1887

Abstract

, the causative agent of melioidosis, has been found to increase its resistance to antibiotics when growing as a biofilm. The resistance is related to several mechanisms. One of the possible mechanisms is the efflux pump. Using bioinformatics analysis, it was found that BPSL1661, BPSL1664 and BPSL1665 were orthologous genes of the efflux transporter encoding genes for biofilm-related antibiotic resistance, PA1874–PA1877 genes in strain PAO1. Expression of selected encoding genes for the efflux transporter system during biofilm formation were investigated. Real-time reverse transcriptase PCR expression of , cytoplasmic membrane protein of AmrAB-OprA efflux transporter encoding gene, was slightly increased, while BPSL1665 was significantly increased during growth of bacteria in biofilm formation. Minimum biofilm inhibition concentration and minimum biofilm eradication concentration (MBEC) of ceftazidime (CTZ), doxycycline (DOX) and imipenem were found to be 2- to 1024-times increased when compared to their MICs for of planktonic cells. Inhibition of the efflux transporter by adding phenylalanine arginine β-napthylamide (PAβN), a universal efflux inhibitor, decreased 2 to 16 times as much as MBEC in biofilms with CTZ and DOX. When the intracellular accumulation of antibiotics was tested to reveal the pump inhibition, only the concentrations of CTZ and DOX increased in PAβN treated biofilm. Taken together, these results indicated that BPSL1665, a putative precursor of the efflux pump gene, might be related to the adaptation of in biofilm conditions. Inhibition of efflux pumps may lead to a decrease of resistance to CTZ and DOX in biofilm cells.

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2016-11-16
2019-12-15
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References

  1. Anderl J. N., Franklin M. J., Stewart P. S.. 2000; Role of antibiotic penetration limitation in Klebsiella pneumoniae biofilm resistance to ampicillin and ciprofloxacin. Antimicrob Agents Chemother44:1818–1824 [CrossRef][PubMed]
    [Google Scholar]
  2. Atkins T., Crossman L. C., Pitt T., Churcher C., Mungall K., Bentley S. D., Sebaihia M., Thomson N. R., Bason N. et al. 2004; Genomic plasticity of the causative agent of melioidosis, Burkholderia pseudomallei. Proc Natl Acad Sci U S A101:14240–14245 [CrossRef][PubMed]
    [Google Scholar]
  3. Bazzini S., Udine C., Sass A., Pasca M. R., Longo F., Emiliani G., Fondi M., Perrin E., Decorosi F. et al. 2011; Deciphering the role of RND efflux transporters in Burkholderia cenocepacia. PLoS One6:e18902 [CrossRef][PubMed]
    [Google Scholar]
  4. Beaudoin T., Zhang L., Hinz A. J., Parr C. J., Mah T.-F.. 2012; The biofilm-specific antibiotic resistance gene ndvB is important for expression of ethanol oxidation genes in Pseudomonas aeruginosa biofilms. J Bacteriol194:3128–3136 [CrossRef]
    [Google Scholar]
  5. Biot F., Lopez M. M., Poyot T., Neulat-Ripoll F., Lignon S., Caclard A., Thibault F. M., Peinnequin A., Pagès J.-M., Valade E.. 2013; Interplay between three RND efflux pumps in doxycycline-selected strains of Burkholderia thailandensis. PLoS One8:e84068 [CrossRef][PubMed]
    [Google Scholar]
  6. Buroni S., Pasca M. R., Flannagan R. S., Bazzini S., Milano A., Bertani I., Venturi V., Valvano M. A., Riccardi G.. 2009; Assessment of three resistance-nodulation-cell division drug efflux transporters of Burkholderia cenocepacia in intrinsic antibiotic resistance. BMC Microbiol9:200 [CrossRef][PubMed]
    [Google Scholar]
  7. Chan Y. Y., Chua K. L.. 2005; The Burkholderia pseudomallei BpeAB-OprB efflux pump: expression and impact on quorum sensing and virulence. J Bacteriol187:4707–4719 [CrossRef][PubMed]
    [Google Scholar]
  8. Chan Y. Y., Chua K. L.. 2010; Growth-related changes in intracellular spermidine and its effect on efflux pump expression and quorum sensing in Burkholderia pseudomallei. Microbiology156:1144–1154 [CrossRef][PubMed]
    [Google Scholar]
  9. Chantratita N., Rholl D. A., Sim B., Wuthiekanun V., Limmathurotsakul D., Amornchai P., Thanwisai A., Chua H. H., Ooi W. F. et al. 2011; Antimicrobial resistance to ceftazidime involving loss of penicillin-binding protein 3 in Burkholderia pseudomallei. Proc Natl Acad Sci U S A108:17165–17170 [CrossRef][PubMed]
    [Google Scholar]
  10. Chen L.. 2011; The role of bacterial biofilm in persistent infections and control strategies. Int J Oral Sci3:66–73 [CrossRef][PubMed]
    [Google Scholar]
  11. Chin C.-Y., Hara Y., Ghazali A.-K., Yap S.-J., Kong C., Wong Y.-C., Rozali N., Koh S.-F., Hoh C.-C. et al. 2015; Global transcriptional analysis of Burkholderia pseudomallei high and low biofilm producers reveals insights into biofilm production and virulence. BMC Genomics16:471 [CrossRef][PubMed]
    [Google Scholar]
  12. Chirakul S., Bartpho T., Wongsurawat T., Taweechaisupapong S., Karoonutaisiri N., Talaat A. M., Wongratanacheewin S., Ernst R. K., Sermswan R. W.. 2014; Characterization of BPSS1521 (bprD), a regulator of Burkholderia pseudomallei virulence gene expression in the mouse model. PLoS One9:e104313 [CrossRef][PubMed]
    [Google Scholar]
  13. DeShazer D., Brett P. J., Carlyon R., Woods D. E.. 1997; Mutagenesis of Burkholderia pseudomallei with Tn5-OT182: isolation of motility mutants and molecular characterization of the flagellin structural gene. J Bacteriol179:2116–2125[PubMed][CrossRef]
    [Google Scholar]
  14. Dussault A. A., Pouliot M.. 2006; Rapid and simple comparison of messenger RNA levels using real-time PCR. Biol Proced Online8:1–10 [CrossRef][PubMed]
    [Google Scholar]
  15. Keren I., Kaldalu N., Spoering A., Wang Y., Lewis K.. 2004; Persister cells and tolerance to antimicrobials. FEMS Microbiol Lett230:13–18 [CrossRef][PubMed]
    [Google Scholar]
  16. Kumar A., Mayo M., Trunck L. A., Cheng A. C., Currie B. J., Schweizer H. P.. 2008; Expression of resistance-nodulation-cell-division efflux pumps in commonly used Burkholderia pseudomallei strains and clinical isolates from northern Australia. Trans R Soc Trop Med Hyg102:S145–S151 [CrossRef][PubMed]
    [Google Scholar]
  17. Lamers R. P., Cavallari J. F., Burrows L. L.. 2013; The efflux inhibitor phenylalanine-arginine beta-naphthylamide (PAβN) permeabilizes the outer membrane of Gram-negative bacteria. PLoS One8:e60666 [CrossRef][PubMed]
    [Google Scholar]
  18. Limmathurotsakul D., Paeyao A., Wongratanacheewin S., Saiprom N., Takpho N., Thaipadungpanit J., Chantratita N., Wuthiekanun V., Day N. P., Peacock S. J.. 2014; Role of Burkholderia pseudomallei biofilm formation and lipopolysaccharide in relapse of melioidosis. Clin Microbiol Infect20:O854–O856 [CrossRef]
    [Google Scholar]
  19. Mima T., Schweizer H. P.. 2010; The BpeAB-OprB efflux pump of Burkholderia pseudomallei 1026b does not play a role in quorum sensing, virulence factor production, or extrusion of aminoglycosides but is a broad-spectrum drug efflux system. Antimicrob Agents Chemother54:3113–3120 [CrossRef][PubMed]
    [Google Scholar]
  20. Moore R. A., DeShazer D., Reckseidler S., Weissman A., Woods D. E.. 1999; Efflux-mediated aminoglycoside and macrolide resistance in Burkholderia pseudomallei. Antimicrob Agents Chemother43:465–470[PubMed]
    [Google Scholar]
  21. Moreno-Hagelsieb G., Latimer K.. 2008; Choosing BLAST options for better detection of orthologs as reciprocal best hits. Bioinformatics24:319–324 [CrossRef][PubMed]
    [Google Scholar]
  22. Nierman W. C., Yu Y., Losada L.. 2015; The in vitro antibiotic tolerant persister population in Burkholderia pseudomallei is altered by environmental factors. Front Microbiol6:1338 [CrossRef][PubMed]
    [Google Scholar]
  23. Okusu H., Ma D., Nikaido H.. 1996; AcrAB efflux pump plays a major role in the antibiotic resistance phenotype of Escherichia coli multiple-antibiotic-resistance (Mar) mutants. J Bacteriol178:306–308[PubMed][CrossRef]
    [Google Scholar]
  24. Pibalpakdee P., Wongratanacheewin S., Taweechaisupapong S., Niumsup P. R.. 2012; Diffusion and activity of antibiotics against Burkholderia pseudomallei biofilms. Int J Antimicrob Agents39:356–359 [CrossRef][PubMed]
    [Google Scholar]
  25. Podin Y., Sarovich D. S., Price E. P., Kaestli M., Mayo M., Hii K., Ngian H., Wong S., Wong I. et al. 2014; Burkholderia pseudomallei isolates from Sarawak, Malaysian Borneo, are predominantly susceptible to aminoglycosides and macrolides. Antimicrob Agents Chemother58:162–166 [CrossRef][PubMed]
    [Google Scholar]
  26. Podnecky N. L., Wuthiekanun V., Peacock S. J., Schweizer H. P.. 2013; The BpeEF-OprC efflux pump is responsible for widespread trimethoprim resistance in clinical and environmental Burkholderia pseudomallei isolates. Antimicrob Agents Chemother57:4381–4386 [CrossRef][PubMed]
    [Google Scholar]
  27. Podnecky N. L., Rhodes K. A., Schweizer H. P.. 2015; Efflux pump-mediated drug resistance in Burkholderia. Front Microbiol6:305 [CrossRef][PubMed]
    [Google Scholar]
  28. Rajendran R., Quinn R. F., Murray C., McCulloch E., Williams C., Ramage G.. 2010; Efflux pumps may play a role in tigecycline resistance in Burkholderia species. Int J Antimicrob Agents36:151–154 [CrossRef][PubMed]
    [Google Scholar]
  29. Renau T. E., Léger R., Flamme E. M., Sangalang J., She M. W., Yen R., Gannon C. L., Griffith D., Chamberland S. et al. 1999; Inhibitors of efflux pumps in Pseudomonas aeruginosa potentiate the activity of the fluoroquinolone antibacterial levofloxacin. J Med Chem42:4928–4931 [CrossRef][PubMed]
    [Google Scholar]
  30. Sam I. C., See K. H., Puthucheary S. D.. 2009; Variations in ceftazidime and amoxicillin-clavulanate susceptibilities within a clonal infection of Burkholderia pseudomallei. J Clin Microbiol47:1556–1558 [CrossRef][PubMed]
    [Google Scholar]
  31. Sarovich P., Peacock C., Vonschulze W., Keim P., Limmathurotsakul D.. 2012a; Development of ceftazidime resistance in an acute Burkholderia pseudomallei infection. Infect Drug Resist5:129[CrossRef]
    [Google Scholar]
  32. Sarovich D. S., Price E. P., Von Schulze A. T., Cook J. M., Mayo M., Watson L. M., Richardson L., Seymour M. L., Tuanyok A. et al. 2012b; Characterization of ceftazidime resistance mechanisms in clinical isolates of Burkholderia pseudomallei from Australia. PLoS One7:e30789 [CrossRef]
    [Google Scholar]
  33. Sawasdidoln C., Taweechaisupapong S., Sermswan R. W., Tattawasart U., Tungpradabkul S., Wongratanacheewin S.. 2010; Growing Burkholderia pseudomallei in biofilm stimulating conditions significantly induces antimicrobial resistance. PLoS One5:e9196 [CrossRef][PubMed]
    [Google Scholar]
  34. Schweizer H. P.. 2012; Mechanisms of antibiotic resistance in Burkholderia pseudomallei: implications for treatment of melioidosis. Future Microbiol7:1389–1399 [CrossRef][PubMed]
    [Google Scholar]
  35. Taweechaisupapong S., Kaewpa C., Arunyanart C., Kanla P., Homchampa P., Sirisinha S., Proungvitaya T., Wongratanacheewin S.. 2005; Virulence of Burkholderia pseudomallei does not correlate with biofilm formation. Microb Pathog39:77–85 [CrossRef][PubMed]
    [Google Scholar]
  36. Tribuddharat C., Moore R. A., Baker P., Woods D. E.. 2003; Burkholderia pseudomallei class A β-lactamase mutations that confer selective resistance against ceftazidime or clavulanic acid inhibition. Antimicrob Agents Chemother47:2082–2087 [CrossRef][PubMed]
    [Google Scholar]
  37. Walters M. C., Roe F., Bugnicourt A., Franklin M. J., Stewart P. S.. 2003; Contributions of antibiotic penetration, oxygen limitation, and low metabolic activity to tolerance of Pseudomonas aeruginosa biofilms to ciprofloxacin and tobramycin. Antimicrob Agents Chemother47:317–323 [CrossRef][PubMed]
    [Google Scholar]
  38. Yabuuchi E., Kosako Y., Oyaizu H., Yano I., Hotta H., Hashimoto Y., Ezaki T., Arakawa M.. 1992; Proposal of Burkholderia gen. nov. and transfer of seven species of the genus Pseudomonas homology group II to the new genus, with the type species Burkholderia cepacia (Palleroni and Holmes 1981) comb. nov. Microbiol Immunol36:1251–1275 [CrossRef][PubMed]
    [Google Scholar]
  39. Ye J., Coulouris G., Zaretskaya I., Cutcutache I., Rozen S., Madden T. L.. 2012; Primer-BLAST: a tool to design target-specific primers for polymerase chain reaction. BMC Bioinformatics13:134 [CrossRef]
    [Google Scholar]
  40. Yu N. Y., Wagner J. R., Laird M. R., Melli G., Rey S., Lo R., Dao P., Sahinalp S. C., Ester M. et al. 2010; PSORTb 3.0: improved protein subcellular localization prediction with refined localization subcategories and predictive capabilities for all prokaryotes. Bioinformatics26:1608–1615 [CrossRef][PubMed]
    [Google Scholar]
  41. Zhang L., Mah T.-F.. 2008; Involvement of a novel efflux system in biofilm-specific resistance to antibiotics. J Bacteriol190:4447–4452 [CrossRef][PubMed]
    [Google Scholar]
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