microorganisms Article Environmental Impact on Differential Composition of Gut Microbiota in Indoor Chickens in Commercial Production and Outdoor, Backyard Chickens Zuzana Seidlerova, Tereza Kubasova, Marcela Faldynova, Magdalena Crhanova, Daniela Karasova, Vladimir Babak and Ivan Rychlik * Veterinary Research Institute, 62100 Brno, Czech Republic; [email protected] (Z.S.); [email protected] (T.K.); [email protected] (M.F.); [email protected] (M.C.); [email protected] (D.K.); [email protected] (V.B.) * Correspondence: [email protected]; Tel.: +420-533331201 Received: 8 April 2020; Accepted: 18 May 2020; Published: 20 May 2020 Abstract: In this study, we compared the caecal microbiota composition of egg-laying hens from commercial production that are kept indoors throughout their whole life with microbiota of hens kept outdoors. The microbiota of outdoor hens consisted of lower numbers of bacterial species than the microbiota of indoor hens. At the phylum level, microbiota of outdoor hens was enriched for Bacteroidetes (62.41 4.47% of total microbiota in outdoor hens and 52.01 6.27% in indoor ± ± hens) and Proteobacteria (9.33 4.99% in outdoor and 5.47 2.24% in indoor hens). On the other ± ± hand, Firmicutes were more abundant in the microbiota of indoor hens (33.28 5.11% in indoor ± and 20.66 4.41% in outdoor hens). Horizontally transferrable antibiotic resistance genes tetO, ± tet(32), tet(44), and tetW were also less abundant in the microbiota of outdoor hens than indoor hens. A comparison of the microbiota composition at the genus and species levels pointed toward isolates specifically adapted to the two extreme environments. However, genera and species recorded as being similarly abundant in the microbiota of indoor and outdoor hens are equally as noteworthy because these represent microbiota members that are highly adapted to chickens, irrespective of their genetics, feed composition, and living environment. Keywords: chicken microbiota; microbiome; caecum; environment; backyard chicken 1. Introduction Chickens in commercial production are hatched without any contact with adult birds, and colonization of their intestinal tract is dependent on environmental sources only. The development of their microbiota is therefore slower than in chicks that have any form of contact with adult hen microbiota [1,2]. The speed of microbiota development is quite important since the level of microbiota development correlates with the chicken’s natural resistance to infection with pathogens like Salmonella or Clostridium perfringens [3–5]. Both the speed and quality of microbiota establishment is therefore key for the production of healthy chickens and, consequently, for improved productivity, reduced need for antibiotic therapy, and increased animal welfare [6,7]. Attempts with administration of microbiota from healthy adults to newly hatched chicks in intensive production systems date back to the 1970s [2]. The concept, termed competitive exclusion due to the exclusion of enteric pathogens from gut colonization, is known in poultry production. Products containing complex gut microbiota obtained by anaerobic culture of fecal microbiota from adult hens, e.g., Aviguard or Broilact, are commercially available. However, hens used as donors of fecal microbiota for the fermentation of competitive exclusion products are hatched in hatcheries, without any contact with microbiota from healthy adult hens. Moreover, to minimize the risk of Microorganisms 2020, 8, 767; doi:10.3390/microorganisms8050767 www.mdpi.com/journal/microorganisms Microorganisms 2020, 8, 767 2 of 11 uncontrolled infection of the donor hens, these hens are kept under highly contained conditions. 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Figure 1. Living environment of indoor and outdoor chickens. Raising of egg layers in commercial Figure 1. Living environment of indoor and outdoor chickens. Raising of egg layers in commercial production in an indoor environment (panel (a)) and egg laying hens in an outdoor, backyard production in an indoor environment (panel (a)) and egg laying hens in an outdoor, backyard environment (panels (b–e)). environment (panels (b–e)). 2. Materials and Methods 2. Materials and Methods 2.1. Experimental Animals 2.1. Experimental Animals Twenty 30–50-week-old hens were obtained from three different egg producing farms using enrichedTwenty battery 30–50-week-old cages for egg hens production. were obtained At any from time three of rearingdifferent or egg egg producing production, farms these using hens hadenriched no access battery to thecages outdoor for egg environment production. andAt any were time kept of inrearing a strictly or egg controlled production, indoor these environment. hens had Anno additionalaccess to the 17 outdoor hens were environment obtained from and sixwere diff kepterent in backyard a strictly poultrycontrolled flocks indoor with environment. the agreement An of additional 17 hens were obtained from six different backyard poultry flocks with the agreement of the owners. These hens were 2–4 years old and were reared