Airborne bacterial species in indoor air and association with physical factors Translated title: Luftbårne bakterier i indekliamet og sammenhæng med fysiske forhold

 Thank you for the relevant comments.

A fascinating piece of work! The assessment of indoor bacterial growth has been rarely done using air sampling, which makes this study highly valuable.

A few minor comments that could help readers better understand the context of this study.

1. Was the environmental setting (i.e., RH, Temperature, activities carried out by occupants, the opening of windows, level of cleanliness) monitored prior to sampling? Could some of the indoor environmental conditions have affected the results reported?

A1

We have now added: Dust samples and data on indoor air change rate (ACR), Relative humidity (RH) and temperature are obtained from other studies and they were all measured by members of the research groups

Yes indoor conditions are expected to affect the bacterial concentrations and composition, but we have no other measurements than the factors included in the paper. It may however be relavant to include cleaniness and occupations of the occupants. 

We have now included area per occupant for the five homes – but not in the statistical analyses as it is a constant for each home and we have home as a random effect. We have written: All homes were located in the Greater Copenhagen area. Sampling has been performed 6 or 7 times evenly distributed throughout one 1 year in five homes (called 1, 2, 3, 4, 5 with 39, 31, 44, 85, and 66 m²/occupant, respectively)

2. Were the results from the living rooms of the 5 homes tested for studies A and B included in study C? If not, would it be of value to add the results of the 5 living rooms to the 52 samples?

a2 No they are not included in the 52 homes. The samples from the 5 living rooms have been taken during the 4 seasons while the samples in the 52 homes were only sampled once per home. This may make it difficult to pool the data.

3 Could you please mention how many of the 52 living rooms in study C were tested during each season? This would allow readers to see the distribution of samples in each season.

a3 Yes, we have now added the numbers of samples per season.

4 Could you please briefly mention the methods used to monitor the RH, T, ACH in the studies you cited (12, 17, 21)?

a4 We have now added: Dust samples and data on indoor air change rate (ACR), Relative humidity (RH) and temperature (temp.) are obtained from other studies and they were all measured by members of the research groups ^{12, 17, 21, 22}. ACR was measured continuously in the 5 homes (studies A and B) over a 2- to 4-day period following the sampling using GSP (Study A) using the constant concentration methods with a target level of 4 ppm of Freon. The concentration of tracer gas was monitored using an Innova Multi-Gas Monitor Type 1302 and an Innova Multipoint Sampler and Doser 1303 (Lumasense Technologies, Santa Clara, CA). The concentration of tracer gas was separately controlled in the different rooms of each home; for further details see references

In the 24 homes (study C) ACR was measured using the Perfluorocarbon tracer-gas method  ²³. In studies A, B and C, RH and temp. were measured using Tinytag Plus Data (Gemini, UK). In studies A and B, the loggers were placed close to the GSP samplers and set to measure once every 5 min for 15 min between 10:00 and 11:00 am on each sampling day, and average temp and rh were used. In the 5 homes, the average, ACR, RH, and temp. were 1.1/h (0.053-5.6), 54.4% (38.9-73.7), and 23.2ºC (18.3-26.5), respectively. The ACR and temp. were affected by season with highest ACR and temp. in summer followed by spring (ps<0.0001). The RH was affected by season with highest RH in autumn followed by summer (p<0.0001). In study C, loggers measured every 10 min throughout the sampling period and average temp. and rh were used. For the 24 homes in study C, the mean area per occupant, ACR, RH, and temp. were 15.6 m² (5.2-39.2), 0.54/h (0.15-1.23), 29.5% (16.7-44.5), and 22.0ºC (20.3-23.8) ²³. For study D, no physical data were obtained.

5. Please insert the axis title in figure 1 

Done

6 It would be very helpful if we could also see the composition of bacteria with different seasons. This would help the readers to visualize the potential changes in the concentration of the most dominant species with seasons.

a6 There are too many species to visualize the composition by e.g. a heatmap, but based on the bacterial diversity we have visualised the seasonality by RDA plotting – and Figure 3 is now in the paper.

7 Would it be possible to report some key features like the temperatures, RH, and ACH measurements for the tested properties? These would help readers to understand better the relationship between the environmental context and the bacterial readings.

a7 We have now added AER, Temperature and RH as a function of room in Table 1. AER, Temperature and RH for living rooms are now in the method section.

Very interesting study. This paper describes the bacterial species found in Danish homes and their link with environmental factors. The methodology used to collect bacterial species is very clear and well-described. However, further information regarding the various studies mentioned, the monitoring of environmental conditions and calculation methods should also be provided in detail. Specific comments below:

1. On the background page, the author mentioned emerging human pathogens; Are there any of the species found in the study considered critical pathogens to be concerned about?

a1 We have now written:

2 Some of the found species are classified as risk class 2 pathogens which means that can cause human disease but are unlikely to spread to the community, and there is usually effective prophylaxis or treatment available ²⁶; e.g. P. yeei which has caused e.g. 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 Brevibacterium casei and areexpected to derive from the occupants. No risk class 3 pathogens were found.  Some species are described as opportunistic pathogens, e.g. Mo. 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 Also, please describe within the paper the term ‘species composition.

a2 We have now called it diversity.

3 Please specify or discuss the likely sources of the species found. 

a3 We have already written som sentences about in which environements they previously been found and likely sources, and we have now added some few more likely sources:

Many of the found species have previously been found in quite 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 other 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, and these genera were also dominating in indoor air in Hong Kong and China (reviewed in (20)).

and

Some of the risk class 2 pathogens are normal skin related bacteria such as Brevibacterium casei, and is expected to derive from the occupants.

and

In this study, there was a tendency towards the highest bacterial concentrations and humidity in the bathrooms, while the 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 is found in Chinese hom…

The bacterial diversity in the living rooms differed between seasons with especially summer having another bacterial diversity, and 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 Paracoccus yeei was found in the highest concentrations in the spring. The habitat of P. yeei seems not to be well characterized, and in research papers, it is mainly described concerning infections. Thus 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 UV light on bacterial survival.

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 associated negatively 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 associated negatively with area per occupant. Therefore, it may also have human or human activity as 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 associated significantly 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 both in soil, dust  (49), airways, and human skin (50). It has also been found in school air (49) in the air and on hand palms in occupational settings (24).

4 Provide details of the three studies mentioned in the Study design section. The author mentioned Study A, B and C.

a4 We have now written:

All homes were located in the Greater Copenhagen area. Sampling has been performed 6 or 7 times evenly distributed throughout one 1 year in five homes (called 1, 2, 3, 4, 5 with 39, 31, 44, 85, and 66 m²/occupant, respectively) in living rooms, bedrooms, and bathrooms, and in 3 homes also in the kitchens, and in two homes also in the basements using Gesamtstaubprobenahme samplers called GSPs (Study A, in total 127 samples). Homes 1 and 2 had pets; homes 1-4 had natural ventilation while home 5 had mechanical ventilation; home 4 had previously had moisture problems. In the same five homes and during the same periods, sampling was also performed during each season in living rooms using EDCs (ZEEMAN, Alphen, Holland; Study B, 20 samples). In study C, EDC samples were taken in another 24 homes in living rooms; these samples were taken in winter (n=18) or spring (n=6), with one sample per home. In study D, samples were taken in another 28 homes also in living rooms using EDC. These samples were taken in the autumn (3 samples), winter (20 samples), and spring (5 samples).   

Dust samples and data on indoor air change rate (ACR), Relative humidity (RH) and temperature (temp.) are obtained from other studies and they were all measured by members of the research groups ^{12, 17, 21, 22}. ACR was measured continuously in the 5 homes (studies A and B) over a 2- to 4-day period following the sampling using GSP (Study A) using the constant concentration methods with a target level of 4 ppm of Freon. The concentration of tracer gas was monitored using an Innova Multi-Gas Monitor Type 1302 and an Innova Multipoint Sampler and Doser 1303 (Lumasense Technologies, Santa Clara, CA). The concentration of tracer gas was separately controlled in the different rooms of each home; for further details see references: ^{12, 22}.

In the 24 homes (study C) ACR was measured using the Perfluorocarbon tracer-gas method  ²³. In studies A, B and C, RH and temp. were measured using Tinytag Plus Data (Gemini, UK). In studies A and B, the loggers were placed close to the GSP samplers and set to measure once every 5 min for 15 min between 10:00 and 11:00 am on each sampling day, and average temp and rh were used. In the 5 homes, the average, ACR, RH, and temp. were 1.1/h (0.053-5.6), 54.4% (38.9-73.7), and 23.2ºC (18.3-26.5), respectively. The ACR and temp. were affected by season with highest ACR and temp. in summer followed by spring (ps<0.0001). The RH was affected by season with highest RH in autumn followed by summer (p<0.0001). In study C, loggers measured every 10 min throughout the sampling period and average temp. and rh were used. For the 24 homes in study C, the mean area per occupant, ACR, RH, and temp. were 15.6 m² (5.2-39.2), 0.54/h (0.15-1.23), 29.5% (16.7-44.5), and 22.0ºC (20.3-23.8) ²³. For study D, no physical data were obtained.

5 Clarify whether the bacterial species reported in the document were collected in 52 homes and five extra homes or if the five homes are part of the 52. 

a5 They are not part of the 52 homes – we call it ’another’ xx homes etc.; and it should now be clear from the method section.

6 Were RH and ACH recorded in all 52 homes or just five?  

a6 From 5 homes (study A) and the 24 homes (study C) – and this is now better explained in the method section.

7 Describe in detail how relative humidity and temperature were monitored and recorded in each study (A, B, C). Please also report on the period recorded (season, yearly, daily, etc.), intervals, locations of sensors within each room, external conditions, constant or one-off measurement (one per season), etc. 

a7 Please see above

8 Were temperature and relative humidity recorded simultaneously as sampling bacterial species?

a8 Yes -please see above.

9 Please provide details about how Air Change Rate was calculated and the period it refers to.

a9 Please see above.

10 It would also be good to present a summary of the environmental conditions (e.g. RH and temperature levels) recorded and analysed within the documents. In the document, the author indicated that bacteria concentration was affected by low ACH or when RH was highest. However, how high is the highest? What levels of RH are you referring to? Furthermore, for how long they remain high. 

 a10 We have added data on Temp., RH and AER/h i Table 1 for the room-room in Study A.

We have added this:

In the 5 homes, the average, ACR, RH, and temp. were 1.1/h (0.053-5.6), 54.4% (38.9-73.7), and 23.2ºC (18.3-26.5), respectively. The ACR and temp. were affected by season with highest ACR and temp. in summer followed by spring (ps<0.0001). The RH was affected by season with highest RH in autumn followed by summer (p<0.0001). In study C, loggers measured every 10 min throughout the sampling period and average temp. and rh were used. For the 24 homes in study C, the mean area per occupant, ACR, RH, and temp. were 15.6 m² (5.2-39.2), 0.54/h (0.15-1.23), 29.5% (16.7-44.5), and 22.0ºC (20.3-23.8)

The aim of this study is to obtain knowledge about which cultivable bacterial species are present in indoor air in homes, and whether the concentration and diversity of airborne bacteria are associated with different factors. Measurements have been performed for one whole year inside different rooms in 5 homes and once in 52 homes. Within homes, a room-to-room variation for concentrations of airborne bacteria was found, but an overlap in bacterial species was found across rooms. Eleven species were found very commonly and included: Acinetobacter lowffii, Bacillus megaterium, B. pumilus, Kocuria carniphila, K. palustris, K. rhizophila, Micrococcus flavus, M. luteus, Moraxella osloensis, and Paracoccus yeei. The concentrations of gram-negative bacteria in general and the species P. yeei were significantly associated with the season with the highest concentrations in spring. The concentrations of P. yeei, K. rhizophila, and B. pumilus were associated positively with relative humidity, and concentrations of K. rhizophila were associated negatively with temperature and air change rate. Micrococcus flavus concentrations were associated negatively with air change rate. Overall, this study identified species which are commonly present in indoor air in homes, and that the concentrations of some species were associated with the factors: season, air change rate, and relative humidity.