Chlorophyllin Supplementation of Medicated or Unmedicated Swine Diets Impact on Fecal Escherichia coli and Enterococci

Abstract

Considering that certain catabolic products of anaerobic chlorophyll degradation inhibit efflux pump activity, this study was conducted to test if feeding pigs a water-soluble chlorophyllin product could affect the antibiotic resistance profiles of select wild-type populations of fecal bacteria. Trial 1 evaluated the effects of chlorophyllin supplementation (300 mg/meal) on fecal E. coli and enterococcal populations in pigs fed twice daily diets supplemented without or with ASP 250 (containing chlortetracycline, sulfamethazine and penicillin at 100, 100 and 50 g/ton, respectively). Trial 2, conducted similarly, evaluated chlorophyllin supplementation in pigs fed diets supplemented with or without 100 g tylosin/ton. Each trial lasted 12 days, and fecal samples were collected and selectively cultured at 4-day intervals to enumerate the total numbers of E. coli and enterococci as well as populations of these bacteria phenotypically capable of growing in the presence of the fed antibiotics. Performance results from both studies revealed no adverse effect (p > 0.05) of chlorophyllin, antibiotic or their combined supplementation on average daily feed intake or average daily gain, although the daily fed intake tended to be lower (p = 0.053) for pigs fed diets supplemented with tylosin than those fed diets without tylosin. The results from trial 1 showed that the ASP 250-medicated diets, whether without or with chlorophyllin supplementation, supported higher (p < 0.05) fecal E. coli populations than the non-medicated diets. Enterococcal populations, however, were lower, albeit marginally and not necessarily significantly, in feces from pigs fed the ASP 250-medicated diet than those fed the non-medicated diet. Results from trial 2 likewise revealed an increase (p < 0.05) in E. coli and, to a lesser extent, enterococcal populations in feces collected from pigs fed the tylosin-medicated diet compared with those fed the non-medicated diet. Evidence indicated that the E. coli and enterococcal populations in trial 1 were generally insensitive to penicillin or chlortetracycline, as there were no differences between populations recovered without or with antibiotic selection. Conversely, a treatment x day of treatment interaction observed in trial 2 (p < 0.05) provided evidence, albeit slight, of an enrichment of tylosin-insensitive enterococci in feces from the pigs fed the tylosin-medicated but not the non-medicated diet. Under the conditions of the present study, it is unlikely that chlorophyllin-derived efflux pump inhibitors potentially present in the chlorophyllin-fed pigs were able to enhance the efficacy of the available antibiotics. However, further research specifically designed to optimize chlorophyll administration could potentially lead to practical applications for the swine industry.

Keywords: antimicrobial resistance; chlorophyll metabolites; efflux pump inhibitors; medicated feed; zoonotic pathogens.

Figure 1

Least square means of fecal E. coli from the Phase 1 trial where pigs were fed non-medicated starter diets without supplemental chlorophyllin (open circles), non-medicated starter with diets supplemented with chlorophyllin (green circles), ASP 250-medicated starter diet without supplemental chlorophyllin (open squares) or ASP 250-medicated starter diet supplemented with chlorophyllin (green squares). Figure (A) presents E. coli populations enumerated on MacConkey agar containing no added antibiotics. Figures (B,C) present E. coli populations enumerated on MacConkey agar containing penicillin or chlortetracycline, respectively. Values are least squares means from diet by days on diet interactions based on a two-way repeated measures analysis of variance (p = 0.0015, 0.0009 and < 0.0001 for Figures (A), (B) and (C), respectively). Means within each figure associated with unlike lowercase letters differ (p < 0.05) as determined via the LSMEANS statement with Tukey–Kramer adjusted p-values and LINES option of SAS/Stat 15.1.

Figure 2

Least square means of fecal enterococci from the Phase 1 trial where pigs were fed non-medicated starter diets without supplemental chlorophyllin (open circles), non-medicated starter with diets supplemented with chlorophyllin (green circles), ASP 250-medicated starter diet without supplemental chlorophyllin (open squares) or ASP 250-medicated starter diet supplemented with chlorophyllin (green squares). Figure (A) presents enterococci populations enumerated on M Enterococcus agar containing no added antibiotics. Figures (B,C) present enterococci populations enumerated on M Enterococcus agar containing penicillin or chlortetracycline, respectively. Values are least squares means from diet by days on diet interactions based on a two-way repeated measures analysis of variance (p = 0.0511, 0.0009 and 0.1522 for Figures (A), (B) and (C), respectively). Means within each figure, when associated with unlike lowercase letters, differ (p < 0.05) or tended to differ (p > 0.05 < 0.10) as determined via the LSMEANS statement with Tukey–Kramer adjusted p-values and the LINES option of SAS/Stat 15.1.

Figure 3

Least square means of fecal E. coli from the Phase 2 trial where pigs were fed non-medicated starter diets without supplemental chlorophyllin (open circles), non-medicated starter with diets supplemented with chlorophyllin (green circles), tylosin-medicated starter diet without supplemental chlorophyllin (open squares) or tylosin-medicated starter diet supplemented with chlorophyllin (green squares). Figure (A) presents E. coli populations enumerated on MacConkey agar containing no added antibiotics. Figure (B) presents E. coli populations enumerated on MacConkey agar containing tylosin. Values are least squares means from diet by days on diet interactions based on a two-way repeated measures analysis of variance (p = 0.2536, and 0.5530 for Figures (A) and (B), respectively). Means within each figure did not differ (p > 0.05) as determined via the LSMEANS statement with Tukey–Kramer adjusted p-values and the LINES option of SAS/Stat 15.1.

Figure 4

Least square means of fecal enterococci from the Phase 2 trial where pigs were fed non-medicated starter diets without supplemental chlorophyllin (open circles), non-medicated starter with diets supplemented with chlorophyllin (green circles), tylosin-medicated starter diet without supplemental chlorophyllin (open squares) or tylosin-medicated starter diet supplemented with chlorophyllin (green squares). Figure (A) presents enterococci populations enumerated on M Enterococcus agar containing no added antibiotics. Figure (B) presents enterococci populations enumerated on M Enterococcus agar containing tylosin. Values are least squares means from diet by days on diet interactions based on a two-way repeated measures analysis of variance (p = 0.0003 and 0.0298 for Figures (A) and (B), respectively). Means within each figure, when associated with unlike lowercase letters, differ (p < 0.05) as determined via the LSMEANS statement with Tukey–Kramer adjusted p-values and the LINES option of SAS/Stat 15.1.