Table 14.

Effect on faecal shedding of antimicrobial‐resistant bacteria due to feeding calves milk artificially spiked with antimicrobials

	Alali et al. (2004)	Berge et al. (2006)	Thames et al. (2012)	Pereira et al. (2014)
Study design	Experimental study feeding milk spiked with antimicrobials	Experimental study feeding milk spiked with antimicrobials	Experimental study feeding milk spiked inoculated with antimicrobials	Experimental study feeding milk spiked with antimicrobials
Country	US	US	US	US
Farm	Two rooms in a Biosafety Level 2 facility	Calf ranch for heifer replacement, veal and dairy beef	Experimental study feeding milk spiked with antimicrobials	Experimental farm
Treatment	1 group fed spiked milk with oxytetracycline and neomycin/1 group control with no drug residue/both groups challenged with a nalidixic acid resistant E. coli O157:H7 strain after 1–2 weeks	1 group fed spiked milk/one group residue free milk replacer	1 group fed subtherapeutic concentrations of drug residues spiked in milk/1 group fed therapeutic concentrations in spiked milk/1 group fed residue free milk replacer	1 group fed spiked milk with drug residues (DR)/1 group control with no drug residue (NR)
Feeding scheme	Spiked milk fed from week 1 till 8 weeks after inoculation	Spiked milk fed from week 1 until week 4	Subtherapeutic group was fed spiked milk from day 1 until weaning; therapeutic group was fed spiked milk from day 37 for 14 days	Spiked milk fed from week 1 till week 6; 7.6 L per day
Antimicrobials/concentration	Oxytetracycline (200 mg/kg or 2 mg/kg body weight daily) and neomycin (400 mg/kg or 4 mg/kg body weight daily)	Tetracycline hydrochloride and neomycin sulfate at concentrations of 22 mg/kg body weight/day, each	Subtherapeutic group: neomycin sulfate and oxytetracyline hydrochloride at concentrations of 10 mg/calf per day, each. Therapeutic group: same Antimicrobials at concentrations each of 1,000 mg/calf per day	Ceftiofur, penicillin, ampicillin, oxytetracycline at concentrations of 0.1, 0.05, 0.01, 0.3 μg/mL, respectively
Amount calves	18 male calves (9 treated; 9 control)	30 treated calves/60 control calves	28 divided over three groups (treated/therapeutic/control) (amount of calves/group is not provided)	30 male calves (15 treated; 15 control) in three individual studies of each 10 calves (5 treated/5 control)
Method and criteria applied to interpret resultsa	Nalidixic acid resistant E. coli O157:H7 from faecal rectal material isolated on MacConkey agar with 20 μg/mL nalidixic acid and detection after enrichment	E. coli from faecal swabs isolated on MacConkey agar; 909 isolates; antimicrobial resistance by disk diffusion according to NCCLS (2002)	Quantitative PCR resistance genes detected in faecal samples	E. coli from faecal samples isolated on MacConkey agar; 270 isolates; antimicrobial resistance tested by disk diffusion according to CLSI (2008)
Antimicrobial resistance	Amount of E. coli O157:H7 shed was compared between treated and non‐treated group	Resistance to 12 antimicrobials tested	Detection by PCR of the following resistance genes tet(C), tet(G), tet(W),tet(X) (tetracycline resistance), ermB, ermF (MLSs resistance), and sul1, sul2 (sulfonamides resistance)	Resistance to 12 antimicrobials
Time animals tested for faecal shedding	Weekly, started from 2 days after inoculation to 8 weeks after inoculation	Day 1, day 14, day 28	Daily for 7 days starting at week 6 and weekly after weaning till week 12	Weekly from week 1 till week 6
Result	A wide variation in the magnitude and duration of faecal shedding of E. coli O157:H7 was observed among individual calves. There was a significantly (p < 0.001) higher proportion of the calves shedding E. coli O157:H7 in the antimicrobial‐fed group than the no‐antimicrobial group on day 6 and day 10; there was no difference later on during 8 weeks. There was no difference between treatment and control groups in the concentration of E. coli O157 in faeces. A comparison of the duration of faecal shedding between treated and untreated calves showed no significant difference between groups	Treated calves shed more antimicrobial‐resistant E. coli at day 14 and day 28 compared to control calves and treated calves at day 1. Also in control calves a shift to an increased shedding of antimicrobial‐resistant E. coli occurred at day 14 and day 28 compared to day 1	All genes except tet(C) were detected on week 6 and week 12. Tet(C) was not detected in any of the calf samples the control group. No effect of subtherapeutic antimicrobial treatment was established. No effect of breed, gender or week of testing was observed	46% of isolates from control group were not antimicrobial resistant and 37% were resistant to at least 3 antimicrobials. 6% of isolates from treatment group were not antimicrobial resistant and 84% were resistant to at least 3 antimicrobials. For most antimicrobial resistances, a peak of resistance in treated and control calves was observed at 1–2 weeks; afterwards there was a gradual decline in resistance
Main relevant results as reported in abstract	‘The percentage of calves shedding nalidixic acid‐resistant E. coli O157:H7 in the feces in the antimicrobial‐fed group was higher (p < 0.001) early in the study period (d 6 and 10) compared with the control group fed no antimicrobials. There was no difference between treatment and control groups in the concentration of E. coli O157 in feces that were positive at quantifiable concentrations. A comparison of the duration of faecal shedding between treated and untreated calves showed no significant difference between groups. Supplementation of milk replacer with antimicrobials may increase the probability of E. coli O157:H7 shedding in dairy calves, but the effect appeared to be of low magnitude and short duration’	In‐feed antimicrobials were associated with higher levels of multiple AMR in faecal E. coli. In calves not receiving in‐feed antimicrobials, older calves had higher levels of resistance compared to day‐old calves'	‘Relative abundance (gene copies normalized to 16S rRNA genes) of tet(O) was higher in claves fed the highest dose of antimicrobial (therapeutic group) than in the other treatments. All genes, except tet(C) and intI1 were detectable in faeces from 6 weeks onward, and tet(W) and tet(G) significantly increased even in control calves'	‘A significantly greater proportion of E. coli resistant to ampicillin, cefoxitin, ceftiofur, streptomycin and tetracycline was observed in DR calves when compared to NR calves. Additionally, isolates from DR calves had a significant decrease in susceptibility to ceftriaxone and ceftiofur when compared to isolates from NR calves. A greater proportion of E. coli isolates from calves in the DR group were resistant to 3 or more antimicrobial drugs when compared to calves in the ND group. These findings highlight the role that low concentrations of antimicrobial drugs have on the evolution and selection of resistance to multiple antimicrobial drugs in vivo’
Main uncertainties and limitations of the study	It is not clear from the information when the calves were inoculated with E. coli O157:H7 (after 1 week or after 2 weeks). The study was carried out in rooms with a Biosafety Level 2 label, with the consequence of a far from normal bacterial environmental challenge. The calves were artificially inoculated with a nalidixic acid resistant E. coli O157:H7 strain. The strain was also resistant to the antimicrobials supplemented in the milk with resistance to oxytetracycline (MIC > 40 μg/mL) and neomycin (MIC > 40 μg/mL). This resistance could have influenced the results increasing the shedding of this resistance strain due to the decrease in competitive non‐resistant gut microflora. So, it is highly uncertain if the increased faecal shedding would also occur for non‐antimicrobial‐resistant E. coli strains	Because the body weight of the calves is not provided, it is not possible to quantify the actual doses of antimicrobials received by the calves fed with spiked milk. So no quantitative comparison with other studies is possible on the relationship between antimicrobial residue concentration and AMR induction	The culture‐independent assay tested only for a subset of AMR genes and not for the AMR phenotype. Testing was only started at week 6–7 while in most studies a peak of resistance induction is seen at week 1–2. It is not clear how many animals were tested in each treatment group	Limitations concerning the representativeness of the results because the animals were housed in an experimental farm and only a limited amount of animals were included in the study

^aSee Appendix E.