Determining the antibacterial potential of a medium-chain fatty acid, caprylic acid, against multidrug-resistant Salmonella enterica enterica serovar Heidelberg in broiler chickens

Abstract

Salmonella is the leading bacterial cause of foodborne illness in the United States of America. Among the various foodborne infections, salmonellosis ranks first in terms of hospitalizations and death. More than 50% of foodborne salmonellosis is attributed to the consumption of contaminated poultry products. Salmonella colonizes the chicken cecum and gets excreted, leading to the contamination of the farm environment and poultry carcass during processing. Among the >2500 serovars of Salmonella causing human infections, 7% are associated with foodborne outbreaks through poultry. Salmonella enterica serovar Heidelberg (SH) has emerged through many foodborne outbreaks and found to be resistant to various commonly used and clinically relevant antibiotics. Multidrug-resistant (MDR) SH is one of the commonly isolated Salmonella from chicken carcasses and is associated with foodborne outbreaks through chicken products. Due to the federal initiatives to curb the use of antibiotic resistance development in animal agriculture, alternative antimicrobial strategies that control all Salmonella, including SH, is considered the urgent need of the poultry food industry. Since the pathogen is resistant to clinically important antibiotics, strategies are required to control them on farms and processing. Medium-chain fatty acids (MCFAs) could be an effective alternative to antibiotics approach as MCFAs have broad-spectrum antimicrobial activity. Among the MCFA’s, caprylic acid (CA) has been reported to have antibacterial potential against Salmonella. In this thesis, two questions were asked: Could a long-term supplementation of CA through feed control cecal colonization of MDR SH in broiler chickens (preharvest strategy), and could CA be effective against MDR SH on chicken drumsticks when applied in scalding water (processing aid). At first, we investigated the efficacy of CA in reducing MDR SH colonization in the cecum of 5-week old broiler chickens. Two independent studies were conducted. In each experiment, day-old Ross 708 broiler chicks were randomly allocated to four different groups (3 chicks/group; two studies). The four groups included in the study were: Negative control (NC), Positive Control (PC), Antibiotic group (AB), and CA group (CA). The birds in NC and PC were fed with a standard basal diet, whereas the broilers in the AB group received a standard diet containing 50g bacitracin methylene disalicylate (BMD)/ ton of feed for 5 weeks. The CA (1% w/w) was supplemented through the feed to the broilers in the CA group for 5 weeks. All birds except those in the NC group were challenged with 3.69 log10 CFU MDR SH (2014 Tennessee correctional facility outbreak strain) by crop gavage. Birds were euthanized 7-days after MDR SH inoculation by CO2 asphyxiation. Cecal samples were collected, and the cecal colonization of Salmonella was determined after plating the homogenates onto xylose lysine deoxycholate agar (XLD) plates. The bacterial counts were transformed to log values, and ANOVA was used for statistical analysis. The BMD supplementation resulted in 3.4 and 4.0 log10 CFU/g reduction of MDR SH in studies 1 and 2, respectively. The CA supplementation also resulted in a comparable reduction in cecal colonization of MDR SH as that of BMD. A reduction of 3.2 and 4.0 log10 CFU/g was observed in studies 1 and 2, respectively, in the CA group compared to birds in PC. Therefore, CA could be used as an effective control strategy against MDR SH colonization in 5-week old broilers. This results corroborate with other studies employing CA to control another major Salmonella serovar in broiler chickens, S. Enteritidis. In the second study, we determined the antimicrobial efficacy of CA against MDR SH on chicken drumsticks in simulated soft scalding conditions. Chicken drumsticks were spot inoculated with MDR SH [either lower (~3.0 log10 CFU/g) or higher (~5.0 log10 CFU/g) inoculum] and immersed in scalding water containing treatments for 2 min at 54oC (USDA-recommended time-temperature combination for soft scalding). The antimicrobial treatments included in the study were 0.5% CA, 1% CA, 0.05% peracetic acid (PAA), 0.5% CA + 0.05% PAA and 1.0% CA + 0.05% PAA. Samples inoculated with or without MDR SH and immersed in scalding water containing neither of the antimicrobial treatments served as the PC and NC groups, respectively. Immediately after scalding, the drumsticks were homogenized in phosphate-buffered saline (PBS), and surviving MDR SH populations were recovered on XLD agar plates (n=6). Similarly, MDR SH populations that survived in scalding water was also determined after surface plating (n=6). Additionally, the efficacy of the scalding treatments against MDR SH survival on drumsticks for a storage period of 48 h at 4oC was determined. Furthermore, the effect of these treatments on the surface color of the drumsticks was also evaluated. The antimicrobial treatments resulted in a significant reduction of MDR SH on drumsticks. For the lower inoculum, 0.5% CA, 1% CA, 0.05% PAA, 0.5% CA + 0.05% PAA and 1.0% CA + 0.05% PAA resulted in 0.7, 1.0, 2.5, 1.4 and 1.5 log10 CFU/g reduction of MDR SH on drumsticks (P<0.05). The same treatments resulted in 0.9, 1.3, 2.5, 2.2, and 2.6 log10 CFU/g reduction of MDR SH when the drumsticks were contaminated with the higher inoculum level (P<0.05). Moreover, the antimicrobial treatments completely inactivated MDR SH in scalding water to undetectable levels, whereas 2.0 to 4.0 log10 CFU/mL MDR SH survived in the PC group (P<0.05). Also, the scalding treatments were effective in inhibiting MDR SH on the drumsticks compared to the respective controls during a storage period of 48 h at 4oC (P<0.05), although the magnitude of reduction remained the same as observed during the scalding treatment. Additionally, none of the treatments affected the color of the drumsticks (P>0.05). The results indicated that CA could be used as an effective intervention strategy against MDR SH on chicken drumsticks at scalding to render safe meat production during subsequent stages of processing. The overall results from the MS studies indicated that CA could be used as an effective natural antimicrobial against MDR SH in the pre- and post-harvest stages in broiler production and could improve the microbiological safety of chicken meat.