Antibiotic Susceptibility Profiles and Bacteriological Risks Associated With Used Toothbrushes: A Case Study of Some Apparently Healthy University Students in Southwestern Nigeria
O. O. Bello, A. Osho, S.A. Bankole, T. K. Bello
American International Journal of Biology, 1(1), pp. 01-12.

Toothbrushes play a significant role in disease transmission and increase the risk of infection since they serve as reservoirs for microorganisms in healthy, oral-diseased and medically ill adults. Investigation was carried out on the antibiotic susceptibility profiles of bacteria isolated from used toothbrushes. Thirty toothbrushes used for at least 5 weeks by thirty University students were collected. Heads of the brushes were soaked in 10 ml of sterile tryptone soya broth (TSB) and agitated by vortex mixing. The bacterial suspension was serially diluted. Plate count agar, MacConkey agar and Mannitol salt agar media were used for the isolation of non-fastidious bacteria, coliforms and staphylococci, respectively, employing the spread plate technique. Biochemical characterization of isolates was carried out using standard methods. Survival ability of bacterial contaminants on the used toothbrushes was also investigated at the 24th hr, 72nd hr and 144th. The disk diffusion method was employed for the determination of the antimicrobial susceptibility profiles of the bacterial isolates. Seven genera of microorganisms were encountered and these include Staphylococcus, Escherichia, Klebsiella, Pseudomonas, Lactobacillus, Leuconostoc and Proteus. Pseudomonas aeruginosa was most prevalent as shown by mean total plate count of 5.0 x 102 CFU ml-1 while E. coli had the lowest prevalence (1.2 x 102 CFU ml-1). It was discovered that S. aureus, S. epidermidis, E. coli and Proteus sp all survived at 144th hr indicating high survival ability, while Lactobacillus sp only survived at 24th hr. There were variations in the susceptibility patterns of the isolates to the various antibiotics. It was determined that 62.5% of the isolates showed susceptibility; twenty percent (20%) of isolates were intermediately susceptible and the remaining 17.5% were resistant. It was concluded that most bacterial isolates from toothbrushes were susceptible to antibiotics but the percentage resistant should be of great concern as it poses high health risk and may generate the spread of antibiotic-resistant bacteria within the family and beyond. Organisms such as some members of the enterobacteriaceae which are not normally associated with oral flora isolated from used toothbrushes investigated in this study should also be of interest.

Key Words: Bacteria, toothbrush, risk, antibiotic, susceptibility, health.

Introduction and Literature Review

Toothbrushes play an essential role in oralhygiene and are commonly found in bothcommunity and hospital settings. Toothbrushesmay play a significant role in diseasetransmission and increase the risk of infectionsince they can serve as a reservoir formicroorganisms in healthy, oral-diseased andmedically ill adults (Glass, 1992a; Downes et al.,2008). Contamination is the retention andsurvival of infectious organisms that occur onanimate or inanimate objects. In healthy adults,contamination of toothbrushes occurs early afterinitial use and increases with repeated use (CDC,2002). Toothbrushes can become contaminatedfrom the oral cavity, environment, hands, aerosolcontamination, and storage containers. Bacteriawhich attach to, accumulate, and survive ontoothbrushes may be transmitted to theindividual causing disease (Caudry et al., 1995;ADA, 2009).

The human oral cavity is colonized by a largervariety of bacteria flora than any other anatomicarea. More than 700 species of bacteria havealready been identified 400 of which were foundin the periodontal pocket adjacent to teeth(Abraham et al., 1990). In the hospital setting,toothbrushes are commonly used for oral care bynurses. There is a need for standardized nursingguidelines to prevent toothbrush contamination,which may increase the risk of infections frompotentially pathogenic microorganisms and isclinically relevant for assessing the risks andbenefits of oral care and informing nursingpractice (Bezirtzogloua et al., 2008). Thetoothbrush is used on a daily basis to clean theoral cavity, so it is a very important piece ofequipment known for proper dental hygiene.Sadly, toothbrushes are most commonly locatednear the bathroom sink, which is a good place toharvest hundreds of microorganisms. No matterhow sanitized the bathroom is, the toothbrushwill still be consistently exposed to the mouthwhich will inevitably result in bacterial growthon the toothbrush. A new toothbrush is usuallynot a favorable habitat for bacteria and fungi, butin some cases, toothbrushes are already slightlyinfected because there is not a regulation thatstates toothbrushes must be sold in a sterilepackage (Glass and Lare, 1986; Efstratiou et al.,2007). Typically, the presence of microbes onthe toothbrush comes from brushing because themouth is a hospitable niche to many kinds ofmicrobes. Therefore, the bacteria will transferfrom the inside of the mouth to the toothbrush(Kozai et al., 1989; Quirynen, 2003). In thisway, the toothbrush is considered a niche formany microbes.

The human body is constantly exposed topotentially harmful microbes. However, the bodyis normally able to defend itself againstinfections through a combination of passive andactive mechanisms (Mehta et al., 2007). Intactskin and mucous membranes function as apassive barrier to bacteria and other organisms.When these barriers are challenged or breached,active mechanisms such as enzymes, digestiveacids, tears, white blood cells and antibodiescome into play to protect the body from disease.Although studies have shown that variousmicroorganisms can grow on toothbrushes afteruse (Fernandes and Cesar, 2006; Devine, 2007),and other studies have examined variousmethods to reduce the level of these bacteria(Bunetel et al., 2000; Quirynen, 2003; Efstratiouet al., 2007), there is insufficient clinicalevidence to support that bacterial growth ontoothbrushes will lead to specific adverse oral orsystemic health effects. In a vulnerablepopulation such as critically ill adults,pathogenic contamination may increase the riskof infection and mortality.

Although some interventions such aschlorhexidine, toothpaste, mouthwash, andultraviolet sanitizers reduce bacterial survival,oral hygiene practices in the hospital setting bynurses vary (Downes et al., 2006). Currently,there are no nursing guidelines related totoothbrush frequency of use, storage, anddecontamination. In the hospital setting, theenvironment as a source of pathogenic bacteria isnow a hot topic and the focus of many currentinfectious disease research studies. Surfaces inclose contact with the patient such as bed frames,countertops, sinks, bedside tables, linens, andmattresses may act as fomites. Toothbrushesmay come into contact with these surfaces priorto or after use thus increasing risk (Fernandesand Cesar, 2006). In clinical practice, Devine(2007) has observed that there is no standardizednursing protocol for the storage or replacementof toothbrushes and that some commonlyobserved nursing practices include storing thetoothbrush in the bath basin with otherbathing/personal supplies and linens, in a papertowel, in a plastic wrapper, on the bedside table,next to the sink, and in an oral rinse cup at thebedside.

These practices may impact the contamination oftoothbrushes. Toothbrushing plays an importanteveryday role for personal oral hygiene andeffective plaque removal. Appropriate toothbrushcare and maintenance are also importantconsiderations for sound oral hygiene. The ADArecommends that consumers replacetoothbrushes approximately every 3–4 months orsooner if the bristles become frayed with use. Inrecent years, scientists have studied whethertoothbrushes may harbor microorganisms thatcould cause oral and/or systemic infection(ADA, 2009). The oral cavity is home tohundreds of different types of microorganisms(Mehta et al., 2007); therefore, it is notsurprising that some of these microorganisms aretransferred to a toothbrush during use.

It may also be possible for microorganisms thatare present in the environment where thetoothbrush is stored to establish themselves onthe brush. Toothbrushes may even have bacteriaon them right out of the box (Dabas, 2008), sincethey are not required to be sold in a sterilepackage. The toothbrush is not naturallyfavorable towards the growth of microbes, butcan sustain bacterial life once they aretransferred onto the toothbrush. Different modesof transfer are responsible for the bacteria on thetoothbrush such as contact with the mouth, crosscontamination, and the bacteria in the toiletcommunity. Organisms that can survive for acertain amount of time on the toothbrush arediverse, ranging from fungus to bacteria to yeast.The environment of the toothbrush is affected bymany conditions whether it is the architecture ofthe toothbrush itself regarding bristles or byadjusting the pH level. These conditions alter thepopulation of bacteria on the toothbrush. Whilethe toothbrush is not the ideal niche for amicrobe, the toothbrush is capable of supportingmicrobial life (Downes et al., 2008). This studyaims at investigating the antibiotic susceptibilityprofiles of bacteria isolated from usedtoothbrushes of apparently healthy Universitystudents in Ago-Iwoye, Southwestern Nigeria.

3.0 Materials and Methods
3.1 Collection of samples

In this study, thirty (30) toothbrushes from thirtydifferent students of Olabisi OnabanjoUniversity, Ago-Iwoye, Ogun State used for toothbrushing for at least 5 weeks were collected for the purpose of determining the microbial population on them

3.2 Isolation of organisms

Toothbrush of every person were rinsed in tap water and transported to the laboratory in sterile bag. Handles of toothbrushes were cut off using aheat sterile scissors, heads of the brushes(containing the bristles) were then soaked in 10ml of sterile tryptone soya broth (TSB) for 60mins This was followed by vortex mixing for 1min to dislodge suspected adherent bacteria. Thebacterial suspension was serially diluted toobtain dilution factors of up to 10-3. The spreadplate technique was employed. One mil (1 ml)each of the dilution factors was obtained using asterile pipette and plated on plate count agar,MacConkey agar and Mannitol salt agar mediafor the isolation of non-fastidious bacteria,coliforms and staphylococci, respectively. Plateswere incubated aerobically at 370C for 24- 48 h(Sammons et al., 2004).

3.3 Identification of isolates

Total viable counts of bacterial population were enumerated. Morphological characteristics of isolates were observed and Gramʼs staining was performed for each isolate.

A. Gram positive cocci of Manitol salt agar were further identified as Staphylococcus aureus and Staphylococcus epidermidis by several biochemical tests such as Catalase test (Collee et al., 1996), Oxidase test (Benson, 2002 ), Coagulase test (Collee et al.,1996), Carbohydrates fermentation test (Stukus,1996; Benson, 2002) and others

B. Gram negative bacilli on MacConkey plates were identified as follows:
a. Gram negative, non lactose fermenting, oxidase positive colonies were considered as Pseudomonas spp (Benson, 2002).
b. Gram negative, lactose fermenting, oxidase negative colonies were considered as Coliform spp.( (Collee et al., 1996)/

Survival of isolates on toothbrushes

Survival ability of bacterial contaminants on used toothbrushes was investigated. Used toothbrushes kept in sterile polythene bag were re-subjected to microbiological assay to determine the natural survival ability of the bacterial contaminants after abandoning the toothbrushes for use for 24 hrs (one day), 72 hrs (three days) and 144 hrs (six days) (Sammons et al., 2004).

Antimicrobial Susceptibility Testing

The Kirby-Bauer (disk diffusion) method wasused to determine the antimicrobial susceptibilityprofiles of the bacterial isolates. Antibioticmultidisks used consisted of Amoxycillin (Amx),Chloramphenicol (Chl), Ciprofloxacin (Cpx),Cloxacillin (Clo), Cotrimoxazole (Cot),Erythromycin (Ery), Gentamycin (Gen),Norfloxacin (Nfx), Rifampicin (Rfp),Streptomycin (Str) and Tetracycline (Tet). Themedium used was Mueller Hinton (MH) agar.Pure cultures of organisms were enriched innutrient broth and incubated at 370C to aturbidity of 0.5 Macfarland standards. The MHagar was inoculated by streaking using sterilecotton swab of each of the cultures. Theantibiotic disks were applied using sterile forcepsand sufficiently separated from each other inorder to prevent overlapping of the zones ofinhibition. The agar plates were left on the benchfor 30minutes to allow for diffusion of theantibiotics and the plates were incubated invertedat 370C for 24 hours. Results were recorded bymeasuring the zone of inhibition and comparingwith the NCCLS interpretive performancestandard for antimicrobial disk susceptibilitytesting (NCCLS, 2004; Bello et al., 2013).

Results and Discussion

Table 1 showed the morphological and biochemical characteristics of bacterial isolates from used toothbrushes.Seven (7) different genera of microorganisms were encountered in the study and these include Staphylococcus, Escherichia, Klebsiella, Pseudomonas, Lactobacillus, Leuconostoc and Proteus. Two staphylococcal species – S. aureus and S. epidermidis were encountered (Table 1).

Table 1: Morphological and biochemical characteristics of bacterial isolates from used toothbrushes

Percentage toothbrush contaminated withdifferent bacterial species was shown in Table 2.Results showed that nineteen of thirty (63%)used toothbrushes investigated were contaminated with Pseudomonas aeruginosamaking the organism the most prevalent in thisstudy. Nine of thirty (30%) used toothbrusheswere found to be contaminated withStaphylococcus aureus; eight of thirty (27%)were contaminated with Leuconostoc sp; sevenof thirty (23%) were contaminated withLactobacillus sp. Other bacterial contaminantsof used toothbrush include Staphylococcusepidermidis which contaminated six of thirty(20%) used toothbrushes; Proteus spcontaminated four of thirty (13.33%), Klebsiellasp also contaminated four of thirty (13.33%) andthe least bacterial contaminant of usedtoothbrushes encountered in this study wasEscherichia coli isolated from three of thirty(10%) of the toothbrushes investigated.

Table 2: Percentage toothbrush contaminated with different species of bacteria

The mean total plate count (in CFU/ml) of bacterial isolates was shown in Figure 1. Results showed that Pseudomonas aeruginosa was most prevalent as shown by mean total plate count of 5.0 x 102 CFU ml-1. This was followed by Staphylococcus epidermidis with mean total plate count of 3.4 x 102 CFU ml-1. The mean total plate counts (CFU ml-1) of Staphylococcus aureus, Leuconostoc sp, Lactobacillus sp, Klebsiella sp, Proteus sp and Escherichia coli were 2.0 x 102 CFU ml-1, 1.9 x 102 CFU ml-1, 1.8 x 102 CFU ml-1, 1.6 x 102 CFU ml-1, 1.4 x 102 CFU ml-1 and 1.2 x 102 CFU ml-1, respectively (Figure 1).

Figure 1:Total bacterial counts of isolates from used toothbrushes

Survival ability of bacterial contaminants on used toothbrushes was investigated and reported (Table 3). Used toothbrushes kept in sterile polythene bag were re-subjected to microbiological assay to determine the natural survival of the bacterial contaminants after abandoning the toothbrushes for use for 24 hrs (one day), 72 hrs (three days) and 144 hrs (six days).

It was discovered that Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli and Proteus sp all survived over a period of six days, though there were reductions in total plate counts but negligible. It was also found that Pseudomonas aeruginosa (which appeared as the most prevalent organism in this study), Leuconostoc sp and Klebsiella sp survived on toothbrushes for 72 hrs but were not isolated on the sixth: they did not survive on toothbrushes over a six-day period.

It was also interesting to find that Lactobacillussp was isolated after one day but did not survivetill 72th hour, and thus 144th hour. Thisestablished that use and re-use of toothbrushesover a long period of time is one of the majorfactors that contribute to the survival ofbacterial contaminants on toothbrushes. This isbecause there could be the tendency that allbacterial contaminants are naturally eliminatedon toothbrushes if not re-used over aconsiderably long period of time and if keptunder aseptic conditions, since bacterialcontaminants could not have possessed theability to survive for that long on nutrient-freesurface.

Table 3: Survival ability of bacterial isolates from used toothbrushes

Table 3 showed the antibiotic susceptibilitypatterns of bacterial isolates from usedtoothbrushes. There were variations in thesusceptibility patterns of the isolates to thevarious antibiotics. Staphylococcus aureus wasfound to be susceptible to ciprofloxacin,erythromycin and norfloxacin but resistant tochloramphenicol and tetracycline. The organismwas, however, intermediately susceptible tostreptomycin, gentamycin, amoxycillin andcloxacillin (Table 4). Staphylococcusepidermidis was susceptible to ciprofloxacin,gentamycin, norfloxacin, streptomycin andtetracycline. It was found to be intermediatelysusceptible to chloramphenicol anderythromycin but resistant to amoxycillin,cloxacillin and cotrimoxazole.

Pseudomonas aeruginosa was susceptible to allbut resistant to three antibiotics namelyerythromycin, gentamycin and streptomycin.Similarly, Leuconostoc sp was found to besusceptible to all but intermediately susceptibleto norfloxacin, stretomycin and tetracyclin. Itwas interesting to find that Lactobacillus spshowed susceptibility to all the antibioticsinvestigated in this study with inhibition zonesranging from 21 + 1.3 mm to 15 + 1.3 mm.Escherichia coli was susceptible tociprofloxacin, erythromycin, gentamycin andstreptomycin; it was intermediately susceptibleto cloxacillin and resistant to amoxycillin,chloramphenicol, cotrimoxazole andtetracycline.

Proteus sp showed no resistance to any of theantibiotics. It showed susceptibility toamoxicillin, cloxacillin, ciprofloxacin,erythromycin, gentamycin, norfloxacin andstreptomycin, and was intermediatelysusceptible to chloramphenicol, cotrimoxazoleand tetracyclin. Klebsiella sp was susceptible tocloxacillin, cotrimoxazole, ciprofloxacin,erythromycin, gentamycin, norfloxacin butresistant to chloramphenicol and tetracyclinewith no zone of inhibition at all. It was,however, intermediately susceptible toamoxycillin and streptomycin. Klebsiella spshowed no zone of inhibition tochloramphenicol and tetracycline, indicatingtheir high level of resistance to the antibiotics(Table 3).

Table 4: Antibiotic susceptibility patterns of bacterial isolates from toothbrush

< 8 = Resistant
9 to 14 = Intermediately susceptible
> 15 = Susceptible
Figure 2 showed the percentage distributions of susceptibilty, intermediate susceptibilty and resistance of bacterial isolates from used toothbrushes.It was determined that 62.5% of the isolates showed susceptibility to the various conventional antibiotics investigated; twenty percent (20%) of isolates were intermediately susceptible and the remaining 17.5 percent were resistant.

Figure 2: Percentage distributions of susceptibilty, intermediate susceptibilty and resistance of bacterial isolate from used toothbrushes to some conventional antibiotics

Organisms such as some members of theenterobacteriaceae which are not normallyassociated with oral flora have been isolatedfrom used toothbrushes investigated in thisstudy. So the infectious microorganismsremaining on the brush can reinfect our mouthagain, some of them can even spread to the restof our body and cause serious health problems,including heart disease, stroke, arthritis,haematogenous, bacterimia and chronic (Warrenet al., 2001; Sammons et al., 2004). A singletoothbrush can be the breeding ground forbillions of bacteria (Abraham et al., 1990;Gabe-Mirkin, 2011). There are attempt toreduce bacterial survival time, detercolonization and inhibit biofilm formation bytoothbrushes containing antibacterial agent havebeen developed and methods for sterilization ofbrushes devised (Caudry et al., 1995; Neal andRippin, 2003).Particular attention was paid to Staphylococciand Pseudomonas like organisms as both ofthese are opportunistic pathogens responsiblefor many nosocomial infections and becausePseudomonas species are also resistant to manydisinfectants in toothpaste including triclosan(Warren et al., 2001). Glass (1992a) found thattoothbrushes from both healthy patients andpatients with oral disease contained potentiallypathogenic bacteria and viruses such asStaphylococcus aureus, E. coli, Pseudomonas spand herpes simplex virus.

He also found toothbrushes contaminated with herpes simplex virus 1 in numbers sufficient to cause an infection in the patient (Glass, 1992b). Bunetel et al. (2000) found that toothbrushes used by patients with existing oral disease quickly became contaminated.between repeated use and bacterial retention ontoothbrushes and that the oral cavity can beinoculated from a contaminated toothbrush.Several of the studies found that toothbrusheswere contaminated before use (Glass and Lare,1986; Glass and Jensen, 1994; Sato et al., 2005).Caudry et al. (1995) found that toothbrushes areheavily contaminated with normal use. Mehta etal. (2007) found that 70% of the toothbrushes intheir study became heavily contaminated withpathogenic microorganisms after use. Studies byboth Taji and Rogers (1998) and Glass (1992b)found extensive toothbrush contamination afteruse except in cases where an oral antiseptic,such as mouthwash, was used immediately priorto brushing. Verran and Leahy-Gilmartin (1996)found that toothbrushes supported manydifferent bacteria and the amount of growth wasvaried.

Conclusion and Recommendation

It was concluded in this study that mostbacterial isolates from used toothbrushes weresusceptible to antibiotics but the percentageresistant should be of great concern as it poseshigh health risk and may generate the spread ofantibiotic-resistant bacteria within the familyand beyond.Organisms such as some members of theenterobacteriaceae which are not normallyassociated with oral flora isolated from usedtoothbrushes investigated in this study shouldalso be of interest. It is recommended in thisstudy that toothbrush should not be shared.Sharing a toothbrush could result in anexchange of body fluids and/or microorganismsbetween the users of the toothbrush, placing theindividuals involved at an increased risk forinfections. This practice could be a particularconcern for persons with compromised immunesystems or existing infectious diseases (Bunetelet al., 2000). Toothbrushes should bethoroughly rinsed with tap water after brushingto remove any remaining toothpaste and debris.Toothbrush should be stored in an uprightposition if possible and allowed to air-dry untilused again. If more than one brush is stored inthe same holder or area, the brushes should beseparated to prevent cross-contamination(Council on Scientific Affairs, 2011).Toothbrushes should not be routinely covered orstored in closed containers. A moistenvironment such as a closed container is moreconducive for the growth of microorganismsthan the open air. Toothbrushes should bereplaced at least every 3–4 months. The bristlesbecome frayed and worn with use and cleaningeffectiveness will decrease (Quirynen, 2003).Children’s toothbrushes often need replacingmore frequently than adult brushes (ADA,2009)


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