title: Assessment of antibiotic resistance in Klebsiella pneumoniae exposed to sequential in vitro antibiotic treatments.
abstract: BACKGROUND
  Bacteria treated with different classes of antibiotics exhibit changes in susceptibility to successive antibiotic treatments. This study was designed to evaluate the influence of sequential antibiotic treatments on the development of antibiotic resistance in Klebsiella pneumoniae associated with β-lactamase and efflux pump activities.
  
  
  METHODS
  The antibiotic susceptibility, β-lactamase activity, and efflux activity were determined in K. pneumoniae grown at 37 °C by adding initial (0 h) and second antibiotics (8 or 12 h). Treatments include control (CON; no first and second antibiotic addition), no initial antibiotic addition followed by 1 MIC ciprofloxacin addition (CON-CIP), no initial antibiotic addition followed by 1 MIC meropenem addition (CON-MER), initial 1/4 MIC ciprofloxacin addition followed by no antibiotic addition (1/4CIP-CON), initial 1/4 MIC ciprofloxacin addition followed by 1 MIC ciprofloxacin addition (1/4CIP-CIP), and initial 1/4 MIC ciprofloxacin addition followed by 1 MIC meropenem addition (1/4CIP-MER).
  
  
  RESULTS
  Compared to the CON, the initial addition of 1/4 MIC ciprofloxacin inhibited the growth of K. pneumoniae throughout the incubation period. The ciprofloxacin treatments (CON-CIP and 1/4CIP-CIP) showed significant reduction in the number of K. pneumoniae cells compared to meropenem (CON-MER and 1/4CIP-MER). The 1/4CIP-CIP achieved a further 1 log reduction of K. pneumoniae, when compared to the 1/4CIP-CON and 1/CIP-MER. The increase in sensitivity of K. pneumoniae to cefotaxime, kanamycin, levofloxacin, nalidixic acid was observed for CON-CIP. Noticeable cross-resistance pattern was observed at the 1/4CIP-CIP, showing the increased resistance of K. pneumoniae to chloramphenicol, ciprofloxacin, kanamycin, levofloxacin, nalidixic acid norfloxacin, sulphamethoxazole/trimethoprim, and tetracycline. The levels of β-lactamase activities were estimated to be 8.4 μmol/min/ml for CON, 7.7 μmol/min/ml for 1/4CIP-CON and as low as 2.9 μmol/min/ml for CON-CIP. Compared to the absence of phenylalanine-arginine-β-naphthylamide (PAβN), the fluorescence intensity of EtBr was increased in K. pneumoniae cells treated at the CON, CON-CIP, and CON-MER in the presence of PAβN. However, the efflux pump activity remained in K. pneumoniae cells treated at the 1/CIP, 1/CIP-CIP, and 1/CIP-MER in the presence of PAβN.
  
  
  CONCLUSIONS
  The results suggest that the pre-exposed antibiotic history, treatment order, and concentrations influenced the development of multiple antibiotic resistant associated with β-lactamase and efflux pump activities. This study highlights the importance of antibiotic treatment conditions, which would be taken into consideration when new antibiotic strategy is designed to prevent antibiotic resistance.
authors.affiliation: Department of Medical Biomaterials Engineering, Kangwon National University, Chuncheon, Gangwon, 24341, South Korea.
authors.forename: Jeongjin
authors.initials: J
authors.lastname: Kim
authors.affiliation: Department of Medical Biomaterials Engineering, Kangwon National University, Chuncheon, Gangwon, 24341, South Korea.
authors.forename: Ara
authors.initials: A
authors.lastname: Jo
authors.affiliation: School of Science, Mae Fah Luang University, Chiang Rai, 57100, Thailand.
authors.forename: Ekachai
authors.initials: E
authors.lastname: Chukeatirote
authors.affiliation: Department of Medical Biomaterials Engineering, Kangwon National University, Chuncheon, Gangwon, 24341, South Korea. juheeahn@kangwon.ac.kr.|Institute of Bioscience and Biotechnology, Kangwon National University, Chuncheon, Gangwon, 24341, South Korea. juheeahn@kangwon.ac.kr.
authors.forename: Juhee
authors.initials: J
authors.lastname: Ahn
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000378:metabolism; D002939:Ciprofloxacin / Q000494:pharmacology; D024881:Drug Resistance, Bacterial; D006801:Humans; D007710:Klebsiella Infections / Q000382:microbiology; D007711:Klebsiella pneumoniae / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics; D064704:Levofloxacin / Q000494:pharmacology; D008826:Microbial Sensitivity Tests; D001618:beta-Lactamases / Q000378:metabolism
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D002939:Ciprofloxacin; D064704:Levofloxacin; D001618:beta-Lactamases
keywords: Antibiotic resistance; Ciprofloxacin; Efflux pump system; Meropenem; β-Lactamase