Table 12.

Effect on faecal shedding of antimicrobial‐resistant bacteria due to feeding calves waste milk in experimental trials

	Aust et al. (2013)	Langford et al. (2003)a	Brunton et al. (2014)
Study design	Experimental study comparing the effect of feeding waste milk and antimicrobial‐free milk to calves	Experimental study comparing the effect of feeding milk from cows treated with penicillin at different concentrations or antimicrobial‐free milk	Experimental study comparing the effect of feeding waste milk and milk replacer to calves
Country	Germany	Not specified	UK
Farm	1 dairy farm	Not specified	1 ESBL positive dairy farm known to use cefquinome
Treatment	1 group fed waste milk 1 group pasteurised waste milk/1 group bulk milk/1 group pasteurised bulk milk	1 group fed milk containing different concentrations of penicillin due to dilution of the milk harvested after treating the cow; 1 control group without antimicrobials	1 group fed waste milk/1 group control
Feeding scheme	Week 1 to‐week 6	3 days/week contaminated milk from day 10 till day 40	Week 1 to week 6
Antimicrobials/concentration	Residues in 17/20 waste milk samples as determined by a microbial inhibitory test	Penicillin present at concentrations of 37.5, 75, 150 and 300 μg/kg milk	Mean cefquinome concentration 746 μg/kg (ranging from 390–1,700 μg/kg)
Number of calves	114 in 4 groups: waste milk, pasteurised waste milk, bulk milk, pasteurised bulk milk	31 calves in total (16 males, 15 females); 7 calves in control group, 6 calves in each treatment group	50‐cows in control group; 50 cows in treatment group
Method and criteria applied to interpret resultsb	Escherichia coli and Enterococcus faecalis from faecal swabs on Coliform Agar Chromocult and Enterococci‐Agar ChromoCULT; identified colonies tested by broth microdilution, clinical breakpoint concentrations published by the CLSI, 2008	Released material from faecal swabs cultured on soy tryptase plates with penicillin disk (10 μg). Inhibition zone was measured and compared between groups; differences in resistance/susceptibility were statistically determined	E coli from faecal swabs on CHROMAgar supplemented with CTX, a subset of isolates confirmed by PCR for the presence of CTX‐M ESBL genes
Antimicrobial resistance	Resistance to 25 antimicrobials	Penicillin resistance	Cefquinome resistance
Time animals tested for faecal shedding	Week 1, week 2, week 4, week 6	Day 10 till day 40	Weekly from week 1 till 6 weeks after weaning
Result	From week 2–week 6, a significant higher amount of antimicrobial‐resistant E. coli detected in calves fed waste milk and pasteurised waste milk than in the bulk milk and pasteurised bulk milk groups. At week 1, only 1 resistant E. coli was multiresistant while from week 2 till week 4 all resistant E. coli were multiresistant; at week 6 the % multiresistant E. coli declined. Highest proportion of resistant E. coli found on week 2 (day 14). No difference in antimicrobial‐resistant E. faecalis observed between the 4 groups. No difference between calves fed waste and pasteurised waste milk	In pretreatment period, statistically significant difference between treatment groups. 300 and 150 groups: clear decreased inhibition (increased resistance) in relation to control and pretreatment period; not in 75 and 37.5 groups. Resistance increased in 300 and 150 groups from day 15 (5 days after treatment) till day 40 (30 days after treatment). Resistance induction in 300 and 150 treatment groups remained stable over time	Week 1, week 2.5 and week 3: significant higher AMR E. coli counts in treatment group compared to control group. Not ≠ at start and 6 weeks after weaning (12 weeks of age) between both groups with slower decline in treatment group
Main relevant results as reported in abstract	‘The proportion of resistant E. coli isolates was significantly higher in calves fed waste milk and in calves fed pasteurised waste milk (most pronounced for cephalosporins) than in calves receiving bulk milk. No differences in resistance were found for Enterococcus spp. Thus, the concerns for selecting resistant faecal bacteria by feeding waste milk seem to be justified’	‘Inhibition was greatest for bacteria from calves fed milk with no penicillin and declined as the penicillin dose provided in the milk increased. In conclusion, resistance of gut bacteria to antimicrobials increased with increasing concentrations of penicillin in the milk fed to dairy calves'	‘Calves in the treatment group shed greater numbers of CTX‐M‐positive E. coli than calves in the control group throughout the study, and shedding decreased at a slower rate in the treatment group. There was no difference between calves fed waste milk with antimicrobial residues or calves fed milk replacer in the proportion of E. coli isolates that were CTX‐M‐positive. Shedding of CTX‐M‐positive E. coli persisted for longer in calves fed waste milk with antimicrobial residues and persisted after weaning’
Main uncertainties and limitations of the study	The waste milk consisted of all unsaleable milk and had therefore a not standardised composition. The presence of antimicrobial activity as determined by the used microbial inhibition tests gives no information on the presence of individual antimicrobials and their concentration. Therefore, the study is not informative on the relationship between antimicrobial residue concentration and antimicrobial resistance induction. The study is limited to resistance of E. coli and E. faecalis	The differences and fluctuations in the resistance in the control group and pretreatment period (variation in baseline levels) leads to uncertainty on small effects, e.g. in the lower dose treatment groups The statistical interpretation of the data is not provided. The graphical presentation of the data indicates only a clear difference in resistance for the two highest doses penicillin and not for the lower ones which is not what the authors concluded. Therefore, it is uncertain if the lower penicillin doses (37.5 and 75 μg/kg) would induce resistance in the gut microbiota. The study is limited to penicillin resistance for bacteria cultivated on soy tryptase plates	The waste milk consisted of all unsaleable milk and had therefore not a standardised composition and a concentration of cefquinome ranging from 390–1,700 μg/kg. Therefore, the study is not informative on the relationship between antimicrobial residue concentration and antimicrobial resistance induction. The study is limited to cefquinome resistance in E. coli leaving uncertainty on the resistance development towards other antimicrobials/bacteria present in waste milk

ESBL: extended‐spectrum beta‐lactamases; PCR: polymerase chain reaction.

^aConcentration (μg/kg) of penicillin were calculated by assuming that 1,670 IU penicillin = 1 mg.

^bSee Appendix E.