Many enteric bacteria are bile resistant, yet none of these resistant species have been found to grow in the healthy gallbladder of any animal, with the exception of Salmonella enterica
serovar Typhi in humans. Typhoid carriers harbor this pathogen in the gallbladder, and typhoid fever is thought to be transmitted from carriers by expulsion of contaminated bile from this organ into the intestine and out of the body in feces. To date, only L. monocytogenes
has been demonstrated to grow in the asymptomatic gallbladder in an animal model. Figure shows that many mice exhibit these signals upon intravenous infection. In this case, 19 of 20 CD1 mice showed increased signals from the gallbladder. We investigated this remarkable ability using imaging to assess bacterial burden and location in the individual animal over time. We sought to image bacteria induced to enter the intestine by causing the contraction of the gallbladder and to therefore directly demonstrate biliary excretion of L. monocytogenes
, as first posited by Briones et al. (3
), and support our previous data demonstrating extracellular replication.
After fasting for several hours, the gallbladder is filled with bile from the liver and becomes distended. The contraction of the organ can then be induced by a fat meal, which causes the small intestine to release cholecystokinin and secretin, hormones that enter the bloodstream and induce the contraction of the muscle layer on the outside of the gallbladder. This process results in the expulsion of bile, which aids in the emulsification of lipids. Mice exhibiting BLI signals from virulent L. monocytogenes
in the gallbladder also often show signals from lower in the abdomen (Fig. ), which suggests that excretion from the gallbladder could possibly result in intestinal infection. Such an infection may be extremely difficult to demonstrate in mice, because oral infection with L. monocytogenes
is well known to be highly inefficient in these animals (14
). The maximum number of CFU found in the gallbladder so far is 108
(data not shown), which is at least an order of magnitude less than the minimum infectious oral dose of L. monocytogenes
in mice, and therefore, the bacteria expelled would not be sufficient to observe reinfection in the mouse model. Humans and larger animals, however, are much more susceptible to listeriosis orally and have gallbladders thousands of times the size of those in mice, so the colonized gallbladders of humans could therefore easily contain numbers of CFU that exceed infectious doses. The nature and origin of gut signals that follow signals from the gallbladder were unknown prior to this report. Lower abdominal signals could be due to sources of the bacteria other than the gallbladder, such as directly from the liver through the common bile duct (3
). In addition, the blood and mesenteric lymph nodes often harbor L. monocytogenes
and could be sources of the abdominal signal. To show a direct correlation between gallbladder contraction and the subsequent bacterial signal in the abdomen, we induced gallbladder distension and contraction using a fast-and-feed procedure. Mice infected with the virulent strain as described in the legend of Fig. were starved for 4 to 8 h to prevent gallbladder contraction. When such animals were then fed whole cow's milk, signals from the abdomen subsequently appeared (Fig. ). Quantification of the signal remaining in the gallbladder and the signal appearing in the lower abdomen could be demonstrated by selecting regions of interest. The results for the representative mouse shown in Fig. reveal a decrease in gallbladder signal and a concomitant increase in the lower abdomen signal. To visualize this process in real time, serial images were obtained. These images revealed the rapid movement of L. monocytogenes
through the abdomen of the animal, such that within 18 min of expulsion, a portion of the signal had reached a point in the lower abdomen where it remained for the duration of the experiment. We interpret this result to be due to the bacteria being prevented from further movement along the intestine, probably due to obstruction by fecal material. Quantification of the data using regions of interest identical to those of Fig. showed that a decrease in the gallbladder signal was accompanied by only a modest increase in the signal from the lower abdomen in this case, although the abdominal signal was clearly evident.
As further evidence that gallbladder contraction was responsible for the observed signals and to increase the signal for serial imaging, we employed the synthetic tyrosine 27-sulfated peptide fragment 26-33 of cholecystokinin (CCK8), which is known to induce gallbladder contraction in a highly specific manner. Using ultrasound, the contraction of the gallbladder was observed to begin within 4 min of intraperitoneal injection of the hormone-derived peptide. CCK8 administration was found to cause more complete contraction than feeding as judged by the greater reduction of gallbladder signal observed, as is clearly shown by the differences between the serial images of the two procedures. Using CCK8, the results showed kinetics of signal movement similar to that of feeding, however. Feeding took much longer to induce contraction, as expected, and was never as complete as CCK8 administration, so animals subjected to fasting and feeding retained most of the gallbladder signal after the procedure. CCK8, however, reduced gallbladder signals to background levels with the virulent strain in CD1 mice and the shedding of increased numbers of bacteria in the feces after the procedure (Fig. ). These data show that the signal from the gallbladders of these mice was almost entirely due to extracellular bacteria, supporting previous observations, because intracellular organisms would not be released by this procedure. The process does not appear to be entirely the same in all animals. This result is expected due to the well-known flexibility of the intestines of mice; however, the overall pattern is much the same in each animal. When the abdominal signal has the greatest intensity in the lower abdomen, the signal travels from the mouse's right to left, consistent with the anatomy of the transverse colon.
During the process of moving through the body, the signal was lost for periods of time corresponding to one or two images, whereupon it reemerged in a different location from that previous to the disappearance (Fig. ). This effect could be due to either the differential light output of the bacteria or their movement through different locations in the body. The former possibility would most likely be due to differences in oxygen availability in the different parts of the alimentary tract rather than other enzyme activity differences in the bacteria. Oxygen tension affects light output from a given amount of luciferase, because O2
is required for the function of the enzyme. The most likely cause of the varying signal intensity in our estimation was the movement of the bacteria through the intestinal tract, which would result in differences in the depths of the signal within the mouse body. As the bacteria transit the intestine, some regions of this heavily folded organ will be closer to the ventral surface of the animal, resulting in a much greater signal intensity because the light has much less tissue to travel through. The effects of tissue depth on photon flux in mice have been carefully determined (18
), revealing that attenuation at different tissue depths can vary by orders of magnitude. The tissue overlaying the gallbladder is the liver, which is dark and attenuates the signal to a greater extent than the tissues overlaying many parts of the intestine. These factors may greatly change the signal as the bacteria move from one part of the body to another.
To determine the location along the intestine of the signal detected in live animals, mice exhibiting various signals during the process of expulsion were sacrificed, and the intestines were removed and imaged ex vivo. These images revealed that the terminal signal was largely localized to one area of the excised intestine (Fig. ) but that at earlier time points, some distribution of bacteria proximal and distal to this concentrated signal was detectable at high-sensitivity settings (Fig. ). We interpret these results to be that the bacteria are expelled over the period of contraction, as observed with ultrasound, but that a detectable bolus is expelled as well. This bolus could possibly be associated with mucus from the gallbladder. The transit of the bolus is impeded by fecal material at some point along the intestine, and the bacteria expelled later in the process catch up with those expelled earlier to form a more localized terminal signal, as shown in Fig. . During periods of reduced signal in the live animal, the subsequently excised intestines revealed signal concentrations (Fig. ) that we interpret as the previously detectable source in the live animal, indicating that the bacteria were still present and emitting light. Thus, it appears that the disappearance of the signal is the result of the location of the bacteria, i.e., deeper in the animal, where the signal is greatly reduced by the attenuation of the tissues. The bacteria then move along the intestine until they reach a more ventral location and the light is detectable once more.
These results have implications for human listeriosis. It is currently unknown if the human gallbladder is a site of extensive replication, but the presence of gallbladder signals in all mice early in infection with 1 LD50
of wild-type L. monocytogenes
, albeit on different days in different animals (12
), as well as the currently presented data shown in Fig. indicate that this phenomenon is the rule in mice rather than a rare exception in some animals. The gallbladder, which is normally sterile and acellular, could provide the bacterium with an anatomical site in which to grow to large numbers without causing disease or eliciting an immune response. This phenomenon may occur in domestic animals as well, which could be particularly ominous if the gallbladder is ruptured during slaughter. In developed countries, listeriosis is often associated with processed foods contaminated at the point of processing, but this bacterium, which is ubiquitous in the environment, may be prevalent in the foods prepared domestically in impoverished areas. Due to the complete absence of careful studies, no data are available on the prevalence of listeriosis in the Third World, but the symptoms of the disease, fever and chills sometimes followed by convulsions and death, are common to a number of infectious diseases, including many viral infections. Thus, listeriosis may be much more common than is currently estimated because diagnosis is difficult. The recent report of the first outbreak of listeriosis in Japan (16
) suggests that we do not know the extent of this disease. Many countries in the western part of Asia depend on milk products for a major portion of the normal diet, including yoghurts and other fermented substances that are known to support the growth of L. monocytogenes
. People in many of these areas are in an almost constant state of hunger, which could cause their gallbladders to remain in the distended state, providing the bacteria with a location in which to multiply. When a meal containing milk fat is consumed, the gallbladder would then contract, and the bacteria would be expelled into the environment and spread via fecal contamination. Our data imply that such a scenario, while still speculative at this time, is possible in the physiological sense.