Frequency of biofilm formation in toothbrushes and wash basin junks


Background: Biofilms are known to be resistant to several antibiotics once they are allowed to form on any surface. Aim: To investigate the biofilm forming ability of some bacterial isolates in toothbrushes and wash basin junks. Materials and Methods: A total of 606 students of Federal University of Technology, Yola were provided with new toothbrushes, which were collected after 1 month of usage and screened for biofilm formation. Another 620 swabs were collected from the wash basins of Federal Medical Centre, Specialist Hospital, Federal University of Technology, and students’ hostels in Yola and from some residence in Jimeta, Yola Metropolis; they were all screened for biofilm formation. Results: A total of 38.3% biofilm formation rate was recorded. Three types of bacterial isolates were identified in the biofilms of toothbrushes and wash basin junks, namely Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa at the prevalence rate of 48.0%, 29.1%, and 22.6%, respectively. Overall, 83.3% of the toothbrush biofilm were identified from female students, while 16.7% were from their male counterparts. Statistically, the frequency of biofilm formation showed a significant difference by gender (X 2 = 10.242, P < 0.05). However, frequency of biofilm formation in students’ toothbrush had no association with age (X 2 = 1.0312, P > 0.05). Conclusion: This study identified three microorganisms namely S. aureus, E. coli, and P. aeruginosa that were involved in wash basin junk biofilm formation. The findings also showed that occurrence of biofilm in females’ toothbrushes were significantly higher than in males’ (X 2 = 10.242, P < 0.05).

Keywords: Bacteria, biofilms, wash basin junks, toothbrushes

How to cite this article:
Abubakar AA, Pukuma MS, Abdulazeez FB. Frequency of biofilm formation in toothbrushes and wash basin junks. Ann Trop Med Public Health 2013;6:55-8
How to cite this URL:
Abubakar AA, Pukuma MS, Abdulazeez FB. Frequency of biofilm formation in toothbrushes and wash basin junks. Ann Trop Med Public Health [serial online] 2013 [cited 2016 Aug 15];6:55-8. Available from:

A biofilm is an aggregate of microorganisms in which cells adhere to each other and/or to a surface. These adherent cells are frequently embedded within a self-produced matrix of extracellular polymeric substance (EPS), which is also referred to as slime. It is a polymeric conglomeration generally composed of extracellular DNA, proteins, and polysaccharides in various configurations. Biofilms may form on living or non-living surfaces, and represent a prevalent mode of microbial life in natural, industrial, and hospital settings. [1] The microbial cells growing in a biofilm are physiologically distinct from planktonic cells of the same organism, which, in contract, are single cells that float on a liquid medium.

Microbes form biofilm in response to many factors according to Karantan and Wetrick, [2] including cellular recognition of specific or non-specific attachment sites on a surface, nutritional cues, or, in some cases, by exposure of planktonic cells to sub-inhibitory concentrations of antibiotics. [3] When a cell switches to the biofilm mode of growth, it undergoes a phenotypic shift in behavior, in which large suites of genes are differentially regulated. [4] There are five stages of biofilm development, namely initial attachment, irreversible attachment, first, second, and dispersion. [5],[6]

Different types of microorganism form biofilms, e.g., bacteria, protozoa, fungi, and algae; each group perform specialized metabolic functions. However, some organisms form mono-species films under certain conditions. When bacteria are organized in biofilms, they produce effective substance that individual bacteria are unable to produce alone. [7]

Bacteria living in a biofilm usually have significantly different properties from free-floating bacteria of the same species as the dense and protected environment of the film allows them to cooperate and interact in various ways. [8] Biofilms may be present on the teeth of most animals and human, most often causing tooth decay and gum diseases.

Biofilms have been found to be involved in a wide variety of microbial infections in the body. Going by one study, 8% of all infections infectious process in which biofilms have been implicated include common problems such as urinary tract infections, catheter infections, middle-ear infections, formation of dental plague, [9] gingivitis, [10] coating contact leases, [11] and less common but more lethal processes such as endocarditis, infections in cystic fibrosis, and infections of permanent in dwelling devices such as joint prostheses and heart valves. [12],[13] Recently, it was noted that bacterial biofilms may impair cutaneous wound healing and reduce tropical antibacterial efficiency in healing or treating infections or skin wounds. [14] Also, it has been shown that biofilms are present on the removed tissue of 80% of patients undergoing surgery for chronic sinusitis. [15],[16]

Conventional methods of killing bacteria such as antibiotics and disinfection are often ineffective with biofilm bacteria. The huge doses of antimicrobials required to rid a system of biofilm bacteria are environmentally undesirable and medically impractical since what it would take to kill biofilm bacteria would have been enough to kill the host. Therefore, new strategies based on a better understanding and characterization of some common biofilms is essential for public health purposes.

Therefore, the aim of this study was to investigate biofilm forming ability of some bacterial isolates in wash basin junk and toothbrushes as a step towards improving public health.

Materials and Methods

Study population

A total of 1,226 samples were collected, comprising of 606 toothbrush scrapings and 620 junk swabs. The junk swabs were collected from the wash basins of Federal Medical Centre, Specialist Hospital, student hostels of Federal University of Technology, Yola, and from some residences in Jimeta Metropolis. Six hundred and six (606) randomly selected students of Federal University of Technology (males: 300, females: 306 females, age range: 18-34 years) were enrolled in the study. Before sample collection, written approval from Research and Ethics Committee of the State Ministry of Health was obtained in accordance with the Helsinki Declaration Guidelines. In addition, written consent of the participating institutions and subjects were sought and obtained.

Collection and processing of samples

To obtain the toothbrush scrapings, new brushes were provided to each students. They were instructed to use the brushes for 1 month, after which the brushes were collected and analyzed for biofilm formation. The top of each brush was aseptically removed with a sterile razor and the remaining part of the brush head was immersed into a big test tube containing phosphate buffer solution minutes so as to allow removal of any sediment adhered to the brush. Thereafter, the solution was properly mixed, and a loopful of it was cultured on MacConkey agar, blood agar, and manitol salt agar media. The cultures were incubated aerobically and anaerobically at 37°C for 24 hours.

The junk swabs were inoculated directly onto the three media and incubated aerobically and anaerobically at 37°C for 24 hours.

Bacterial identification and detection of biofilm formation

Overnight cultures on the plate were examined and identified based on colonial appearance, gram staining reaction, and biochemical/serological test results as described by Chessbrough [17] and Bakar and Silverton. [18] To detect biofilm formation, a loopful of isolates from the overnight culture was inoculated into 10 ml of trypticase soya broth with 1% glucose and incubated for 24 hours at 37°C. The tubes were decanted and washed with phosphate buffer saponin and dried. The dried tubes were stained with 0.1% crystal violet, excess stain was removed by inverting the tube, and it was observed for biofilm formation characterized by ring formation.


[Table 1] shows the characterization of bacterial isolates associated with toothbrush and junk biofilm formation. Three pathogenic bacteria were isolated and identified, namely Staphylococcus aureus,  Escherichia More Details coli, and Pseudomonas aeruginosa

Table 1: Identification of bacterial isolates associated with toothbrush and wash basin junk biofilms
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The break down of frequency of bacterial isolates in biofilm formation is depicted in [Table 2]. Out of the total 606 samples cultured from toothbrushes, 122 biofilms were recorded. Of this, 81 (83.5%) was due to S. aureus alone, 16 (16.5%) was due to E. coli, and 25 (20.1%) was formed from interaction between the bacterial isolates. Similarly, 70.0% 20.9%, 26.1%, and 16.7% of the 287 biofilms recorded from 620 junk swabs were caused by S. aureus, E. coli, P. aeruginosa, and mixed isolates, respectively. Statistical analysis of frequency of biofilm formation in relation to the type of bacterial isolate showed no significant difference in distribution (X 2 = 1.093, P > 0.05).

Table 2: Frequency of biofilm formation in relation to bacteria isolates
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The distribution of bacterial isolates in toothbrush biofilm by age and gender is shown in [Table 3]. The highest rate (50.5%) of the biofilm was recorded within the age bracket 18-22 years with 34.0% caused by S. aureus and 16.5% by E. coli. No biofilm formation was recorded within the age group 33-37 years. Statistically, the frequency of bacterial isolates in toothbrush biofilm showed no significant difference with respect to age (X 2 = 1.312, P > 0.05).

Table 3: Distribution of bacterial isolates in toothbrush biofilms by age and gender
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Statistical analysis however showed a significantly higher (83.3%) rate of biofilm in the toothbrushes of the females than in their male counterparts with 16.5% biofilm formation (X 2 =10.241, P < 0.05).


This study revealed that bacterial isolates from wash basin junks are more predisposed to biofilm formation than the isolates from toothbrush scrapings. This observation is evidenced from the overall higher rate (58.8%) of biofilm formation recorded for junk swabs than 25.2% recorded for brush scrapping. The finding is consistent with those of George and Mary, [19] in which wash basin junk was reported to be a better source of biofilm formation.

The reason for higher colonization of biofilm forming bacteria in junks could be attributed to higher nutrients in the junk emanating from the remnants of waste products and water disposal through the wash basin. Microbial populations are found higher in almost all moist environments, and this is an initial step in biofilm formation. Assessing the distribution of bacterial isolates associated with the biofilm, the highest (68.9%) of the overall biofilm formation was due to the activities of S. aureus, while the least (17.8%) was due to mixed isolates.

The reason for higher frequency of S. aureus in biofilm was attributed to higher affinity of the organism for surface polysaccharide, which plays a vital role in biofilm formation. [19],[20] Statistically, Chi square test showed no significant difference in the frequency of biofilm formation with respect to bacterial isolate (X 2 = 1.143, P > 0.05). The frequency of bacterial isolates in toothbrush biofilm by gender showed a higher rate (83.5%) among the females than among their male counterparts (16.5%). Statistical analysis of -Chi square test showed significant difference in biofilm distribution by gender (X 2 = 10.242, P < 0.05). The reason for the difference could probably be attributed to the feeding habit, as the female students are used to food items like sweet candy and chewing gum, while this is less common among males. The sugar deposit of these food items enhances the growth of microorganism, thereby leading to plaque formation.

Similarly, this study revealed a higher prevalence among age bracket 18-22 years. Statistical analysis by Chi square however showed no significant difference in the distribution of biofilm forming bacterial isolate in toothbrush with respect to age (X 2 = 1.0312, P > 0.05). Owing to the ability of bacteria in biofilms to resist antibiotics, [8],[19] we suggest the use of toothpastes that contain antibiotics tufts that will prevent formation of biofilm in the toothbrushes, especially among female students. Such toothpastes should be used regularly after the last meal of the day and sugary diets should be reduced to minimum or avoided completely.


Bacterial isolates commonly associated with biofilm formation in toothbrushes and wash basin in this side of the globe are S. aureus, E. coli, and P. aeruginosa with frequency rates of 68.9%, 18.6%, and 18.3%, respectively. Also, occurrence of biofilm formation in females’ toothbrushes was found to be significantly higher than in their male counterparts.

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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/1755-6783.115198


[Table 1], [Table 2], [Table 3]

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