Introduction

Particulate matter (PM), a major component of air pollution (Kelly and Fussell, 2011; Kelly and Fussell, 2012), has a detrimental impact on both human and environmental health (Janssen et al., 2012; Kelly and Fussell, 2012; Thurston and Lippmann, 2015; Xu et al., 2016). Current WHO guidelines set limits for both the 24‐hour and annual mean concentrations of different size fractions of PM. PM 10 , that is, particulate matter with an aerodynamic diameter of less than 10 µm, should be kept below a 20 µg/m3 annual mean, and a 50 µg/m3 24‐hour mean. Stricter limits are set for PM 2.5 as this size fraction is associated with more damaging health effects, and should be kept below an annual mean of 10 µg/m3, and 25 µg/m3 over a 24 hour period. However despite WHO and EU legislation to reduce air pollution, PM levels exceed these recommended guidelines (WHO, 2006, 2016), particularly in industrialised regions (Janssen et al., 2012; Wang et al., 2014).

PM and other air pollutants spread in the atmosphere and cross national boundaries, settling on plants and soils, as well as contaminating fresh and marine water (Forbes et al., 2006; Cattaneo et al., 2010). PM therefore has the potential to affect multiple essential ecosystems globally, as well as having a known major impact on human health and morbidity. PM exposure causes increased respiratory and cardiovascular disease (Faustini et al., 2012; Shah et al., 2015; Thurston and Lippmann, 2015; Costello et al., 2016), and are strongly associated with increased acute respiratory infections, including pneumonia (Brugha and Grigg, 2014; MacIntyre, 2014; Qiu et al., 2014; Chang et al., 2015; Xu et al., 2016). Indeed, air pollution is responsible for an eighth of all global deaths per year (WHO, 2014).

Many studies have shown that inhalation of PM causes host tissue damage, inflammation, oxidative stress and alteration in cardiovascular functioning, as well as significantly impacting the immune response by impairing macrophage function (Host et al., 2007; Lundborg et al., 2007; Kelly and Fussell, 2011, 2012; Heal et al., 2012; Rylance et al., 2015). However, these host‐focused studies do not fully account for all the observations of PM‐related diseases in humans. Importantly, there is a major omission in our understanding of the impact of PM because there have been no studies on the direct impact of PM on the behaviour of bacteria. This is surprising considering that bacteria are directly responsible for respiratory infections, and play a key role in the diversity and functioning of the normal microbiome, which is crucial for maintaining the health of the host. Therefore it is essential to further understand the role bacteria play in the detrimental impacts of air pollution.

A major component of PM is black carbon (BC), a by‐product of fossil fuel combustion. In developed countries diesel exhaust fumes are the major source of BC, whereas in the developing world BC mostly arises from indoor burning of biomass for heat and fuel. BC is a chemically and biologically active pollutant that can generate oxidative stress, induce inflammation, and be mutagenic (Janssen et al., 2012; Butterfield et al., 2015). In 2014, black carbon (BC), a major component of PM, levels ranged from 1 to 7 µg/m3 across the UK, and the country‐wide average was 1.6 µg/m3 (Butterfield et al., 2015). In general, higher concentrations were recorded at the roadside in comparison to other urban environments. However, Europe and North America only account for about 13% of global BC emissions, whereas developing countries are responsible for ∼80%, with the biggest global contributors to BC being China and India (USEPA, 2012; Ni et al., 2014; Wang et al., 2014). BC exposure is strongly implicated in predisposition to respiratory infectious disease, which is particularly damaging to children under 5 years old (Janssen et al., 2012; Brugha and Grigg, 2014).

To address whether bacteria are an unexplained mechanism for BC induced morbidity, we investigated the impact of BC on two model bacterial species; Streptococcus pneumoniae and Staphylococcus aureus. Both are important respiratory tract commensals intermittently carried by a large section of the population without signs of disease as part of the normal microbiome. However they are also globally important human pathogens; S. pneumoniae is the leading bacterial cause of pneumonia, and S. aureus is a significant cause of respiratory and skin and tissue disease (Wertheim et al., 2005; Edwards et al., 2012; Shak et al., 2013).

We report here that BC significantly affects the behavior of S. pneumoniae and S. aureus. Our data show that BC impacts biofilm formation, an essential aspect of bacterial colonisation and environmental survival. Exposure to BC induced significant changes in S. pneumoniae and S. aureus biofilm structure, composition and function. Importantly, BC differentially altered the tolerance of biofilms to proteolytic degradation and multiple antibiotics, increasing S. pneumoniae survival against penicillin, the front line treatment of bacterial pneumonia. Furthermore this work shows that BC does indeed impact bacterial colonization in vivo. In a murine colonisation model, black carbon induced S. pneumoniae to spread from the nasopharynx to the lungs, which is a prerequisite for invasive disease in a susceptible host. Therefore, if extrapolated these data show, for the first time, that air pollution could have a significant effect on human bacterial infection that has been largely overlooked.