title: Impact of zinc on the gut bacterial microbiota and enterotoxigenic E. coli pathotypes causing post-weaning diarrhoea in porcine populations; an in vitro study
Creators: name: Munsi, Md Nuruzzaman
Publisher: name: University of Reading
Year: 2025

description: Zinc oxide provides a positive effect on the intestinal microflora of pigs in controlling 
or preventing diarrhoea caused by both enterotoxigenic and Shiga-toxin-producing E. coli 
pathotypes during weaning, but it remains unclear how zinc controls or prevents gut 
pathogen infection. This study investigated the effects of both therapeutic and prophylactic 
zinc levels on the gut microbiota of pre-weaned piglets to understand how zinc treatment 
overcomes post-weaning diarrhoea (PWD) in pigs. An in vitro gut model approach was 
employed where faecal specimens from piglets were inoculated into batch gut culture 
vessels (n=36) in five runs. Moreover, cultivations were performed to determine the specific 
effects of zinc on E. coli laboratory F4 and F18 strains – the most common causal agents of 
postweaning diarrhoea in swine populations. For each inoculum in the case of a therapeutic 
zinc trial, three conditions were maintained: the control, and zinc treatment (13.2 mM) using 
piglet donors receiving dietary iron (FeSO4 at 150 mg/kg body weight) or piglet donors 
receiving intramuscular iron regime (Iron Dextran® at 200 mg iron/piglet) in addition to 
dietary iron. For each inoculum in the case a of sub-therapeutic zinc trial, four conditions 
were employed: a non-treatment (control) and three zinc treatments (0.26, 0.52 and 0.78 
mM) reflecting prophylactic zinc levels (piglet donors received identical iron regimes). 
Samples were harvested at 0, 8, 24 and 48 hours for analysis by flow-cytometry 
fluorescence in situ hybridisation (Flow-FISH), short chain fatty acids (SCFAs) analysis, 
elemental assay and microbiota community profiling. Interestingly, the Flow-FISH results 
revealed that the therapeutic zinc level caused a minimum 100-fold reduction in gut bacterial 
population levels with no significant difference between high and low iron treatment groups 
at a 5% significance level. In contrast, the prophylactic zinc levels (0.52 and 0.78 mM), at 24 
h, caused a significant reduction (~60-fold) in gut bacterial population levels without a 
significant difference between 0.52 and 0.78 mM at a 5% significance level. However, 0.26 
mM zinc resulted in only ~8-fold decline. The Flow-FISH results are supported by the 
findings of SCFAs data revealing no production at 0.52-13 mM zinc. However, zinc addition 
resulted in a 20-fold lower production of SCFAs than that noticed in the absence of added 
Zn. The variations in total SCFA production induced by Zn treatment were highly significant 
(Student’s t test, p = 0.001) at 24 h in the cases of both high and low iron regimes. The ICP-OES metal analysis indicated the likelihood of Zn-mediated interference with the distribution 
of transition metals such as Fe, Cu, Ni, Co and Mn within the gut model between the pellet 
and supernatant fractions, in the case of therapeutic zinc, while little effect was exerted by 
the prophylactic zinc. Regarding application of therapeutic zinc, significant differences in Fe 
levels (as compared to the corresponding 0 h) were found in pellets of the Zn-treated 
cultures for the -Fe group at 24 and 48 h (increase from 5.1 to 7.7 µM, p &lt;0.01), 
supernatants of Zn-free cultures in the +Fe group at both 8 and 24 h (reductions of ~0.62 
and ~0.59 fold, p &lt;0.01 and &lt;0.05, respectively) and pellets of the Zn-free cultures in the -Fe 
group at 24 h (fall from 120 to 31.4 µM, p &lt;0.05). Also, significant variations in Cu contents 
(with respect to the corresponding 0 h) were noticed in supernatants of Zn-treated cultures in 
the -Fe group at 48 h (2.5-fold rise; p &lt;0.05), the supernatants of Zn-free cultures in the +Fe 
group at the 24 h (1.7-fold fall, p &lt;0.05), supernatants of Zn-free cultures in -Fe group at 
both 8 h (decease of ~1.7-fold, p &lt;0.05) and pellets of Zn-free cultures in -Fe group at 24 h 
(elevation of ~1.8-fold, p &lt;0.001). Again, significant differences in Ni contents (in regards to 
the corresponding 0 h) were found in pellets of Zn-treated cultures of the +Fe group at 24 h 
(~1.12-fold rise, p ≤0.05), supernatants of Zn-treated cultures for the +Fe group at 8 h (~60-
fold reduction, p =0.05) and supernatants of Zn-free cultures in the -Fe group at 24 h (~1.13-
fold increase, p =0.01). Further, significant differences of Co concentrations (with regards to 
the corresponding 0 h) were found in pellets of Zn-treated cultures of the +Fe group at 24 h 
(~1.06-fold e, p &lt;0.05) and supernatants of Zn-treated cultures of the -Fe group at both 24 
and 48 h (1.3- and 1.4-fold increases, p &lt;0.05 and p ≤0.05, respectively). Furthermore, Mn 
contents (as compared to the corresponding 0 h) showed significant differences in the 
pellets and supernatants of Zn-free cultures in the +Fe group at 48 h (elevation of ~1.34-fold 
at p &lt;0.05 and reduction of ~1.6-fold at p =0.01) as well as pellets of Zn-free cultures in the -
Fe group at 24 h (increase of ~1.94-fold, p &lt;0.05). From the next generation sequencing 
(NGS) data analysis, it was evident that at 24 h, therapeutic Zn resulted in reduced 
proportions of Bacteroidetes (~1.7-fold) and Firmicutes (~1.4-fold), while a raised level for 
Proteobacteria (~3.3-fold). On the contrary, in the case of prophylactic Zn, 0.26 mM caused 
a decrease in Bacteroidetes (~3-fold, FDR p &lt;0.05) and Proteobacteria (~2.5-fold, FDR p
&gt;0.05) but a significant (FDR p &lt;0.01) increase in Firmicutes (~4.6-fold) at 24 h, whereas 
0.52 mM causes a significantly (FDR p &lt;0.001) lower level of Proteobacteria (~48.5-fold)
and insignificantly lower levels of Bacteroidetes (~1.15-fold) but insignificantly higher 
Firmicutes (~6-fold) at the same time point; a similar trend was seen in the case of 0.78 mM 
Zn. These results were reflected at the genus level. In laboratory pure cultures of the F4 and 
F18 E. coli strains using either 96-well microtitre plates or shake flasks, almost complete 
growth inhibition was observed for 0.52 and 0.78 mM zinc. In addition, it was endeavoured to 
determine the effects of Zn on the ETEC pathogens of PPWD in an in vitro gut model co-culture. However, the designed Flow-FISH probes failed to detect the ETEC strains and 
therefore, a Taqman qPCR approach was designed (but not executed due to time 
constraints). To conclude, both therapeutic (13.2 mM) and prophylactic doses of zinc exert 
major effects in reducing the total number of the gut bacterial microbiota of pre-weaned 
piglets as well as F4 and F18 E. coli strains in laboratory cultivations which highlight the 
antibacterial impact of zinc. Therefore, this PhD study has offered novel insights into 
understanding the effects of Zn on the PPWD-causing ETEC pathogens and suggests that 
the nutritional levels of Zn can be applied as prophylactic doses to prevent PWD in porcine 
populations. Also, ZnCl2 appears to be more potent as an antibacterial, while ZnO seems to 
be less potent, possibly because it is less soluble. This suggests that future experiments 
should be performed to compare ZnO and ZnCl2, with/without gastric predigestion.