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Relative quantification of the recA gene for antibiotic susceptibility testing in response to ciprofloxacin for pathogens of concern

ABSTRACT: Antibiotic resistance (AR) is one of the greatest threats to global health and is associated with higher treatment costs, longer hospital stays, and increased mortality. Current gold standard antibiotic susceptibility tests (AST) are dependent on organism growth rates resulting in prolonged diagnostic answers for slow growing organisms. Changes in the cellular transcriptome can be instantaneous in the presence of stressors such as antibiotic pressure. Here, we demonstrate that relative quantification of the recA gene in response to breakpoint concentrations of ciprofloxacin is an indicator of pathogen susceptibly. For this purpose, we developed seven duplex RT-qPCR assays targeting the recA and 16S rRNA gene, as response and housekeeping genes respectively, for biothreat and ESKAPE pathogens. Surrogate biothreat agents Y. pestis and B. anthracis saw increases in relative recA gene expression, independent of growth rate after 15 minutes of exposure to ciprofloxacin with maximal expression seen after 60 minutes. Treatment with doxycycline also demonstrated an increase in relative recA fold changes compared to no treatment controls. Final evaluation of all seven duplex assays tested across 125 strains, including Tier 1 pathogens, from broth culture demonstrated an overall categorical agreement compared to gold standard microbroth dilution of 97.56% with major error (ME) rates of 1.59% and very major error (VME) rates of 3.23%. Testing on pathogen strains commonly associated with urinary tract infections in contrived clinical sample sets demonstrated an overall categorical agreement of 95.8% with a ME rate of 0.0% and VME rate of 7.69%. These data indicate that relative quantification of a single highly conserved gene accurately predicts susceptibility for multiple bacterial species in response to ciprofloxacin.

ORGANISM(S): Bacillus anthracis Yersinia pestis

PROVIDER: GSE241489 | GEO | 2024/02/07

REPOSITORIES: GEO

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Project description:Kaiser2014 - Salmonella persistence after ciprofloxacin treatment The model describes the bacterial tolerance to antibiotics. Using a mouse model for Salmonella diarrhea, the authors have found that bacterial persistence occurs in the presence of the antibiotic ciprofloxacin because Salmonella can exist in two different states. One, the fast-growing population that spreads in the host's tissues and the other, slow-growing "persister" population that hide out inside dendritic cells of the host's immune system and cannot be attacked by the antibiotics. However, this can be killed by adding agents that directly stimulate the host's immune defense. This model is described in the article: Cecum lymph node dendritic cells harbor slow-growing bacteria phenotypically tolerant to antibiotic treatment. Kaiser P, Regoes RR, Dolowschiak T, Wotzka SY, Lengefeld J, Slack E, Grant AJ, Ackermann M, Hardt WD. PLoS Biol. 2014 Feb 18;12(2):e1001793. Abstract: In vivo, antibiotics are often much less efficient than ex vivo and relapses can occur. The reasons for poor in vivo activity are still not completely understood. We have studied the fluoroquinolone antibiotic ciprofloxacin in an animal model for complicated Salmonellosis. High-dose ciprofloxacin treatment efficiently reduced pathogen loads in feces and most organs. However, the cecum draining lymph node (cLN), the gut tissue, and the spleen retained surviving bacteria. In cLN, approximately 10%-20% of the bacteria remained viable. These phenotypically tolerant bacteria lodged mostly within CD103⁺CX₃CR1⁻CD11c⁺ dendritic cells, remained genetically susceptible to ciprofloxacin, were sufficient to reinitiate infection after the end of the therapy, and displayed an extremely slow growth rate, as shown by mathematical analysis of infections with mixed inocula and segregative plasmid experiments. The slow growth was sufficient to explain recalcitrance to antibiotics treatment. Therefore, slow-growing antibiotic-tolerant bacteria lodged within dendritic cells can explain poor in vivo antibiotic activity and relapse. Administration of LPS or CpG, known elicitors of innate immune defense, reduced the loads of tolerant bacteria. Thus, manipulating innate immunity may augment the in vivo activity of antibiotics. This model is hosted on BioModels Database and identified by: MODEL1312170001. To cite BioModels Database, please use: BioModels Database: An enhanced, curated and annotated resource for published quantitative kinetic models. To the extent possible under law, all copyright and related or neighbouring rights to this encoded model have been dedicated to the public domain worldwide. Please refer to CC0 Public Domain Dedication for more information.

Project description:Background: Antibiotic resistance is an urgent threat to public health. Prior to the evolution of antibiotic resistance, bacteria frequently undergo response and tend to develop a state of adaption to the antibiotic. Ciprofloxacin is a broad-spectrum antibiotic by damaging DNA. With the widespread clinical application, the resistance of bacteria to ciprofloxacin continues to increase. This study aimed to investigate the transcriptome changes under the action of high concentration of ciprofloxacin in Escherichia coli. Results: We identified 773 up-regulated differentially expressed genes (DEGs) and 645 down-regulated DEGs in ciprofloxacin treated cells. Enriched biological pathways reflected the up-regulation of biological process such as DNA damage and repair system, toxin/antitoxin systems, formaldehyde detoxification system, peptide biosynthetic process and cellular protein metabolic process. With KEGG pathway analysis, up-regulated DEGs of kdsA and waa operon were associated with “LPS biosynthesis”. rfbABC operon was related to “streptomycin biosynthesis” and “polyketide sugar unit biosynthesis ”. Down-regulated DEGs of thrABC and fliL operons were associated with “flagellum-dependent cell motility” and “bacterial-type flagellum” in GO terms, and enriched into “biosynthesis of amino acids” and “flagellar assembly” in KEGG pathways. After treatment of ciprofloxacin, bacterial lipopolysacchride (LPS) release was increased by two times, and the mRNA expression level of LPS synthesis genes, waaB, waaP and waaG were elevated (P < 0.05). Conclusions: Characterization of the gene clusters by RNA-seq showed high dose of ciprofloxacin not only lead to damage of bacterial macromolecules and components, but also induce protective response against antibiotic action by up-regulating the SOS system, toxin/antitoxin system and formaldehyde detoxification system. Moreover, genes related to biosynthesis of LPS were also upregulated by the treatment indicating that ciprofloxacin can enhance the production of endotoxin on the level of transcription. These results demonstrated that transient exposure of high dose ciprofloxacin is double edged. Cautions should be taken when administering the high dose antibiotic treatment for infectious diseases.

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Relative quantification of the recA gene for antibiotic susceptibility testing in response to ciprofloxacin for pathogens of concern

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