Folia Microbiol. 51 (5), 387–391 (2006)
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Incidence of Lysogenic, Colicinogenic and Siderophore-Producing Strains among Human Non-Pathogenic Escherichia coli J. ŠMARDA, D. ŠMAJS, S. HORYNOVÁ Department of Biology, Faculty of Medicine, Masaryk University, 602 00 Brno, Czechia fax +420 549 497 070 e-mail
[email protected] Received 20 December 2005 Revised version 20 April 2006
ABSTRACT. The current incidence of Escherichia coli strains in healthy humans capable of producing the inhibitory exoproducts, such as temperate bacteriophages, corpuscular or HMW (high-molar mass) and proteinaceous or LMW (low-molar mass) colicins and siderophores was determined. Fifty-three E. coli strains were collected from the colons of 53 healthy human volunteers in Brno (Czechia) and tested for spontaneous and induced production of inhibitory exoproducts in a cross-test against each other. Of the strains tested, 37.7 % produced bacteriophages, 41.5 % produced from one to several LMW colicins, 11.3 % formed HMW colicins and 15.1 % (eight strains) produced exocellular siderophores different from enterochelin. Of these, seven strains formed aerobactin and one strain formed an untyped siderophore. E. coli strains differ greatly in the incidence of colicinogeny and lysogeny from its closest systemic relatives in the genus Escherichia and therefore should not be regarded as a model bacterium in this respect.
During the 20th century Escherichia coli developed a reputation as the standard model in bacteriology. In fact, most classical phenomena and processes in physiology, immunology, morphology and genetics, as well as host–virus relationships in bacteria, were discovered in E. coli. This happened in large part due to the easily recognizable markers and unique capabilities of this species, together with its innocuous, saprophytic way of living in the gut of homeotherms, including humans. The phenomena of lysogeny (Bordet and Ciuca 1921) and of colicinogeny (Gratia 1925) have also been described in E. coli. Since these early observations were made, the synthesis and release of several antimicrobial compounds including various colicins, HMW colicins, microcins and some siderophores, has been described among strains of E. coli; in many strains, colicinogeny, lysogeny, and the ability to synthesize microcins and siderophores are independent of each other (e.g., Šmarda 1960). Therefore, the question emerged from these observations what is the current incidence of human E. coli strains capable of producing the inhibitory exoproducts previously mentioned. This question becomes even more interesting when we consider the dearth of reliable data published so far. This lack of data is likely due to the differences in the indicator strains used, differences in the host health conditions, variation in the geographic and historical background of the hosts etc. From 1993 to 1999, Šmarda and Obdržálek (2001) found that 41.4 % of E. coli strains in humans with healthy colons were colicinogenic; however, the incidence varied from 22.4 to 56.1 %, based on enteric diagnoses. In Escherichia hermanii, E. vulneris and E. fergusonii, the incidence of colicinogenic strains was, surprisingly, 0, 0 and 12 %, respectively (Šmarda et al. 2002). Interestingly, colicins produced by the E. fergusonii strains were remarkably similar to the colicins produced by E. coli strains (Šmajs et al. 2002).
MATERIALS AND METHODS Bacterial strains. Fifty-three strains of E. coli were isolated, determined, and supported by Prof. V. Obdržálek (Department of Microbiology, Faculty of Medicine, Masaryk University in Brno). All were selected randomly during the summer of 2004 from the daily diagnostic practice of the Department of Microbiology. Each strain was taken from a healthy, human, adult volunteer (both sexes). None of the volunteers suffered from any type of intestinal problem. All donors lived in the city of Brno or its close surroundings.
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Additionally, four strains of E. coli (widely accepted as universal indicators) were used as phage and colicin indicators: K12-Row, Φ, 5K and P400 (all coming from our in-house collection; Šmarda et al. 2002). Culture media. The nutrient broth was TY medium consisting of (in g/L) tryptone (Hi-Media) 8, yeast extract (Hi-Media) 5, sodium chloride 5; the nutrient agar consisted of a base layer (1.5 %, W/V, solid agar) nutrient (beef-peptone) agar no. 2 (Imuna) 40 g/L, and a top layer (0.95 %, W/V, soft agar) agar no. 2 (Imuna) 25.3 g/L. For the cross-feeding experiments we used iron-limited nutrient agar plates (in g/L): nutrient broth (Difco Laboratories) 8, NaCl 5; agar 15) with 200 μmol/L 2,2´-dipyridyl and 100 μmol/L nitrilotriacetate as the iron-chelating compounds. For induction of bacteriophages and HMW and LMW colicin production, the top agar layer was supplemented with 0.01 % (W/V) mitomycin C. For enhancement of siderophore inhibitive action, 200 μmol/L 2,2´-bipyridyl and 100 μmol/L nitrilotriacetate were added to the top layer of agar to limit the supply of iron. To test the proteinase sensitivity of the inhibitory agents, 0.1 % (W/V) trypsin was added to the soft top layer of agar. Experimental procedure. The basic protocol was a cross test: each of the 53 strains was tested both as a possible producer and as a possible sensitive strain to any of the four inhibitory factors followed. For this testing, a modified Fredericq’s (1946) agar stab test for colicinogeny was applied in a 53 by 57 matrix. Agar plates were inoculated by needle stab with fresh broth cultures and incubated for 20 h at 37 °C. The plated bacteria were then killed by exposure to chloroform vapor and each plate was overlaid with a thin layer of soft agar containing 107 cells per mL of a fresh indicator strain broth culture. After solidification, the plates were incubated at 37 °C overnight. All 53 strains were cross-tested – as possible producers and as possible indicators – and further tested against 4 additional indicator strains. Each pair of strains was tested repeatedly. The presence of LMW colicin appeared as a growth-inhibition zone that formed around the macrocolony of the producer strain; no LMW bacteriocin zone appeared on agar that contained trypsin. The presence of lysogenic producers appeared as phage plaques (mostly tiny ones) that were scattered around the macrocolonies. The phages were auto-reproducible in subcultures. The presence of HMW colicin appeared as a growth-inhibition zone that was conspicuously narrow, compared to LMW colicin zones and, unlike LMW colicin, it was not sensitive to trypsin. The presence of siderophore appeared as a growth inhibition zone that was usually very wide, only partially hampered growth, was not delineated by a distinct (sharp) edge, and was not sensitive to trypsin. However, the zone was much more distinct on iron-limited agar, i.e. on nutrient agar containing the iron-chelating compounds (see nutrient media). The siderophore strain specificity was in some cases very low. Each test was performed at least three times; tests with inconclusive results were repeated until the ambiguity was cleared. Aerobactin production was tested in a cross-feeding test (Braun et al. 1983) on iron-limited nutrient agar plates. E. coli H1887 was used as an aerobactin indicator. Strain H1886 was used as a negative control while strain K311 was used as a positive control in the aerobactin assay (Podschun et al. 2000). Enterobactin was detected in a cross-feeding experiment with E. coli RK4349 (CGSC, E. coli Genetic Stock Center, Yale University, New Haven, USA). (RK4349 is a strain which is unable to synthesize enterobactin but which has functional receptors for the enterobactin–iron complex.)
RESULTS A summary is given in Table I. Of the strains tested, 30 strains of 53 (56.6 %) produced 1–3 (generally two) specific antibacterial factors. Temperate bacteriophages were produced by 20 strains (37.7 %), each of them killing 1–6 other strains of the 57 strains analyzed. Six strains (11.3 %) produced HMW colicins that were active against 1–12 other strains. 22 strains (41.5 %) produced LMW colicins that were active against 1–28 strains. For those strains capable of killing ≥9 other strains, there was a high probability that they produced at least two different types of colicin. Finally, eight strains (15.1 %) produced siderophores different from enterochelin, seven of which producing aerobactin and one producing an untyped siderophore that was different from both enterobactin and aerobactin. The siderophores normally hampered the growth of only one or two strains; the aerobactin of the strains 1 and 35 were exceptions, decreasing the growth of 13 and 10 strains, respectively (see Table I). Concerning the sensitive strains (omitting the classical indicators), it was confirmed that the sensitivity to a single inhibitory agent were independent of each other. Nevertheless, strain 40 which had the broadest sensitivity spectrum to colicins (being sensitive to the colicins of 14 other producers) was, at the same time, also sensitive to 2 bacteriophages; the opposite holds for strain 47. It had receptors for phages of five producer strains along with receptors for colicins from two other producers (no. 13 and 39). Strain 30 turned out to be sensitive to three HMW colicins (produced by strains 1, 11 and 43), as well as to the LMW colicins of two producers (no. 19 and 24). At the same time, strain 30 was depressed by aerobactin (of strain 1) and by
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the untyped siderophore from the strain 18. Similarly, strain 53 was sensitive to HMW colicin of the strain 11 and was broadly depressed by aerobactin from three producers. Strains 40, 26 and 51 are sensitive to colicins from the 14, 6 and 4 producers, respectively. Table I. Escherichia coli strains producing bacteriophages, HMW and LMW colicins, and siderophores (different from enterochelin)a Producer strain no. 1 2 5 6 7 8 9 10 11 13 18 19 20 21 22 24 27 28 32 34 35 36 37 39 40 42 43 45 49 52
Number of strains sensitive to its bacteriophages
HMW colicins
– 1 3 1 – – 2 2 – 6 1 – 1 – 1 3 – 2 1 3 2 – 2 – 4 1 1 1 – 1
9 – – – – 1 – 1 12 – – – – – – – – – – – 1 – – – – – 1 – – –
LMW colicins 8 1 – 4 1 – – – 6 3 4 6 1 10 1 5 1 – 6 1 – 14 2 29 5 1 – 7 1 –
siderophoresb 13 – – – 1 1 1 1 – – 1 – – 2 – – – – – – 10 – – – – – – – – –
aStrains producing no detectable inhibitory exoproduct are not shown. bIn most strains aerobactin, in strain 18 untyped siderophore.
Of the universal indicators, strain Φ was sensitive to bacteriophages of six of the producers and to LMW colicins of five others. Strain K12-Row was sensitive to phages of three of the producers and to LMW colicins of nine others. Strain P400 was sensitive to phages of three producers and to LMW colicins of 11 other producers. The most interesting appeared the production of two or three factors by one and the same strain targeted against a certain sensitive strain (e.g., the strain 1 formed simultaneously HMW colicin, LMW colicin and aerobactin again 6 sensitive strains). The most frequent combination of factors was the simultaneous production of LMW colicins and bacteriophages, though not against the same indicator (13 producers), LMW colicins and siderophores (4 producers), and HMW colicins and aerobactin (also 4 producers).
DISCUSSION Šmarda and Obdržálek (2001) found that 41.4 % of E. coli strains from healthy human colons were colicins producers. In addition, about the same proportion of colicinogenic E. coli was found in patients with
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salmonelloses, malignant tumors of the colon or among nonhemolytic uropathogenic strains. The incidence increased further to 47.5 % in patients with Crohn’s disease and climbed to 56.1 % in patients with colitis ulcerosa. There was a decrease to 22.4 % in patients with hemolytic uropathogenic strains. Before this comprehensive study, the published data on the incidence of colicinogenic E. coli in humans ranged from 9 % (Lorkiewicz et al. 1964) to 82 % (Šmarda 1960), with the variation likely due to differences in both ecological and methodological (bacteriological) approaches. Our study revealed the current incidence of colicinogenic E. coli strains in a healthy Central-European adult population is 41.5 %, which corresponds to result of Šmarda and Obdržálek (2001). However, our value is about 20 % less than the 62.5 % stated by Bureš et al. (1983). The high incidence of colicinogenic E. coli strains is actually more surprising than the absence of bacteriocinogenic E. hermanii and E. vulneris strains, and the only 12 % bacteriocinogenic incidence in E. fergusonii (Šmarda et al. 2002). It is clear that E. coli differs greatly in the incidence of colicinogeny (43.4 % to 0, 0, and 12 %) from the three closest systemic relatives in the genus Escherichia and, therefore, probably should not be regarded as a model bacterium in this respect. This finding could reflect the unique conditions facing intestinal commensal E. coli strains which need to achieve a selective advantage over other E. coli strains. This advantage can be obtained either by producing one of antimicrobial compounds we have discussed or by producing a combination of these. Alternatively, this situation could be explained by the ever increasing number of experimental recombinant applications of E. coli, especially plasmid-coded transfers, which is then followed by subsequent escape of the manipulated bacteria from the laboratory. It is not surprising that so few (in fact three) E. coli producers of HMW bacteriocins were found. So far, just a single strain of E. coli producing a HMW bacteriocin (defective phage) has been described (strain 15; Mennigmann 1965). Lysogenic strains of Escherchia found were as follows (Šmarda et al. 2002): E. hermanii (57 %), E. vulneris (10), and E. fergusonii (10). We are not aware of any statement on the incidence of lysogeny in E. coli but our finding that 37.7 % of the strains of this species are lysogenic, falls within the same limits. Clearly, the genus Escherichia with its high incidence of lysogeny is again in no way a good model of a Gramnegative bacterium. Among its close relatives in the family Enterobacteriaceae, lysogenic strains are found in the genus Citrobacter (18 %) (Šmarda and Slováčková 2004), Leclercia (10 %), Enterobacter (8 %) and in Kluyvera (only 2 %) (J. Šmarda, to be published). Of course, substantial differences in incidence appear between individual species of these genera. Pathogenic E. coli strains are often found to produce aerobactin (41 % – Cercenado et al. 1986, 71 % – Koczura and Kaznowski 2003). In contrast, fecal E. coli strains isolated from healthy volunteers produce aerobactin with a lower frequency (13.6 % – Bollmann et al. 1997, 21% – Yamamoto et al. 1995). In this respect, our finding of 13.2 % aerobactin-positive strains supports previous values found in the colons of healthy volunteers. The commensal bacterium E. coli occupies a critical ecological niche close to the surface of the intestinal mucosa and through its oxygen utilization, creates the strict anaerobic conditions required by gastrointestinal anaerobes (Sonneborn and Greinwald 1991). In addition, some non-pathogenic E. coli strains can control the growth of other microorganisms and can therefore display probiotic properties (Vančíková et al. 2003). There is no doubt that production of exoproducts endowed with profound antibiotic capabilities (mostly bactericidal) against other bacteria with undesirable properties, is the main virtue of a probiotic. The authors thank Prof. V. Obdržálek for his kind support of the experimental E. coli strains. This work was supported by grants from the Grant Agency of the Czech Republic (310/01/0013 and 310/03/1091) and by the institutional support of the Ministry of Education, Youth and Sports of the Czech Republic (MŠM 002 162 2415). 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