Antibiotics Related Alteration in Gut Microbiota and Compromised Immunity in Broiler Chickens

Animal health benefits from a stable intestinal microenvironment, for which proper development and functioning of the intestinal microbiota and immune system are essential [1]. The gastrointestinal microbiota has one of the highest cell densities of any ecosystem and in poultry, it ranges from 108 to 1010 bacteria per gram of gut content [2]. Colonization of the gut microbiota in young animals occurs simultaneously with the development of the gut tissues [3,4]. Immediately after hatch, the microbiota, mainly comprising bacteria, co-evolve with their host to form dynamic and unique microbial communities along the intestine and form a synergistic relationship with their poultry host [5,6]. Due to differences in morphology, functionality, metabolic interactions, and microenvironment, regional heterogeneity in community composition is observed along the different gastrointestinal (GI) segments [7,8]. However, the composition of the bacterial communities is believed to be influenced mainly by age, diet, and gut location. However, host genetics, rearing environment, stress, immune status, and interactions within bacterial communities are also important influencing factors [9-13].

It is believed that microorganisms can also directly interact with the lining of the gastrointestinal tract [14]. This may alter the physiology of the tract and immunological status of the bird [15]. During the first week post-hatch, the chicken small intestine grows rapidly and gets readily affected by post hatch feeding. Post hatch feeding thus alters development of gut microflora within the first two weeks post hatch [16]. It is generally known that the diversity of gut microbiota plays essential role in host metabolism, nutrient digestion, growth performance and overall health of the host [17]. Microbial flora is also believed to protect against colonization of the intestines by pathogens and to stimulate the immune response [18].

The widespread use of antibiotics during the livestock production process [19], not only changes the gut micro-ecosystem but also leads to the emergence of pathogenic bacteria resistant to antimicrobials. This issue has seriously threatened animal husbandry and human health [20]. In relation to productivity, it becomes important to have an understanding about their impact on gut microbiota. Phylogenetic analyses of antibiotic resistance genes and analyses of bacterial genome content suggest widespread and rapid distribution of antibiotic resistance genes among hostassociated (especially intestinal) bacteria after ampicillin, penicillin, ciprofloxacin, and carbadox treatments [21]. Antibiotic disruption of commensal microbiomes may remove the protective barrier, leaving the host susceptible to colonization by pathogens from food and other sources [22]. Gene expression experiments have revealed that most antibiotics, including the rifampicin, erythromycin, and tetracycline, at sub inhibitory concentrations, modulate bacterial gene expression [23].

Recent molecular approaches allow studying the biodiversity, composition and growth dynamics of gut microflora through T-RFLP (Terminal restriction fragment length polymorphism) and quantitative PCR (real-time polymerase chain reaction) [24]. In addition, advances in ribosomal DNA-based molecular techniques have made it possible to obtain new information by identifying different bacterial populations in intestinal contents and mucosal samples [25]. These techniques may prove helpful for monitoring the effect of diets and other variables on the microbial communities of the GI tract under commercial conditions [26].

Pyrosequencing approach was used by Choi et al. [27] to investigate the bacterial communities in separate GIT regions of broiler chicken. In this study, the DNA samples extracted from seven different regions along the GIT were subjected to bacterialcommunity analysis by pyrosequencing of the V1-V3 region of 16S rRNA gene. The results revealed that major bacterial phyla in the chicken-gut microbiota included Firmicutes, Proteobacteria, Bacteroidetes, Actinobacteria, and Acidobacteria, but Firmicutes were mostly dominant (67.3±16.1% of the total sequence reads identified). This study indicated further that microbial communities of main segments in the chicken GIT were distinctive according to both individuals and the different segments of GIT, but their stability was maintained along the GIT [27].

Another recent study [28], elaborated and confirmed the understanding of the chicken intestinal microbial composition. Microbiota inhabiting five different intestinal locations (duodenum, jejunum, ileum, cecum, and colon) of 42-day-old broiler chickens was detected through 16S rRNA gene sequence analysis. As a result, 1,502,554 sequences were clustered into 796 operational taxonomic units (OTUs) at the 97% sequence similarity value and identified into 15 phyla and 288 genera. Firmicutes, Bacteroidetes, Proteobacteria, Actinobacteria, and Cyanobacteria were the major microbial groups and Firmicutes was the dominant phylum in duodenum, jejunum, ileum and colon accounting for > 60% of sequences, while Bacteroidetes was the dominant phylum in cecum (>50% of sequences), but had less occurrence in the other gut sections [28].

Gut microbiota development studies in chickens by sequencing 16S rRNA gene and T-RFLP revealed that complexity of gut microbiota increased gradually with age [29], and that the most dramatic development occurred during the first week of life [30]. Likewise, it was shown that complexity of chicken microbiota gradually developed from day 1 to day 19 and among younger birds, there was greater individual variation in microbiota composition as compared to older birds from the same flock [31]. It has been also established that changes in microbial colonization in early life (the first 2wk post hatch) influence the functioning of the adult gut in broiler chickens [1].

Short-term (24h) early life exposure of day 1 old broiler chickens to orally administered amoxicillin has been shown to affect microbial colonization, mucosal gene expression and intestinal immune development over a period of 2 weeks. These data support that early life colonization of the gut by microbiota is an important driver of immune development and/or immune programming [32]. The effect of dietary supplementation with a combination of avilamycin and salinomycin on the bacterial community in the ileum of broiler chickens at different ages (7,14,21, and 35 days) was studied using PCR with denaturing gradient gel electrophoresis (DGGE) analysis and bacteriological culture. Enumeration of bacteria by culture demonstrated an effect of antibiotics on the total Lactobacillus population and C. perfringens population in ileal contents of broilers. Both PCR-DGGE and bacterial counts revealed that the composition of the microflora was age dependent and influenced by dietary antibiotic supplementation [33].

Caecal microbiota has been characterized by sequencing of bacterial 16S rRNA gene amplicons. In 15-day-old Cobb broiler, in which zinc bacitracin and avilamycin were administered over a 10-day period. However, avilamycin produced no effect on growth performance and exhibited little significant disturbance in the microbiota structure. On the other, zinc bacitracin reduced the feed conversion ratio (FCR) in treated birds, changed the composition and increased the diversity of caecal microbiota by reducing the dominant species. Avilamycin only produced minor reductions in the abundance of two microbial taxa, whereas zinc bacitracin produced relatively large shifts in a number of taxa, primarily Lactobacillus species [34].

Administration of antibiotics later in life (day 15 to day 20 post hatch) in broiler chicken have also studied the microbiota and immune parameters. Chickens received enrofloxacin through drinking water from 15d post hatch. Before and at 6, 16, and 27d after start of the administration of antibiotics, the composition of the microbiota in the jejunum was determined using a 16S ribosomal RNA gene-targeted DNA microarray, the CHICKChip. The results of this study indicated that the antibiotics affect the composition of microbiota and immune parameters in older birds only temporarily [35]. Neumann & Suen [36] compared the effects of two in-feed antibiotics i.e. virginiamycin and bacitracin methylene disalicylate (BMD), which are typically used by commercial poultry producers in the United States, on the chicken gastrointestinal microbiota. Quantitative PCR and 454 pyrosequencing of the V6-V8 region of the 16S rRNA gene were employed to examine the bacterial microbiota and Clostridium perfringens, respectively, in the jejunum and caecum of market-age broiler chickens. Their results suggested that virginiamycin had a more pronounced impact on broiler gastrointestinal tract bacterial communities, relative to BMD, and it manifested primarily through significant enrichments in the genus Faecalibacterium in the caecum and a distinct population of Lactobacillus [36].

The effects of avilamycin, zinc bacitracin, and flavophospholipol on broiler gut microbial community colonization and bird performance in the first 17 days post hatch were identified by T-RFLP. Significant differences were found in the gut microbiota associated with gut sections, dietary treatment, and age. Results also showed that interbird variabilities in ileal bacterial communities were reduced (3 to 7 days posthatch) in chicks fed with feed containing antimicrobial agents, while avilamycin and flavophospholipol had the most consistent effects on gut microbial communities. Identification of key bacterial species influenced by antimicrobial-supplemented feed immediately post hatch may therefore assist in the formulation of diets that facilitate beneficial gut microbial colonization and, hence, the development of alternatives to current antimicrobial agents in feed for sustainable poultry production [37].

In our laboratory, we administered enrofloxacin, a potent veterinary antibiotic, to broiler chicken (1 and 15 days) at various doses for a period of 7 days. In addition to microflora, the antibiotic radically affected the delicate balance of micro- and macro minerals in the gut tissues and as well other organs [38]. Considering our results, future studies as to how altered mineral composition as a result of antibiotics use affects the microbiota and its performance should be conducted. Dietary interventions at young age, such as the usage of (pre) starter feeds, prebiotics, probiotics and antibiotics, are considered to affect the crosstalk between microbiota and host mucosal cells in the intestinal tract, which may result in a change of immune development [39,40]. Ban on use of antibiotics as growth promotors in the European Union (EC Regulation, No. 1831/2003), and their potential restriction in other countries, has resulted in a reduction of animal performance and in an increase of enteric pathologies [41]. As a consequence, non antibiotic feed additives are a subject of increasing interest [42]. Prebiotics are defined as non-digestible dietary compounds that modulate the composition and/or activity of gut microbiota, conferring a beneficial effect on the host [43]. Successful productive results have been reported in poultry by using dry whey powder [44] and inulin [45].

In this scenario, it is clear that the antimicrobials disrupt the gut microbiota population. Thus, under these conditions, prebiotics may be selectively used as substrate for the growth of beneficial bacteria. Further work should focus not only the caecal bacterial profile in response to additive supplementation at some time points of the broiler lifespan, but should also include the characterization of different sections of the upper gastrointestinal tract. It would also be relevant to investigate, through metagenomics, the functional properties of the microbiota when broilers are fed with different non-antibiotic additives. Subsequently, Next-generation highthroughput sequencing (HT-NGS) technologies have been developed to overcome the limitations of first-generation technology that included higher speed, less labor, and low costs. Various platforms that have been developed include: sequencing-by-synthesis 454 Life Sciences, Illumina (Solexa) sequencing, SOLID sequencing (among others), and the Ion Torrent semiconductor sequencing technologies that use different detection principles. As technology advances, progress that has been made toward third generation sequencing technologies is being reported. This includes Nanopore Sequencing and real-time monitoring of PCR activity through fluorescent resonant energy transfer. The advantages of these technologies include scalability, simplicity, with increasing DNA polymerase performance and yields, being less error prone, and economically cost-effective with the eventual goal of obtaining realtime results. These technologies can be directly applied to improve poultry production and enhance food safety. For instance, sequencebased (determination of the gut microbial community, genes for metabolic pathways, or presence of plasmids) and function-based (screening for function such as antibiotic resistance, or vitamin production) metagenomic analysis can be highly informative. Gut microbialflora/communities of poultry can be readily sequenced to determine the changes that affect health and disease along with efficacy of methods to control pathogenic growth [46].

In conclusion, previous research has provided ample evidence that the administration of various antibiotics, at different doses regimes, has a clear impact on microbiota composition and performance measures in broiler chicken of different age groups. A detailed knowledge of broiler gut microbiota colonization and succession immediately post hatch and later in age, and how these bacteria influence bird productivities, are essential to understand impact of different generations of antibiotics for variable time periods. Moreover, studies on the effect of altered microbiota post antibiotics usage on the immune system of birds are scant. It is therefore pertinent that future studies should take this aspect into consideration. Further research is also needed to understand the effects resulting from contrasting concentrations of antibiotics, particularly by looking into possible interactions between the concentration of antibiotics, the age of the birds, the dietary treatment period and the pre-existing microbiota. Such kind of knowledge will assist in beneficial manipulation of gut microbiota via diet or additives or isolating organisms that might be used as probiotics. This would also help to develop high-productivity strategies in the poultry sector that could rely more on the use of probiotics and less on in-feed antibiotics.

1. Wisselink HJ, Cornelissen JBWJ, Mevius DJ, Smits MA, Smidt H, et al. (2017) Antibiotics in 16-day-old broilers temporarily affect microbial and immune parameters in the gut. Poult Sci 96(9): 3068-3078.
2. Apajalahti J, Kettunen A, Graham H (2004) Characteristics of the gastrointestinal microbial communities, with special reference to the chicken. World’s Poultry Science Journal 60(2): 223-232.
3. Lu J, Idris U, Harmon B, Hofacre C, Maurer JJ, et al. (2003) Diversity and succession of the intestinal bacterial community of the maturing broiler chicken. Appl Environ Microbiol 69(11): 6816-6824.
4. Chung H, Pamp SJ, Hill JA, Surana NK, Edelman SM, et al. (2012) Gut immune maturation depends on colonization with a host-specific microbiota. Cell 149(7): 1578-1593.
5. Brisbin JT, Gong J, Sharif S (2008) Interactions between commensal bacteria and the gut-associated immune system of the chicken. Anim Health Res Rev 9(1): 101-110.
6. Simon K, Verwoolde MB, Zhang J, Smidt H, de Vries R, et al. (2016) Longterm effects of early life microbiota disturbance on adaptive immunity in laying hens. Poultry science 95(7): 1543-1554.
7. Yeoman CJ, Chia N, Jeraldo P, Sipos M, Goldenfeld ND, et al. (2012) The microbiome of the chicken gastrointestinal tract. Animal Health Research Reviews 13(1): 89-99.
8. Choi JH, Kim GB, Cha CJ (2014) Spatial heterogeneity and stability of bacterial community in the gastrointestinal tracts of broiler chickens. Poult Sci 93(8): 1942-1950.
9. Wei S, Morrison M, Yu Z (2013) Bacterial census of poultry intestinal microbiome. Poult Sci 92(3): 671-683.
10. Yeoman CJ, Chia N, Jeraldo P, Sipos M, Goldenfeld ND, et al. (2012) The microbiome of the chicken gastrointestinal tract. Animal Health Research Reviews 13(1): 89-99.
11. Choi JH, Kim GB, Cha CJ (2014) Spatial heterogeneity and stability of bacterial community in the gastrointestinal tracts of broiler chickens. Poult Sci 93(8): 1942-1950.
12. Gabriel I, Lessire M, Mallet S, Guillot JF (2006) Microflora of the digestive tract: critical factors and consequences for poultry. Worlds Poultry Science Journal 62(3): 499-511.
13. Yeoman CJ, White BA (2014) Gastrointestinal tract microbiota and probiotics in production animals. Annual Review of Animal Biosciences 2(1): 469-486.
14. Van LP, Mouwen JMVM, Van DKJD, Verstegen MWA (2004) Morphology of the small intestinal mucosal surface of broilers in relation to age, diet formulation, small intestinal microflora and performance. Br Poult Sci 45(1): 41-48.
15. Smirnov A, Tako E, Ferket PR, Uni Z (2006) Mucin gene expression and mucin content in the chicken intestinal goblet cells are affected by in ovo feeding of carbohydrates. Poult Sc 85(4): 669-673.
16. Smirnov A, Perez R, Amit-RE, Sklan D, Uni Z (2005) Mucin dynamics and microbial populations in chicken small intestine are changed by dietary probiotic and antibiotic growth promoter supplementation. J Nutr 135(2): 187-192.
17. Ohimain EI, Ofongo RT (2012) The effect of probiotic and prebiotic feed supplementation on chicken health and gut microflora: a review. International Journal of Animal and Veterinary Advances 4(2): 135-143.
18. Mead GC (2000) Prospects for ‘competitive exclusion’ treatment to control salmonellas and other foodborne pathogens in poultry. Vet J 159(2): 111-123.
19. Pfaller MA (2006) Flavophospholipol use in animals: Positive implications for antimicrobial resistance based on its microbiologic properties. Diagnostic microbiology and infectious disease 56(2): 115- 121.
20. Gong J, Yin F, Hou Y, Yin Y (2013) Chinese herbs as alternatives to antibiotics in feed for swine and poultry production: Potential and challenges in application. Canadian Journal of Animal Science 94(2): 223-241.
21. Gaskins HR, Croix JA, Nakamura N, Nava GM (2008) Impact of the intestinal microbiota on the development of mucosal defense. Clinical infectious diseases 46(Supplement_2): 80-86.
22. Kohanski MA, De Pristo MA, Collins JJ (2010) Sublethal antibiotic treatment leads to multidrug resistance via radical-induced mutagenesis. Molecular Cell 37(3): 311-320.
23. Martínez JL (2012) Natural antibiotic resistance and contamination by antibiotic resistance determinants: the two ages in the evolution of resistance to antimicrobials. Frontiers in Microbiology 3: 1.
24. Deusch S, Tilocca B, Camarinha-SA, Seifert J (2015) News in livestock research-use of Omics-technologies to study the microbiota in the gastrointestinal tract of farm animals. Computational and Structural Biotechnology Journal 13: 55-63.
25. Hiergeist A, Reischl U, Gessner A (2016) Multicenter quality assessment of 16S ribosomal DNA-sequencing for microbiome analyses reveals high inter-center variability. International Journal of Medical Microbiology 306(5): 334-342.
26. Luo YH, Peng HW, Wright ADG, Bai SP, Ding XM, et al. (2013) Broilers fed dietary vitamins harbor higher diversity of cecal bacteria and higher ratio of Clostridium, Faecalibacterium, and Lactobacillus than broilers with no dietary vitamins revealed by 16S rRNA gene clone libraries. Poult sci 92(9): 2358-2366.
27. Choi JH, Kim GB, Cha CJ (2014) Spatial heterogeneity and stability of bacterial community in the gastrointestinal tracts of broiler chickens. Poult Sci 93(8): 1942-1950.
28. Xiao Y, Xiang Y, Zhou W, Chen J, Li K, et al. (2016) Microbial community mapping in intestinal tract of broiler chicken. Poult Sci 96(5): 1387- 1393.
29. Gong J, Yu H, Liu T, Gill JJ, Chambers JR, et al. (2008) Effects of zinc bacitracin, bird age and access to range on bacterial microbiota in the ileum and caeca of broiler chickens. J Appl Microbiol 104(5): 1372-1382.
30. Torok VA, Ophel-KK, Hughes RJ, Forder R, Ali M, et al. (2007) Environment and age: impact on poultry gut microflora. In Proceedings of the 19th Australian Poultry Science Symposium, Poultry Research Foundation, Sydney, New South Wales, Australia pp. 149-152.
31. Crhanova M, Hradecka H, Faldynova M, Matulova M, Havlickova H, et al. (2011) Immune response of chicken gut to natural colonization by gut microflora and to Salmonella enterica serovarenteritidis infection. Infection and immunity 79(7): 2755-2763.
32. Schokker D, Jansman AJ, Veninga G, De Bruin N, Vastenhouw SA, et al. (2017) Perturbation of microbiota in one-day old broiler chickens with antibiotic for 24 hours negatively affects intestinal immune development. BMC genomics 18(1): 241.
33. Mountzouris KC, Palamidi I, Tsirtsikos P, Mohnl M, Schatzmayr, et al. (2015) Effect of dietary inclusion level of a multi-species probiotic on broiler performance and two biomarkers of their caecal ecology. Animal Production Science 55(4): 484-493.
34. Crisol-ME, Stanley D, Geier MS, Hughes RJ, Moore RJ (2017) Understanding the mechanisms of zinc bacitracin and avilamycin on animal production: linking gut microbiota and growth performance in chickens. Applied Microbiology and Biotechnology 101(11): 4547-4559.
35. Wisselink HJ, Cornelissen JBWJ, Mevius DJ, Smits MA, Smidt H, et al. (2017) Antibiotics in 16-day-old broilers temporarily affect microbial and immune parameters in the gut. Poult Sci 96(9): 3068-3078.
36. Neumann AP, Suen G (2015) Differences in major bacterial populations in the intestines of mature broilers after feeding virginiamycin or bacitracin methylene disalicylate. J Appl Microbiol 119(6): 1515-1526.
37. Torok VA, Allison GE, Percy NJ, Ophel-KK, Hughes RJ (2011) Influence of antimicrobial feed additives on broiler commensal posthatch gut microbiota development and performance. Appl Environ Microbiol 77(10): 3380-3390.
38. Ayesha R, Irfan Z (2015) Effect of orally administered enrofloxacin on tissue trace metals concentration in broiler chicken. Oral presentation on PSA Annual Meeting 27th - 30th July 2015, Galt House Hotel Kentucky, USA.
39. Nauta AJ, Amor KB, Knol J, Garssen J, Van der BEM (2013) Relevance of pre-and postnatal nutrition to development and interplay between the microbiota and metabolic and immune systems. Am J Clin Nutr 98(2): 586S-593S.
40. Mulder IE, Schmidt B, Lewis M, Delday M, Stokes CR, et al. (2011) Restricting microbial exposure in early life negates the immune benefits associated with gut colonization in environments of high microbial diversity. PLoS One 6(12): e28279.
41. Dibner JJ, Richards JD (2005) Antibiotic growth promoters in agriculture: history and mode of action. Poult sci 84(4): 634-643.
42. Roberts T, Wilson J, Guthrie A, Cookson K, Vancraeynest D, et al. (2015) New issues and science in broiler chicken intestinal health: Emerging technology and alternative interventions. Journal of Applied Poultry Research 24(2): 257-266.
43. Bindels LB, Delzenne NM, Cani PD, Walter J (2015) Towards a more comprehensive concept for prebiotics. Nature reviews Gastroenterology & hepatology 12(5): 303-310.
44. Pineda-QC, Atxaerandio R, Zubiria I, Gonzalez-PI, Hurtado A, et al. (2017) Productive performance and cecal microbial counts of floor housed laying hens supplemented with dry whey powder alone or combined with Pediococcusacidilactici in the late phase of production. Livestock Science 195: 9-12.
45. Velasco S, Ortiz LT, Alzueta C, Rebolé A, Treviño J, et al. (2010) Effect of inulin supplementation and dietary fat source on performance, blood serum metabolites, liver lipids, abdominal fat deposition, and tissue fatty acid composition in broiler chickens. Poult Sci 89(8): 1651-1662.
46. Diaz-SS, Hanning I, Pendleton S, D’Souza D (2013) Next-generation sequencing: the future of molecular genetics in poultry production and food safety. Poult Sci 92(2): 562-572.