Investigating the Potential of Lemongrass Essential Oil Against Multidrug-Resistant Salmonella Heidelberg in Broiler Chickens

Abstract

Foodborne illness continues to persist as a global public health concern, despite technological advances. In particular, Salmonella has remained a major foodborne illness-causing pathogen in the United States. Epidemiological studies indicate that poultry are among the major source of Salmonella in developed nations, as food-producing animals like chickens often serve as natural hosts for the pathogen. Among the many serovars of Salmonella enterica, Salmonella enterica serovar Heidelberg has emerged as a pathogen of concern as it causes infections of greater severity in humans and possesses high potential for multidrug resistance. As Salmonella colonization in broiler chickens increases the risk of contamination during subsequent stages of processing, antimicrobial intervention strategies are warranted to ensure food safety and prevent outbreaks from occurring. Decades of efforts to control Salmonella in food systems have indicated that the problem must be approached holistically, with the best intervention strategy ideally applied at multiple stages of production. In that regard, the current study aimed to investigate the efficacy of lemongrass essential oil (LGEO), a generally recognized as safe (GRAS) compound that is obtained from plants of the Cymbopogon species, in reducing MDR S. Heidelberg in vitro, in broiler chicken, and on broiler chicken carcass. The direct effects of LGEO against MDR S. Heidelberg virulence factors, which are important for colonization, survival, and transmission, were first assessed through a series of in vitro assays. Additionally, the potential quorum sensing modulating properties of LGEO against S Heidelberg was indirectly assessed using the biosensor organism, Chromobacterium violaceum. C. violaceum produces violacein, a violet pigment under the vio operon regulation, which is easily observable and quantifiable. Effect of LGEO against S. Heidelberg multiplication was examined through macrodilution assays performed in either tryptic soy broth (TSB) or poultry cecal contents. The effect of LGEO on S. Heidelberg motility was assessed using a motility assay where S. Heidelberg was spot inoculated on the center of semi-solid agar plates supplemented with LGEO. Assays that investigated the effect of LGEO against S. Heidelberg biofilms formation and inactivation of mature biofilms were performed using 96-well microtiter plate assays and 24-well tissue culture plate assays, respectively. Finally, the quorum sensing modulation of LGEO was assessed based on violacein synthesis by C. violaceum. At concentrations of 0.5% (vol/vol) or higher, LGEO was effective in inhibiting multiplication S. Heidelberg by at least 4.0 log10 CFU/mL after 24 hours in TSB and poultry cecal contents (P ≤ 0.05). Addition of 0.15% LGEO in semi-solid motility agar yielded complete inhibition of S. Heidelberg motility (P ≤ 0.05). Concentrations of 0.15% and 1% LGEO were also found effective against MDR S. Heidelberg biofilm formation and inactivation of mature MDR S. Heidelberg biofilms (P ≤ 0.05). Additionally, a reduction in violacein production by C. violaceum was observed with 0.5% and 1% LGEO (P ≤ 0.05). Results of the in vitro assays indicate the potential of LGEO to mitigate the persistence and dissemination of MDR S. Heidelberg in poultry production systems. Subsequently, in vivo experiments were performed to determine the efficacy of LGEO as an antimicrobial supplement either through feed or drinking water to reduce S. Heidelberg colonization in broilers. The efficacy of in-feed supplementation of LGEO against MDR S. Heidelberg was evaluated in 3-week-old broiler chickens. Efficacy of LGEO supplementation through drinking water was first examined in 5-week-old broiler chickens and then in 7-week-old broiler chickens. All birds, except those in NC groups, in all studies, were challenged by crop gavage with 105, 104, and 108 CFU/mL MDR S. Heidelberg, at 2-, 4-, and 6-weeks of age, respectively. For both feed studies where 1% LGEO was supplemented through feed, no significant reduction in S. Heidelberg populations was observed (P > 0.05). Likewise, when supplemented through drinking water, 0.5% LGEO resulted in no significant reduction in both 5- and 7-week old broiler chickens (P > 0.05). However, a significant reduction of 2.4 log10 CFU/gram cecal S. Heidelberg was observed in one replicate involving 5-week old broilers (P < 0.05). In conclusion, the results of this study indicated that LGEO supplementation in either feed or water was not effective in reducing MDR S. Heidelberg colonization in broiler chickens at the current concentrations tested. Finally, the last set of experiments examined the effect of LGEO dip treatments on MDR S. Heidelberg attachment to broiler chicken skin and meat samples. Skin from drumsticks and breast meat samples were spot-inoculated with 4.0 log10 CFU MDR S. Heidelberg per sample and dipped in water containing 0.5, 1, or 2% LGEO for 2, 3, or 5 minutes at either 54°C or 4°C, individually, as well as at 54°C and 4°C in sequence. At 2%, LGEO consistently resulted in significant reduction of MDR S. Heidelberg on both skin and meat samples, though the magnitude of reduction was lesser on meat than on skin (P < 0.05). Sequential dip treatments performed on skin yielded comparable results to that observed in individual dip treatments at scalding temperatures. However, subsequent dip treatments were also not as effective on meat compared to skin samples. All concentrations of LGEO effectively inactivated S. Heidelberg from the scalding and chilling treatment waters for both skin and meat samples (P < 0.05). The results indicate that LGEO could be utilized as a natural antimicrobial intervention in scalding and chilling waters to reduce S. Heidelberg on poultry carcasses. In conclusion, the results are indicative of the direct antibacterial effects which LGEO exerts on S. Heidelberg in vitro and on broiler chicken skin samples. However, results of the in vivo study suggest that additional studies must examine the possible factors that may have impeded LGEO’s efficacy against foodborne pathogens in broiler chickens. Overall, the efficacy of LGEO observed on retail broiler chicken samples is indicative of its potential use to control MDR S. Heidelberg transmission in poultry production systems.