Media effects of sugars on pigmentation and antibiotic susceptibility in Serratia marcescens.

Abstract

Serratia marcescens produces a red pigment called prodigiosin, and lack of production has been associated with reduced antibiotic susceptibility. Many patients are hospitalized with infections caused by this microbe each year. Prodigiosin synthesis on Tryptic Soy and Nutrient Agar supplemented with glucose, lactose, sucrose, or sorbitol was evaluated and colonies were subjected to growth on Triple Sugar Iron slants. Antibiotic sensitivities to 12 antibiotics on supplemented media was measured. Further, motility of bacteria on each media was determined since certain sugars have been implicated in repression of flagellar synthesis. Results indicated that glucose and sorbitol have a repressive effect on prodigiosin synthesis and TSI slants may also cause selective repression. At 25[degrees]C, antibiotic inhibition zones decreased on TSA with sorbitol, whereas all bacteria exhibited motility, irregardless of the media they were grown on.

Key words: antibiotic, pigment, prodigiosin, Serratia marcescens

1. Introduction

Serratia marcescens is a common environmental microbe often associated with human diseases, including respiratory and urinary tract infections (Daschner 1980), and colonizes immunocompromized patients (Tsuji et al. 1992). Extensive studies of its secondary metabolite production of the red pigment 2-methyl-3-pentyl-6-methoxyprodigiosin (prodigiosin) (Williams et al. 1971), has shown antimicrobial and immunosuppressive properties (Tsuji et al. 1992, Williams and Qadri 1980, respectively). Correlation between prodigiosin and flagellar production (Goluszko et al. 1995, Kobayashi and Ichikawa 1990, Paruchuri and Harshey 1987), prodigiosin and carbapenem synthesis (Thomson et al. 2000), as well as prodigiosin synthesis, motility, and growth conditions (Lai et al. 1997, Liu et al. 2000, Loren and Guinea 1978, Williams et al. 1971) has produced conflicting results. Malouin et al. (1991) found that increased glucose concentrations decreased susceptibility of Escherichia coli to ceftazidime. This study documents catabolite repression of prodigiosin synthesis, motility, and antibiotic susceptibility properties based upon different sugar supplemented growth conditions using S. marcescens D1 American Type Culture Collection (ATCC) 14756.

2. Materials and Methods

Serratia marcescens ATCC 14756 was grown on nutrient agar (NA), tryptone agar, and tryptic soy agar (TSA) (Difco) plates at 25[degrees]C and then a "lawn of growth" was inoculated to NA and TSA plates supplemented with one sugar (10% by weight/volume addition of glucose, sucrose, lactose, or sorbitol to traditional media) and incubated at 25[degrees]C, 30[degrees]C and 37[degrees]C for 24 hours. Antibiotic disks (BBL) containing chloramphenicol, ciprofloxacin, gentamicin, kanamycin, lincomycin, nitrofurantoin, moxalactam, polymyxin B, rifampin, streptomycin, sulfa/trimethoprim, and tetracycline were added to the sugar supplemented plates. Plates containing no antibiotics served as a control of growth and prodigiosin production. Unsupplemented TSA and NA plates also served as controls. Sulfur-Indole-Motility (SIM) tubes and wet mounts were utilized for the motility study, and Triple Sugar Iron (TSI) slants determined sugar fermentation. This study was performed in duplicate or triplicate to ensure reproducibility. Photographs were taken with a Nikon 990E digital camera.

3. Results

Growth on the supplemented media produced different hues of prodigiosin production, which was dependent upon temperature or carbon source. Pigmentation at 30[degrees]C was less than at 25[degrees]C, and colonies incubated at 37[degrees]C appeared white, which supported observations by Williams et al. (1971). Growth on sugar-supplemented media ranged from dark pink (TSA with sucrose) to completely colorless (NA with glucose). Paruchuri and Harshey (1987) correlated hue visualization with prodigiosin production level, concluding that significantly different levels were synthesized on various media. The lighter-colored colonies appeared on the media supplemented with glucose, which has been shown to inhibit prodigiosin synthesis (Loren and Guinea 1978) and repress phospholipase production (Givskov and Molin 1992). Tryptic soy agar plates appeared more orange than pink when compared to the NA plates, especially on the lactose and glucose supplemented media. Colonies grown on TSA with sorbitol were the lightest shade seen with TSA. Conversely, lighter pink colonies were visualized on NA supplemented with lactose, but the sorbitol appeared dark pink.

Antibiotic susceptibility of Serratia was measured after 24 hours of growth. Plates were rechecked at 48 hours, with no inhibition zone differences observed. Serratia appeared resistant to cefaclor, colistin, lincomycin, penicillin, rifampin, streptomycin, and tetracycline since no inhibition zones were observed. Chloramphenicol, ciprofloxacin, gentamicin, moxalactam, and sulfa/trimethoprim all showed a difference in the antibiotic susceptibility from one designation (sensitive, intermediate, or resistant) to another. Sensitivity category designation to kanamycin, nitrofurantoin, and polymyxin B did not change, even though inhibition zones ranged from 12-17 mm, 0-11 mm, and 0-7.5 mm, respectively. Thus, sugar substrate appears to change inhibition zone size whether or not a difference in category assignment is seen. Surprisingly, Serratia grown on TSA media supplemented with sorbitol continually exhibited the smallest zone of inhibition, whereas NA zones were variable. The results of the other antibiotics are shown in Table I.

Growth on sugar and antibiotic supplemented plates demonstrated traditional lighter Serratia rings around the antibiotic disks. This phenomenon has previously been demonstrated in pigmented organisms, especially in response to chloramphenicol. However, on different media types, darker rings were observed around the antibiotics (Figure 1). Since the pH of NA plates is 6.8, and the pH of TSA is 7.3, the pH may cause a physiological response in prodigiosin regulation due to antibiotic stress.

[FIGURE 1 OMITTED]

Motility results showed that the organisms grown on control and supplemented plates were motile in SIM tubes. Wet mounts made from each plate confirmed that each culture was very motile, which also supports observations by Liu et al. (2000).

Finally, organisms from the 25[degrees]C plates were subcultured onto TSI slants and grown at 25[degrees]C for 24 h (Fig. 2). Colonies from NA with sorbitol (dark pink on plates), TSA with lactose (orange), and the original tryptone (medium orange) strain all exhibited prodigosin synthesis and a result of K/A (not simply due to red pigmentation--no yellow was seen on the slant). The NA with sucrose grown at 37[degrees]C appeared white, but when it was transferred to the TSI slant and grown at 25[degrees]C, prodigosin synthesis resumed, which also supports previous observations (Williams et al. 1971).

[FIGURE 2 OMITTED]

Tryptic soy agar with sucrose (light to medium pink), NA with sucrose (light-medium pink) TSA with glucose (medium pink), NA with glucose (dark orange), TSA with sorbitol (white), and NA with lactose (light orange) exhibited white colonies and showed an A/A result, even if they were previously pigmented. The glucose in the butt of the TSI slants was fermented by each strain, but the fermentation of lactose and/or sucrose was variable.

4. Conclusions and Discussion

These results appear to support catabolite repression by glucose and sorbitol with prodigiosin production. However, the results do not necessarily support an inverse proportion of prodigiosin production and flagellar production as demonstrated by Kobayashi and Ichikawa (1990). Further, antibiotic susceptibility appears to be affected by prodigiosin synthesis levels, with higher prodigiosin levels increasing antibiotic susceptibility, whereas inhibited catabolite repression seems linked to antibiotic resistance.

Liu et al. (2000) determined that prodigiosin is not regulated by the flhDC gene, and they did not observe flagellar inhibition with glucose supplemented up to 0.5%. In fact, they found that an increased glucose level enhanced flhDC activity, which is responsible for nuclease A and phospholipase expression. Williams et al. (1971) linked casein and yeast extract as enabling prodigiosin to be synthesized. Based upon the variation in pigment hues between the NA, TSA, and TSI slants, the possibility of peptones, ferrous sulfate, and sodium thiosulfate serving as repressors exists since they are the ingredients which differ between media. The peptone, ferrous sulfate, and sodium thiosulfate may also interact with lactose or sucrose to cause prodigiosin repression. Future explorations could involve both media and molecular expression observations.

Acknowledgements

Thank you to William Kirby and Glenda Carmack for their laboratory and technician support.

Literature Cited

Daschner, F.D. 1980. The epidemiology of Serratia marcescens. In The Genus Serratia, Von Graevenitz, A., and Rubin, S.J. (eds.). Boca Raton, FL: CRC Press pp. 187-194.

Givskov, M., and S. Molin. 1992. Expression of extracellular phospholipase from Serratia liquifaciens is growth-phase-dependent, catabolite repressed and regulated by anaerobiosis. Mol. Microbiol. 6:1363-74.

Goluszko, P., B. Nowicki, E. Goluszko, S. Nowicki, A. Kaul, and T. Pham. 1995. Association of color variation in Serratia marcescens with the differential expression of protease and type 1 fimbriae. FEMS Microbiol. Lett. 133: 41-45.

Kobayashi, N. and Y. Ichikawa. 1990. Inverse relationship between the flagella formation and prodigiosin synthesis in Serratia marcescens. Microbiol. Immunol. 34:347-54.

Lai, H., J. Shu, S. Ang, M. Lai, B. Fruta, S. Lin, K. Lu, and S. Ho. 1997. Effect of glucose concentration on swimming motility in Enterobacteria. Biochem. Biophys. Res. Comm. 231: 692-5.

Liu, J., M. Lai, S. Ang, J. Shu, P. Soo, Y. Horng, W. Yi, H. Lai, K. Luh, S. Ho., and S. Swift. 2000. Role of flhDC in the expression of the nuclease gene nucA, cell division and flagellar synthesis in Serratia marcescens. J. Biomed. Sci. 7:475-483.

Loren, J.G. and J. Guinea. 1978. Effect of glucose concentration on the biosynthesis of prodigiosin by Serratia marcescens. Revista Espanola de Fisiologia. 34:247-52.

Malouin, F, S. Chamberland, N. Brochu, and T.R. Parr, Jr. 1991. Influence of growth media on Escherichia coli cell composition and ceftazidime susceptibility. Antimicrob. Agents. Chemother. 35:477-83.

Paruchuri, D.K. and R.M. Harshey. 1987. Flagellar variation in Serratia marcescens is associated with color variation. J. Bacteriol. 169: 61-5.

Thomson, N.R., M.A. Crow, S.J. McGowan, A. Cox, and G.P.C. Salmond. 2000. Biosynthesis of carbapenem antibiotic and prodigision pigment in Serratia is under quorum sensing control. Mol. Microbiol. 36:539-556.

Tsuji, R.F., J. Magae, M. Yamashita, K. Nagai, and M. Yamasaki. 1992. Immunomodulating properties of prodigiosin 25-C, an antibiotic which preferentially suppresses induction of cytotoxic T cells. J. Antibiot. 45:1295-1302.

Williams, R.P. and S.M. Qadri. 1980. The pigment of Serratia. In The Genus Serratia. Von Graevenitz, A., and Rubin S.J. (eds.). Boca Raton, FL: CRC Press pp. 31-75.

Williams, R.P., C.L. Gott, S.M.H. Qadri, and R.H. Scott. 1971. Influence of temperature of incubation and type of growth medium on pigementation of Serratia marcescens. J. Bacteriol. 106: 438-443.

Anna R. Oller

Department of Biology

Central Missouri State University, WC Morris 306, Warrensburg, MO 64093

TABLE 1. Antibiotic Sensitivity of Serratia to various antibiotics on
different media. The numbers represent the inhibition results measured
in millimeters and the letters I (Intermediate), R (Resistant), and S
(Sensitivity) designate the assigned antibiotic sensitivity according
to the BBL insert. The bolded results indicate the smallest zone (most
resistant) measured. Sulfa/trimethoprim and Chloramphicol results were
rechecked for growth due to the zone variability.

Media
Antibiotic           TSA   Glu       Suc       Lac       Sorb

Chloramphenicol            11(R)     10(R)     15(I)     0(R)#
Ciprofloxacin              28(S)     28(S)     29(S)     25(S)#
Gentamicin                 15(S)     15(S)     16(S)     9(R)#
Sulfa/Trimethoprim         13(I)     21(S)     0(R)#     0(R)#
Moxalactam                 28(S)     22(I)#    27(S)     22(I)#

Media
Antibiotic           NA    Glu       Suc       Lac       Sorb

Chloramphenicol            12(R)     10(R)#    11(R)     11(R)
Ciprofloxacin              20(I)#    28(S)     28(S)     26(S)
Gentamicin                 10(R)#    11.5(R)   14(l)     12(R)
Sulfa/Trimethoprim         17(S)     0(R)#     0(R)#     0(R)#
Moxalactam                 22(I)     24(S)     24(S)     21(I)#

Note: The bolded results indicate the smallest
zone (most resistant) measured indicated with #.