GB2585132A - Composition for reducing antimicrobial resistance - Google Patents

Abstract

The use of a carvacrol and thymol composition for inhibiting an increase in the prevalence of cephalosporin-resistant Enterobacteriaceae in a bacterial population exposed to a cephalosporin antimicrobial is provided. The cephalosporin-resistant Enterobacteriaceae may be exposed to a cephalosporin antimicrobial in a milk product wherein the milk product is preferably milk, colostrum, transitional milk or calf milk replacer. The carvacrol and thymol composition may preferably be for inhibiting the increase in the prevalence of cephalosporin-resistant Enterobacteriaceae in a bacterial population exposed to a cephalosporin antimicrobial in the gut of a pre-ruminant or ruminant animal. Preferably the cephalosporin-resistant Enterobacteriaceae is E. coli. Preferably the carvacrol and thymol composition comprises oregano essential oil. The cephalosporin antimicrobial is preferably a third or fourth generation cephalosporin. An animal feed, feed supplement or feed ingredient comprising the carvacrol and thymol composition is also provided.

Description

Composition for Reducing Antimicrobial Resistance Antimicrobial resistance is a global health concern, with many bacteria now developing the ability to grow in the presence of antimicrobial drugs designed to prevent or limit their growth. The use of antibiotics in animals is a contributing factor to this problematic increase in antimicrobial resistance.

Antibiotics are commonly used in agricultural industries and the use is particularly prevalent in the dairy industry where dairy cows are commonly treated for mastitis and other diseases.

When antibiotics are administered during a dry period of the lactation cycle, colostrum can still contain antibiotic residues. Similarly, when antibiotics are required to treat infection during lactation, milk will contain antibiotic residues. This "waste milk" is a major by-product of the dairy industry. It cannot be sold for human consumption and so instead, it is commonly fed to calves.

Colostrum and milk contain bacteria that contribute to the progressive microbial colonisation of the ruminant gastrointestinal tract. These bacteria include a number of beneficial microbes for metabolism and immune development but waste milk can contain bacteria that may be harmful to cattle and also humans. It can be processed by pasteurisation and/or acidification to reduce its pathogenic load, but antimicrobial residues remain in the milk, resulting in a low concentration of antibiotics passing into the calf gut, which affects the innate microbial population and facilitates the emergence and spreading of antimicrobial resistance.

In early life, calves depend entirely on liquid feeds of milk or milk replacers. A number of studies have found waste milk to contain significantly higher numbers of bacteria than calf milk replacer or colostrum. Common bacteria found in waste milk include Streptococcus, Enterobacteriaceae, Staphylococcus and E. coli. Microbial load can increase further if waste milk is kept at room temperature, for example between milkings.

Common antibiotics used in the dairy industry include penicillin and cephalosporins. These are sometimes combined with aminoglycosides. Other important antimicrobials include Cefquinome (e.g., Cobactan/Caphaguard), 4th generation cephalosporins, Tylosin (e.g., Tylofen, Tylan), Macrolide, Amoxycillin (e.g., Amoxi-Mast), Penicillin, Streptomycin (e.g., Ubro Yellow), Aminoglycoside, Oxytetracycline (e.g., Terramycin) and Tetracycline.

Third and higher generation cephalosporins select for cephalosporin-resistant Salmonella and E. coli in animals. These third-and higher generation cephalosporins are also one of the few available therapies for serious Salmonella and E. con infections in humans (especially in children).

Feeding of "waste milk" containing antimicrobial residues to calves in the dairy industry is therefore contributing to the dangerous increase in development of antibiotic resistance in bacteria and presents a global problem not only for these animals but also human health.

It would be advantageous to provide a way to reduce the development of antimicrobial resistance in bacteria.

One aspect of the invention provides the use of a carvacrol composition for inhibiting cephalosporinresistant Enterobacteriaceae in a bacterial population exposed to a cephalosporin antimicrobial.

The composition may be for inhibiting an increase in the prevalence of cephalosporin-resistant Enterobacteriaceae in a bacterial population.

The composition may comprise as an active ingredient one or more of thymol, carvacrol and pcymene.

The composition may comprise thymol. The composition may comprise p-cymene.

The cephalosporin-resistant Enterobacteriaceae may be exposed to a cephalosporin antimicrobial present in a milk product or in a lactating animal.

The cephalosporin antimicrobial may comprise an antimicrobial residue.

The antimicrobial residue may be a subinhibitory/sub-lethal concentration of the antimicrobial. This may be lower than the Minimum Inhibitory Concentration (MIC).

Optionally, the method is for inhibiting the increase in the gut of a pre-ruminant or ruminant animal.

In certain embodiments, the bacterial population comprises the gastrointestinal microbiome of a pre-weaned calf.

The method may be for inhibiting cephalosporin-resistant Enterobacteriaceae in a milk product. This may be for inhibiting the increase in a milk product or precurser thereof.

The milk product may comprise milk, colostrum, transitional milk or calf milk replacer.

Optionally, the inhibiting comprises reducing antimicrobial resistance in the gut or milk product.

Optionally, the inhibiting comprises delaying onset of antimicrobial resistance in the gut or milk product.

In certain embodiments, the cephalosporin-resistant Enterobacteriaceae comprise E. coli.

Advantageously, the use may reduce the presence or percentage of E. coli resistant to cephalosporins.

In one embodiment, the composition comprises oregano essential oil.

The cephalosporin may comprise a 15 or 2"d generation cephalosporin.

Optionally, the cephalosporin antimicrobial comprises a 3rd generation cephalosporin or higher. Optionally, the cephalosporin antimicrobial comprises a 4th generation cephalosporin or higher.

Another aspect of the invention provides an animal feed, feed supplement or feed ingredient comprising the composition.

The animal feed, feed supplement or feed ingredient may be for pre-ruminant or ruminant animals. Optionally, it may be for pre-weaned dairy calves.

In certain embodiments the composition or animal feed is for use in a method of inhibiting an increase in the prevalence of cephalosporin-resistant Enterobacteriaceae in a bacterial population exposed to a cephalosporin antimicrobial in the gut of a pre-ruminant or ruminant animal.

Another aspect of the invention provides a method of inhibiting an increase in the prevalence of cephalosporin-resistant Enterobacteriaceae in a bacterial population exposed to a cephalosporin antimicrobial in the gut of a pre-ruminant or ruminant animal by administering a carvacrol and thymol composition to the animal in an effective amount to inhibit growth of the cephalosporinresistant Enterobacteriaceae in the gastrointestinal tract of the animal.

Optionally, the method comprises administering the composition in a dosage of between about 4m1 and 10m1 per day.

The method may comprise administering a dosage of around 10m1 per day. This may be administered for about 10 days.

Optionally, the composition is administered in a milk or milk replacer feed comprising the cephalosporin antimicrobial.

The composition may also comprise a drench. This may comprise the solution diluted in water. The drench may comprise water to composition in a ratio of about 9:1.

In certain embodiments, the composition is administered before the animal is 3 days old.

The composition may be administered to the animal for 10 days or less.

One aspect of the invention provides a kit comprising the composition or animal feed in any of claims and optionally instructions for use.

One aspect of the invention provides the use of a carvacrol and thymol composition for reducing antimicrobial resistance in bacteria.

The use of composition may reduce the proportion of bacteria in a population that are resistant to antimicrobials.

The composition may be an oregano essential oil composition The population may comprise the gastrointestinal microbiome of a ruminant or pre-ruminant animal.

One aspect of the invention provides the use of a carvacrol and thymol composition for delaying onset of antimicrobial resistance in bacteria.

The bacteria may comprise gram negative bacteria. The bacteria may comprise Garnmaproteobacteria The bacteria may comprise Enterobacteriaceae. In one embodiment, the bacteria comprise E. colt The composition may comprise oregano essential oil.

In certain embodiments, the antimicrobial resistance comprises resistance to cephalosporin antimicrobials.

The cephalosporin antimicrobials may be 3td generation cephalosporins or higher. The cephalosporin antimicrobials may be 4th generation cephalosporins or higher.

Another aspect of the invention provides the use of oregano essential oil composition for inhibiting cryptosporidium oocytes in a pre-weaned calf.

Another aspect of the invention provides the use of oregano essential oil composition for increasing the growth rate of a pre-weaned calf.

In certain embodiments, the invention comprises reducing antimicrobial resistance in bacteria in the gut of a ruminant or pre-ruminant animal.

The gut may comprise the gastrointestinal tract.

The use may comprise reducing the presence and/or activity of antibiotic resistant bacteria in the gut of the ruminant or pre-ruminant animal.

In certain embodiments, the invention comprises use of the composition for and a method of reducing antimicrobial resistance in bacteria in a milk composition.

The milk composition may be for feeding to a ruminant or pre-ruminant animal. The animal may be a bovine animal. Optionally, the animal is a calf.

In one embodiment, the animal gut comprises antimicrobial residues from contaminated milk.

One aspect of the invention provides the use of a composition comprising oregano essential oil for delaying onset of antimicrobial resistance in bacteria.

The invention may comprise use for delaying onset of antimicrobial resistance in bacteria and reducing antimicrobial resistance in bacteria.

The composition may further comprise milk and/or colostrum.

Another aspect of the invention provides a milk or milk replacer composition comprising oregano essential oil for reducing antimicrobial resistance and/or delaying onset of antimicrobial resistance in bacteria.

In one embodiment the composition comprises oregano essential oil composition and milk in a ratio of about 1 to 500.

In certain embodiments, the composition comprises more than about 0.05% of oregano essential oil composition, more preferably the composition comprises more that about 0.1% oregano essential oil and more preferably more than about 0.15% oregano essential oil. Even more preferably, the composition comprises about 0.2% oregano essential oil.

The composition may comprise a dietary supplement which may be an animal feed. Optionally, the animal feed comprises 2m1 of oregano essential oil per litre of feed.

In one embodiment the animal feed comprises about 2.5 litres of milk and about 5m1 of oregano essential oil composition.

In one embodiment the animal feed comprises 5 litres of milk and about 10m1 of oregano essential oil composition.

Another aspect of the invention provides a milk or milk replacer composition comprising carvacrol and/or thymol for reducing antimicrobial resistance and/or delaying onset of antimicrobial resistance in bacteria.

The milk may comprise antibiotic residues.

Optionally, the milk replacer is a calf milk replacer. The milk may comprise colostrum.

Another aspect of the invention provides a method of reducing antimicrobial resistance in a pre-ruminant or ruminant animal comprising administering a carvacrol composition to the pre-ruminant or ruminant animal in an effective amount to reduce the number of antimicrobial resistant bacteria in the animal gut.

The carvacrol composition may further comprise and thymol. It may comprise oregano essential oil.

The pre-ruminant or ruminant animal may be fed milk, colostrum, transitional milk or calf milk replacers comprising antimicrobial residues.

Optionally, the animal is a pre-weaned calf.

In one embodiment, the method comprises adding the carvacrol composition to a milk feed. The milk feed may comprise cows milk, colostrum, transitional milk or calf milk replacers. The milk feed may comprise antimicrobial residues.

The milk feed may comprise a calf milk replacer.

In certain embodiments, the milk feed comprises cephalosporin residues.

Optionally, the method comprises adding the carvacrol and thymol composition to the milk feed substantially immediately before feeding the milk feed to the animal.

In one embodiment the method comprises adding the carvacrol and thymol composition to the milk feed 1 or more hours before feeding the animal.

In one embodiment, the method comprises administering the carvacrol and thymol composition to the pre-ruminant or ruminant animal before the pre-ruminant or ruminant animal is about 3 days old.

Optionally, the method comprises administering the carvacrol and thymol composition to the pre-ruminant animal before the pre-ruminant animal is about 2 days old.

Optionally, the method comprises administering the carvacrol and thymol composition to the pre-ruminant animal before the pre-ruminant animal is about 1 day old.

Optionally, the method comprises administering the carvacrol and thymol composition to the pre-ruminant animal from birth.

Optionally, the method comprises administering the carvacrol and thymol composition to the pre-ruminant animal from birth.

In one embodiment, the method comprises administering the carvacrol and thymol composition to the pre-ruminant or ruminant animal for a period of around 10 days.

In one embodiment, the method comprises administering the carvacrol and thymol composition to the pre-ruminant or ruminant animal for a period of less than 10 days.

In one embodiment, the method comprises administering the carvacrol and thymol composition to the pre-ruminant or ruminant animal for a period of less than 5 days.

In one embodiment, the method comprises administering the carvacrol and thymol composition to the pre-ruminant or ruminant animal for a period of between about 4 and 10 days.

In one embodiment the method comprises providing oregano essential oil and milk in a ratio of about 1 to 500 in the composition.

In certain embodiments, the composition comprises more than about 0.05% of oregano essential oil, more preferably the composition comprises more that about 0.1% oregano essential oil and more preferably more than about 0.15% oregano essential oil. Even more preferably, the composition comprises about 0.2% oregano essential oil.

Another aspect of the invention provides an oregano essential oil composition for use in reducing microbial resistance and/or delaying onset of antimicrobial resistance in bacteria wherein the composition is administered to a pre-ruminant animal at a dosage of more than about 5m1 per day.

The composition may be administered to the pre-ruminant animal at a dosage of more than about 10m1 per day.

The composition may be administered to the pre-ruminant animal at a dosage of more than about 5m1 per day.

The composition may be administered to the pre-ruminant animal at a dosage of more than about 4m1 per day.

Optionally, the composition is administered in a milk or milk replacer feed. The milk feed may comprise antimicrobial residues.

Optionally, the composition is administered before the animal is 3 days old. Optionally, the composition is administered to the animal for 10 days or less. Optionally, the composition is administered to the animal for 5 days or less. In one embodiment, the composition is administered to the animal for around 50 days.

In one embodiment, the composition is administered to the animal for around 56 days or until weaning.

The composition, milk feed, use or method may be for modulating the gut microbiota in a pre-ruminant or ruminant animal to inhibit antimicrobial resistance.

In the Figures, which illustrate embodiments of the invention by way of example only: Figure 1 is a bar chart showing the proportion of E. Coli resistant to 4th generation cephalosporin antibiotics in control and experimental groups.

Figure 2 is a line graph showing the proportion of E. Coli resistant to 4th generation cephalosporin antibiotics in control and experimental groups over time.

Figure 3 is a bar chart showing number of cryptosporidia oocysts in control and experimental groups.

Figure 4 is a line graph showing total E coli count over time.

Figure 5 is a line graph showing total Enterococcus count over time.

Material and methods Experimental Holstein calves were recruited to the trial within 48 hours of birth on a rolling basis and divided into two groups (5 calves/group) and assigned to one of two dietary treatments: control (fed waste milk with antibiotic residues) and Essential Oil (Orego-Stim Liquid, Anpario plc, UK; EO); fed waste milk + Oregano EO for the first 10 days of life).

After 10 days EO supplementation all calves received the same diet and were exposed to the same housing and management conditions. Faecal grab samples were collected on day 0, 3, 7, 10, 14 and 21 and then at weaning. All calves were housed in individual pens for first 21 days and then group housed in a pen for remaining period of the study (2 weeks post weaning).

Faecal samples were diluted in sterile phosphate-buffered saline solution and plated on MacConkey and Bile Esculin Azide agar, respectively, to isolate and count E. coli and Enterococcus colonies.

Antibiotics used were from five different classes: penicillin, tetracycline, aminoglycoside, macrolide and 4th generation cephalosporin. A specific concentration of each antibiotic was used to prepared antibiotic-supplemented MacConkey and Bile Esculine Azide agar plates.

Replica plating technique was used to determine the proportion of E. coli colonies resistant to different antibiotics: a moxicillin, oxytetracycline, streptomycin, tylosin and cefquinome (penicillin, tetracycline, aminoglycoside, macrolide and 4th generation cephalosporin class of antibiotics). Total number of Enterococcus colonies resistant to different antibiotics were determined by plating faecal samples directly onto antibiotic supplemented Bile Esculine Azide agar plates.

Data (bacterial count, prevalence of resistant bacteria and average daily gain) were subjected to ANOVA using a General Linear Model (Minitab); the model included treatment and day as fixed effects and calves nested within treatment as a random effect. The model for average daily gain included only treatment as a fixed effect Both groups received 5 litres per day of waste milk, 2.5 litres morning and evening following regular calf feeding protocol on the trial farm (CEDAR). The waste milk was pooled from cows treated with antibiotics for mastitis. EO group calves also received 10m1/day of the Essential Oil liquid added to the milk before each feeding. 5m1 was added to the milk substantially immediately prior to each feeding, morning and night. Thereafter, all calves were fed the same waste milk and starter feed diet up to weaning. Faecal samples were taken on days 0, 3, 7, 10, 14, 21 & weaning at 56 days of age.

Water was provided ad libitum and calves were treated as per the usual husbandry practices on the farm.

Blood samples (10 mL) were collected into non-heparinised tubes (no additive) once from each calf within 72 hrs after birth (after at least one or two colostrum feeding). Calves were weighed at 4 time points; on entry to the study, on day 10 (of the study), at weaning and at the end of the study.

Faecal grab samples were collected from all calves frequently during first 2 weeks on day 0 (before feeding supplement), 3, 7, 10 and 14. After 14 days of enrolment faecal samples were collected 3 more times: day 21, weaning and 2-3 weeks post weaning. All faecal samples were collected directly into sterile containers.

Blood samples were centrifuged at 3,000 xg for 10 min at 4°C to separate serum. Total protein concentration in serum was determined using a veterinary refractometer (Vetlab Suplies, Pulborough, UK). Cryptosporidium eggs in faeces were counted using the floatation technique and McMaster counting chambers.

Results All experimental calves had serum total protein concentration >5.5 g/dL (6.0 to 9.0 g/dL), which indicated adequate passive transfer of immunity to all calves.

As calves grew, faecal count of E. coli (log CFU: 10.0 on day 0 to 6.30 after 2 weeks of weaning) and Enterococcus (log CFU: 9.64 on day 0 to 4.92 after 2 weeks of weaning) declined in both treatment and control calves.

Total counts of Gram positive bacteria in the faeces was not influenced by EC/ supplementation. As such, supplementation of calf diet (waste milk) with oregano essential oil did not influence Enterococcus count in the faeces (log CFU/g faeces: 7.28 vs 6.82; P= 0.19).

Faecal count of Gram negative bacteria (eg E. coil) tended to decrease (log CFU/g faeces: 8.31 vs 8.79; P = 0.10) in calves fed Oregano essential oil compared to control calves fed waste milk containing antibiotic residues (8.58 vs 9.13; P = 0.08).

Proportions of total E. coli colonies resistant to penicillin (61.5 vs 65.5%), tetracycline (54.5 vs 63.2%), aminoglycoside (63.4 vs 68.4%) and macrolide (90.7 vs 81.5%) classes of antibiotics were similar in both essential oil supplemented and control calves. Thus, the proportions of total E. coli colonies that were resistant to penicillin, tetracycline, aminoglycoside and macrolide classes of antibiotics were not affected by treatment. As shown in Figure 4, proportion of E. coil colonies resistant to a moxicillin, oxytetracycline, streptomycin and tylosin was not different between control and EO groups.

Faecal abundance of Gram positive Enterococcus resistant to penicillin, tetracycline, aminoglycoside, macrolide and 4th generation cephalosporin was not affected by supplementation of calf diet with oregano essential oil.

Surprisingly, it was found that the effect of supplementation of calf milk with the oregano essential oil composition had a significant effect on resistance of bacteria to cephalosporin antibiotics. In particular, supplementation of calf diet with oregano essential oil reduced the proportion of total E. coil colonies that were resistant to a 4th generation cephalosporin (oregano essential oil vs control: 12.6 vs 41.2%; P <0.001).

Figures 1 and 2 illustrate the effect of the formulation on resistance to cephalosporin antibiotics. Figure 1 shows the percentage of E coli tested that was found to be resistant to 4th generation cephalosporin antibiotics, from faecal samples taken from both the experimental group and the control group calves. As shown in Figure 1, 41.2% of colonies from the control group were resistant to 4th generation cephalosporin antibiotics, with only 12.6% of colonies from the experimental group exhibiting resistance.

Figure 2 illustrates the effect of the formulation on the emergence of onset of resistance to cephalosporin bacteria over time.

As the calves grew (and particularly following weaning at 56 days) faecal counts of both total and resistant E. coil declined in both groups. Pre-weaning, there was a tendency in the control group for an increase over time in the proportion of E. coli resistant to cephalosporin antibiotics.

As shown in Figure 2 and with reference to Table 1 below, early dietary supplementation with E0 delayed the emergence of resistance to 4th generation cephalosporin in the experimental group (day 7 rather than day 3) and the proportion of E. coil colonies resistant to 4th generation cephalosporin was significantly lower at weaning (day 56) in the faeces of E0 supplemented calves compared with control calves (17 vs 66%; P = 0.04).

Age of calves influenced antimicrobial resistance (AMR) in faecal E. coil irrespective of classes of antibiotics. Proportions of total E. coli colonies resistant to five different classes of antibiotics used declined as calves matured (P < 0.001).

Resistance of E. coil to 4th generation cephalosporin was tended to be influenced by treatment x calf age (P = 0.07) with a prominent effect of early life dietary supplementation of essential oil on faecal count of cephalosporin resistant E. coil at the age of weaning.

Table 1

Cephalosporin Resistant E coli Day Control EO Standard Error 1 Standard Error 2 0 0 0 8.32 8.32 3 1 0 9.3 9.3 7 52.1 26.5 8.32 8.32 49.5 6.8 9.3 8.32 14 49.3 11.4 8.32 9.3 21 60.7 23.1 10.7 10.7 66 16.3 8.32 9.3 78 50.7 17.6 9.2 9.2 With reference to Tables 2 and 4 below, growth rates in the experimental group were increased. Average daily gain during EO supplementation tended to be higher in EO fed calves compared with control calves (597 vs 411 g; P = 0.11).

Table 2

ADG (days 1-10) Control EO SE1 5E2 411 597 69 77 Average daily gain (ADG) in calves fed diet supplemented with oregano essential oil tended to improve during the first 10 days (597 vs 411 g; P = 0.11). However, the effect of essential oil supplementation on ADG disappeared as supplementation stopped and calves matured.

Although, oregano essential oil is known to have some anti-bacterial and anti-fungal properties, anti-parasitic properties are poorly understood. With reference to Tables 3 and 4 below, faecal shedding of Cryptosporidium eggs declined in calves fed essential oil supplemented diet (206 vs 546 epg; P = 0.02). Cryptosporidium oocysts were not detected on day 3 and after day 21.

Table 3

Cryptosporidium Oocysts Control EO SE1 5E2 546 206 148 83.8 As illustrated in Table 4 below, resistance to cephalosporins emerged early in control calves compared to the experimental (EO) group calves (on day 3 in control calves vs on day 7 in essential oil supplemented calves). The onset of emergence of antimicrobial resistance includes any antimicrobial resistance found ie ability of the bacteria to survive in the presence of antimicrobial concentrations at which they cannot ordinarily survive. The use of ECOFFS may be used to determine which isolates are resistance.

Table 4

"\;. "\". *\,... *"*.,\ Experimental 0 Sp Value:4 logCFU/g 8.79 aaa 8.31 0.10 N 564 200 0.02 k Eggs/gram * ki 44.1 12.6 <0.001 k Days 7 ns g/day 411 597 0.11 \ Faecal E. coil Faecal Cryptosporidia E. Coll with AMR 4th Generation Cephalasporins hEmergence of A R iiiii iii N Growth Rate d k L. A further study into the early life modulation of animal gut microbiota and antibiotic resistance showed that feeding the oregano essential oil composition to young calves modulates the gut microbiota such that growth of antibiotic resistant bacteria and parasite colonization is reduced.

This study aimed to investigate the modulation in the gut microbiota and associated changes in antibiotic resistance in calves fed Oregano essential oil (EO).

Ten Holstein bull calves were divided into 2 groups (5 calves/group) and assigned to one of two dietary treatments: control (fed waste milk) and EO (Orego-Stim Liquid, Anpario plc, UK); fed waste milk with EO for the first 10 days of the study) within 48h after birth.

Fecal grab samples were collected on day 0, 3, 10, 21 and at weaning. Replica plating technique was used to determine the proportion of E. colt colonies resistant to a 4th generation cephalosporin (cefquinome). Genomic DNA was extracted, 165 rRNA (V3-V4) was amplified and sequenced using Illumina MiSeq platform. Data were processed in R using DADA2 Pipeline and analyzed using Minitab. Firmicutes and Actinobacteria were two most predominant phyla across all samples.

While faecal abundance of Firmicutes was higher in control calves compared to EO-fed calves (66 vs 44%; P = 0.01), Actinobacteria was more abundant in EO-fed calves than in control calves (39 vs 15%; P = 0.05). Relative abundance of genus Butyricicoccus in the faeces was higher in control calves compared to EO-fed calves (19 vs 9%; P = 0.04). Arthrobacter and Escherichia tended to be less abundant (3.8 vs 3.9%; P = 0.10; 0.4 vs 0.6%; P = 0.05) and Ruminococcus tended to be more abundant in E0-fed calves compared to control calves (2.1 vs 1.5%; P = 0.05).

Feeding EO did not influence overall species richness and evenness. However, both richness and evenness were greater in control calves compared to EO-fed calves on day 3 and 10, but not after day 10. Relative abundance of Arthrobacter was positively correlated to the faecal abundance of cefquinome resistant E. colt (p = 0.56; P < 0.01).

Fecal count of Cryptosporidium eggs tended to increase with increasing abundance of Arthrobacter (p = 0.40; P = 0.09) and decreasing abundance of Ruminococcus (p = 0.40; P = 0.10).

The invention provides new and surprising insight into the colonization of the ruminant gut by antimicrobial resistant bacteria in the early weeks following birth. This is a complex environment, with many reactions taking place during early development and establishment of the microbial community. Supplementation of calf diet (waste milk with antibiotic residues) with essential oil did not lead to increased AMR in gram-negative and gram-positive commensal bacteria in the calf gut. Importantly, Oregano essential oil has the potential, as a dietary supplement, to delay the emergence of AMR or reduce AMR in bacteria present in the calf gut (and/or in waste milk).

A newborn calf's intestinal tract contains very limited bers of bacteria. After birth, colonisatio with bacteria m the environment occurs rapidly. Some milk fed to newborn calves may pass into the developing rumen and affect the microbial community of the rumen, which can have health implications in later life.

Feeding waste milk containing antimicrobial residues to calves is associated with a decrease ire the diversity f bacteria in colon and faeces. Thus the invention provides a formulation for use in influencing 'olonizaticin of the rumen and maintaining diversity in the calf gut microhicita.

The level of residues in the waste milk depends on numerous factors including the type of drug, the dosage and timing of administration relative to the milking, and the route of administration.

It has been found that oregano essential oil (having bioactive compounds carvacrol and thymol) has the potential to reduce the load of opportunistic pathogens such as E. coil and Cryptosporidium in the calf gut, and also improve daily body weight gain. Exposure to the plant bioactive compounds in oregano essential oil in early life of the calf leads to a delay in the onset of emergence of resistance and a significant reduction at weaning in the number of resistant bacteria as compared with the control group.

Although the results refer to the effect of oregano essential oil in milk, antibiotic resistance due to antimicrobial residues in agricultural settings can develop through contaminated water and feeds. Thus, the utility of the invention is not be restricted to milk feeds and may also apply to other feeds and water. Similarly, the invention is not restricted to oregano essential oil, since any composition comprising the main bioactive compound carvacrol found in oregano essential oil, and/or thymol, and/or p-cymene, or another bioactive in oregano essential oil could produce the same effect.

The invention comprises synthetic versions of oregano essential oil or similar oils such as thyme oil, which comprise the same active components.

Administration of the oregano essential oil together with the milk containing antimicrobial residues, or separately, affects the calf microbiome to inhibit the emergence of antimicrobial resistance ie inhibiting proliferation of resistant E coli in the gut. This may be by reducing, preventing or otherwise abrogating resistance to antibiotics. The composition may exert a sensitizing action or affect bacterial virulence factor to inhibit growth or kill the resistant bacteria. It may also provide beneficial effects for the developing immune system of the animals.

The increase in the prevalence of antimicrobial resistant bacteria (ie increase in antimicrobial resistance) can occur because bacteria from the waste milk or colostrum that are already resistant are taken up by the cat or antimicrobial residues in the milk select for resistant variants in the gut microbiota. Selection pressure from the low level (sub-MIC) antimicrobial can amplify the effect of taking up resistant bacteria from the milk or colostrum. Antimicrobial residues may also cause de novo development of AMR by upregulating gene expression and selecting for spontaneous mutations. When a mixed microbiota is exposed to low level antimicrobials, pre-existing resistant mutants will outgrow the susceptible population. These low levels of antimicrobials have been shown to increase homologous recombination rates and horizontal gene transfer and activate integrating genetic elements.

Although the invention has been described with reference to use in bovine animals, the problem of antibiotic resistance and E. coil resistance to cephalosporin antibiotics extends to poultry and swine, seafood and fish. The effect in the pre-ruminant animal can be extrapolated to those species and as such, the invention also applies for use for those species.

Claims (19)

1. Claims 1. The use of a carvacrol and thymol composition for inhibiting an increase in the prevalence of cephalosporin-resistant Enterobacteriaceae in a bacterial population exposed to a cephalosporin antimicrobial.
2. 2. The use according to claim 2, wherein the cephalosporin-resistant Enterobacteriaceae is exposed to a cephalosporin antimicrobial in a milk product.
3. 3. The use according to claim 1 or 2, for inhibiting the increase in the gut of a pre-ruminant or ruminant animal.
4. 4. The use according to claim 1 or 2, for inhibiting the increase in a milk product.
5. 5. The use according to claim 4, wherein the milk product comprises milk, colostrum, transitional milk or calf milk replacer.
6. 6. The use according to any of claims 3 to 5, wherein the inhibiting comprises reducing antimicrobial resistance in the gut or milk product.
7. 7. The use according to any of claims 3 to 5, wherein the inhibiting comprises delaying onset of antimicrobial resistance in the gut or milk product.
8. 8. The use according to any preceding claim, wherein the cephalosporin-resistant Enterobacteriaceae comprise E. coli.
9. 9. The use according to any preceding claim, wherein the carvacrol and thymol composition comprises oregano essential oil.
10. 10. The use according to any preceding claim, wherein the cephalosporin antimicrobial comprises a 3rd generation cephalosporin or higher.
11. 11. The use according to any preceding claim, wherein the cephalosporin antimicrobial comprises a 4th generation cephalosporin or higher.
12. 12. An animal feed, feed supplement or feed ingredient comprising the composition of any of claims 1 to 11.
13. 13. An animal feed according to claim 12, wherein the animal feed, feed supplement or feed ingredient is for pre-ruminant or ruminant animals.
14. 14. A method of inhibiting an increase in the prevalence of cephalosporin-resistant Enterobacteriaceae in a bacterial population exposed to a cephalosporin antimicrobial in the gut of a pre-ruminant or ruminant animal by administering a carvacrol and thymol composition to the animal in an effective amount to inhibit growth of the cephalosporinresistant Enterobacteriaceae in the gastrointestinal tract of the animal.
15. 15. A method according to claim 14, wherein the method comprises administering the composition in a dosage of between about 4m1 and 10m1 per day.
16. 16. A method according to claim 14 or 15, wherein the composition is administered in a milk or milk replacer feed comprising the cephalosporin antimicrobial.
17. 17. A method according to any of claims 14 to 16, wherein the composition is administered before the animal is 3 days old.
18. 18. A method according to any of claims 14 to 17, wherein the composition is administered to the animal for 10 days or less.
19. 19. A kit comprising the composition or animal feed in any of claims and optionally instructions for use.