Airborne bacteria in indoor environments are confirmed or presumed causal agents of various infectious diseases [1,2]. In addition, airborne bacteria are inflammogenic [3] and seem to be involved in either an increase or decrease in the risk of developing asthma and atopy [4,5]. Indoor work activities such as, for example, bed making [6,7] can aerosolise bacteria, but whether concentrations of airborne bacteria in a home are related to room type is not clear. Concentrations of airborne bacteria are often higher in homes than in offices [8], and since the Covid-19 pandemic, more office work is occurring from homes. Generally, studies comparing the indoor and outdoor levels of bacteria have found that the indoor : outdoor ratios are above 1 [9–14].

Exposure assessment using personal samplers, for example, the Gesamtstaubprobenahme (GSP) (CIS by BGI, Inc., Waltham, MA, USA), actively sampling airborne inhalable dust, is expected to be a good measure of personal exposure [15]. However, the sampling activity may interfere with everyday life in a home, due to, for example, noise of the sampling pump, and the necessity of the presence of a technician; thus only a few studies of this kind have been performed. Instead, bacteria in surface dust are studied; however, the exact age of surface dust is unknown, and some of it may not have been airborne. Alternatively, Electrostatic Dust Collector (EDC) (ZEEMAN, Alphen, Holland) sampling airborne dust by sedimentation on a cloth has been used in homes, offices and social rooms at workplaces [15–19].

Air is an important transmission route for bacteria and therefore it is important to obtain knowledge about which bacteria are present in the indoor air and which factors affect this presence. In one review paper it was suggested that outdoor bacteria from plants may enter the building through ventilation systems, doors, windows, attached to people, pets and other objects, and as a result, affects the indoor concentration level – but also that information on these factors is still not well understood [20].

Potential health risks of bacteria are for many species evaluated at species level, and matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF MS) is revealed as a reliable and useful method for identification of bacteria from the indoor environment [1,18]. Using MALDI-TOF MS for identification, it has been shown that the concentration of indoor Staphylococcus is associated with the indoor air change rate (ACR) and area per occupant indicating that it might be possible to affect the presence of Staphylococcus in indoor air [18]. In contrast, relative humidity (RH) of indoor air was not significantly associated with the concentration of viable bacteria in general [12] and with the commonly present genera Staphylococcus, Bacillus, Kocuria and Micrococcus [18].

The aim of this study is to obtain knowledge about which bacterial species (non-Staphylococcus species) are present in indoor air in Danish homes, and whether the factors: season, ACR, RH and occupants per area affect the concentration of the most abundant bacterial species in living rooms. To obtain knowledge about whether the concentration of bacteria is related to room type we also study room-to-room variation.

In this study, we show that cultivable K. palustris, K. rhizophila, M. luteus, M. flavus, B. pumilus, K. megaterium, K. carniphila, A. lowffii, M. osloensis and P. yeei are common in indoor air in homes in Greater Copenhagen, in addition to the previously found Staphylococcus species [18]. These results were stable and unaffected by the sampling method. Thus, people seem to be exposed to these bacteria via inhalation on daily basis.

Some of the found species are classified as risk class 2 pathogens, which means that they can cause human disease but are unlikely to spread to the community, and there is usually an effective prophylaxis or treatment available [26]; for example, P. yeei, which has caused, for example, keratitis and conjunctivitis [27,28] and B. cereus, which is a food-poisoning agent [26]. Examples of other risk class 2 pathogens found in the homes are: Aerococcus viridans, Acinetobacter lwoffii, Bacillus mycoides, Brevibacterium casei, Enterococcus casseliflavus and several Kocuria species. Some of the risk class 2 pathogens are normal skin-related bacteria such as B. casei and are expected to derive from the occupants. No risk class 3 pathogens were found. Some species are described as opportunistic pathogens, for example, M. osloensis (e.g., in the airways of an elderly patient [29]) and Rhizobium radiobacter (e.g., pneumonia in a cancer patient [30]). An underlying mechanism explaining many health effects of exposure to airborne particles is the ability to induce the formation of reactive oxygen species (ROS) within the airways. There is limited knowledge on the ability of different species to induce ROS production (without infection), but few studies indicate differences at the species level [31,32].

Many of the species found have previously been found in very different environments. The bacteria found in high concentrations, P. yeei, B. megatarium and M. luteus, have previously been found on indoor surfaces [33,34], but also as airborne bacteria in totally different environments such as wastewater treatment plants [35], pigeon coops [36] and on workers’ clothes [37]. We have found many different Bacillus species, and a high species richness of Bacillus has also been found in air samples from different occupational settings [36,38] as well as on indoor surfaces [33]. When considering the bacteria at the genus level, the genera Kocuria, Micrococcus, Bacillus and Paenibacillus were among the dominating ones, and these genera were also dominating in indoor air in Hong Kong and China (reviewed in [20]).

This study found a within-home variation (room-to-room) for concentrations of airborne bacteria. Thus, airborne bacterial concentrations were not uniform throughout the homes during the time of sampling, which is in agreement with a study in homes in the UK [39]. In spite of this, no significant variation was observed for general room type (e.g., bathroom vs. living room) except for a lower concentration in basements. Furthermore, the same bacterial species were found in the highest concentrations in all rooms within the same home. In a study from the USA, bacterial concentrations in cellars were lower than in bathrooms, while cellars, kitchens and bedrooms did not differ significantly [14]. In this study, there was a tendency towards the highest bacterial concentrations and humidity in bathrooms, while bedrooms also had high bacterial concentrations but low temperatures. This may indicate a larger contribution of skin-related bacteria such as Corynebacterium xerosis and Dermacoccus sp. to the airborne bacteria in these rooms. However, it was only a tendency, and the lack of a general effect of room type for bacterial concentration is in accordance with what was found in Chinese homes [40]. This indicates, on the one hand, that the variation in exposure found between rooms may not be attributable to what the rooms are used for in general, or that it is also affected by other factors such as, for example, ACR. In this study, the repeated sampling using GSP samplers in the five homes was done in the daytime while for the most part the occupants were not at home. Previous studies have shown that bacterial concentrations in indoor air are higher in the presence of occupants [21].

The bacterial diversity in the living rooms differed between seasons with summer especially having another bacterial diversity, this might be caused by the high ACR in the summer. For some bacterial species, associations between concentrations in living rooms and seasons were found. Thus, P. yeei was found in the highest concentrations in the spring. The habitat of P. yeei seems not to be well characterised, and in research papers it is mainly described concerning infections. Therefore, we do not know the source of exposure to this bacterium. Paracoccus yeei is a Gram-negative bacterium, and for Gram-negative bacteria in general lower concentrations were found in summer than in winter. This may be related to the impact of ultraviolet (UV) light on bacterial survival.

The two species B. pumilus and K. palustris were found in the highest concentrations in the autumn. These bacteria have previously been found in soil. At the genus level, we have previously observed that Kocuria is present in the lowest concentrations in summer [18]. Kocuria palustris, K. rhizophila and M. flavus were associated negatively with indoor temperature. For the two Kocuria species, this is in accordance with what has previously been found for the genus and in contrast to what is found for the genus Staphylococcus in Danish homes [18] and bacteria in general in Greek homes [41].

Human skin is shed into the indoor air [42], and therefore it could be expected that the concentration of skin-related bacterial species is negatively associated with ACR. The two species K. rhizophila and M. flavus were negatively associated with ACR and thus seem not to enter by open windows or ventilation systems. These species are not described as skin-related bacteria, but K. rhizophila has been found on the skin [43]. Another transmission route to the home environment may be clothing and in particular work clothing from environments with high exposure to bacteria. Thus, a recent study has shown that bacteria accumulate on work clothing in high amounts during a workday and that bacteria may be released from the clothes to the home air; in fact, cultivable K. rhizophila has been found on work clothes together with more than 200 different cultivable bacterial species [37,44]. In Study C, the K. palustris concentration was negatively associated with area per occupant. Therefore, it may also have human or human activity as a source. The habitats of this species are not well described, but it has been isolated from very different environments including human skin [45], workers’ hands [24], human noses [46] and marine algae [47]. In a study about bacterial genera in outdoor air, Bacillus and Acinetobacter but not Kocuria were among the most frequently found genera [48].

Micrococcus luteus was very common in indoor air in this study, and it is described as a skin-related bacterium. Despite that, it was not significantly associated with ACR or area per occupant. Furthermore, the species did not show seasonality. The lack of association between the studied factors and concentrations of M. luteus might be because this species has several sources as it is found in soil, dust [49], airways and on human skin [50]. It has also been found in the air in schools [49] in the air and on hand palms in occupational settings [24].

Across homes and room types within homes, occupants are potentially exposed to some of the same cultivable bacterial species typically including: K. palustris, K. rhizophila, M. luteus, M. flavus, B. pumilus, B. megaterium, K. carniphila, A. lowffii, M. osloensis and P. yeei. Seasonality in bacterial diversity was found, and concentrations of P. yeei were significantly associated with the season. Bacterial concentrations were not uniform throughout the homes investigated, but no significant variation was observed for the general room type except for the lower concentration in the basements. The concentrations of P. yeei, K. rhizophila and B. pumilus were positively associated with RH, and concentrations of K. rhizophila were associated negatively with temp while K. rhizophila and M. flavus were negatively associated with ACR, and K. palustris negatively with area per occupant. Thus decreasing the RH, and increasing the ACR and area per occupant might be a strategy to reduce the exposure to some airborne bacterial species.