Naturally drying bacterial droplets on inanimate surfaces representing fomites are the most consequential mode for transmitting infection through oro-fecal route. We provide a multiscale holistic approach to understand flow dynamics induced bacterial pattern formation on fomites leading to pathogenesis. The most virulent gut pathogen, Salmonella Typhimurium (STM), typically found in contaminated food and water, is used as model system in the current study. Evaporation-induced flow in sessile droplets facilitates the transport of STM, forming spatio-temporally varying bacterial deposition patterns based on droplet medium’s nutrient scale. Mechanical and low moisture stress in the drying process reduced bacterial viability but interestingly induced hyper-proliferation of STM in macrophages, thereby augmenting virulence in fomites. In vivo studies of fomites in mice confirm that STM maintains enhanced virulence. This work demonstrates that stressed bacterial deposit morphologies formed over small timescale (minutes) on organic and inorganic surfaces, plays a significant role in enhancing fomite’s pathogenesis over hours and days.

Salmonella systemic infections claim thousands of lives worldwide even today. Certain cases lead to an infection in the brain culminating in meningitis and associated neurological abnormalities. Multiple reports have indicated neurological manifestations in patients suffering from typhoid fever during the course of infection and afterwards. While the meanderings of Salmonella systemic infections are fairly well studied, the flow of events in the brain is very poorly understood. We investigated the colonization of various brain parts by Salmonella in mice. It was observed that the bacterium is frequently able to invade various brain parts in mice. Selected mutants namely deletion mutants of key proteins encoded by the Salmonella pathogenicity islands (SPIs) 1 and 2 and ompA gene were also used to decipher the roles of specific genes in establishing an infection in the brain. Our results suggest roles for the Salmonella pathogenicity island (SPI) 1 and outer membrane protein A gene in enabling blood-brain barrier penetration by the pathogen. We further investigated behavioral abnormalities in infected mice and used an antibiotic treatment regime in an attempt to reverse the same. Results show some mice still display behavioral abnormalities and a high bacterial burden in brain despite clearance from spleen and liver. Overall, our study provides novel insights into S. Typhimurium's capacity to invade the mouse brain and the effectiveness of antibiotic treatment on behavioral manifestations due to infection. These observations could have important implications in understanding reported neurological manifestations in typhoid patients.

Bacterial porins are highly conserved outer membrane proteins used in the selective transport of charged molecules across the membrane. In addition to their significant contributions to the pathogenesis of Gram-negative bacteria, their role(s) in salmonellosis remains elusive. In this study, we investigated the role of outer membrane protein A (OmpA), one of the major outer membrane porins of Salmonella, in the pathogenesis of Salmonella Typhimurium (STM). Our study revealed that OmpA plays an important role in the intracellular virulence of Salmonella. An ompA deficient strain of Salmonella (STM ΔompA) showed compromised proliferation in macrophages. We found that the SPI-2 encoded virulence factors such as sifA and ssaV are downregulated in STM ΔompA. The poor colocalization of STM ΔompA with LAMP-1 showed that disruption of SCV facilitated its release into the cytosol of macrophages, where it was assaulted by reactive nitrogen intermediates (RNI). The enhanced recruitment of nitrotyrosine on the cytosolic population of STM ΔompAΔsifA and ΔompAΔssaV compared to STM ΔsifA and ΔssaV showed an additional role of OmpA in protecting the bacteria from host nitrosative stress. Further, we showed that the generation of greater redox burst could be responsible for enhanced sensitivity of STM ΔompA to the nitrosative stress. The expression of several other outer membrane porins such as ompC, ompD, and ompF was upregulated in STM ΔompA. We found that in the absence of ompA, the enhanced expression of ompF increased the outer membrane porosity of Salmonella and made it susceptible to in vitro and in vivo nitrosative stress. Our study illustrates a novel mechanism for the strategic utilization of OmpA by Salmonella to protect itself from the nitrosative stress of macrophages.

Salmonella is a facultative intracellular pathogen that has co-evolved with its host and has also developed various strategies to evade the host immune responses. Salmonella recruits an array of virulence factors to escape from host defense mechanisms. Previously chitinase A (chiA) was found to be upregulated in intracellular Salmonella. Although studies show that several structurally similar chitinases and chitin-binding proteins (CBP) of many human pathogens have a profound role in various aspects of pathogenesis, like adhesion, virulence, and immune evasion, the role of chitinase in the intravacuolar pathogen Salmonella has not yet been elucidated. Therefore, we made chromosomal deletions of the chitinase encoding gene (chiA) to study the role of chitinase of Salmonella enterica in the pathogenesis of the serovars, Typhimurium, and Typhi using in vitro cell culture model and two different in vivo hosts. Our data indicate that ChiA removes the terminal sialic acid moiety from the host cell surface, and facilitates the invasion of the pathogen into the epithelial cells. Interestingly we found that the mutant bacteria also quit the Salmonella-containing vacuole and hyper-proliferate in the cytoplasm of the epithelial cells. Further, we found that ChiA aids in reactive nitrogen species (RNS) and reactive oxygen species (ROS) production in the phagocytes, leading to MHCII downregulation followed by suppression of antigen presentation and antibacterial responses. Notably, in the murine host, the mutant shows compromised virulence, leading to immune activation and pathogen clearance. In continuation of the study in C. elegans, Salmonella Typhi ChiA was found to facilitate bacterial attachment to the intestinal epithelium, intestinal colonization, and persistence by downregulating antimicrobial peptides. This study provides new insights on chitinase as an important and novel virulence determinant that helps in immune evasion and increased pathogenesis of Salmonella.

Objectives Salmonella enterica serovar Typhimurium is one of the significant non-typhoidal Salmonella serovars that causes gastroenteritis. The rapid development of antimicrobial resistance necessitates studying new antimicrobials and their therapeutic targets in this pathogen. Our study aimed to investigate the role of four prominent outer membrane porins of S. Typhimurium, namely OmpA, OmpC, OmpD and OmpF, in developing resistance against ceftazidime and meropenem. Methods The antibiotic-mediated inhibition of bacterial growth was determined by measuring the absorbance and the resazurin assay. DiBAC4 (Bis-(1,3-Dibutylbarbituric Acid)Trimethine Oxonol), 2,7-dichlorodihydrofluoroscein diacetate (DCFDA) and propidium iodide were used to determine the outer membrane depolarization, reactive oxygen species (ROS) generation and subsequent killing of Salmonella. The expression of oxidative stress-response and efflux pump genes was quantified by quantitative RT–qPCR. HPLC was done to determine the amount of antibiotics that entered the bacteria. The damage to the bacterial outer membrane was studied by confocal and atomic force microscopy. The in vivo efficacy of ceftazidime and meropenem were tested in the C57BL/6 mouse model. Results Deleting ompA reduced the survival of Salmonella in the presence of ceftazidime and meropenem. Massive outer membrane depolarization and reduced expression of oxidative stress-response genes in S. Typhimurium ΔompA hampered its growth in the presence of antibiotics. The enhanced uptake of antibiotics and decreased expression of efflux pump genes in S. Typhimurium ΔompA resulted in damage to the bacterial outer membrane. The clearance of the S. Typhimurium ΔompA from C57BL/6 mice with ceftazidime treatment proved the role of OmpA in rendering protection against β-lactam antibiotics. Conclusions OmpA protects S. Typhimurium from two broad-spectrum β-lactam antibiotics, ceftazidime and meropenem, by maintaining the stability of the outer membrane.