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Infection and Immunity, August 2005, p. 5198-5203, Vol. 73, No. 8
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.8.5198-5203.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Biofilm Formation by Salmonella enterica Serovar Typhimurium and Escherichia coli on Epithelial Cells following Mixed Inoculations

Cristina L. C. Esteves, Bradley D. Jones, and Steven Clegg^*

Department of Microbiology, College of Medicine, University of Iowa, Iowa City, Iowa 52242

Received 10 March 2005/ Returned for modification 29 March 2005/ Accepted 4 April 2005

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Biofilms were formed by inoculations of Salmonella enterica serovar Typhimurium and Escherichia coli on HEp-2 cells. Inoculations of S. enterica serovar Typhimurium and E. coli resulted in the formation of an extensive biofilm of S. enterica serovar Typhimurium. In experiments where an E. coli biofilm was first formed followed by challenge with S. enterica serovar Typhimurium, there was significant biofilm formation by S. enterica serovar Typhimurium. The results of this study indicate that S. enterica serovar Typhimurium can outgrow E. coli in heterologous infections and displace E. coli when it forms a biofilm on HEp-2 cells.

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Salmonella enterica serovar Typhimurium is a frequent cause of gastroenteritis in humans. It is estimated that every year, in the United States alone, there are 2 to 4 million cases of salmonellosis, resulting in a loss of over $2 billion to the economy (13). The bacteria are usually acquired by ingestion of contaminated food, commonly beef, poultry, or eggs (9).

Salmonellosis is associated with the small intestine, although some studies report colonic involvement during the acute phases of the disease (7, 8, 12). Both the distal small intestine and the large intestine have diverse, abundant normal flora populations that provide a barrier to infection by pathogens. Large numbers of S. enterica serovar Typhimurium, 10⁷ to 10⁹ organisms, are required to cause clinical illness in humans, and in order to colonize the intestine, S. enterica serovar Typhimurium has to compete with the natural flora (13). Indeed, both in humans and in mice, it has been shown that treatment with antibiotics, which reduces the natural flora of the intestine, resulted in higher susceptibility to salmonellosis (2, 11). Therefore, in order to establish infection, S. enterica serovar Typhimurium has to effectively compete with the resident bacteria during the process of attachment to the intestinal epithelial cells and subsequent growth on those cells.

In some instances, biofilm formation seems to be associated with the ability to cause disease, and it has been suggested that biofilms play a role in the pathogenesis of numerous bacterial species (4, 5, 14). The most abundant gram-negative facultative anaerobe in the colon is Escherichia coli. In an effort to establish a system for studying the interactions of pathogens with normal flora organisms, we describe herein investigations to assess the ability of S. enterica serovar Typhimurium to compete with the resident E. coli during colonization of and growth on epithelial cells in biofilm development.

In the present work, we examined the competition between S. enterica serovar Typhimurium BJ2710, UK-1, and 986 and the gastrointestinal E. coli isolates 3.14 and IA52 while forming biofilms on the HEp-2 epithelial cells. For this purpose a flowthrough continuous culture system was used, as described previously by our group (1). The use of this system has the advantage of allowing the biofilm to form in a dynamic rather than static environment that can be directly visualized by confocal scanning laser microcopy (CSLM). To the best of our knowledge, the present study is the first report on mixed bacterial biofilm formation, using this system, by S. enterica serovar Typhimurium and E. coli on eukaryotic cells.

Growth and biofilm formation by S. enterica serovar Typhimurium and E. coli strains on HEp-2 cells. The S. enterica serovar Typhimurium strains used in this study were BJ2710, UK-1 (6), and 986. S. enterica serovar Typhimurium BJ2710 is a derivative of strain SL1344 (15) possessing the fimH gene isolated from the LT2 fim gene cluster that mediates binding to HEp-2 cells (1). The E. coli strains 3.14 and IA52 were both isolated from the natural flora of the gastrointestinal tract. Growth and biofilm formation by S. enterica serovar Typhimurium and E. coli strains on HEp-2 cells were assayed using a flowthrough continuous culture system that has the advantage over batch cultures of providing a dynamic environment, where bacteria grow attached to eukaryotic cells, compared to the static conditions of batch cultures.

In these experiments, HEp-2 cells were grown as a confluent layer attached to a glass coverslip in the flow chambers and were then inoculated with bacteria. S. enterica serovar Typhimurium and E. coli were transformed with plasmids expressing green fluorescent protein (GFP) and red fluorescent protein (RFP). The flow chambers used allowed the direct visualization of bacterial growth on HEp-2 cells by CSLM, thus minimizing any disturbance of the biofilm formed.

Initially, we investigated the ability of S. enterica serovar Typhimurium BJ2710 or E. coli 3.14 alone to grow and form biofilms on the HEp-2 cells over a 48-h period. When inoculated alone, either S. enterica serovar Typhimurium BJ2710 or E. coli 3.14 grew and formed extensive biofilms on HEp-2 cells, and by 12 h of incubation, each bacterial strain had already established a biofilm on the HEp-2 cells (Fig. 1A and B). S. enterica serovar Typhimurium UK-1 and 986 and E. coli IA52 were also grown on HEp-2 cells in the flow chambers, and after 24 h incubation of monoinoculated chambers, each strain formed an extensive biofilm on the HEp-2 cells (data not shown). We have previously demonstrated that S. enterica serovar Typhimurium efficiently colonizes and grows on HEp-2 cells using this system (1).

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FIG. 1. Biofilms produced by S. enterica serovar Typhimurium BJ2710 (A) and E. coli 3.14 (B) expressing GFP and RFP, respectively. The CSLM composite images were obtained 12 h after bacterial incubation in the flowthrough chambers coated with HEp-2 cells.

In another series of experiments, S. enterica serovar Typhimurium BJ2710 and E. coli 3.14 were individually inoculated in chambers not containing HEp-2 cells and with glass coverslips as the substrate for bacterial attachment. By 12 h of inoculation, no growth was detected for either S. enterica serovar Typhimurium BJ2710 or E. coli 3.14, and after 24 h of incubation a limited patchy growth on the coverslips was observed for S. enterica serovar Typhimurium BJ2710. No growth was observed in the chambers inoculated with E. coli 3.14. Consequently, S. enterica serovar Typhimurium BJ2710 and E. coli 3.14 were unable to form a biofilm when cultured on the glass coverslips of the chambers not possessing HEp-2 cells. Thus, the tissue culture cells are necessary as a substrate in the chambers to support bacterial biofilm growth and were used in all subsequent experiments.

In the experiments described above, the HEp-2 cells were not stained but cell confluence was confirmed using the transmitted light mode of the microscope. However, in preliminary assays, biofilms of S. enterica serovar Typhimurium BJ2710 and E. coli 3.14 were allowed to form on HEp-2 cells prestained with a fluorescent probe requiring cell viability. In those experiments, all bacteria were labeled with GFP. Both S. enterica serovar Typhimurium BJ2710 and E. coli 3.14 formed extensive biofilms on HEp-2 cells either in the presence or absence of the cell tracker probe. In all subsequent experiments, therefore, the use of cell tracker probe was discontinued to avoid cross fluorescence signals between the probe and RFP expressed by E. coli strains.

Bacterial biofilms were formed on HEp-2 epithelial cells, a cell line previously used as the substratum in studies of biofilm formation by enteric bacteria (1). In a second series of experiments, the intestinal epithelial cell line Int 407 was used to coat the flowthrough chambers. However, Int 407 cells attached poorly to the glass coverslips of the flow chamber and were, therefore, not used in subsequent experiments.

Effect of mixed bacterial inoculation on biofilm formation by S. enterica serovar Typhimurium and E. coli strains on HEp-2 cells. Since S. enterica serovar Typhimurium has to compete with host flora, including E. coli, during intestinal colonization, we investigated the ability of S. enterica serovar Typhimurium and E. coli to form biofilms on HEp-2 cells using mixed inoculations, to begin to investigate the interactions between these two organisms.

Equal numbers (5 x 10⁸ CFU/ml) of S. enterica serovar Typhimurium BJ2710 and E. coli 3.14 were used in a mixture to inoculate the biofilm chambers. After 24 h of inoculation, S. enterica serovar Typhimurium BJ2710 was able to establish a biofilm on the HEp-2 cells, whereas only very limited attachment and growth of E. coli 3.14 was observed (Fig. 2). In addition to visualization of fluorescent bacteria, viable counts of harvested bacteria from the chambers were obtained by plating on differential MacConkey agar, and those counts consistently indicated that significantly higher numbers of S. enterica serovar Typhimurium BJ2710 than E. coli 3.14 were growing on the cells in the chambers (Table 1).

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FIG. 2. Biofilm produced by a coinoculum of S. enterica serovar Typhimurium BJ2710 and E. coli 3.14 containing equal numbers of each bacterial species. S. enterica serovar Typhimurium BJ2710 and E. coli 3.14 expressed GFP and RFP, respectively. The CSLM composite images were obtained 24 h after bacterial incubation in the flowthrough chambers coated with HEp-2 cells.

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TABLE 1. Coinoculation of S. enterica Serovar Typhimurium BJ2710 and E. coli 3.14 in batch culture and in flowthrough chambers coated with HEp-2 cellsa

Under these experimental conditions, E. coli 3.14 was not able to develop a biofilm in the presence of S. enterica serovar Typhimurium BJ2710. This was not due to the inability of the E. coli strain to form a biofilm on the HEp-2 cells since, as demonstrated earlier, chambers inoculated with only E. coli 3.14 were rapidly colonized and an extensive biofilm was observed. In additional experiments, the number of S. enterica serovar Typhimurium BJ2710 was decreased so that inoculation ratios of 1:10 and 1:1,000 (S. enterica serovar Typhimurium:E. coli) were tested. At the inoculation ratio of 1:10, both bacterial species showed considerable growth when analyzed by microscopy (data not shown), and the numbers of S. enterica serovar Typhimurium BJ2710 were slightly higher than those observed for E. coli 3.14 growing on the cells (Table 1). At a ratio of 1:1,000 (S. enterica serovar Typhimurium:E. coli) E. coli 3.14 outgrew S. enterica serovar Typhimurium BJ2710, although confocal micrographs indicated that the S. enterica serovar Typhimurium strain could still form small patches of growth on the HEp-2 cells. Therefore, even when the inoculum consisted of higher numbers of E. coli 3.14 (1:1,000, S. enterica serovar Typhimurium:E. coli), S. enterica serovar Typhimurium BJ2710 could establish growth on limited areas of the HEp-2 cells (data not shown). However, when equal numbers of S. enterica serovar Typhimurium BJ2710 and E. coli 3.14 were present in the inoculum, the growth of E. coli 3.14 was significantly reduced on the HEp-2 cells.

In another set of experiments S. enterica serovar Typhimurium BJ2710 and UK-1 and E. coli 3.14 and IA52 were dual-inoculated in the ratio 1:1 (S. enterica serovar Typhimurium:E. coli) on the HEp-2 cells. The flowthrough chambers were analyzed by CSLM 24 h after inoculation, and in all assays S. enterica serovar Typhimurium formed extensive biofilm, whereas E. coli growth was very limited and scattered (Fig. 3). These results demonstrate that, when equal amounts of each bacterium was used in the assay, the outgrowth of S. enterica serovar Typhimurium over E. coli was strain independent.

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FIG. 3. Biofilms produced by a coinoculum of S. enterica serovar Typhimurium and E. coli strains containing equal numbers of each bacterial species and expressing GFP and RFP, respectively. The CSLM composite images show the growth of S. enterica serovar Typhimurium UK-1 and E. coli 3.14 (A), S. enterica serovar Typhimurium 986 and E. coli 3.14 (B), S. enterica serovar Typhimurium BJ2710 and E. coli IA52 (C), and S. enterica serovar Typhimurium UK-1 and E. coli IA52 (D) 24 h after bacterial incubation in the flowthrough chambers coated with HEp-2 cells.

In the biofilm experiments described above, both S. enterica serovar Typhimurium and E. coli strains were transformed with plasmids encoding fluorescent proteins so that the bacterial strains could be visualized by CSLM. These plasmids also conferred resistance to ampicillin, and to maintain the transformants in the biofilm assays, this antibiotic was used in the flow medium. When S. enterica serovar Typhimurium interacts with E. coli in the intestine, the bacteria are not normally exposed to antibiotics. Therefore, the mixed-inoculum experiments were repeated using the plasmidless S. enterica serovar Typhimurium BJ2710 and E. coli 3.14 in the absence of antibiotic, and viable plate counts alone were used to determine the number of bacteria growing on the HEp-2 cells. The results, expressed as the proportion of CFU/ml of S. enterica serovar Typhimurium to E. coli, were 770:1 and were consistent with the fluorescence experiments, as S. enterica serovar Typhimurium BJ2710 significantly outcompeted E. coli 3.14, confirming that S. enterica serovar Typhimurium BJ2710 could effectively compete with and outgrow E. coli 3.14 on epithelial surfaces in mixed inoculations.

In order to demonstrate that the results obtained using mixed inoculations were not simply due to the ability of S. enterica serovar Typhimurium to replicate more rapidly than E. coli, we grew S. enterica serovar Typhimurium and E. coli strains in batch culture in the same medium as used in the biofilm assays. After 24 h of incubation in RPMI broth, viable counts of S. enterica serovar Typhimurium and E. coli were obtained by plating on MacConkey agar to differentiate between the two species. The numbers of S. enterica serovar Typhimurium and E. coli in mixed cultures were equivalent, and Table 1 shows the results for S. enterica serovar Typhimurium BJ2710 and E. coli 3.14. In these assays, the S. enterica serovar Typhimurium strains displayed the same growth rate as the E. coli strains. The ability of S. enterica serovar Typhimurium to outgrow E. coli during biofilm formation on HEp-2 cells may be due to several factors involving the attachment and subsequent growth by each bacterial species. However, the molecular mechanism(s) of this observed phenomenon has yet to be elucidated.

Growth and biofilm formation by S. enterica serovar Typhimurium BJ2710 on an established E. coli 3.14 biofilm on HEp-2 cells. When S. enterica serovar Typhimurium reaches the intestine after being ingested with contaminated food, it encounters an established natural flora. Under these conditions, S. enterica serovar Typhimurium would have to establish itself in the presence of the resident E. coli in order to colonize the intestine. In the experiments presented above, S. enterica serovar Typhimurium and E. coli strains were mixed in the inoculum and then allowed to compete to form a biofilm on the HEp-2 cells. To further explore the interaction of those two bacterial species growing on HEp-2 cells in vitro, we performed experiments in which S. enterica serovar Typhimurium BJ2710 was inoculated to a chamber possessing a preformed E. coli 3.14 biofilm.

In this series of experiments, the E. coli strain was cultured in the biofilm chambers on the HEp-2 cells for 12 h prior to inoculation with S. enterica serovar Typhimurium BJ2710. Under these conditions, an extensive E. coli 3.14 biofilm is present (Fig. 4A). The biofilms were subsequently monitored immediately after S. enterica serovar Typhimurium BJ2710 inoculation and then 4, 24, and 36 h following S. enterica serovar Typhimurium BJ2710 inoculation. By 4 h of S. enterica serovar Typhimurium BJ2710 inoculation on the E. coli 3.14 biofilm, incipient and random S. enterica serovar Typhimurium BJ2710 growth was already detectable by CSLM. As shown in Fig. 4A, S. enterica serovar Typhimurium BJ2710 was able to establish itself in the presence of an E. coli 3.14 biofilm. Similar results were obtained with plasmid-free bacteria, in which plate counts of the harvested bacteria from the chambers confirmed the ability of S. enterica serovar Typhimurium BJ2710 to grow on a fully developed E. coli 3.14 biofilm (Table 2).

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FIG. 4. Growth of S. enterica serovar Typhimurium BJ2710 on a preformed E. coli 3.14 biofilm (A) and growth of E. coli 3.14 on a preformed S. enterica serovar Typhimurium BJ2710 biofilm (B). Flowthrough chambers coated with HEp-2 cells were inoculated with one of the bacterial species for 12 h, followed by inoculation of the other bacterial species for 36 h. The biofilms were monitored by CSLM immediately after the second bacterial inoculation and then at 4, 24, and 36 h following the second bacterial inoculation. S. enterica serovar Typhimurium BJ2710 and E. coli 3.14 expressed GFP and RFP, respectively.

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TABLE 2. Growth of one bacterial species on an established biofilm of the other bacterium in flowthrough chambers coated with HEp-2 cellsa

In additional experiments, S. enterica serovar Typhimurium BJ2710 was inoculated on an E. coli 3.14 biofilm that had developed for 24 h, and the biofilm was then monitored 12 and 24 h after S. enterica serovar Typhimurium inoculation (data not shown). These results were similar to those obtained when S. enterica serovar Typhimurium BJ2710 was inoculated on an E. coli 3.14 biofilm grown for 12 h.

Next we tested the growth of E. coli 3.14 on an established S. enterica serovar Typhimurium BJ2710 biofilm of 12 h (Fig. 4B). The biofilm was observed at 0, 4, 24, and 36 h following E. coli 3.14 inoculation, and the results showed no evident growth of the E. coli strain and an extensive S. enterica serovar Typhimurium BJ2710 biofilm. These results were confirmed by bacterial plate counts of biofilms formed with plasmid-free bacteria, which indicated S. enterica serovar Typhimurium BJ2710 was present in significantly higher numbers than E. coli 3.14 (Table 2). These results again suggested that in the presence of E. coli 3.14, the S. enterica serovar Typhimurium strain could effectively compete with E. coli 3.14. Furthermore, if S. enterica serovar Typhimurium BJ2710 is established on the HEp-2 cells, the E. coli 3.14 strain is a poor colonizer of the surfaces.

There are indications that multispecies biofilm development depends on the bacterial species involved, the surface composition, and the sequence of attachment (10). Several factors may contribute to the ability of S. enterica serovar Typhimurium to grow and establish a niche on an E. coli biofilm. In the beginning of infection, S. enterica serovar Typhimurium may have the ability to simply displace E. coli or attach to locations not fully occupied by the E. coli biofilm on the HEp-2 cells. We hypothesize that S. enterica serovar Typhimurium adheres to the HEp-2 cells and increases in number, displacing E. coli and resulting in partial replacement of the E. coli biofilm by an S. enterica serovar Typhimurium biofilm. A similar phenomenon may occur when S. enterica serovar Typhimurium colonizes a host intestinal tract prior to causing infection.

 
We thank J. Jagnow for help in establishing the biofilm chambers.

During this research C. L. C. Esteves was supported by Fundação para a Ciência e a Tecnologia, Portugal. The research was supported, in part, by grant AI50011 from the NIH to S. Clegg.

 
* Corresponding author: Department of Microbiology, University of Iowa College of Medicine, 51 Newton Road, 3334 -BSB, Iowa City, IA 52242. Phone: (319) 335-7778. Fax: (319) 335-9006. E-mail: steven-clegg{at}uiowa.edu.

Editor: A. D. O'Brien

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Infection and Immunity, August 2005, p. 5198-5203, Vol. 73, No. 8
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.8.5198-5203.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Esteves, C. L. C. Articles by Clegg, S. Search for Related Content PubMed PubMed Citation Articles by Esteves, C. L. C. Articles by Clegg, S.