Korean Journal of Microbiology (미생물학회지)

The Microbiological Society of Korea (한국미생물학회)
권호 표지
DOI QR코드

DOI QR Code

Characteristics of Klebsiella pneumoniae exposed to serial antibiotic treatments

항생제 노출에 따른 Klebsiella pneumoniae의 내성 특성

  • Jung, Lae-Seung (Department of Medical Biomaterials Engineering, Kangwon National University) ;
  • Jo, Ara (Department of Medical Biomaterials Engineering, Kangwon National University) ;
  • Kim, Jeongjin (Department of Medical Biomaterials Engineering, Kangwon National University) ;
  • Ahn, Juhee (Department of Medical Biomaterials Engineering, Kangwon National University)
  • 정래승 (강원대학교 의생명과학대학 생물의소재공학과) ;
  • 조아라 (강원대학교 의생명과학대학 생물의소재공학과) ;
  • 김정진 (강원대학교 의생명과학대학 생물의소재공학과) ;
  • 안주희 (강원대학교 의생명과학대학 생물의소재공학과)
  • Received : 2016.12.02
  • Accepted : 2016.12.26
  • Published : 2016.12.31
Download PDF
⟨ Previous Next ⟩

Abstract

The emergence of antibiotic-resistant bacteria has been increased and become a public health concern worldwide. Many bacterial infections can be sequentially treated with different types of antibiotics. Thus, this study was designed to evaluate the changes in survival, antibiotic susceptibility, mutant frequency, ${\beta}$-lactamase activity, biofilm formation, and gene expression in Klebsiella pneumoniae after exposure to sequential antibiotic treatments of ciprofloxacin and meropenem. Treatments include control (CON; no addition), 1/2 MIC ciprofloxacin addition (1/2CIP), 2 MIC ciprofloxacin addition (2CIP), initial 1/2 MIC ciprofloxacin addition followed by 1/2 MIC meropenem (8 h-incubation) and 2 MIC ciprofloxacin (16 h-incubation) (1/2CIP-1/2MER-2CIP), initial 1/2 MIC ciprofloxacin addition followed by 1/2 MIC meropenem (8 h-incubation) and 2 MIC meropenem (16 h-incubation) (1/2CIP-1/2MER-2MER), and initial 1/2 MIC ciprofloxacin addition followed by 2 MIC ciprofloxacin(8 h-incubation) and 2 MIC meropenem(16 h-incubation) (1/2CIP-2CIP-2MER). No growth of K. pneumoniae was observed for the 2CIP throughout the incubation period. The numbers of planktonic cells varied with the treatments (7~10 log CFU/ml), while those of biofilm cells were not significantly different among treatments after 24-h incubation, showing approximately 7 log CFU/ml. Among the sequential treatments, the least mutant frequency was observed at the 1/2CIP-1/2MER-2CIP (14%). Compared to the CON, 1/2CIP-2CIP-2MER decreased the sensitivity of K. pneumoniae to piperacillin, cefotaxime, and nalidixic acid. The highest ${\beta}$-lactamase activity was 22 nmol/min/ml for 1/2CIP-1/2MER-2CIP, while the least ${\beta}$-lactamase activity was 6 nmol/min/ml for 1/2CIP-2CIP-2MER. The relative expression levels of multidrug efflux pump-related genes (acrA, acrB, and ramA) were increased more than 2-fold in K. pneumoniae exposed to 1/2CIP-1/2MER-2MER and 1/2CIP-2CIP-2MER. The results suggest that the sequential antibiotic treatments could change the antibiotic resistance profiles in K. pneumoniae.

항균제에 대한 내성 증가는 국내뿐만 아니라 세계적으로도 인류 건강에 큰논란이 되고 있다. 박테리아에 의한 감염을 치료하기 위해 같은 혹은 다른 계열의 항생제에 순차적으로 노출된다. 따라서, 본 연구는 ciprofloxacin과 meropenem의 순차적 처리에 따른 폐렴간균(Klebsiella pneumoniae)의 생육, 항생제 민감성, 돌연변이 빈도, ${\beta}$-lactamase activity, 생물막 형성 및 내성 관련 유전자 발현을 평가하기 위해 설계되었다. 처리군은 대조군(control; CON), 1/2 MIC ciprofloxacin (1/2CIP), 2 MIC ciprofloxacin (2CIP), 1/2 MIC ciprofloxacin+1/2 MIC meropenem+2 MIC ciprofloxacin (1/2CIP-1/2MEM-2CIP), 1/2 MIC ciprofloxacin+1/2 MIC meropenem+2 MIC meropenem(1/2CIP-1/2MEM-2MEM), 1/2 MIC ciprofloxacin+2 MIC ciprofloxacin+2 MIC meropenem (1/2CIP-2CIP-2MEM)을 포함한다. 24시간의 배양 동안 2CIP처리군에서 K. pneumoniae의 생육이 관찰되지 않았다. 모든 처리군에서 planktonic cell의 수는 7에서 10 log CFU/ml의 유의적인 차이를 보였으나 biofilm cell의 수는 7 log CFU/ml로 비슷하였다. 돌연변이 빈도는 1/2CIP-1/2MEM-2CIP에서 가장 낮은 14%을 보였다. 대조군과 비교하여 1/2CIP-2CIP-2MEM 처리 K. pneumoniae는 piperacillin, cefotaxime, nalidixic에 대한 민감도가 감소되었다. 1/2CIP-1/2MEM-2CIPrk 가장 높은 ${\beta}$-lactamase activity(22 nmol/min/ml)을 보인 반면 1/2CIP-2CIP-2MEM은 가장 낮은 ${\beta}$-lactamase activity (6 nmol/min/ml)을 보였다. Multidrug efflux pump 관련 유전자(acrA, acrB, and ramA)의 발현은 1/2CIP-1/2MER-2MER and 1/2CIP2CIP-2MER 처리된 K. pneumoniae에서 2배 이상 증가하였다. 따라서 순차적 항생제의 처리는 K. pneumoniae의 항생제 내성 양상을 변화시킬 수 있다.

Keywords

References

  1. Baran, I. and Aksu, N. 2016. Phenotypic and genotypic characteristics of carbapenem-resistant Enterobacteriaceae in a tertiary-level reference hospital in Turkey. Ann. Clin. Microbiol. Antimicrob. 15, 20. https://doi.org/10.1186/s12941-016-0136-2
  2. Chen, L.X., He, S., Li, C., and Ryu, J. 2009. Sublethal kanamycin induced cross resistance to functionally and structurally unrelated antibiotics. J. Exp. Microbiol. Immunol. 13, 53-57.
  3. Drago, L., Nicola, L., Mattina, R., and De Vecchi, E. 2010. In vitro selection of resistance in Escherichia coli and Klebsiella spp. at in vivo fluoroquinolone concentrations. BMC Microbiol. 10, 119. https://doi.org/10.1186/1471-2180-10-119
  4. Du, J., Li, P., Liu, H., Lu, D., Liang, H., and Dou, Y. 2014. Phenotypic and molecular characterization of multidrug resistant Klebsiella pneumoniae isolated from a University teaching hospital, China. PLoS ONE 9, e95181. https://doi.org/10.1371/journal.pone.0095181
  5. George, A.M., Hall, R.M., and Stokes, H.W. 1995. Multidrug resistance in Klebsiella pneumoniae: a novel gene, ramA, confers a multidrug resistance phenotype in Escherichia coli. Microbiology 141, 1909-1920. https://doi.org/10.1099/13500872-141-8-1909
  6. Gutierrez, A., Laureti, L., Crussard, S., Abida, H., Rodriguez-Rojas, A., Blazquez, J., Baharoglu, Z., Mazel, D., Darfeuille, F., Vogel, J., et al. 2013. ${\beta}$-Lactam antibiotics promote bacterial mutagenesis via an RpoS-mediated reduction in replication fidelity. Nat. Commun. 4, 1610. https://doi.org/10.1038/ncomms2607
  7. Jacobs, M.R. 2001. Optimisation of antimicrobial therapy using pharmacokinetic and pharmacodynamic parameters. Clin. Microbiol. Infect. 7, 589-596. https://doi.org/10.1046/j.1198-743x.2001.00295.x
  8. Latifpour, M., Gholipour, A., and Damavandi, M.S. 2016. Prevalence of extended-spectrum ${\beta}$-lactamase-producing Klebsiella pneumoniae isolates in nosocomial and community-acquired urinary tract infections. Jundishapur J. Microbiol. 9, e31179.
  9. Lopez, E. and Blazquez, J. 2009. Effect of subinhibitory concentrations of antibiotics on intrachromosomal homologous recombination in Escherichia coli. Antimicrob. Agent. Chemother. 53, 3411-3415. https://doi.org/10.1128/AAC.00358-09
  10. Mulcahy, H., Charron-Mazenod, L., and Lewenza, S. 2008. Extracellular DNA chelates cations and induces antibiotic resistance in Pseudomonas aeruginosa biofilms. PLoS Pathog. 4, e1000213. https://doi.org/10.1371/journal.ppat.1000213
  11. Nichol, D., Jeavons, P., Fletcher, A.G., Bonomo, R.A., Maini, P.K., Paul, J.L., Gatenby, R.A., Anderson, A.R.A., and Scott, J.G. 2015. Steering evolution with sequential therapy to prevent the emergence of bacterial antibiotic resistance. PLoS Comput. Biol. 11, e1004493. https://doi.org/10.1371/journal.pcbi.1004493
  12. Pages, J.M., Lavigne, J.P., Leflon-Guibout, V., Marcon, E., Bert, F., Noussair, L., and Nicolas-Chanoine, M.H. 2009. Efflux pump, the masked side of ${\ss}$-lactam resistance in Klebsiella pneumoniae clinical isolates. PLoS ONE 4, e4817. https://doi.org/10.1371/journal.pone.0004817
  13. Pakzad, I., Karin, M.Z., Taherikalani, M., Boustanshenas, M., and Lari, A.R. 2013. Contribution of AcrAB efflux pump to ciprofloxacin resistance in Klebsiella pneumoniae isolated from burn patients. GMS Hyg. Infect. Control 8, 1-6.
  14. Penesyan, A., Gillings, M., and Paulsen, I. 2015. Antibiotic discovery: Combatting bacterial resistance in cells and in biofilm communities. Molecules 20, 5286-5298. https://doi.org/10.3390/molecules20045286
  15. Perron, G.G., Kryazhimskiy, S., Rice, D.P., and Buckling, A. 2012. Multidrug therapy and evolution of antibiotic resistance: When order matters. Appl. Environ. Microbiol. 78, 6137-6142. https://doi.org/10.1128/AEM.01078-12
  16. Poole, K. 2004. Resistance to ${\beta}$-lactam antibiotics. Cell. Mol. Life Sci. 61, 2200-2223.
  17. Rice, L.B., Yao, J.D.C., Klimm, K., Eliopoulos, G.M., and Moellering, R.C. 1991. Efficacy of different ${\beta}$-lactams against an extendedspectrum ${\beta}$-lactamase-producing Klebsiella pneumoniae strain in the rat intra-abdominal abscess model. Antimicrob. Agent. Chemother. 35, 1243-1244. https://doi.org/10.1128/AAC.35.6.1243
  18. Shaikh, S., Fatima, J., Shakil, S., Rizvi, S.M.D., and Kamal, M.A. 2015. Antibiotic resistance and extended spectrum ${\beta}$-lactamases: Types, epidemiology and treatment. Saudi J. Biol. Sci. 22, 90-101. https://doi.org/10.1016/j.sjbs.2014.08.002
  19. Shannon, K. and Phillips, I. 1986. The effects on ${\beta}$-lactam susceptibility of phenotypic induction and genotypic derepression of ${\beta}$-lactamase synthesis. J. Antimicrob. Chemother. 18, 15-22. https://doi.org/10.1093/jac/18.Supplement_E.15
  20. Tolun, V., Kucukbasmaci, O., Torumkuney-Akbulut, D., Catal, C., Ang-Kucuker, M., and Aug, O. 2004. Relationship between ciprofloxacin resistance and extended-spectrum ${\beta}$-lactamase production in Escherichia coli and Klebsiella pneumoniae strains. Clin. Microbiol. Infect. 10, 72-75. https://doi.org/10.1111/j.1469-0691.2004.00723.x
  21. Tuomanen, E., Durack, D.T., and Tomasz, A. 1986. Antibiotic tolerance among clinical isolates of bacteria. Antimicrob. Agent. Chemother. 30, 521-527. https://doi.org/10.1128/AAC.30.4.521
  22. Vestergaard, M., Paulander, W., Marvig, R.L., Clasen, J., Jochumsen, N., Molin, S., Jelsbak, L., Ingmer, H., and Folkesson, A. 2016. Antibiotic combination therapy can select for broad-spectrum multidrug resistance in Pseudomonas aeruginosa. Int. J. Antimicrob. Agent 47, 48-55. https://doi.org/10.1016/j.ijantimicag.2015.09.014
  23. Warren, D.K., Hill, H.A., Merz, L.R., Kollef, M.H., Hayden, M.K., Fraser, V.J., and Fridkin, S.K. 2004. Cycling empirical antimicrobial agents to prevent emergence of antimicrobial-resistant Gramnegative bacteria among intensive care unit patients. Crit. Care Med. 32, 2450-2456. https://doi.org/10.1097/01.CCM.0000147685.79487.28
  24. Williamson, R. and Tomasz, A. 1985. Inhibition of cell wall synthesis and acylation of the penicillin binding proteins during prolonged exposure of growing Streptococcus pneumoniae to benzylpenicillin. Eur. J. Biochem. 151, 475-483. https://doi.org/10.1111/j.1432-1033.1985.tb09126.x
  25. Zhong, H.Q., Zhang, S., Pan, H., and Cai, T. 2013. Influence of induced ciprofloxacin resistance on efflux pump activity of Klebsiella pneumoniae. J. Zhejiang Univ. Sci. B 14, 837. https://doi.org/10.1631/jzus.B1200221

Cited by

  1. 태안군 이원면 육상오염원 배출수에서 분리한 그람음성균의 항생제 내성 특성 vol.54, pp.4, 2016, https://doi.org/10.5657/kfas.2021.0377