The study included 20 healthy volunteers (9 men, 11 women) with ages ranging from 19 to 62 years and an average age of 35.1 years who correctly followed the instructions for the periocular hygiene for five days. The hygienic protocol consisting on wiping their eyelids twice a day with an independent cleansing wipe for each eye (right and left eye were included). The cleansing wipe served both to wipe the eyelid and to collect the sample eyelid bacterial load of each eye of the volunteers was collected and further analyzed on day 1, 3 and 5. (Fig. 1). The first wipe employed on day 1 was used as internal control
Eyelid microbiota recovered from the right and left eyes separately of 20 volunteers were grown both in TSA and MSA medium in order to both assess bacterial load and observe colony morphology, which denote the heterogeneity of the sample. TSA is a non-specific or differential medium that allows the growth of white colonies of varied morphology belonging to various taxonomic groups. However, the morphologies observed in TSA in this study were quite homogeneous (Fig. 2), suggesting that there was not much variability within the analyzed microbiota that should be composed by few predominant bacterial groups. On the contrary, the MSA is specific for Staphylococcus spp. and allowed the growth of white small opaque colonies showing in general a uniform morphology (Fig. 2). The fact that the phenol red contained in the MSA plates did not turn yellow suggests that most Staphylococcus spp. recovered were catalase negative Staphylococcus spp. /Staphylococcus epidermidis.
Comparison between both eyes
We evaluated the data obtained from both the right and left eye of each volunteer to statistically evaluate significant differences.
Results showed that there were almost no differences between the right and left eyes for either of the evaluated microorganisms at all time points (Fig. 3). Those data indicate that each eye can be evaluated independently, therefore rendering a n = 40.
Total values for total aerobic bacteria and Staphylococcus spp. counts on day 1, 3 and 5 are shown in Table 1. For all the assessed days, the average of CFU/ml per day (n = 40) showed that load of total aerobes was higher than the one corresponding to Staphylococcus spp. (day 1: 3.5·104 vs 2.0·104 respectively, day 3: 1.1·104 vs 7.8·103, and day 5: 4.7·103 vs 2.7·103).
The results led to the inference that the great majority of flora detected belonged to the genus Staphylococcus and only a small fraction belonged to other bacterial genera. This is supported by the observation of the morphology of the colonies grown in the TSA medium, which allows the growth of different bacterial genera, however, the morphology of the colonies appeared quite homogeneous, indicating that mostly the same genera of bacteria were isolated.
Data showed that already on day 3, total aerobes and Staphylococcus spp. load were reduced by 58.71% and 56.50% respectively. This bacterial load was further reduced by 42.15% (aerobes) and 50.37% (Staphylococcus spp.) from day 3 to 5, achieving a total reduction from day 1 to 5 of 80.76% and 82.71% for total aerobes and Staphylococcus spp. respectively. These results which are depicted in Table 1 can also be visualized in Figure 4.
Moreover, it could be observed that on day 5, the percentage of eyes harboring > 90% reduced bacterial load was 42.5% for total aerobes and 42.5% for Staphylococcus spp. (Table 1 and Fig 5).
Positive and negative controls indicate that the employed methodology is valid to collect and determine eyelid bacterial load. The contamination which can be carried to the wipe through the previously disinfected hands is insignificant, since the values obtained for total aerobes from control wipes are several orders of magnitude below the values obtained from eyelid samples (data not shown).
Results visualized according to each individual (Fig. 6) showed differences between the initial levels of microorganisms at day 1, with bacterial loads ranging between 2 orders of magnitude (volunteers 7, 8 and 20 harbored a bacterial load almost 2 orders of magnitude above the bacterial load present in the eyes from volunteer 15).
Shapiro-Wilk’s test of normality and Cullen and Frey’s chart revealed that not all data could be considered normal. Therefore, non-parametric statistics were used to evaluate both parameters (total aerobic bacteria and Staphylococcus spp.). A significant decrease (Mann Whitney Test, p < 0.05) in bacterial load was observed, with values consistently close to 90% for both microorganisms between days 1 and 5, despite inter-individual differences. Some eyes presented a greater reduction between days 1 to 3 (volunteer nº 5 RE, volunteer nº 6 LE, and volunteers nº 14, 16 and 19 both eyes) while in others the greatest reduction occurred between days 3 to 5 (volunteer nº 8 RE and volunteer nº 17 RE and LE) (Fig. 6).
When assessed by individuals, bacterial load quantification on day 1 and 5 showed that 3 individuals (15%) (volunteers nº 5, 7 and 14) showed ≥ 90% reduction for the two microorganisms in both eyes and 4 more individuals (volunteers nº 1, 6, 8 and 17) showed reduction values ≥ 89% for the two microorganisms in both eyes. Moreover, the percentage of individuals showing either total aerobic bacteria or Staphylococcus spp. load reduction of ≥ 90% in both eyes were 40% (volunteers nº 1, 5, 6, 7, 8, 12, 14 and 17). Additionally, the percentage of individuals who showed a reduction of ≥ 90% for aerobes or for Staphylococcus spp. in at least one eye was 65% on day 5 (volunteers nº 1, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 17 and 20). Furthermore, 50% of individuals showed more than a 90% reduction for aerobes in one of the two eyes, and this percentage increased to 65% in the case of Staphylococcus spp. (Fig. 5 and Table 2). On day 3, the percentages of individuals showing bacterial load reduction ≥ 90% were lower but still significant (Table 2).
All individuals showed some reduction in total aerobes and Staphylococcus spp. bacterial load after 5 days of eyelid wiping. The lowest reduction observed corresponded to 13.5% (volunteer nº 11, RE). Volunteers nº 3, 4, 11 and 18 had the lowest values of reduction (Fig. 5).
Moreover, significant reduction of bacterial load for both total aerobic bacteria and Staphylococcus spp. occurred not only between the initial (day 1) and the last day (day 5) of the study, but also between days 1 and 3, as indicated by Mann-Whitney Test (p < 0.05).
These results show a significant decrease in bacterial load on day 3, which further decrease on day 5, demonstrating that eyelid cleansing wipes are effective in reducing bacterial load in both periods of time.