Research Article

Effect of Bacteriophages on Intestinal Colonization of Escherichia coli, Cecal Microbiota Composition, Intestinal Morphology, and Growth Performance in Nursery Pigs from Commercial Pig Farms

Pawinee Kingkan1, Thanathip Supcharoenkul1, Chaowit Rakangthong1, Chaiyapoom Bunchasak1, Komwit Surachat2,3, Wiriya Loongyai1*

1Department of Animal Science, Faculty of Agriculture, Kasetsart University, Bangkok 10900, Thailand; 2Department of Biomedical Sciences and Biomedical Engineering, Faculty of Medicine, Prince of Songkla University, Songkhla 90110, Thailand; 3Translational Medicine Research Center, Faculty of Medicine, Prince of Songkla University, Songkhla 90110, Thailand.

Received | Februay 20, 2023; Accepted | March 20, 2023; Published | May 06, 2023

*Correspondence | Wiriya Loongyai, Department of Animal Science, Faculty of Agriculture, Kasetsart University, Bangkok 10900, Thailand; Email: agrwyl@ku.ac.th

Citation | Kingkan P, Supcharoenkul T, Rakangthong C, Bunchasak C, Surachat K, Loongyai W (2023). Effect of bacteriophages on intestinal colonization of Escherichia coli, cecal microbiota composition, intestinal morphology, and growth performance in nursery pigs from commercial pig farms. Adv. Anim. Vet. Sci. 11(6): 960-967.

DOI | http://dx.doi.org/10.17582/journal.aavs/2023/11.6.960.967

ISSN (Online) | 2307-8316

 

 

Copyright: 2023 by the authors. Licensee ResearchersLinks Ltd, England, UK.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

INTRODUCTION

Gut infection of nursery pigs caused by multidrug-resistant (MDR) pathogenic bacteria immediately after weaning is closely associated with farm and feed management and the available health care system (Bruun et al., 2009; Heo et al., 2013). The infection also imposes a significant decrease in nursery pig growth performance and mortality (Hermann-Bank et al., 2015). Enterotoxigenic E. coli (ETEC) infection in piglets results in growth retardation, diarrhea, and mortality (Bogere et al., 2019). In swine, ETEC F4 and F18 produce enterotoxins that cause diarrheal diseases (Fairbrother et al., 2005, Nagy et al., 2005, Yang et al., 2017).

In general, the disease is commonly prevented or alleviated by adding antibiotics to the feed of weaned piglets for 3–6 weeks, but the use of antibiotics as a feed additive has been banned in the European Union (EU) since 2006. Thailand’s National strategic plan on antimicrobial resistance 2017–2021 was proposed and subsequently endorsed by the Cabinet Resolution on 17 August 2016. Therefore, the Ministry of Agriculture and Cooperatives is responsible for (1) the control and prevention of antimicrobial resistance (AMR) genes in agriculture and animals and (2) the regulation of antimicrobial distribution. In a recent systematic review, Lekagul et al. (2021) reported that in the pig sector of Thailand, antibiotics are commonly used during the suckling and post-weaning stages of production under the regulation of antimicrobial distribution. In addition, antibiotics have led to adverse effects on the beneficial microbiota and the emergence of AMR genes, which is a major threat to human health through drug resistance gene transfer (Ghaisas et al., 2016). Correa-Fiz et al. (2019) reported that antimicrobial treatments change the gut microbiota structure and reduce the health and productivity of piglets. Hence, reducing antibiotic use in commercial pig farms may potentially enhance swine health.

Bacteriophages are proposed as a way to control pathogenic bacteria in the swine farming industry (Kim et al., 2017; Oh et al., 2022). Several studies have shown that bacteriophage therapy can significantly reduce the E. coli and Salmonella spp. counts in the gastrointestinal tract of pigs (Kim et al., 2014; Seo et al., 2018). Lee et al. (2016) found that dietary supplementation of bacteriophages to nursery pigs with post-weaning diarrhea (PWD) caused by ETEC (F4) reduced E. coli adhesion in the ileum and cecum. The effect of bacteriophage dietary supplementation on growth performance has been studied; the inclusion of apramycin and anti-Salmonella bacteriophages did not affect the growth performance of growing pigs compared with the basal diet (Yan et al., 2012). The application of a bacteriophage cocktail can inhibit pathogen growth, and weaned pigs fed a bacteriophage cocktail diet had an increased (p < 0.05) average daily gain (ADG), gain to feed (G: F) ratio, and fecal score (Kim et al., 2017).

Given the lack of studies investigating the effects of bacteriophages as potential substitutes for antibiotic use in piglets on pig farms, we aimed to study the existing situation in commercial pig farms in Thailand. Moreover, we aimed to identify combinations of dietary supplementation with bacteriophages and amoxycillin compared with the use of in-feed colistin and amoxycillin in nursery pigs. We analyzed the diarrhea score and E. coli intestinal colonization to determine the effect on the cecal microbiota composition and growth performance of nursery pigs on commercial pig farms.

MATERIALS AND METHODS

Experimental animal feeding and sampling

The management and design of the experiment followed the animal care rules approved by the Animal Ethics Committee of Kasetsart University (Thailand). The Ethics Committee agreement number is U1-03970-2559. The study was carried out in a commercial pig farm, the study included eight-hundred weaned (6 weeks of age) and even-weight (Large White x Landrace x Duroc) piglets from a commercial pig farm in Saraburi Province, Thailand.

The pigs were assigned into two treatments with 8 replicates per treatment (50 pigs/pen). With an initial body weight of 8.54–8.58 kg, one group was routinely raised (Amox-Co) and the other group was raised without colistin (Amox-Phage). In the Amox-Co group, 300 ppm amoxycillin and 160 ppm colistin were added to the standard diet. This combination of antibiotics is commonly used in Thailand in feed for fattening pigs. In the Amox-Phage group (300 ppm amoxycillin and 1,000 mg bacteriophage cocktail/kg feed), the product contained a cocktail of bacteriophages of Salmonella (S. Typhimurium, S. Enteritidis, and S. Cholerasuis), E. coli (k88, F18, and Stx2e [ETEC] and EPEC, enteropathogenic E. coli), and C. perfringens types A and C. The titre of each bacteriophage in the bacteriophage cocktail was 109 pfu/g bacteriophage cocktail. Piglets were fed the same commercial feed and drank water ad libitum for a period of 6 weeks. Three piglets per pen (n = 12/treatment) were slaughtered, and the gastrointestinal tract was immediately removed. The duodenum, jejunum, ileum, and cecal contents were collected into 5 mL tube and immediately stored at -80 C until further microbial analysis. In these pigs, small sections were taken from the duodenum, jejunum, and ileum for histological examination to examined and analysed villus height and crypt depth.

Growth performance measurement and diarrhea incidence

The pigs were weighed individually at the start and at the end of experiment, recording the average daily feed intake (ADFI) and the ADG. The feed conversion ratio (FCR) was also determined. All piglets were checked for the consistency of fecal samples as an indicator of their health status. Fecal scores were based on diarrhea incidence (Perez et al., 2019), determined by using a consistency score system with three categories.

 

Table 1: DNA primer sequences for analyzed bacterial populations.

Bacteria		DNA sequence	Annealing temperature (C)	Source
Escherichia coli	F	5’-GACCTCGGTTTAGTTCACAGA-3’	60	Rekha et al., 2006
	R	5’-CACACGCTGACGCTGACCA-3’
ETEC (F18)*	F	5’-GGTTTGACCACCTTTCAGTTGAGCAG-3'	63	Seo et al., 2018
	R	5’-TGGCACTGTAGGAGATACCATTCAGC-3'
EHEC (hlyA) **	F	5'-ACGATGTGGTTTATTCTGGA-3'	50	(Kargar and Homayoon, 2015)
	R	5'-CTTCACGTCACCATACATAT-3'

* Enterotoxigenic E.coli, ETEC

**Enterohemorrhagic E.coli, EHEC

 

Table 2: The Effect of Bacteriophage supplementation on the growth performance of nursery pigs.

Items	Amox-Co	Amox-Phage	P-value
Overall, 42 days
Initial body weight, kg	8.58 ± 0.47	8.54 ± 0.48	0.874
Final body weight, kg	24.82 ± 1.17	25.25 ± 1.36	0.510
ADG (g)	396 ± 28	407 ± 28	0.454
ADFI (g)	587 ± 36	606 ± 38	0.326
FCR	1.48 ± 0.037	1.48 ± 0.040	0.742
BWG (kg)	16.25 ± 1.28	16.75 ± 1.38	0.466
Mortality+ Lost (%)	9.71 ± 2.29	7.46 ± 2.60	0.0875

Amox-Co: Amoxicillin + Colistin,

Amox-Phage: Amoxicillin + Bacteriophage.

DNA Isolation and 16S rRNA Gene Sequencing and Data Analysis

Total DNA was extracted from 0.25 gram of cecal content with a commercial extraction kit (QIAamp PowerFecal Pro DNA Kit, Qiagen, Hilden, Germany) in accordance with the manufacturer’s instructions. DNA extracts were subjected to amplicon sequencing using Illumina. The sequencing library was constructed using a MetaVX Library Preparation Kit (GENEWIZ Inc., South Plainfield, NJ). The forward primers 5‘-CCTACGGRRBGCASCAGKVRVGAAT-3’ and the reverse primers 5‘-GGACTACNVGGGTWTCTAATCC-3’ amplicons that cover V3 and V4 regions of the 16s rRNA gene of bacteria were used in the PCR reaction. Next generation sequencing was conducted on the Illumina Miseq/Novaseq Platform (Illumina, San Diego, USA). The resulting 16S-rRNA sequences were imported and analysed by QIIME2 version 2022.11. The q2-diversity plugin was used to perform diversity analyses including alpha- and beta-diversity analyses. In addition, Bray Curtis and unweighted Unifrac metrics were used to construct distance matrices for beta-diversity. Linear discriminant analysis (LDA) effect size (LEfSe) was used to determine significant differences in abundance among different sample groups. LEfSe analysis was conducted using an LDA score of 2.

Real-Time PCR

PCR-based detection was used to confirm the presence/absence of E. coli, ETEC, and EHEC (enterohemorrhagic E. coli). Total DNA was extracted from 0.25 g of the intestinal contents with a commercial extraction kit (QIAamp PowerFecal Pro DNA Kit, Qiagen, Hilden, Germany) in accordance with the manufacturer’s instructions. The DNA was used as a template for real-time PCR with specific primers (Table 1). Amplification was conducted using the HOT FIREPol® EvaGreen® qPCR Mix Plus (Solid Biodye, Inc.) at the following conditions: initially 15 min at 95 C, 30 cycles with 15 s at 95 C, and 60 s at 60 C for product amplification. The number of bacteria were reported as log cfu/g of digesta.

Statistical analysis

Data were subjected to statistical analysis with Student t-test using the SAS software (version 9.4; SAS Institute Inc., Cary, NC) with pigs being the experimental units. P-value ≤0.05 is considered statistically significant.

RESULTS

Growth performance and piglet diarrhea

Table 2 showed the effect of bacteriophages used to replace

 

Table 3: The Effect of Bacteriophage supplementation on fecal score of nursery pigs.

Fecal score	Amox-Co	Amox-Phage	P-value
Week 1	1.38 ± 0.24	1.28 ± 0.14	0.325
Week 2	1.40 ± 0.21	1.38 ± 0.13	0.856
Week 3	1.46 ± 0.14	1.45 ± 0.18	0.842
Week 4	1.57 ± 0.22	1.61 ± 0.24	0.708
Week 5	1.56 ± 0.11	1.42 ± 0.22	0.121
Week 6	1.39 ± 0.05a	1.24 ± 0.13b	0.015

Amox-Co: Amoxicillin + Colistin, Amox-Phage: Amoxicillin + Bacteriophage.

Fecal score: 0= normal faeces, 1= soft faeces, 2 = mild diarrhoea, and 3 = severe diarrhoea a,b Means in a row with no common superscripts differ (p≤0.05)

 

Table 4: The Effect of Bacteriophage supplementation on small intestinal morphology

Items	Amox-Co	Amox-Phage	P value
Duodenum
Villus height (μm)	503.1 ± 105.9	500.3 ± 115.5	0.9127
Crypt depth (μm)	310.9 ± 71.3	327.6 ± 83.3	0.3637
VH:CD ratio	1.63 ± 0.087a	1.55 ± 0.082b	<0.0001
Jejunum
Villus height (μm)	535.7 ± 75.58a	410 ± 75.37b	<0.0001
Crypt depth (μm)	243.6 ± 43.53a	212.4 ± 25.63b	0.0088
VH:CD ratio	2.218 ± 0.14	1.9234 ± 0.15	<0.0001
Ileum
Villus height (μm)	449.4 ± 106.7	417.3 ± 54.23	0.2383
Crypt depth (μm)	226.9 ± 56.22	215.9 ± 43.33	0.4906
VH:CD ratio	1.98 ± 0.16	1.96 ± 0.16	0.6433

Amox-Co: Amoxicillin + Colistin, Amox-Phage: Amoxicillin + Bacteriophage. VH:CD: Villus height (μm)/ Crypt depth (μm)

a,b Means in a row with no common superscripts differ (p≤0.05)

 

Table 5: The Effect of Bacteriophage supplementation on E. coli, ETEC (F18) and EHEC population (log CFU/g) in nursery pigs.

E. coli population		Amox-Co	Amox-Phage	P-value
Total E. coli
Duodenum		10.36 ± 1.08	10.51 ± 0.67		0.635
Jejunum		9.74 ± 0.70	9.80 ± 0.74		0.543
Ileum		10.55 ± 1.12	11.14 ± 1.56		0.079
Cecum		9.83 ± 0.20	9.91 ± 0.44		0.190
ETEC (F18)
Duodenum		ND	ND
Jejunum		ND	ND
Ileum		ND	ND
Cecum		ND	ND
EHEC
Duodenum		ND	ND
Jejunum		ND	ND
Ileum		ND	ND
Cecum		ND	ND

Amox-Co: Amoxicillin + Colistin, Amox-Phage: Amoxicillin + Bacteriophage.

ND = Non-detect

antibiotics (colistin) (Amox-Phage group) in nursery pigs for 6 weeks. The ADG, ADFI, and FCR did not differ compared with the Amox-Co group. Furthermore, the mortality of piglets in the bacteriophage-supplemented group was 7.46%, while the mortality in the Amox-Co group was 9.71%. The effect of bacteriophage supplementation on the fecal score of nursery pigs is shown in Table 3, represented by the diarrhea incidence. The fecal scores were not significantly different between the groups during weeks 1–5. At the end of the experiment (week 6), there were lower fecal scores in the Amox-Phage group (p < 0.05).

Morphology of the small intestine

The effect of bacteriophage supplementation on small intestinal morphology is shown in Table 4. The villus, jejunum, and ileum of the Amox-Co group were significantly higher than the Amox-Phage group (p < 0.05); however, the height of the duodenum was not different between the groups. The crypt depth was significantly higher in the Amox-Co group than in the Amox-Phage group (p < 0.05). Representative images of hematoxylin-eosin-stained small intestine are shown in Figure 1. The Amox-Phage group had a normal appearance with regular intestinal villus structure when compared with the Amox-Co group; there was no obvious damage. In the duodenum, the Amox-Co group had epithelial shedding. In addition, the duodenum of the Amox-Phage group contained more goblet cells (black arrow) than the Amox-Co group.

 

Gut microbiota richness and diversity

Overall, 2,171,459 quality-filtered sequences from the cecum of nursery pigs showed similarity to sequences in the SILVA database. There was no significant difference (p > 0.05) in species richness, as represented by the Observed_species values in the two treatments (Fig. 2A). There was a high Shannon diversity index value in the Amox-Phage group; however, there was no significant difference (p > 0.05) in species diversity compared with the Amox-Co group. The evenness of the cecal microbiota, as evaluated by Pielou’s evenness index, changed but not significantly (p > 0.05) throughout the experiment.

Cecal microbiota composition and Real-Time PCR detection

We observed the effects of dietary bacteriophage supplementation on the cecal microbial composition of piglets at different taxonomic levels. The overall bacterial compositions of the cecal samples at the phylum level are shown in Figure 2B. Firmicutes was predominant in all groups, with a relative abundance of 92.89% and 95.74% in the Amox-Phage and Amox-Co groups, respectively. The relative abundance of Bacteroidetes increased slightly upon the addition of bacteriophages (1.41% in the Amox-Phage group and 0.28% in the Amox-Co group). The Proteobacteria phylum was predominant (p < 0.05) in the Amox-Phage group (0.84%) compared with the Amox-Co group (0.06%). At the family level, LEfSe analysis showed a remarkable difference between the groups in terms of the relative abundances of cecal microbiota (Fig. 2C). The relative abundances of the Lactobacillaceae, Pseudomonadaceae, and Enterobacteriaceae families were significantly higher (p < 0.05) in the Amox-Phage group, while the relative abundances of the Leuconostocaceae, Ruminococcaceae, and Anaerovoracaceae families were significantly higher in the Amox-Co group. There were eight genera with significantly dominant bacterial populations in the Amox-Phage group: Pediococcus, Vagococcus, Enterococcus, Escherichia, Shigella, Pseudomonas, Aerococcus, and Clostridium_sensu_stricto_13.

 

Table 5 showed the quantification/ detection of E. coli, ETEC, and EHEC. The E. coli population in the duodenum, jejunum, ileum, and cecum did not differ between groups (p > 0.05), and ETEC (F18) and EHEC were not detected.

DISCUSSION

In pig production, stressful changes in weaned pigs leads to immune system dysfunctions and decreased piglet health and growth performance (Campbell et al., 2013). The findings of this study are consistent with previous reports (Yan et al., 2012; Oh et al., 2022). Supplementing bacteriophages in the diet of nursery pigs to replace colistin did not affect the ADFI, the ADG, or the FCR. These results are different from those of Kim et al. (2017), who stated that the ADG or the G:F ratio were improved after bacteriophage supplementation. The current study revealed that bacteriophage supplementation did not affect the growth performance of pigs, even when they lacked the effects of colistin. Moreover, pigs fed bacteriophages had a significantly decreased diarrhea incidence compared with the antibiotic group at the late period of the experiment. A fecal score greater than 3 is defined as a clinical sign of diarrhea (Luise et al., 2019; Perez et al., 2019). Kim et al. (2017) reported a reduced fecal diarrhea score in weaned pigs fed bacteriophages; the bacteriophage cocktail resulted in a greater fecal score compared with the control.

We did not infect pigs with ETEC in this study because the experiment was performed on a commercial farm. E. coli, ETEC, and EHEC were quantified in the digesta by using real-time PCR. The ETEC and EHEC were not detected in the control or Amo-Phage groups. Because E. coli occurs as part of the normal intestinal bacterial flora and has hundreds of serotypes, we also measured total E. coli (based on 16S rRNA); the E. coli counts did not differ between the groups. This finding suggests that pigs treated with bacteriophages show similar total E. coli counts in the digestive tract compared with those fed colistin (Yan et al., 2012; Kim et al., 2014).

The high-throughput 16S rRNA sequencing was used to evaluate the abundance and diversity of the cecal microbial community in pigs. The diversity of the Amox-Phage group was higher than that of the Amox-Co group (p > 0.05). These findings indicate that the use of bacteriophages to replace colistin can enhance microbial diversity (Abeles et al., 2016; Correa-Fiz et al., 2019). Overall, long-term bacteriophage supplementation (more than 6 weeks) can improve gut microbial diversity and increase species richness and OTUs. In the cecal gut microbiota of piglets, Firmicutes (92.89%) was the dominant bacterial phylum; however, Proteobacteria, which contains several genera with known animal pathogens—Escherichia, Shigella, Salmonella, and Yersinia—was not be reduced in the Amox-Phage group (colistin replacement). The bacteriophages against Shigella and Yersinia as well as other E. coli and Salmonella serotypes were not included in the bacteriophage cocktail used in this study, and therefore, Proteobacteria was the dominant phylum. Song et al. (2019) stated that a characteristic feature of bacteriophages is their ability to recognize proteins present on pathogenic strains; hence, they can only kill the pathogen that they can recognize. In a recent metagenomic analysis of the fecal microbiota, the genera Prevotella, Sutterella, and Campylobacter, and the family Fusobacteriaceae dominated in diarrheic piglets (Yang et al., 2017); we did not observe these changes in the Amox-Phage group. A few members of Bacteroidetes showed decreased relative abundances in the cecum of the Amox-Co group (control); these bacteria provide protection against pathogens and supply nutrients to the microbiota (Zafar and Saier, 2019).

The Amox-Phage group displayed much higher relative abundances of the families Lactobacillaceae and Enterobacteriaceae. This explains the significant increase in the abundances of the genera Pediococcus and Enterococcus in this group. Lactobacillaceae is considered one of the most promising replacements and, therefore, represents a safe alternative to substitute antibiotics in pigs (Dowarah et al., 2017). According to recent studies, the gut microbiota of healthy piglets has a higher abundance of Lactobacillaceae, the members of which regulate maturation of the innate immune system after weaning (Dou et al., 2017). In addition, Enterobacteriaceae (including the genus Enterococcus) includes commensal gut bacteria isolated for food, feed, and probiotic use (Hanchi et al., 2018). The genus Pediococcus, which increased significantly in the Amox-Phage group, could modulate microbial communities related to the intestinal health of piglets (Wang et al., 2019). In their recent review, Lekang et al. (2022) discussed how amoxicillin disturbs the overall bacterial diversity and richness. This phenomenon may clarify how the bacteriophage cocktail altered the gut microbiota.

The changes in small intestinal morphology are indications of gut health and digestive efficiency of pigs. In the present study, enterocyte proliferation was not accelerated by bacteriophage supplementation. Researchers have observed that the intestinal morphology and proliferation of epithelial cells that increase the villus height are impaired by dominant pathogenic bacteria due to partial pathogen colonization and damage to villus cells (Mourao et al., 2005; Kitamoto et al., 2016). Shorter villi were observed than those found in the jejunum and ileum in the Amox-Phage group, probably because most phages are inhibited by intestinal glycan in the mucus layer (Green et al., 2021). Kim et al. (2017) reported that supplementation with bacteriophages and probiotics increased the villus height in weaned pigs. These results, however, are somewhat different from the findings of our study, where the structural variables of the villus did not change in the Amox-Phage group. A normal intestinal villus structure and no obvious damage were observed in the Amox-Phage group. Moreover, the number of goblet cells increased in the duodenum because the abundance of Lactobacillus in the Amox-Phage group induced an intestinal mucosal immune response, and mucus is secreted by intestinal goblet cells (Tassell et al., 2011; Peng et al., 2021). The density of goblet cells we found in this study are similar to those observed by Jang et al. (2014). Therefore, bacteriophages and antibiotics improve different aspects of nursery pig performance, but supplementation with a bacteriophage cocktail and amoxicillin have a greater ability to reduce diarrhea than supplementation with antibiotics alone.

Conclusion

Dietary supplementation with amoxicillin and a bacteriophage cocktail controlled the E. coli population and altered the abundance of Lactobacillaceae in the cecum. This treatment may also help to improve intestinal morphology, but it does not improve the growth performance of nursery pigs.

ACKNOWLEDGEMENT

The authors gratefully acknowledge a commercial pig farm, Saraburi Province, Thailand as well as Department of Animal Science, Faculty of Agriculture, Kasetsart University, Bangkok, Thailand. This work was funded by Pathway Intermediates (Thailand) Co., Ltd. This work was also supported by Innovation and Technology Assistance Program (ITAP), National Science and Technology Development Agency (NSTDA), Thailand.

conflict of interest

No conflict of interest.

novelty statement

The main objective of this paper is to find out the influence of bacteriophages on the diarrhea score and E. coli intestinal colonization, cecal microbiota composition and growth performance, performance in commercial pigs is published for the first time.

authors contribution

KP - carried out the experiment, preparation of an article for publication. ST – performed the study of intestinal microbiota.

RC - designed the model, verified the analytical methods, helped supervise the project. BC - helped supervise the project. SK – Microbiota data analysis. LW- supervised the project, preparation of an article for publication.

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