Conventional laboratory agar media provide an iron-limited environment for bacterial growth

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1 FEMS Microbiology Letters 50 (1988) Published by Elsevier FEM Conventional laboratory agar media provide an iron-limited environment for bacterial growth 1. SUMMARY I.A. Critchley and M.J. Basker Beecham Pharmaceuticals Research Division, Brockham Park, Betchworth, Surrey, ~K. Received 7 December 1987 Accepted 10 December 1987 Key words: Iron limitation; Siderophore; Outer membrane The outer membrane proteins of Escherichia coli and Pseudomonas aeruginosa grown in a number of conventional laboratory media were examined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) Highmolecular-weight proteins similar to those produced by these strains in an iron-limited chemically defined medium were detected in cells grown on the surface of various agar media. In contrast, these proteins were not produced or were only poorly expressed by the corresponding broth cultures or by cells grown an agar supplemented with iron. A catecholic substance could be detected in DST agar extracts subsequent to bacterial growth which was produced to a lesser extent in IST agar and in broth cultures. 2. INTRODUCTION Nutrient limitation is known to have a profound effect on the composition and surface structure of bacteria [1,2]. For example, under conditions of iron limitation, many bacteria induce a Correspondence to: M.J. Basker, Beecham Pharmaceuticals Research Division, Brocldaam Park, Betchworth, Surrey, RH3 7AJ, U.K. high-affinity iron transport system involving the production of iron-chelating agents (sidetophores) and several high-molecular-weight iron-regulated outer membrane proteins (IROMPs) which act as receptors for the siderophore-iron complexes [3]. Iron-starved bacteria are produced in vitro by cultivation either in an iron-deficient chemically defined liquid medium or in a conventional broth which has been rendered iron-limited by passing through an ion-exchange resin [4] or by incorporating an iron-chelating agent such as bipyridyl or desferal [5]. Few investigators have examined outer membrane protein profiles of organisms grown on the surface of agar media, presumably because of difficulties in obtaining a sufficient number of cells for analysis. In this study we compared the outer membrane proteins of bacteria grown in iron-limited chemically defined media with.those in conventional laboratory media, and noted surprising differences in the profiles between agarand browth-grown cells. 3. MATERIALS AND METHODS 3.1. Strains E. coli NCTC10418 and P. aeruginosa NCTC10662 are standard reference strains; the other cultures were recent clinical isolates /88/$ Federation of European Microbiological Societies

2 Media Outer membrane proteins were prepared from bacteria grown in iron-sufficient and iron-limited chemically defined liquid media (CDM + Fe and CDM - Fe) [6], tryptone soya broth (TSB, Oxoid), iso-sensitest broth and agar (IST, Oxoid), Mueller-Hinton broth and agar (BBL and Difco) and diagnostic sensitivity test agar (DST, Oxoid). The initial cultures were grown overnight in CDM - Fe at 37 C in shaken cultures and 0.5 ml used to inoculate batches of broth media (50 ml) in 250-ml Erlenmeyer flasks or spread over the surface of agar media (50 ml) in 140 mm-diameter Petri dishes. All media were incubated at 37 C for 5 h to ensure that cells were in the mid- to late exponential growth phase Preparation and examination of outer membrane proteins Cells were harvested by centrifugation at x g for 10 rain at 4, either directly in the case of broth cultures or after removal from the surface of agar into cold phosphate buffer (0.01 M, ph 7.0). The pellets were washed twice and finally resuspended in 2.5 ml of buffer. Cell suspensions were subjected to ultrasonic treatment (MSE, Soniprep 150) for 5 min in 6 equal periods allowing 30-s intervals for cooling. Undisrupted cells and large fragments were removed by centrifugation at x g for 10 min at 4 C. The supernatant was then centrifuged twice at x g for 60 rain at 8 C to produce a gelatinous membrane pellet. The protein content of these pellets were determined by the method of Bradford [7]. Membrane preparations were diluted with phosphate buffer to give a protein concentration of 7 mg/ml and mixed with n-lauroylsarcosine (Sigma) at a final concentration of 1%. The insoluble outer membrane and peptidoglycan layer were obtained by centrifugation at x g for 20 min at 8 C. Outer membrane protein suspensions (100 kd) were mixed with gel sample buffer (50 #1) consisting of 0.2 M Tris-HC1, ph 6.8, 3% (w/v) SDS, 30% (v/v) glycerol and 0.002% bromophenol blue, followed by 2-mercaptoethanol (20/~1). Each sample was heated to 100 C for 4 min and then fractionated by SDS-PAGE (BioRad 'Protean' gel system) as described by Laemmli and Favre [8]. Gels were fixed in fresh 50% (w/v) methanol/10% (v/v) acetic acid containing 0.1% (w/v) PAGE blue 83 (BDH) for 1 h and then left to destain overnight in 15% (v/v) methanol/10% (v/v) acetic acid Siderophore assays The presence of catecholic substances in laboratory media was determined by the method of Rioux [9]. For broth media, bacteria were harvested by centrifugation and the culture supernatant fluids were assayed directly. For agar media, bacterial growth was scraped off the surface of the plate, the agar was washed twice with sterile deionised water and then left to equilibrate with sterile deionised water (10 ml) for 1 h at room temperature before assay. A modification of the universal chemical assay of Schwyn and Neilands [10] was also used to detect siderophore production in agar media. This involved adding the reagents chrome azurol S and hexadecyl-trimethyl ammonium bromide HDTMA, (Aldrich) directly to molten agar. Orange 'halos' around the colonies indicated siderophore production. 4. RESULTS The outer membrane profiles of E. coli NCTC10418 and P. aeruginosa NCTC10662 cultivated in a variety of conventional laboratory media are shown in Figs. 1 and 2. The cells grown on the surface of DST, IST and Mueller-Hinton agars synthesised additional outer membrane proteins which had the same molecular weight as the IROMPs produced by those organisms when grown in CDM-Fe medium. In contrast these proteins were not seen or were only poorly expressed in the outer membrane of cells grown in the corresponding broth media or in TSB. It was also noted that, with both organisms, the intensity of staining of the high-molecular-weight proteins was greatest with ceils grown on DST agar than on other agar media, suggesting some variation in the degree of iron-limitation and hence in the quantities of protein synthesised. Furthermore, the

3 _/74K 72K n68k Fig. 1. Outer membrane proteins of E. cob NCTC10418 grown in different media. CDM + Fe and CDM- Fe (1 and 2), IST broth and agar (3 and 4), BBL Mueller-Hinton broth and agar (5 and 6), Difco Mueller-Hinton broth and agar (7 and 8), TSB (9), DST (10) and DST supplemented with 0.05 mm FeSO 4 (11). addition of iron (0.05 ram) to DST suppressed the expression of these proteins. Similar outer membrane profiles have been obtained for other strains of E. coli and P. aeruginosa and for a strain of Klebsiella pneumoniae (not shown). Several members of the family Enterobacteriaciae produce a catecholic siderophore (en- Table 1 Production of Catecholic Substances by bacteria grown in different media Results represent mean from 2 independent estimations. Organism Catechol concentration equivalence (#g/ml) a Agar extracts IST DST DST mm FeSO4 E. coli E96 R K. pneumoniae Va2 R K. pneumoniae T K. pneumoniae E P, aeruginosa Charlier a Quantified using catechol as a standard! Fig. 2. Outer membrane proteins of P. aeruginosa NCTC grown in different media. CDM + Fe and CDM- Fe (1 and 2), IST broth and agar (3 and 4), BBL Mueller-Hinton broth and agar (5 and 6), Difco Mueller-Hinton broth and agar (7 and 8), TSB (9), DST (10) and DST supplemented with 0.05 mm FeSO 4 (11). terochelin) in response to iron stress [3], which is released into the surrounding medium. Indeed, it proved possible to detect a catecholic substance in agar extracts particularly DST after growth of several bacteria which was produced to a lesser extent if iron was added to the medium (Table 1). Liquid media supernatants IST TSB CDM CDM Broth + Fe - Fe ~

4 38 Fig. 3. Bacterial siderophore production on conventional agar media. E. coli E96 R+ grown on IST and DST (3a and b), K pneumoniae VA2 R+ grown on IST and DST (3c and d), P. aeruginosa Horton R+ grown on IST and DST (3e and f).

5 Similarly only low concentrations were found after growth in liquid media other than CDM-Fe. The P. aeruginosa strains examined gave negative results because their siderophore (pyochelin) is not catecholic and cannot be measured using the same methods [11]. However, it was possible to show that P. aeruginosa produced siderophores whilst growing on agar, particularly DST, as a pronounced colour change occurred in the agar medium containing an iron-chelating dye (Fig. 3). Similar results were seen also with E. coli and K. pneumoniae. 5. DISCUSSION Complex laboratory media have been widely acknowledged to be iron-sufficient. This certainly appears to be true for conventional broth media but, from the results of these studies, not necessarily for the corresponding agar formulations. Presumably agar itself has some iron-chelating properties. Bacteria isolated from human infections have been shown to possess iron-uptake systems [4,12,13] that are repressed on transfer into iron-rich broth media. Direct subculture onto agar, therefore, may be of value for clinical isolates as this would allow for continued growth in an iron-limited environment. The expression of IROMPs by a surface-grown culture of K. pneumoniae has been reported previously [14], however, these workers used a specially prepared iron-depleted medium rather than a conventional agar for bacterial growth. The results of this study also have implications for antibiotic susceptibility testing of compounds able to utilise the iron-uptake pathway. For example, Watanabe et al. [15] provided evidence that the catecholic cephalosporin, E-0702, was transported into E. coli via a tonb-dependent iron transport system. Similarly, the catecholic penicillin, BRL35560A, appeared much more active when tested in agar, particularly in DST, than in a broth medium [16]. Presumably the compound is taken into the cells by both porin proteins and IROMPs in agar but only the porin channels in broth. REFERENCES [1] Brown, M.R.W. and Williams P. (1985) Annu. Rev. Microbiol. 39, [2] Brown, M.R.W. (1977) J. Antimicrob. Chemother. 3, [3] Neilands, J.B. (1982) Annu. Rev. Microbiol. 36, [4] Brown, M.R.W., Anwar, H. and Lambert, P.A. (1984) FEMS Microbiol. Lett. 21, [5] Chart, H., Buck, M., Stevenson, P. and Griffiths, E. (1986) J. Gen. Microbiol. 132, [6] Kadurugamuwa, J.L., Anwar, H., Brown, M.R.W. and Zak, O. (1985) Antimicrob. Agents Chemother. 27, [7] Bradford, M.M. (1976) Anal. Biochem. 72, [8] Laemmli, U.K. and Favre, M. (1973) Y.. Mol. Biol. 80, [9] Rioux, C., Jordan, D.C. and Rattray, J.B.M. (1983) Anal. Biochem. 133, [10] Schwyn, B. and Neilands, J.B. (1987) Anal. Biochem. 160, [11] Cox, C.D. and Graham, R. (1979) J. Bacteriol. 137, [12] Lam, C., Turnowsky, F., Schwarginger, E. and Neruda, W. (1984) FEMS Microbiol. Lett. 24, [13] Shand, G.H., Anwar, J., Kadurugamuwa, J., Brown, M.R.W., Silverman, S.H. and Melting, J. (1985) Infect. Immun. 48, [14] Kadurugamuwa, J.L., Anwar, H., Brown, M.R.W., Shand, G.H. and Ward, K.H. (1987) J. Clin. Microbiol. 25, [15] Watanabe, N., Nagasu, T., Katsu, K. and Kitoh, K. (1987) Antimicrob. Agents Chemother. 31, [16] Basker, M.J., Edmondson, R.A., Knott, S.J., Ponsford, R.J., Slocombe, B., and White, S.J. (1984) Antimicrob. Agents Chemother. 26,