China is the largest egg producer, accounting for approximately 40% of the global share in 2020, and egg production continues to grow worldwide (Dai et al., 2022). Many frequent animal feeding operations may produce high concentrations of bioaerosols, which are generally defined as aerosolized particles containing microorganisms (Heederik et al., 2007; Jiang et al., 2022; Millner, 2009; Viegas et al., 2020; Wang et al., 2019). Antibiotics are often used to prevent animal diseases and improve the performance of layers (Bushen et al., 2021). Increasing antibiotic abuse may contribute to the evolution of antibiotic-resistant bacteria, which may significantly reduce the effects of antibiotics and cause an increased number of deaths each year due to aerosol pollution (Castro-Vargas et al., 2020; Gao, 2018; Zeineldin et al., 2019). Exposure can occur through inhalation or skin contact with this bioaerosol (Hoppin et al., 2014; Paton et al., 2015). Some studies have shown that agricultural workers and residents living near farms have a higher risk of respiratory diseases and bacterial infections (Audi et al., 2017; Hedelin et al., 2016; Viegas et al., 2013). The allergic immune reaction of broiler workers was higher than that of nonagricultural workers (Gautam et al., 2018). Specific microbial composition and accumulation of antibiotic resistance genes in the aerosols of poultry farms were two key factors that induced high exposure risks for agricultural workers. On the one hand, with the rapid development of the breeding industry, the development and spread of ARGs in breeding systems have received increasing attention. The levels of ARGs in the feces of chickens are higher than those of pig and cattle farms, and ARG contamination in the feces of layer hens is more severe than that of broilers (Gu et al., 2020; Hubbard et al., 2020). On the other hand, Bacteroides, Corynebacterium and Fusobacterium, which are widely distributed inside poultry farms, have been identified as risk pathogens (Brennan and Garrett, 2019; Hoefer et al., 2021b; Hong et al., 2012; Zafar and Saier, 2021). More importantly, the chicken house has a long operation cycle, and the bacterial diversity and antibiotic resistance genes in different periods may be different. Therefore, it is very important to understand the microbial composition and resistance gene contamination during different laying periods. The egg production of layer hens varies greatly during the laying period, which can be divided into three periods, including the early laying (EL) period, peak laying period (PL) and late laying (LL) period (Zhu et al., 2021). Although antibiotic use is not a sustainable strategy, farmers in some areas still use several antibiotics in the EL period to reduce the immune stress that may occur after repeated vaccination. Antibiotic use is reduced during PL periods, while it is increased again in LL periods due to the rapid decline in egg production caused by problems such as chronic inflammation. These antibiotics often included beta-lactams, tetracyclines, macrolides, and aminoglycosides. Farms that overuse antibiotics are more likely to have problems with resistant bacteria than those with normal usage, and resistant bacteria can persist in the farm environment even after selection pressures have dissipated(Xu et al., 2020).

It has been reported that the main source of microbial pollution in the air of closed livestock and poultry houses is feces (Chien et al., 2011; Clark et al., 1983; Cormier et al., 1990; Donham et al., 1986; Nehme et al., 2008). Until now, most studies of antibiotic resistance in chicken farming have focused mainly on feces (Qiu et al., 2021; Sergeant et al., 2014). Information on antibiotic resistance in the air of layer hen farming systems is still very limited. At the same time, there are also few studies on the microbial composition and potential zoonotic pathogens in the environment of layer houses during different laying periods (Zhu et al., 2021). Indoor air in layer houses is an important reservoir of ARGs (Wychodnik et al., 2020). There is an urgent need for risk assessment of microorganisms and antibiotics in the layer hen house environment to distinguish the environmental impacts of antibiotic use at different periods of eventual egg production.

In this study, bacterial community and resistance genomes in bioaerosols from layer houses at different laying periods were analyzed to obtain taxonomic and compositional changes, as well as their co-occurrence networks in different ecological niches. This can extend our understanding of the link between bioaerosol microbes and ARGs, the key driving forces shaping resistance genes in the air, and facilitate targeted interventions to avoid antibiotic residues and improve egg safety in layer hens.