Abstract
The pool of antimicrobial resistance determinants in the environment and in the gut flora of cattle is a serious public health concern. In addition to being a source of human exposure, these bacteria can transfer antibiotic resistance determinants to pathogenic bacteria and endanger the future of antimicrobial therapy. The occurrence of antimicrobial resistance genes on mobile genetic elements, such as plasmids, facilitates spread of resistance. Recent work has shown in vitro anti-plasmid activity of menthol, a plant-based compound with the potential to be used as a feed additive to beneficially alter ruminal fermentation. The present study aimed to determine if menthol supplementation in diets of feedlot cattle decreases the prevalence of multidrug-resistant bacteria in feces. Menthol was included in diets of steers at 0.3% of diet dry matter. Fecal samples were collected weekly for 4 weeks and analyzed for total coliforms counts, antimicrobial susceptibilities, and the prevalence of tet genes in E. coli isolates. Results revealed no effect of menthol supplementation on total coliforms counts or prevalence of E. coli resistant to amoxicillin, ampicillin, azithromycin, cefoxitin, ceftiofur, ceftriaxone, chloramphenicol, ciprofloxacin, gentamicin, kanamycin, nalidixic acid, streptomycin, sulfisoxazole, and sulfamethoxazole; however, 30 days of menthol addition to steer diets increased the prevalence of tetracycline-resistant E. coli (P < 0.02). Although the mechanism by which menthol exerts its effects remains unclear, results of our study suggest that menthol may have an impact on antimicrobial resistance in gut bacteria.
Introduction
The rise of antimicrobial-resistant bacteria has been observed worldwide [1] and is a growing concern because of its potential to endanger the future of antimicrobial drug therapy [2]. Excessive use of therapeutic and non-therapeutic antimicrobials in human, animal health, and animal husbandry contributes to the emergence and dissemination of antimicrobial resistance (AMR; [3, 4]) in our environment (soil, water, etc…). Livestock, and more specifically ruminants’ gut flora, represents a large reservoir of antibiotic-resistant bacteria and resistance gene determinants, which can spread to the environment and to humans [5, 6]. The genes encoding for AMR, including multidrug resistance (MDR), are often carried on mobile genetic elements such as plasmids, transposons, and integrons [7, 8, 9], which facilitate horizontal transfer [10] from commensal to pathogenic bacteria and from livestock to human bacterial flora. Escherichia coli, a common gut bacterium in most mammal species and prevalent in feces of cattle [11], is often used to assess the impact of antimicrobial agents as it carries antibiotic resistance genes. Multiple antimicrobial resistance determinants have been found in Escherichia coli on the same plasmid, further facilitating their propagation and co-selection. For instance, the multidrug resistance plasmid IncA/C found in enteric bacteria, such as Salmonella enterica and Escherichia coli, often encodes for resistance to common antimicrobial agents such as tetracycline (tetA), chloramphenicol/florfenicol (floR), streptomycin/spectinomycin (aadA2), sulfonamides (sul1 and sul2), and extended spectrum β-lactamases (blaCMY-2; [12]), and its spread to pathogenic bacteria may limit antibacterial means to fight infections caused by these bacteria. Therefore, compounds capable of limiting or preventing emergence of AMR and/or eliminating or inactivating mobile genetic elements may be of use in controlling antibiotic resistance dissemination, as well as MDR bacteria, and preserving antimicrobial efficacy.
Interest is considerable in using growth-promoting feed additives, such as probiotics, prebiotics, and plant-based compounds, as alternatives to antimicrobial agents to minimize the role of livestock as a reservoir of AMR bacteria [13, 14]. The impact of these non-antibiotic alternatives on prevalence and persistence of AMR bacteria in the gut has not yet been investigated in cattle in vivo. Menthol, a plant-based compound, is a monoterpene alcohol with known cooling and anesthetic properties, anti-pruritic activity, and antibacterial and antifungal activities [15]. Menthol has been shown to increase body weight gains in poultry [16–18]. In ruminants, inclusion of 3.3% menthol in a digestion trial [19] or 0.2% of peppermint oil in an in vitro ruminal fermentation assay [20] reduced protozoal, fungal, and bacterial populations in the rumen fluid. Direct addition of menthol to ruminal fluid in in vitro fermentation at concentrations greater than 0.1% was also shown to reduce total volatile fatty acid (VFA) concentrations [20]. However, nutrient digestibility tended to increase with 2.9% menthol in steers [19] and decrease with 5% menthol in lactating cows [21]. In addition to menthol’s inconsistent effect on animal growth performance, another interesting characteristic of menthol is its plasmid-curing activity. Schelz et al. [22] investigated the effects of peppermint oil and menthol in vitro on bacteria and their plasmids and demonstrated anti-plasmid activity similar to sodium dodecyl sulfate, which is a known plasmid-curing compound [23]. Because of menthol’s anti-plasmid activity, we postulated that inclusion of menthol in cattle diets could lead to reduction in the prevalence of MDR bacteria in the gut. Our objectives were to investigate the effects of menthol inclusion in the diet of feedlot cattle on fecal coliform populations and on AMR Escherichia coli in feces.
Materials and Methods
Procedures for this study were approved by the Kansas State University Institutional Animal Care and Use Committee.
Animals
Twenty-six Holstein steers (568.8 ± 55 kg body weight) were housed in individual pens within three barns containing 5, 5, and 3 steers representing each treatment. Barns had concrete-surfaced pens (1.5 m × 6 m), were covered with corrugated roofing and equipped with individual feed bunks. Water fountains were shared between adjacent pens. Two treatments, a control and a menthol group, were randomly assigned to steers and were equally represented in each of the three barns. Crushed menthol (99.7% purity, Prinova USA LLC, Carol Stream, IL) was included at 0.3% on a dry matter basis in a basal diet consisting of 50% steam flaked corn, 33% corn gluten feed, and 10% corn silage. Diets were manufactured daily to avoid excess volatilization of menthol. Steers received 300 mg of monensin (Elanco Animal Health, Greenfield, IN) and 90 mg of tylosin (Elanco Animal Health) per animal daily and were fed ad libitum with free access to water for 30 days.
Sample collection and processing
Fecal samples were obtained from each animal from the rectum, before feeding, on days 0 (before inclusion of menthol), 16, 23, and 30. Samples were placed in plastic bags, kept on ice, and transported to the Kansas State University Preharvest Food Safety Laboratory. Fecal samples were stored at -80°C before analysis.
Total coliform counts
Fecal samples obtained on days 0 and 30 were thawed and homogenized in a stomacher, and 1 g of each sample was suspended in 9 mL phosphate buffered saline (PBS) in a tube and vortexed. Fecal suspensions were allowed to settle, and a 50-μL of supernatant was spiral-plated, in duplicate, onto MacConkey agar using an Eddy Jet spiral plater (IUL instruments, Barcelona, Spain) and incubated overnight at 37°C. Lactose-fermenting colonies (coliform bacteria) were counted following spiral plating guidelines to determine coliform concentrations. If additional dilutions were needed, PBS was used to dilute the initial fecal suspension.
E. coli isolation
A 100-μL volume of each fecal suspension from each of the sampling days (day 0, 16, 23, and 30) was spread-plated on MacConkey agar (BD Diagnostic Systems, Franklin Lakes, NJ) and incubated overnight at 37°C. A single lactose-fermenting colony was selected randomly from each plate and re-plated onto tryptic soy agar (TSA; Thermo Fisher Scientific, Lenexa, KS). After an overnight incubation at 37°C, colonies were tested for indole production by a spot indole test. Indole-positive colonies were stored on cryobeads (Cryocare; Key Scientific Products, Stamford, TX) at -80°C until further analysis.
Minimum inhibitory concentration (MIC) determinations
Isolates from control and menthol groups were used to determine MIC for amoxicillin, ampicillin, azithromycin, cefoxitin, ceftiofur, ceftriaxone, chloramphenicol, ciprofloxacin, gentamicin, kanamycin, nalidixic acid, streptomycin, sulfisoxazole, tetracycline, and trimethropim/sulfamethoxazole (Gram-negative National Antimicrobial Resistance Monitoring System [NARMS] panel) using the broth microdilution method. Isolates stored in beads were grown on blood agar plates (Thermo Fisher Scientific Remel Products), and colonies were suspended in demineralized water (Trek Diagnostics Systems, Cleveland, OH) to obtain a suspension of 0.5 McFarland turbidity. A 50-μL aliquot of the suspension added to cation-adjusted Mueller-Hinton broth (Trek Diagnostics Systems) served as the inoculum. Tubes were vortexed and placed in the Sensititre automated inoculation delivery system (Trek Diagnostics Systems) to inoculate the Gram-negative NARMS panel plates (CMV2AGNF, Trek Diagnostics Systems). Plates were incubated for 18 h at 37°C and read manually using the Sensititre manual viewer (Sensitouch, Trek Diagnostics Systems). E. coli 25922 and Staphylococcus aureus 29213 strains (American Type Culture Collection, Manassas, VA) were used as quality control strains. Resistance or susceptibility of the isolates was determined based on CLSI guidelines [24].
PCR detection of tetracycline resistance genes
Isolates that were phenotypically resistant to tetracycline were tested for tetA and tetB genes. DNA extraction was performed by suspending a single colony from a blood agar plate in 500 μL of deionized water in 1.5 mL microcentrifuge tube, boiling for 10 min at 100°C, and centrifuging at 10,000 x g for 5 min. A duplex PCR assay for tetA and tetB was performed as described by Harvey et al. [25] with E. coli ATCC 47042 (positive for tetB) and XL1-Blue E. coli strain (positive for tetA) as positive controls. The DNA in 96-well plates were amplified using an Eppendorf Mastercycler gradient thermal cycler (USA Scientific, Inc., Ocala, FL), and PCR products were then transferred to the Automated QIAxcel System (QIAgen, Valencia, CA). Microcapillary electrophoresis was performed using a QIAxcel DNA screening cartridge (QIAgen), a QX alignment marker (15bp/1 kb; QIAgen), and a 50 to 800 bp QX size marker (QIAgen). The electrophoresis was documented and analyzed for the presence of specific bands.
Statistical analysis
Total coliform colony counts were log10 transformed and normality of the results was verified. Results were then analyzed with a generalized linear mixed model using the GLIMMIX procedure of SAS (9.2, Cary, NC). Treatment (control or menthol), sampling days (day 0, 16, 23, and 30), and their interaction were included in the model as fixed effects, and animal ID was used as the random effect. Unbiased least square means and standard errors were calculated using the LSMEANS statements of SAS and used to produce graphs.
Frequency analyses of resistant E. coli isolates to the multiple antibiotics tested were performed using the FREQ procedure of SAS with a chi-square test. Tetracycline resistance data were further analyzed with generalized linear mixed model using the GLIMMIX procedure of SAS with a binomial distribution, where treatment, sampling day, and their interaction were included as fixed effects, animal ID nested within treatment was used as the random effect, and tetracycline resistance status on day 0 was used as a covariate. Unbiased least square means and standard errors were calculated using the LSMEANS statements of SAS.
E. coli isolates were considered multidrug-resistant when resistant to 5 or more of the antibiotics tested. Multidrug-resistant bacteria, MDR phenotypes, tetA or tetB-positive genotypes, and the number of isolates resistant to tetracycline but not carrying tetA or tetB genes were analyzed with a chi-square test using the FREQ procedure of SAS. Further analysis of tetB genes was performed using a generalized linear mixed model in a GLIMMIX procedure with a binomial distribution where treatment and sampling day were included as fixed effects, animal ID nested within treatment was used as the random effect, and tetB resistance status on day 0 was used as a covariate. Unbiased least square means and standard errors were calculated using the LSMEANS statements of SAS. Low prevalence of tetB-positives genotypes precluded us from including in the model the interaction between sampling day and treatment.
Differences in least square means or frequencies were considered significant if the P-value was < 0.05.
Results
Total colony counts of coliform bacteria in fecal samples of cattle fed diets with or without 0.3% menthol were 1.2 x 103 and 6.0 x 102 CFU/g on day 0 and 1.2 x 103 and 3.6 x 103 CFU/g on day 30, respectively. Total coliform counts were not affected by the day of sampling (P = 0.231) or the inclusion of menthol in the diet (P = 0.841), and there was no significant interaction between the day of sampling and the inclusion of menthol (P = 0.254). A total of 103 E. coli isolates (52 from the control and 51 from menthol groups) were tested to determine antimicrobial susceptibility patterns. The number and proportions of isolates resistant to antimicrobial agents, as determined by CLSI guidelines, are shown in Table 1. Frequency analyses showed that all isolates, regardless of sampling day or menthol treatment, were susceptible to azithromycin, ceftriaxone, ciprofloxacin, gentamicin, nalidixic acid, and sulfamethoxazole. In addition, overall E. coli isolates resistant to cefoxitin, amoxicillin, ceftiofur, or kanamycin were equal to or lower than 3.9%, and no differences in frequencies were observed between isolates originating from animals fed diets with and without menthol on any of the sampling days (P > 0.05). Chloramphenicol-, ampicillin-, and streptomycin-resistant isolates were found in 5.8, 5.8, and 7.7% of the control group samples and 3.9, 9.8, and 3.9% of the menthol group samples, respectively, but were not significantly different among treatments (P > 0.05). Of the E. coli isolates from steers fed the control and menthol diets, 94.4% and 88.2% were resistant to sulfisoxazole, respectively (P > 0.05). Tetracycline resistant isolates were found in 32.7%, respectively, of the control group and 56.9% of the menthol group (P = 0.014). Glimmix analysis for tetracycline resistance revealed no sampling day effect (P = 0.480), no menthol treatment effect (P = 0.093), but an interaction between sampling day and menthol treatment (P = 0.044).
Table 1. Antimicrobial susceptibilities of fecal Escherichia coli isolates from steers fed diets supplemented with or without 0.3% menthol.
| Number of resistant isolates/total isolates tested | |||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| Control | Menthol, 0.3% | Control | Menthol, 0.3% | ||||||||
| Antimicrobials | Day 0 | Day 16 | Day 23 | Day 30 | Day 0 | Day 16 | Day 23 | Day 30 | Total (%) | ||
| Amoxicillin† | 0/13 | 0/13 | 0/13 | 0/13 | 0/13 | 0/12 | 1/13 | 1/13 | 0/52 (0) | 2/51 (3.9) | |
| Ampicillin | 1/13 | 1/13 | 1/13 | 0/13 | 1/13 | 0/12 | 2/13 | 2/13 | 3/52 (5.8) | 5/51 (9.8) | |
| Azithromycin | 0/13 | 0/13 | 0/13 | 0/13 | 0/13 | 0/12 | 0/13 | 0/13 | 0/52 (0) | 0/51 (0) | |
| Cefoxitin | 0/13 | 0/13 | 0/13 | 0/13 | 0/13 | 0/12 | 1/13 | 1/13 | 0/52 (0) | 2/51 (3.9) | |
| Ceftiofur | 0/13 | 0/13 | 0/13 | 0/13 | 0/13 | 0/12 | 1/13 | 0/13 | 0/52 (0) | 1/51 (2.0) | |
| Ceftriaxone | 0/13 | 0/13 | 0/13 | 0/13 | 0/13 | 0/12 | 0/13 | 0/13 | 0/52 (0) | 0/51 (0) | |
| Chloramphenicol | 1/13 | 1/13 | 1/13 | 0/13 | 0/13 | 0/12 | 2/13 | 0/13 | 3/52 (5.8) | 2/51 (3.9) | |
| Ciprofloxacin | 0/13 | 0/13 | 0/13 | 0/13 | 0/13 | 0/12 | 0/13 | 0/13 | 0/52 (0) | 0/51 (0) | |
| Gentamicin | 0/13 | 0/13 | 0/13 | 0/13 | 0/13 | 0/12 | 0/13 | 0/13 | 0/52 (0) | 0/51 (0) | |
| Kanamycin | 1/13 | 0/13 | 0/13 | 0/13 | 0/13 | 0/12 | 0/13 | 0/13 | 1/52 (1.9) | 0/51 (0) | |
| Nalidixic acid | 0/13 | 0/13 | 0/13 | 0/13 | 0/13 | 0/12 | 0/13 | 0/13 | 0/52 (0) | 0/51 (0) | |
| Streptomycin | 1/13 | 2/13 | 1/13 | 0/13 | 0/13 | 0/12 | 2/13 | 0/13 | 4/52 (7.7) | 2/51 (3.9) | |
| Sulfamethoxazoleф | 0/13 | 0/13 | 0/13 | 0/13 | 0/13 | 0/12 | 0/13 | 0/13 | 0/52 (0) | 0/51 (0) | |
| Sulfisoxazole | 11/13 | 11/13 | 12/13 | 13/13 | 13/13 | 9/12 | 12/13 | 11/13 | 47/52 (90.4) | 45/51 (88.2) | |
| Tetracycline | 6/13 | 5/13 | 3/13 | 3/13a | 8/13 | 3/12 | 8/13 | 10/13b | 17/52 (32.7) | 29/51 (56.9) | |
ф with trimethoprim
† with clavulanic acid
a, bValues with different superscript letters are different; P < 0.02.
CLSI thresholds: Amoxicillin-clavulanic acid, R ≥ 32 μg/mL; ampicillin, R ≥ 32 μg/mL; azithromycin, R ≥ 16 μg/mL; ciprofloxacin, R ≥ 4 μg/mL; cefoxitin, R ≥ 32 μg/mL; ceftiofur, R ≥ 8 μg/mL; ceftriaxone, R ≥ 64 μg/mL; chloramphenicol, R ≥ 32 μg/mL; gentamicin, R ≥ 16 μg/mL; kanamycin, R ≥ 64 μg/mL; nalidixic acid, R ≥ 32 μg/mL; streptomycin, R ≥ 64 μg/mL; sulfamethoxazole, R ≥ 4 μg/mL; sulfisoxazole, R ≥ 256 μg/mL; tetracycline, R ≥ 16 μg/mL.
The proportion of isolates resistant to tetracycline was not different between treatments on day 16 (38.5% in the control group and 25% in the menthol group; P = 0.447), but tended to be greater in the menthol group on day 23 (23% in the control group and 61.5% in the menthol group; P = 0.084) and was greater on day 30 (P = 0.020; 23% in the control group and 76.9% in the menthol group).
Table 2 summarizes the prevalence of tetA and tetB genes in fecal E. coli phenotypically resistant to tetracycline in steers fed diets with or without menthol.
Table 2. Prevalence of tetA or tetB in fecal Escherichia coli phenotypically resistant to tetracycline in steers fed diets supplemented with or without 0.3% menthol.
| Genes and treatment groups | Number of tetA- or tetB-positive/total number of isolate resistant to tetracycline (%) | ||||
|---|---|---|---|---|---|
| Day 0 | Day 16 | Day 23 | Day 30 | Total | |
| tetA | |||||
| Control | 2/6 (33.3) | 2/5 (40.0) | 2/3 (66.7) | 1/3 (33.3) | 7/17 (41.2) |
| Menthol, 0.3% | 0/8 (0.0) | 2/3 (66.7) | 3/8 (37.5) | 1/10 (10) | 6/29 (20.7) |
| tetB | |||||
| Control | 2/6 (33.3) | 1/5 (20.0) | 0/3 (0.0) | 1/3 (33.3) | 4/17 (23.5) |
| Menthol, 0.3% | 7/8 (87.5) | 0/3 (0.0) | 2/8 (25.0) | 4/10 (40.0) | 13/29 (44.8) |
Overall, 41.2% of the isolates from the control group and 20.7% of the isolates from the menthol group carried tetA. Frequency analysis showed no difference in proportion of tetA positive isolates regardless of sampling day or menthol treatment (P = 0.592). Frequency analysis did, however, reveal an increase in proportion of tetB-positives isolates in the menthol treatment (44.8%) compared to the control (23.5%; P = 0.015). Isolates from animals fed menthol had greater prevalence of tetB than the control group on day 0 (87.5 and 33.3%, respectively; P = 0.039), but no significant difference among treatment were observed on day 16, 23, or 30 (P > 0.1). Those results were further investigated using the Glimmix analysis where tetB prevalence on day 0 was used as a covariate in the model. Results showed that number of tetB-positive isolates was not influenced by sampling day (P = 0.206) or by menthol treatment (P = 0.379).
All isolates carrying tetA or tetB were phenotypically resistant to tetracycline (MIC ≥ 16 μg/mL). Overall, 64.7% of the isolates resistant to tetracycline in the control group and 65.5% of the isolates resistant to tetracycline in the menthol group were found to carry tetA or tetB (P > 0.9). Conversely, 6 isolates from the control group (35.3%) and 10 from the high menthol group (34.5%) were resistant to tetracycline but did not carry tetA or tetB (Table 3; P > 0.9). On day 30, 50% of isolates in the menthol group and 33.3% of isolates in the control group were resistant to tetracycline and did not carry either tetA or tetB, but the difference was not a statistically significant (P = 0.850).
Table 3. Prevalence of tetracycline resistant isolates that do not carry tetA or tetB genes in fecal Escherichia coli in steers fed diets supplemented with or without 0.3% menthol.
| Isolates resistant to tetracycline that do not carry either tetA or tetB/ Number of tetracycline resistant isolates (%) | |||||
|---|---|---|---|---|---|
| Treatment | Day 0 | Day 16 | Day 23 | Day 30 | Total |
| Control | 2/6 (33.3) | 2/5 (40.0) | 1/3 (33.3) | 1/3 (33.3) | 6/17 (35.3) |
| Menthol, 0.3% | 1/8 (12.5) | 1/3 (33.3) | 3/8 (37.5) | 5/10 (50.0) | 10/29 (34.5) |
Table 4 presents the percentages of MDR (≥ 5 antimicrobial agents) E. coli isolates in each treatment group. Frequency analysis showed that overall prevalence of MDR isolates was not different in the control group (3.8%) or in the 0.3% menthol (5.9%; P > 0.631). Additionally, there was no difference in MDR frequency between treatments on days 0, 16, 23, and 30 (P > 0.3).
Table 4. Multidrug resistance (MDR; ≥ 5 antimicrobials) prevalence (%) in fecal Escherichia coli from steers fed diets supplemented with or without 0.3% menthol.
| Number of MDR isolates/total isolates tested (%) | |||||
|---|---|---|---|---|---|
| Treatment | Day 0 | Day 16 | Day 23 | Day 30 | Total |
| Control | 0/13 (0) | 1/13 (7.7) | 1/13 (7.7) | 0/13 (0) | 2/52 (3.8) |
| Menthol, 0.3% | 0/13 (0) | 0/12 (0) | 2/13 (15.4) | 1/13 (7.7) | 3/51 (5.9) |
Table 5 illustrates the various antibiotic resistance phenotypes among the E. coli isolates tested. Only 4.8% of the total isolates were pan-susceptible. Of the isolates tested, 50.5% were resistant to sulfisoxazole only, and 30.1% were resistant to both sulfisoxazole and tetracycline. Only one isolate from the menthol group was found to be resistant to eight antibiotics.
Table 5. Prevalence of antimicrobial-resistant fecal Escherichia coli isolates from cattle fed diets with or without 0.3% menthol.
| Phenotypes | Control | Menthol, 0.3% | Total (%) |
|---|---|---|---|
| _AMP_CHL_STR_FIS_TET | 2 | 1 | 3 (2.9) |
| _AMP_FIS_TET | 0 | 2 | 2 (1.9) |
| _AMP_STR_FIS_TET | 1 | 0 | 1 (1.0) |
| _AUG_AMP_FOX_FIS_TET | 0 | 1 | 1 (1.0) |
| _AUG_AMP_FOX_XNL_CHL_STR_FIS_TET | 0 | 1 | 1 (1.0) |
| _CHL_KAN_FIS_TET | 1 | 0 | 1 (1.0) |
| _FIS | 32 | 20 | 52 (50.5) |
| _FIS_TET | 11 | 20 | 31 (30.1) |
| _TET | 1 | 4 | 5 (4.8) |
| Pan susceptible | 3 | 2 | 5 (4.8) |
Amoxicillin-clavulanic acid, AUG; ampicillin, AMP; azithromycin, AZI; ciprofloxacin, CIP; cefoxitin, FOX; ceftiofur, XNL; ceftriaxone, AXO; chloramphenicol, CHL; gentamicin, GEN; kanamycin, KAN; nalidixic acid, NAL; streptomycin, STR; sulfamethoxazole, SXT; sulfisoxazole, FIS; tetracycline, TET.
Discussion
Based on the known antimicrobial activity of menthol against E. coli, we hypothesized that menthol would impact total coliform counts in cattle fecal samples. Menthol metabolism in the rumen is poorly understood, leading us to investigate the impact of 0.3% dietary menthol on commensal coliform populations in feedlot cattle. Menthol concentration in ruminal contents was not measured in this experiment, but can be roughly estimated at 2.7 mM considering a 50 L ruminal volume and 7 kg daily dry matter intake [26]. Previous studies have demonstrated inhibitory effects with concentrations of 75 mM for E. coli O157:H7 [27] and as low as 16 mM with E. coli ATCC15221 [28]. The lack of difference between total fecal coliform counts in the control group and menthol-supplemented groups suggests either that the level of menthol reaching the hindgut was insufficient to inhibit the organism or that bacteria were able to adapt to menthol presence within the gut. Landau and Shapira [27] recently showed enterohemorrhagic E. coli (EHEC) to have the ability to adapt to increasing levels of subinhibitory concentration of menthol, and a similar adaptation processes could be anticipated for other E. coli.
Our main objective was to investigate if menthol inclusion in feedlot diets would affect E. coli resistance to antibiotics and the prevalence of MDR organisms. Although total E. coli populations and MDR E. coli were not affected by 30 days of menthol supplementation, we analyzed individual minimum inhibitory concentration (MIC) of fecal E. coli isolates from the control group and the group receiving 0.3% of menthol daily after 0, 16, 23, and 30 days of exposure to treatments. Results of MIC evaluations revealed that all 103 isolates were susceptible to azithromycin, ceftriaxone, ciprofloxacin, gentamicin, and nalidixic acid, sulfamethoxazole, and only a small percentage of isolates were resistant to amoxicillin, cefoxitin, ceftiofur, and kanamycin, regardless of sampling day and of treatment received by the animals. Similar observations were made by Mirzaagha et al. [29], who found all 531 E. coli isolates collected from feedlot cattle fed diets with and without chlortetracycline and/or sulfamethazine susceptible to ceftriaxone, cefoxitin, gentamicin, and nalidixic acid. Gow et al. [30] also failed to detect any fecal E. coli resistance to ceftriaxone, ciprofloxacin, or nalidixic acid among the 207 isolates collected from cow-calf herds in western Canada and observed that only 1% were resistant to gentamicin, 1.5% to ceftiofur, and 4.8% to amoxicillin and cefoxitin. They did, however, observe greater resistance rates for kanamycin (15%) and sulfamethoxazole (55.1%) compared with our study. Chloramphenicol resistance, like previous antibiotics, was not affected by menthol treatment in our experiment. The presence of resistant isolates (4.8% overall) was somewhat surprising, as chloramphenicol use in animal production systems was banned more than 30 years ago [31]. Our observations are, however, not unusual; others have reported even higher prevalence (14.5 to 31%) in commensal E. coli from cattle [30, 32, 33]. Persistence of chloramphenicol resistance in the environment is thought to be due to the use of closely related antibiotics, such as florfenicol, or to a co-selection phenomenon [34]. Unfortunately, low prevalence of chloramphenicol-resistant isolates in this study did not allow us to reveal a resistance pattern associated with the presence of chloramphenicol resistance. Like chloramphenicol, the prevalence of isolates resistant to streptomycin was not affected by sampling day or menthol treatment. Overall, 5.8% of E. coli isolates tested in this experiment were resistant to streptomycin. Gow et al. [30] reported 41.6% E. coli resistant isolates from cow-calf herds, and Ma et al. [32] found that 89.1% of E. coli isolates from dairy cows were resistant. Differences in animal production system practices could explain the lower prevalence observed in our study, as animal exposures to antimicrobials are likely to be different. Resistance to sulfisoxazole was found in 89.3% of the E. coli isolates tested in our study and was not influenced by menthol inclusion in the diet. A large-scale study conducted in a feedlot in Texas also reported high resistance rate, with 65% of the tested 7,097 E. coli isolates resistant to sulfisoxazole. Prevalence in the Texas study was not influenced by the type of growth promotants received by the animals [35], which further underscores the widespread nature of sulfisoxazole resistance determinants in commensal bacteria. Overall ampicillin resistance, 7.8%, was not affected by the inclusion of menthol in the diets. This prevalence was lower than previously observed prevalence in E. coli from cattle, which has ranged from 18 to 48% [30, 33, 36]. Menthol supplementation did, however, have a significant effect on tetracycline resistance. After 30 days of menthol treatment, 76.9% of the isolates from the menthol group tested resistant to tetracycline compared with only 23.1% in the control group. Moreover, E. coli isolates resistant to tetracycline within the menthol group increased from day 16 to day 23 (25% and 61.5%, respectively) and from day 23 to day 30 (61.5% and 76.9%, respectively), while control group remained fairly constant (38.5% on day 16 and 23.1% on day 23 and 30). These observations underlined an effect of menthol on tetracycline resistance phenotypes, which compelled us to further investigate tetracycline genotype profiles of these isolates.
There are 40 known tet resistance determinants most of which are found on mobile genetic elements that encode for efflux pump [37]. TetA and tetB are the most prevalent genes in tetracycline resistant E. coli [38], which is why we chose to focus on these two. TetA and tetB encode for an efflux pump in the lipid bilayer of the bacteria, which removes the tetracycline/cation complex from the cell by exchanging a proton [39]. TetB is usually more predominant than tetA and is linked to higher MIC [40]. Out of the 103 isolates investigated in our experiment, 16.5% were found to carry tetB and 13.6% to carry tetA. Moreover, no isolates were found to carry both determinants, which corroborates previous findings [40, 41]. TetA and tetB are believed to be located on plasmids, though from different incompatibility groups [41]. This potentially explains why they rarely are detected together in bacteria. The absence of an effect of menthol inclusion on tetA seems to exclude the implication of tetA in the difference observed in tetracycline phenotypes in E. coli isolates from steers fed diets with and without menthol. In addition, the greater prevalence of tetB in the menthol group compared to the control on day 0 and the absence of an effect of menthol inclusion on tetB prevalence also exclude the implication of tetB in the difference observed in tetracycline phenotypes. Despite the greater prevalence of tetracycline resistance in the menthol group, frequency of E. coli isolates that were phenotypically resistant to tetracycline, though not carrying either tetA or tetB, was not significantly affected by the menthol treatment. Based on our results, the observed increase in tetracycline resistance also was not explained by the presence of other tet resistance determinants in E. coli isolates from steers fed 0.3% menthol; however, the small sample size of phenotypically tetracycline resistant isolates (46 isolates) may have limited our ability to detect statistically meaningful differences.
In conclusion, menthol supplementation of feedlot diets at a 0.3% rate for 30 days did not alter the total coliform population in fecal samples and did not affect prevalence of resistance to azithromycin, ceftriaxone, ciprofloxacin, gentamicin, nalidixic acid, cefoxitin, amoxicillin, ceftiofur, sulfamethoxazole, kanamycin, streptomycin, sulfisoxazole, chloramphenicol, and ampicillin. Menthol supplementation did, however, increase the prevalence of tetracycline-resistant E. coli isolates, but did not affect tetA and tetB gene-positive E. coli or the number of MDR bacteria. The underlying mechanism associated with this increase in tetracycline resistance is unknown; nevertheless, this study demonstrates a possible effect of menthol on bacterial antimicrobial resistance. If antibiotic substitutes, such as menthol, do indeed influence prevalence of AMR in gut bacteria, this raises questions concerning the appropriateness of these compounds as alternatives to traditional antibiotics.
Supporting Information
(XLS)
Acknowledgments
The authors wish to express their gratitude to Jordan Eder and Maggie Stephens for their assistance with sample processing, MIC analysis, and PCR work. This is contribution 14-342-J of the Kansas Agricultural Experiment Station.
Data Availability
All relevant data are within the paper and its Supporting Information file. For more information, please contact the corresponding author (jdrouill@ksu.edu).
Funding Statement
The authors received no specific funding for this work.
References
- 1.WHO. WHO global strategy for containment of antimicrobial resistance. In Department of communicable disease surveillance and response WHO, Geneva, Switzerland; 2001. [Google Scholar]
- 2.Marshall BM, Levy SB. Food animals and antimicrobials: Impacts on human health. Clin. Microbiol. Rev. 2011; 24:718–733 10.1128/CMR.00002-11 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Levy SB. Antibiotic resistance: Consequences of inaction. Clin. Infect. Dis. 2001; 33:S124–S129. 10.1086/321837 [DOI] [PubMed] [Google Scholar]
- 4.Chopra I. New developments in tetracycline antibiotics: glycylcyclines and tetracycline efflux pump inhibitors. Drug Resis. Update. 2002; 5:119–125. [DOI] [PubMed] [Google Scholar]
- 5.van den Bogaard AE, Stobberingh EE. Epidemiology of resistance to antibiotics—Links between animals and humans. Int. J. Antimicrob. Agents. 2000; 14:327–335. [DOI] [PubMed] [Google Scholar]
- 6.Alexander TW, Reuter T, Sharma R, Yanke LJ, Topp E, McAllister TA. Longitudinal characterization of resistant Escherichia coli in fecal deposits from cattle fed subtherapeutic levels of antimicrobials. Appl. Environ. Microbiol. 2009; 75:7125–7134. 10.1128/AEM.00944-09 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Medeiros AA. Evolution and dissemination of beta-lactamases accelerated by generations of beta-lactam antibiotics. Clin. Infect. Dis. 1997; 24(Suppl 1):19–45. [DOI] [PubMed] [Google Scholar]
- 8.Chopra I, Roberts M. Tetracycline antibiotics: Mode of action, applications, molecular biology, and epidemiology of bacterial resistance. Microbiol. Mol. Biol Rev. 2001; 65:232–260. 10.1128/MMBR.65.2.232-260.2001 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Ochman H, Lawrence JG, Groisman EA. Lateral gene transfer and the nature of bacterial innovation. Nature. 2000; 405:299–304. 10.1038/35012500 [DOI] [PubMed] [Google Scholar]
- 10.Martinez JL, Fajardo A, Garmendia L, Hernandez A, Linares JF, Martinez-Solano L, et al. A global view of antibiotic resistance. FEMS Microbiol. Rev. 2009; 33:44–65. 10.1111/j.1574-6976.2008.00142.x [DOI] [PubMed] [Google Scholar]
- 11.Nuru S, Osbaldiston GW, Stowe EC, Walker D. Fecal microflora of healthy cattle and pigs. Cornell Vet. 1972; 62:242–253. [PubMed] [Google Scholar]
- 12.Fernandez-Alarcon C, Singer RS, Johnson TJ. Comparative genomics of multidrug resistance-encoding IncA/C plasmids from commensal and pathogenic Escherichia coli from multiple animal sources. Plos One. 2011; 6:1–9. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.Benchaar C, Calsamiglia S, Chaves AV, Fraser GR, Colombatto D, McAllister TA, et al. A review of plant-derived essential oils in ruminant nutrition and production. Animal Feed Sci. Tech. 2008; 145: 209–228. [Google Scholar]
- 14.Gaggia F, Mattarelli P, Biavati B. Probiotics and prebiotics in animal feeding for safe food production. Int. J. Food Microbiol. 2010; 141(Suppl 1):15–28. [DOI] [PubMed] [Google Scholar]
- 15.Patel T, Ishiuji Y, Yosipovitch G. Menthol: A refreshing look at this ancient compound. J. Am. Acad. Dermatol. 2007; 57:873–878. 10.1016/j.jaad.2007.04.008 [DOI] [PubMed] [Google Scholar]
- 16.Ocak N, Erener G, Burak Ak F, Sungu M, Altop A, Ozmen A. Performance of broilers fed diets supplemented with dry peppermint (Mentha piperita L.) or thyme (Thymus vulgaris L.) leaves as growth promoter source. Czech J. Anim. Sci. 2008; 53:169–175. [Google Scholar]
- 17.Toghyani M, Toghyani M, Gheisari A, Ghalamkari G, Mohammadrezaei M. Growth performance, serum biochemistry and blood hematology of broiler chicks fed different levels of black seed (Nigella sativa) and peppermint (Mentha piperita). Livest. Sci. 2010; 129: 173–178. [Google Scholar]
- 18.Sharifi SD, Khorsandi SH, Khadem AA, Salehi A, Moslehi H. The effect of four medicinal plants on the performance, blood biochemical traits and ileal microflora of broiler chicks. Vet. Arhiv. 2013; 83:69–80. [Google Scholar]
- 19.Ando S, Nishida T, Ishida M, Hosoda K, Bayaru E. Effect of peppermint feeding on the digestibility, ruminal fermentation and protozoa. Livest. Prod. Sci. 2003; 82:245–248. [Google Scholar]
- 20.Agarwal N, Shekkar C, Kumar R, Chaudhary LC, Kamra DN. Effect of peppermint (Mentha piperita) oil on in vitro methanogenesis and fermentation of feed with buffalo rumen liquor. Anim. Feed Sci. Technol. 2009; 148:321–327. [Google Scholar]
- 21.Hosoda K, Nishida T, Park WY, Eruden B. Influence of Mentha×piperita L. (Peppermint) Supplementation on Nutrient Digestibility and Energy Metabolism in Lactating Dairy Cows. Asian-Aust. J. Anim. Sci. 2005; 18:1721–1726 [Google Scholar]
- 22.Schelz Z, Molnar J, Hohmann J. Antimicrobial and antiplasmid activities of essential oils. Fitoterapia. 2006; 77:279–285. 10.1016/j.fitote.2006.03.013 [DOI] [PubMed] [Google Scholar]
- 23.El-Mansi M, Anderson KJ, Inche CA, Knowles LK, Platt DJ. Isolation and curing of the Klebsiella pneumoniae large indigenous plasmid using sodium dodecyl sulphate. Res. Microbiol. 2000; 151:201–208. [DOI] [PubMed] [Google Scholar]
- 24.Clinical and Laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing; 21sd informational supplement. In CLSI M100-S21 Clinical and Laboratory Standards Institute, Wayne, PA; 2011.
- 25.Harvey R, Funk J, Wittum TE, Hoet AE. A metagenomic approach for determining prevalence of tetracycline resistance genes in the fecal flora of conventionally raised feedlot steers and feedlot steers raised without antimicrobials. Am. J. Vet. Res. 2009; 70:198–202. 10.2460/ajvr.70.2.198 [DOI] [PubMed] [Google Scholar]
- 26.Van Bibber-Krueger CL, Miller KA, Aperce CC, Alvarado-Gilis CA, Higgins JJ, Drouillard JS. Effects of crystalline menthol on blood metabolites in Holstein steers and in vitro volatile fatty acid and gas production. J. Anim. Sc. 2016; 94(3):1170–1178. [DOI] [PubMed] [Google Scholar]
- 27.Landau E, Shapira R. Effects of subinhibitory concentrations of menthol on adaptation, morphological, and gene expression changes in enterohemorrhagic Escherichia coli. Appl. Environ. Microbiol. 2012; 78:5361–5367. 10.1128/AEM.00894-12 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 28.Trombetta D, Castelli F, Sarpietro MG, Venuti V, Cristani M, Daniele C, et al. Mechanisms of antimicrobial action of three monoterpenes. Antimicrob. Agents Chemother. 2005; 49:2472–2478. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 29.Mirzaagha P, Louie M, Sharma R, Yanke LJ, Topp E, McAllister TA. Distribution and characterization of ampicillin-and tetracycline-resistant Escherichia coli from feedlot cattle fed subtherapeutic antimicrobials. BMC Microbiol. 2011; 11:78 10.1186/1471-2180-11-78 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 30.Gow SP, Waldner CL, Harel J, Boerlin P. Associations between antimicrobial resistance genes in fecal generic Escherichia coli isolates from cow-calf herds in western Canada. Appl. Environ. Microbiol. 2008; 74:3658–3666. 10.1128/AEM.02505-07 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 31.Gilmore A. Chloramphenicol and the politics of health. Can. Med. Assoc. J. 1996; 134:423–435. [PMC free article] [PubMed] [Google Scholar]
- 32.Ma YP, Chang SK, Chou CC. Characterization of bacterial susceptibility isolates in sixteen dairy farms in Taiwan. J. Dairy Sci. 2006; 89:4573–4582. 10.3168/jds.S0022-0302(06)72507-3 [DOI] [PubMed] [Google Scholar]
- 33.Sawant AA, Hegde NV, Straley BA, Donaldson SC, Love BC, Knabel SJ, et al. Antimicrobial-resistant enteric bacteria from dairy cattle. Appl. Environ. Microbiol. 2007; 73:156–163. 10.1128/AEM.01551-06 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 34.White DG, Hudson C, Maurer JJ, Ayers S, Zhao SH, Lee MD, et al. Characterization of chloramphenicol and florfenicol resistance in Escherichia coli associated with bovine diarrhea. J. Clin. Microbiol. 2000; 38:4593–4598. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 35.Branham LA. Antimicrobial susceptibility of generic Escherichia coli following administration of subtherapeutic antimicrobial drugs to feedlot cattle. Ph.D. thesis. Texas Tech University, Lubbock, TX; 2007.
- 36.Medina A, Horcajo P, Jurado S, De La Fuente R, Ruiz-Santa-Quiteria JA, Dominguez-Bernal G, et al. Phenotypic and genotypic characterization of antimicrobial resistance in enterohemorrhagic Escherichia coli and atypical enteropathogenic E. coli strains from ruminants. J. Vet. Diagn. Invest. 2011; 23:91–95. [DOI] [PubMed] [Google Scholar]
- 37.Thaker M, Spanogiannopoulos P, Wright GD. The tetracycline resistome. Cell. Mol. Life Sci. 2010; 67:419–431. 10.1007/s00018-009-0172-6 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 38.Bryan A, Shapir N, Sadowsky MJ. Frequency and distribution of tetracycline resistance genes in genetically diverse, nonselected, and nonclinical Escherichia coli strains, isolated from diverse human and animal sources. Appl. Environ. Microbiol. 2004; 70:2503–2507. 10.1128/AEM.70.4.2503-2507.2004 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 39.Yamaguchi A, Ono N, Akasaka T, Noumi T, Sawai T. Metaltetracycline/H+ antiporter of Escherichia coli encoded by a transposon, Tn10. J. Biol. Chem. 1990; 265:15525–15530. [PubMed] [Google Scholar]
- 40.Blake DP, Humphry RW, Scott KP, Hillman K, Fenlon DR, Low JC. Influence of tetracycline exposure on tetracycline resistance and the carriage of tetracycline resistance genes within commensal Escherichia coli populations. J. Appl. Microbiol. 2003; 94:1087–1097. [DOI] [PubMed] [Google Scholar]
- 41.Jones CS, Osborne DJ, Stanley J. Enterobacterial tetracycline resistance in relation to plasmid incompatibility. Mol. Cell. Probe. 1992; 6:313–317. [DOI] [PubMed] [Google Scholar]
Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
(XLS)
Data Availability Statement
All relevant data are within the paper and its Supporting Information file. For more information, please contact the corresponding author (jdrouill@ksu.edu).