World J Microbiol Biotechnol (2014) 30:1251–1260 DOI 10.1007/s11274-013-1538-3 ORIGINAL PAPER Involvement of outer membrane proteins and peroxide-sensor genes in Burkholderia cepacia resistance to isothiazolone Gang Zhou • Qing-shan Shi • You-sheng Ouyang • Yi-ben Chen Received: 23 June 2013 / Accepted: 22 October 2013 / Published online: 6 November 2013 Ó Springer Science+Business Media Dordrecht 2013 Abstract Isothiazolones are used as preservatives in Keywords Burkholderia cepacia Á Kathon various modern industrial products. Although microor- preservative Á Biocide resistance properties Á Outer ganisms that exhibit resistance towards these biocides have membrane proteins Á Peroxide-sensor genes been identified, the underlying resistance mechanisms are still unclear. Therefore, we investigated the resistance properties of the following Burkholderia cepacia strains to Kathon (a representative of isothiazolones): a wild-type Introduction (WT) strain; a laboratory resistance strain (BC-IR) induced from WT; and an isolated strain (BC-327) screened from Isothiazolone biocides are widely used as antimicrobial industrial contamination samples. The bacterial cell struc- agents to control microbial growth and biofouling in ture was disrupted by 50 lgml-1 Kathon treatment. BC- industrial applications such as cooling towers, metal- IR and BC-327 did not display resistance in the presence of working fluids, paper systems and cosmetics (Xu et al. 1mlL-1 Tween 80, 1 ml L-1 Triton X-100, 0.1 % 2009; Speksnijder et al. 2010). Isothiazolones possess sodium dodecyl sulfate or 1 mmol L-1 EDTA-2Na. broad-spectrum antimicrobial efficacy against gram-posi- Additionally, BC-IR and BC-327 exhibited lower relative tive and gram-negative bacteria (Pucci et al. 2007, 2011), conductivity from 10 to 180 min. The types as well as the fungi (Pedras and Suchy 2006; Adibpour et al. 2007; Vi- levels of outer-membrane proteins (OMPs) were altered centini et al. 2011), viruses, and algae (Sharmeen et al. among WT, BC-IR and BC-327. Finally, the two Kathon- 2001; Khalaj et al. 2004). resistance strains BC-IR and BC-327 presented higher Isothiazolones were thought to interact oxidatively with resistance capacity to H2O2. We measured the levels of accessible thiols such as glutathione and cysteine within peroxide-sensor genes and observed that the transcriptional the cell (Collier et al. 1990b) resulting in inhibition of activator oxyR, superoxide dismutase sod1, sod2, catalase growth and metabolism and loss of viability (Williams cat1 and cat3 were all up-regulated under oxidative con- 2007). Moreover, one of the most important properties of ditions for all strains. Taken together, OMPs and peroxide- isothiazolone biocides is that they are not specifically tar- sensor genes in B. cepacia contributed to isothiazolone geted to envelope or cytosolic proteins (Collier et al. resistance; However, the laboratory strain BC-IR exhibited 1990a). Several reports demonstrate biocide activity of a different resistance mechanism and properties compared isothiazolone by inhibition of transferases (El Abdellaoui to the isolated strain BC-327. et al. 2006; Furdas et al. 2011; Reddy et al. 2011), oxidoreductases (Collier et al. 1991), isomerases (Cheng et al. 2007; Pucci et al. 2011), ligases (Gedi et al. 2011), G. Zhou Á Q. Shi (&) Á Y. Ouyang Á Y. Chen hydrolases (King et al. 2009), growth factor receptors State Key Laboratory of Applied Microbiology, South China (Kiselyov et al. 2009), and even mouse fibroblast cell (The Ministry-Province Joint Development), Guangdong growth (Adibpour et al. 2010). Institute of Microbiology, Guangzhou 510070, Guangdong, People’s Republic of China To exert antibacterial action, bactericides must penetrate e-mail: [email protected] the cell envelope or accumulated therein at a sufficiently 123 1252 World J Microbiol Biotechnol (2014) 30:1251–1260 high concentration. Adaptation of the microbial cell Materials and methods envelope may contribute to the mechanism allowing for resistance to antimicrobial agents (Cloete 2003). As Bacterial strains and chemicals expected, different outer membrane proteins (OMPs) expression patterns between sensitive and resistant strains B. cepacia ATCC25416 (WT), kindly provided by were observed upon isothiazolones treatment. A 35 kDa Guangdong Culture Collection Center (Guangzhou, OMP was detectable in wild-type (WT) cells of Pseudo- China), was routinely incubated in M9 minimal medium monas aeruginosa but not in cells resistant to isothiazo- (Howard-Flanders and Theriot 1966) containing 1 g L-1 -1 -1 lone. Therefore, it was proposed that this protein was the NH4Cl, 11 g L Na2HPO4Á7H2O, 3gL KH2PO4, -1 -1 -1 channel for isothiazolone (Bro¨zel and Cloete 1994). Sim- 5gL NaCl, 4 g L glucose, 120 mg L MgSO4, and -1 ilarly, in a previous study, upon examination of OMP 10 mg L CaC12 at 37 °C in a bathing rotator with 120 profiles of methylchloroisothiazolone (MCI)-resistant iso- revolutions per minute (rpm). A representative isothiazo- lates of P. aeruginosa, it was found that each of the isolates lone product Kathon, consisting of a 3:1 (volume:volume) lacked the 42 kDa protein which is believed to be a porin ratio of 5-chloro-2-methyl-4-isothiazolin-3-one (CMIT) known as OprD. This result indicates the role of the outer and 2-methyl-4-isothiazolin-3-one (MIT) at a final con- membrane as a permeability barrier allowing for MCI centration of 14 % total active ingredient, was obtained resistance (Chapman et al. 1998). In addition, a T-OMP in from Guangdong Dimei Biology Technology Co., LTD P. aeruginosa PAO1 was found from biocide-exposed (Guangzhou, China). Meantime, all other chemicals used in preparations but not from resistance-induced cultures, this study were of analytical grade and purchased from which were passaged in the absence of biocide. This result Sigma Chemical Co. (St. Louis, MO, USA) unless other- suggests that the disappearance of T-OMP is associated wise stated. with the onset of resistance to isothiazolones (Winder et al. 2000). Isolation and identification of B. cepacia In general, preservative resistance occurs when a for- from a contaminated sample merly effective preservative system no longer inhibits microbial growth (Chapman et al. 1998). It is important A contaminated sample of shampoo, obtained from to completely understand properties and mechanisms of Guangdong Jiangmen Investment Industry CO., LTD (Ji- industrial biocides that confer resistance for optimizing angmen, China) and contained antimicrobial agents practical application (Williams 2007). However, previous including isothiazolones, was diluted with sterile distill studies of the isothiazolone resistance mechanism were water and placed onto MacConkey agar (Oxoid Ltd., nearly all performed by analyzing an induced laboratory Basingstoke, UK), and then repeatedly cultured at 37 °C. strain, whereas little attention has been devoted to an When the morphology of colony appeared to be homolo- environmental strain isolated from a contaminated sam- gous, pure bacterium was considered to be obtained and ple. We hypothesized that the two strains have different preserved at -80 °C with 30 % glycerol. Subsequently, the resistance mechanisms or properties. Meanwhile because biochemical characteristics of isolated strains were analy- of widespread and long-term use, an increasing number sized using API 20NE system (API-BioMerieux, La Balme of isothiazolone resistance cases have been reported. Les Grottes, France) according to the manufacter’s direc- Recently a resistant strain of Burkholderia cepacia,a tions. Meanwhile, genome of selected strain was extracted major industrial contaminant in cosmetic and pharma- with TIANamp Bacteria DNA Kit (Tiangen, Beijing, ceutical raw materials and even finished products (Jime- China) based on the manusfacter’s instructions. The 16 s nez and Smalls 2000), was isolated and identified from rRNA gene was amplified with a primer pair of 16s-F (50- industrial putrefaction in our laboratory. The new genus AGAGTTTGATCATGGCTCAG-30) and 16s-R (50-TAG- Burkholderia was first assigned in 1992 (Yabuuchi et al. GGTTACCTTGTTACGACTT-30) in an Eppendorf ther- 1992), and it was identified to be resistant to most anti- mocycler 5330 (Eppendorf AG, Hamburg, Germany) using microbial agents (Vermis et al. 2003) including isot- the above genome as templates. The PCR product was hiazolones (Rushton et al. 2013). The objective of this cloned into a pMD18-T vector (TaKaRa, Dalian, China) paper is to elucidate the difference of the resistance and sequenced at Beijing Genomics Institute (Guangzhou, features between induced and isolated strains of B. China). The resultant sequence was aligned using Clustal X cepacia. Our results will contribute to the knowledge of 1.83 with corresponding sequences of representative spe- the bacteria resistance mechanism to isothiazolone bio- cies of the genus of Burkholderia. Phylogenetic trees were cides and inform users about the corrected method of constructed using neighbour-joining algorithms from biocide application. MEGA 4 (Tamura et al. 2007). The identified B. cepacia 123 World J Microbiol Biotechnol (2014) 30:1251–1260 1253 with higher resistance level to Kathon was desiganed as external structure by transmission electron microscopy BC-327. (TEM; H-7650, Hitachi, Japan) at 80 kV. Inducement of the resistance strain Determination of cell conductivity Inducement of resistance to Kathon biocide was performed Aliquots of 1 ml overnight culture of WT, BC-IR and BC- according to the method previously reported by Bro¨zel and 327 were pipetted into 50 ml fresh M9 medium respectively, Cloete (1994) with some modifications. Briefly, the WT and then the flasks were placed in a rotary incubator at 37 °C -1 strain was first sub-cultured at 0.5 lgml of Kathon. with 180 rpm shaking until the optical density (OD600) Then the culture was transferred into a fresh M9 medium reached about 2.5. After centrifugation at 5,000 rpm, the supplemented with 1 lgml-1 of Kathon, and the concen- pellets were washed three times with deionized water, tration of Kathon was increased by 0.5 lgml-1 until the transferred into new tubes and resuspended with 5 ml sub-culture could not grow within 48 h.