A susceptibility assay for salmonella to commercial and household biocides

Authors: Humayoun, Shaheen; Hiott, Lari; GUPTA, SUSHIM - Orise Fellow; Barrett, John; Woodley, Tiffanie; JOHNSTON, JOHN - Food Safety Inspection Service (FSIS); Jackson, Charlene; Frye, Jonathan

Submitted to: PLOS ONE
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/4/2018
Publication Date: 12/20/2018
Citation: Humayoun, S.B., Hiott, L.M., Gupta, S., Barrett, J.B., Woodley, T.A., Johnston, J., Jackson, C.R., Frye, J.G. 2018. A susceptibility assay for salmonella to commercial and household biocides. PLoS One. https://doi.org/10.1371/journal.pone.0209072.
DOI: https://doi.org/10.1371/journal.pone.0209072

Interpretive Summary: The food industries use a wide variety of antimicrobial interventions, biocides, to reduce bacterial contamination during processing of retail meat. All of these compounds are generally regarded as safe (or GRAS) for food use and can reduce bacterial contamination with pathogens such as Salmonella. However, little is known about how bacteria may develop resistance to these compounds and there is no systematic way to detect changes in susceptibility to these compounds that may indicate the development of resistance. Therefore a high throughput, 96 well plate of 17 common household and commercially used biocides was designed to determine the minimum inhibitory concentration (MIC) of Salmonella for these compounds, and to determine if resistance to biocides correlates with resistance to antibiotics. The assay contained serial dilutions of the chemicals to be tested, these included: Dodecyltrimethylammonium chloride (DC), Benzalkonium chloride (BKC), Cetylpyridinium chloride (CPC), Hexadecyltrimethylammonium bromide (HB), Hexadecyltrimethylammonium chloride (HC), Acetic acid (AA), Lactic acid (LA), Citric acid (CA), Peroxyacetic acid (PXA), Acidified sodium chlorite (ASC), Sodium hypochlorite (SHB), 1,3 dibromo, 5,5 dimethylhydantoin (DBH), Chlorhexidine (CH), Sodium metasilicate (SM), Trisodium phosphate (TP), Arsenite (ARI), and Arsenate (ARA). The panel was used to test 88 multi-drug resistant (MDR) Salmonella isolates to calculate the minimum inhibitory concentration (MIC) of Salmonella to each of the chemicals. For the MDR Salmonella isolates tested, the MICs to each compound (in ug/mL) were: 256 for DC, 80 for CPC, 40 for BKC, 80 for HB and HC, 1640 for AA, 3776 for LA, 3156 for CA, 880 for PXA, 320 for ASC, 1.6 for CHX, 1248 for DBH, 138752 for SHB, 60320 for SM, 37712 for TP, 56 for ARI and 832 for ARA. No correlation was detected between susceptibility of Salmonella to the biocides and their antibiotic resistance. As the overall aim in the food industry is to reduce antimicrobial use, this assay and the MIC data will be important to help monitor the effect of biocides on both biocide and antimicrobial resistance.

Technical Abstract: Poultry and meat products contaminated with Salmonella enterica are a major cause of foodborne illness in the United States. The food industries use a wide variety of antimicrobial interventions to reduce bacterial contamination. However, little is known about Salmonella susceptibility to these compounds and some studies have shown a concerning link between biocide resistance and antibiotic resistance. To investigate this, a 96 well panel of 17 common household and commercially used biocides was designed to determine the minimum inhibitory concentrations (MIC) of these compounds for Salmonella. The panel contained two-fold serial dilutions of chemicals including Dodecyltrimethylammonium chloride (DC), Benzalkonium chloride (BKC), Cetylpyridinium chloride (CPC), Hexadecyltrimethylammonium bromide (HB), Hexadecyltrimethylammonium chloride (HC), Acetic acid (AA), Lactic acid (LA), Citric acid (CA), Peroxyacetic acid (PXA), Acidified sodium chlorite (ASC), Sodium hypochlorite (SHB), 1,3 dibromo, 5,5 dimethylhydantoin (DBH), Chlorhexidine (CHX), Sodium metasilicate (SM), Trisodium phosphate (TP), Arsenite (ARI), and Arsenate (ARA). The assay was used to test the susceptibility of 88 multidrug resistant (MDR) Salmonella isolates from animal sources. The concentration of biocide at which =50% of the isolates could not grow was designated as the minimum inhibitory concentration or MIC50 and was used as the breakpoint in this study. The MIC50 (µg ml-1) for the tested MDR Salmonella was 256 for DC, 80 for CPC, 40 for BKC, 80 for HB and HC, 1,640 for AA, 3,776 for LA, 3,156 for CA, 880 for PXA, 320 for ASC, 3.2 for CHX, 1,248 for DBH, 3,152 (6%) for SHB, 60,320 for SM, 37,712 for TP, 56 for ARI and 832 for ARA. A few isolates were not susceptible at the MIC50 breakpoint to some chemicals indicating possible resistance. Biocides with isolate MICs two 2-fold dilutions above the MIC50 included DC (n=1 isolate), CPC (2), ASC (6), CHX (22), ARA (16), and ARI (4). There was no correlation detected between the biocide susceptibility of Salmonella isolates and antibiotic resistance. This assay can determine the MICs of bacteria to several biocides in a single test and will be useful in evaluating the efficacy of biocides and to detect the development of resistance to them.