The effect of environmental conditions on Salmonella fimbriae and biofilm formation

Date
2012
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University of Delaware
Abstract
Salmonella typhimurium is a Gram-negative enteric pathogen that may cause salmonellosis in individuals that consume contaminated food. Triclosan, a broad-spectrum bisphenol, is an antimicrobial agent commonly added to a broad range of products including hand soaps, cleaners and surfaces. Overuse of such products can result in the development of resistant strains able to withstand higher concentrations of the agent than the original parental (Wild Type) strain. We developed two strains of Salmonella with reduced susceptibly (SRS) that had triclosan minimum inhibitory concentrations (MIC) of approximately 400 ppm compared to the parental MIC of 1 ppm, indicating increased resistance. Biofilms produced by bacteria are a public health concern. While in a biofilm, microbes are protected from harmful agents, such as antibiotics and antimicrobials. Abiotic and biotic factors such as temperature and presence of cell structures influence biofilm formation. We used two growth conditions in our study; condition one, 37ºC on Luria Bertani (LB) agar and condition two, 28ºC on LB agar without NaCl. Parental and SRS growth, flagella, motility, fimbriae and biofilm formation were studied under these two conditions. Cellular structures such as fimbriae and flagella initiate biofilm formation. S. typhimurium produces a variety of fimbriae. The type we examined in our study was curli fimbriae. Using transmission electron microscopy and growth on Congo Red agar we observed the lack of curli fimbriae under both growth conditions. Quantitative real time PCR analysis of fimA (type 1 fimbriae) and csgG (curli fimbriae) genes indicated that growth conditions used did not have an effect on the presence or absence of fimbriae. All strains possessed flagella. Unlike the parental and SRS strains tested, one SRS strain was more motile at the lower temperature. Motility of all strains was affected by temperature. A crystal violet assay was used to quantify the amount of biofilm produced as a function of time. Fluorescence microscopy of acridine orange stained biofilms was also carried out. Both studies were done under the two growth conditions. Initially, biofilm formation for all strains was greater in condition one. By hour six, there were no differences between any of the strains under either condition. Seven-hour biofilms of all strains were challenged with 200 ppm triclosan, the concentration of residual triclosan commonly found on surfaces and 2000 ppm triclosan, the concentration found in antimicrobial products. After one hour of exposure, the biofilms were stained with the Live Dead Stain®. All cells in the parental biofilms exposed to both triclosan concentrations were red, indicating cell death. Approximately half of the cells in both SRS biofilms were green, indicating viability at 200 ppm, but all were red at the higher concentration. This indicates that biofilms of SRS strains were resistant to residual levels of triclosan that are found on surfaces. Higher concentrations of triclosan in commercial products are effective in killing microorganisms. However, residual levels left on surfaces may foster the growth of SRS strains. These resistant organisms may develop cross-resistance to other antimicrobials.
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