The presence of bacteria in the cosmetic production environment is often connected with non-sterile raw materials, inappropriate production lines disinfection or cross contamination. Among bacteria isolated from the environment, opportunistic pathogens can be also found, posing a risk to patients with lowered immunity. Moreover, their susceptibility to antibiotics and disinfectants is frequently decreased as they develop more complex forms - biofilms. As hydrophobicity and adhesive properties play a vital role in the colonization process the aim of this research was to determine hydrophobic, aggregative and adhesive properties of bacteria isolated from the cosmetics.Bacteria used in the research were isolated from the body balm and the cosmetic preservative (three strains of Pseudomonas aeruginosa and four strains of Pseudomonas cedrina) and identified using 16S rRNA gene sequencing. For those strains and also two reference strains (P. aeruginosa ATCC15442 and P. cedrina DSM17516) an aggregation test, hydrophobicity by two different methods (SAT and MATH) and adhesion to polystyrene by crystal violet binding assay were performed.According to the SAT method more than half of the tested strains were strongly hydrophobic. Using MATH test, it was proved that four strains (P. cedrina DSM17516 and three isolates of P. aeruginosa) were strong hydrophobes, however, the rest of the strains expressed moderate hydrophobicity. Moreover, self-aggregation was also observed and for P. aeruginosa CFII was more than 20%. All of the strains were able to adhere to polystyrene after 30 minutes contact, almost all of them (excluding P. cedrina DSM17516) indicated a moderate adhesion already after four hours of incubation. These results indicate that environmental Pseudomonas strains possess strong hydrophobic and adhesive properties, that may results in a colonization of abiotic surfaces

Kunicka-Styczyńska, Alina

Otlewska, Anna

Zabielska, Julia

English

Akademicka Platforma Czasopism

http://dx.doi.org/10.12775/EQ.2017.037Ecological Questions 28 (2017) 4:  41–46Cosmetics may be the source of microorganisms, both pathogenic and non-pathogenic. Apart from the secondary contamination in the course of usage, microbial contamina-tion may take also place during the production processes. Microorganisms may be introduced withraw materials and also from the production line due to itsimproper disinfec-tion and cross-contaminations. Those irregularities may lead to biodeterioration of products, revealing a change of pH, color, smell, consistency, viscosity, phase separation Pseudomonas sp. is a group of Gram-negative bacte-ria, frequently causing nosocomial infections of patients Adhesive and hydrophobic properties  of Pseudomonas aeruginosa and Pseudomonas cedrina associated with cosmetics* Lodz University of Technology, Wolczanska 171/173 St, 90-924 Lodz, Poland,  *e-mail: julia.zabielska@dokt.p.lodz.pl Received: 08 October 2017 /Accepted: 24 November 2017 TSB, Trypticase Soya Broth; PBS, phosphate buffered saline; TSA, Trypticase Soya Agar; PUM, phosphate urea magnesium sulfate buffer; ATTC, American Type Culture Collection; DSM, Deutsche Sammlung von Mikroorganismen; ISO, Inter-national Organization for Standardization; RAPEX, Rapid Alert System. The presence of bacteria in the cosmetic production environment is often connected with non-sterile raw materials, inap-propriate production lines disinfection or cross contamination. Among bacteria isolated from the environment, opportunistic pathogens can be also found, posing a risk to patients with lowered immunity. Moreover, their susceptibility to antibiotics and disinfectants is frequently decreased as they develop more complex forms - biofilms. As hydrophobicity and adhesive properties play a vital role in the colonization process the aim of this research was to determine hydrophobic, aggregative and adhesive properties of bacteria isolated from the cosmetics.Bacteria used in the research were isolated from the body balm and the cosmetic preservative (three strains of Pseudomonas aeru- ginosa and four strains of Pseudomonas cedrinareference strains (P. aeruginosa ATCC15442 and P. cedrina DSM17516) an aggregation test, hydrophobicity by two different methods (SAT and MATH) and adhesion to polystyrene by crystal violet binding assay were performed.According to the SAT method more than half of the tested strains were strongly hydrophobic. Using MATH test, it was proved that four strains (P. cedrina DSM17516 and three isolates of P. aeruginosa) were strong hydrophobes, however, the rest of the strains expressed moderate hydrophobicity. Moreover, self-aggregation was also observed and for P. aeruginosaAll of the strains were able to adhere to polystyrene after 30 minutes contact, almost all of them (excluding P. cedrina DSM17516) indicated a moderate adhesion already after four hours of incubation. These results indicate that environmental Pseudomonas strains possess strong hydrophobic and adhesive properties, that may results in a colonization of abiotic surfaces. Pseudomonas, cosmetic contamination, hydrophobicity, adhesion, biofilm.42 Julia Zabielska, Alina Kunicka-Styczyńska, Anna Otlewskawith reduced immunity. P. aeruginosa belonging to this group is one of the common species associated with infec-tions and are usually very tough to eradicate. Among many virulence factors of P. aeruginosa the ones connected with the process of adhesion and biofilm formation (capabil-ity of aggregation, cell hydrophobicity, rhamnolipid and EPS production, motility etc.) can be (Briendstien et al., 2011). The ability of colonization of abiotic surfaces by P. aeruginosa et al. (2015) and the biofilm formed reached more than 6 2 after 24 hours of incubation. Once the biofilm is developed, its removing is much more complicated due to numerous mechanisms such as the presence of matrix or “super resistant” cells – metabolically inactive, grown in the nutrition and oxygen deficiency (Matusiak, 2014).The purpose of this study was to identify microorgan-isms isolated from a cosmetic product and a cationic sur-factant used as a cosmetic ingredient and determine their hydrophobic, aggregative and adhesive properties, crucial for the biofilm development.A total of seven strains were isolated: three P. aeruginosa to P. cedrina (DH1, DH2, DN3, DH4) from cetyltrimethyl ammonium chloride, a commercial cationic surfactant used as a cosmetic ingredient. One ml of appropriate product was diluted in 10 ml of saline solution with Tween 80 to neutralize the sample. One ml of each sample was spread on cetrimide agar (BTL, Poland) to allow Pseudomonas sp. growth. Strains were initially identified by biochemi-reference strains were used – P. aeruginosa ATCC15442 (American Type Culture Collection) and P. cedrina DSM17516 (Deutsche Sammlung von Mikroorganismen). Bacteria were activated in TSB (Merck, Germany) for 24 of the following reference strains were used: P. cedrina C37, G25, CI-10 originated from milk and clausen (CI-10); P. aeruginosa -and sputum.Bacterial strains were identified based on 16S rRNA gene sequencing. Briefly, genomic DNA was extracted employ-according to manufacturer’s protocol. 16S rRNA gene was amplified with universal primers 27f (5’-AGAGTTT-GATCCTGGCTCAG-3’) and 1492r (5’-GGTTACCTT-GTTACGACTT-3’). PCR reactions were performed using 1.5 U RedTaqReadyMix DNA polymerase (Sigma-Al--dient Thermal Cycler (Bio-Rad, USA). The amplification (2014). The PCR fragments were purified using Clean Up sequences were obtained using the BigDye Terminator -tems, USA) and analyzed with the Applied Biosystems model 3730 Genetic Analyzer (Genomed S.A., Poland). The nucleotide sequences of 16S rRNA were proofread, assembled and compared with sequences available in The National Center for Biotechnology Information (NCBI) us-et al., 2010). The nucleotide sequences of 16S rRNA gene were deposited in GenBank of the NCBI under the fol-P. ce-drina P. aeruginosa).The phylogenetic tree was constructed by neighbor-joining method using the MEGA7 software (Saitou & Nei, 1987; -puted using the Maximum Composite Likelihood method and are in the units of the number of base substitutions per site (Tamura et al., 2004). The analysis involved 13 nucleotide sequences of 16S rRNA genes from the NCBI database. All positions containing gaps and missing data were eliminated. There was a total of 1384 positions in the final dataset.Aggregation of strains was evaluated by the method pro-posed by Del Re et al. (2000). Bacteria were grown in TSB (Merck, Germany) for 24 hours at 30ºC. Two ml of the culture was centrifuged for 10 minutes (5000 rpm). The precipitate was suspended in two ml of 10 μM PBS buffer and the absorbance at the wavelength 600 nm was meas-ured (A). Subsequently, the suspension was incubated for two hours at 30ºC and the absorbance of the upper layer was measured (A0). Aggregation was calculated according to the formula:43Adhesive and hydrophobic properties of Pseudomonas aeruginosaHydrophobic properties of the cell surface were deter-mined by two different methods SAT (Salt Aggregation Test) and MATH (Microbial Adhesion to Hydrocarbon).In the SAT method, activated bacteria were inoculat-ed on TSA (Merck, Germany) and incubated for 24 hours at 30ºC. After that, bacteria inoculum in 2 μM phosphate buffer saline (PBS, Merck, Germany) was prepared (ap-sulfate of different molarity (from 0.1 to 4) in a ratio 1:1. The control included bacteria inoculum in PBS buffer. Af-ter 15–20 minutes, the aggregates formation was evalu-ated by macroscopic observations and classified according to the scale proposed by Nwanyanwu and Abu (2013), in which strains were divided into three categories: of high hydrophobicity (<1.0 M), of moderate hydrophobicity (1.0–2.0 M) and of low hydrophobicity (>2.0 M).In the MATH assay with some modifications (Rosen-berg, 1984), bacteria were activated on TSA slants (in-cubation 24 hours, 30ºC). Then, inoculum (A1) was pre-pared either in 10 μM phosphate buffer saline (PBS), or in phosphate urea magnesium sulfate buffer (PUM, Merck, Germany). Two and half ml of inoculum was mixed with half ml of p-xylene and incubated for 10 minutes at 37ºC. Thereafter, samples were homogenized and incubated once again for 45 minutes. The samples were allowed to be sep-arated into two phases (aqueous and hydrocarbon). The aqueous phase (A2) was measured spectrophotometrically at the wavelength 520 nm. The hydrophobicity index (per-centage of cell adhesion to hydrocarbon) was calculated upon the following formula:  -am et al., 2009): strongly hydrophobic (>50%), moderate hydrophobic (20–50%) and low hydrophobic (<20%).Adhesion to polystyrene was carried out by the method proposed by Stepanovic et al. (2000) with some modifica-tions. Bacteria were cultivated in TSB (Merck, Germany) for 24 hours at 30ºC. 20 μl of bacteria was carried into 230 μl of TSB on 96-well microtitration plate. Plates were incubated in different periods of time – for 0.5, 4 and 24 hours, then the content was poured out and wells were rinsed with sterile water. The plates were dried and adhered bacteria cells were fixed with 96% ethanol for 20–30 min-utes. Afterwards, ethanol was removed and bacteria were stained with 0.5% crystal violet. The dye was removed and the plates were again rinsed with water. After drying, each well was decolorized with 96% ethanol and the absorb-the growth medium.The tests were conducted in triplicate and SD (standard deviation) was calculated using ORIGIN LAB 6.1 version (Northampton, USA). The correlation between buffers used in MATH method was evaluated by Pearson’s coef-ficient analysis.Seven strains belonging to Pseudomonas species were iso-lated from the body balm and the cosmetic preservative. Genetic identification based on 16S rRNA gene sequenc-classified as P. aeruginosa and four (DH1, DH2, DH3 and DH4) as P. cedrina. Phylogenetic analysis, presented in The presence of unfavorable microflorawas described also by Campana et al. (2006). They investigated 91 com-mercial cosmetics and opportunistic strains such as Pseu-domonas putida and Staphylococcus aureus were found in some samples. According to the different source - RAPEX data for non-food products in the EU, only in the last dec-ade over 100 cases of microbiological contaminations were noted, including the presence of opportunistic pathogens such as P. aeruginosa, Burkholderia cepacia and S. aureus. Also Birketsoz et al. (2013) indicated the contamination of P. aeruginosa, P. putida and Pseudomonas fluorescens in commercial, unopened cosmetic products.In the course of the study aggregative, adhesive and hydrophobic properties of the identified Pseudomonas sp. strains were assessed. Aggregation and hydrophobic abili-ties expressed by Pseudomonas strains were presented in was presented by environmental P. aeruginosa and it reached 20.74%. The aggregation of the majority of strains fluctuated from 9 to 13%. The least aggregative strain was the reference strain P. aeruginosa ATCC15442 (4.82%). Aggregation of different pathogens such as Sal-monella typhimurium, Listeria monocytogenes, Staphylo-coccus aureus and Shigella boydii was described by Xu et al. (2010). The values obtained by Xu et al. (l.c.) were similar and oscillated from 5 to 23% for bacteria in PBS buffer, however after addition of NaCl in different con-centration aggregation were much higher. The aggrega-tion is connected with cell-to-cell binding and as it was showed, environmental conditions have a great influence on it.44 Julia Zabielska, Alina Kunicka-Styczyńska, Anna OtlewskaHydrophobicity of isolated bacteria was determined by two methods. The first one – Salt Aggregation Test – indicated that all of the strains were high or moderate hydrophobes. In comparison to environmental isolates, reference strains were less hydrophobic (Table 1). The same method was used by Wolska et al. (2002) for clini-cal strains of P. aeruginosa. More than half of the tested bacteria expressed strong hydrophobic properties. Result of Pseudomonas sp. hydrophobicity using MATH method P. cedrina was characterized by the highest hydrophobicity index, which reached respec-tively 88.74% in PBS buffer and 89.62% in PUM buffer, and was classified as strongly hydrophobic. P. aeruginosa index values oscillated between 60–80%. The lowest index values were obtained for P. cedrina strains (DH 1–4) and for the reference strain P. aeruginosa ATCC15442, what classified them as moderate hydrophobes.Moreover, the degree of hydrophobicity evaluated by Pearson’s coefficient analysis showed strong correlation Pseudomonas sp. cosmetic contaminant: P. aeruginosa P. cedrina DH1, DH2, DN3, DH4 isolated from cetyltrimethyl ammonium chloride; P. cedrina C37, G25, CI-10 origi-nated from milk and clausen (CI-10); P. aeruginosaTable 1. Pseudomonas sp. strains aggregation and degree of cell surface hydrophobicity assessed by *SAT methodMicroorganism (%) bicity*Pseudomonas cedrinaDSM17516 11.57±3.64 moderateDH1 11.80±3.43 highDH2 10.50±4.11 moderateDH3 9.43±0.86 highDH4 10.58±1.47 highPseudomonas aeruginosaATCC15442 4.82±0.11 moderate20.74±1.66 high12.94±0.47 high11.00±1.87 moderate45Adhesive and hydrophobic properties of Pseudomonas aeruginosabetween both buffers tested. Norouzi et al. (2010) also pre-sented data regarding P. aeruginosa strains hydrophobicity assessed by the MATH method and the majority of strains demonstrated moderate hydrophobic properties. Research P. aeruginosa and P. fluorescens indicated that both strains are moderate hydrophobic in PBS buffer (28.8% and 36.4% respectively) and low hydrophobic in PUM buffer (18% and 15.2% respectively). Tyfa et al. (2015) tested Gram-positive Alicyclobacillus sp. using the same method and almost all of them were strong hydrophobes in PUM buffer and ma-jority of strains were moderate hydrophobes in PBS buffer.The crystal violet binding method was used to assess Pseudomonas sp. strains capability of adhering to polysty-rene and creating biofilm, thus to evaluate their adhesive the polystyrene surface after 0.5 hour contact and the high-est absorbance values were reached for P. cedrina isolates (DH1–4). After 4 hours of contact for almost all strains absorbance ranged from 0.12 to 0.22 (excluding P. cedrina DSM17516), which indicated on their moderate adherence. 24-hour incubation showed that strains P. cedrina: DH3, DH1 and P. aeruginosa ATCC15442 expressed a great ad-P. cedrina DSM17516 reference strain the ab-Pseudomonas sp. cosmetic contaminants hydrophobicity index estimated by the MATH test in PBS and PUMbuffers, clas-sified as: S – strongly hydrophobic, M – moderate hydrophobic and L – low hydrophobicPseudomonas sp. cosmetic contaminants adhesion to polystyrene within 0.5–24 hours of incubation46 Julia Zabielska, Alina Kunicka-Styczyńska, Anna Otlewskasorbance reached 0.097 which indicated on weak adhesion. The rest of strains expressed moderate adhesion. Similar re-sults to P. cedrina et al. (2011) for Pseudomonas fluorescens reaching after 0.5 hour incubation the absorbance equal 0.087.All in all, cosmetics can be the source of opportunistic pathogens which pose a health threat to humans with im-paired immune system. In pursuance of the ISO standard (PN-EN ISO 17516: 2014-11), the presence of microbes such as P. aeruginosa, S. aureus and Candida albicans is not allowed in one gram of the cosmetic product, however these microorganisms are often found in cosmetics or their ingredients, which was proved in this report. What is more, such bacteria often present a great hydrophobic and adhe-sive properties which can lead to colonization of abiotic surfaces and biofilms creation.Birteksoz A.R., Tuysuz M. & Otuk G., 2013, Investiga-tion of preservative efficacy and microbiological con-tent of some cosmetics found on the market. Pakistan Journal of Pharmaceutical Sciences 26: 153–157.R.E.W., 2011, Pseudomonas aeruginosa: all roads lead to resistance. Trends in Microbiology 19: 419–426.W., 2006, Microbiological study of cosmetics product during their use by consumers: health risk and efficacy of preservative systems. Letters in Applied Microbiol-ogy 43: 301–306.Del Re B., Sgorbati B., Miglioli M. & Palenzona D., 2000, Adhesion, autoaggregation and hydrophobicity of 13 strains of Bifidobacterium longum. Letters in Applied Microbiology 31: 438–442.-26–29.-nanath G., 2009, Determination of the degree of hydro-phobicity – A technique to assess bacterial colonization on leaf surface and root region of lotus plant. Asian Journal of Experimental Sciences 23(1): 135–139.of Asaia bogorensis originating in fruit-flavored water to packaging materials. BioMed Research Internation-al. (http://dx.doi.org/10.1155/2014/514190).-lecular Evolutionary Genetics Analysis version 7.0 for bigger datasets. Molecular Biology and Evolution 33: 1870–1874.Proteus mirabilis – rola biofilmu i in- -tions caused by Proteus mirabilis – role of the biofilm Mikrobiologii 53(2): 173–181.Nwanyanwu C. & Abu G., 2013, Influence of growth media on hydrophobicity of phenol-utilizing bacteria found in petroleum refinery effluent. International Re-search Journal of Biological Sciences 2: 6–10.Comparison of cell surface hydrophobicity and bio-film formation among ESBL- and non-ESBL-produc-ing Pseudomonas aeruginosa clinical isolates. African Journal of Microbiology Research 4(11): 1143–1147.PN-EN ISO 17516: 2014-11 Cosmetics-Microbiology-Mi-crobiological limits.Rosenberg M., 1984, Bacterial adherence to hydrocarbons: a useful technique for studying cell surface hydropho-Saitou N. & Nei M., 1987, The neighbor-joining method: A new method for reconstructing phylogenetic trees. Molecular Biology and Evolution 4: 406–425.2011, Cell surface characteristic of Asaia bogorensis – spoilage microorganism of bottled water. Czech Journal for quantification of staphylococcal biofilm formation. Journal of Microbiological Methods 40(2): 175–179.-ferring very large phylogenies by using the neighbor-joining method. Proceedings of the National Academy of Sciences (USA) 101:11030–11035.Evaluation of hydrophobicity of quantitative analysis of biofilm formation by Alicyclobacillus sp. Acta Bio-chimica Polonica 62(4): 785–790.on the biofilm formation by foodborne pathogens. Jour--cell surface hydrophobicity of Pseudomonas aerugi-nosa61–66.& Tyfa A., 2015, Opportunistic Gram-negative rods’ capability of creating biofilm structures on polivynyl chloride and styrene-acronitrile copolymer surfaces. Acta Biochimica Polonica 62(4): 733–737.A greedy algorithm for aligning DNA sequences. Jour-nal of Computational Biology 7: 203–214.

Adhesive and hydrophobic properties of Pseudomonas aeruginosa and Pseudomonas cedrina associated with cosmetics

https://apcz.umk.pl/EQ/article/download/EQ.2017.037/14724

Adhesive and hydrophobic properties of Pseudomonas aeruginosa and Pseudomonas cedrina associated with cosmetics

Abstract

Similar works

Full text

Available Versions

Akademicka Platforma Czasopism