Abstract
Oral infection of mice with Salmonella Typhimurium is an important model system. In particular C57Bl/6 mice, which are susceptible to Salmonella, are used to study both systemic and gastrointestinal pathogenesis. Pretreatment with streptomycin disrupts the intestinal microbiota and results in colitis resembling human intestinal Salmonellosis. Oral gavage is typically used for delivery of both antibiotic and bacteria. Although convenient, this method requires a moderate level of expertise, can be stressful for experimental animals, and may lead to unwanted tracheal or systemic introduction of bacteria. Here, we demonstrate a simple method for oral infection of mice using small pieces of regular mouse chow inoculated with a known number of bacteria. Mice readily ate chow pieces containing up to 108 CFU Salmonella, allowing for a wide range of infectious doses. In mice pretreated with streptomycin, infection with inoculated chow resulted in less variability in numbers of bacteria recovered from tissues compared to oral gavage, and highly consistent infections even at doses as low as 103 Salmonella. Mice not treated with streptomycin, as well as resistant Nramp1 reconstituted C57Bl/6J mice, were also readily infected using this method. In summary, we show that foodborne infection of mice by feeding with pieces of chow inoculated with Salmonella results in infection comparable to oral gavage but represents a natural route of infection with fewer side effects and less variability among mice.
Introduction
Bacteria belonging to the genus Salmonella enterica subsp. enterica are one of the most common causes of self-limiting foodborne diarrheal disease in humans and other animals [1] and a leading cause of death due to foodborne pathogens globally [2] and in the US [3]. Salmonella enterica serovar Typhimurium (hereafter Salmonella) is one of the serovars most commonly isolated from human gastrointestinal infections. Salmonella infects new hosts in a fecal-to-oral manner and is often the cause of foodborne disease outbreaks when present in contaminated food, such as fresh produce [4] and poultry and egg products [5]. Salmonella has been thoroughly studied and is one of the best characterized human pathogens. This, combined with its simple growth requirements, has led to its frequent use as a model organism for in vivo studies of the pathogenesis of gastrointestinal infections.
The most widely used animal model for Salmonella is the mouse [6]. Strains of mice differ in their susceptibility to Salmonella infection, with C57Bl/6J and BALB/c mice being highly susceptible and 129/Sv strains of mice being very resistant [7–10]. Susceptibility is multifactorial, but one important resistance factor is the Nramp1 protein encoded by the Slc11a1 gene [11]. Nramp1 is an ion transporter responsible for the transport of divalent cations out of phagosomes, thus limiting the availability of iron and other ions for ingested microbes and impairing their growth in phagocytes [12]. Many susceptible mouse strains, including C57Bl/6J, harbor a point mutation in the Slc11a1 gene resulting in a non-functional Nramp1 protein [13, 14]. Oral infection of susceptible mouse strains eventually leads to a lethal systemic infection but without diarrhea and only diffuse enteritis [15]. However, following disruption of the intestinal microbiota by antibiotic treatment, mice develop intestinal inflammation more similar to human intestinal Salmonellosis, although Salmonella do still go systemic [16, 17].
In the preferred murine model of oral Salmonella infection, C57Bl/6 mice are treated with antibiotics and infected by oral gavage using blunt end gavage needles. This method of delivery is intragastric rather than oral, since substances are delivered directly into the stomach. While the use of gavage needles allows for the delivery of precise amounts of inoculum and timing of delivery, there are several drawbacks to their use, which have been recognized mostly in toxicological studies [18]. Performing oral gavage requires a moderate degree of technical expertise and can induce stress, e.g. raising corticosteroid levels in the blood or increasing blood pressure, which may affect study outcome [19–21]. Furthermore, mice may regurgitate delivered substances or infectious agents following gavage, resulting in tracheal or nasal administration [22, 23]. Lastly, gavage may induce pharyngeal or esophageal trauma, leading to the inadvertent delivery of substances or infectious agents directly into the blood stream or, in rare cases, death [23, 24].
Improvements to oral gavage have been suggested, such as precoating needles with sucrose, which improved gavage success rate and reduced stress of animals [25]. An alternative method to gavage would be ingestion of food or water containing a pathogen. This would circumvent many of the drawbacks with oral gavage and mimics a natural route of infection for Salmonella. Recently, a paper described a method of delivering Salmonella orally in drinking water [26], and a similar approach was used for the oral delivery of a Salmonella vaccine to sheep [27]. Inoculated food (pieces of bread) has been used for Listeria monocytogenes infections [28]. However, to our knowledge, food as a vehicle of delivery for Salmonella infection has not been reported.
In this paper, we describe an oral infection method using pieces of regular mouse chow inoculated with Salmonella. Preparation of inoculated chow is simple and mice readily consume chow containing high numbers of bacteria. This mode of infection leads to a very consistent disease progression among mice, less variability in bacterial load, in both systemic and gastrointestinal tissues, and eliminates many of the possible drawbacks with oral gavage. Importantly, this method represents a natural route of infection with Salmonella.
Results and Discussion
Salmonella remains viable on mouse chow
We hypothesized that regular mouse chow could be used to inoculate mice with Salmonella to establish a simple, stress-free and natural route of Salmonella infection. At our facility, mice typically receive 2016 Teklad Global 16% Protein Rodent Diet (Envigo, Madison, Wisconsin USA), chow commonly used by research institutions (see e.g. [29]). To confirm that Salmonella does not lose viability on chow, pieces of chow (approximately 5 mm in diameter) were prepared from pellets (Fig 1A) and inoculated with known numbers of bacteria. Following incubation at room temperature for 1 or 3 h, the chow pieces were homogenized, diluted and plated to enumerate colony forming units (CFUs). As a comparison, bacteria were also diluted and inoculated in sterile pharmaceutical grade saline (SPGS). Salmonella showed no decrease in viability in food or saline over the course of 3 h (Figs 1B and C), irrespective of the initial numbers of bacteria.
Delivery of Salmonella using chow results in infection similar to gavage delivery and has less variability
Gavage is the method traditionally used to deliver Salmonella in oral infection of mice and for streptomycin treatment. To investigate how oral infection with chow compared to the commonly used procedure for infection by gavage, streptomycin treated (hereafter referred to as strep+) C57Bl/6J mice were infected with 104 CFU Salmonella using either method (Fig 2A). For oral gavage, mice were gavaged with streptomycin (20 mg) 24 h prior to being gavaged with Salmonella diluted in SPGS (100 μl). Mice were fasted for 4 h prior to each gavage. For mice infected with chow, to avoid gavage altogether, streptomycin was added to the drinking water (final dilution of 5 mg/ml) for 24 h [30]. C57Bl/6J mice drink approximately 6 ml of water per 24 h [31], which results in an approximate total dose of 30 mg streptomycin. After 24 h normal drinking water was returned, mice were fasted for approximately 20 h, then fed pieces of chow inoculated with Salmonella diluted in SPGS. Since we were concerned that high levels of Salmonella might affect the palatability of chow, we initially used a low inoculum of Salmonella, although the dose most frequently used is approximately 108 Salmonella [16, 29, 32]. Mice readily ate chow pieces inoculated with approximately 104 CFU. At 3 days post infection (p.i.), mice displayed very mild, if any, clinical signs of disease, although feces was frequently found on the walls of the cage, indicating wet stool. Despite the low inoculum, all mice were infected although those infected with inoculated chow had higher bacterial loads in tissues, especially in the intestines, compared to those infected by gavage (Fig 2B). While this could be a result of the inoculation method itself, it could also be due to either the prolonged streptomycin treatment in the drinking water or the prolonged fasting period [33, 34]. Bacterial numbers were more consistent between the mice in the chow infection group when compared to mice in the gavage group. This consistency was most notable in the feces, where bacterial numbers in gavaged mice ranged from 0 (below the limit of detection) to 3.4×108 and in chow infected mice from 1.6×108 to 1.3×1010. The variability in infection due to oral gavage has been observed previously [26]. Since oral gavage may induce regurgitation and subsequent unintended tracheal delivery of substances [22] we also examined the lungs for Salmonella.Mice infected by either method contained low levels of Salmonella in the lungs, again with more variability in mice infected with gavage (0 to 8.7×103) compared to those infected via chow (8.9×101 to 5.4×102) (Fig 2B). The presence of bacteria in the lungs is not overly surprising considering the susceptibility of strep+ C57Bl/6J mice to disseminated Salmonella infection, however, two mice infected by oral gavage contained considerably (>1 log) higher bacterial numbers in the lungs than the rest of the mice. This may indicate unintended tracheal administration of Salmonella as a complication of the gavage process. Altogether, these findings indicate that infection with inoculated chow results in more consistent disease with less complications compared to the traditional gavage method.
Mice readily eat chow pieces inoculated with high loads of Salmonella and show consistent infection
Having shown that chow inoculated with a low dose (104 CFU) of Salmonella results in consistent infection we next compared a dose range. Pieces of chow were inoculated with 103 to 106 CFU and fed to strep+ C57Bl/6J mice. For doses of 105 bacteria and higher, the bacteria were pelleted by centrifugation and then diluted in SPGS (washed) before dilution since mice were hesitant to eat chow inoculated with these high doses if they were not washed (data not shown). This may be due to the intrinsic smell or taste of the growth medium or the presence of high amounts of bacterial products. After introducing the wash step, mice readily ate chow pieces containing up to 108 CFU. By 3 days p.i. all mice had developed systemic infection even at the lowest dose (103) and bacterial loads were strikingly similar (Fig 3A), indicating no dose dependence for the development of disseminated systemic disease, correlating with previous reports [30]. Bacterial loads were consistent in systemic tissues but showed more variability in gastrointestinal tissues with the greatest variability seen in the ileum (3.0×105 to 5×108 CFU/g). Bacterial loads were highest in the cecum (approximately 109 CFU/g) and lowest in the blood (approximately 102 CFU/ml). To investigate if chow infection works in a resistant mouse strain, we used C57Bl/6J mice reconstituted with a functional Nramp1 (hereafter referred to as Nramp1+/+) [11, 13, 14]. Mice with a functional Nramp1 protein are more resistant to oral Salmonella infection both with and without prior streptomycin treatment [8, 17]. For the Nramp1+/+ mice we tested doses of 104 and 105 bacteria. A dose of 104 resulted in inconsistent infection in systemic tissues, while 105 resulted in a consistent systemic infection, indicating that 105 is the minimum dose required for 100% systemic infection. As expected, Nramp1+/+ mice were substantially more resistant to infection, compared to C57Bl/6J mice, with lower bacterial loads in all tissues but particularly in systemic organs (Fig 3B). Nonetheless, bacterial loads in Nramp1+/+ mice followed the same trends as in C57Bl/6J mice with the highest number of bacteria in the feces followed by the cecum, ileum, spleen, liver and blood. Interestingly, bacterial loads were also lower in the feces of Nramp1+/+ mice, indicating that functional Nramp1 is important for limiting bacterial numbers in intestinal luminal contents as well as systemically.
Next we infected non-streptomycin treated (hereafter referred to as strep-) C57Bl/6J and Nramp1+/+ mice using pieces of chow. Since these mice are much less susceptible to oral infection, we used a dose of 108 bacteria, the standard dose used in oral infections of mice with Salmonella (see e.g. [16, 32, 35]). After washing the bacteria, mice readily ate pieces of chow containing this high inoculum. C57Bl/6J mice had consistent bacterial loads in intestinal tissues, while Nramp1+/+ mice showed more variability in loads which were also sometimes below the level of detection (Fig 4). In systemic tissues, no bacteria were detected in any of the Nramp1+/+ mice and bacterial numbers were variable in C57Bl/6J mice. In the intestinal tract, the differences in bacterial loads between C57Bl/6J and Nramp1+/+ mice were statistically significant in the ileum and feces.
In summary, infections of strep+ and strep- C57Bl/6J and Nramp1+/+ mice with pieces of chow inoculated with Salmonella are consistent with studies using oral gavage. While it is difficult to determine the dose of nontyphoidal Salmonella required to cause gastroenteritis in humans, reports indicate doses as low as 10 organisms [36, 37]. In this infection model we achieved consistent infection of strep+ mice using only 103 organisms, indicating that this model can be used to investigate a range of relevant infection doses.
Mice infected using pieces of chow succumb to Salmonella infection similar to mice infected by oral gavage
Next, we sought to compare the development of clinical disease of C57Bl/6J and Nramp1+/+ mice infected using inoculated chow. Strep+ and strep- mice were inoculated with 104 or 108 CFU of Salmonella, respectively, and monitored for overt clinical signs at which point they were euthanized. As expected, strep+ mice succumbed faster to disease compared to strep- mice (Figs 5A and B). Only one strep- Nramp1+/+ mouse developed clinical signs and was euthanized 15 days p.i.; all others were euthanized at the experimental end point of 21 days p.i. This is consistent with an intact microbiota and the ability of Nramp1+/+ mice to effectively combat infection. The mouse euthanized 15 days p.i. showed very high bacterial loads in all tissues except the liver and blood (open symbols in Fig 5F). Strep+ mice developed very high bacterial loads in the intestinal tissues, especially C57Bl/6J mice, which also developed high loads in systemic tissues (Figs 5C and D). This indicates that Nramp1 contributes to controlling Salmonella infection both in systemic and intestinal sites. At the time of euthanasia, strep- C57Bl/6J mice had higher and more consistent bacterial loads in all tissues compared to strep- Nramp1+/+ (Figs 5E and F). It is interesting to note that although strep- C57Bl/6J mice have lower intestinal bacterial loads compared to strep+ mice, they do go on to develop higher systemic bacterial loads. This is probably a result of the prolonged survival of strep- mice allowing additional time for the bacteria to replicate at systemic sites. These findings correlate with other studies of oral infection of strep- susceptible and resistant mouse strains [8, 35, 38], and show the reduced survival of strep+ mice.
Shorter periods of fasting increases hesitancy of mice to eat inoculated chow
For practical reasons, it would be advantageous to have the option of varying the fasting time although mice fasted for shorter times are less likely to eat inoculated chow. Therefore, to determine how changing the duration of fasting would impact the method, we fasted mice for 4, 8 or 14 h and then measured the time taken to completely consume inoculated pieces of chow. These mice were not streptomycin treated. After 14 h (o/n) of fasting, mice consumed chow within 2 min, while shorter fasting resulted in longer consumption times, up to 23 min (Fig 6A). The inoculum did not seem to affect consumption time, since mice fasted for 4 h and offered chow inoculated with 108 CFU consumed the chow in a time frame similar to mice fed 104 CFU (Fig 6A). The very low consumption time after 14 h may be due to fasting taking place overnight when mice are more active. Shorter fasting times required a modification to the feeding procedure, where mice were moved into individual clean cages, offered a piece of chow and then left undisturbed until consuming the whole chow piece. When comparing the organ loads 3 days p.i. of mice fed 108 CFU after fasting for 4 h (Fig 6B) or 20 h (Fig 4), the shorter fasting period led to lower bacterial loads in systemic tissues and in some of the intestinal tissues. This indicates that shorter fasting times results in a delayed, or less efficient, dissemination of Salmonella. In a study describing oral infection of mice with Listeria monocytogenes the authors noted that mice sometimes had to be left undisturbed for up to 2 h to eat the offered food, even after 24 h of fasting [28]. The reason for the mice more readily eating the inoculum in this study may be due to the inoculated pieces of chow are derived from their regular chow, a type of food they are already familiar with.
In summary, oral infection of mice using pieces of their regular chow inoculated with Salmonella mimics the natural route of infection, is technically very simple and results in reproducible infection while avoiding the stress and potential adverse side effects of oral gavage. Further refinements to the method are possible, such as; adjusting fasting times; the concentration of streptomycin in drinking water; and the time allowed for mice to access water containing streptomycin. We expect that this approach would work for other strains of mice and intestinal pathogens.
Materials and Methods
Ethics statement
All animal studies were carried out following the recommendations in the Guide for the Care and Use of Laboratory Animals, 8th Edition (National Research Council), and the animal study protocol was approved by the Rocky Mountain Laboratories Animal Care and Use Committee. Protocol number 2017-021-E. Animals were either euthanized before the development of clinical disease, at specified time points, or at the defined humane endpoint (development of clinical disease: ruffled fur, hunched posture, lethargy).
Bacterial strains and growth conditions
Salmonella Typhimurium strain SL1344 was used for all experiments. For infections, bacteria were grown in a 125 ml Erlenmeyer flask in 10 ml LB-Miller containing 100 μg/ml streptomycin for 18 h at 37°C, with shaking at 225 RPM, and diluted in sterile SPGS to get the correct inoculum in 10 μl (e.g. to get 104 CFU an overnight culture was diluted 1:5000), the volume added to pieces of chow. For inoculum of 105 CFU and higher, a wash step was included prior to dilution. 1 ml of the overnight culture was centrifuged at 8000 G for 2 min, the supernatant was aspirated, and the bacterial pellet resuspended in 1 ml SPGS. In order to achieve a final concentration of 108 CFU in 10 μl, the bacterial pellet was resuspended in 0.5 ml SPGS.
Preparation of chow pieces for infection
Mouse chow pellets (2016 Teklad Global 16% Protein Rodent Diet, Envigo, Madison, Wisconsin USA), were broken into smaller pieces of about 4-5 mm in diameter by gentle tapping with a small hammer followed by trimming with forceps. Selected pieces were gently tested for physical integrity, by dropping from a height of 4-5 inches, before 10 μl of inoculum was pipetted onto the surface. Prepared pieces were kept separated in a petri dish during transport to the animal facility. One piece of inoculated chow was retained for estimation of the CFU by plating.
Mouse chow infections
Except where specified, mice had unlimited access to food and water. For Streptomycin pretreatment the antibiotic (5 mg/ml) was added to drinking water 42 – 46 h prior to infection for 24 h. Mice were then moved to a clean cage (to limit coprophagy and access to cached food), containing normal drinking water but no chow. After a period of 18 – 22 h (typically 20) individual mice were put in a clean empty cage (without bedding material) and were then offered a piece of chow. Typically, mice ate the piece of chow immediately or within a couple of min. For short fasting times (4 and 8 h) mice were left undisturbed until the chow was eaten. Immediately after the inoculated chow was consumed, mice were returned to their cage with unlimited access to food and water.
Mouse oral gavage infections
Mice were streptomycin treated 24 h before infection, using a blunt end gavage needle with 100 μl SPGS containing 200 mg/ml streptomycin. For Salmonella infection, mice were gavaged with bacteria in 100 μl SPGS. Mice were fasted for 4 h prior to all gavages. For infections without streptomycin treatment, mice were only fasted prior to feeding.
Tissue collection and processing
Mice were euthanized by isoflurane inhalation followed by exsanguination. Tissues were collected in screwcap tubes containing 500 μl SPGS and 3-4 2.0 mm zirconia beads (BioSpec Products) and homogenized using a Bead Mill 24 (Fisher Scientific, 4.85 m/s for 20 seconds). Tubes were weighed before and after organ collection. CFUs were estimated by 10 μl spot plating of 10-fold dilutions on LB agar plates containing the appropriate antibiotic.
Acknowledgments
We thank the members of the Steele-Mortimer laboratory, Karin Peterson and Clayton Winkler for critical review of the manuscript, and Ryan Kissinger for assistance with figures. This research was supported by the Intramural Research Program of the NIH, NIAID.