J. Microbiol. Infect. Dis., (2026), Vol. 16(2): 72–81

Research Article

10.5455/JMID.2026.v16.i2.2

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Examination of microorganisms in Wukari public transportation systems and public health risk

Dawn Ify Agwaranze¹, Blessing Chioma Nwaneri², Ikechukwu Daniel Emmanuel¹, Ikrima Mohammed Usman^1* and Cynthia Sylvester Odinchefu¹

¹Department of Microbiology, Faculty of Biosciences, Federal University Wukari, Wukari, Nigeria

²Department of Microbiology, Faculty of Biological Sciences, Federal University of Technology Oweri, Oweri, Nigeria

*Corresponding Author: Ikrima Mohammed Usman. Department of Microbiology, Faculty of Biosciences, Federal University Wukari, Wukari, Nigeria. Email: ikrimah25 [at] gmail.com

Submitted: 17/07/2025 Revised: 11/02/2026 Accepted: 22/02/2026 Published: 04/04/2026

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ABSTRACT

Background: Public transportation systems are essential components of urban and semi-urban environments, providing daily affordable and efficient mobility to millions of people daily.

Aim: This study aimed to examine microorganisms in Wukari’s public transport system, specifically buses and Sienna vehicles.

Methods: A total of 18 samples were collected from high-contact surfaces (seats, handrails, and door handles) of each vehicle using sterile cotton swabs moistened with normal saline aseptically. These samples were analysed for microbial presence of public health implications at different times of the day. The total bacterial and fungal counts were determined, and bacterial isolates were characterized using colonial morphology, Gram staining, and biochemical tests, such as the catalase, oxidase, indole, citrate, and triple sugar iron tests, while the fungi were characterized using lactophenol cotton blue stain.

Results: The total viable bacterial count was high, especially in the evening. In the morning, bacterial counts ranged from 1.4 × 10⁷ cfu/m³ to 5.4 × 10⁷ cfu/m³, with the highest levels recorded on bus handrails. The bacterial isolates included Proteus mirabilis, Escherichia coli, Pseudomonas aeruginosa, Bacillus spp., Acinetobacter baumannii, and Enterobacter aerogenes. P. mirabilis (64.8%) was the most prevalent species across all surfaces. Bacillus spp., Pseudomonas aeruginosa, and Escherichia coli were more commonly found in the morning samples but were absent in the evening sample. The spore-forming unit ranged from 0.1 x 10⁷ to 1.0 × 10⁷ sfu/m³. Fungal contamination with Penicillium spp. (50%) and Aspergillus flavus (50%) was observed only in the evening. Both fungi were found exclusively in the buses.

Conclusion: Public transport vehicles can harbor harmful microorganisms, highlighting the importance of regular cleaning and disinfection.

Keywords: Bacteria, Fungi, Microbial contamination, Microorganisms, Public health.

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Introduction

The built environment (BE) harbors a diverse assemblage of microorganisms (Bruno et al., 2022). Advances in scientific research have significantly enhanced the understanding of how environmental, geographical, and anthropogenic factors influence BE microorganisms (Ahn and Hayes, 2021). These insights shed light on the functional roles, adaptive mechanisms, and resistance capabilities of indoor microbial communities, as well as the potential for microbial transmission between BEs and their occupants (Leung et al., 2021).

Public transit systems are among the most common infrastructures among different urban built environments, through which more than 160 million individuals pass every day, exchanging microorganisms with each other as well as with public transit surfaces (Leung et al., 2021). As urbanization and modernization occur, the number of individuals traveling on global public transit systems will surely increase for decades to come (Leung et al., 2021). Public places, such as restaurants, day-care centers, public transport, playgrounds, and education centers, can aid the spread of microorganisms to several people (Brilliant et al., 2023). Contact with surfaces contaminated with infectious microorganisms is a way to spread diseases such as colds, flu, and diarrhea (da Silva et al., 2024).

Public transportation systems are essential components of urban and semi-urban environments, providing daily affordable and efficient mobility to millions of people (Jaiswal et al., 2024). However, the high volume of human traffic and the frequent contact with shared surfaces make public transportation a potential hub for the transmission of microorganisms, including pathogenic bacteria, viruses, and fungi (Cave et al., 2021). These microorganisms can be introduced into the transportation system through various sources, such as passengers, air, food particles, and contaminated objects, posing a significant risk to public health (Ly et al., 2024). This interaction has pinched great responsiveness from public health researchers as pathogenic microorganisms have developed an enhanced way of multiplication, which is quicker and more extensive in quantity than before (Abdulai et al., 2020).  The high occupant density within a typical public transit environment may present a public health concern by facilitating the transmission of microorganisms between commuters via fomites or airborne routes (Cummings et al., 2024). Therefore, a better understanding of the assembly mechanisms of public transit microorganisms, as well as potential factors governing the relationships between the public transit environment, commuters, and microbial community, will pave the way toward minimizing pathogen transmission in public transit (Leung et al., 2021).

Microorganisms are ubiquitous in the environment and colonize various surfaces (Abdulai et al., 2020). High-touch surfaces such as handrails, seats, door handles, and steering wheels are hotspots for microbial contamination in public transportation systems (Cave et al., 2021). The confined nature of transportation vehicles, such as buses, trains, and taxis, further facilitates the spread of microorganisms through air circulation, direct contact, and fomites (Abdulai et al., 2020; Vega et al., 2024). This is especially concerning in densely populated areas where overcrowding is common, increasing the likelihood of microbial transmission among passengers.

Microorganisms commonly found in public transportation systems cause serious infections (Dewi et al., 2024). Escherichia coli and Staphylococcus aureus, Cutibacterium acnes, Micrococcus luteus, Propionibacterium granulosum, and Staphylococcus hominis, which are frequently isolated from such environments, can cause gastrointestinal and skin infections, respectively. In addition, antibiotic-resistant strains such as methicillin-resistant S. aureus (MRSA) and multidrug-resistant Pseudomonas aeruginosa have been reported, emphasizing the potential role of public transportation systems in the dissemination of antimicrobial resistance (Abdulai et al., 2020; Dewi et al., 2024). The presence of these pathogens on shared surfaces poses significant public health challenges, particularly for immunocompromised individuals, children, and the older adults (Aliu Olalekan Olatunji et al., 2024).

The public health risks associated with microbial contamination in transportation systems are further compounded during infectious disease outbreaks. Occurrences during the COVID-19 pandemic emphasized the critical role of public transportation in the spread of respiratory pathogens (Campisi et al., 2024). Crowded and poorly ventilated spaces within transit vehicles provided ideal conditions for transmission, emphasizing the urgent need for enhanced sanitation measures and hygiene awareness (Du et al., 2024). Despite the evident risks, comprehensive studies examining the extent of microbial contamination in public transportation systems, particularly in low- and middle-income countries (LMICs), are lacking. Many urban areas in LMICs experience inadequate maintenance and poor public transport and sanitation, creating an ideal environment for microbial growth and spread (Abdulai et al., 2020). Therefore, this study aimed to examine the presence of microorganisms in public transportation systems in Wukari and their public health risk.

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Materials and Methods

Study area

This study was conducted in Wukari. It is one of the sixteen local government areas of Taraba State, located at latitude 7.53′ 43′′N, longitude 9.47′ 59′′E, with a population ranging from 5000 to 10,000. The location of Wukari in the Times Comprehensive Atlas of the World is plate 86F8. It is one of the major towns in Taraba State and has an area of 4,308 km² and a population of 241,545 according to the 2006 census (Agwaranze et al., 2024). It is bordered by Takum to the south, Donga to the east, Ibi to the north, the Benue State Ukum Local Government Area to the west, and Gassol to the northeast. It is an important town in Taraba State, characterized by agricultural and commercial activities (Brown et al., 2022).

Sample collection

Before sample collection, permission was sought from each of the park’s chairmen, and drivers were duly informed about the study’s aim and objectives. Sampling was conducted multiple times (during peak and off-peak hours) in the morning and evening. Eighteen (Hasssan et al., 2022) samples were collected from (Bitew and Bati, 2021) vehicles (2 buses, 3 taxis, and 1 washed car). The high-contact surfaces (seats, handrails, and door handles) of each vehicle were sampled using sterile cotton swabs moistened with normal saline, which were used to aseptically collect samples; the washed car served as the control. The moistened swabs are firmly rubbed over a predetermined 20 cm² surface area using parallel strokes with slow rotation to ensure uniform coverage. A sample was also collected from a washed yet unused vehicle, which served as the control.  After collection, the swabs were securely capped, appropriately labelled, and stored in ice boxes, and promptly transported to the microbiology laboratory for analysis as described by John et al. (John and Adegoke, 2018). A total of 15 swab samples were collected, with five samples randomly taken from each of the following parks: Wukari Main Park (New Market), Takum Junction Park, and Izala Mosque Park. This sampling strategy ensures a diverse representation of microbial contamination across different public transportation hubs in Wukari, providing a comprehensive basis for microbial analysis.

Media preparation

The media preparation followed the manufacturer’s guidelines (Brown et al., 2023). First, 46.53 g of Sabroud Dextrose Agar was accurately weighed using a precision weighing balance. The agar was then dissolved in 1000 ml of distilled water. The mixture was homogenized using a hot plate and stirrer. Finally, the media were sterilized in an autoclave for 15 minutes at a temperature of 121 °C and a pressure of 15 pounds per square inch. Every medium was created according to the manufacturer’s instructions.

Serial dilution and inoculation of the bacteria

The media preparation followed the manufacturer’s guidelines (Brown et al., 2023). First, 46.53 g of Sabroud Dextrose Agar was accurately weighed using a precision weighing balance. The agar was then dissolved in 1000 ml of distilled water. The mixture was homogenized using a hot plate and stirrer. Finally, the media were sterilized in an autoclave for 15 minutes at a temperature of 121 °C and a pressure of 15 pounds per square inch. Every medium was created according to the manufacturer’s instructions. Swabs were immersed in 9 ml (0.1%) sterile saline for 5 minutes while constantly shaking to ensure proper mixing of samples. A 10-fold serial dilution of the samples was prepared. Each test tube was vigorously shaken before being transferred. Subsequent serial dilutions were made to 10 (Bruno et al., 2022) (Vega et al., 2024). Each 0.1 ml of the seventh dilution (107) of each of the sample dilutions was taken aseptically and inoculated on a solidified Nutrient agar and Sabouraud dextrose agar (SDA) for the isolation of bacteria and fungi, respectively. The inoculated plates were inverted and incubated at 37 °C for 24 hours and 48–72 hours for bacteria and fungi, respectively (Abdulai et al., 2020). Total viable bacteria counts were enumerated based on a colony-forming unit per cubic meter (cfu/m³).

Characterization and identification of fungi

Pure fungal isolates were identified by studying their macroscopic and microscopic characteristics. The pigmentation of the front and the reverse side, texture, topography, and growth rate of each culture were considered for macroscopic identification as described by Bitew and Bati (2021). Fungal isolates were microscopically examined after staining with Lactophenol Cotton Blue (LCB).

Lactophenol cotton blue staining

An LCB staining technique was employed to ascertain the microscopic characteristics of fungal mycelia. A small drop of a small piece of the fungal colony was placed on a grease-free glass slide containing lactophenol, cotton blue. A coverslip was placed over the slide, and the excess lactophenol blue stain was blotted out using blotting paper. The slide was examined under the microscope using 10× and 40× objectives. Microscopic features, such as macro- and micro-conidia, chlamydospores, reproductive structure morphology, and hyphae nature, were examined. Using mycology atlases, features observed in the stained slide were compared to known fungal features (Agwaranze et al., 2024).

Statistical analysis

Data were entered into Microsoft Excel Office 2021 and presented as frequency and percentages in tables.

Ethical approval

Before collecting the samples, the Head of Department Microbiology provided a letter of introduction that was submitted to the Education Secretary Wukari Local Government, through the administration of National Union of Road Transport Workers Wukari for clearance. Samples were gathered, as well as collection schedules, at the Wukari motor parks.

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Results

The total viable bacteria count is presented in Table 1. The seat samples showed bacterial counts of 1.4 × 10⁷ cfu/m³ in the Sienna and 3.6 × 10⁷ cfu/m³ in the bus. The handrails in the bus had a greater bacterial concentration, with 5.4 × 10⁷ cfu/m³, while handrails in the Sienna had a high bacterial count. In the evening, seats, handrails, and door handles were high in both the Sienna and the bus. The control sample showed no bacterial growth on the seats. However, the control handrail and door handle samples recorded 1.0 × 10⁷ cfu/m³. Fungal contamination was observed in different areas of public transit vehicles during the morning and evening. The results indicate no fungal growth (NG) in most of the sampled surfaces, including the seats, handrails, and door handles of both the Sienna and bus during the morning period. Similarly, no fungal growth was detected on the seats and handrails of both vehicles, as well as on the door handle of the Sienna, in the evening. However, fungal growth was observed on the bus seats and door handle in the evening, both recording a fungal count of 1.0 × 10⁷ cfu/m³.

The phenotypic identification of bacterial isolates is shown in Table 2. These bacteria were characterized based on colonial morphology, Gram staining reactions, and a series of biochemical tests, including catalase, oxidase, citrate utilization, indole production, and sugar fermentation patterns. Bacillus spp., Proteus mirabilis, Pseudomonas aeruginosa, E. coli, Acinetobacter baumannii, and Enterobacter aerogenes were identified.

The data presented in Table 3 illustrate the distribution of bacterial isolates on vehicle surfaces at different times of the day, specifically in the morning and evening. The results reveal a notable shift in bacterial presence over time, with most species declining by the evening, except for Proteus mirabilis, which showed a significant increase. All six bacterial species were detected on vehicle surfaces in the morning. However, by the evening, Bacillus spp., Pseudomonas aeruginosa, E. coli, A. baumannii, and E. aerogenes were no longer detected.

Table 1. Total viable bacteria count and fungal count.

Table 2. Phenotypic identification of bacterial isolates.

Table 3 also shows the distribution of bacterial isolates in 2 types of vehicles, specifically buses and Sienna cars. The results indicate variations in the presence of bacteria between the 2 vehicle types, suggesting differences in contamination levels and possible environmental factors influencing bacterial survival. The most dominant bacterial species was Proteus mirabilis (35.5%). Bacillus spp., P. aeruginosa, and E. coli had occurrences of 5.9%. However, Acinetobacter baumannii and Enterobacter aerogenes were absent from the bus samples. Conversely, in the Sienna, Proteus mirabilis (29.4%) was again the most prevalent species, followed by A. baumannii (5.9%), while Bacillus spp., P. aeruginosa, E. coli, and E. aerogens were not detected.

The distribution of various bacterial isolates on different vehicle surfaces, including handrails, seats, and door handles, is shown in Table 3. The findings indicate varying levels of bacterial contamination across these commonly touched areas, suggesting that public transport is a potential source of microbial transmission. Proteus mirabilis exhibited the highest prevalence across all surfaces; Bacillus spp. was only detected on seats, with no presence on handrails or door handles. Pseudomonas aeruginosa and E. aerogenes were detected only on the handrails. Escherichia coli was present on handrails and door handles, where as Acinetobacter baumannii was detected solely on seats.

Table 4 shows the prevalence of bacterial isolates within each vehicle type and time of day (Fig. 1).  This study demonstrates that public transportation vehicles in Wukari serve as reservoirs for bacterial pathogens, with the time of day, vehicle type, and microbial ecology influencing the contamination patterns. Morning samples exhibited higher bacterial diversity, including Bacillus spp., Proteus mirabilis, P. aeruginosa, E. coli, and A. baumannii, reflecting residual contamination from previous passengers and environmental deposition. By evening, Proteus mirabilis dominated (100% prevalence in both buses and Sienna), indicating cumulative contamination and selective survival driven by biofilm formation and resilience to environmental stress.

Buses showed higher morning diversity due to greater passenger load and surface area, whereas Sienna vehicles had slightly lower morning diversity but similar evening dominance of Proteus mirabilis, highlighting that microbial persistence depends more on organism traits than vehicle size. The control samples contained E. coli and Enterobacter aerogenes, suggesting potential cross-contamination from cleaning procedures.

Table 3. Microbial distribution of bacteria isolates from vehicles according to the time of the day, type of vehicles and sampling parts of vehicle.

Table 4. Prevalence (%) of bacterial isolates within each vehicle type and time of day.

Figure 1. Prevalence (%) of bacterial isolates bar chart

Table 5 presents the prevalence of bacterial isolates within each vehicle part and time of day. Bacterial contamination on the surfaces of public transport exhibits clear temporal and ecological patterns. In the morning, high-touch surfaces such as handrails, seats, and door handles showed moderate diversity, including Proteus mirabilis, Bacillus spp., Pseudomonas aeruginosa, Escherichia coli, Acinetobacter baumannii, and Enterobacter aerogenes. Prevalence within surfaces ranged from 25% to 50%, reflecting residual environmental deposition, passenger contact, and potential cross-contamination. The door handles were particularly contaminated, indicating their critical role in fomite transmission.

By evening, Proteus mirabilis dominated all surfaces at 100% prevalence, whereas other organisms were absent, demonstrating selective persistence driven by microbial resilience, biofilm formation, and cumulative passenger contact. The frequently touched handrails and door handles consistently supported this persistent pathogen, whereas seats showed lower contamination and transient colonization.

Table 6 presents the macroscopic features of fungal isolates based on colony, color, edge type, size, elevation, and texture. The colonies of Penicillium spp. were light green in color, with entire and small edges and a flat, cottony texture. Aspergillus flavus colonies exhibited a dark brown coloration; the edges were entire, small, and flat.

Table 7 shows the fungal distribution based on time of day, indicating the presence of Penicillium spp. and Aspergillus flavus at different times of the day. No fungal isolates were detected during the morning period (0% occurrence); however, in the evening period, both fungal isolates where detected, with Penicillium spp. and Aspergillus flavus each accounting for 50% of the total fungal isolates. The analysis of fungal isolates across different vehicle types revealed that Penicillium spp. and Aspergillus flavus were only present in buses, whereas no fungal isolates were detected in Sienna vehicles. The distribution of fungal isolates across different vehicle parts indicates the selective presence of fungi in specific areas. Penicillium spp. was found exclusively on door handles, whereas Aspergillus flavus was detected only on seats. Interestingly, no fungal isolates were observed on handrails.

Table 5. Prevalence (%) of bacterial isolates within each vehicle parts and time of day.

Table 6. Macroscopic and microscopic characterization of fungi isolates.

Table 7. Distribution of fungi based on the time of the day, type of vehicle and vehicle parts sampled.

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Discussion

This study demonstrates substantial bacterial and fungal contamination of public transportation vehicles in Wukari, particularly on high-contact surfaces such as door handles, handrails, and seats. While the results clearly indicate that public transportation vehicles serve as reservoirs of microorganisms, a critical interpretation of these findings must account for methodological limitations, potential biases, and broader public health implications.

The consistently higher microbial loads observed in the evening than in the morning strongly suggest that cumulative contamination is driven by repeated human contact, a trend that has been widely reported in public transportation studies (Birteksoz and Erdogdu, 2017). However, the study relied on culture-based methods, which may underestimate total microbial diversity and fail to detect viable but non-culturable organisms. Molecular techniques such as 16S rRNA sequencing or metagenomic analysis would have provided a comprehensive microbial profile and allowed strain-level identification, particularly for opportunistic and antimicrobial-resistant pathogens (Bai et al., 2023).

Another key limitation is the control design. Although control samples were obtained from a washed vehicle, the detection of E. coli and measurable bacterial loads on control handrails and door handles indicates possible cross-contamination, contamination of cleaning water, or inadequate sterilization of cleaning tools. Ribeiro et al. (2024) raised similar concerns, emphasizing that improper sanitation practices can introduce rather than remove microbial contaminants. This limitation weakens the causal attribution strength but does not negate the observed differences between active public transport vehicles and controls, which remained substantially higher.

The predominance of Proteus mirabilis is one of the most notable findings of this study. Unlike other isolates that declined by evening, P. mirabilis increased in prevalence, suggesting enhanced persistence under public transportation conditions. This observation with the findings of Dike et al. (2020) and Abdulai et al. (2020) who reported Staphylococcus aureus and E. coli, respectively, as dominant isolates. The discrepancy may reflect differences in local hygiene behavior, passenger density, cleaning frequency, or climatic conditions. The ability of P. mirabilis to form biofilms and survive on dry surfaces likely contributed to its dominance (Chakkour et al., 2024), raising concerns about its role in environmental persistence and dissemination of AMR.

The isolation of fecal indicator organisms, such as E. coli, E. aerogenes, and Proteus mirabilis, strongly suggests poor hand hygiene among commuters, consistent with reports from Nigeria and Ghana (Abdulai et al., 2020). However, the absence of these organisms in some evening samples may reflect environmental stressors, such as surface drying, temperature changes, or UV exposure, which have been shown to reduce bacterial survival on fomites (Hu et al., 2020). These dynamics highlight the complex interaction between human activity and environmental factors in shaping microbial contamination patterns.

Detection of opportunistic pathogens such as Pseudomonas aeruginosa and Acinetobacter baumannii, even at lower frequencies, is of particular public health concern. Both organisms are strongly associated with healthcare-associated infections and exhibit high levels of AMR (Kyriakidis et al., 2021; Roy et al., 2022). Their presence on public transportation surfaces suggests that community settings may act as bridges for pathogen transfer between hospitals and the general population, a risk often overlooked in infection control policies.

Fungal contamination, which was limited to evening samples, was dominated by Aspergillus flavus and Penicillium spp., supporting previous reports that enclosed transport systems facilitate airborne fungal accumulation (Kim et al., 2022). However, the absence of fungal isolates in morning samples may reflect methodological sensitivity rather than true absence, underscoring the need for air sampling and molecular fungal identification in future studies.

These findings emphasize the urgent need for evidence-based sanitation strategies in public transport systems from a public health perspective. Studies have shown that cleaning high-touch surfaces at least two to three times daily, combined with the use of alcohol-based or chlorine-containing disinfectants, significantly reduces microbial load (Abdulai et al., 2020; Hasssan et al., 2022). Additionally, public awareness campaigns promoting hand hygiene before and after transport use have been demonstrated to lower fomite-mediated transmission in crowded urban settings (Ribeiro et al., 2024).

Overall, this study provides important baseline data on microbial contamination of public transport vehicles in Wukari despite its limitations. The findings highlight public transportation systems as under-recognized environments for pathogen transmission and underscore the need for integrated interventions combining improved vehicle sanitation, behavioral change, and policy enforcement.

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Conclusion

This study focused on examining microorganisms in public transportation systems and their public health risk. This study reveals the presence of bacterial and fungal contaminants in PTVs, highlighting potential health risks. Proteus mirabilis was the most prevalent bacterium, especially in buses and during the evening, while E. coli, P. aeruginosa, and A. baumannii were also detected, particularly on frequently touched surfaces such as door handles and seats. Fungal isolates showed Penicillium spp. on door handles and Aspergillus flavus on seats, indicating contamination through human contact and airborne spores. The public transportation system serves as a reservoir for pathogenic microorganisms. The findings emphasize the need for regular cleaning and disinfection of PTVs to reduce microbial contamination and protect public health.

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Acknowledgments

None.

Conflict of interest

The authors have no competing interests to declare.

Funding

The research did not receive any specific grant from funding agencies in the public, commercial, or non-profit sectors.

Authors' contributions

Conceptualization: A.D.I and O.C.S, methodology: A.D.I, U. I.M. and O.C.S, validation: A.D.I, U. I.M., N.C.B, E.I.D., and O.C.S., formal analysis: U. I.M. and O.C.S, investigation: A.D.I, U. I.M. and E.I.D, resources: A.D.I, U. I.M., N.C.B, E.I.D and O.C.S, data curation, U. I.M., N.C.B, E.I.D and O.C.S, writing the original draft preparation: A.D.I and O.C.S, writing review and editing: U. I.M., and E.I.D final review: All authors.

Data availability

All data were provided in the manuscript.

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