Investigating the Roles of Active Site Residues in Mycobacterium tuberculosis Indole-3-glycerol Phosphate Synthase, a Potential Target for Antitubercular Agents.

Mycobacterium tuberculosis drug resistance is emerging and new drug targets are needed. Tryptophan biosynthesis is necessary for M. tuberculosis replication and virulence. Indole-3-glycerol phosphate synthase (IGPS) catalyzes a step in M. tuberculosis tryptophan biosynthesis and has been suggested as a potential anti-infective target, but our understanding of this enzyme is limited. To aid in inhibitor design and gain a greater mechanistic picture of this enzyme, there is a need to understand the roles of active site amino acids in ligand binding and catalysis. In this work, we explored the roles of conserved active site amino acids Glu57, Lys59, Lys119, Glu168, and Glu219. Mutation of each to Ala results in loss of all detectable activity. The Glu57Gln, Lys59Arg, Lys119Arg, Glu168Gln, and Glu219Asp mutations result in large activity losses, while Glu219Gln has enhanced activity. Analysis of the enzymatic data yields the following main conclusions: (A) Lys119 is the likely catalytic acid in the CdRP ring closure step. (B) Glu168 stabilizes a charged reaction intermediate and may also be the catalytic base. (C) Glu57, Glu219, and Lys119 form a closely arranged triad in which Glu57 and Glu219 modulate the pKa of Lys119, and thus overall activity. This increased understanding of inter- and intramolecular interactions and demonstration of the highly coordinated nature of the M. tuberculosis IGPS active site provide new mechanistic information and guidance for future work with this potential new drug target.

Crystal structure of dihydrofolate reductase from the filarial nematode W. bancrofti in complex with NADPH and folate.

Lymphatic filariasis is a debilitating illness with an estimated 50 million cases as of 2018. The majority of cases are caused by the parasitic worm W. bancrofti and additional cases by the worms B. malayi and B. timori. Dihydrofolate reductase (DHFR) is an established target in the treatment of cancer, bacterial, and protozoal infections and may be a potential target for drugs targeting parasitic worm infections, including filariasis. Recent studies have shown that known antifolate compounds, including methotrexate, inhibit the activity of W. bancrofti DHFR (WbDHFR). However, the absence of structural information for filarial DHFRs has limited the study of more in-depth structure-function relationships. We report the structure of WbDHFR complexed with NADPH and folate using X-ray diffraction data measured to 2.47 Å resolution. The structure of WbDHFR reveals the usual DHFR fold and is currently only the second nematode DHFR structure in the Protein Data Bank. The equilibrium dissociation constants for NADPH (90 ± 29 nM) and folate (23 ± 4 nM) were determined by equilibrium titrations. The interactions of known antifolates with WbDHFR were analyzed using molecular docking programs and molecular dynamics simulations. Antifolates with a hydrophobic core and extended linker formed favorable interactions with WbDHFR. These combined data should now facilitate the rational design of filarial DHFR inhibitors, which in turn can be used to determine whether DHFR is a viable drug target for filariasis and whether existing antifolates may be repurposed for its treatment.

Mycorrhizal Infection Can Ameliorate Abiotic Factors in Urban Soils.

Once abandoned, urban and post-industrial lands can undergo a re-greening, the natural regeneration and succession that leads to surprisingly healthy plant communities, but this process is dependent upon microbial activity and the health of the parent soil. This study aimed to evaluate the effects of arbuscular mycorrhizal fungi (AMF) in facilitating plant production in post-industrial soils. In so doing, we helped to resolve the mechanism through which AMF ameliorate environmental stress in terrestrial plants. An experiment was established in which rye grass (Lolium perenne) was grown in two heavy metal-contaminated soils from an urban brownfield in New Jersey, USA, and one non-contaminated control soil. One set of the treatments received an AMF inoculum (four species in a commercial mix: Glomus intraradices, G. mosseae, G. etunicatum and G. aggregatum) and the other did not. Upon harvest, dried plant biomass, root/shoot ratio, AMF colonization, and extracellular soil phosphatase activity, a proxy for soil microbial functioning, were all measured. Plant biomass increased across all treatments inoculated with AMF, with a significantly higher average shoot and root mass compared to non-inoculated treatments. AMF colonization of the roots in contaminated soil was significantly higher than colonization in control soil, and the root/shoot ratio of plants in contaminated soils was also higher when colonized by AMF. Mycorrhizal infection may help plants to overcome the production limits of post-industrial soils as is seen here with increased infection and growth. The application of this mechanistic understanding to remediation and restoration strategies will improve soil health and plant production in urban environments.

Artificial root exudates restore microbial functioning in a metal contaminated, barren, inactive soil.

Restoring enzyme function in barren, brownfield soils using green strategies can improve microbial functioning and enable phytoremediation. It is known that adding simple, readily metabolized substrates secreted by growing plant roots (root exudates) or a laboratory prepared solution of root exudates (artificial root exudates) can stimulate soil microbial function. It is not known whether and how well this strategy works in a contaminated, low functioning soil from an industrial barren site because contaminants in the barren soil might inhibit microbial survival and functioning, or the microbial community might not be adapted to functionally benefit from root exudates. The objective of this study was to determine whether artificial root exudates stimulate microbial function in a barren soil. We collected soils from a barren brownfield (25R) site and an adjacent vegetated brownfield site (25F), with low and high enzyme activities, respectively. We subjected both soils to three treatments: switchgrass (native to the site), artificial root exudates, and a combination of switchgrass and artificial root exudates. We measured enzymatic activity, plant growth, soil moisture, organic matter content, and easily extractable glomalin content over 205 days. By day 157, artificial root exudates increased the phosphatase activity by 9-fold in previously vegetated brownfield soil and by 351-fold in barren brownfield soil. When exudates were added to the barren soil, the plant shoot mass was higher (52.2 ± 2.5 mg) than when they were not (35.4 ± 3.6 mg). In both soils, adding artificial root exudates significantly increased the percent moisture, organic matter, and glomalin content. Treating contaminated, barren soil with artificial root exudates resulted in increased soil microbial function and improved soil properties that might promote a hospitable habitat to support vegetation in such extreme environments. Summary: We added artificial root exudates to stimulate enzymatic function in two contaminated soils. Plant shoot mass, soil percent moisture, glomalin content, and organic matter content significantly increased due to the addition of artificial root exudates to the study soils. Microbially-mediated phosphatase activity was established in a barren, previously inactive, polluted soil.

Characterization of Competitive Inhibitors of Plasmodium falciparum cGMP-Dependent Protein Kinase.

Plasmodium falciparum cGMP-dependent protein kinase (PfPKG) is an enticing antimalarial drug target. Novel chemotypes are needed because existing inhibitors have safety issues that may prevent further development. This work demonstrates isoxazole-based compounds are potent ATP competitive inhibitors of PfPKG and discloses a new analogue in this series. Isoxazoles 3 and 5 had Ki values that are comparable to a known standard, 4-[2-(4-fluorophenyl)-5-(1-methylpiperidine-4-yl)-1H pyrrol-3-yl] pyridine. They also exhibited excellent selectivity for PfPKG over the human orthologue and the gatekeeper mutant T618Q PfPKG, which mimics the less accessible binding site of the human orthologue. The human orthologue's larger binding site volume is predicted to explain the selectivity of the inhibitors for the P. falciparum enzyme.

Indole-3-Glycerol Phosphate Synthase From Mycobacterium tuberculosis: A Potential New Drug Target.

Tuberculosis (TB), caused by the pathogen Mycobacterium tuberculosis, affects millions of people worldwide. Several TB drugs have lost efficacy due to emerging drug resistance and new anti-TB targets are needed. Recent research suggests that indole-3-glycerol phosphate synthase (IGPS) in M. tuberculosis (MtIGPS) could be such a target. IGPS is a (ß/α)8 -barrel enzyme that catalyzes the conversion of 1-(o-carboxyphenylamino)-1-deoxyribulose 5'-phosphate (CdRP) into indole-glycerol-phosphate (IGP) in the bacterial tryptophan biosynthetic pathway. M. tuberculosis over expresses the tryptophan pathway genes during an immune response and inhibition of MtIGPS allows CD4 T-cells to more effectively fight against M. tuberculosis. Here we review the published data on MtIGPS expression, kinetics, mechanism, and inhibition. We also discuss MtIGPS crystal structures and compare them to other IGPS structures to reveal potential structure-function relationships of interest for the purposes of drug design and biocatalyst engineering.

Distal Regions Regulate Dihydrofolate Reductase-Ligand Interactions.

Protein motions play a fundamental role in enzyme catalysis and ligand binding. The relationship between protein motion and function has been extensively investigated in the model enzyme dihydrofolate reductase (DHFR). DHFR is an essential enzyme that catalyzes the reduction of dihydrofolate to tetrahydrofolate. Numerous experimental and computational methods have been used to probe the motions of DHFR through the catalytic cycle and to investigate the effect of distal mutations on DHFR motions and ligand binding. These experimental investigations have pushed forward the study of protein motions and their role in protein-ligand interactions. The introduction of mutations distal to the active site has been shown to have profound effects on ligand binding, hydride transfer rates and catalytic efficacy and these changes are captured by enzyme kinetics measurements. Distal mutations have been shown to exert their effects through a network of correlated amino acids and these effects have been investigated by NMR, protein dynamics, and analysis of coupled amino acids. The experimental methods and the findings that are reviewed here have broad implications for our understanding of enzyme mechanisms, ligand binding and for the future design and discovery of enzyme inhibitors.

Plants mitigate restrictions to phosphatase activity in metal contaminated soils.

Soil anthropogenic contaminants can limit enzymatic nutrient mineralization, either by direct regulation or via impacts on the microbial community, thus affecting plant growth in agricultural and non-agricultural soils. The impact on phosphatase activity of mixing two contaminated, post-industrial rail yard soils was investigated; one was vegetated and had high phosphatase function, the other was barren and had low enzymatic function. The two soils had different abiotic properties, including contaminant load, vegetation cover, soil aggregate size distribution, and phosphatase potential. An experimental gradient was established between the two soils to systematically vary the abiotic properties and microbial community composition of the two soils, creating a gradient of novel ecosystems. The time dependence of extracellular phosphatase activity, soil moisture, and organic matter content was assessed along this gradient in the presence and absence of plants. Initially, mixtures with higher percentages of functional, vegetated soil had higher phosphatase activities. Phosphatase activity remained unchanged through time (65 days) in all soil mixtures in unplanted pots, but it increased in planted pots. For example, in the presence of plants, phosphatase activity increased from 0.6 ± 0.1 to 2.4 ± 0.3 µmol•h-1•gdry soil-1 from day one to day 65 in the 1:1 functional:barren soil mixture. The presence of plants also promoted moisture retention. Inoculation of poorly functioning soil with 10% of the functional soil with its microbial community did not, over 65 days, revitalize the poorly functioning soil. The findings showed that abiotic limitations to enzymatic activity in barren brownfield soils could be mitigated by establishing primary production but not by the addition of enzymatically active microbial communities alone.

Mutational analysis confirms the presence of distal inhibitor-selectivity determining residues in B. stearothermophilus dihydrofolate reductase.

Many antibacterial and antiparasitic drugs work by competitively inhibiting dihydrofolate reductase (DHFR), a vital enzyme in folate metabolism. The interactions between inhibitors and DHFR active site residues are known in many homologs but the contributions from distal residues are less understood. Identifying distal residues that aid in inhibitor binding can improve targeted drug development programs by accounting for distant influences that may be less conserved and subject to frequent resistance causing mutations. Previously, a novel, homology-based, computational approach that mines ligand inhibition data was used to predict residues involved in inhibitor selectivity in the DHFR family. Expectedly, some inhibitor selectivity determining residue positions were predicted to lie in the active site and coincide with experimentally known inhibitor selectivity determining positions. However, other residues that group spatially in clusters distal to the active site have not been previously investigated. In this study, the effect of introducing amino acid substitutions at one of these predicted clusters (His38-Ala39-Ile40) on the inhibitor selectivity profile in Bacillus stearothermophilus dihydrofolate reductase (Bs DHFR) was investigated. Mutations were introduced into these cluster positions to change sidechain chemistry and size. We determined kcat and KM values and measured KD values at equilibrium for two competitive DHFR inhibitors, trimethoprim (TMP) and pyrimethamine (PYR). Mutations in the His38-Ala39-Ile40 cluster significantly impacted inhibitor binding and TMP/PYR selectivity - seven out of nine mutations resulted in tighter binding to PYR when compared to TMP. These data suggest that the His38-Ala39-Ile40 cluster is a distal inhibitor selectivity determining region that favors PYR binding in Bs DHFR and, possibly, throughout the DHFR family.

Environmental forensic characterization of former rail yard soils located adjacent to the Statue of Liberty in the New York/New Jersey harbor.

Identifying inorganic and organic soil contaminants in urban brownfields can give insights into the adverse effects of industrial activities on soil function, ecological health, and environmental quality. Liberty State Park in Jersey City (N.J., USA) once supported a major rail yard that had dock facilities for both cargo and passenger service; a portion remains closed to the public, and a forest developed and spread in this area. The objectives of this study were to: 1) characterize the organic and inorganic compounds in Liberty State Park soils and compare the findings to an uncontaminated reference site (Hutcheson Memorial Forest); and 2) identify differences between the barren low-functioning areas and the forested high-functioning areas of the brownfield. Soil samples were solvent-extracted, fractionated, and analyzed by gas chromatography-mass spectrometry and subjected to loss-on-ignition, pyrolysis-gas chromatography-mass spectrometry, inductively-coupled-plasma mass spectrometry, and optical microscopy analyses. Compared to soil from the reference site, the forested soils in Liberty State Park contained elevated percentages of organic matter (30-45%) and more contaminants, such as fossil-fuel-derived hydrocarbons and coal particles. Microscopy revealed bituminous and anthracite coal, coke, tar/pitch, and ash particles. Barren and low-functioning site 25R had a similar organic contaminant profile but contained a higher metal load than other Liberty State Park sites and also lacked higher plant indicators. These can obscure the signatures of contaminants, and data from adjacent barren and vegetated sites are valuable references for soils studies. A deeper understanding of the chemistry, biochemistry, and ecology of barren soils can be leveraged to prevent land degradation and to restore dysfunctional and phytotoxic soils.

Soil microbial response to metal contamination in a vegetated and urban brownfield.

The ubiquity of urban brownfields presents not only a challenge for environmental managers but also an opportunity to study the functional aspects of degraded ecosystems that are in close contact with human habitation. In this study, we investigate the soil microbial community response to heavy metal contamination at Liberty State Park (LSP), an urban brownfield in Jersey City, NJ, USA. Heavy metal contamination of the soils at LSP is heterogeneous, varying widely across site and among metals. We collected soils along a previously mapped gradient of metal contamination at LSP and sampled soil from a local and uncontaminated reference site (Hutcheson Memorial Forest (HMF)) for comparison. For all soils, we measured soil heavy metal concentrations, soil organic carbon content, bacterial density, and extracellular phosphatase activity as a proxy of ecosystem functioning. Additionally, we analyzed the microbial community composition using high-throughput sequencing. Data show that some sites within LSP have significantly higher phosphatase activity compared to HMF, indicating that some heavily contaminated LSP soils are highly functional. We also found that soil organic carbon and bacterial density have a significant and positive relationship with phosphatase activity. The microbial community analyses showed that the bacterial communities were sensitive to heavy metals and that the composition was significantly affected in particular by copper, zinc, and lead. The fungal communities, however, did not vary significantly with heavy metals. Our results shed important light on the composition and functioning of urban brownfield soils. A deeper understanding of these unique ecosystems is required for successful remediation, restoration and urban sustainability.

Abiotic factors determine functional outcomes of microbial inoculation of soils from a metal contaminated brownfield.

Whole community microbial inoculation can improve soil function in contaminated environments. Here we conducted a case study to investigate whether biotic factors (inoculum) or abiotic factors (soil base) have more impact on the extracellular enzymatic activities in a whole community microbial inoculation. To this end, we cross-inoculated microbial communities between two heavy metal-contaminated soils, with high and low extracellular enzyme activities, respectively. We measured extracellular phosphatase activity, a proxy for soil function, after self- and cross-inoculation of microbial communities into sterilized soils, and all activities were normalized to non-inoculated controls. We found that inoculation increased phosphatase activity in the soils. For soils treated with different inocula, we found significant differences in the microbial community compositions but no significant differences in the extracellular phosphatase activities normalized to their respective sterilized, non-inoculated controls (4.7 ±â€¯1.8 and 3.3 ±â€¯0.5 for soils inoculated with microbial communities from 146 to 43, respectively). On the other hand, normalized phosphatase activities between the two soil bases were significantly different (4.1 ±â€¯0.12 and 1.9 ±â€¯0.12 for soil bases 146 and 43, respectively) regardless of the source of the inoculum that did not vary between soil bases. The results indicate that the abiotic properties of the soils were a significant predictor for phosphatase activity but not for the end-point composition of the microbial community. The findings suggest that targeted microbial inocula from metal contaminated soils can increase phosphatase activity, and likely soil functioning in general, but the degree to which this happens depends on the abiotic environment, in this case, metal contamination.

Expression, purification, and inhibition profile of dihydrofolate reductase from the filarial nematode Wuchereria bancrofti.

Filariasis is a tropical disease caused by the parasitic nematodes Wuchereria bancrofti and Brugia malayi. Known inhibitors of dihydrofolate reductase (DHFR) have been previously shown to kill Brugia malayi nematodes and to inhibit Brugia malayi DHFR (BmDHFR) at nanomolar concentrations. These data suggest that BmDHFR is a potential target for the treatment of filariasis. Here, protocols for cloning, expression and purification of Wuchereria bancrofti DHFR (WbDHFR) were developed. The Uniprot entry J9F199-1 predicts a 172 amino acid protein for WbDHFR but alignment of this sequence to the previously described BmDHFR shows that this WbDHFR sequence lacks a crucial, conserved 13 amino acid loop. The presence of the loop in WbDHFR is supported by a noncanonical splicing event and the loop sequence was therefore included in the gene design. Subsequently, the KM for dihydrofolate (3.7 ± 2 µM), kcat (7.4 ± 0.6 s-1), and pH dependence of activity were determined. IC50 values of methotrexate, trimethoprim, pyrimethamine, raltitrexed, aminopterin, (-)-epicatechin gallate, (-)-epicatechin, and vitexin were measured for WbDHFR and BmDHFR. Methotrexate and structurally related aminopterin were found to be effective inhibitors of WbDHFR, with an KI of 1.2 ± 0.2 nM and 2.1 ± 0.5 nM, respectively, suggesting that repurposing of known antifolate compound may be an effective strategy to treating filariasis. Most compounds showed similar inhibition profiles toward both enzymes, suggesting that the two enzymes have important similarities in their active site environments and can be targeted with the same compound, once a successful inhibitor is identified.

Mechanism of inhibition of botulinum neurotoxin type A light chain by two quinolinol compounds.

Quinolinol-based compounds are a promising starting point for discovery of effective inhibitors of the clostridial neurotoxin, botulinum neurotoxin type A light chain (BoNT/A LC). Insights into the mechanism of inhibition by quinolinol compounds facilitate interpretation of docking data and inhibitor optimization. In this study, a fluorogenic substrate of BoNT/A, SNAPtide, was used to study the mechanism by which two new quinolinol compounds, MSU58 and MSU84, with IC50 values of 3.3 µM and 5.8 µM, respectively, inhibit BoNT/A LC. Kinetic studies and model discrimination analysis showed both compounds to be competitive inhibitors of BoNT/A LC with inhibition constants (KI) 3.2 µM and 6.2 µM for MSU58 and MSU84, respectively. These data indicate that the inhibitors bind in the BoNT/A LC active site and that inhibitor binding is mutually exclusive with the binding of the substrate. This is the first study to report the competitive inhibition of BoNT/A LC by quinolinol compounds. These data help define the inhibitor binding pocket and, along with structure activity relationship studies, provide immediate direction for further compound synthesis.

Expression, purification and enzymatic characterization of Brugia malayi dihydrofolate reductase.

Brugia malayi (B. malayi) is one of the three causative agents of lymphatic filariasis, a neglected parasitic disease. Current literature suggests that dihydrofolate reductase is a potential drug target for the elimination of B. malayi. Here we report the recombinant expression and purification of a ∼20 kDa B. malayi dihydrofolate reductase (BmDHFR). A His6-tagged construct was expressed in E. coli and purified by affinity chromatography to yield active and homogeneous enzyme for steady-state kinetic characterization and inhibition studies. The catalytic activity kcat was found to be 1.4 ± 0.1 s(-1), the Michaelis Menten constant KM for dihydrofolate 14.7 ± 3.6 µM, and the equilibrium dissociation constant KD for NADPH 25 ± 24 nM. For BmDHFR, IC50 values for a six DHFR inhibitors were determined to be 3.1 ± 0.2 nM for methotrexate, 32 ± 22 µM for trimethoprim, 109 ± 34 µM for pyrimethamine, 154 ± 46 µM for 2,4-diaminoquinazoline, 771 ± 44 µM for cycloguanil, and >20,000 µM for 2,4-diaminopyrimidine. Our findings suggest that antifolate compounds can serve as inhibitors of BmDHFR.

Views from academia and industry on skills needed for the modern research environment.

Reports from employers of higher education graduates indicate the existence of a considerable gap between the skills required by employers and those possessed by recent graduates. As a first step toward closing this gap, this study aims to determine its origin. Interviews with nine research-active biochemistry professionals were used to identify the most important skills for biochemistry students to succeed in research positions postgraduation. The results of these interviews were used to develop a survey, which was then administered to a larger group of biochemistry faculty and industry professionals. The output of the survey was a list of 52 skills valued by biochemistry professionals and rated by perceived importance. Importantly, the survey results also afford a comparative look at the prioritization of skills by two key populations: the academic faculty training students and the industry professionals hiring them. While there are many areas of agreement between these two populations, the survey also reveals areas were priorities diverge. The discrepancies found here suggest that the skills gap manifest at the point of employment may stem directly from differences in prioritization between the academic and industrial environments. This article aims to provide insight into the needs and requirements of the modern biochemical research environment, and invites debate concerning the preparation students receive in academia. Moreover, the results presented herein point to a need for further exploration of the possible misalignment of these two critical environments for young scientists.

Temporally overlapped but uncoupled motions in dihydrofolate reductase catalysis.

Temporal correlations between protein motions and enzymatic reactions are often interpreted as evidence for catalytically important motions. Using dihydrofolate reductase as a model system, we show that there are many protein motions that temporally overlapped with the chemical reaction, and yet they do not exhibit the same kinetic behaviors (KIE and pH dependence) as the catalyzed chemical reaction. Thus, despite the temporal correlation, these motions are not directly coupled to the chemical transformation, and they might represent a different part of the catalytic cycle or simply be the product of the intrinsic flexibility of the protein.

Kinetic mechanism of indole-3-glycerol phosphate synthase.

The (ßα)(8)-barrel enzyme indole-3-glycerol phosphate synthase (IGPS) catalyzes the multistep transformation of 1-(o-carboxyphenylamino)-1-deoxyribulose 5-phosphate (CdRP) into indole-3-glycerol phosphate (IGP) in tryptophan biosynthesis. Mutagenesis data and crystal structure analysis of IGPS from Sulfolobus solfataricus (sIGPS) allowed for the formulation of a plausible chemical mechanism of the reaction, and molecular dynamics simulations suggested that flexibility of active site loops might be important for catalysis. Here we developed a method that uses extrinsic fluorophores attached to active site loops to connect the kinetic mechanism of sIGPS to structure and conformational motions. Specifically, we elucidated the kinetic mechanism of sIGPS and correlated individual steps in the mechanism to conformational motions of flexible loops. Pre-steady-state kinetic measurements of CdRP to IGP conversion monitoring changes in intrinsic tryptophan and IGP fluorescence provided a minimal three-step kinetic model in which fast substrate binding and chemical transformation are followed by slow product release. The role of sIGPS loop conformational motion during substrate binding and catalysis was examined via variants that were covalently labeled with fluorescent dyes at the N-terminal extension of the enzyme and mobile active site loop ß1α1. Analysis of kinetic data monitoring dye fluorescence revealed a conformational change that follows substrate binding, suggesting an induced-fit-type binding mechanism for the substrate CdRP. Global fitting of all kinetic results obtained with wild-type sIGPS and the labeled variants was best accommodated by a four-step kinetic model. In this model, both the binding of CdRP and its on-enzyme conversion to IGP are accompanied by conformational transitions. The liberation of the product from the active site is the rate-limiting step of the overall reaction. Our results confirm the importance of flexible active loops for substrate binding and catalysis by sIGPS.

An exploratory investigation of the risk of pathogenic contamination at selected New Jersey skilled nursing and assisted living residences.

Elderly populations are vulnerable and generally have the highest incidence of morbidity related to foodborne illnesses and this problem may be aggravated in institutional or communal eating settings. The objective of this investigation was to examine the potential risk of food contamination in selected skilled nursing and assisted-living residences using bacteria indicator tests for Listeria spp., Salmonella spp. and E. coli. Of the 45 samples tested for Listeria, three (6.67 %) were found to be contaminated; Salmonella or E. coli contamination was not found in any of the samples. Reported incidents of foodborne illnesses are increasing in institutional settings, therefore there is an urgent need to collect information on practices that can prevent bacterial contamination of food served in elderly residences.