Sections were obtained using a microtome. Considering the presence of bacteria in the cortex, medulla, cerebellum and midbrain, we were curious to know whether S. Typhimurium could infect neurons. We used a murine neuronal cell line Neuro2A N2A as a model and carried out invasion and intracellular survival assays.
Results indicated that both stationary phase and log phase S. Typhimurium can very successfully invade and proliferate massively within the neuronal cell line Figure 4A and at a rate much higher than in other cells like macrophages Figure 4B. Immunofluorescence studies indicated the presence of S. Typhimurium in neurons from early through late time points of infection Figures 4C—F. Figure 4. Salmonella can invade neuronal cells. A Fold proliferation of log phase or stationary phase S.
Typhimurium in N2A cells. B Comparison between fold proliferation of stationary phase S. Typhimurium in N2A cells vs. RAW Typhimurium at 30 min, 4, 8, and 24 h post-infection respectively. G Quanitation of colocalization between S. Typhimurium and LAMP1. A majority of pathogenic characteristics of Salmonellae can be attributed to one or more of its virulence factors such as the SPIs, virulence plasmids, flagella and others.
Some encode secretion systems while others code for effectors and a variety of virulence factors. Two most prominent pathogenicity islands are Salmonella pathogenicity island SPI 1 and 2. Both these islands code for type three secretion systems. While SPI-1 sends effectors across the host cell membrane and has a prominent role in epithelium invasion, SPI-2 translocates effectors across the SCV membrane and promotes intracellular replication.
In trying to understand whether the SPIs can spy into the host brain, we used various mutants that are deficient in either forming the T3SS or in an important effector protein.
The results suggest that an intact SPI-1 aids in effective translocation of the bacteria across the blood-brain barrier. However, their presence in various parts of the brain despite deletions in important SPI-I genes indicates the importance of other factors in blood-brain barrier penetration.
Figure 5. These results indicate that an unrelated and unknown dedicated mechanism besides SPI-1 and essential for crossing the blood-brain barrier exists in S. This intriguing question led us to explore various candidates for a role in penetrating the blood-brain barrier. Outer membrane protein A OmpA is a constituent protein of the outer membrane of several Gram negative bacteria.
Interactions between proteins on the outer membrane of bacteria would conceivably be essential for binding and invasion through the blood-brain barrier. We thus asked whether an outer membrane protein OmpA is essential for blood-brain barrier penetration in case of S.
We created a deletion strain with the ompA gene knocked out via one step deletion, and infected mice at a dose of 10 7 via the oral route. We observed a reduction in bacterial burden in the cortex, cerebellum, midbrain and the medulla Figures 6A—D. Since the bacterial burden in liver and spleen also showed lower levels Figures 6E,F , we further used the intraperitoneal mode of infection to bypass the intestinal route. Upon intraperitoneal infection, we observed a significant decrement in the bacterial concentration in mouse brain between wild-type S.
Figure 6. Role of ompA in brain invasion. Figure 7. Student's T -test and Mann Whitney U -post-test. We have consistently observed that infected mice occasionally develop prominent neurological abnormalities such as rolling and rotatory motions. We carried out water-maze experiments to estimate the time taken by infected mice to reach a platform in water.
All mice were first trained for the task following which their performances were recorded both pre and post-infection. We observed a significant increase in the time taken by certain mice infected at either 10 7 or 10 8 CFU dosage orally, to remember and swim to the dedicated platform with a few absolutely unable to do so.
Figures 8A—D. We could not however, directly correlate the bacterial load in any particular brain part among those tested, with the behavioral deficiencies. Figure 8. Behavioral changes in mice infected with S.
Typhoid patients manifest neurological symptoms during and even years after the disease incidence. This led us to investigate the behavior of infected mice after antibiotic Figure S1 treatment and the corresponding bacterial burden.
While some mice showed a restoration in their ability to find the water maze platform, there were animals which were still strikingly incapable of performing the task. Moreover, bacteria were still present in the brains of even the better performing mice Figures 9A—F. Figure 9. Neurological manifestations of Salmonella brain invasion and effect of antibiotics.
B Time taken by the mice in A to swim to a platform in a water maze experiment pre and post-infection. D Time taken by the mice in C to swim to a platform in a water maze experiment pre and different stages post-infection.
F Time taken by the mice in E to swim to a platform in a water maze experiment pre and different stages post-infection. Each point in the plots represents an animal. Few animals died prior to the designated test days. These results could have serious implications in the general neurological health of the host in the long run. Salmonella infections are frequent in developing nations around the world. Neuro-psychiatric symptoms related to typhoid fever have been observed for years Khosla et al.
Alarmingly, multidrug resistant typhoid incidences have also been found to be associated with neurologic manifestations Lutterloh et al. While we have looked at S. Typhimurium infection in mice which mimics human typhoid fever, the effect of non-typhoidal infections in this regard cannot be ignored. Tracking and data collection regarding self-limitting gastrointestinal diseases would be considerably more challenging to understand the interaction with brain in that regard.
Consequently, reports are expected to be limiting Rizek et al. Recently, Bauler et al. Typhimurium burden in the brain Bauler et al. Our results corroborate the observations made by Bollen et al. Further, we observed an enhanced thickness of the granular layer in infected cerebellum. Enhanced granular layer thickness has been associated with age related degeneration and might be an important sign of neurological deficit Zhang et al.
Typhimurium genes like ompA and SPI-1 genes, that take part in mediating host brain invasion. Here, we show that ompA is essential for brain invasion by S. Apart from its structural roles, OmpA also serves a pathogen-associated molecular pattern in bacteria belonging to Enterobacteriaceae Jeannin et al. Besides its other roles, OmpA has been shown to be an important player in blood-barrier penetration of a number of bacteria especially E. OmpA of C. Enterobacter sakazakii requires OmpA for invasion of brain endothelial cells via induction of microtubule condensation Singamsetty et al.
The implications of Salmonella burden in various parts of the brain could be far reaching. Proliferation of the pathogen in various brain parts could be associated with an enormous variety of neurological symptoms. Moreover, we found a very high proliferation capacity in neuronal cell lines bolstering our hypothesis that the neurological manifestations post-infection are effects of possible cell death or modulation in the brain.
Further, cellular endotoxins are known to induce inflammation in substantia nigra dopaminergic neurons. Hippocampal slice cultures were found to undergo neurodegeneration in presence of Salmonella LPS Johansson et al.
Interestingly, sepsis has been found to make the blood-brain barrier leaky Brandtzaeg et al. Effects mediated by endotoxins could therefore lead to a battery of symptoms in context of the neurological manifestations.
Glial cells could also serve as a niche for Salmonella in the brain. Conceivably, Salmonella would be less proliferative in glial cells, however, they may act as a niche harboring persister populations. With the average life-span of humans getting extended every decade and the complexities in modern lifestyles, we are also witnessing an increase in the incidences of neurological abnormalities ranging from depression, anxiety, bipolar disorders to diseases like Parkinson's disease and Alzheimer's disease.
In view of the enhanced incidences, the possible causes such as external pathogens are of outstanding importance. For people from Canada oral revaccination is recommended after seven years while Americans revaccinate after five years.
Typhoid fever is widespread across the globe, but it is less common in industrialized parts of the world such as Western Europe, the United States, Canada, Japan, and Australia.
Part of the world with the highest risk of infection is in South Asia. However, infections also occur in other parts of Asia, Africa, and Latin America. A person can get typhoid fever throughout the year, but the infection is particularly common during summer. It could be due to the fact typhoid mainly affects regions with poor sanitation and bacteria spread faster in warmer weather. According to the WHO , last typhoid outbreak occurred in Uganda in At the beginning of the said year, the outbreak started in Kampala City, and by March 5, about cases of the infection were identified.
From Kampala, typhoid fever outbreak spread to neighboring cities and regions affecting primarily men ages 20 to In November , typhoid outbreak started in a city called Hyderabad, Pakistan. The biggest problem regarding this latest outbreak is the fact it was caused by drug-resistant superbug strain.
Despite the lacking of official data, it is estimated that cases of the infection were detected. Typhoid fever is a common disease that is caused by bacteria we introduce to our body through the intake of contaminated food and water primarily.
Blood sample with typhoid positive. What organs can typhoid affect? What is the minimum age requirement for typhoid fever vaccine? The attack resulted in cases of salmonellosis with no deaths. In New York City from to , Mary Mallon, also known as Typhoid Mary, spread typhoid fever to 47 people, killing three, through food that she handled. Approximately different sub-species, or serotypes, of salmonella can sicken humans.
The bacteria cause salmonellosis, an illness commonly associated with food poisoning. The flu-like typhoid fever, which only infects humans, is caused by the Typhi serotype. Transmission of the salmonella bacteria occurs through ingestion of contaminated food and drinks. A bioterrorist could simply drop samples of the bacteria onto food or drinks ready to be consumed. Outbreaks of salmonella in the U. Different salmonella serotypes are found worldwide and in a variety of different environments.
These locations include water, soil, insects, factory and kitchen surfaces, animal feces, and raw meats, poultry, and seafood. The bacteria can be isolated from these sources and grown.
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