tightly packed biofilms with decreased numbers of culturable bacteria but increased biomass. The ratio of protein per unit weight of biofilm decreased as a result of antibiotic exposure. Antibiotic-stimulated biofilms had altered ultrastructure, and genes involved in glycogen production and transporter function were up regulated in response to antibiotic exposure. Down-regulated genes were linked to multiple metabolic processes but not those involved in stress response. Antibiotic-stimulated biofilm bacteria were more resistant to a lethal dose (10M-BM-5g/mL) of cefuroxime. Our results suggest that beta-lactam antibiotic exposure may act as a signaling molecule that promotes transformation into the biofilm phenotype. Loss of viable bacteria, increase in biofilm biomass and decreased protein production coupled with a concomitant up-regulation of genes involved with glycogen production might result in a biofilm of sessile, metabolically inactive bacteria sustained by stored glycogen. These biofilms may protect surviving bacteria from subsequent antibiotic challenges, and act as a reservoir of viable bacteria once antibiotic exposure has ended. 12 samples
Project description:Variations in daptomycin-beta-lactam synergy and enterococcal species - Daptomycin-ß-lactam synergy depends on enterococcal species and specific mutation
Project description:Non-typeable Haemophilus influenzae (NTHi) is a common acute otitis media pathogen, with an incidence that is increased by previous antibiotic treatment. NTHi is also an emerging causative agent of other chronic infections in humans, some linked to morbidity, and all of which impose substantial treatment costs. In this study we explore the possibility that antibiotic exposure may stimulate biofilm formation by NTHi bacteria. We discovered that sub-inhibitory concentrations of beta-lactam antibiotic (i.e., amounts that partially inhibit bacterial growth) stimulated the biofilm-forming ability of NTHi strains, an effect that was strain and antibiotic dependent. When exposed to sub-inhibitory concentrations of beta-lactam antibiotics NTHi strains produced tightly packed biofilms with decreased numbers of culturable bacteria but increased biomass. The ratio of protein per unit weight of biofilm decreased as a result of antibiotic exposure. Antibiotic-stimulated biofilms had altered ultrastructure, and genes involved in glycogen production and transporter function were up regulated in response to antibiotic exposure. Down-regulated genes were linked to multiple metabolic processes but not those involved in stress response. Antibiotic-stimulated biofilm bacteria were more resistant to a lethal dose (10µg/mL) of cefuroxime. Our results suggest that beta-lactam antibiotic exposure may act as a signaling molecule that promotes transformation into the biofilm phenotype. Loss of viable bacteria, increase in biofilm biomass and decreased protein production coupled with a concomitant up-regulation of genes involved with glycogen production might result in a biofilm of sessile, metabolically inactive bacteria sustained by stored glycogen. These biofilms may protect surviving bacteria from subsequent antibiotic challenges, and act as a reservoir of viable bacteria once antibiotic exposure has ended.
Project description:Activity-based protein profiling data from Mycobacterium tuberculosis (Mtb) H37Rv grown under replicating and carbon-starvation conditions with beta lactam (meropenem) probe labeling or no probe control (npc). Proteins were obtained from Mtb cultures lysed by bead beating, digested with trypsin, and then analyzed by LC-MS/MS. Data was searched with MS-GF+ using PNNL's DMS processing pipeline.