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Environmental Microbiology (2015) 17(11), 4459–4468

doi:10.1111/1462-2920.12890

Presence of Pseudomonas aeruginosa influences biofilm formation and surface protein expression of Staphylococcus aureus Amit Kumar and Yen Peng Ting* Department of Chemical and Biomolecular Engineering, National University of Singapore, Singapore. Summary Although Staphylococcus aureus and Pseudomonas aeruginosa can individually colonize and infect their hosts, the commensalistic effect of the two is more tenacious and lethal. In this study, it was shown that in co-culture with P. aeruginosa, a sub-population of S. aureus exhibited improved resistance to kanamycin by selection of small colony variant (SCV) phenotype. Additionally, biofilm formation by the two bacteria was denser in the co-culture, compared with biofilm formed in individual pure cultures. Using Atomic Force Microscope (AFM) force spectroscopy for single cells, it was demonstrated that S. aureus cultured in the presence of P. aeruginosa bound more tenaciously to substrates. Surface-shaved peptides were isolated and identified using ultra-performance liquid chromatography-quadrupole-time of flight and a homology search program SPIDER. Results indicated that serine-rich adhesin, extracellular matrix binding protein and other putative adhesion proteins could be responsible for the enhanced attachment of S. aureus in the co-culture. Besides, several other proteins were differentially expressed, indicating the occurrence of a range of other interactions. Of particular interest was a multidrug resistant protein named ABC transporter permease which is known to expel xenobiotics out of the cells. Positive regulation of this protein could be involved in the SCV selection of S. aureus in the co-culture. Introduction Since its foundation, microbiology has mainly been a science of pure culture. In laboratory experiments, analyses are usually made on monocultures. In nature, however, bacteria seldom exist as pure cultures; in most Received 19 October, 2014; accepted 23 April, 2015. *For correspondence. E-mail [email protected]; Tel. 0065 6516 2190; Fax 0065 6779 1936.

© 2015 Society for Applied Microbiology and John Wiley & Sons Ltd

instances, bacteria occupy a common niche with other microorganisms and interact with the species of the same or different kind. Clearly, any such interactions occurring between two or more organisms are completely overlooked in pure culture studies. Although there have been some notable studies on mixed cultures of bacteria in the last few decades, the focus has mainly been on the contradiction between competition and coexistence (Shank and Kolter, 2009; Peters et al., 2010; Freilich et al., 2011; Mitri et al., 2011; Elias and Banin, 2012). Staphylococcus aureus and Pseudomonas aeruginosa are recognised as two extremely opportunistic pathogens that have been implicated in a number of infectious diseases. While S. aureus is renowned for developing antibiotic resistance and its immune evasion strategies, P. aeruginosa is naturally resistant to a number of antibiotics and readily infects immune-compromised hosts. Individually, S. aureus is capable of colonizing almost all types of artificial implant devices, and P. aeruginosa has the ability to infect compromised live tissues in a host. However, it is recognised that the commensalistic effect of the two is even more tenacious and lethal. Studies have shown that P. aeruginosa and S. aureus share a common habitat and synergistically interact with one another (Mashburn et al., 2005; Fazli et al., 2009; Dalton et al., 2011; Korgaonkar et al., 2013). Staphylococcus aureus has frequently been co-isolated with P. aeruginosa from the skin, eyes and catheter infections and from the lungs of patients with cystic fibrosis (Biswas et al., 2009; Willner et al., 2012). In the lungs of cystic fibrosis patients, a sub-population of the staphylococcal cells survives in the presence of P. aeruginosa by the selection of small colony variants (SCVs). Small colony variants usually have defective electron transport chain and can survive elevated levels of aminoglycoside treatment. In recent years, tandem mass spectrometry (MS/MS) has emerged as a standard method for protein identification. In the current practice of protein identification, purified proteins are digested into short peptides with proteolytic enzymes such as trypsin before they are separated using electrophoretic or chromatographic techniques. The mass spectra of the separated fractions are measured with a tandem mass spectrometer and interpreted using software to identify the amino acid

4460 A. Kumar and Y. P. Ting sequences of the peptides and proteins (Zhang et al., 2012). Two approaches exist for the software to interpret the data, namely the database search and the de novo sequencing. The database search approach is effective only if the protein sequence exists in database, whereas a homology search program such as SPIDER can be used with the de novo approach to identify proteins that are not present in the database. Researchers have reported that the de novo approach provides better results than the database search approach for the MS/MS data produced with certain instruments (Mann and Wilm, 1994; Searle et al., 2004). To the best of our knowledge, no in vitro study has been carried out which examines the changes in surface properties of S. aureus in the presence of P. aeruginosa in the culture. The objective of this study was to investigate the changes arising on the surface of S. aureus cells in the presence of P. aeruginosa in the co-culture. In particular, surface attachment of S. aureus in the co-culture was compared with that in the pure culture of S. aureus. Single-cell Atomic force Microscope (AFM) force spectroscopy measurements were carried out to quantify the direct force of interaction between bacteria and a colonizing surface. In addition, surface shaved peptides were isolated and identified using ultra-performance liquid chromatography-quadrupole-time of flight and a homology search program SPIDER.

Materials and methods

Quantification of planktonic and sessile cells Stainless steel 316 (SS316) coupons of 10 mm diameter were polished using sand paper of grit sizes P400, P800, P1200 and P2500. Coupons were subsequently washed with copious amount of water before sonicating with acetone and finally rinsed with 70% ethanol. The cleaned coupons were stored in 70% ethanol prior to use. The co-culture and pure cultures of bacteria were incubated with stainless steel coupons hung in the culture flasks. Coupons were removed at 48 h and gently rinsed with PBS thrice to remove any loosely adhering bacterial cells. In addition, vigorous washing of the coupons was performed by vortexing the coupons for 10 s followed by washing with copious amount of PBS thrice. Gently and vigorously washed coupons were separately immersed in 1 ml of fresh TSB in 10 ml vials. The vial containing the coupon and TSB was sonicated in a water bath for 3 min at 50 KHz to detach the sessile cells. The samples were spiral plated onto agar plates containing selective media. The plates were incubated at 37°C for 24–48 h, followed by automatic counting of the colony forming units (cfu) using an automatic colony counter (aCOLyte, Synbiosis, Frederick, MD). Mannitol salt agar and P. aeruginosa agar (Oxoid, Basingstoke, UK) were used as selective media for S. aureus and P. aeruginosa respectively. For enumeration of planktonic cells, serial dilutions of the samples were spirally plated on selective media followed by automatic counting of the colonies. Colony diameter was estimated with SigmaScan software from Systat Software Inc.

Bacterial strains and growth conditions The two bacterial strains employed in this study were S. aureus ATCC 25923 and P. aeruginosa NRRL-B 3509. Pseudomonas aeruginosa was obtained from Agricultural Research Service culture collection, United States Department of Agriculture (USDA). Tryptic soy broth (TSB) media and tryptic soy agar (TSA) media were purchased from Merck Millipore. Bacterial cells were streaked in Petri plates on solid TSA media and grown for 24 h at 37°C. Cells from a single colony were then transferred to a culture flask containing TSB media under sterile conditions. Cultures were grown in a shaker incubator at 37°C and 100 r.p.m. until stationery phase was reached. For mixed culture experiments, stationary phase cultures of both bacterial species were mixed in equal volume and diluted with 2X TSB. For pure culture experiments, stationary phase bacterial cultures were diluted with 4X TSB. Both types of cultures were incubated at 37°C and 100 r.p.m. in a shaker incubator for 48 h before further analysis. The minimum inhibitory concentration (MIC) of kanamycin (SigmaAldrich) was determined according to the Clinical and Laboratorial Standards Institute (CLSI) microbroth dilution for S. aureus in the pure culture and the co-culture.

Imaging of surface attached cells The sessile cells on SS316 coupons were fixed overnight with 3% gluteraldehyde (in PBS), rinsed with PBS and dehydrated with an ethanol gradient (25%, 50%, 70%, 90% and 99%). The dehydrated samples were stored in desiccators. Prior to analysis, the coupons were coated with platinum at a voltage of 30 mV for 60 s. A field emission scanning electron microscope (FESEM) (JEOL JSM5600) with 15 kV beam voltage was used to visualize the bacteria attached on SS316 coupons.

Preparation of cell probes and AFM force measurement To prepare cell probes, silicon nitride tip-less cantilever (Veecoprobes Nanofabrication Center, CA) was irradiated under UV light for 30 min. The cantilever was then treated with 4 mg ml−1 dopamine hydrochloride in 10 mM Tris buffer (pH 8.5) solution for 1 h to coat the inner surface before it was washed with deionized water and dried under vacuum (Kang and Elimelech, 2009). A single bacterial cell was immobilized under the functionalized cantilever by

© 2015 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology, 17, 4459–4468

Bacterial biofilm formation in co-culture moving it onto a single bacterium spread using an Agilent AFM system coupled to an upright microscope. The cantilever was maintained in contact with the bacterium for 60 s to ensure cell attachment (Harimawan et al., 2011). The presence of bacterial cell on the cantilever was further confirmed by FESEM. Polydopamine functionalized bare AFM tip was used as control in the AFM force measurement. The spring constant of cantilevers was re-calibrated after the attachment of a bacterium by thermal tune method. Force curves were obtained on SS316 surface buffered with PBS solution. Data were sampled from at least six locations on the surface of the coupon. The total duration of each experiment was kept less than an hour to ensure cell viability at the end of the experiment. Data were analysed with the software PICOVIEW and MATLAB. Plots were generated with MICROSOFT EXCEL 2010. Isolation of surface peptides Cells were harvested by centrifugation at 3000 r.p.m. for 15 min at 4°C. Harvested cells were gently washed three times with ice cold wash solution (20 mM Tris-HCl, 150 mM NaCl, pH 7.6) and were re-suspended in one hundredth volume re-suspension solution (20 mM TrisHCl, 150 mM NaCl, 10 mM CaCl2, 1M D-arabinose, pH 7.6). Digestions were carried out with 20 mg of Trypsin and 8 mg of proteinase K (Sigma Aldrich) for 30 min at 37°C with gentle shaking. The digestion mixtures were centrifuged at 5000 r.p.m. for 10 min at 4°C, and the supernatants (containing the peptides) were filtered using 0.22 mm pore-size filters (Solis et al., 2010). Protease reactions were stopped with formic acid at 0.1% final concentration. Before analysis, salts were removed by off-line ultra-performance liquid chromatography (UPLC), with a 7 min gradient of 2–80% acetonitrile in 0.1% formic acid. Peptide fractions were concentrated under vacuum and kept at −20°C until further analysis (Rodriguez-Ortega et al., 2006). All samples were collected in biological duplicates. Identification of proteins Extracted peptide samples were separated by reversephase UPLC (Waters Corp.) coupled to a microTOF-Q (Bruker Daltonics, Germany). Samples were eluted at 1 μl min−1 over an increasing linear gradient of acetonitrile [0–40% Buffer B (90% acetonitrile, 0.5% acetic acid)] over 60 min. For each MS scan, the five most intense peptide ions were automatically selected for fragmentation by MS/MS. A peptide separation column (Acquity UPLC BEH300 C18 1.7 um, 2.1 × 100 mm) from Waters was used, and the samples were run in positive ionization mode.

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Identification of proteins was carried out with a homology search program SPIDER (Han et al., 2005; Ma and Johnson, 2012). For the de novo sequencing, the parent ion tolerance was set to 1 Da and the fragment ion tolerance to 0.6 Da. For the homology search in SPIDER, the parent ion tolerance was set to 1 Da. A SPIDER score above 30 was considered a confident hit. Homology search was carried out as the genomes of both strains used in this study have not been sequenced, and therefore the corresponding proteins do not exist in the database. The peptide database was obtained from HAMAP resource, Aureus DB and BioCyc. Protein databases were downloaded for a total of 19 strains of S. aureus.

Subcellular localization and structure prediction To filter off the contaminant cytosolic proteins, subcellular localization of the identified proteins was determined by PSORT v.3.0 (Yu et al., 2010). Only those surface proteins that were identified as a confident hit in all replicate samples are reported in this study. Structure prediction was performed with RAPTOR (Xu et al., 2003). Protein cell adhesion receptors were predicted using the software program ADHEN (Wang et al., 2011).

Statistical analysis Quantification of cells was carried out in biological triplicates and technical duplicates (n = 6). Statistical significance (P < 0.05) was determined using unpaired, two tailed t-test.

Results Planktonic interactions The results for the number of planktonic cells in the pure cultures and the co-culture are presented in Table 1. In the pure culture, the final population of planktonic S. aureus was not significantly different (P > 0.05) from its initial population, whereas P. aeruginosa exhibited about 30% reduction in its population at the end of 48 h. In comparison, the planktonic populations of S. aureus and P. aeruginosa in the co-culture were markedly reduced (by 72% and 66% respectively). In this study, SCV phenotype was monitored by plating the bacterial cells from the pure and co-culture on TSA plates supplemented with 30 gl−1 NaCl (to suppress the growth of P. aeruginosa on the plates). After 48 h of incubation, the diameter of the cells from the co-culture [at 0.5 ± 0.2 mm, 95% confidence interval (CI): 0.3–0.7] was significantly lower (P < 0.05) than the diameter of the cells from the pure culture (at 1.0 ± 0.1 mm, 95% CI: 0.9–1.1) (Fig. 1). The S. aureus cells in the co-culture also

© 2015 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology, 17, 4459–4468

4462 A. Kumar and Y. P. Ting Table 1. Comparison of planktonic cell population (cfu/ml) in pure culture and co-culture. Culture type

Stage

S. aureus

P. aeruginosa

Total

Pure culture

Initial population Final population Initial population Final population

2.84 ± 0.3 × 109,a 2.65 ± 0.3 × 109,a 2.84 ± 0.3 × 109,a 5.12 ± 0.9 × 108,b

4.98 ± 0.9 × 109,a 3.46 ± 0.6 × 109,b 4.98 ± 0.9 × 109,a 1.69 ± 0.2 × 109,c

7.8 × 109 6.1 × 109 7.8 × 109 2.2 × 109

Co-culture

Different letters within a column (a, b, c) indicate that the means are significantly different (P < 0.05). The experiments were performed in biological triplicates and technical duplicates (n = 6).

exhibited improved resistance to the antibiotic kanamycin. At the end of 48 h, the MIC of S. aureus cells from the co-culture was observed at 32 mg l−1 in comparison to the MIC of S. aureus cells in the pure culture at 6 mg l−1.

Biofilm formation Surface attachment of bacteria in the co-culture was denser compared with the pure cultures (Table 2). On the gently washed coupons, the attachment of S. aureus cells to SS316 coupons in the co-culture (3.65 × 103 cfu mm−2) was higher by more than an order of magnitude compared with that by pure culture cells (2.9 × 102 cfu/mm2). Similarly on the vigorously washed coupons, S. aureus cells were approximately 10X lower in the pure culture (8 × 101 cfu/mm2) compared with the co-culture (7.6 × 102 cfu mm−2). Surface attachment of P. aeruginosa was also significantly higher (P < 0.05) in the co-culture compared with the pure culture regardless of the coupon washing technique. Scanning electron microscope imaging of the sessile cells on SS316 coupons revealed that S. aureus cells from the pure culture were only occasionally detected on SS316 coupons, most often as a cluster of two cells (Fig. 2A). In the co-culture, S. aureus cells attached in higher numbers and were found in close contact with P. aeruginosa (Fig. 2B) on a number of occasions. The number of S. aureus cells in contact with one P. aeruginosa cell ranged from 0–16 (7 ± 6).

nique as the previous method of glutaraldehyde fixation led to cross-linking of proteins and amino acids in the exocellular polymeric layer and significantly influenced the interaction between the bacteria and the surface. On the other hand, the polydopamine technique is noninvasive and does not affect cell viability (Kang and Elimelech, 2009). Instead of one, two cells were attached to AFM cantilever as a single individual cell of S. aureus was rarely observed. Hence, the AFM force spectroscopy results illustrate the interaction due to two bacteria rather than a single bacterium. Figure 3B shows AFM force curves between SS316 coupons and S. aureus in the pure culture and the co-culture. It is evident that the maximum attractive force between S. aureus and SS316 surface increased significantly from about 8 nN [standard deviation (SD) < 5%] in the pure culture to about 35 nN (SD < 10%) in the co-culture. It is also noteworthy that the range of interaction between S. aureus and SS316 surface also

A

B

SCV

AFM force spectroscopy Figure 3A presents SEM image of an AFM cell probe. AFM tips were functionalized with polydopamine tech-

Fig. 1. Colonies of cells (on TSA + 30 g NaCl) after 48 h from (A) pure culture and (B) co-culture. The arrow points to an SCV on the plate with cells from co-culture.

Table 2. Comparison of sessile cell population (cfu/mm2) in pure culture and co-culture. Culture type

Washing technique

S. aureus

P. aeruginosa

Total

Pure culture

Gentle washing Vigorous washing Gentle washing Vigorous washing

2.93 ± 0.89 × 102,a 7.67 ± 1.50 × 101,b 3.65 ± 0.57 × 103,c 7.57 ± 1.49 × 102,d

4.76 ± 0.75 × 103,a 3.33 ± 1.63 × 101,b 1.01 ± 0.21 × 104,c 2.63 ± 1.94 × 102,d

5.05 × 103 1.10 × 102 1.36 × 104 1.02 × 103

Co-culture

Different letters within a column (a, b, c, d) indicate that the means are significantly different (P < 0.05). The experiments were performed in biological triplicates and technical duplicates (n = 6).

© 2015 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology, 17, 4459–4468

Bacterial biofilm formation in co-culture

A

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Fig. 2. Scanning electron microscope images of cells attached on SS316 coupons in (A) S. aureus pure culture and (B) co-culture.

B

increased from about 160 nm in the pure culture to approximately 300 nm in the co-culture (Fig. 3).

binding/activation, iron/sulfur storage, regulation of gene expression and enzyme activity (Johnson et al., 2005).

Surface proteins

Discussion

Figure 4 shows the relative distribution of different classes of surface proteins upregulated in S. aureus in the presence of P. aeruginosa in the culture. The individual proteins are listed in Table 3. It is observed that the major family of proteins upregulated are transport proteins which mediate passive and active transport of small solutes across cell membrane. We hypothesize that the presence of P. aeruginosa alters the extracellular environment for S. aureus in the co-culture, and a change in the expression of transport proteins occurs in order for the S. aureus cells to adapt to the new environment. In addition, the upregulation of transport proteins in S. aureus indicates that the biosynthetic pathways within the cells may also be affected. For example, Aminobenzoyl-glutamate transport protein (Table 3) is involved in transport of aminobenzoyl-glutamate (Hussein et al., 1998) and is also known to increase the sensitivity of cells to low levels of aminobenzoyl-glutamate. This protein is a precursor in folic acid biosynthesis which is required for the synthesis of deoxyribonucleic acid (DNA), RNA, amino-acids, etc. It was found that formyltetrahydrofolate was also absent in the extracellular environment in the co-culture indicating that the folic acid biosynthesis was affected (data not shown). In addition to signalling proteins, resistance and virulence proteins were other major class of proteins positively regulated in the co-culture (Fig. 4). Positive regulation of these proteins in the co-culture has significant implications in industry, in particular food and medicine. Other notable proteins were an enzyme similar to Chitin synthase and iron–sulfur cluster binding protein (Table 3). The former has been known to play a role in biofilm formation by Staphylococcus epidermis (Heilmann et al., 1996), and the latter is involved in many biochemical activities including electron transfer, substrate

In comparison to no significant reduction in the population of S. aureus in the pure culture, a five to sixfold reduction in its population in the co-culture indicates the occurrence of strong antagonistic interactions between the two pathogens (Table 1). Other researchers have also observed the occurrence of antagonistic interactions in the co-culture of

A

B

5 0 0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

-5

Force (nN)

-10 -15 -20 -25 -30 -35 Pure culture -40

Symbioc culture

Seperaon distance (μm)

Fig. 3. (A) Scanning electron microscope image of AFM cell probe. (B) Force curves for S. aureus from the pure culture and the co-culture (P < 0.05).

© 2015 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology, 17, 4459–4468

B

4464 A. Kumar and Y. P. Ting

S. aureus

Transport Proteins 32%

Adhesion proteins 14% Reistance proteins 7% Virulence proteins 11%

Putave proteins 18% Signalling proteins 7% Other proteins 11% Fig. 4. Different classes of surface proteins positively regulated on S. aureus cell surface in the co-culture.

the two bacteria. Some authors have described the interaction as parasitic (Mashburn et al., 2005) for the reason that P. aeruginosa kills S. aureus to use it as iron source. Others have considered the interaction between the two pathogens as competitive (Harrison et al., 2007). However, it should also be noted that at some level, synergistic relationship between the two species has not been ruled out by researchers (Pastar et al., 2013).

SEM images reveal that the two bacteria were found in the vicinity of one another (Fig. 2). Therefore, the interaction of S. aureus with P. aeruginosa was not limited to the planktonic phase but also extended to the sessile form. The information on the interaction between the two pathogens in the biofilms is limited. Using confocal laser scanning microscopy, it was shown that P. aeruginosa PAO1 facilitates attachment of S. aureus in flow chambers and both species coexist in the firmly packed microcolonies of the biofilm (Yang et al., 2011). The results in the present study are in agreement with this observation, although the strains used by Yang and colleagues were different than those in the present study. It is important to realize that the attachment of the two pathogens on the colonizing surface increased significantly (P < 0.05) in spite of large reduction in their planktonic cell population in the co-culture (Tables 1 and 2). It appears that the antagonistic interactions in the planktonic phase lead to denser biofilm formation as the latter life form may offer superior resistance to adverse conditions. On the coupons rinsed gently with PBS, the pure culture P. aeruginosa sessile cells were much higher in number compared with the pure culture S. aureus cells. However on the vigorously washed coupons, S. aureus outnumbered P. aeruginosa indicating that both cell types did not adhere to SS316 with the same affinity. On vigorously washed coupons, a 10-fold increase in the number

Table 3. List of S. aureus surface proteins positively regulated in co-culture. Accession no.

Name/Recommended name

Localization

Q99WE0 Q99RG0 Q2FH04 Q931S5 Q99QZ2 Q99UA2 Q932F5 Q99W38 D2NAN6 Q99R97 Q7A2V4 D2N760 Q7A2S1 D1GPZ1 Q99XA7 Q99T22 P0A0J8 Q99VX4 Q931I3 D2NAQ0 Q99US7 D2N884 Q99WC0 D9RQQ6 D9RN08 Q2YVG3 Q6GDP1 Q6GE15 Q99RA9

Multidrug resistant ABC transporter permease Similar to aminobenzoyl-glutamate transport protein Extracellular matrix-binding protein ebh Gamma-aminobutyrate permease Putative uncharacterized protein Putative oligopeptide transport ATP-binding protein Putative anti-porter subunit mnhA2 Putative uncharacterized protein Serine-rich adhesin for platelets PTS system glucoside-specific EIICBA component Fructose 1-phosphate kinase EbhA protein Phosphate-binding protein pstS Phage protein Myosin-cross-reactive streptococcal antigen homologue Similar to iron-sulfur cluster-binding protein Antiseptic resistance protein ATP-binding cassette transporter A Similar to Na+ transporting ATP synthase Chitin synthase Cell division protein SepF Putative uncharacterized protein Putative uncharacterized protein Putative uncharacterized protein Extracellular enterotoxin type I Sec-independent protein translocase protein Copper-exporting P-type ATPase A Immunoglobulin-binding protein Sbi Similar to ABC transporter homologue yydI

Membrane Membrane Cell wall Membrane Cell wall Membrane Membrane Unknown Cell wall Membrane Unknown Cell wall Membrane Transmembrane Unknown Membrane Membrane Membrane Membrane Membrane Unknown Unknown Unknown Unknown Extracellular Membrane Membrane Transmembrane Unknown

ATP, Adenosine triphosphate.

© 2015 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology, 17, 4459–4468

Bacterial biofilm formation in co-culture

A

B

of surface attached S. aureus cells in the co-culture compared with the pure culture indicates that the cells in the co-culture not only attached more densely to the colonizing surface but also more tenaciously. A fourfold increase in the force of interaction (measured by AFM force spectroscopy) corroborates the observed increase in biofilm formation by S. aureus and explains its tenacious attachment to SS316 coupons in the co-culture (Fig. 3B). In a separate study, similar observations were made; AFM force of detachment between bacteria under external antibacterial stress (norfloxacin at sub-inhibitory concentration) and SS316 surface increased (Kumar and Ting, 2013). It was hypothesized that the increase in AFM force of detachment is a result of altered expression of proteins on the bacterial cell surface. Other studies have also observed that surface proteins play a crucial role in biofilm formation and the initial attachment of bacteria to surfaces is mediated by surface proteins (Cucarella et al., 2001; Bogino et al., 2013). The increase in the AFM force of detachment for S. aureus cells in the co-culture culture over the pure culture (i.e. by more than 3 fold) may be attributed to the positive regulation of adhesion proteins. For example, Serine rich glycoproteins (Fig. 5A) are known to mediate adhesion to platelets (Plummer et al., 2005). Similarly, extracellular matrix binding protein ebh helps in adhering S. aureus to extracellular matrix of other hosts. Ebh is a cell envelope-associated protein and is proposed to form a specialized surface structure involved in cellular adhesion (Clarke et al., 2002). Using a protein structure prediction program RAPTOR (Xu et al., 2003), it was found that the extracellular matrix binding is a rod-like protein which extends more than 300 nm in length (Fig. 5B). Similar observations have been made in another study that extracellular matrix binding protein ebh is a rod-like protein of about 320 nm with some plasticity at module junctions (Tanaka et al., 2008). The positive regulation of this protein also explains the increase in the range of interaction between S. aureus cells and SS316 surface

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Fig. 5. Structure prediction of (A) serine-rich adhesins and (B) extracellular matrix binding protein ebh using RAPTOR.

from 160 nm in the pure culture to about 300 nm in the co-culture. In addition, out of the five uncharacterized proteins identified on S. aureus surface (Table 3), three (i.e. D9RQQ6, Q99WC0, Q99W38) contain adhesive integrin domains. Using a program named ‘ADHEN’ (Wang et al., 2011), it was found that the adhesive integrin domains may enable these proteins to act as adhesion proteins. Thus, it is likely that the positive regulation of adhesion proteins in the co-culture aids in preferential attachment of S. aureus cells to the substrates. Of particular interest was a protein named multidrug resistant ABC transporter permease that was positively regulated on S. aureus surface in the co-culture. In general, ABC transporter permeases are involved in the import and export of a variety of substances, including multidrug exporters (Kuroda et al., 2001; Ohta et al., 2004; van Pée and Patallo, 2006). However, multidrug resistant ABC transporter permeases are involved exclusively in efflux of xenobiotics from the cell. In the lungs of cystic fibrosis patients, P. aeruginosa selects small colony variants (SCVs) of S. aureus cells by killing the more susceptible ones (Biswas et al., 2009). Thus, P. aeruginosa bolsters the fitness of S. aureus as SCVs are relatively fitter cells and less susceptible to antibiotic treatment. In this study, the marked difference in the colony size of S. aureus form the pure culture and the co-culture indicated SCV selection (Fig. 1). This was further corroborated by higher MIC value of kanamycin against S. aureus cells in the co-culture (32 mg l−1) compared with the pure culture (6 mg l−1). The positive regulation of multidrug resistant ABC transporter permease in the co-culture indicates that this protein probably plays a role in SCV selection. It is appealing to hypothesize that P. aeruginosa release molecules (xenobiotics) which find their way to penetrate S. aureus cells and cause harm. Staphylococcus aureus cells which express this protein in large enough numbers expel the xenobiotics out and survive, whereas those which fail to do so in adequate numbers die (Fig. 6).

© 2015 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology, 17, 4459–4468

4466 A. Kumar and Y. P. Ting Fig. 6. Role of multidrug resistant-ABC transporter in small colony variant (SCV) selection. Multidrug resistant-ABC transporter was not detected in the pure culture and upregulated in the co-culture. It is hypothesized that SCV cells express a relatively larger number of mdr-ABC transporter which expel the xenobiotics out of the cells and help them survive in the presence of P. aeruginosa in the culture, leading to SCV selection.

(SCV)

P

AT

xenobiotics

AT

P

mdr-ABC transporter

(normal cell)

Conclusion In pure cultures, S. aureus attached to surfaces in miniscule numbers and formed sparse biofilms. However, in the presence of P. aeruginosa in a co-culture, attachment of S. aureus to solid surfaces increased significantly. Using AFM force spectroscopy, a comparison of the detachment forces of S. aureus in a pure and a co-culture (with P. aeruginosa) revealed that cells in the latter were much more difficult to detach upon initial attachment on SS316. Isolation and identification of surface shaved peptides revealed that S. aureus in the co-culture upregulated the expression of a number of proteins. Apart from resistance, virulence and signalling proteins, many transporter proteins were positively regulated in the co-culture. The higher force of detachment of S. aureus from SS316 surface in co-culture may be due to the positive regulation of adhesion proteins. Additionally, positive regulation of extracellular matrix binding protein ebh may also explain the increase in the range of interaction between the bacteria and the surface in a co-culture. Of particular interest was a protein expressed by S. aureus in the co-culture, named multidrug resistant ABC transporter permease, the positive regulation of which indicates that P. aeruginosa secretes compounds harmful to S. aureus and penetrates inside the surface barrier of S. aureus. It is likely that this protein plays a key role in small colony variant selection of

S. aureus cells in the presence of P. aeruginosa, thereby bolstering the fitness of the former against antibiotics. Acknowledgements The authors are sincerely grateful to Ms. Chen Sicong for providing training on the AFM force spectroscopy measurements and to Agricultural Research Services (ARS) Culture Collection, USDA for providing the P. aeruginosa strain.

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Presence of Pseudomonas aeruginosa influences biofilm formation and surface protein expression of Staphylococcus aureus.

Although Staphylococcus aureus and Pseudomonas aeruginosa can individually colonize and infect their hosts, the commensalistic effect of the two is mo...
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