Isolation of Fungi and Gram Negative Bacteria from Toothbrushes and Bathroom Bioaerosols

Objective: To identify, using phenotypic methods, FGNB, NFGNB and Candida sp. in toothbrushes, and environmental samples of bathroom air in a group of students from the Dentistry School of the Universidad Central de Venezuela. Material and Methods: Thirty-four toothbrushes were supplied to the cohort during a 60-day period; environmental samples were collected in the rooms where toothbrushes were kept during this period. All samples were processed by traditional methods of microbiological counting isolation and phenotypic identification using selective and differential agar based on the international guidelines of the United States Pharmacopeia (USP) 38. Results: 82.36% of the toothbrush samples were positive to bacteria and fungi and 91.17% of the environmental samples were positive to enterobacteria. Conclusion: It is necessary to establish antiseptic protocols for the management, storage and disinfection of toothbrushes. The high rate of contamination may represent an opportunity for enterobacteria colonization of oral biofilms, reservoir to infection foci and metastatic infections in certain populations.


Introduction
Toothbrushes are conventional and reliable accessories used for oral health, including control and elimination of oral biofilms.However, because of its storage and synthetic composition, the bristles are prone to be contaminated by microorganisms from the oral cavity, surrounding environment and water lines.The contamination begins immediately after its first use by individuals.
The extent of contamination and the amount of microorganisms culture-recovered will depend on the period of time it is used, water quality and cleansing habits of the host [1][2][3][4].
Previous authors reported that toothbrushes stored in dry environments are less contaminated in comparison to humid conditions such as those in bathrooms [2]; a wet environment provides nutrition and appropriate conditions for the aggregation of microorganisms and viability on the bristles.The proximity between the toilet and the toothbrushes provides to microorganisms from fecal bioaerosoles adherence to the surface.Also, the quality of water is an important contamination factor to be considered.It is known that toilet flushing produces bioaerosols capable of surface contamination within the toilet area and bathroom items.Many enteric pathogens are spread by the fecal-oral route and it has been suggested that the fallout of droplets containing fecal material, is an important infection hazard in the bathroom [2,5,6].
In recent years, there has been a remarkable interest in understanding the implications of enterobacteria, not only due to sanitary quality but also because it could represent a risk factor for public health.Another relevant aspect of these bacteria is its resistance mechanisms and their ability to be transmitted among other groups.Some of these mechanisms are the production of β-lactamases A class (TEM, SHV, CTX) and D class (OXA), enzyme expression, mutation and high rate transference plasmids which provides to bacteria the possibility to survive the action of several antibiotics [7].

Study Design
A cross-sectional and descriptive study was developed from January to March 2017, 34 dentistry students were selected to participate.Inclusion criteria were the following: between 18 and 24 years old, healthy individuals (no pre-existent medical conditions) and no oral prosthetic device use.Persons with any pre-existent disease, active periodontitis lesions and use of any oral prosthetic device were excluded.

Toothbrush Samples
Each participant received 1 new sterile Biodent toothbrush TM (Naturovision Bangladesh Ltd., Dhanmondi, Bangladesh) and instructions its use for a continuous 60-day period as their previous toothbrush.They also received a closed survey to indicate the storage habits and the distance from the toilet (in centimeters) of the toothbrushes.After this period, the toothbrushes were collected in sterile plastic bags (Izy pack™; Izy Products LLC, Florida, USA) with 5mL of deionized sterile water.
The active part of the toothbrush was submerged inside the plastic bag and transported for its microbiologic processing.Toothbrushes were incubated inside the plastic bags for 24hrs at 37°C in aerobic conditions.After the incubation, an aliquot (McFarland pattern) of each bag was collected and cultured in two compartment petri dishes with Sabouraud Dextrose Agar (Oxoid™; Thermo Fisher Scientific Inc., Hampshire, United Kingdom) and Endo Agar (HiMedia Laboratories, Mumbai, India).Each medium was prepared and sterilized following the manufacturer's instructions.
All petri dishes were incubated again for 48hrs at 37°C.Then, the colonies were purified by sub-culturing in selective mediums as Chromogenic Brilliance Candida Agar (Oxoid™; Thermo Fisher Scientific Inc., Hampshire, United Kingdom), Hypertonic NaCl 6.5% Sabouraud Agar (Thermo Fisher Scientific Inc., Hampshire, United Kingdom) and Endo Agar (HiMedia Laboratories, Mumbai, India).All colonies were microscopically observed at 400X and 1000X and colored, with cotton blue lactophenol (BD™) for fungus colonies and Gram stain for the bacterial colonies.

Environmental Samples
The bathroom air was analyzed following the passive method of settling plate sedimentation described in chapter 1116 of the United States Pharmacopeia (USP) 38 [9]; each participant received 1 closed Petri dish with sterile Endo Agar (HiMedia Laboratories, Mumbai, India) inside a sterile plastic bag (Izy Products LLC, Florida, USA) and they were instructed to place it uncovered on the top of the toilet tank for a two-hour lapse in the same room where toothbrushes were used an stored.
Once finished this lapse, the participants recapped the Petri dish and place it inside the plastic bag and it was collected for its microbiological processing.The dishes were incubated at 37°C for 48hrs in aerobic conditions.After incubation, the colonies suspected were sub-cultured in Endo Agar for a pure colony recovery and transferred to essay tubes with Kligler Agar (Merck™ KgaA, Darmstadt, Germany) following the manufacturer instructions for its preparation and inoculation.
The samples were verified from 24hrs to 48hrs to assess carbohydrates fermentation, gas and sulfuric acid production.
The control strains used for this study (positive and negative controls) were Pseudomonas aeruginosa ACTT 9027, Escherichia coli ATCC 8739, Candida albicans ATCC 10231 and Staphylococcus aureus ATCC 6538.

Data Analysis
Data was processed and organized following a descriptive analysis and percentual method.

Ethical Aspects
This study was reviewed and approved by the Bioethics Committee of the Dentistry School of the Universidad Central de Venezuela (UCV).Informed consent was obtained from all participants.

Results
The wide range of habits associated to toothbrushes management is shown in this study, specifically in table 1.Up to 90% of toothbrushes were kept inside the bathroom, specifically on top of the sink overexposed to bathroom environment and bioaerosols; another significant percentage (29.4%)were cap covered and 3% of the sample's individuals kept their toothbrush outside the bathroom.In addition, a high percentage of the sample kept their toothbrush in a close range to the toilet (below 1.2 mts); all these variables affect the results After all data was analyzed, over 80% reported positive cultures to bacteria and fungus as expressed in Table 2.The results obtained from the passive method of settling plate sedimentation are shown in Table 3 and the quality of the air inside the bathroom.

Discussion
In this study, toothbrushes were found to be extensively contaminated with a variety of microorganisms.Thirty one (91.1%) of the participants conventionally kept their toothbrushes inside the toilet rooms, same as described by other investigators who observed that 85% of the participants also kept their toothbrushes inside the bathroom.A study conducted in 2012, determined this contamination began with the manipulation and the close distance with toilets; more than 700 bacterial species have been recovered in bathrooms and bioaerosols generated from sink and toilet activation, which increases the total amount of microorganisms in toothbrushes.Also, the family members usually kept these items very close in small recipients, which may represent a cross contamination source between individuals [1,2,10].
Up to manipulation, 52.9% the toothbrushes were used twice per day; in 2008, a group of investigators reported that children commonly brush their teeth twice per day in 39 European countries of the World Health Organization (WHO) evaluated.Toothbrush contamination rate increases with the frequency of use and initially do not have the amount of microorganisms conventionally recovered after its use, which confirms the high rate of contamination in this study.
Since 1977, several reports referred that toothbrushes caps and the wet environment of bathrooms, are determinant factors to increase the isolation of microorganisms in comparison to the capped off items.In this investigation, the amount of bacteria recovered in toothbrushes with or without capping was proportional.Toothbrushes are reservoirs for several microorganisms that may conform mature biofilms on the synthetic bristles and colonize oral structures [1,11,12].
The capacity of Candida spp. to colonize different types of synthetic materials is largely known.In 1981 a group of investigators, recovered Candida albicans in 58% of the samples collected from this items in secluded patients at the Hospital Universitario de Caracas; this finding may be related to the adhesion ability of C. albicans to epithelial cells in oral mucosa of immunocompromised patients by means of proteinase and other virulence factors, as well as resistance to host's inflammatory reaction and presence of phospholipases.In our case, only 8/34 (23.5%) of the toothbrushes were positive; however, the prevalence of C. albicans on toothbrushes was 7/8 (87.5%) [13,14].
Regarding the gram-negative bacteria isolation percentage, 82.3% of the cultures were positive, similar to other results who also obtained 100% of positive cultures in their study.The percentage variation, may respond to the pre-treatment of the collected samples: these authors preenriched the recovered toothbrushes in a brain heart infusion (BHI) solution that could affect the density of microorganisms recovered; however, both investigations concur in the high rate of bacteria recovered from this items [2].
As microbial growth occurred in clumps, the determination of pathogenicity level/infectious dose was not possible.Nonetheless, it is important to emphasize that tooth brushing with contaminated items might lead to the development of diseases depending on the type and quantity of bacteria isolated from toothbrushes and the host's immunologic condition [10,15].
The phenotypic characterization of the toothbrush samples demonstrated 89.2% of the recovered colonies were red, compatible with fermenting gram negative bacilli and 85.2% light pink or colorless, indicating the presence of non-fermenting gram negative bacilli; our results are similar to previous investigators [2,7,10].
Concerning environmental samples, 91.1% of the Petri dishes were positive to gram negative fermenting and non-fermenting bacteria; toothbrushes may get contaminated by initial contact with droplets from fecal bioaerosols, due to sink or toilet activation.However, survival of microorganisms in the toothbrushes bristles is determined by the storage and replacement frequency.Several studies point that this item should be replaced every 3 months, nevertheless the toothbrushes of this study were only used during a 2-month period and heavy contamination was widely demonstrated.For this reason, disinfection of toothbrushes should be a key point to oral health.Previous investigations have determined an efficient disinfection method for these items.In 2016, a study compared diverse chemical agents as disinfectants such as clorhexidine gluconate, sodium hypochlorite, white vinegar and a mouth rinse with essential oils (Listerine ® ); their results pointed towards white vinegar as the most effective method for disinfecting toothbrushes [1,2,6,16,17].
E. coli and Enterobacter spp.were also isolated with 56.2% for each germ.The possibility that aerosols containing enteric pathogens could lead to infection after being swallowed following deposition in the nose or pharynx was suggested by other investigators, who also reported that bacteria carrying droplets produced by flushing a toilet remained airborne for up to 12 min before settling on surfaces throughout a bathroom.Subsequent studies implied coliform bacteria isolation in domestic and hospital toilets, aerolization and deposition of these bacteria on adjacent surfaces.It was demonstrated that large numbers of E. coli remained in the toilet bowl after flushing with the lid open due to the adsorption of organisms to the porcelain surfaces; this findings concur with our results and may explain the high rate of E. coli isolation in this study [17][18][19].
E. coli and Pseudomonas sp, are opportunistic pathogens with multi-drug resistance mechanisms previously described by other investigators.Regarding E. coli, it is important to mention that besides its low-counting in some samples, there are serotypes responsible for enterohemorragic diseases, as O157:H7 serotype transmitted by contaminated water or food.Also, the entero-virulent and entero-pathogenic serotypes may produce diarrheic conditions with histopathologic implications of the intestine.Other pathogens that might be naturally present in the environment may be able to cause diseases in vulnerable subpopulations: the elderly or the very young, patients with burns or extensive wounds, those undergoing immunosuppressive therapy or those with HIV/AIDS [6,7,10,17,19].
The humidity in bathrooms is a key point that allows to bacteria to remain for long periods in the surfaces.In a previous study, Clostridium difficile was isolated on settle plates placed on the floor, cistern and toilet seat during the 90 min after flushing, demonstrating that relatively large droplets are released and can contaminate the immediate environment [20].Other bacteria were also isolated in the same study, such as Salmonella spp.and Pseudomonas aeruginosa, both capable of biofilms formation and present in bathrooms bioaerosols.
Water lines activation including shower hoses, offers an excellent bacterial growth environment in close proximity to a critical end-user exposure route within building drinking water plumbing.However, the health risks associated with and processes underlying the development of biofilms in shower hoses are poorly studied as well as the presence and role of pathogens as P.
aeruginosa.The unique bacterial growth environment and potential relevance to human health demands a better understanding of the biofilms that develop inside water lines [21].
Although the aim of this study was not related to gram negative bacteria isolation from individuals, it is important to quote that recent epidemiological studies have provided convincing evidence to support the hypothesis of host's colonization by fecal bacteria through bathroom aerosols [10,17].Other investigators carried out toilet seeding experiments using Salmonella enteritidis and were able to isolate the organism from the air following flushing with the lid open.Multiple trips to the toilet during diarrhea are likely to result in large numbers of pathogens persisting in the toilet, both on the porcelain surfaces and in the bowl water [5].Although none of the participants of this study had any gastroenteritis condition undergoing, this may be considered a risk factor to airborne contamination and could help to explain the high level of secondary spread of pathogens such as Salmonella sp., Shigella sp. and E. coli species.
There are several reports about isolation of enterobacteria and NFGNB from oral cavity; the colonization of these bacteria may occur at early states of life and can be sustained for the rest of the host's life as it was explained by a previous study when Enterobacteriaceae family bacteria were isolated from children with nail biting habit [22][23][24].
Contaminated toothbrush can be an appropriate item for retention, growth and microbial transportation.Reinfection of oral cavity may occur through any pre-existent injuries of the oral tissue as periodontal diseases, ulcers, or any continuity solution inside the mouth.Also, brushing with a contaminated toothbrush introduces new microorganisms to the oral microbiome while simultaneously reducing the existing normal flora.Fluids and food debris can be drawn into the spaces between the bristles representing a bacterial growth factor [15,17,25].
Previous authors determined that bacteria from Enterobacteriaceae and Pseudomonaceae family are isolated in tongue dorsum in 43% of the studied sample.Some reserachers isolated yeast and gram negative bacilli as resident mouth bacteria in a young and elderly healthy population of Lhasa, but other studies relate the high rate of enterobacteria isolation to patients medically compromised in intensive care units (ICU) and undergoing with cytotoxic therapy.Interestingly, in 2017 a study associated the presence of NFGNB in patients with oral leukoplakia and their role in severe dysplasia cases [22,[26][27][28][29][30][31].

Conclusion
Besides the problem of constant mouth colonization and probable long-term systemic infection, contaminated toothbrushes behaves as reservoir for other bacteria and fungi from the surrounding environment.Toothbrushes coliforms found in this study came from the water and toilet bioaerosols.The health staff and specially de dentist should consider the presence and increasing prevalence of enterobacteria and NFGNB in oral biofilms among healthy and medical compromised patients.Likewise, procedures for decontamination of toothbrushes would prevent the risks of reinfection or infection by other pathogenic microorganisms from the environment and water.