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Effect of Lemongrass (Cymbopogon citratus) Essential Oil on the Survival of Multidrug-Resistant Salmonella enterica serovar Heidelberg in Contaminated Poultry Drinking Water1
C 2019 Poultry Science Association Inc.
C. Peichel,∗,2 D. V. T. Nair,∗,2 G. Dewi,∗ A. M. Donoghue,† K. M. Reed,‡ and A. Kollanoor Johny∗,3 ∗
Department of Animal Science, University of Minnesota, Saint Paul, MN – 55108, USA; † Poultry Production and Product Safety Research Unit, ARS, USDA, University of Arkansas, Fayetteville AR 72701, Arkansas; and ‡ Department of Veterinary and Biomedical Sciences, University of Minnesota, Saint Paul MN – 55108, USA
Primary Audience: Flock Supervisors, Quality Assurance and Laboratory Personnel, Researchers and Veterinarians SUMMARY Drinking water contaminated with Salmonella could serve as a source for cecal colonization of the pathogen in birds. In this study, we investigated the efficacy of a generally recognized as safe—status essential oil (lemongrass essential oil, LGEO) against multidrug-resistant Salmonella enterica serovar Heidelberg (S. Heidelberg) in poultry drinking water. Farm water with and without added droppings, litter, or feed [0.5% (w/v)] inoculated with 6.0 log10 cfu/mL S. Heidelberg was treated with 0, 0.03, 0.06, 0.125, 0.25, or 0.5% (v/v) LGEO and incubated at 12.5◦ C or 22◦ C for up to 7 d. The number of viable S. Heidelberg populations was determined on d 0, 1, 3, 5, and 7. At 12.5◦ C, all concentrations of LGEO inactivated S. Heidelberg to non-detectable levels in water alone and water contaminated with droppings or litter by d 7 (>6 log10 cfu/mL; P < 0.05). The highest LGEO concentration tested (0.5%) resulted in >5.0 log10 cfu/mL reduction of S. Heidelberg on d 7 in water contaminated with feed (P < 0.05). At 22◦ C, all concentrations of LGEO resulted in the reduction of S. Heidelberg to non-detectable levels in water alone by d 5 (>6 log10 cfu/mL; P < 0.05). Concentrations of LGEO ≥0.125% resulted in inhibition of S. Heidelberg to non-detectable levels in water contaminated with droppings from d 5 onwards (P < 0.05). The LGEO at the highest tested concentration (0.5%) resulted in >5.0 log10 cfu/mL reduction of S. Heidelberg in water contaminated with litter on d 7 (P < 0.05) but did not result in reduction of the pathogen in water contaminated with feed at 22◦ C. Results indicate the
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The research was supported by the USDA National Institute of Food and Agriculture (Hatch) Project [Accession#1016910 (MIN-16-120); PI – A. Kollanoor Johny] and the USDA National Needs Fellowship (# 2016-38420-25285; PD – K. M. Reed). 2 Contributed equally to the conduct of experiments and manuscript preparation. 3 Corresponding author: [email protected]
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Effect of Lemongrass (Cymbopogon citratus) Essential Oil on the Survival of Multidrug-Resistant Salmonella enterica serovar Heidelberg in Contaminated Poultry Drinking Water1
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Key words: lemongrass essential oil, Salmonella Heidelberg, poultry drinking water, contaminants, natural disinfectant 2019 J. Appl. Poult. Res. 0:1–10 http://dx.doi.org/10.3382/japr/pfz076
DESCRIPTION OF PROBLEM Salmonella accounts for 1.2 million illnesses, 23,000 hospitalizations, and 450 deaths annually in the United States (U.S.) of which about a million illnesses are attributed to the consumption of contaminated food [1]. The pathogen leads to an estimated $3.3 billion loss to the U.S. economy in costs associated with the illness [2]. A variety of food commodities, including dairy products, raw fruits and vegetables, nuts, spices, fresh produce, seafood, poultry, beef, and pork serve as vehicles for Salmonella-associated foodborne illness [3]. Among these food commodities, eggs and poultry meat and products contribute more than half of Salmonella-associated foodborne outbreaks [4]. Poultry serve as carriers for the pathogen where excretion leads to the contamination of new incoming flocks and thus carcasses and products during processing [5]. Animal grazing and other agricultural practices, and sewage waste disposal can pose an environmental risk through contamination of groundwater, rivers, lakes, and other reservoirs with non-typhoidal Salmonella [6–10]. Multiple sources such as rodent and insect vectors, personnel, feed, water, litter, trucks, contaminated surfaces, and feces allow Salmonella to enter and persist in broiler and laying hen houses [11–12]. Among these, water tanks and drinkers pose a heightened source of Salmonella exposure to the birds [13–16]. Salmonella serovars, including the multiple antibiotic resistant clones, have been previously isolated from groundwater systems [6]. Salmonella attaches to the water flowing systems, forms biofilms, and may act as a constant source of exposure [17–18]. Poultry drinking water, especially those in small-scale grow-out facilities and backyard flocks, can be further contaminated with litter, droppings, or feed which provide a nutrient-rich environment promoting Salmonella survival and
multiplication at temperatures common in production facilities [19]. Organic contaminants and alterations in pH at different locations in the farm water supply often reduce the efficacy of water treatments, such as chlorine, that are aimed at reducing the Salmonella load [20–22]. Ingestion of Salmonella through drinking water may lead to the colonization of Salmonella in day-old or young chicks [19]. Therefore, effective strategies have to be developed to control Salmonella in drinking water to avoid the introduction into the flocks. The use of natural antimicrobials as antibiotic alternatives against pathogenic bacteria are receiving increased consideration due to heightened concerns over antibiotic resistance in pathogens encountered in food animal production. Plant-based, natural, and environmentally-friendly approaches, especially essential oils and their components, are effective options against foodborne pathogens such as Salmonella, Campylobacter, and Escherichia coli O157:H7 [23–28] and are traditionally being used to preserve or flavor foods [23]. Lemongrass (Cymbopogon citratus) is traditionally used in Asian cuisine as a flavoring agent [29]. Lemongrass essential oil (LGEO) is extracted from lemongrass [30] and is classified as a generally recognized as safe (GRAS) compound by the U.S. Food and Drug Administration (FDA; 21CFR182.20) [31]. The essential oil contains citral as an active component which is found as a combination of isomeric forms such as geranial (α-citral) and neral (β-citral) [30]. The essential oil also contains other compounds such as limonene, citronellal, ß-myrcene, and geraniol at low concentrations [30, 32]. Previous studies have demonstrated the antimicrobial property of LGEO against foodborne pathogens, including Salmonella [33–36]. For example, LGEO and citral were found to increase the sensitivity of antibiotic streptomycin
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potential of LGEO against S. Heidelberg in water alone or water containing droppings or litter, indicating its efficacy as a natural disinfectant in poultry drinking water.
PEICHEL ET AL.: LEMONGRASS INHIBITS SALMONELLA
MATERIALS AND METHODS S. Heidelberg and Growth Conditions Two S. Heidelberg isolates (N13F0000466 and NI3×001904; 2014 Tennessee correctional facility outbreak), kindly provided by Dr. James Gibson [37] were used for the experiment. Working cultures of each isolate were prepared separately in 10 mL trypticase soy broth [TSB; 38] by transferring 100 μL of culture from glycerol stock stored at -80◦ C. Broth cultures were incubated at 37◦ C for 24 h. After 3 successive subculture passages in TSB, the isolates were made resistant to 50 μg/ml nalidixic acid [NA; 39] for selective enumeration of the pathogen. The population of NA-resistant isolates in the overnight broth cultures was determined in xylose lysine desoxycholate agar [XLD; 40] plates containing 50 μg/mL NA (XLD-NA). Overnight (18 h) broth cultures containing 8 log10 cfu/mL S. Heidelberg were centrifuged at 3,500 Xg for 15 min, re-suspended in 10 mL of phosphate buffered saline (pH 7.2) and equal portions (5 mL) from each S. Heidelberg isolate were combined to prepare an inoculum containing 8 log10 cfu/mL S. Heidelberg. From this inoculum, 1 mL was used for the experiments [41]. Experimental Design Water collected from the Poultry Teaching and Research Facility (PTRF) at the Univer-
sity of Minnesota was used for the experiment. The selected temperatures (12.5◦ C or 22◦ C) have been previously used to study effects of a plant compound, trans-cinnamaldehyde, in poultry drinking water [25], and are in the lower and upper limits of the thermoneutral zone for poultry [42]. Our preliminary tests with the PTRF water revealed that the viability of NA-resistant S. Heidelberg declined in the control samples devoid of any antimicrobial when incubated at 12.5◦ C or 22◦ C. Therefore, water was autoclaved to kill any potential background microorganisms. In studies involving water with no added contaminants, different polypropylene container jars containing 99 mL water was inoculated with 1 mL of 6.0 log10 cfu/mL of NA-resistant S. Heidelberg. This step was followed by adding LGEO at 0%, 0.03%, 0.06%, 0.125%, 0.25%, or 0.5% (v/v) to the water and vortexing the contents in the jar to mix. In studies involving water added with contaminants, a similar protocol mentioned above was followed, however, with added contaminants. In order to simulate the natural contamination of poultry drinking water on farms, grow-out house organic matter, litter, droppings or feed (0.5 g of each) was added to water in the container jars. Fresh wood shavings from PTRF and fecal material collected from broilers (5-wk-old) in the farm was used as litter and dropping samples, respectively. Non-medicated starter crumble feed procured from Famo feeds Inc. [43] was used to simulate the contamination of water with feed. We ensured that these samples were negative for Salmonella before the experiments. To each container jar with 99 ml autoclaved water with contaminants, 1 mL of S. Heidelberg (final inoculum; 6.0 log10 cfu/mL) was added followed by LGEO at 0, 0.03, 0.06, 0.125, 0.25, or 0.5% (v/v). The samples were vortexed to mix the contents in the jar. For all studies, separate water samples inoculated with S. Heidelberg and without any LGEO treatments (0% LGEO) served as the positive control whereas samples without S. Heidelberg or LGEO were used as negative controls. After the addition of treatments, the containers were incubated for 7 d at either 12.5 or 22◦ C. Survival of S. Heidelberg was determined on d 0, 1, 3, 5 and 7 of incubation with repeated sampling from each treatment. All experiments included
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towards S. Typhimurium [36]. Additionally, LGEO showed concentration and timedependent reduction of S. Newport attached on the leafy greens when used as a dip treatment [35]. However, the potential use of LGEO in poultry production, including its efficacy in controlling foodborne Salmonella in poultry drinking water and the compounding effects of contaminants such as feed, litter, or droppings, have not been explored. Therefore, the objective of this study was to determine the efficacy of LGEO in reducing survival of a major serovar colonizing poultry, Salmonella enterica serovar Heidelberg (S. Heidelberg), in poultry drinking water with or without added contaminants during a 7-d storage period at 12.5 or 22◦ C.
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Determination of S. Heidelberg Survival The effectiveness of LGEO on S. Heidelberg survival in the treatment groups was determined for 7 d incubation. A 100 μL sample was taken from each treatment container on d 0, 1, 3, 5, and 7, serially diluted (1:10) and appropriate dilutions plated on XLD-NA plates and surviving S. Heidelberg populations were enumerated after 24 h incubation at 37◦ C [41]. Samples from which no S. Heidelberg was detected by direct plating were enriched in selenite cysteine broth [SCB; 44]. Briefly, 1 mL of each sample was incubated in 10 ml SCB for 8–12 h at 37◦ C. Presence of S. Heidelberg in enriched samples was then detected after streaking on XLD and XLD-NA plates with incubation for 24 h at 37◦ C [41]. The surviving S. Heidelberg populations were expressed in colony forming units per mL (cfu/mL). Statistical Analysis The experiment followed a completely randomized design with a 6 × 2 × 5 factorial treatment structure. The factors included 6 treatments, 2 temperatures, and 5 time points. There were 3 experiments, and all treatments groups in each experiment included duplicate samples. S. Heidelberg populations (cfu/mL) were transformed to log values (log10 cfu/mL) to achieve homogeneity of variance. A value of 0.95 was included in the statistical analysis in cases where no S. Heidelberg growth was detected by direct surface plating (except in negative controls) but where bacterial growth was observed after enrichment. A mixed procedure of SAS (PROCMIXED) was used for analysis, and the difference in means was contrasted using Tukey’s studentized range test. Statistical significance was determined at P-values less than or equal to 0.05.
RESULTS Effects of the treatment of drinking water with LGEO at 12.5◦ C and 22◦ C are given in
Figures 1A–1D and Figures 2A–D, respectively. At 12.5◦ C, the higher concentrations of LGEO (0.25 and 0.5%) resulted in inactivation of S. Heidelberg to non-detectable levels (by surface plating and enrichment steps) after 24 h of incubation (Figure 1A). Thereafter, no S. Heidelberg growth was detected for these treatments until the end of the study. However, 6.0 log10 cfu/mL S. Heidelberg survived in the positive controls on all the sampling days. At 0.06% and 0.125% LGEO concentrations, 5.0- and 5.8- log10 cfu/mL reductions in S. Heidelberg were observed at d 1 treatments incubated at 12.5◦ C, respectively (P < 0.05). From d 3 onwards, these concentrations resulted in complete inhibition of S. Heidelberg. All tested concentrations of LGEO inactivated S. Heidelberg populations at 7 d after incubation to non-detectable levels (Figure 1A). The LGEO treatments at 12.5◦ C significantly reduced S. Heidelberg in drinking water contaminated with 0.5% droppings (Figure 1B). Similar to water without any contaminants, the 0.25% and 0.5% LGEO treatments at 12.5◦ C inactivated S. Heidelberg to non-detectable levels from water contaminated with droppings after 24 h incubation. On d 1, the remaining LGEO treatments (0.03%, 0.06%, and 0.125%) resulted in 4.2-, 5.1-, and 5.8- log10 cfu/mL reduction of S. Heidelberg compared to the positive control, respectively (P < 0.05). On d 3, 5.3-, 5.6-, and 6.2- log10 cfu/mL reduction of S. Heidelberg was observed in the same LGEO treatments, respectively (P < 0.05). From d 5 onwards, all LGEO treatments at 12.5◦ C resulted in inactivation of S. Heidelberg in drinking water with droppings to non-detectable levels whereas 5.5 to 6.2 log10 cfu/mL S. Heidelberg survived in the positive control (Figure 1B). The LGEO treatments at 12.5◦ C were also effective against S. Heidelberg in water contaminated with 0.5% litter (Figure 1C). On d 1, the LGEO treatments resulted in significant reduction of S. Heidelberg in water contaminated with litter, although at varying levels. On d 1, LGEO at 0.06%, 0.125%, 0.25%, and 0.5%, resulted in 1.5-, 2.7-, 4.4-, and 4.5- log10 cfu/mL reductions of S. Heidelberg compared to the positive control, respectively (P < 0.05). The inhibitory activity of LGEO improved after d 3 of incubation and resulted in 3.8-, 3.7-, 5.1-, and 5.9- log10
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duplicate samples for each treatment (n = 6) and were repeated three times [25].
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cfu/mL reductions of S. Heidelberg, respectively (P < 0.05). From d 5, the higher LGEO concentrations (0.25 and 0.5%) resulted in inactivation of S. Heidelberg in water containing litter to non-detectable levels. In addition, the lower LGEO concentrations (0.03, 0.06, and 0.125%) resulted in >4.0 log10 cfu/mL reduction of S. Heidelberg. By d 7, all LGEO treatments at 12.5◦ C resulted in inactivation of S. Heidelberg from water contaminated with litter to nondetectable levels whereas ∼5.0 log10 cfu/mL S. Heidelberg survived in the positive control (Figure 1C). The LGEO was also effective against S. Heidelberg in water contaminated with 0.5% feed (Figure 1D). On d 5, 0.25 and 0.5% LGEO resulted in ∼5.0 log10 cfu/mL reduction of S. Heidelberg, compared to the positive controls.
On d 7, LGEO at 0.5% resulted in 5.4 log10 cfu/mL reduction of S. Heidelberg (P < 0.05) compared to the positive control, although other concentrations also significantly reduced S. Heidelberg populations (Figure 1D). Unlike the other experiments described previously, LGEO treatment of the feed-contaminated water did not result in inactivation of S. Heidelberg to non-detectable levels after 7 d of incubation. Similar to the anti-S. Heidelberg effect at 12.5◦ C, LGEO was also effective at reducing S. Heidelberg at 22◦ C (Figures 2A–D). At 22◦ C and 0.5%, LGEO inactivated S. Heidelberg (>6 log10 cfu/mL; non-detectable levels) in water after 24 h, compared with the positive control (Figure 2A). From d 3 onward, 0.125% and 0. 25% LGEO resulted in inactivation of S. Heidelberg in the water to non-detectable
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Figure 1. Effect of lemongrass essential oil (LGEO) against Salmonella enterica serovar Heidelberg (S. Heidelberg) in (A) water, (B) water contaminated with 0.5% poultry droppings, (C) water contaminated with 0.5% litter, and (D) water contaminated with 0.5% feed, and incubated at 12.5◦ C. Water was inoculated with 6.0 log10 cfu/mL of S. Heidelberg, treated with 0%, 0.03%, 0.06%, 0.125%, 0.25%, or 0.5% (v/v) LGEO and incubated for 7 d at 12.5◦ C. Survival of S. Heidelberg was determined on d 0, 1, 3, 5, and 7 of incubation with each treatment (n = 6). The LGEO treatments 0%, 0.03%, 0.06%, 0.125%, 0.25%, and 0.5% (v/v) are depicted as: positive control (), 0.03% LGEO (), 0.06% LGEO (), 0.125% LGEO ( ), 0.25% LGEO ( r), and 0.5% LGEO (), respectively. D 0 time point is ∼30 s immediately after the broth dilution and plating. Data are presented as mean with SE bars at each sampling point.
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levels compared to control (>5 log10 cfu/mL reduction; P < 0.05) until the end of the experiment. Although not completely inactivating, 0.06% LGEO resulted in 5.2 log10 cfu/mL reduction of S. Heidelberg on d 3 (P < 0.05). On d 5 and 7, all LGEO treatments resulted in inactivation of S. Heidelberg to non-detectable levels in water whereas 5.9 log10 cfu/mL S. Heidelberg survived in the positive control (Figure 2A). The LGEO treatments at 22◦ C significantly reduced S. Heidelberg in drinking water contaminated with 0.5% droppings (Figure 2B). On d 1, LGEO treatments at 0.125%, 0.25%, and 0.5% resulted in 5.2-, 3.5-, and 3.9- log10 cfu/mL reduction of S. Heidelberg, respectively compared to positive control (P < 0.05). On d 3, the same LGEO treatments resulted in 5.7-, 5.2-, and 6.4- log10 cfu/mL S. Heidelberg reduction, respectively (P < 0.05). From d 5 onwards,
these concentrations resulted in S. Heidelberg inactivation (∼7 log10 cfu/mL reduction; nondetectable levels). The lower LGEO concentrations (0.03% and 0.06% LGEO) resulted in ∼4 log10 cfu/mL reduction on d 5 and 7 (P < 0.05; Figure 2B). The LGEO treatments at 22◦ C were also effective against S. Heidelberg in water contaminated with litter, although the magnitude of reduction varied with concentration (Figure 2C). On d 3, ∼3 log10 cfu/mL reduction of S. Heidelberg populations was obtained with 0.5% LGEO treatment (P < 0.05). Likewise, 0.5% LGEO resulted in 4.0- and 5.3- log10 cfu/mL reductions of S. Heidelberg, respectively on d 5 and 7 (P < 0.05) (Figure 2C). The LGEO treatments did not result in significant S. Heidelberg reductions in feed-contaminated drinking water incubated at 22◦ C (Figure 2D).
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Figure 2. Effect of lemongrass essential oil (LGEO) against Salmonella enterica serovar Heidelberg (S. Heidelberg) in (A) water, (B) water contaminated with 0.5% poultry droppings, (C) water contaminated with 0.5% litter, and (D) water contaminated with 0.5% feed, and incubated at 22◦ C. Water was inoculated with 6.0 log10 cfu/mL of S. Heidelberg, treated with 0%, 0.03%, 0.06%, 0.125%, 0.25%, or 0.5% (v/v) LGEO and incubated for 7 d at 22◦ C. Survival of S. Heidelberg was determined on d 0, 1, 3, 5 and 7 of incubation with each treatment (n = 6). The LGEO treatments 0%, 0.03%, 0.06%, 0.125%, 0.25%, and 0.5% (v/v) are depicted as: positive control (), 0.03% LGEO (), 0.06% LGEO (), 0.125% LGEO ( ), 0.25% LGEO ( r), and 0.5% LGEO (), respectively. D 0 time point is ∼30 s immediately after the broth dilution and plating. Data are presented as mean with SE bars at each sampling point.
PEICHEL ET AL.: LEMONGRASS INHIBITS SALMONELLA
Contamination of poultry drinking water with Salmonella could be a serious concern due to the potential for long-term exposure of the flock to the pathogen, resulting in intestinal colonization [14–16, 19]. Newly-hatched chicks are particularly vulnerable as they are usually susceptible to a low infectious dose of Salmonella due to low microbial diversity and immature gut microbiota [45–46]. As the birds mature, they can act as a source of infection to the other birds through the excretion of the pathogen via fecal droppings [47]. An increase in bird density associated with the current production practices and vertical integration of poultry production are also reported to facilitate transmission of serovars such as S. Enteritidis in poultry flocks [48]. In order to reduce potential microbial contamination, the use of chemical water disinfectants is a common practice [49]. For example, some commonly used chemical oxidizers are sodium hypochlorite, chlorine gas, and calcium hypochlorite. However, in enclosed watering systems, at higher concentrations, these can lead to the corrosion of the equipment, and can be easily neutralized by organic matter such as feed, litter, and droppings in bell drinkers [20–22]. With consumer preference for natural choices over synthetic compounds [50–52], use of plantbased options could improve the value of the product given their potential to reduce foodborne pathogens in production and processing, including poultry operations. In this regard some botanicals, essential oils or their major components have been tested for their efficacy against Salmonella by supplementing through feed [53]. However, a major obstacle with this approach is the requirement of large concentrations of essential oils (mostly >1%) to result in pathogen reduction which could be potentially attributed to interactions of the essential oils with fats and proteins in the feed [25, 54]. To overcome these potential interactions, water could be used as a medium to administer essential oils to poultry. Additionally, in a similar study, it was reported that trans-cinnamaldehyde, a major component of cinnamon oil, could be effective against S. Enteritidis in poultry drinking water [25]. In this study, we are determining the potential of LGEO
against S. Heidelberg, supplemented through water. The LGEO treatments were most effective in water, followed by water contaminated with droppings and then by water with litter (Figures 1A-D, 2A-D). The greater efficacy of LGEO in the presence of droppings and litter could be due to the nutrient-deficient environment of these contaminants in comparison with the feed. Lowered activity of LGEO against S. Heidelberg in water containing feed could be due to interactions of the essential oil with fat, carbohydrates, and proteins present in the feed. The fats in the feed could also provide protection to Salmonella from environmental or physiological stresses [25, 54], and this could include the destructive effect of antimicrobials. Similar observations were reported with trans-cinnamaldehyde, a major active component in cinnamon oil when tested against S. Enteritidis in drinking water contaminated with feed or chicken feces at similar temperatures [25]. In the same study, S. Enteritidis survived in feed for a longer duration compared to droppings. However, LGEO treatments in water containing feed were not consistently effective and was temperature dependent (Figures 1D and 2D). For example, a more or less concentration dependent inhibition of S. Heidelberg was observed at 12.5◦ C, with lower concentrations (0.03% and 0.06%) yielding 1 to 2 log reduction on d 5 and 7, whereas, the higher concentrations (0.25% and 0.5%) resulting in 4 to 5 log reductions of the pathogen during the same time (Figure 1D). However, none of the concentrations proved effective against the pathogen in the water contaminated with feed at 22◦ C by the end of the study (Figure 2D). A few studies have reported the antimicrobial activity of LGEO [33–36, 55]. For example, the antibacterial activity of LGEO against S. Newport, S. Cholerasuis, Listeria monocytogenes, E. coli, and Staphylococcus aureus on leafy vegetables have been reported previously [35, 55]. The antibacterial activity of LGEO against S. Newport at 4◦ C or 8◦ C was time and concentration dependent [35]. Moreover, the reduction observed with the Salmonella species was in the range of 0.5 to 4 logs, compared to the controls [35, 55]. Similarly, 0.3 g% of LGEO essential oil resulted in 0.8 and 1.0 log10 cfu/g reduction of
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DISCUSSION
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CONCLUSIONS AND APPLICATIONS 1. LGEO showed the highest reduction of S. Heidelberg in water without contaminants, indicating the importance of cleaning bell waterers for better antimicrobial activity.
2. The efficacy of LGEO decreased in the order of water contaminated with droppings>litter>feed at both tested temperatures. This is highly relevant as fecal matter and litter could be the most common contaminants in waterers on farms employing bell drinkers. 3. LGEO at higher concentrations (0.25% and 0.5%) resulted in 5 log10 cfu/ml reduction of S. Heidelberg in water with feces by d 1 at both temperatures. 4. LGEO at 0.5% resulted in 5 log10 cfu/ml reduction of the pathogen in water contaminated with litter on d 1 at 12.5◦ C, but needed 7 d for pathogen reduction at 22◦ C. 5. LGEO at 0.5% resulted in 5 log10 cfu/ml reduction of the pathogen in water contaminated with feed by d 5 at 12.5◦ C, but was not effective at 22◦ C. 6. LGEO could be used as a natural disinfectant to reduce S. Heidelberg in water in poultry production within the thermoneutral zone.
REFERENCES AND NOTES 1. CDC. 2019. Salmonella. Available online at https: //www.cdc.gov/salmonella/index.html. 2. Hoffmann, S., M. B. Batz, and J. G. Morris. 2012. Annual cost of illness and quality-adjusted life year losses in the United States due to 14 foodborne pathogens. J. Food Prot. 75:1292–1302. 3. CDC. 2018. Outbreaks involving Salmonella. Available online at https://www.cdc.gov/salmonella /outbreaks.html. 4. CDC. 2013. Morbidity and mortality weekly report: Surveillance for foodborne disease outbreaks United States, 1998–2008. Available online at https: //www.cdc.gov/mmwr/preview/mmwrhtml/ss6202a1.htm. 5. FAO/WHO. 2009. Salmonella and Campylobacter in chicken meat. microbiological risk assessment series-19: Meeting Report, Rome. Available online at http://www.fao.org/3/a-i1133e.pdf. 6. Kovaˇci´c, A., Z. Huljev, and E. Suˇsi´c. 2017. Ground water as the source of an outbreak of Salmonella Enteritidis. J. Epidemiol. Global Health 7:181–184. 7. CDC. 2018. Water-related diseases and contaminants in public water system. Available online at https://www.cdc.gov/healthywater/drinking/public/water diseases.html. 8. Levantesi, C., L. Bonadonna, R. Briancesco, E. Grohmann, S. Toze, and V. Tandoi. 2012. Salmonella in surface and drinking water: Occurrence and water-mediated transmission. Food Res. Int. 45:587–602. 9. Pandey, P. K., P. H. Kass, M. L. Soupir, S. Biswas, and V. P. Singh. 2014. Contamination of water resources by pathogenic bacteria. AMB Expr. 4:51.
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S. Cholerasuis on spinach and lettuce, respectively [55]. Furthermore, it is also reported that LGEO alone or in combination with 2 antibiotics, streptomycin and kanamycin could result in synergistic or additive effects in vitro [36]. Not much information is available on the mechanism of antimicrobial action of LGEO against the aforementioned pathogens, including Salmonella species. Antibacterial activity of LGEO could reasonably be attributed to its biologically active major constituent, citral [30]. It is present as a mixture of geranial and neral which are 2 isomeric acyclic monoterpene aldehydes [34]. These isomers have been reported to elicit antibacterial activity against Gram positive and negative organisms in vitro [34]. Citral is lipophilic in nature and diffuses through the cell membrane. Then, citral acts as an alkylating agent to the cells due to the presence of conjugated α, β carbons and carbonyl groups. Further, these molecules modify the microbial cellular functions by potentially binding with nucleophiles present in the bacterial cells [56,57]. These studies have focused on Penicillium species and L. monocytogenes. Similarly, citral exhibits its antimicrobial effect against pathogens such as Cronobacter sakazakii via multitude of mechanisms including altering membrane potential, affecting cellular pH, depleting intracellular ATP concentration, and damaging cellular integrity [58]. Additionally, citral is found to be inhibiting the mixed species biofilm formed by S. aureus and S. Enteritidis by interrupting the quorum sensing system [59]. However, the specific mechanisms by which LGEO inhibits S. Heidelberg growth have yet to be explored. Overall results of this study indicate that LGEO could be used as a natural disinfectant against S. Heidelberg in poultry drinking water. However, the efficacy of LGEO against Salmonella in birds has to be determined before recommending for industry usage.
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52. Smid, E. J., and L. G. M. Gorris. 1999. Natural antimicrobials for food preservation. Pages 285–308 in Food Science and Technology. Marcel Dekker. New York. 53. Diaz-Sanchez, S., D. D’Souza, D. Biswas, and I. Hanning. 2015. Botanical alternatives to antibiotics for use in organic poultry production. Poult. Sci. 94:1419–1430. 54. Jones, F. T. 2011. A review of practical Salmonella control measures in animal feed. J. Appl. Poult. Res. 20:102– 113. 55. Ortega-Ramirez, L. A., B. A. Silva-Espinoza, I. Vargas-Arispuro, G. A. Gonzalez-Aguilar, M. R. CruzValenzuela, F. Nazzaro, and J. F. Ayala-Zavala. 2017. Combination of Cymbopogon citratus and Allium cepa essential oils increased antibacterial activity in leafy vegetables. J. Sci. Food Agric. 97:2166–2173. 56. Wuryatmo, E., A. Klieber, and E. S. Scott. 2003. Inhibition of citrus postharvest pathogens by vapor of citral and related compounds in culture. J. Agric. Food Chem. 51:2637–2640. 57. Hadjilouka, A., M. Polychronopoulou, S. Paramithiotis, P. Tzamalis, and E. Drosinos. 2015. Effect of lemongrass essential oil vapors on microbial dynamics and Listeria monocytogenes survival on rocket and melon stored under different packaging conditions and temperatures. Microorganisms 3:535–550. 58. Shi, C., K. Song, X. Zhang, Y. Sun, Y. Sui, Y. Chen, Z. Jia, H. Sun, Z. Sun, and X. Xia. 2016. Antimicrobial activity and possible mechanism of action of citral against Cronobacter sakazakii. PLoS One 11:e0159006. 59. Zhang, H., W. Zhou, W. Zhang, A. Yang, Y. Liu, Y. Jiang, S. Huang, and J. Su. 2014. Inhibitory effects of citral, cinnamaldehyde, and tea polyphenols on mixed biofilm formation by foodborne Staphylococcus aureus and Salmonella Enteritidis. J. Food Prot. 77:927–933.
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42. Anderson, K., and T. A. Carter. 2004. Hot weather management of poultry. Available online at https://thepoultrysite.com/articles/hot-weather-management -of-poultry 43. Famo feeds Inc. 446 Industrial Dr, Freeport, MN 5633. 44. SCB; catalog no. C6921 Criterion, Hardy Diagnostics, Santa Maria, CA, USA. 45. Oakley, B. B., H. S. Lillehoj, M. H. Kogut, W. K. Kim, J. J. Maurer, A. Pedroso, M. D. Lee, S. R. Collett, T. J. Johnson, and N. A. Cox. 2014. The chicken gastrointestinal microbiome. FEMS Microbiol. Lett. 360: 100–112. 46. Pan, D., and Z. Yu. 2014. Intestinal microbiome of poultry and its interaction with host and diet. Gut. Microbes 5:108–119. 47. Barrow, P. A., J. M. Simpson, and M. A. Lovell. 1988. Intestinal colonisation in the chicken by food-poisoning Salmonella serotypes; microbial characteristics associated with faecal excretion. Avian Pathol. 17:571–588. 48. Foley, S. L., R. Nayak, I. B. Hanning, T. J. Johnson, J. Han, and S. C. Ricke. 2011. Population dynamics of Salmonella enterica serotypes in commercial egg and poultry production. Appl. Environ. Microbiol. 77:4273– 4279. 49. United States Department of Agriculture Food Safety Inspection Service. Chlorine: health hazard information sheet. Available online at https://www.fsis.usda.gov/wps/ wcm/connect/84bb772a-d53d-4734-92d2-f375d39f7ab7/ Chlorine.pdf?MOD=AJPERES. 50. Winter, C. K., and S. F. Davis. 2006. Organic foods. J. Food Sci. 71:R117–R124. 51. Pokorn´y, J. 1991. Natural antioxidants for food use. Trends Food Sci. Technol. 2:223–227.
JAPR: Research Report
C. Peichel,∗,2 D. V. T. Nair,∗,2 G. Dewi,∗ A. M. Donoghue,† K. M. Reed,‡ and A. Kollanoor Johny∗,3 ∗
Department of Animal Science, University of Minnesota, Saint Paul, MN – 55108, USA; † Poultry Production and Product Safety Research Unit, ARS, USDA, University of Arkansas, Fayetteville AR 72701, Arkansas; and ‡ Department of Veterinary and Biomedical Sciences, University of Minnesota, Saint Paul MN – 55108, USA
Primary Audience: Flock Supervisors, Quality Assurance and Laboratory Personnel, Researchers and Veterinarians SUMMARY Drinking water contaminated with Salmonella could serve as a source for cecal colonization of the pathogen in birds. In this study, we investigated the efficacy of a generally recognized as safe—status essential oil (lemongrass essential oil, LGEO) against multidrug-resistant Salmonella enterica serovar Heidelberg (S. Heidelberg) in poultry drinking water. Farm water with and without added droppings, litter, or feed [0.5% (w/v)] inoculated with 6.0 log10 cfu/mL S. Heidelberg was treated with 0, 0.03, 0.06, 0.125, 0.25, or 0.5% (v/v) LGEO and incubated at 12.5◦ C or 22◦ C for up to 7 d. The number of viable S. Heidelberg populations was determined on d 0, 1, 3, 5, and 7. At 12.5◦ C, all concentrations of LGEO inactivated S. Heidelberg to non-detectable levels in water alone and water contaminated with droppings or litter by d 7 (>6 log10 cfu/mL; P < 0.05). The highest LGEO concentration tested (0.5%) resulted in >5.0 log10 cfu/mL reduction of S. Heidelberg on d 7 in water contaminated with feed (P < 0.05). At 22◦ C, all concentrations of LGEO resulted in the reduction of S. Heidelberg to non-detectable levels in water alone by d 5 (>6 log10 cfu/mL; P < 0.05). Concentrations of LGEO ≥0.125% resulted in inhibition of S. Heidelberg to non-detectable levels in water contaminated with droppings from d 5 onwards (P < 0.05). The LGEO at the highest tested concentration (0.5%) resulted in >5.0 log10 cfu/mL reduction of S. Heidelberg in water contaminated with litter on d 7 (P < 0.05) but did not result in reduction of the pathogen in water contaminated with feed at 22◦ C. Results indicate the
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The research was supported by the USDA National Institute of Food and Agriculture (Hatch) Project [Accession#1016910 (MIN-16-120); PI – A. Kollanoor Johny] and the USDA National Needs Fellowship (# 2016-38420-25285; PD – K. M. Reed). 2 Contributed equally to the conduct of experiments and manuscript preparation. 3 Corresponding author: [email protected]
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Effect of Lemongrass (Cymbopogon citratus) Essential Oil on the Survival of Multidrug-Resistant Salmonella enterica serovar Heidelberg in Contaminated Poultry Drinking Water1
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Key words: lemongrass essential oil, Salmonella Heidelberg, poultry drinking water, contaminants, natural disinfectant 2019 J. Appl. Poult. Res. 0:1–10 http://dx.doi.org/10.3382/japr/pfz076
DESCRIPTION OF PROBLEM Salmonella accounts for 1.2 million illnesses, 23,000 hospitalizations, and 450 deaths annually in the United States (U.S.) of which about a million illnesses are attributed to the consumption of contaminated food [1]. The pathogen leads to an estimated $3.3 billion loss to the U.S. economy in costs associated with the illness [2]. A variety of food commodities, including dairy products, raw fruits and vegetables, nuts, spices, fresh produce, seafood, poultry, beef, and pork serve as vehicles for Salmonella-associated foodborne illness [3]. Among these food commodities, eggs and poultry meat and products contribute more than half of Salmonella-associated foodborne outbreaks [4]. Poultry serve as carriers for the pathogen where excretion leads to the contamination of new incoming flocks and thus carcasses and products during processing [5]. Animal grazing and other agricultural practices, and sewage waste disposal can pose an environmental risk through contamination of groundwater, rivers, lakes, and other reservoirs with non-typhoidal Salmonella [6–10]. Multiple sources such as rodent and insect vectors, personnel, feed, water, litter, trucks, contaminated surfaces, and feces allow Salmonella to enter and persist in broiler and laying hen houses [11–12]. Among these, water tanks and drinkers pose a heightened source of Salmonella exposure to the birds [13–16]. Salmonella serovars, including the multiple antibiotic resistant clones, have been previously isolated from groundwater systems [6]. Salmonella attaches to the water flowing systems, forms biofilms, and may act as a constant source of exposure [17–18]. Poultry drinking water, especially those in small-scale grow-out facilities and backyard flocks, can be further contaminated with litter, droppings, or feed which provide a nutrient-rich environment promoting Salmonella survival and
multiplication at temperatures common in production facilities [19]. Organic contaminants and alterations in pH at different locations in the farm water supply often reduce the efficacy of water treatments, such as chlorine, that are aimed at reducing the Salmonella load [20–22]. Ingestion of Salmonella through drinking water may lead to the colonization of Salmonella in day-old or young chicks [19]. Therefore, effective strategies have to be developed to control Salmonella in drinking water to avoid the introduction into the flocks. The use of natural antimicrobials as antibiotic alternatives against pathogenic bacteria are receiving increased consideration due to heightened concerns over antibiotic resistance in pathogens encountered in food animal production. Plant-based, natural, and environmentally-friendly approaches, especially essential oils and their components, are effective options against foodborne pathogens such as Salmonella, Campylobacter, and Escherichia coli O157:H7 [23–28] and are traditionally being used to preserve or flavor foods [23]. Lemongrass (Cymbopogon citratus) is traditionally used in Asian cuisine as a flavoring agent [29]. Lemongrass essential oil (LGEO) is extracted from lemongrass [30] and is classified as a generally recognized as safe (GRAS) compound by the U.S. Food and Drug Administration (FDA; 21CFR182.20) [31]. The essential oil contains citral as an active component which is found as a combination of isomeric forms such as geranial (α-citral) and neral (β-citral) [30]. The essential oil also contains other compounds such as limonene, citronellal, ß-myrcene, and geraniol at low concentrations [30, 32]. Previous studies have demonstrated the antimicrobial property of LGEO against foodborne pathogens, including Salmonella [33–36]. For example, LGEO and citral were found to increase the sensitivity of antibiotic streptomycin
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potential of LGEO against S. Heidelberg in water alone or water containing droppings or litter, indicating its efficacy as a natural disinfectant in poultry drinking water.
PEICHEL ET AL.: LEMONGRASS INHIBITS SALMONELLA
MATERIALS AND METHODS S. Heidelberg and Growth Conditions Two S. Heidelberg isolates (N13F0000466 and NI3×001904; 2014 Tennessee correctional facility outbreak), kindly provided by Dr. James Gibson [37] were used for the experiment. Working cultures of each isolate were prepared separately in 10 mL trypticase soy broth [TSB; 38] by transferring 100 μL of culture from glycerol stock stored at -80◦ C. Broth cultures were incubated at 37◦ C for 24 h. After 3 successive subculture passages in TSB, the isolates were made resistant to 50 μg/ml nalidixic acid [NA; 39] for selective enumeration of the pathogen. The population of NA-resistant isolates in the overnight broth cultures was determined in xylose lysine desoxycholate agar [XLD; 40] plates containing 50 μg/mL NA (XLD-NA). Overnight (18 h) broth cultures containing 8 log10 cfu/mL S. Heidelberg were centrifuged at 3,500 Xg for 15 min, re-suspended in 10 mL of phosphate buffered saline (pH 7.2) and equal portions (5 mL) from each S. Heidelberg isolate were combined to prepare an inoculum containing 8 log10 cfu/mL S. Heidelberg. From this inoculum, 1 mL was used for the experiments [41]. Experimental Design Water collected from the Poultry Teaching and Research Facility (PTRF) at the Univer-
sity of Minnesota was used for the experiment. The selected temperatures (12.5◦ C or 22◦ C) have been previously used to study effects of a plant compound, trans-cinnamaldehyde, in poultry drinking water [25], and are in the lower and upper limits of the thermoneutral zone for poultry [42]. Our preliminary tests with the PTRF water revealed that the viability of NA-resistant S. Heidelberg declined in the control samples devoid of any antimicrobial when incubated at 12.5◦ C or 22◦ C. Therefore, water was autoclaved to kill any potential background microorganisms. In studies involving water with no added contaminants, different polypropylene container jars containing 99 mL water was inoculated with 1 mL of 6.0 log10 cfu/mL of NA-resistant S. Heidelberg. This step was followed by adding LGEO at 0%, 0.03%, 0.06%, 0.125%, 0.25%, or 0.5% (v/v) to the water and vortexing the contents in the jar to mix. In studies involving water added with contaminants, a similar protocol mentioned above was followed, however, with added contaminants. In order to simulate the natural contamination of poultry drinking water on farms, grow-out house organic matter, litter, droppings or feed (0.5 g of each) was added to water in the container jars. Fresh wood shavings from PTRF and fecal material collected from broilers (5-wk-old) in the farm was used as litter and dropping samples, respectively. Non-medicated starter crumble feed procured from Famo feeds Inc. [43] was used to simulate the contamination of water with feed. We ensured that these samples were negative for Salmonella before the experiments. To each container jar with 99 ml autoclaved water with contaminants, 1 mL of S. Heidelberg (final inoculum; 6.0 log10 cfu/mL) was added followed by LGEO at 0, 0.03, 0.06, 0.125, 0.25, or 0.5% (v/v). The samples were vortexed to mix the contents in the jar. For all studies, separate water samples inoculated with S. Heidelberg and without any LGEO treatments (0% LGEO) served as the positive control whereas samples without S. Heidelberg or LGEO were used as negative controls. After the addition of treatments, the containers were incubated for 7 d at either 12.5 or 22◦ C. Survival of S. Heidelberg was determined on d 0, 1, 3, 5 and 7 of incubation with repeated sampling from each treatment. All experiments included
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towards S. Typhimurium [36]. Additionally, LGEO showed concentration and timedependent reduction of S. Newport attached on the leafy greens when used as a dip treatment [35]. However, the potential use of LGEO in poultry production, including its efficacy in controlling foodborne Salmonella in poultry drinking water and the compounding effects of contaminants such as feed, litter, or droppings, have not been explored. Therefore, the objective of this study was to determine the efficacy of LGEO in reducing survival of a major serovar colonizing poultry, Salmonella enterica serovar Heidelberg (S. Heidelberg), in poultry drinking water with or without added contaminants during a 7-d storage period at 12.5 or 22◦ C.
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Determination of S. Heidelberg Survival The effectiveness of LGEO on S. Heidelberg survival in the treatment groups was determined for 7 d incubation. A 100 μL sample was taken from each treatment container on d 0, 1, 3, 5, and 7, serially diluted (1:10) and appropriate dilutions plated on XLD-NA plates and surviving S. Heidelberg populations were enumerated after 24 h incubation at 37◦ C [41]. Samples from which no S. Heidelberg was detected by direct plating were enriched in selenite cysteine broth [SCB; 44]. Briefly, 1 mL of each sample was incubated in 10 ml SCB for 8–12 h at 37◦ C. Presence of S. Heidelberg in enriched samples was then detected after streaking on XLD and XLD-NA plates with incubation for 24 h at 37◦ C [41]. The surviving S. Heidelberg populations were expressed in colony forming units per mL (cfu/mL). Statistical Analysis The experiment followed a completely randomized design with a 6 × 2 × 5 factorial treatment structure. The factors included 6 treatments, 2 temperatures, and 5 time points. There were 3 experiments, and all treatments groups in each experiment included duplicate samples. S. Heidelberg populations (cfu/mL) were transformed to log values (log10 cfu/mL) to achieve homogeneity of variance. A value of 0.95 was included in the statistical analysis in cases where no S. Heidelberg growth was detected by direct surface plating (except in negative controls) but where bacterial growth was observed after enrichment. A mixed procedure of SAS (PROCMIXED) was used for analysis, and the difference in means was contrasted using Tukey’s studentized range test. Statistical significance was determined at P-values less than or equal to 0.05.
RESULTS Effects of the treatment of drinking water with LGEO at 12.5◦ C and 22◦ C are given in
Figures 1A–1D and Figures 2A–D, respectively. At 12.5◦ C, the higher concentrations of LGEO (0.25 and 0.5%) resulted in inactivation of S. Heidelberg to non-detectable levels (by surface plating and enrichment steps) after 24 h of incubation (Figure 1A). Thereafter, no S. Heidelberg growth was detected for these treatments until the end of the study. However, 6.0 log10 cfu/mL S. Heidelberg survived in the positive controls on all the sampling days. At 0.06% and 0.125% LGEO concentrations, 5.0- and 5.8- log10 cfu/mL reductions in S. Heidelberg were observed at d 1 treatments incubated at 12.5◦ C, respectively (P < 0.05). From d 3 onwards, these concentrations resulted in complete inhibition of S. Heidelberg. All tested concentrations of LGEO inactivated S. Heidelberg populations at 7 d after incubation to non-detectable levels (Figure 1A). The LGEO treatments at 12.5◦ C significantly reduced S. Heidelberg in drinking water contaminated with 0.5% droppings (Figure 1B). Similar to water without any contaminants, the 0.25% and 0.5% LGEO treatments at 12.5◦ C inactivated S. Heidelberg to non-detectable levels from water contaminated with droppings after 24 h incubation. On d 1, the remaining LGEO treatments (0.03%, 0.06%, and 0.125%) resulted in 4.2-, 5.1-, and 5.8- log10 cfu/mL reduction of S. Heidelberg compared to the positive control, respectively (P < 0.05). On d 3, 5.3-, 5.6-, and 6.2- log10 cfu/mL reduction of S. Heidelberg was observed in the same LGEO treatments, respectively (P < 0.05). From d 5 onwards, all LGEO treatments at 12.5◦ C resulted in inactivation of S. Heidelberg in drinking water with droppings to non-detectable levels whereas 5.5 to 6.2 log10 cfu/mL S. Heidelberg survived in the positive control (Figure 1B). The LGEO treatments at 12.5◦ C were also effective against S. Heidelberg in water contaminated with 0.5% litter (Figure 1C). On d 1, the LGEO treatments resulted in significant reduction of S. Heidelberg in water contaminated with litter, although at varying levels. On d 1, LGEO at 0.06%, 0.125%, 0.25%, and 0.5%, resulted in 1.5-, 2.7-, 4.4-, and 4.5- log10 cfu/mL reductions of S. Heidelberg compared to the positive control, respectively (P < 0.05). The inhibitory activity of LGEO improved after d 3 of incubation and resulted in 3.8-, 3.7-, 5.1-, and 5.9- log10
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duplicate samples for each treatment (n = 6) and were repeated three times [25].
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cfu/mL reductions of S. Heidelberg, respectively (P < 0.05). From d 5, the higher LGEO concentrations (0.25 and 0.5%) resulted in inactivation of S. Heidelberg in water containing litter to non-detectable levels. In addition, the lower LGEO concentrations (0.03, 0.06, and 0.125%) resulted in >4.0 log10 cfu/mL reduction of S. Heidelberg. By d 7, all LGEO treatments at 12.5◦ C resulted in inactivation of S. Heidelberg from water contaminated with litter to nondetectable levels whereas ∼5.0 log10 cfu/mL S. Heidelberg survived in the positive control (Figure 1C). The LGEO was also effective against S. Heidelberg in water contaminated with 0.5% feed (Figure 1D). On d 5, 0.25 and 0.5% LGEO resulted in ∼5.0 log10 cfu/mL reduction of S. Heidelberg, compared to the positive controls.
On d 7, LGEO at 0.5% resulted in 5.4 log10 cfu/mL reduction of S. Heidelberg (P < 0.05) compared to the positive control, although other concentrations also significantly reduced S. Heidelberg populations (Figure 1D). Unlike the other experiments described previously, LGEO treatment of the feed-contaminated water did not result in inactivation of S. Heidelberg to non-detectable levels after 7 d of incubation. Similar to the anti-S. Heidelberg effect at 12.5◦ C, LGEO was also effective at reducing S. Heidelberg at 22◦ C (Figures 2A–D). At 22◦ C and 0.5%, LGEO inactivated S. Heidelberg (>6 log10 cfu/mL; non-detectable levels) in water after 24 h, compared with the positive control (Figure 2A). From d 3 onward, 0.125% and 0. 25% LGEO resulted in inactivation of S. Heidelberg in the water to non-detectable
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Figure 1. Effect of lemongrass essential oil (LGEO) against Salmonella enterica serovar Heidelberg (S. Heidelberg) in (A) water, (B) water contaminated with 0.5% poultry droppings, (C) water contaminated with 0.5% litter, and (D) water contaminated with 0.5% feed, and incubated at 12.5◦ C. Water was inoculated with 6.0 log10 cfu/mL of S. Heidelberg, treated with 0%, 0.03%, 0.06%, 0.125%, 0.25%, or 0.5% (v/v) LGEO and incubated for 7 d at 12.5◦ C. Survival of S. Heidelberg was determined on d 0, 1, 3, 5, and 7 of incubation with each treatment (n = 6). The LGEO treatments 0%, 0.03%, 0.06%, 0.125%, 0.25%, and 0.5% (v/v) are depicted as: positive control (), 0.03% LGEO (), 0.06% LGEO (), 0.125% LGEO ( ), 0.25% LGEO ( r), and 0.5% LGEO (), respectively. D 0 time point is ∼30 s immediately after the broth dilution and plating. Data are presented as mean with SE bars at each sampling point.
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levels compared to control (>5 log10 cfu/mL reduction; P < 0.05) until the end of the experiment. Although not completely inactivating, 0.06% LGEO resulted in 5.2 log10 cfu/mL reduction of S. Heidelberg on d 3 (P < 0.05). On d 5 and 7, all LGEO treatments resulted in inactivation of S. Heidelberg to non-detectable levels in water whereas 5.9 log10 cfu/mL S. Heidelberg survived in the positive control (Figure 2A). The LGEO treatments at 22◦ C significantly reduced S. Heidelberg in drinking water contaminated with 0.5% droppings (Figure 2B). On d 1, LGEO treatments at 0.125%, 0.25%, and 0.5% resulted in 5.2-, 3.5-, and 3.9- log10 cfu/mL reduction of S. Heidelberg, respectively compared to positive control (P < 0.05). On d 3, the same LGEO treatments resulted in 5.7-, 5.2-, and 6.4- log10 cfu/mL S. Heidelberg reduction, respectively (P < 0.05). From d 5 onwards,
these concentrations resulted in S. Heidelberg inactivation (∼7 log10 cfu/mL reduction; nondetectable levels). The lower LGEO concentrations (0.03% and 0.06% LGEO) resulted in ∼4 log10 cfu/mL reduction on d 5 and 7 (P < 0.05; Figure 2B). The LGEO treatments at 22◦ C were also effective against S. Heidelberg in water contaminated with litter, although the magnitude of reduction varied with concentration (Figure 2C). On d 3, ∼3 log10 cfu/mL reduction of S. Heidelberg populations was obtained with 0.5% LGEO treatment (P < 0.05). Likewise, 0.5% LGEO resulted in 4.0- and 5.3- log10 cfu/mL reductions of S. Heidelberg, respectively on d 5 and 7 (P < 0.05) (Figure 2C). The LGEO treatments did not result in significant S. Heidelberg reductions in feed-contaminated drinking water incubated at 22◦ C (Figure 2D).
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Figure 2. Effect of lemongrass essential oil (LGEO) against Salmonella enterica serovar Heidelberg (S. Heidelberg) in (A) water, (B) water contaminated with 0.5% poultry droppings, (C) water contaminated with 0.5% litter, and (D) water contaminated with 0.5% feed, and incubated at 22◦ C. Water was inoculated with 6.0 log10 cfu/mL of S. Heidelberg, treated with 0%, 0.03%, 0.06%, 0.125%, 0.25%, or 0.5% (v/v) LGEO and incubated for 7 d at 22◦ C. Survival of S. Heidelberg was determined on d 0, 1, 3, 5 and 7 of incubation with each treatment (n = 6). The LGEO treatments 0%, 0.03%, 0.06%, 0.125%, 0.25%, and 0.5% (v/v) are depicted as: positive control (), 0.03% LGEO (), 0.06% LGEO (), 0.125% LGEO ( ), 0.25% LGEO ( r), and 0.5% LGEO (), respectively. D 0 time point is ∼30 s immediately after the broth dilution and plating. Data are presented as mean with SE bars at each sampling point.
PEICHEL ET AL.: LEMONGRASS INHIBITS SALMONELLA
Contamination of poultry drinking water with Salmonella could be a serious concern due to the potential for long-term exposure of the flock to the pathogen, resulting in intestinal colonization [14–16, 19]. Newly-hatched chicks are particularly vulnerable as they are usually susceptible to a low infectious dose of Salmonella due to low microbial diversity and immature gut microbiota [45–46]. As the birds mature, they can act as a source of infection to the other birds through the excretion of the pathogen via fecal droppings [47]. An increase in bird density associated with the current production practices and vertical integration of poultry production are also reported to facilitate transmission of serovars such as S. Enteritidis in poultry flocks [48]. In order to reduce potential microbial contamination, the use of chemical water disinfectants is a common practice [49]. For example, some commonly used chemical oxidizers are sodium hypochlorite, chlorine gas, and calcium hypochlorite. However, in enclosed watering systems, at higher concentrations, these can lead to the corrosion of the equipment, and can be easily neutralized by organic matter such as feed, litter, and droppings in bell drinkers [20–22]. With consumer preference for natural choices over synthetic compounds [50–52], use of plantbased options could improve the value of the product given their potential to reduce foodborne pathogens in production and processing, including poultry operations. In this regard some botanicals, essential oils or their major components have been tested for their efficacy against Salmonella by supplementing through feed [53]. However, a major obstacle with this approach is the requirement of large concentrations of essential oils (mostly >1%) to result in pathogen reduction which could be potentially attributed to interactions of the essential oils with fats and proteins in the feed [25, 54]. To overcome these potential interactions, water could be used as a medium to administer essential oils to poultry. Additionally, in a similar study, it was reported that trans-cinnamaldehyde, a major component of cinnamon oil, could be effective against S. Enteritidis in poultry drinking water [25]. In this study, we are determining the potential of LGEO
against S. Heidelberg, supplemented through water. The LGEO treatments were most effective in water, followed by water contaminated with droppings and then by water with litter (Figures 1A-D, 2A-D). The greater efficacy of LGEO in the presence of droppings and litter could be due to the nutrient-deficient environment of these contaminants in comparison with the feed. Lowered activity of LGEO against S. Heidelberg in water containing feed could be due to interactions of the essential oil with fat, carbohydrates, and proteins present in the feed. The fats in the feed could also provide protection to Salmonella from environmental or physiological stresses [25, 54], and this could include the destructive effect of antimicrobials. Similar observations were reported with trans-cinnamaldehyde, a major active component in cinnamon oil when tested against S. Enteritidis in drinking water contaminated with feed or chicken feces at similar temperatures [25]. In the same study, S. Enteritidis survived in feed for a longer duration compared to droppings. However, LGEO treatments in water containing feed were not consistently effective and was temperature dependent (Figures 1D and 2D). For example, a more or less concentration dependent inhibition of S. Heidelberg was observed at 12.5◦ C, with lower concentrations (0.03% and 0.06%) yielding 1 to 2 log reduction on d 5 and 7, whereas, the higher concentrations (0.25% and 0.5%) resulting in 4 to 5 log reductions of the pathogen during the same time (Figure 1D). However, none of the concentrations proved effective against the pathogen in the water contaminated with feed at 22◦ C by the end of the study (Figure 2D). A few studies have reported the antimicrobial activity of LGEO [33–36, 55]. For example, the antibacterial activity of LGEO against S. Newport, S. Cholerasuis, Listeria monocytogenes, E. coli, and Staphylococcus aureus on leafy vegetables have been reported previously [35, 55]. The antibacterial activity of LGEO against S. Newport at 4◦ C or 8◦ C was time and concentration dependent [35]. Moreover, the reduction observed with the Salmonella species was in the range of 0.5 to 4 logs, compared to the controls [35, 55]. Similarly, 0.3 g% of LGEO essential oil resulted in 0.8 and 1.0 log10 cfu/g reduction of
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DISCUSSION
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CONCLUSIONS AND APPLICATIONS 1. LGEO showed the highest reduction of S. Heidelberg in water without contaminants, indicating the importance of cleaning bell waterers for better antimicrobial activity.
2. The efficacy of LGEO decreased in the order of water contaminated with droppings>litter>feed at both tested temperatures. This is highly relevant as fecal matter and litter could be the most common contaminants in waterers on farms employing bell drinkers. 3. LGEO at higher concentrations (0.25% and 0.5%) resulted in 5 log10 cfu/ml reduction of S. Heidelberg in water with feces by d 1 at both temperatures. 4. LGEO at 0.5% resulted in 5 log10 cfu/ml reduction of the pathogen in water contaminated with litter on d 1 at 12.5◦ C, but needed 7 d for pathogen reduction at 22◦ C. 5. LGEO at 0.5% resulted in 5 log10 cfu/ml reduction of the pathogen in water contaminated with feed by d 5 at 12.5◦ C, but was not effective at 22◦ C. 6. LGEO could be used as a natural disinfectant to reduce S. Heidelberg in water in poultry production within the thermoneutral zone.
REFERENCES AND NOTES 1. CDC. 2019. Salmonella. Available online at https: //www.cdc.gov/salmonella/index.html. 2. Hoffmann, S., M. B. Batz, and J. G. Morris. 2012. Annual cost of illness and quality-adjusted life year losses in the United States due to 14 foodborne pathogens. J. Food Prot. 75:1292–1302. 3. CDC. 2018. Outbreaks involving Salmonella. Available online at https://www.cdc.gov/salmonella /outbreaks.html. 4. CDC. 2013. Morbidity and mortality weekly report: Surveillance for foodborne disease outbreaks United States, 1998–2008. Available online at https: //www.cdc.gov/mmwr/preview/mmwrhtml/ss6202a1.htm. 5. FAO/WHO. 2009. Salmonella and Campylobacter in chicken meat. microbiological risk assessment series-19: Meeting Report, Rome. Available online at http://www.fao.org/3/a-i1133e.pdf. 6. Kovaˇci´c, A., Z. Huljev, and E. Suˇsi´c. 2017. Ground water as the source of an outbreak of Salmonella Enteritidis. J. Epidemiol. Global Health 7:181–184. 7. CDC. 2018. Water-related diseases and contaminants in public water system. Available online at https://www.cdc.gov/healthywater/drinking/public/water diseases.html. 8. Levantesi, C., L. Bonadonna, R. Briancesco, E. Grohmann, S. Toze, and V. Tandoi. 2012. Salmonella in surface and drinking water: Occurrence and water-mediated transmission. Food Res. Int. 45:587–602. 9. Pandey, P. K., P. H. Kass, M. L. Soupir, S. Biswas, and V. P. Singh. 2014. Contamination of water resources by pathogenic bacteria. AMB Expr. 4:51.
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S. Cholerasuis on spinach and lettuce, respectively [55]. Furthermore, it is also reported that LGEO alone or in combination with 2 antibiotics, streptomycin and kanamycin could result in synergistic or additive effects in vitro [36]. Not much information is available on the mechanism of antimicrobial action of LGEO against the aforementioned pathogens, including Salmonella species. Antibacterial activity of LGEO could reasonably be attributed to its biologically active major constituent, citral [30]. It is present as a mixture of geranial and neral which are 2 isomeric acyclic monoterpene aldehydes [34]. These isomers have been reported to elicit antibacterial activity against Gram positive and negative organisms in vitro [34]. Citral is lipophilic in nature and diffuses through the cell membrane. Then, citral acts as an alkylating agent to the cells due to the presence of conjugated α, β carbons and carbonyl groups. Further, these molecules modify the microbial cellular functions by potentially binding with nucleophiles present in the bacterial cells [56,57]. These studies have focused on Penicillium species and L. monocytogenes. Similarly, citral exhibits its antimicrobial effect against pathogens such as Cronobacter sakazakii via multitude of mechanisms including altering membrane potential, affecting cellular pH, depleting intracellular ATP concentration, and damaging cellular integrity [58]. Additionally, citral is found to be inhibiting the mixed species biofilm formed by S. aureus and S. Enteritidis by interrupting the quorum sensing system [59]. However, the specific mechanisms by which LGEO inhibits S. Heidelberg growth have yet to be explored. Overall results of this study indicate that LGEO could be used as a natural disinfectant against S. Heidelberg in poultry drinking water. However, the efficacy of LGEO against Salmonella in birds has to be determined before recommending for industry usage.
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