The Potential Health Risk of Acinetobacter Baumannii as a Foodborne Pathogen and Attenuating its Antibiotic Resistance Using Plant-Derived Antimicrobials

Non-Technical Summary:<br/>
Multi-drug resistant (MDR) Acinetobacter baumannii is a major cause of hospital-associated infections in humans, and recent evidence indicates that it has emerged as a community-associated pathogen. Being ubiquitous in distribution, A. baumannii has been isolated from soil, water and a variety of foods, including meat, fish, fruits and vegetables, thereby raising concerns that food could be a potential source of infection to humans, particularly in health-care settings. In addition, isolation of genotypically similar strains of Acinetobacter strains from food, environment and clinical specimens further underscores the concerns on the role of food and water as a potential source of human infections. However, currently very limited information exists on the prevalence of A. baumannii in foods, especially in the United States. Therefore, the goal of this proposal is to determine the potential of foods, especially fresh produce as a vehicle of A. baumannii transmission to humans. Moreover, the efficacy of several food-grade, plant-derived antimicrobials (PDAs) in reducing antibiotic resistance in A. baumannii will be investigated.
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Approach:<br/>
1. Determine the prevalence of A. baumannii on lettuce and spinach in Connecticut. One hundred grams each of lettuce and spinach will be separately added to 150 ml of saline solution and shaken in Whirl-packs for 15 min. Duplicate 0.5 ml volumes will be spread plated on Minimal Salt Agar plates. The plates will be incubated at 30C and will be examined daily for 3 days. A. baumannii colonies will be identified using Gram's staining, cell and colony morphology, and biochemical tests. The antibiotic resistance profiles of each A. baumannii isolate will be determined using methods approved by the Clinical and Laboratory Standards Institute.
<br/>2. Development and optimization of A. baumannii-specific PCR. Acinetobacter strains, including A. baumannii will be cultured separately and genomic DNA extracted. Based on the BLAST analysis of the whole genome sequence data available for A. baumannii strains in the NCBI database, we designed primers specifically targeting A. baumannii hypothetical hydrolase and multiple drug resistance protein B. The PCR reactions will be repeated with at least 20 f A. baumannii isolates, other Acinetobacter species, and non-Acinetobacter isolates significant in human health.
<br/>3. Determine the efficacy of sub-inhibitory concentrations (SICs) of PDAs for decreasing resistance of A. baumannii to four classes of antibiotics. Three different strains of MDR A. baumannii with different resistance profiles will be used in this study. The SIC of PDAs against A. baumannii will be determined by broth dilution assay. The highest concentration of each PDA that did not inhibit bacterial growth after 24 h of incubation will be selected as the SIC of the molecule. To determine if the PDAs increased the sensitivity of A. baumannii to antibiotics, the SICs of each PDA will be added separately to duplicate wells of 24-well tissue culture plates containing 2 ml TSB inoculated with 5-log CFU of the bacterial culture and supplemented with each antibiotic at the respective breakpoint for resistance. The plates will be incubated at 37C for 24 h, and bacterial growth monitored.
<br/>4. Determine the potential of A. baumannii to develop resistance to PDAs. Tubes containing 10 ml of TSB will be inoculated with A. baumannii strains separately at 4.0 log CFU/ml. A range of concentration of PDAs will be added to the medium and incubated at 37C for 24 h. Continuous passages for 40-50 generations will be carried out at the SICs of each plant molecule. The stability of the developed resistance if any will be checked by passaging the cultures 20-25 times through PDA-free medium and testing the MIC at the end. An increase in the MIC will be taken as a measure of development of resistance. <br/>5. Characterize the potential mechanisms by which PDAs decrease antibiotic resistance in A. baumannii. The transcriptome of A. baumannii exposed to each antibiotic and PDA combination will be compared to the same strain exposed only to the respective antibiotic or PDA or none. The 454 Life Sciences pyrosequencing platform (Roche) will be used to characterize the transcriptome A. baumannii.