Household insect pests as transient vectors of multidrug-resistant Gram-negative bacteria in Dutsin-Ma, Nigeria: a cross-sectional study

¹Department of Microbiology, Federal University Dutsin-Ma, Dutsin-Ma, Katsina State, Nigeria, ²Department of Biology Education, Federal University of Education, Kano, Kano State, Nigeria

^&Corresponding author
Emmanuel Dayo Alabi, Department of Microbiology, Federal University Dutsin-Ma, Dutsin-Ma, Katsina State, Nigeria

Introduction    

Antimicrobial resistance (AMR) is a concerning global public health threat affecting human, animal, and environmental safety. AMR refers to the ability of microorganisms, often pathogenic, to develop physical or biochemical mechanisms that make antimicrobial agents, including antibiotics, ineffective [1]. In 2016, AMR was responsible for 700,000 annual deaths globally. By 2019, this number had nearly doubled to an estimated 1.27 million. Projections suggest that AMR could lead to 10 million deaths per year by 2050 [2,3].

Houseflies (Musca domestica) are among the synanthropic insects (i.e., common insect pests associated with human settlements), especially in areas with poor sanitation, and are known to spread opportunistic pathogens, such as E. coli, E. faecium, K. pneumoniae, P. aeruginosa, Shigella, Salmonella, and S. aureus [4-7]. Furthermore, reports worldwide have described antibiotic resistance patterns and profiles in antibiotic-resistant bacteria (ARB) isolated from cockroaches collected from households, public spaces, and hostels [8-12]. Household insect pests often inhabit damp areas, such as sewers and decaying matter, including carcasses, garbage, and faeces, where they acquire pathogens, making them ideal vectors for transmitting these pathogens to humans. Their exoskeletons and guts serve as reservoirs for ARB, antibiotic resistance genes (ARGs), and mobile genetic elements (MGEs), whereas saliva, vomit, and faeces contribute to the transmission of ARB [4].

Studies have shown that AMR is present in insects, both edible and those commonly found in household environments, such as cockroaches, houseflies, ants, and mosquitoes, which have been reported to carry ARB [13-16]. AMR in synanthropic insects poses a significant public health risk, as they can transmit difficult-to-treat pathogens to humans through direct contact or contaminated food [17]. This risk is heightened in low-and middle-income countries (LMICs) due to various challenges, such as a high prevalence of household insect pests (houseflies and cockroaches) in tropical environments, poor sanitation, and inadequate resources for effective infection prevention and control (IPC) measures.

Due to the significance of houseflies and cockroaches as vectors of foodborne pathogens and ARB, surveillance of these household insect pests is essential in low-resource settings for understanding their role in the transmission of infections and their contribution to the spread of AMR, particularly in high-risk settings such as food processing areas and residential areas. Pathogenic bacteria have been isolated from houseflies in Southwest Nigeria [16,18]; however, the role of household insects as transient carriers of ARB in Dutsin-Ma town has not been extensively studied. This exploratory study was conducted to elucidate the role of household insect pests as transient carriers of antibiotic-resistant Gram-negative bacteria in Dutsin-Ma town. The objectives of this study were to isolate, biochemically characterize, determine the prevalence, and assess the antibiogram of multidrug-resistant (MDR) Gram-negative bacteria on houseflies (Musca domestica) and household cockroaches (Periplaneta americana) collected from various eateries and residential areas in and around Dutsin-Ma, Katsina State, Nigeria.

Methods     

Study design: random sampling of houseflies and cockroaches was conducted to determine the distribution, prevalence, and antibiogram of Gram-negative bacteria associated with synanthropic insects in Dutsin-Ma.

Study setting and population: Nigeria is situated in the western region of Africa, and features varied topography and climate, ranging from arid to equatorial. The country's population comprises numerous ethnic groups with more than 400 dialects. Some of these languages are Yoruba, Hausa, Igbo, Bini, Ibibio, and Tiv. Nigeria shares borders with the Niger Republic to the north, Chad and Cameroon to the east, the Gulf of Guinea of the Atlantic Ocean to the south, and the Benin Republic to the west [19].

This study was conducted in Dutsin-Ma, a local government area in Katsina State, Northwest Nigeria. The town is situated at 12°27'18''N and 7°29'29''E. Dutsin-Ma covers an area of 527 km² with a population of 169,671, according to the 2006 census. The area is primarily inhabited by the Hausa and Fulani communities, which are predominantly engaged in farming, animal husbandry, and trading. The town is located near Zobe Dam, a significant water supply resource in the region [20]. Households in the suburban areas of Dutsin-Ma lack access to improved toilet facilities, leading to widespread open defecation. Waste disposal is primarily managed through designated open refuse dumping, although undesignated refuse dumping is common. These practices create hotspots for houseflies and cockroaches to feed and breed because of their unsanitary behaviors and indiscriminate feeding on waste and food. This leads to increased populations of synanthropic insects in the study area and a heightened risk of disease transmission.

Sample size, sample collection, and microbiological analysis: due to the scarcity of prior prevalence data on insect-derived Gram-negative bacteria in Dutsin-Ma, random sampling of household insects was performed in this pilot study. The study was conducted between the 1^st to 30^th of August 2024; a total of 66 insect vectors were randomly collected, comprising 41 houseflies (Musca domestica) and 25 cockroaches (Periplaneta americana). The houseflies were sampled from various eateries using a sterile aerial net swept across the top of a prepared fruit trap at the sampling sites [21,22], whereas the cockroaches were collected independently from kitchens, toilets, and bathrooms by handpicking with sterile gloves. Samples were obtained from the following locations (Darawa, Kadangaru, Gidan Radio, Opposite the Federal University Dutsin-Ma Take-Off Site (FUDMA), and Miami) within Dutsin-Ma, Katsina State, Nigeria.

The captured insects were transferred into sterile plastic bags and transported to the Microbiology Laboratory at the Federal University Dutsin-Ma, Katsina State, Nigeria, for further processing. The collected insects were placed in sterile airtight bags containing cotton wool soaked in chloroform to suffocate and kill them. Each housefly was aseptically transferred to a sterile test tube containing 2 mL sterile distilled water. The mixture was shaken for 2-5 min to dislodge bacteria from the external body surface, including appendages, to form a stock solution. Ten-fold serial dilutions of the samples were prepared. Test tubes were labeled as 10¹, 10², 10³, 10⁴, and 10⁵, each containing 9 mL of sterile distilled water. From the stock solution, 1 mL was transferred into the 10¹ test tube and mixed thoroughly. Subsequently, 1 mL was transferred from the 10¹ test tube to the 10² test tube, and this process was repeated until the final dilution of 10⁵. Then, 1 mL was discarded into the sink at 10⁵ dilution [23]. As for the cockroaches, each specimen was aseptically placed into a sterile conical flask containing 20 mL of sterile distilled water and shaken for 2-5 minutes to dislodge bacteria from the body surface [21,22]. Ten-fold serial dilutions were then carried out using the resulting stock solutions following the same procedure as described above.

Isolation of bacteria: after carrying out the serial dilutions, 1 mL aliquots from the 10² and 10⁴ dilutions were plated using the pour plate method on Eosin Methylene Blue (EMB) agar and MacConkey agar, and incubated at 37°C for 18 to 24 hours. The media were then observed for growth after incubation, specifically by looking for lactose fermentation on MacConkey Agar and the formation of a green metallic sheen on EMB Agar.

Biochemical characterization of bacteria: distinct colonies (pure cultures) of presumptive Enterobacteriaceae were picked and subjected to Gram staining and biochemical tests, which included: indole, methyl red, Voges-Proskauer, citrate utilization, oxidase, and triple sugar iron tests. Afterward, the pure cultures were stored on double-strength nutrient agar slants at 4°C in the refrigerator for further analyses.

Antibiotics susceptibility testing: antibiotic susceptibility testing (AST) of pure bacterial isolates was performed on Mueller-Hinton agar using the Kirby-Bauer disk diffusion method with standard antibiotic disks (RapidLabs, UK). The bacterial inoculum was prepared in sterile Mueller-Hinton broth (MHB) and standardized to an optical density of 0.5 (OD₆₀₀), equivalent to 1 x 10⁸ CFU/mL. Then, using sterile swabs, 0.5 mL of each inoculum was evenly spread on sterile Mueller-Hinton Agar (MHA) plates. After drying for 5 minutes, antibiotic disks were placed aseptically on the plates which included: ampicillin (25 μg), cephalexin (10 μg), nalidixic acid (30 μg), ciprofloxacin (10 μg), streptomycin (30 μg), gentamicin (10 μg), augmentin (amoxicillin plus clavulanic acid 20 μg+10 μg), ofloxacin (10 μg), septrin (30 μg), and pefloxacin (10 μg). After 24 hours of incubation, the zones of inhibition around the disks were measured and interpreted as resistant, intermediate, or susceptible following the Clinical and Laboratory Standards Institute (CLSI) guidelines [24]. Isolates resistant to three or more antibiotic classes were classified as multidrug-resistant strains.

Data analysis: descriptive and inferential statistics were used to analyze the data in Microsoft Excel (Microsoft Office 2021) and IBM SPSS Statistics version 31.0.1.0 (49), respectively. A two-way analysis of variance (ANOVA) was conducted to assess the types of insect vectors, bacterial species, and their interaction on antimicrobial susceptibility (p ≤ 0.05).

Ethical consideration: this study involved the environmental collection of synanthropic insects, with no involvement of humans or animals as research subjects. Prior to sample collection, verbal informed consent was obtained from the eatery owners and local residents. The study protocol was conducted in accordance with the ethical principles outlined in the Declaration of Helsinki and relevant national guidelines governing environmental research ethics.

Results     

In this study, a total of 66 insect samples were collected, comprising 41 houseflies (62.1%) and 25 cockroaches (37.9%). Across the sampling locations, houseflies were most (24.4% each) frequently collected from Kadangaru and Opposite FUDMA Take-off Campus, while cockroaches were most (32.0%) commonly collected in Darawa and least (0%) from Opposite FUDMA Take-off Campus (Table 1).

A total of 109 Gram-negative isolates were recovered from the samples. Houseflies accounted for 58 isolates (53.2%), while 51 (46.8) isolates were from cockroaches. Escherichia coli was the most (22/25; 80%) prevalent species recovered from cockroaches, while Klebsiella spp. was the most (21/41; 51.2%) frequently isolated from houseflies. Salmonella and Pseudomonas species were evenly (i.e., 6.4% each) distributed in houseflies. Overall, E. coli was the most (41/109; 37.6%) predominant species recovered from both insects. In contrast, Proteus spp. (8 /41; 7.3%) had the lowest frequency of occurrence (Table 2).

Antibiotic resistance patterns varied among the tested isolates. The highest (100%) resistance was observed against nalidixic acid in all bacteria isolated from houseflies. Additionally, Proteus and Pseudomonas species from cockroaches showed complete (100%) resistance to both ampicillin and augmentin (amoxicillin-clavulanic acid). Similarly, high resistance rates were observed in Klebsiella spp. (82.4%) and E. coli (83.4%) recovered from cockroaches against ampicillin and augmentin, respectively. In contrast, isolates from houseflies showed high susceptibility to ciprofloxacin, with rates ranging from 70% to 100%, while those from cockroaches exhibited high susceptibility to pefloxacin, with rates ranging from 71.4% to 100 % (Table 3). However, percentage resistance varied by bacterial species (Figure 1).

Discussion     

This exploratory study investigated the role of household insect pests as transient carriers of antibiotic-resistant Gram-negative bacteria in Dutsin-Ma, Nigeria (Table 1). Our findings showed that both houseflies and cockroaches harbored diverse Gram-negative bacterial species, with E. coli being the dominant species recovered from cockroaches, whereas Klebsiella spp. were the most frequently recovered isolate from houseflies, many of which exhibited resistance to multiple classes of antibiotics. Insect types significantly influenced antimicrobial susceptibility (p = 0.012), whereas the bacterial species did not (p = 0.869). Housefly-associated isolates were generally more susceptible to antibiotics than cockroach-associated isolates, and this pattern was consistent across all bacterial species examined. These findings confirm that synanthropic insects may contribute to the dissemination of ARB within the community and underscore their potential public health significance in low-resource settings.

Enterobacteriaceae was the most prevalent species recovered from cockroaches and houseflies (Table 2). A similar observation was reported in Southwest Nigeria, where Escherichia coli, Klebsiella spp., and Salmonella spp. were isolated from the bodies of houseflies (Musca domestica) in Akure [16]. However, our study reported a lower prevalence than that reported in the study, which recorded 46.3% and 25.4% prevalence rates for E. coli and Klebsiella spp., respectively [16]. Furthermore, Gebreyohans et al. [24] documented the isolation of Enterobacteriaceae from cockroaches in Bonga Town, Ethiopia. The differences in the prevalence of the isolates could be attributed to variations in the study design, environmental conditions, and the broader range of sample collection sources employed in their study, including slaughterhouses, human residences, hospitals, eateries, and refuse dumping sites. In addition to serving as transient vectors for pathogenic bacteria, the housefly microbiome has been reported to be significantly influenced by random environmental events [25,26]. Thus, the frequent contact of synanthropic insects with animals, manure, food, waste dumps, and humans makes them potential mechanical or biological carriers of pathogenic ARB [17,27,28]. The bacterial isolates recovered in this study revealed that Enterobacteriaceae were dominant in houseflies and cockroaches (Table 2). No other group of bacteria currently recognized has had a more profound impact on public health, medicine, and veterinary science worldwide than Enterobacteriaceae [29]. The hairy legs of houseflies are known to trap pathogens, acting as reservoirs that facilitate their transmission to animals and humans [17,30]. This is consistent with our findings, as houseflies accounted for more bacterial isolates (53.2%) than did cockroaches (46.8%).

The multiple antibiotic resistance (MAR) indices of Gram-negative bacterial isolates recovered from houseflies and cockroaches in Dutsin-Ma, Katsina State, Nigeria. All isolates exhibited MAR index values greater than 0.20, ranging from 0.37 to 0.67, indicating that the bacteria originated from environments with frequent antibiotic exposure and substantial antimicrobial selection pressure (Table 3). A comparison between insect vectors revealed that bacterial isolates from cockroaches generally exhibited higher MAR indices than those from houseflies. Overall, the consistently elevated MAR indices across most tested bacteria indicate a high burden of AMR within the study environment and highlight the potential role of these isolates as reservoirs of AMR. Landolsi et al. [12] and Popoola et al. [18] reported a high prevalence of antibiotic-resistant isolates from cockroaches in Tunisia and in Ago-Iwoye, Southwest Nigeria, respectively. Notably, antibiotic resistance was higher in cockroach-derived isolates across different bacterial species than in housefly-derived isolates, indicating that cockroach-derived isolates may serve as a more significant reservoir and vector for the dissemination of resistance determinants in the environment than housefly-derived isolates. Cockroaches have traditionally been considered potential carriers of human enteropathogens because of their unhygienic habits and tendency to feed indiscriminately on waste and human food [27].

Furthermore, the antibiotic resistance profiles of the bacterial isolates showed that E. coli and Klebsiella spp. had the highest cumulative resistance across the tested antibiotics, indicating a substantial burden of AMR among the dominant insect-associated bacterial species (Figure 1). The predominance of resistant E. coli and Klebsiella spp. is of particular public health concern, given their recognized roles in community-and healthcare-associated infections. Complete resistance to nalidixic acid was observed among housefly-derived isolates in the present study. This finding is consistent with the high level of fluoroquinolone resistance (95.2%) reported by Songe et al. [31] among housefly-derived bacterial isolates recovered from fish markets in Zambia. However, studies from other settings have reported lower levels of quinolone resistance; for example, a survey conducted in Mymensingh City, Bangladesh, found that 78% of housefly-derived isolates were susceptible to ciprofloxacin [32]. These findings suggest that household insects may serve as reservoirs of MDR Enterobacterales and other clinically important bacteria. The variability in resistance phenotypes observed in this study reflects the diversity of AMR patterns among bacteria recovered from household insects. Although cockroaches and houseflies may act as environmental reservoirs or carriers of ARB, their role in transmitting AMR to humans was not assessed. Continued surveillance of AMR in insect-associated bacteria is therefore important for understanding the environmental ecology and distribution of AMR.

The limitations of the study include the use of randomly selected sampling areas, a relatively small sample size, and phenotypic (biochemical characterization) of isolates. In addition, the exclusive use of external washing techniques limited the ability to distinguish between surface contamination and true internal colonization in insects. Furthermore, the study design did not fully account for temporal and environmental variability, which may affect the robustness and generalizability of the findings. Future studies should therefore incorporate larger, longitudinal sampling across multiple seasons and ecological settings, as well as insect dissection, gut microbiota analysis, and advanced molecular techniques, to better characterize bacterial pathogens and circulating ARGs, and to clarify the role of household insects in the transmission of AMR in Dutsin-Ma town.

Conclusion     

This study highlights the potential role of household insect pests in the dissemination of ARB and MDR strains in Dutsin-Ma town. However, the distribution and transmission of AMR may also be influenced by environmental and epidemiological factors, such as sanitation conditions, waste disposal practices, antibiotic exposure sources, and food handling behaviors. Therefore, comparisons with findings from different ecological settings should be interpreted with caution. These results reinforce the need for improved hygiene practices, environmental contamination control, antibiotic stewardship, and one health interventions. Future studies should incorporate the gut microbiome of these insects, species-specific antimicrobial susceptibility testing panels, molecular resistance characterization, ESBL confirmation, whole-genome sequencing, and integrated one health surveillance frameworks to better elucidate the role of synanthropic insects in the maintenance and dissemination of AMR across the human-animal-environment interface.

Competing interests     

The authors declare no competing interests.

Authors' contributions     

Ayodele Timilehin Adesoji conceptualized and designed the study; Joseph Oluwajuwonlo Olowoporoku, Mercy Agbo, Kamala Abdullahi, and Emmanuel Dayo Alabi performed laboratory analyses; Ayodele Timilehin Adesoji and Emmanuel Dayo Alabi analyzed and interpreted the data; Emmanuel Dayo Alabi wrote the draft and final manuscript; Kamala Abdullahi, Ayodele Timilehin Adesoji, and Simeon Ipinlaye Johnson reviewed the manuscript. All the authors read and approved the final version of this manuscript.

Tables and figure     

Table 1: distribution of household insect sampled across sampling locations in Dutsin-Ma, Katsina State, Nigeria from 1^st to 30^th of August 2024 (n=66)

Table 2: prevalence of Gram-negative bacteria in household insect pests within Dutsin-Ma, Katsina State, Nigeria (n=109)

Table 3: multiple antibiotic resistance (MAR) index of Gram-Negative bacterial isolates recovered from household insects within Dutsin-Ma, Katsina State, Nigeria

Figure 1: percentage resistance of bacterial isolates from household insects to antibiotics

References