DOI: 10.5061/dryad.4qrfj6qks
title: The impact of antibiotic induction on virulence and antibiotic resistance in Klebsiella pneumoniae

description: Background: Klebsiella pneumoniae is an opportunistic
 pathogen causing nosocomial infections, classified into
 carbapenem-sensitive and carbapenem-resistant strains. Understanding the
 virulence factors and antibiotic resistance of these strains is
 essential for effective clinical management. Objective: This study
 compared the virulence genes and antibiotic resistance profiles of 50
 carbapenem-sensitive Klebsiella pneumoniae (CSKP)
 strains and 50 carbapenem-resistant Klebsiella
 pneumoniae (CRKP) strains, examining their expression under antibiotic
 pressure and the mechanisms contributing to their pathogenicity. Methods:
 Virulence genes (rmpA, rmpA2, iucA, iutA, Peg-344, ybts, iroB) were
 detected in both strains using polymerase chain reaction (PCR). Antibiotic
 susceptibility testing established minimum inhibitory concentrations
 (MICs) for key antibiotics. Gene expression analysis was performed with
 quantitative reverse transcription PCR (qRT-PCR) after 10 days of
 antibiotic exposure. Results: CSKP strains exhibited significantly higher
 positivity rates for virulence genes compared to CRKP strains. CRKP
 strains predominantly expressed resistance genes KPC, SHV, and CTX-M3,
 whereas no resistance genes were found in CSKP. Antibiotic susceptibility
 tests showed increased MICs, particularly for ciprofloxacin and imipenem,
 following antibiotic induction. CSKP demonstrated elevated expression of
 rmpA and rmpA2, while CRKP showed increased expression of SHV,
 and KPC after antibiotic exposure. Conclusion: This study
 highlights the intricate relationship between virulence and resistance
 in Klebsiella pneumoniae. CSKP strains show strong virulence
 factor expression, while CRKP strains adapt to antibiotic pressure through
 altered gene expression patterns. These findings underscore the urgent
 need for continuous surveillance and innovative therapeutic strategies to
 combat multidrug-resistant Klebsiella pneumoniae infections
description: We performed conventional PCR for virulence and resistance
 genes in bacterial strains. DNA was extracted by cell lysis in buffer and
 heating (100 °C, 10 min). After centrifugation (12,000 rpm, 5 min), the
 supernatant with ~100 ng/µL DNA was collected. PCR
 (25 µL) had 2.0 µL DNA, 1.0 µL each of primers, 12.5 µL 2X Taq MasterMix,
 and 8.5 µL water. Thermal cycling: 94°C 3 min, 30 cycles (94°C 30 s, 55°C
 30 s, 72°C 1 min), and 72°C 10 min. Agarose gel electrophoresis analyzed
 amplified products. Eight carbapenem - sensitive and - resistant strains
 were selected. Antibiotic Induction
 Experiment<br>Eight carbapenem-resistant Klebsiella pneumoniae
 (CRKP) strains (with resistance and virulence genes) and eight
 carbapenem-sensitive Klebsiella pneumoniae (CSKP) strains (same virulence
 genes) strains were chosen. Strains were inoculated into LB broth and
 incubated at 37°C to logarithmic phase. The antibiotic - induction
 protocol was adjusted (Wistrand - Yuen et al., 2018). Induction: Day 1,
 1/2 MIC; Day 2, 1 MIC; Day 3, 2 MIC, increasing for 10 days to 512 MIC or
 64 μg/mL (if initial MIC &gt; 64 μg/mL). Daily treatment with
 antibiotics to assess susceptibility and resistance changes.
  Real - Time Quantitative PCR (qPCR) Analysis<br>Three
 time points (baseline, Day 4, Day 7, Day 10) were selected after
 antibiotic - induction (Gaudin et al., 2013; Viers et al., 2014). Total
 RNA was isolated using a kit (Bioteke, Shanghai).
 After RNA extraction, reverse transcription generated cDNA.
 qPCR with SYBR Green Master Mix and primers measured gene expression
 before and after antibiotic exposure. qPCR protocol: 95°C 3 min, 40 cycles
 (95°C 3 s, 60°C 30 s). Primer sequences are in
 Supplementary Table 2. Each gene was analyzed in triplicate. The 16S rRNA
 gene was the internal control, and data were quantified by 2 - ΔΔCT
 method.
description: # The impact of antibiotic induction on virulence and antibiotic
 resistance in Klebsiella pneumoniae
 [https://doi.org/10.5061/dryad.4qrfj6qks
 ](https://doi.org/10.5061/dryad.4qrfj6qks) ## Description of the data and
 file structure ## 1. Introduction This README provides essential
 information about the data uploaded to Dryad. The data presented here is
 related to "a study on bacterial gene expression under antibiotic
 exposure". ## 2. Data Overview ### 2.1 Data Sources * **Bacterial
 Strains**: The data was obtained from the strains were retrieved from a
 preserved culture collection at the microbiology laboratory. *
 **Experimental Procedures**: Multiple experimental procedures were carried
 out, including conventional PCR, antibiotic induction experiments, and
 real - time quantitative PCR (qPCR). ### 2.2 Data Types * **Genetic
 Data**: * DNA sequences related to virulence and antimicrobial resistance
 genes were analyzed. The results of conventional PCR for detecting these
 genes are included in the dataset. * RNA data was also collected for qPCR
 analysis. After RNA extraction and reverse transcription to cDNA, the
 expression levels of virulence and resistance genes were measured. *
 Descriptions of data in each column are below: ROX: Roxithromycin ROXA:
 Roxithromycin acetylsalicylate AKN: Amikacin AMC: Amoxicillin - Clavulanic
 Acid CAZ: Ceftazidime CFP: Cefoperazone CRO: Ceftriaxone CXT: Cefuroxime
 Axetil FEP: Cefepime IMI: Imipenem LVX: Levofloxacin TSU: Tosufloxacin
 TZP: Piperacillin - Tazobactam ESBL_NM: Extended Spectrum Beta - Lactamase
 - Non - Metallic ETP: Ertapenem * Day 4 CIP: The strains induced by
 Ciprofloxacin for 4 days * Day 4 IPM: The strains induced by Imipenem for
 4 days * Day 4 PB: The strains induced by Polymyxin B for 4 days * Day 4
 CAZ-AVP: The strains induced by Ceftazidime - Avibactam for 4 days * Day 7
 CIP: The strains induced by Ciprofloxacin for 7 days * Day 7 IPM: The
 strains induced by Imipenem for 7 days * Day 7 PB: The strains induced by
 Polymyxin B for 7 days * Day 7 CAZ-AVP: The strains induced by Ceftazidime
 - Avibactam for 7 days * Day 10 CIP: The strains induced by Ciprofloxacin
 for 10 days * Day 10 IPM: The strains induced by Imipenem for 10 days *
 Day 10 PB: The strains induced by Polymyxin B for 10 days * Day 10
 CAZ-AVP: The strains induced by Ceftazidime - Avibactam for 10 days *
 **Experimental Metadata**: * Information about the bacterial strains, such
 as whether they are carbapenem - sensitive or - resistant, and their
 growth phases. * The experimental data include the presence or absence of
 virulence gene and drug resistance gene detection, the minimum inhibitory
 concentration (MIC, with the unit of μg/ml) values of bacteria at each
 stage, and the qPCR expression values of representative virulence genes
 and drug resistance genes at each stage. ## 3. File Structure *
 **`Antibiotic_induction` Directory**: * Files here document the data from
 the antibiotic induction experiment. * **`qPCR_data` Directory**: * Holds
 data related to qPCR analysis. This includes files with the Ct values for
 each gene analyzed in triplicate, as well as the calculated expression
 levels using the 2 - ΔΔCT method. The "+" and "-"
 simply represent whether the gene was detected or not. ## 4. Data Quality
 Assurance * **Experimental Repetition**: All key experiments (PCR,
 antibiotic induction, and qPCR) were repeated multiple times to ensure the
 reliability of the results. For example, each gene was analyzed in
 triplicate during qPCR. * **Quality Control in Experimental Procedures**:
 * In DNA extraction, the concentration of the obtained DNA was measured
 and verified to be approximately 100 ng/µL. * For PCR and qPCR, standard
 protocols were followed, and positive and negative controls were included
 in each experiment to check for false - positive and false - negative
 results. ## 5. Contact Information If you have any questions or need
 further clarification regarding the data, please contact
 [keda419@163.com](mailto:keda419@163.com). ### Files and variables ####
 File: data\_1498779.xlsx **Description:** PCR MIC qPCR ## Code/software
 Excel can be used to view the file.