International Research Journal of Public and Environmental Health Vol.8 (2),pp. 87-97, March 2021 Available online at https://www.journalissues.org/IRJPEH/ https://doi.org/10.15739/irjpeh.21.012 Copyright © 2021 Author(s) retain the copyright of this article ISSN 2360-8803

Original Research Article

Microbiological contamination of the air at two high-traffic-volume roundabouts in Cotonou, Benin

Received 4 December, 2020 Revised 20 January, 2021 Accepted 28 January, 2021 Published 10 February, 2021

Lucrèce Marie Karine Codjo-Seignon*1,

Emmanuel Ghislain Sopoh1, Cyriaque

Degbey1,2, Raoul Zinhota2 and

Martin Pépin Aina3

1Regional Institute of Public Health, University of Abomey-

Calavi, Benin. 2University Hospital Hygiene Clinic of National University

Hospital Center Hubert Koutoukou Maga in Cotonou,

Benin. 3Water Science and Technology

Laboratory of the National Water Institute, University of Abomey-

Calavi, Benin.

*Corresponding Author Email: karineseignon@yahoo.fr

Little has been documented about microbiological air pollution in Africa though it is vital to understanding our environment and the related health risks. This study has assessed the bacterial and fungal contamination of the air at two high-traffic-volume roundabouts in Cotonou as well as to assess the intensity of exposure at these two locations. Air samples were taken at the Akpakpa PK3 roundabout (roundabout A) and the Vèdokô Cica-Toyota roundabout (roundabout B) using a microbial air sampler (BAOSHISHAN FKC-1) and then grown on different media to identify fungi, staphylococci, enterococci, streptococci and enterobacteria. Staining and microbiological tests (Gram stain, catalase test, oxidase test and deoxyribonuclease test) helped identify the microbes. The intensity of exposure was estimated based on the volume of air aspirated and the duration of exposure. The air samples from the two roundabouts contained a similar number of fungi (approximately 400 CFU (colony-forming units)/m3). There were, however, differences in their contents (Syncephalastrum sp. in the samples from the roundabout A; Rhizopus sp. and Chrysoniliasp. in the samples from the roundabout B; and Fusarium sp., Aspergillus niger, Aspergillus flavus, Cladosporium sp. and Penicillium sp. in the samples from both roundabouts B). There were twice as many Gram-positive bacteria (Staphylococcus aureus, Staphylococcus sp., Bacillus sp., Lactobacillus sp. and Micrococcus sp.) in the roundabout A samples (163 CFU/m3) than in the roundabouts B samples (76 CFU/m3).This study shows the presence of pathogenic bacteria and fungi in the ambient air at the two roundabouts, thus constituting a potential health risk. Keywords: Pollution, traffic volume, microorganism, outdoor air, environment, Health risk.

INTRODUCTION Air pollution is defined as the contamination of an indoor or outdoor environment by a chemical, physical or biological agent that modifies the natural characteristics of the atmosphere and is likely to cause discomfort or have a harmful effect on humans and/or the environment as a whole (“OMS | Pollution de l’air” WHO). In this regard, it is one of the major concerns facing our world this decade. The WHO (World Health Organization) reports that in 2012,

approximately 7 million people died prematurely as a result of air pollution exposure worldwide, including Africa (OMS, 2014).

In fact, Africa is the continent most affected by air contamination after Asia. Approximately one million Africans die from it every year according to the WHO (Africa News 2019). More than 90% of air pollution-related deaths occur in low- and middle-income countries, mainly

Int. Res. J. Pub. Environ. Health 88 in Asia and Africa, followed by low- and middle-income countries of the Eastern Mediterranean region, Europe and the Americas (OMS, 2018).

Air contamination has been linked in several studies to cardiac, neurological and allergic respiratory diseases (Levy 2015; Shutt et al., 2017; Wang et al., 2016).The WHO recognizes that air pollution is a critical risk factor for noncommunicable diseases, causing an estimated one-quarter (24%) of all adult deaths from heart disease, 25% from stroke, 43% from chronic obstructive pulmonary disease and 29% from lung cancer (OMS, 2018).

Many studies on chemical air pollution have already been conducted in Benin. They mainly focused on the population’s exposure (specifically motorcycle taxi drivers) to toxic chemicals such as carbon monoxide, volatile organic compounds and particles. These studies showed that this exposure was linked to respiratory, muscle, bone, and eye diseases, laryngeal infections and DNA (deoxyribonucleic acid) damage (Avogbe, 2004; Ayi-Fanou et al., 2011; Herve et al., 2018; Joachim, 2003; Tohon et al., 2015).

However, microbiological air contamination has not yet been investigated in Benin. This pollution can come in the form of fungi or bacteria carried in pollen with human activities as their primary source. According to Pignol et al. (1992), “The germ content is low when the population density is low, and it increases considerably in cities polluted by industrialization”. In fact, humans produce and transport microbes that can become airborne through breathing, touch, movement, hair and clothing. The consequences of this contamination are mostly health-related, with an increase in allergies, asthma and respiratory illnesses such as hypersensitivity pneumonitis Pignol et al. (1992). MATERIALS AND METHODS Study locations This study was conducted at two roundabouts in the city of Cotonou, which were identified on the basis of the fixed and/or mobile sources of pollution present there. These were the roundabouts at Akpakpa PK3 (roundabout A) and Vèdokô Cica-Toyota (roundabout B) (Figure 1). An earlier study by Akiyo et al. identified roundabout B (also known as the Akossombo roundabout) as one of the main centers of traffic congestion in Cotonou (Akiyo 2016). Moreover, in 2019, the Road Data Division of the General Directorate of Infrastructure carried out an hourly count of the number of light, heavy-duty and two-wheel vehicles circulating at multiple locations in Cotonou, including these two roundabouts. This count revealed that roundabouts A and B were the most crowded, with 157517 and 235589 vehicles, respectively (Division des Données Routières du la Direction Générale des Infrastructures 2019).

Their geographic coordinates are 6°22'01''N 2°27'52''E and 6°22'37"N 2°23'23"E, respectively (Figure 2). There are

many factories and industries in the vicinity of the Akpakpa PK3 roundabout (paint manufacturing, cement works, thermal power plant, gas manufacturing, brewery, bakery and grain mill). The Vèdokô Cica-Toyota roundabout does not have any factories in its surrounding area but rather a cluster of stores (sale and maintenance of cars; sale of portable devices; sale of men’s and women’s wear; sale of food and sundries). Study type and target period This is a cross-sectional study that took place on March 23, 2020 and focused on air samples taken at the Akpakpa PK3 roundabout and the Vèdokô Cica-Toyota roundabout. Measured variables and sampling Measured variables The measured variables are quantity and identification of fungi, Staphylococci, Enterobacteria and Streptococci in air samples from the two roundabouts and also the intensity of exposure to these microbes. sampling The FKC-1 microbial air sampler (BAOSHISHAN FKC-1) chosen for this study uses the aerosol impaction principle or Andersen collision principle to assess air quality. First, a 90 mm diameter petri dish is inserted into the air sampler. Then, for 1 minute, air is pulled into the device through hundreds of micropores. The chosen sampling rate for this study was 100 L of air per minute and the microbial air sampler was sterilized with 70% alcohol prior to the insertion of each petri dish (Jacob et al., 2016). PCA medium (plate count agar) was used to quantify the total flora; CHAP medium (Chapman mannitol salt agar) was used to identify staphylococci; BEA medium (bile esculin azide agar) was used to identify enterococci and streptococci; EMB medium (eosin methylene blue agar) was used to identify enterobacteria; and SAB medium (Sabouraud chloramphenicol agar) was used to identify and quantify fungi.

In total, 30 samples were taken at the two roundabouts (15 samples per roundabout) a rate of 3 repetitions per growing medium. Since ultraviolet (UV) radiation destroys nucleic acids and disrupts DNA strands, it is sometimes used as a disinfectant or sterilizer to kill harmful microorganisms (Fondation contre le Cancer 2017). The samples in this study were taken between 12 PM and 2 PM, when UV radiation is most intense (Hart 1938). by doing that, , is the to access at the minimal concentration of airborne microorganisms which is aimed at . The average temperature was 29°C, and the average humidity was 40.45%.

After exposure, the petri dishes were placed in a container, taken to the laboratory and placed in an incubator. After incubation, the dishes were collected, and

Codjo-Seignon et al. 89

Figure 1: Traffic at the Akpakpa PK3 roundabout (roundabout A) (left) and the Vèdokô Cica-Toyota roundabout (roundabout B) (right)

Figure 2: Map of the city of Cotonou with the roundabouts Akpakpa PK3 and Vèdokô Cica-Toyota

the colonies were fixed onto slides using heat. The colonies were then Gram stained (Beveridge, 2001), and the slides were observed under an optical microscope. Catalase tests using oxygenated water and slide drop method was used (Jacob et al., 2016). Also, oxidase tests were made using oxidase slide. Material, method and technique of sample analysis Material The materials used in this study consisted of a microbial air

sampler (BAOSHISHAN FKC-1); 90 mm diameter petri dishes; a GPS application (Compass); an application for measuring temperature and humidity (Weather); 3 incubators (BINDER BD 115 and GENLAB MINO/50); a Bunsen burner; slides for microscopic examination; a BE5 binocular microscope connected to a camera (IMX185) and to Mv Image software; a Gram staining kit (gentian violet, Lugol's solution, 90% alcohol and fuchsin counterstain); oxygenated water (hydrogen peroxide); and oxidase slides (DIFCO Dry Slide).

The following growth media were used in this study: PCA medium; CHAP medium; BEA medium; EMB medium; and

Int. Res. J. Pub. Environ. Health 90

Figure 3: Concentration of microbes at crossroads A and B according to growth media.

Roundabout A: Akpakpa PK3 roundabout; roundabout B: Vèdokô Cica-Toyota roundabout; BEA: bile esculin azide agar (growth of Enterobacteria); EMB: eosin methylene blue agar (growth of Streptococci and Enterococci); CHAP: Chapman mannitol salt agar (growth of Staphylococci); CFU: colony-forming unit.

SAB medium’ Incubation, enumeration and isolation of microorganisms After collection, the samples on the SAB medium were incubated for 5 days at 25°C; those on the PCA medium were incubated for 3 days at 30°C. The samples taken on the EMB, CHAP and BEA media were placed at 37°C for 24 hours.

After incubation, the flora on the PCA medium were counted . Colonies on the EMB, CHAP and BEA media were enumerated, and those differing in morphology were isolated on new media for identification. For the fungi, their total count was performed on SAB medium. Microorganism identification The identification of bacteria was performed by first performing Gram staining and then testing for catalase and oxidase. Whenever there was suspicion of Staphylococcus aureus, the deoxyribonuclease (DNase) test was completed as well.

The fungi were identified by a combination of macroscopic and microscopic observation. For microscopic examination, the low-distortion tape touch method was used (Harris, 2000); then, the slides were stained with lactophenol for observation under an optical microscope. Assessment of the intensity of microbial exposure To assess the intensity of exposure to airborne microorganisms at the two roundabouts, it is necessary to take into account the fact that a healthy human being

breathes approximately 0.5 L of air per inspiration (volume of air inhaled at one time), 16 times per minute (number of breaths), and approximately 23,000 times in 24 hours (CNEWS 2016).

The volume of gases can be expressed in liters or cubic decimeters: 1 L = 1 dm3 = 1 x 10-3 m3.

The following equation was used to assess the number of microorganisms inhaled:

Microbes inhaled = (number of CFU/m3 x number of inspirations) x volume of air inhaled in m3

Data analysis

Data was analyzed using the Excel spreadsheet. The calculation of averages and the bar chart was also performed in Excel spreadsheet. RESULTS

Bacterial flora

All bacteria found are Gram-positive. Figure 3 illustrates the bacterial distribution at the two study sites according to the culture media used. The results were different for each culture medium. Of the three culture media used (CHAP, EMB and BEA), the CHAP medium seemed to be the most favorable for the growth of the airborne bacteria at both roundabouts, as it had the most colonies. The EMB and BEA media show respectively the fewest number of bacterial colonies and on any culture media, the roundabout A present the most colonies.

A total of 163 CFU/m3 was counted in roundabout A. The

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Table 1. Microbial identification and count

Microbes identified/ Microbial count in CFU/m3

Roundabout A Roundabout B

Bacteria Microbes identified Lactobacillus sp.

Micrococcus sp. Bacillus sp. Staphylococcus aureus Staphylococcus sp.

- Micrococcus sp. Bacillus sp. - Staphylococcus sp.

Total microbial count 163 76 Fungi Microbes identified

Fusarium sp. Aspergillus flavus Aspergillus niger Cladosporium sp. Penicillium sp. Syncephalastrum sp. - -

Fusarium sp. Aspergillus flavus. Aspergillus niger Cladosporium sp. Penicillium sp. - Rhizopus sp. Chrysonilia sp.

Total microbial count 460 430

Table 2. Estimated calculation of the number of bacteria and fungi inhaled as a function of the duration of exposure Roundabouts Microbes CFU inhaled in 1 min CFU inhaled in 1 h CFU inhaled in 8 h CFU inhaled in 24 h RoundaboutA Bacteria 1 78 626 1878 Fungi 3 206 1651 4954 Total - 5 284 2277 6831 RoundaboutB Bacteria <1 36 292 875 Fungi 4 221 1766 5299 Total - 4 257 2058 6174

CFU: colony-forming units

bacteria identified were Lactobacillus sp., Micrococcus sp., Bacillus sp., Staphylococcus aureus and Staphylococcus sp. For roundabout B, the bacteria identified were Micrococcus sp., Bacillus sp., Staphylococcus aureus and Staphylococcus sp., with a total of 76 CFU/m3 (Table 1 and Figure 3).

The quantity and quality of bacteria was different between the two roundabouts with approximatively 2 times more bacteria on A roundabout compare to the B. This suggested that environmental conditions of roundabout A could be more conducive for those bacteria. Fungal flora All fungi were filamentous. The fungal flora found at the two roundabouts were composed of two species (Aspergillus niger and Aspergillus flavus) and six genera (Fusarium sp., Cladosporum sp., Syncephalastrum sp., Chrysonilia sp., Rhizopus sp. and Penicillium sp.). Roundabout A had 430 CFU/m3 of fungal flora, whereas roundabout B had 460 CFU/m3 (Table 2 and Figure 3).

The fungi quantity is approximatively the same on the two localizations, but they differ by their composition. In addition, quantity of fungi is also higher (2.6 times) than bacteria. This implies that fungi have environmental

conditions more favorable compare to bacteria. Total microbial flora The total microbial flora was enumerated on the PCA medium. The number of microorganisms present in roundabout A was approximately 1.2 times greater than that in roundabout B, with values of 660 CFU/m3 in roundabout A versus 557 CFU/m3 in roundabout B (Figure 3).

This numeration is show that there are probably some fungi, bacterial and other germs present in those area which could not be reached with the methods used.

Assessment of the intensity of exposure at the two roundabouts

Table 2 illustrates the number of microorganisms inhaled by an individual at each roundabout according to the duration of exposure. For two individuals X and Y placed at roundabouts A and B, the individual X would inhale on average 2,277 CFU (approximately 2.3 x 103 colony-forming units) versus 2,058 CFU (approximately 2.1 x 103 colony- forming units) for individual Y during 8 hours.

Int. Res. J. Pub. Environ. Health 92

Figure 4: Fungal flora identified at roundabouts A and B (low-distortion tape touch method and lactophenol cotton blue staining) A: Fusarium sp.; B: Aspergillus flavus; C: Syncephalastrum sp.; D: Chrysonilia sp.; E: Cladosporium sp.; F: Aspergillus niger; G: Rhyzopus sp.; H: Penicillium sp. Magnification: A, G, H: x 40; B, C, D, E, F: x 100

There were differences in the number of airborne microbes found at each roundabout. Individual B would be exposed to a higher number of fungal CFUs than individual A, with values of 1,766 CFU for individual B versus 1,651 CFU for individual A. Conversely, it is rather the individual placed at roundabout A who would be most exposed to bacteria (626 CFU for individual A versus 292 CFU for individual B).

In general, individuals A and B are exposed to more airborne fungi than bacteria on average, as individuals A and B are exposed to 102 CFU of bacteria versus 103 CFU of fungi.

DISCUSSION

The goal of this study was to estimate the level of microbiological air contamination at two high-traffic-volume roundabouts in the city of Cotonou and to assess the exposure intensity of the populations at these points.

In this study, the different culture media used didn’t always allow to find staphylococci, enterococci, streptococci or enterobacteria but other genera and species. Five genera and species of bacteria were identified in the two roundabouts. The following genera and species were identified in roundabout A: Lactobacillus sp., Micrococcus sp.,Bacillus sp., Staphylococcus aureus and Staphylococcus sp. For roundabout B, the following bacteria were identified: Micrococcus sp., Bacillus sp. and Staphylococcus sp. (Figure 5). The highest level of bacteria was found in the atmosphere of roundabout A with 163 UFC/m3. The fundamental difference between these two zones lies in the traffic flow and the sources of fixed and mobile pollution. Indeed, it is the roundabout B which

presents a more abundant traffic flow compared to crossroads A, whereas the bacterial density is higher at the level of roundabout A. This observation is different from that of Jacob et al, where the highest concentration was found at the level of the zone with the highest density of human flow. Here the human flow does not seem to be at the origin of the concentration differences found. An explanation can be found in the fixed sources of pollution or in chemical air pollutants produced by vehicle exhaust gases. In fact, crossroads A differs from B by the presence of many factories. Liu et al., (2018) are study the effect of air pollution on the total and pathogenic bacteria in different sizes of particulate matter in China and found that density of bacteria was highest on heavily and moderately polluted air. Also, the humidity, ozone and carbon monoxide have an impact on this density(Liu et al. 2018). Roundabout A would have more particulate matter than B roundabout. A further analysis of chemical pollution on those roundabouts could help to confirm this hypothesis.

Also, the results of this study indicate that there are more Gram-positive than Gram-negative bacteria in outdoor air. As has been reported in several earlier studies of Jacob et al., 2016 as example, it seems that the majority of airborne bacteria are Gram-positive even if Gram-negative bacteria were also identified in those studies (Tsai and Macher 2005).Gram-positive bacteria differ from Gram-negative bacteria in the constitution of their cell walls. As Gram-positive bacteria have a much thicker layer of peptidoglycan than Gram-negative bacteria, this feature gives them an increased resistance to ultraviolet rays compared to Gram-negative bacteria (Silhavy, Kahne, and Walker 2010; McKinney and Pruden 2012). In addition, some bacterial species, such as those belonging to the genus

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Figure 5 : Bacterial flora identified at roundabout A and B after Gram staining.

Magnification times 100. A : Lactobacillus sp. ; B : S. aureus ; C : Micrococcus sp. ; D : Staphylococcus sp. ; E : Bacillus sp.

Bacillus, are able to form endospores (dormant forms of the bacteria) in environment stress conditions as pH variation (Duport et al., 2016; Tan and Ramamurthi, 2014). These structures allow them to withstand dehydration and heat (Atrih and Foster, 1999). Finally, all of these bacteria have already been found in the air, and for good reason, most of them are ubiquitous. Each of these bacteria has characteristics that make it possible to distinguish them from one another and associate them with the sampling site.

Bacillus is ubiquitous bacteria, and some species are known to cause serious diseases, such as Bacillus anthracis, which causes anthrax, Bacillus cereus and Bacillus thuringiensis(Ehling-Schulz et al., 2019). Although they appear to be abundant outdoors and they are generally not pathogenic to humans. They are involved in other processes, such as the biogeochemical cycle of phosphorus (Bacillus subtilis and Bacillus megaterium) or are used in biotechnology (Bacillus amyloliquefaciens and Bacillus coagulans) (Khan et al., 2020). This research helps to identify the Bacillus bacteria down to the species, unlike the study conducted by Jacob et al. 2016, in which a Gallery API specific to Gram-positive bacteria combined with 16S rRNA

PCR made it possible for them to identify Bacillus species with precision, but none were found to be pathogenic.

In our study, the genus Staphylococcus was found at both roundabouts; however, the species Staphylococcus aureus was found only at roundabout A. Staphylococci are known for their pathogenicity. They are ubiquitous saprophytic bacteria and can therefore be found almost everywhere in our environment. They can also be found in a commensal interaction in humans, especially in the nasal cavity (30% carriage in humans) (Krismer et al., 2017), or on the epidermis. . Certain species, including aureus, epidermidis and saprophyticus, are well known to cause infections in humans.

The genus Micrococcus is more generally associated with human or animal presence. It is considered a commensal and saprophytic organism, but under certain conditions, it can be an opportunistic bacterium (Rodriguez-Nava et al., 2020). Previous studies have reported its presence in air samples (Yang et al., 2019; Faridi et al., 2015).

Finally, the genus Lactobacillus was identified. Lactobacillus groups is represented by ubiquitous bacteria present on the surface of plants together and is in abundance on decaying plant matter and fruits. They are

Int. Res. J. Pub. Environ. Health 94 also found in humans and animals (vaginal, intestinal, buccal and rectal flora) (Tailliez, 2004).

This study’s results support the data in the literature. The bacterial genera and species that found in this study have already been found to be associated with air, earth, vegetation and humans. The two roundabouts had all of these characteristics (Figures 1 and 2).

For fungi, five main genera and species were found at the two roundabouts: Fusarium sp., Aspergillus niger, Aspergillus flavus, Cladosporium sp. and Penicillium sp. Some fungi were found only at roundabout A (Syncephalastrum sp.) or roundabout B (Rhizopus sp. and Chrysonilia sp.) (Table 1 and Figure 4). All of them are filamentous fungi. The CFU were similar at both roundabouts. All of the fungi identified in this study had already been associated with air in earlier studies (Jacob et al., 2016; Levetin et al., 2016; Shabankarehfard et al., 2017).

In the literature, people with existing medical conditions seemed most likely to be infected with these fungi. These were immunocompromised people, such as individuals taking chemotherapeutic agents or immunosuppressants, transplant recipients or patients with HIV infection (Antinori et al., 2018; Köhler et al., 2017), Specific diseases and particular traits are associated with fungi depending on the genera or species.

The fungi in genus Fusarium are anamorphic. They can be soil saprophytes, devastating plant pathogens and human pathogens (Levetin et al., 2016; Munkvold, 2017). Fusarium species can cause sinusitis, bronchopulmonary allergies, keratitis, onychomycosis and many other infections (Levetin et al., 2016).

The genus Aspergillus is generally not associated with human or animal pathologies, with the exception of the species fumigatus, terreus, nidulans and niger (Levetin et al., 2016). In some special cases, this genus has also been associated with allergic bronchopulmonary aspergillosis (ABPA) and allergic fungal sinusitis (Simon-Nobbe et al., 2008).

Other better-known fungi, such as Penicillium, were also identified in this study. They are ubiquitous, saprophytic and grow mainly on biodegradable organic matter (Levetin et al., 2016). Although they are mainly known for their role in the manufacturing of penicillium antibiotics, they may be involved in superficial infections such as keratitis or very exceptionally associated with invasive opportunistic infections (Lavarde and Hennequin 2006) or allergic manifestations such as asthma (Levetin et al., 2016).

The fungi Cladosporium sp. are commonly found on living and dead plants. Many species are phytopathogenic agents, while some parasitize other fungi (Levetin et al., 2016). Some species of this fungus (Cladosporiumherbarum) produce spores, which are inhalable allergens mainly associated with asthma and allergies (Breitenbach and Simon-Nobbe 2002; Simon-Nobbe et al., 2008).

The fungi of genus Chrysonilia sp. can also be considered allergens (Levetin et al., 2016). in particular, the Chrysoniliacitophila species has been associated with asthma (Monzón et al., 2009).

The genus Syncephalastrum sp. has been isolated in a few infections, such as infectious rhinosinusitis (Mathuram et al., 2013), skin infections (Ribes et al., 2000) and otomycosis (Rich, 2000).

Finally, the fungi of the genus Rhizopus are saprophytic and grow on soil and decaying plant matter. On very rare occasions, they have been associated with infectious rhinosinusitis (Mathuram et al., 2013) and rhinocerebral mucormycosis (Rhizopusoryzae) (Bausset et al., 2011).

During the sampling, the temperature was 29˚C and relative humidity was 40,45%. Those parameters are important to explain the microbial abundance. In fact, temperature and humidity can impact airborne microorganism concentration (Islam et al., 2020; Jones and Harrison 2004; Hai et al., 2019). Here 29˚C can be considered as an optimal growth temperature for several microorganisms included fungi and bacteria (Betts 2006; Marín et al., 1995; Onyango et al., 2012; Toqeer et al., 2006), Also, the relative humidity (RH) 40,45% measured in his study appear to be favorable for their development.

Regarding all this information’s, it appears that the main ailments that could be caused by the airborne microorganisms that were found are allergies and asthma. What is the proportion of these ailments in Cotonou?

In Cotonou in 2017, there were a large cases number of respiratory, dermatological and ocular diseases (Hounnakan et al. 2017) with respectively 40% , 38% and 22% of cases. It is not possible to exclude the possibility that these diseases have a bacterial, fungal or even viral origin. Further research in this field is necessary to determine the type of microorganism and the species responsible for those pathologies.

Because the presence of fungi and bacteria in the air could lead to diseases provided that the individual is in a weakened state of health (Tailliez, 2004; Shabankarehfard et al., 2017; Köhler et al., 2017), it would therefore be wise to first study the real health impact of this exposure on the surrounding populations, second to monitor at-risk groups (such as immunocompromised patients) who have these diseases to determine the real share of airborne contamination on their state of health; and finally to propose adequate solutions (example: sanitation of the population’s living environment).

Also, the intensity of exposure to the airborne microbes at the two roundabouts would depend on the duration of exposure. On a working day (8 hours), exposure to airborne microorganisms is approximately 2.1 x 103 CFU. The intensity of exposure is quite low when an individual is only passing through, approximately 4 CFU per minute (Table 2). A study by Akiyo et al revealed that when there was a traffic jam on the Stade de l’Amitié-Vèdokô Cica-Toyota (Akossombo) roundabout stretch, the Vèdokô Cica-Toyota (Akossombo) roundabout-Étoile Rouge stretch or the Akossombo roundabout-Houeyiho interchange stretch, the duration of an individual’s transit through these traffic jams varied between 0.3 min and 6.5 min(Akiyo 2016). This would suggest that exposure to airborne microbes is negligible when passing through these roundabouts, which

is not the case for individuals residing in these areas or practicing their profession in the areas surrounding the roundabouts. Further study is needed to support this hypothesis.

In short, this study showed that there are both bacteria and fungi in outdoor air that are potentially pathogenic to humans and that the intensity of exposure increases with the duration of exposure.

This kind of study is the first conducted in Benin. It provides the initial guidelines for further studies. Although it has some weaknesses, this study nevertheless shows that with minimal equipment and reagents, it is possible to make an identification at least down to the genus. Unfortunately, there are not guidelines for microbial outdoor air quality but only for indoor air quality like in Dang et al study where, the standards of the European Protection Agency were used to evaluate microbiological air quality (Dang et al. 2020). These kinds of research can help government to take a decision about this lack.

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