Environmental and Structural Factors Associated with Bacterial Diversity in Household Dust Across the Arizona-Sonora Border

We previously reported that asthma prevalence was higher in the United States (US) compared to Mexico (MX) (25.8% vs 8.4%). This investigation assessed differences in microbial dust composition in relation to demographic and housing characteristics on both sides of the US-MX Border. Forty homes were recruited in the US and MX. Home visits collected floor dust and documented occupants’ demographics, asthma prevalence, and housing structure and use characteristics. US households were more likely to have inhabitants who reported asthma when compared with MX households (30% vs 5%) and had significantly different flooring types. The percentage of households on paved roads, with flushing toilets, with piped water and with air conditioning was higher in the US, while dust load was higher in MX. Significant differences exist between countries in the microbial composition of the floor dust. Dust from US homes was enriched with Geodermatophilus, whereas dust from Mexican homes was enriched with Alishewanella and Chryseomicrobium. A predictive metagenomics analysis identified 68 significantly differentially abundant functional pathways between US and MX. This study documented multiple structural, environmental, and demographic differences between homes in the US and MX that may contribute to significantly different microbial composition of dust observed in these two countries.

neighborhoods within Nogales, MX, including a traditionally high SES neighborhood and low SES neighborhood.

Study Population
Overall, 20 homes from TUS, 20 homes from NUS, and 40 homes from NMX (20 high SES and 20 low SES) were recruited into the study. The household and environmental characteristics were analyzed and compared between the countries (US vs MX) as well as between neighborhoods (TUS, NUS, high SES NMX, and low SES NMX). There was no signi cant difference in age or gender of the respondents between the US and MX, but the respondents in the US had signi cantly more years of education (Supplemental Table S1). US households had higher incomes and cleaned their oors less frequently ( Table 1). The source of drinking water was also signi cantly different between homes in MX and homes in the US, with US homes utilizing public tap water (Table 1). US families were signi cantly more likely to have inhabitants with asthma than those in MX (Table 1). There were no signi cant differences in smoking, pets, or number of inhabitants between the US and high SES NMX, but households in low SES NMX had more inhabitants and fewer animals per household (Table 1).

Household Characteristics
Although all cities were close to the US-MX Border and within 100 km of each other, there were striking differences between the homes by country. All the homes in high SES NMX had the same oor plan, as it was a planned subdivision. The percentage of homes with paved roads, ushing toilets, and piped water was signi cantly lower in low SES NMX (Table 2) compared with the other neighborhoods. The homes in the US differed from those in MX in type of structure (Table 2), with 33% of homes in US being apartments or trailers, while 100% of the homes in MX were detached or duplex houses (Table 2). Homes in the US had more rooms and more bathrooms than in MX. All homes in the US had either air conditioning, evaporative cooling, or both, whereas only a few of the homes in MX (10%) had either type of cooling (Table 2). Flooring also differed. Most of the rooms where the dust samples were collected in MX had smooth oors (93%), whereas 42% of samples in the US were collected from carpet or rugs (Table 2). Signi cantly more homes in TUS had mildew or moisture present than the other sites ( Table 2).
In-home oor dust loading As presented in Table 3, we found signi cantly greater amounts of dust loading in MX homes (p=0.04) compared to US homes. In addition, the homes located on a dirt road had signi cantly higher (p=0.02) dust load than the homes located on an asphalt road. Signi cantly more dust was retrieved in homes with at least one pet (p=0.01), more than two children (p=0.01), and more than 4 residents (p=0.01) ( Table  3). There were no signi cant differences in dust loading based on the number of rooms or number of adults living in the home. Finally, although we did not nd any signi cant differences between the dust load by oor type in homes located in the US versus MX, there was a signi cantly higher (p=0.03) dust load within US homes that had some sort of carpet, in comparison to homes with smooth ooring (Supplemental Fig. S1). There were no differences in dust loading in relation to whether there were asthmatic inhabitants.

Dust microbiome
The microbiome compositions, as measured by unweighted UniFrac, of the dust in the US samples and MX samples were signi cantly different prior to multiple comparisons correction, and nearly signi cant after multiple comparisons correction (PERMANOVA pseudo-F = 3.02 p < 0.001, q = 0.051; Fig. 1). We trained a random forest classi er to differentiate the neighborhoods using the dust microbiome compositions, and the classi er achieved an overall accuracy of 0.94, which was 1.9 times better than baseline accuracy (assigning the most common category to all samples; Fig. 1). US samples were accurately labeled 100% of the time, and MX samples were accurately labeled 88% of the time (Fig. 1). Our random forest model and ANCOM each reported many different genera, but both methods identi ed two of the same genera that were enriched in the MX dust (A. Alishewanella, and Chryseomicrobium)and one genus that was enriched in the US dust (Geodermatophilus) (Fig. 2).
The dust microbial composition between the four sites (two US cities and two MX neighborhoods) was signi cantly different (PERMANOVA pseudo-F = 2.33, p < 0.001; because all tests results reported in this paragraph were the same, we will report the results as F, p, and q-values for readability). The microbial composition in the dust from the higher SES neighborhood in NMX was signi cantly different from the US cities' (NUS versus high SES NMX): F = 1.90, p = .002, q = 0.0024; TUS versus high SES NMX): F = 2.83, p < 0.001, q = 0.0015) and signi cantly different from low SES NMX: (F = 2.57, p < 0.001, q = 0.0015). The low SES NMX neighborhood dust microbiome composition was also signi cantly different compared to the US cites (NUS versus low SES NMX): F = 2.48, p < 0.001, q = 0.0015; TUS versus low SES NX: F = 2.74, p < 0.001, q = 0.0015) (Fig. 1). The US cities were not signi cantly different from each other (TUS versus NUS: F = 1.36, p = 0.071, q =0.0710 ( Fig. 1). A sub-analysis showed that the house dust microbial composition differed when comparing the homes by road type (F = 2.59, p < 0.001, q < 0.001) and presence of air conditioning (air conditioning vs none: F = 2.33, p = 0.002, q = 0.012) (Supplemental Fig. S2a and Supplemental Fig. S2b). Another sub-analysis showed the house dust microbial composition was approaching but not signi cantly different in houses that had an asthmatic vs those that did not have an asthmatic present in the home (Supplemental Fig. S2c) (p=0.135).We trained a random forest classi er to differentiate the neighborhoods using the dust microbiome compositions, and the classi er achieved an overall accuracy of 0.78, which was 2.8 times better than baseline accuracy (assigning the most common category to all samples). The model predicted NUS accurately 75% of the time; TUS 60% of the time: high SES NMX 75% of the time; and low SES NMX 100% of the time (Fig. 1). When the model predicted the wrong neighborhood, it predicted the correct country. Our random forest model and ANCOM for comparing neighborhoods both found two genera that were enriched in the dust microbiome of both the low SES NMX and high SES NMX groups (Aeromonas, and Dysgonomonas) and one genus and family that was enriched in just the dust microbiome samples from the low SES NMX (Ornithinimicrobium and Intrasporangiaceae, respectively) (Supplemental Fig. S3).
The PICRUSt analysis predicted that there were 68 signi cantly differentially abundant functional pathways in the house dust microbial communities present when comparing the US to MX (Supplemental Fig. S4). Twenty-two signi cantly different functional pathways of the microbial communities in the dust existed between high SES NMX vs low SES NMX vs NUS, as compared to TUS (Supplemental Fig. S5). The PICRUSt analysis predicts that the microbes present in the house dust in low SES NMX had super pathway of polyaminine biosynthesis III capabilities. When comparing the low SES NMX to the high SES NMX, the microbes present in the house dust in the low SES NMX neighborhood had greater pyrimidine deoxy ribonucleotises biosynthetic, benzoyl CoA anaerobic degradation, nitrate reduction, and spirilloxanthin and 2.2-diketo-spirilloxanthin biosynthetic capabilities (Supplemental Fig. S5).

Discussion
This study examined differences in household characteristics, environmental factors, socio-economic factors, and microbial composition of dust from households in neighborhoods across the US-MX Border, in which we previously documented differential rates of asthma prevalence [29]. We identi ed multiple structural, environmental, and human factor differences between the US and MX homes, which may have led to the differences seen in dust loading and microbial diversity across the dust samples collected from the homes. The bacterial genera A. Alishewanella and Chryseomicrobium were found to be enriched in the dust from homes within MX, while Geodermatophilus was found to be enriched in the dust from homes within the US.
This study found multiple housing characteristics that differed between MX and the US, differences that may contribute to the higher prevalence of asthma in children of Mexican descent living in the US compared to MX [29]. Homes in the US were more likely to be on paved roads and to have ushing toilets, piped/municipal water, more rooms and bathrooms, and evaporative cooling and/or air conditioning compared to MX homes. Although only 26.3% of individuals in the US drank their tap water, this was signi cantly higher than in MX (2.6%). It has been shown that municipal water has fewer commensal microbes present and that drinking of municipal water is associated with higher prevalence of asthma and allergies [21,30]. Floors were more likely to be carpeted in the rooms where the dust was collected in the US. Carpets in the home may be an important risk for asthma development as they can be a reservoir for mildew, mold, allergens, and chemical hazards (e.g., pesticides, metals, ame retardants, per-and poly uoroalkyl substances (PFAS), and can contribute to poor indoor air quality [31,32]. Although carpets typically have greater dust loading than hard oors, the homes in MX had signi cantly greater dust loading than the ones in the US, despite the lack of carpet (Tables 2 and 3). This suggests that differences in the household density (particularly the number of children), the presence of pets, and other household structural factors between these communities might account for the greater dust loading in MX.
Microbial populations in indoor environments, where we live and eat, play an important role in human health. Environmental dust exposure early in life appears to in uence what bacteria colonize the gut, skin, and nasal microbiome [33][34][35][36]. It has been proposed that part of the reason for increasing asthma and allergy prevalence worldwide is due to shifts in our lifestyles towards more Western or Modernized ways of living, which has led to a decrease in microbial exposure during a critical period of immune development [37]. Individuals with exposure to more diverse bacteria have lower rates of allergic diseases [38,39]. We found signi cant differences in the microbiome composition of dust collected from homes in MX compared to the US (Fig. 1). Previous studies have shown a link between house dust microbial composition and risk of allergic asthma development. Children raised in Amish communities have lower rates of allergic sensitization and asthma than those from Hutterite communities. Stein et al demonstrated that house dust from an Amish community had a different microbial composition than the house dust from a Hutterite community. Further, mice that received the dust from the Hutterite houses intranasally had decreased airway reactivity and eosinophilia, which are markers of allergic asthma [22].
There was no difference in microbial dust composition between homes with and without an occupant that reports having asthma. There are likely multiple reasons for this nding. First, the study was not powered to nd a difference based on asthma prevalence, and the study simply asked if a person in the household had asthma and not whether they grew up in that house. Also, the study did not use a validated tool to screen for asthma in all household occupants, so there may be a difference in asthma detection between the cities.
House dust microbial composition is affected by outdoor and indoor environments, including structural characteristics, as well as household occupants and their activities in the home. Any combination of the signi cant differences between the homes in MX and the US may have led to the differences observed in the microbial composition of the house dust. A sub-analysis showed that there was a difference in beta diversity (unweighted UniFrac) of the dust microbiome when comparing the homes by road type and presence of air conditioning. Using an air conditioner changes the indoor environment by changing the temperature and humidity, which would lead to differences in microbial growth indoors [40], as well as through ltering of the air. Homes with air conditioners are less likely to have windows open, and therefore less dust is likely to blow into the home. Furthermore, it has been shown that as regions become more industrialized and homes are constructed in a manner where they are more tightly sealed, the microbiome diversity decreases [41]. The structural differences and occupant behavioral differences between US and MX are likely to have led to the differences in microbial diversity in the household dust.
We have previously shown that these two regions have different prevalence of childhood asthma, and this study also found differences in house dust composition between the two regions. The dust from the US homes was more enriched with the genus Geodermatophilus, whereas the dust from Mexican homes was more enriched with A Alishewanella and Chryseomicrobium. While the genera of bacteria found in house dust in both the US and MX have not been previously identi ed as related to asthma prevalence, Ornithinimicrobium (more abundant in the low SES NMX neighborhood) and Geodermatophilus are part of the phylum Actinobacteria, which has been shown to be both negatively and positively associated with allergic disease [25,[42][43][44][45][46]. To our knowledge, Geodermatophilus has not been speci cally linked to being a risk for asthma development previously, but its presence in school dust has been linked to "Sick Building Syndrome" [47]. The genus Alishwaneela is a gram-negative species that have endotoxin present in their cell walls. Previous studies have shown that higher levels of endotoxin in a child's environment are related to lower incidence of atopic asthma and allergic diseases [22,48,49]. Alishwenella is also in the phylum of Proteobacteria, which, much like Actinobacteria, has been shown to be both negatively and positively associated with allergic disease in different studies [42,43,46,50]. Chryseomicrobium is in the family Planococcaceae. In a study by Ta et al. it was shown that Pamnomicrobium, which is also in the family Planococcaceae, was protective against developing allergic eczema [44]. The inconsistencies in the direction of these relationships may be explained by the need to classify beyond the phylum level, which is very broad, or the presence of critical time points at which exposure protects against onset of asthma but may be harmful once asthma has developed. In any case, the differences found in microbial exposure between the sites suggest promising areas for future research related to asthma in the Border region.
Although some microbes in the dust are unlikely to be active metabolically, others are living and metabolically active, and so may play a critical role in alteration of host microbiome. They can colonize the host, where they can become metabolically active and play a key role in risk of asthma development.
A PICRUSt analysis was done to identify potentially relevant metabolic pathways present in the house dust from homes located in MX vs US. There were 23 biosynthetic pathways, 38 degradation/utilization/assimilation pathways, and 7 pathways involved in generation of precursor metabolites that were higher in the house dust from MX. Some of the pathways found to be more prevalent in the house dust from MX are involved in biosynthesis of molecules that are known to be protective against asthma (Supplemental Fig. S4), such as short chain fatty acids and pyrimidine [51,52]. There have been multiple studies that show the importance of metabolites produced by the microbiota that colonize humans in protecting against asthma via alterations in the epithelial barrier function and immune system regulations. Many of the possibly up-regulated functional pathways present in the house dust from MX have been examined in other studies and have been shown to be protective against asthma or helpful in asthma control. For example, the UDP − N−acetyl − D−glucosamine and the mycolate biosynthetic pathways that were higher in the house dust from MX have been shown to down-regulate allergic airway in ammation [53][54][55][56] (Supplemental Fig. S4).
Limitations of this study include that collection of the house dust microbiome samples occurred at a single time-point and during a single season. There was signi cant differences in the ambient temperatures during sample collection across the border and this was likely related to more of the MX samples being collected in the spring/summer months and more of the US sample being collected in the winter. Future studies would bene t by looking at multiple time points to assess the variability of household microbiomes. The high SES NMX homes were all part of a subdivision with identical oor plans, which could drive some of the differences observed between the low and high SES neighborhoods in NMX. We used 16S rRNA amplicon sequencing to characterize the house dust, which generally supports taxonomic resolution only at approximately the genus level, although species level differences may affect human health. Shallow or deep shotgun metagenomics, although more expensive, would improve microbial identi cation, and deep shotgun metagenomics would provide more accurate functional pro les of the samples. PICRUSt uses 16S rRNA data to extrapolate metagenome composition and provides relatively lower con dence functional pathway pro les of samples. Given that asthma is a common reason for presentation to outpatient clinics and household recruitment was through outpatient clinics, there may have been selection bias because families that had an asthmatic child in the home may have been more likely to enroll in the study. However, this bias could have been represented on both sides of the Border. We did not investigate differences in pollutants in the collected dust, such as metals or pesticides, which could affect the dust composition and microbial diversity, along with the growth of opportunistic bacterial pathogens [57].
In conclusion, despite TUS, NUS, and NMX being geographically close (< 100 km) and having similar climates, homes across this Border region differ in ways that lead to signi cantly different indoor environments. Mexican and US households differed in years of education; household income; the percentage of homes that had paved roads, ushing toilets, and piped water; the number rooms and bathrooms present in the home; and presence and type of cooling and ooring. Some of these household differences may have led to the signi cant differences we observed in the microbial composition of the house dust collected from MX or US homes. The dust from the US homes was more enriched with the genus Geodermatophilus, whereas the dust from Mexican homes was enriched with A. alishewanella and Chryseomicrobium. Future research should assess whether exposure to these bacteria during critical windows in early life may offer protection from development of asthma or allergic disease.

Study Population
Patients at each of three clinics were approached from September to December of 2016 to participate in this study: El Rio Community Health Center in TUS (n = 20), Mariposa Community Health Center in NUS (n = 20), and the Secretaría de Salud de Sonora in NMX (n = 40). In NMX, 20 households were recruited from a traditionally high SES neighborhood (high SES) and 20 from a traditionally low SES neighborhood (low SES). All of the households that were recruited from the high SES neighborhood in NMX were in a subdivision neighborhood where the houses were constructed with identical layouts. Families were eligible to participate in this study if at least one parent was of Mexican descent and had at least one child younger than 5 years old. The University of Arizona Human Subjects Protection Program approved all study materials (IRB approval number: 1607687201), in addition to all necessary permissions and reviews from the US/Mexico Border Commission, the Secretaría de Salud de Sonora, Mariposa Community Health Center and El Rio Community Health Center.

Questionnaire
During the home visit, a questionnaire was administered orally in English or Spanish by a trained research assistant to obtain information on household demographics, asthma prevalence, sanitation measures, drinking water sources, and pets in the home. Multiple household characteristics were assessed (e.g., mildew, water damage, structural characteristics, and type and number of bathrooms).

Dust Sample Collection
House dust was collected using a Hoover CH3000 vacuum cleaner equipped with a pre-weighed sterilized X-Cell 100 dust collection sock (Midwestern Filtration, Cincinnati, OH) inserted in the crevice tool. To collect oor dust, a one-meter square template was laid on the oor in the child's room and vacuumed for ve minutes. If a child did not have their own room, then the sample was collected in the room where the child regularly spends most of their time. The sock lter holding the collected dust was placed in a plastic bag sterilized under UV light in a hood. The vacuum and its accessories were cleaned with disinfectant wipes and sprayed with isopropyl alcohol between sampling each house. Collected dust samples were transported in a cooler with ice packs to the laboratory in the Medical Research Building at the University of Arizona in Tucson, AZ.
In the laboratory, each dust sample was transferred from the lter sock to a pre-weighted 50 ml sterilized centrifuged tube. The centrifuge tube with the dust sample was weighed three times using a Mettler Toledo AB54 Precision Balance Weight Scale (Mettler Toledo International, Inc., Columbus, OH). The average of three measurements was then recorded. All environmental samples were stored in a -80 C freezer until analyzed. Frozen dust samples were shipped to the Pathogen and Microbiome Institute at Northern Arizona University for amplicon library preparation and sequencing.

DNA Extraction
DNA was extracted using the MoBio Powersoil DNA isolation kit (Qiagen) with an additional mechanical lysis. Brie y, samples were placed in a lysing matrix E tube (MP Biomedical) with 600 µl of Buffer RLT Plus and lysed in 30 second increments for a total of 6 minutes at 10,000 x g. Samples were sat resting for 30 seconds between each bead beating to prevent heating. Extraction continued following the manufacturer's protocol. DNA was quanti ed using a NanoDrop 2000. Extraction blanks, which did not contain any sample during the extraction, were carried throughout the entire extraction and 16S rRNA gene sequencing.