Salmonella is a food-borne pathogen that leads to substantial illness worldwide. The clinical syndromes associated with Salmonella infection are enteric (typhoid) fever and gastroenteritis, in healthy humans. Typhoid fever is caused by host-adapted S. Typhi and S. Paratyphi. Gastroenteritis is caused by serovars usually referred to as non typhoidal Salmonellae (NTS). In recent years, an increasing number of outbreaks due to NTS, despite increased efforts in food safety, were reported because of persistence of Salmonella in the food chain. Thus I hypothesized that Salmonella is able to withstand stresses in the environment and treatments used during food processing for its elimination and thereby able to develop resistance against subsequent stress encounters. The effect of cold, peroxide, and acid was tested on survival of S. Typhimurium and the survival was persistent under cold stress (5°C) for up to 240 h. Pre-adaptation to cold stress (5°C, 5 h) also increased survival of S. Typhimurium during subsequent exposure to acid stress (pH 4.0, 90 min) by repressing hydroxyl radical formation. Cold stress (5°C, 48 h) to S. Typhimurium significantly (p < 0.05) increased its adhesion and invasion in intestinal iv epithelial cells. This phenotype was attributed to a pair of protein-protein interactorsacting as receptors on microbial (STM2699) and host cell surface (SPTAN1). Cold stress significantly (q < 0.05) induced STM2699 in S. Typhimurium and SPTAN1 was significantly (q < 0.05) induced in pithelial cells upon infection with cold-stressed S. Typhimurium. Cold stress to S. Typhimurium also significantly (q < 0.05) induced genes related to virulence such as type 3 secretion system apparatus and effectors genes, prophage genes, and plasmid genes and they remain induced upon infection of epithelial cells with additional induction of spv genes on the plasmid. Infection of epithelial cells with cold-stressed S. Typhimurium significantly (p < 0.05) increased activation of caspase 9 and 3/7. Cold-stressed S. Typhimurium switched metabolism from aerobic respiration to fermentation and it persisted during infection of epithelial cells. As a result, short chain fatty acids formate and acetate, which act as diffusible signal for invasion, were detected in significantly (q < 0.05) high amounts in extracellular media of cells infected with cold-stressed S. Typhimurium supporting the phenotype of high adhesion and invasion of cold-stressed S. Typhimurium in epithelial cells.

Bacterial pathogens on the surfaces of raw produce may be difficult to remove for identification and enumeration. The first part of this project examined whether ultrasonic treatment (40 kHz) of a rinse solution would enhance recovery of Salmonella spp. from various produce surfaces. Strawberries, apples, and cantaloupe were surface inoculated with a five-strain cocktail of nalidixic acid resistant Salmonella spp. Samples were subjected to one of six different treatments using different combinations of agitation methods (manual shaking or ultrasound), diluent temperatures (25°C and 40°C), and agitation times (60 and 120 seconds). After treatment, diluent was spiral plated onto tryptic soy agar supplemented with 50 ppm of nalidixic acid and plates were incubated at 37°C for 48 hours. Results from this study indicate that ultrasonic treatment of a rinse solution did not enhance or diminish recovery of Salmonella spp. from produce surfaces, as compared to manual agitation. The effects of diluent temperature and exposure time appeared to have a significant effect on recovery, depending on the type of produce. The second part of this project used a computer imaging system to determine the surface area of various types of produce. The imaging system acquired and stored multiple images of the produce samples. From these images, surface fitting and approximation of a 3-D wire frame model were used to calculate surface area. From these measurements, it was determined that there were statistical relationships between surface area and weight. Surface area measurements were used to develop equations to predict surface area from weight measurements. / Master of Science

Salmonella is often associated with retail poultry products. Our research evaluated the effect of temperature on the biofilm formation by Salmonella spp. and the efficacy of essential oils in controlling these biofilms on stainless steel surfaces. The sublethal concentrations of thyme, oregano and carvacrol at 0.006-0.012% suppressed biofilm formation by Salmonella spp. while concentrations at 0.05-0.1% reduced the biofilms of a three-strain mixture of Salmonella spp. by 7 logs. Carvacrol was evaluated in combination with lauric arginate for controlling the three-strain mixture of Salmonella spp. in ground turkey containing 1%, 7% or 15% fat. Higher concentrations of carvacrol (1%) or lauric arginate (2000 ppm) when applied individually did not reduce Salmonella counts in ground turkey containing 7% fat. The combined mixture of carvacrol and lauric arginate at these higher concentrations was found to be synergistic in reducing the Salmonella counts by 4 log CFU/g in ground turkey containing 7% fat.

No description available.

Salmonella enterica serovar Typhimurium (Salmonella) is a Gram-negative bacterial pathogen capable of causing both gastroenteritis and bacteraemia in human hosts. During infection, Salmonella invokes a complex network of virulence factors, regulatory systems, and metabolic pathways to promote immune evasion, sometimes demanding antibiotic treatment for resolution. Unfortunately, antibiotic resistance has reached critical levels in this and other pathogens, necessitating the discovery of new anti-infective targets and treatment options. Herein, we have sought to exploit the dynamic interactions between Salmonella and the host immune system to identify new, conditionally active anti-Salmonella therapies. In chapter 2, we aim to identify chemical compounds that are selectively antimicrobial against intracellular Salmonella, and discover that the anxiolytic drug metergoline inhibits Salmonella survival in cultured macrophages and systemically infected mice. In chapter 3, we screen for anti-virulence compounds that target regulatory signaling in Salmonella, and characterize the inhibitory activity of methyl-3,4-dephostatin, which perturbs SsrA/B and PmrB/A signaling and enhances sensitivity to colistin in vitro and in vivo. In chapter 4, we identify several host-directed compounds that modulate macrophage immunity and investigate their ability to attenuate a multidrug resistant Salmonella infection. Together, the work presented in this thesis demonstrates the potential for drug screening in infection-relevant conditions to identify new anti-infectives with non-traditional targets. / Thesis / Doctor of Philosophy (PhD)

The interaction of bacterial pathogens and the human host is a complex process that has shaped both organisms on a molecular, cellular and population level. When pathogenic bacteria infect the human body, a battle ensues between the host immune system and the pathogen. In order to escape an immune response and to colonize the host, pathogenic bacteria have developed diverse virulence strategies and some pathogens even replicate within host cells. For survival and propagation within the dynamic environment of a host cell, these bacteria interfere with the regulation of host pathways, such as the cell cycle, for their own benefit. The intracellular pathogen Salmonella Typhimurium invades eukaryotic cells and resides and replicates in a modified vacuolar compartment in which it is protected from the innate immune response. To this end, it employs a set of virulence factors that help to invade cells (SPI-1 effectors) and to hijack and modify the host endolysosomal system, in order to stabilize and mature its vacuolar niche (SPI-2 effectors). Previous studies have shown that Salmonella arrests host cells in G2/M phase and that Salmonella infected cells progress faster from G1 into S phase, suggesting that the G1 phase is disadvantageous for Salmonella infection. In fact, it has already been observed that Salmonella replication is impaired in G1 arrested cells. However, the reason for this impairment remained unclear. The current study addressed this question for the first time and revealed that the highly adapted, intracellular lifestyle of Salmonella is drastically altered upon G1 arrest of the host cell. It is shown that proteasomal degradation in G1 arrested cells is delayed and endolysosomal and autophagosomal trafficking is compromised. Accordingly, processing of lysosomal proteins is insufficient and lysosomal activity is decreased; resulting in uneven distribution and accumulation of endolysosomes and autophagosomes, containing undegraded cargo. The deregulation of these cellular signaling pathways affects maturation of the Salmonella containing vacuole (SCV). For the first time it is shown that acidification of SCVs is impaired upon G1 arrest. Thus, an important environmental factor for the switch from SPI-1 to SPI-2 gene expression is missing and the SPI-2 system is not activated. Consequently, targeting and modification of host cell structures by SPI-2 effectors e.g. recruitment of endolysosomal membrane proteins, like LAMP1, or exchange of endosomal cargo, is compromised. In addition, degradation of Salmonella SPI-1 effectors by the host proteasome is delayed. Their prolonged presence sustained the recruitment of early endosomes and contributed to the SCV remaining in an early, vulnerable maturation stage. Finally, it was shown that SCV membrane integrity is compromised; the early SCV ruptures and bacteria are released into the cytoplasm. Depending on the host cell type, SPI-2 independent, cytoplasmic replication is promoted. This might favor bacterial spreading, dissemination into the tissue and provide an advantage in host colonization. Overall, the present study establishes a link between host cell cycle regulation and the outcome of Salmonella infection. It fills the gap of knowledge as to why the host cell cycle stage is of critical importance for Salmonella infection and sheds light on a key aspect of host-pathogen interaction. / Die Interaktion zwischen bakteriellen Krankheitserregern und dem menschlichen Wirt ist ein komplexer Prozess, der beide Organismen auf molekularer, zellulärer und Populationsebene geprägt hat. Wenn pathogene Bakterien den menschlichen Körper infizieren, kommt es zu einem Kampf zwischen dem Immunsystem des Wirtes und dem Krankheitserregers. Um einer Immunantwort zu entgehen und den Wirt zu besiedeln, haben pathogene Bakterien diverse Strategien entwickelt und einige Erreger vermehren sich sogar innerhalb von Wirtszellen. Zum Überleben und zur Vermehrung innerhalb der dynamischen Umgebung einer Wirtszelle, manipulieren diese Bakterien die Regulation zellulärer Netzwerke, wie zum Beispiel den Zellzyklus, zu ihrem eigenen Vorteil. Salmonella Typhimurium, ein intrazelluläres Bakterium, dringt in eukaryotische Wirtszellen ein und vermehrt sich in einem modifizierten, vakuolären Kompartiment, welches gleichzeitig vor der angeboren Immunantwort des Wirtes schützt. Zu diesem Zweck entwickelten Salmonellen eine Reihe von Virulenzfaktoren. Diese sind zum einen für die Invasion von Zellen verantwortlich (SPI-1 Faktoren), zum anderen greifen sie das endolysosomale System der Wirtszelle an und modifizieren es, mit dem Ziel die intrazelluläre Salmonellen-enthaltende Vakuole (SCV) zu stabilisieren und reifen zu lassen (SPI-2 Faktoren). Frühere Studien haben gezeigt, dass Salmonellen ihre Wirtszellen in der G2/M Phase blockieren. Zudem gehen Salmonellen-infizierte Zellen schneller von der G1 in die S-Phase über, was auf einen Nachteil der G1-Phase für die Salmonelleninfektion hindeutet. In der Tat wurde bereits beobachtet, dass die Vermehrung von Salmonellen in G1-arretierten Zellen beeinträchtigt war. Der Grund für diese Beeinträchtigung blieb jedoch unklar. Die vorliegende Studie befasst sich zum ersten Mal mit dieser Frage und zeigt auf, dass der hoch angepasste, intrazelluläre Lebensstil von Salmonellen während des G1-Arrest der Wirtszelle dramatisch verändert wird. Im Rahmen der hier vorgelegten Arbeit wurde gezeigt, dass der proteasomale Abbau in G1-arretierten Zellen verzögert und die endolysosomalen und autophagosomalen Transportnetzwerke beeinträchtigt sind. Dementsprechend ist die Prozessierung lysosomaler Proteine unzulänglich und die lysosomale Aktivität herabgesetzt; was zu einer ungleichmäßigen Verteilung und Anreicherung von Endolysosomen und Autophagosomen führt, die nicht abgebaute Stoffwechselprodukte akkumulieren. Die Deregulierung der genannten zellulären Signalwege beeinflusst die Reifung der SCV. Es konnte hier zum ersten Mal gezeigt werden, dass die Ansäuerung der SCV in G1-arretierten Zellen inhibiert ist. Somit fehlt ein essentieller Faktor für den Wechsel von SPI-1 zu SPI-2-Genexpression und das SPI-2 System wird nicht aktiviert. Folglich findet keine Modifikation der Wirtszelle durch SPI-2-Effektoren, z.B. die Rekrutierung endolysosomaler Membranproteine, wie LAMP1 oder der Austausch endosomaler Fracht statt. Zudem ist der Abbau von bakteriellen SPI-1-Effektoren durch das Wirtsproteasom verzögert. Die verlängerte Präsenz der SPI-1 Effektoren fördert eine anhaltende Rekrutierung von frühen Endosomen und trägt zum Verbleib der SCV in einem frühen, sehr instabilen Reifestadium bei. Schließlich wurde gezeigt, dass die Integrität der SCV Membran kompromittiert ist, die Vakuole aufbricht und die Bakterien ins Zytoplasma entlassen werden. In Abhängigkeit des Wirtszelltyps wird eine SPI-2 unabhängige, zytoplasmatische Vermehrung begünstigt, was möglicherweise die Ausbreitung der Bakterien ins Gewebe erleichtert und somit einen Vorteil bei der Besiedelung des Wirtes darstellt. Insgesamt etabliert die vorliegende Studie einen Zusammenhang zwischen der Regulation des Wirtszellzyklus und dem Ergebnis einer Salmonelleninfektion. Es wird aufgezeigt, warum der Zellzyklus der Wirtszelle von entscheidender Bedeutung für den Verlauf der Salmonelleninfektion ist und beleuchtet somit einen essentiellen Aspekt der Wirt-Pathogen-Interaktion.

Small proteins, often defined as shorter than 50 amino acids, have been implicated in fundamental cellular processes. Despite this, they have been largely understudied throughout all domains of life, since their size often makes their identification and characterization challenging. This work addressed the knowledge gap surrounding small proteins with a focus on the model bacterial pathogen Salmonella Typhimurium. In a first step, new small proteins were identified with a combination of computational and experimental approaches. Infection-relevant datasets were then investigated with the updated Salmonella annotation to prioritize promising candidates involved in virulence. To implement the annotation of new small proteins, predictions from the algorithm sPepFinder were merged with those derived from Ribo-seq. These were added to the Salmonella annotation and used to (re)analyse different datasets. Information regarding expression during infection (dual RNA-seq) and requirement for virulence (TraDIS) was collected for each given coding sequence. In parallel, Grad-seq data were mined to identify small proteins engaged in intermolecular interactions. The combination of dual RNA-seq and TraDIS lead to the identification of small proteins with features of virulence factors, namely high intracellular induction and a virulence phenotype upon transposon insertion. As a proof of principle of the power of this approach in highlighting high confidence candidates, two small proteins were characterized in the context of Salmonella infection. MgrB, a known regulator of the PhoPQ two-component system, was shown to be essential for the infection of epithelial cells and macrophages, possibly via its stabilizing effect on flagella or by interacting with other sensor kinases of twocomponent systems. YjiS, so far uncharacterized in Salmonella, had an opposite role in infection, with its deletion rendering Salmonella hypervirulent. The mechanism underlying this, though still obscure, likely relies on the interaction with inner-membrane proteins. Overall, this work provides a global description of Salmonella small proteins in the context of infection with a combinatorial approach that expedites the identification of interesting candidates. Different high-throughput datasets available for a broad range of organisms can be analysed in a similar manner with a focus on small proteins. This will lead to the identification of key factors in the regulation of various processes, thus for example providing targets for the treatment of bacterial infections or, in the case of commensal bacteria, for the modulation of the microbiota composition. / Kleine Proteine, oft definiert als kürzer als 50 Aminosäuren, sind in fundamentale zelluläre Prozesse involviert. Trotzdem sind sie in allen Domänen des Lebens noch weitgehend unerforscht, da ihre Größe ihre Identifizierung und Charakterisierung oft schwierig macht. Diese Arbeit adressiert die Wissenslücke um kleine Proteine mit einem Fokus auf das bakterielle Modellpathogen Salmonella Typhimurium. In einem ersten Schritt wurden neue kleine Proteine mit einer Kombination aus bioinformatischen und experimentellen Ansätzen identifiziert. Anschließend wurden infektionsrelevante Datensätze mit der aktualisierten Salmonella-Annotation untersucht, um vielversprechende Kandidaten zu priorisieren, die an der Virulenz beteiligt sind. Um die Annotation neuer kleiner Proteine zu implementieren, wurden die Vorhersagen aus dem Algorithmus sPepFinder mit denen aus Ribo-seq kombiniert. Diese wurden der Salmonella-Annotation hinzugefügt und zur (Re-)Analyse verschiedener Datensätze verwendet. Für jede gegebene kodierende Sequenz wurden Informationen zur Expression während der Infektion (duale RNA-seq) und zum Beitrag zur Virulenz (TraDIS) gesammelt. Parallel dazu wurden Grad-seq-Daten ausgewertet, um kleine Proteine zu identifizieren, die an intermolekularen Interaktionen beteiligt sind. Die Kombination von dualer RNA-seq und TraDIS führte zur Identifizierung von kleinen Proteinen mit Merkmalen von Virulenzfaktoren, nämlich einer hohen intrazellulären Induktion und einem Virulenz-Phänotyp nach Transposon- Insertion. Als Beweis für die Leistungsfähigkeit dieses Ansatzes Identifikation von vielversprechenden Kandidaten wurden zwei kleine Proteine im Kontext einer Salmonella-Infektion charakterisiert. MgrB, ein bekannter Regulator des PhoPQ-Zweikomponentensystems, erwies sich als ein für die Infektion von Epithelzellen und Makrophagen essentielles Protein, möglicherweise über seine stabilisierende Wirkung von Flagellen oder durch Interaktion mit Sensorkinasen von Zweikomponentensystemen. YjiS, das in Salmonella bisher nicht charakterisiert wurde, hatte eine entgegengesetzte Rolle bei der Infektion, wobei seine Deletion Salmonella hypervirulent macht. Der Mechanismus, der dem zugrunde liegt, ist zwar noch unklar, beruht aber wahrscheinlich auf der Interaktion mit inneneren Membranproteinen. Insgesamt liefert diese Arbeit eine globale Beschreibung der kleinen Salmonella- Proteine im Kontext der Infektion mit einem kombinatorischen Ansatz, der die Identifizierung interessanter Kandidaten beschleunigt. Verschiedene Hochdurchsatz- Datensätze, die für ein breites Spektrum von Organismen verfügbar sind, können auf ähnliche Weise mit einem Fokus auf kleine Proteine analysiert werden. Dies wird zur Identifizierung von Schlüsselfaktoren in der Regulation verschiedener Prozesse führen und damit z. B. Targets für die Behandlung bakterieller Infektionen oder, im Falle kommensaler Bakterien, für die Modulation der Mikrobiota- Zusammensetzung liefern.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Abstract: COVID-19 has impressively shown how quickly an emerging pathogen can have a massive impact on our entire lives and show how infectious diseases spread regardless of national borders and economic stability. We find ourselves in a post-antibiotic era and have rested too long on the laurels of past research, so today more and more people are dying from infections with multi-resistant germs. Infections are highly plastic and heterogeneous processes that are strongly dependent on the individual, whether on the host or pathogen side. Improving our understanding of the pathogenicity of microorganisms and finding potential targets for a completely new class of drugs is a declared goal of current basic research. To tackle this challenge, single-cell RNA sequencing (scRNA-seq) is our most accurate tool. In this thesis we implemented different state of the art scRNA-seq technologies to better understand infectious diseases. Furthermore, we developed a new method which is capable to resolve the transcriptome of a single bacterium. Applying a poly(A)-independent scRNA-seq protocol to three different, infection relevant growth conditions we can report the faithful detection of growth-dependent gene expression patterns in individual Salmonella Typhimurium and Pseudomonas aeruginosa bacteria. The data analysis shows that this method not only allows the differentiation of various culture conditions but can also capture transcripts across different RNA species. Furthermore, using state of the art imaging and single-cell RNA sequencing technologies, we comprehensively characterized a human intestinal tissue model which in further course of the project was used as a Salmonella enterica serovar Typhimurium infection model. While most infection studies are conducted in mice, lacking a human intestinal physiology, the in vitro human tissue model allows us to directly infer in vivo pathogenesis. Combining immunofluorescent imaging, deep single-cell RNA sequencing and HCR-FISH, applied in time course experiments, allows an unseen resolution for studying heterogeneity and the dynamics of Salmonella infection which reveals details of pathogenicity contrary to the general scientific opinion. / Zusammenfassung: COVID-19 hat eindrucksvoll gezeigt, wie schnell ein neu auftretender Erreger massive Auswirkungen auf unser aller Leben haben kann und wie sich Infektionskrankheiten unabhängig von Landesgrenzen und wirtschaftlicher Stabilität ausbreiten. Wir befinden uns in einer post-antibiotischen Ära und haben uns zu lange auf den Lorbeeren der vergangenen Forschung ausgeruht, so dass heute immer mehr Menschen an Infektionen mit multiresistenten Keimen sterben. Infektionen sind sehr plastische und variable Prozesse, die stark vom Individuum abhängen, sei es auf Seiten des Wirts oder des Erregers. Die Pathogenität von Mikroorganismen besser zu verstehen und potenzielle Angriffspunkte für eine völlig neue Klasse von Arzneimitteln zu finden ist ein erklärtes Ziel der aktuellen Grundlagenforschung. Um diese Herausforderung zu meistern, ist die Einzelzell-RNA-Sequenzierung (scRNA-seq) unser präzisestes Werkzeug. In dieser Arbeit haben wir verschiedene hochmoderne scRNA-seq-Technologien eingesetzt, um Infektionskrankheiten besser zu verstehen. Darüber hinaus haben wir eine neue Methode entwickelt, die in der Lage ist, das Transkriptom eines einzelnen Bakteriums aufzulösen. Durch die Anwendung eines poly(A)-unabhängigen scRNA-seq-Protokolls unter drei verschiedenen, infektionsrelevanten W achstumsbedingungen konnten wir die wachstumsabhängigen Genexpressionsmuster in einzelnen Salmonella Typhimurium- und Pseudomonas aeruginosa- Bakterien zuverlässig nachweisen. Die Datenanalyse zeigt, dass diese Methode nicht nur die Differenzierung verschiedener Kulturbedingungen ermöglicht, sondern auch Transkripte über verschiedene RNA-Spezies hinweg erfassen kann. Darüber hinaus haben wir unter Verwendung modernster Bildgebungs- und Einzelzell-RNA- Sequenzierungstechnologien ein menschliches Darmgewebemodell umfassend charakterisiert, das im weiteren Verlauf des Projekts als Salmonella Typhimurium-Infektionsmodell verwendet wurde. Während die meisten Infektionsstudien in Mäusen durchgeführt werden, denen die menschliche Darmphysiologie fehlt, ermöglicht uns das in vitro Modell des menschlichen Gewebes direkte Rückschlüsse auf die Pathogenese in vivo. Die Kombination aus immunfluoreszierender Bildgebung, deep single-cell RNA Sequenzierung und HCR-FISH, angewandt in Zeitverlaufsexperimenten, ermöglicht eine bisher ungesehene Auflösung zur Untersuchung von Heterogenität und Dynamik einer Salmonella Infektion, welche Details der Pathogenität entgegen der allgemeinen wissenschaftlichen Meinung offenbaren.

Bacteria frequently encounter changes in their environment and must adapt accordingly. When these changes are predictable, there is evidence of anticipatory gene regulation. For example, the model enteric pathogen Salmonella has a well-defined natural history, typically only encountering iron in the lumen of the gut. However, this bacterium responds to the presence of iron not by upregulating genes needed to thrive in the lumen, but rather the iron-deplete epithelium, the subsequent environment it encounters. This may be similar to Pavlovian conditioning, a type of associative learning that involves pairing two unrelated stimuli and anticipatory behavioural changes. Since conditioning has not been well explored in bacteria, we are investigating whether Salmonella can learn new conditioned responses by pairing two unrelated carbon sources, citrate and maltose. We leveraged a prototype of the Microbial Evolution and Growth Arena (MEGA)-plate motility assay to define a new natural history. By pairing stimuli across a physical landscape, we can select for bacteria that learn to use citrate to anticipate maltose and can quickly deplete the second carbon source. Time-series imaging of bacteria as they swim across the plate shows evidence of emerging variants capable of swimming faster through maltose and unique swimming behaviours through repeated passaging. This approach selects for the fastest swimming bacteria, not necessarily bacteria that have acquired anticipatory regulation. As such, further genetic and transcriptional analysis of the variants are necessary. Similar passaging of Salmonella in broth allowed us to compare anticipatory regulation across a physical and non-physical landscape. Learning to anticipate environmental changes will provide a bacterium with a selective advantage, allowing it to outcompete its conspecifics which are slower to respond. From this investigation, we hope to provide insight into the learning capacity of bacteria and further understand how bacteria exploit memory to problem-solve. / Thesis / Master of Science (MSc) / Bacteria that encounter predictable changes in their environment can acquire anticipatory gene regulation. This may be similar to Pavlovian conditioning, a type of associative learning that involves pairing two unrelated stimuli and anticipatory behavioural changes. Since conditioning has not been well explored in bacteria, we investigate whether Salmonella can learn new behaviours by pairing two unrelated carbon sources, citrate and maltose. We leveraged a motility plate assay to define a new natural history. By pairing stimuli across a physical landscape, we can select for bacteria that learn to use citrate to anticipate maltose. Time-series imaging shows evidence of variants capable of swimming faster through maltose and unique swimming behaviours through repeated passaging. Similar passaging of Salmonella in broth allowed us to compare anticipatory regulation across physical and non-physical landscapes. From this investigation, we hope to further understand the learning capacity of bacteria and how bacteria exploit memory to solve problems.