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Communication

Gentamicin Susceptibility and Comparison of Adhesion and Invasion of Caco-2 and HD11 Cell Lines by Salmonella enterica Serotypes

by
Juliany Rivera Calo
1,†,
Peter M. Rubinelli
1 and
Steven C. Ricke
2,*
1
Center for Food Safety and Department of Food Science, University of Arkansas, Fayetteville, AR 72701, USA
2
Meat Science and Animal Biologics Discovery Program, Department of Animal and Dairy Sciences, University of Wisconsin, 1933 Observatory Drive, Madison, WI 53706, USA
*
Author to whom correspondence should be addressed.
†
Current address: Ardent Mills, Denver, CO 80202, USA.
Appl. Sci. 2024, 14(8), 3305; https://doi.org/10.3390/app14083305
Submission received: 26 September 2023 / Revised: 26 March 2024 / Accepted: 12 April 2024 / Published: 14 April 2024
(This article belongs to the Special Issue Applied Microbial Biotechnology for Poultry Science)
Versions Notes

Abstract

:
Foodborne Salmonella serovars are important facultative intracellular pathogens that cause gastroenteritis in humans. Four strains from three of the more predominant Salmonella serovars in poultry were studied: Typhimurium, Enteritidis, and Heidelberg. Gentamicin susceptibility was determined using an agar disc diffusion test and minimum inhibitory concentration (MIC) assays for S. Typhimurium ATCC 14028 and S. Heidelberg ARI-14. Both strains were susceptible to gentamicin in disc diffusion. The MIC of gentamicin was approximately 125 mg/ml for all strains tested. These strains’ adhesion and invasion abilities were determined with two different cell lines, a human intestinal epithelial cell line (Caco-2) as well as a chicken macrophage cell line (HD11). Attachment percentages for each Salmonella strain were greater than the strain’s ability to invade cells. Similar attachment percentages to Caco-2 cells were observed for S. Typhimurium and S. Heidelberg. Attachment percentages were lower in HD11 cells than in Caco-2 cells, although Salmonella exhibited higher apparent HD11 invasion, likely from HD11 phagocytosis. Salmonella Enteritidis showed lower rates of adhesion and invasion in HD11 cells compared to Salmonella Typhimurium. Developing a better understanding of Salmonella virulence mechanisms is critical to reducing Salmonella infections.

1. Introduction

Salmonella infections is considered one of the foremost causes of foodborne gastroenteritis in humans [1]. It is estimated that nearly a million Americans contract Salmonella annually, and yearly costs of Salmonella control efforts have been estimated to reach several billion dollars [2,3,4]. Foodborne Salmonella have been associated with poultry consumption [5]. Salmonella Enteritidis, S. Typhimurium, and S. Heidelberg originating from poultry have been identified as being three of the more frequent serovars recovered from humans [5,6,7,8].
Some genes necessary for the invasion of intestinal epithelial cells and initiation of intestinal secretory and inflammatory responses are contained within Salmonella Pathogenicity Island 1 (SPI-1) [9]. Salmonella Pathogenicity Island 2 (SPI-2) is necessary for systemic infection and establishment beyond the intestinal epithelium and encodes genes essential for intracellular replication [9,10,11]. Intestinal epithelial surface adhesion is the initial Salmonella pathogenesis step and is central to its colonization. After Salmonella attaches to the intestinal epithelium, a multiprotein complex known as Type 3 Secretion System (T3SS) containing virulence genes involved in adhesion, invasion, and toxicity is expressed, facilitating endothelial update and invasion [12,13,14]. Over the past few decades, the human intestinal Caco-2 cell line originating from a human colon adenocarcinoma has been extensively employed as an intestinal barrier model. The cell line undergoes a spontaneous differentiation process, forming a monolayer of cells and expressing several morphological and functional characteristics specific to mature enterocytes [15]. HD11 cells, a chicken macrophage virus-transformed cell line was also included in the current study [16]. Salmonella serovar adhesion and invasion responses to Caco-2 and HD11 cell lines were compared. Several pathogens and host factors may be necessary in determining the mechanisms of different Salmonella responses within various cell types [13,17]. Characterizing the ability and mechanisms of this pathogen to attach and invade different cell lines may offer further insight for understanding Salmonella infections.

2. Materials and Methods

2.1. Bacterial Strains for Antibiotic Susceptibility Testing

One colony each of S. Typhimurium ATCC 14028, S. Heidelberg ARI-14 [18], and E. coli ATCC 25,922 (control) were inoculated into Luria–Bertani (5 mL, LB) broth (Alfa Aesar, Ward Hill, MA, USA) followed by incubation for 16 h at 37 °C with shaking at 190 revolutions per minute (rpm).

2.2. Antibiotic Susceptibility Testing

The susceptibility of Salmonella Typhimurium ATCC 14,028 and S. Heidelberg ARI-14 to gentamicin was determined via an agar disc diffusion test along with a modified tube dilution assay. During the agar disc diffusion test, a sterile cotton swab was inserted into the respective overnight bacterial culture and squeezed gently against each tube to remove excess fluid. Cotton swabs were subsequently streaked onto Mueller–Hinton agar plates (BD Biosciences, Franklin Lakes, NJ, USA) employing several angles to promote even growth. Gentamicin paper discs (6-mm, Becton Dickson, Sparks, MD, USA) were aseptically placed at the center of each agar plate, followed by incubation at 37 °C for 16 to 24 h. Inhibition diameter zones were measured for each respective plate, and averages were calculated. The plates were streaked in triplicate, and each experiment was repeated three times.
A modified tube dilution assay determined the minimum inhibitory concentration (MIC) for each Salmonella serovar. Serial dilutions were generated by adding nutrient broth (10 mL, EMD Millipore, Billerica, MA, USA) to the first tube and 5 mL to the remaining tubes, resulting in a total of five tubes. A total of 100 μL of gentamicin (Life Technologies, Grand Island, NY, USA) from a 500 μg/mL stock concentration was added to the first tube (10 mL), and serial dilutions were generated by a transfer of 5 mL of the solution to each subsequent tube. This procedure was repeated for all five dilutions. Five milliliters were removed and discarded from the final tube to ensure identical volumes (5 mL). Tubes were inoculated with 50 μL of the bacterial overnight culture, along with 50 μL of triphenyl tetrazolium chloride (TTC) to act as a growth indicator. Tubes were incubated for 24 h at 37 °C, with a color change from light yellow to pink/red being used as an indication of bacterial growth. The MIC had the lowest concentration of gentamicin, which showed no growth or color in the medium. After incubation for 24 h, 100 μL of each dilution for the respective bacterial serovar was inoculated onto Mueller–Hinton agar (BD Biosciences) plates to enumerate CFU/mL and for confirmation of the MIC results. Plates were conducted in triplicate, and each experiment was repeated three times.

2.3. Cell Cultures

Caco-2 and HD11 cells were sustained in D10F medium (Dulbecco’s Modified Eagles Medium (MEM), High Glucose (Thermo Scientific, Logan, UT, USA) combined with 10% fetal bovine serum (FBS) and nonessential amino acids. Cells were subsequently grown in a 75 cm2 flask at 37 °C held in an (5% CO2 atmosphere) incubator (New Brunswick, Eppendorf, Enfield, CT, USA). Once the cells in the flask reached approximately 80% confluence, they were subjected to 0.25% trypsin–EDTA (Life Technologies) for the release of attached cells, followed by the inoculation (104 cells per mL) of new stock cultures. To conduct the adhesion and invasion assays, 104 Caco-2 and HD11 cells per mL were inoculated into 24-well tissue culture plates (Greiner Bio-One, Monroe, NC, USA) followed by 37 °C incubation in an incubator (5% CO2 atmosphere) until a semi-confluent monolayer was obtained.

2.4. Bacterial Cultures for Adhesion and Invasion Assays

The Salmonella isolates used for this study are listed in Table 1. One colony of each strain was inoculated into 8 mL of LB broth and subsequently placed in the incubator (16 to 18 h at 37 °C).

2.4.1. Adhesion Assays

Bacterial enumeration in each of three representative wells was performed by trypsinizing each respective well with 0.3 mL of trypsin–EDTA, followed by incubation at 37 °C in an incubator (5% CO2 atmosphere) for 5 min and the subsequent addition of 0.7 mL of D10F. Bacterial cultures grown overnight were subsequently washed 3 times with PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, pH 7.4). Bacterial cells were diluted to an MOI ratio of 10:1 (106 Salmonella: 105 HD11 or Caco-2 cells). Washed bacteria cells were subsequently diluted 10−6 with PBS followed by plating 100 μL on LB agar plates to enumerate CFU/mL. The diluted bacteria were added to the respective cell lines, Caco-2 or HD11, and plates were incubated at 37 °C in an incubator (5% CO2 atmosphere, Thermo/Forma Scientific, Marietta, OH, USA) for 2 h. Cells were subsequently washed 3 times with cell PBS (137 mM NaCl, 5.4 mM KCl, 3.5 mM Na2HPO4, 4.4 mM NaH2PO4, 11 mM glucose, pH 7.2) followed by treatment with 1 mL of 0.1% Triton X-100 in cell PBS. Plates were incubated for 10 min at 37 °C in an incubator (5% CO2 atmosphere). The disrupted cells were collected in duplicate, serially diluted, and plated on LB agar to determine the adhesion percentage. Plates were subsequently incubated for 16 h at 37 °C. To determine CFU/mL, all LB agar plates were inoculated for each duplicate. Each strain was subsequently tested in triplicate in three independent experiments.

2.4.2. Invasion Assays

The enumeration of cells in each of the three representative wells was performed by trypsinizing each respective well with 0.3 mL of trypsin–EDTA, incubating at 37 °C in an incubator (5% CO2 atmosphere) for 5 min, and adding 0.7 mL of D10F. Overnight bacterial cultures were washed 3 times with PBS and subsequently diluted to obtain an MOI ratio of 10:1 (Salmonella: animal cells). Washed bacteria were diluted 10−6 with PBS, followed by plating 100 μL on LB agar plates for CFU/mL enumeration. The diluted bacteria were added to the cell lines, Caco-2 or HD11, and the plates were incubated at 37 °C in an incubator (5% CO2 atmosphere) for two hours. The cells were washed 3 times with cell PBS followed by treatment with 1 mL of DMEM containing 100 μg/mL gentamicin per well in order to kill extracellular bacteria considered to be adherent. The plate was incubated for 2 h at 37 °C in an incubator (5% CO2 atmosphere). The cells were subsequently washed 3 times with cell PBS and subjected to treatment with 1 mL of 0.1% Triton X-100 in cell PBS. The plate was incubated for 10 min at 37 °C in an incubator (5% CO2 atmosphere). Serial dilutions of suspensions were conducted in PBS followed by inoculation onto LB agar plates, in duplicate, to determine the number of organisms that survived treatment with gentamicin and, hence, had invaded the Caco-2 or HD11 cells. Plates were incubated at 37 °C for 16 h. Each strain was tested in triplicate in three independent experiments.

2.5. Statistical Analysis

The JMP Pro Software Version 11.0 (SAS Institute Inc., Cary, NC, USA) program was used to conduct all statistical analyses. Mean ± standard deviation was determined for each antibiotic susceptibility test. One-way ANOVA and the Tukey–Kramer HSD test were applied to each bacterial strain’s adhesion and invasion percentages to Caco-2 and HD11 cells. Statistical significance was established at p < 0.05.

3. Results and Discussion

Gentamicin has been shown to inhibit bacterial protein synthesis and eliminate Salmonella tissue cell culture invasion [19]. This makes it a useful treatment for differentiating between external bacterial cells and internalized cells. All three strains were observed to be susceptible to gentamicin. Both Salmonella Heidelberg ARI-14 and S. Typhimurium ATCC 14,028 yielded average inhibition zones of 20.7 ± 1.1 and 20.7 ± 0.1, respectively. Escherichia coli strain 25922 was used as a control and exhibited a zone of inhibition of 19.0 ± 0. To further quantify susceptibility, the gentamycin was serially diluted in a growth medium, cells were added, and aliquots were plated for enumeration. Growth was detected on agar plates for all three organisms after the third dilution. The MIC for gentamicin was 125 μg/mL for all three strains. These results validated using gentamicin during invasion assays as a means to kill cell-adherent extracellular bacteria. Expected MIC ranges have been reported by Andrews [20] for determining the susceptibility of several bacteria to a wide selection of antibiotics, as well as a list of appropriate controls for inclusion when determining MIC responses. As a control, their suggested MIC range for Enterobacteriaceae susceptibility against gentamicin is 0.03 to 128 mg/L using E. coli 25,922 [20]. Our results are consistent with this range. When Shah et al. [21] determined the cell invasion responses of several poultry-associated S. Enteritidis isolates, they noted a gentamicin MIC of <0.125 μg for all the isolates and used 100 μg per mL for their Caco-2 invasion assays. Menashe et al. [22] examined the MIC of S. Typhimurium 14,028 and S. Virchow and reported MIC values of 125 μg per mL for both serovars.
In the current study, we examined the ability of Salmonella strains from different serovars to attach and invade two cell lines, namely, Caco-2 and HD11. We only evaluated two serovars for the Caco-2 study, the well-characterized S. Typhimurium serovar strain and the previously uncharacterized S. Heidelberg serovar. Our rationale for the Caco-2 cell study was to only compare the S. Heidelberg directly with the standard S. Typhimurium typically used in these types of tissue culture studies to assess whether the invasion response of the S. Heidelberg isolate was similar to S. Typhimurium. As expected, the percentage attachment for each respective Salmonella strain was greater than the ability of the respective strain to invade the cells. Salmonella Heidelberg and S. Typhimurium yielded percentages of adhesion of 28.8 ± 6.37 and 18.1 ± 6.25, respectively, to Caco-2 cells (Table 2). However, the Caco-2 invasion ability of these respective strains was lower, 1.37 ± 0.25 for S. Heidelberg and 1.52 ± 0.02 for S. Typhimurium (Table 3). The underlying detailed mechanisms that intracellular pathogens such as Salmonella use to penetrate the host epithelium continue to be investigated. Cultured mammalian cells as in vitro models have been used to study the interaction and internalization of Salmonella [23,24,25]. The invasion of cultured epithelial cells is commonly used to measure Salmonella pathogenicity [21,26,27]. Previous studies report that several environmental stimuli such as osmolarity [28,29], carbohydrate availability [30], and oxygen availability [31,32,33] influence Salmonella invasion of cultured mammalian cells. For example, Durant et al. [25] reported that Salmonella exposed high concentrations of short-chain fatty acids (SCFAs) at levels similar to the lower regions of the gastrointestinal tract, along with pH changes, could influence the association and invasion of Hep-2 cells. Shah et al. [21] reported that in cultured Caco-2 cells, isolates with high invasiveness could invade and/or survive more extensively within chicken macrophage cells compared to low-invasive isolates.
Three Salmonella serovars, S. Typhimurium (two strains, ATCC 14028, and UK-1), S. Enteritidis, and S. Heidelberg, were compared in HD11 cell culture experiments (Table 4 and Table 5). For HD11 cells, attachment percentages were generally greater (Table 4) for S. Heidelberg and S. Typhimurium ATCC 14028 than those observed for these strains in Caco-2 cell cultures (Table 2) and exhibited higher rates for invasion, ranging from 2.9 to 17.6% (Table 5) compared to Caco cell invasion percentages (Table 3). S. Heidelberg, S. Typhimurium ATCC 14,028, and S. Typhimurium UK-1 exhibited the greatest levels of percent attachment (p < 0.05), with S. Enteritidis attachment being less than half the other serovars (18.6 ± 2.42%). Salmonella Typhimurium strain UK-1 invaded HD11 cells at higher percentages, 17.6 ± 3.29, likely due to the strain’s high virulence phenotype. Salmonella Typhimurium UK-1 (abbreviated for “universal killer”) is a highly virulent strain initially isolated in 1991 from an infected horse and passaged through a chicken [34]. Salmonella Typhimurium ATCC 14028 (9.3 ± 0.57) and UK-1 (17.6 ± 3.29)’s respective invasion levels of HD11 cells support that strain differences can occur within the same serovar. Luo et al. [34] conducted an extensive genome analysis comparing UK-1 and other S. Typhimurium strains. They reported that virulence factors from one strain may increase or decrease virulence from another [35]. The identification of polymorphic genomic regions of the strains and further analyses revealed that even highly similar strains of S. Typhimurium could be distinguished [35]. Bhomik et al. [35] reported that mutants of S. Typhimurium UK-1 were less invasive of HD11 cells than the wildtype strain.
Salmonella Enteritidis exhibited the lowest adhesion (Table 4, 18.6 ± 2.42%) and invasion percentage (Table 5, 2.9 ± 1.48%) of all serovars compared in this study. When the Salmonella cell invasion and intracellular survival of five different poultry-associated serovars was compared by He et al. (S. Heidelberg, S. Enteritidis, S. Typhimurium, S. Senftenberg, and S. Kentucky), S. Enteritidis appeared to be more resistant than the other serovars to intracellular killing, leading them to hypothesize that the serovar’s intracellular survival ability may be associated with specific systemic invasion capabilities in chickens [36]. Matulova et al. [37] reported lower invasion after pre-treating the HD11 cells with avidin before S. Enteritidis infection. Shah et al. [21] suggested that not every isolate of S. Enteritidis recovered from poultry may be equally pathogenic or have a similar potential to invade cells. Saeed et al. [38] reported that unlike S. Enteritidis isolates recovered from chicken ceca, isolates from chicken eggs or human clinical cases exhibited greater adherence and invasion of chicken ovarian granulosa cells.

4. Conclusions

Cell line type and different serovars appear to be factors that can lead to detectable differences in adhesion and invasion. However, further studies are necessary that involve more Salmonella serovars and strains to elucidate the properties that enable some serovars to be able to attach and invade tissue culture cells to a greater extent than others. Understanding the ability of different serovars of this pathogen to adhere and invade specific cell lines could be helpful for further understanding Salmonella infections.

Author Contributions

S.C.R. and P.M.R. oversaw design and J.R.C. generated the experimental data. J.R.C. wrote the manuscript and conducted statistical analyses. S.C.R. and P.M.R. edited the final manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

Author J.R.C.’s graduate assistantship was supported by the Dept. of Food Science, University of Arkansas, Fayetteville, AR.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The raw data supporting the conclusions of this article will be made available by the authors on request.

Acknowledgments

We wish to thank the Department of Food Science, University of Arkansas, Fayetteville, AR, for providing the graduate stipend supporting author J.R.C. We thank Margaret K. Costello for editing input. We also thank Dana Dittoe, Dept. of Animal Science, University of Wyoming, for assistance with the data presentation.

Conflicts of Interest

The authors declare no conflicts of interest.

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Table 1. Salmonella strains used in this study.
Table 1. Salmonella strains used in this study.
Adhesion and Invasion to Caco-2 CellsAdhesion and Invasion to HD11 Cells
Salmonella Typhimurium ATCC 14028Salmonella Typhimurium ATCC 14028
Salmonella Heidelberg ARI-14Salmonella Typhimurium UK-1
Salmonella Heidelberg ARI-14
Salmonella Enteritidis ATCC 13076
Table 2. Salmonella Typhimurium and S. Heidelberg adhesion to human epithelial Caco-2 cells.
Table 2. Salmonella Typhimurium and S. Heidelberg adhesion to human epithelial Caco-2 cells.
Bacterial StrainLog Dilution# of Colonies# Bacteria Added# Bacteria Adhering% Adhesion
S. Typhimurium ATCC 14028417.67 ± 2.319.83 × 106 ± 01.78 × 106 ± 0.2518.1 ± 6.25
S. Heidelberg ARI-14426.0 ± 2.839.13 × 106 ± 02.63 × 106 ± 0.3228.8 ± 6.37
Adhesion between S. Heidelberg and Typhimurium was not significantly different (p > 0.05).
Table 3. Salmonella Typhimurium and S. Heidelberg adhesion and invasion to human epithelial Caco-2 cells.
Table 3. Salmonella Typhimurium and S. Heidelberg adhesion and invasion to human epithelial Caco-2 cells.
Bacterial StrainLog Dilution# of Colonies# Bacteria Added# Bacteria Invading% Invasion
S. Typhimurium ATCC 140282124.75 ± 10.698.26 × 106 ± 01.25 × 105 ± 0.011.52 ± 0.02
S. Heidelberg ARI-14263.17 ± 17.545.24 × 106 ± 05.96 × 104 ± 1.421.37 ± 0.25
Invasion between S. Heidelberg and Typhimurium was not significantly different (p > 0.05).
Table 4. Adhesion of four Salmonella strains from different serovars to chicken macrophage HD11 cells.
Table 4. Adhesion of four Salmonella strains from different serovars to chicken macrophage HD11 cells.
Bacterial StrainLog Dilution# of Colonies# Bacteria Added# Bacteria Adhering% Adhesion *
S. Heidelberg459.6 ± 15.961.3 × 107 ± 0.475.5 × 106 ± 1.9843.4 ± 7.46 a
S. Typhimurium ATCC 14028461.8 ± 17.581.6 × 107 ± 0.476.2 × 106 ± 1.1538.7 ± 4.57 a
S. Typhimurium UK-1469.2 ± 19.851.8 × 107 ± 0.717.0 × 106 ± 1.9338.9 ± 9.24 a
S. Enteritidis439.2 ± 9.242.1 × 107 ± 03.9 × 106 ± 0.5018.6 ± 2.42 b
* Percentages not connected by the same letter are significantly different between each assay (p < 0.05).
Table 5. Invasion of four Salmonella strains from different serovars to chicken macrophage HD11 cells.
Table 5. Invasion of four Salmonella strains from different serovars to chicken macrophage HD11 cells.
Bacterial StrainLog Dilution# of Colonies# Bacteria Added# Bacteria Invading% Invasion *
S. Heidelberg433.2 ± 9.046.91 × 107 ± 03.3 × 106 ± 0.504.8 ± 0.73 c
S. Typhimurium ATCC 14028437.3 ± 4.194.0 × 107 ± 0.283.7 × 106 ± 0.089.3 ± 0.57 b
S. Typhimurium UK-1425.3 ± 4.991.4 × 107 ± 0.152.5 × 106 ± 0.4017.6 ± 3.29 a
S. Enteritidis44.05 ± 3.061.7 × 107 ± 0.044.9 × 105 ± 2.302.9 ± 1.48 c
* Percentages not connected by the same letter are significantly different between each assay (p < 0.05).
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Rivera Calo, J.; Rubinelli, P.M.; Ricke, S.C. Gentamicin Susceptibility and Comparison of Adhesion and Invasion of Caco-2 and HD11 Cell Lines by Salmonella enterica Serotypes. Appl. Sci. 2024, 14, 3305. https://doi.org/10.3390/app14083305

AMA Style

Rivera Calo J, Rubinelli PM, Ricke SC. Gentamicin Susceptibility and Comparison of Adhesion and Invasion of Caco-2 and HD11 Cell Lines by Salmonella enterica Serotypes. Applied Sciences. 2024; 14(8):3305. https://doi.org/10.3390/app14083305

Chicago/Turabian Style

Rivera Calo, Juliany, Peter M. Rubinelli, and Steven C. Ricke. 2024. "Gentamicin Susceptibility and Comparison of Adhesion and Invasion of Caco-2 and HD11 Cell Lines by Salmonella enterica Serotypes" Applied Sciences 14, no. 8: 3305. https://doi.org/10.3390/app14083305

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