Introduction

The conjunctival sac is an area in direct contact with the external environment, located between the bulbar and palpebral conjunctiva. It is inhabited by a variety of aerobic and anaerobic flora that coexist to maintain the ocular surface microenvironment. However, the normal flora in the conjunctival sac is a potential pathogen, as it can cause severe eye infections under certain circumstances, such as surgery, trauma, or weakened immunity [1]. Diagnosing eye infections in children and infants is challenging, and ensuring compliance with local medication can be difficult. Additionally, intraocular surgeries and postoperative care in children can be complicated and are associated with a higher risk of complications. Therefore, understanding the distribution characteristics of conjunctival sac flora, drug susceptibility, and timely use of antibiotics can help prevent serious eye infections and guide perioperative medication in children. While there have been numerous studies on conjunctival sac flora in normal adults [2,3,4], research on children is comparatively scarce. Hence, the objective of this study was to culture conjunctival sac microorganisms from a large sample of 1258 normal children aged 0–18 years and to analyze the drug susceptibility of commonly isolated strains.

Research object and methods

Research object

Microorganism cultures of conjunctival sac were analyzed for 1258 children (male 643, female 615, P > 0.05; age, 6.21 ± 3.78 years) at Qingdao Eye Hospital of Shandong First Medical University in 2019. They visited the hospital for eye examination and refraction, and did not exhibit any clinical signs of ocular infection, such as conjunctival congestion or edema. Children with eye infections and other infectious symptoms were excluded from the study, as were children who had used antibiotics locally or systemically to control infection in the past month. Informed consent was obtained from the families of all children before the examination, and they were informed about the purpose and process of the study.

Methods

Conjunctival sac sampling

Operators with clean hands or sterile inspection gloves cleaned the eyelids and surrounding skin with sterile normal saline. Participants were instructed to look up while the operator used their index finger to press down on the upper eyelid and their thumb to press down on the lower eyelid skin, fully exposing the conjunctival sac. Next, sterile artificial fiber swabs (3–5 laps) were rolled in the conjunctival sac across the inferior fornix of the bulbar conjunctiva for 3–5 s. The swabs with the specimens were then dipped into a sterile enrichment broth medium after breaking the plastic rod of the swab (2 cm shorter than the tube) and cultured at 37 °C as soon as possible. During the sampling process, the operators took special care to avoid touching the eyelid skin and eyelashes. Additionally, they made sure that the children did not blink during the sample collection. All the sampling procedures were performed by specialists to ensure accuracy and consistency.

Bacterial culture and drug susceptibility test

The collected specimens were inoculated, cultured, identified for strains, and tested for drug susceptibility at Qingdao Eye Hospital of Shandong First Medicine University. In brief, specimens were inoculated onto blood and chocolate plates and cultured in a 5% CO2 environment at 35 ± 2 °C for 24 h. The cultures were smeared and stained to identify the bacteria with a microscope, while the bacterial strains were identified using the Micro Scan Autoscan-4 system (Siemens Healthcare, Germany).

An antimicrobial susceptibility test was performed using the Automatic TDR-200B Bacteria and the antibiotics susceptibility analyzer (Jinyang Science & Technology, Beijing, China) according to the Clinical and Laboratory Standards Institute (CLSI) protocols [5]. Additionally, the broth microdilution method was used to determine the bacterial strain’s susceptibility to antibiotics. Staphylococcus aureus (ATCC29213), Streptococcus pneumoniae (ATCC49619), Haemophilus influenzae (ATCC49247), Pseudomonas aeruginosa (ATCC27853), Escherichia coli (ATCC25922), and Enterococcus (ATCC29212) were used as the quality control strains. The procedures for developing and analyzing the conjunctival sac flora species and their drug susceptibility were conducted according to the M-38A protocol (microdilution method) of the Clinical and Laboratory Standards Institute (CLSI), and the investigators read the minimum inhibitory concentration (MIC) values.

Statistical methods

SPSS 13.0 (IBM) was used for statistical analyses. The normality of continuous variables was assessed using the single-sample Kolmogorov–Smirnov test. Normally distributed variables were presented as mean ± standard deviation and were compared using two independent sample t-tests. Pearson correlation analysis was used for correlation analysis. A p-value of < 0.05 was considered statistically significant.

Results

Distribution characteristics of conjunctival sac flora

Basic information

A total number of 541 children (827 eyes) were found with a positive culture of conjunctival sac flora (32.87%); 293 males (54.15%) and 248 females (45.84%), the difference was not statistically significant (P > 0.05). There were 255 children (47.13%) with a positive monocular conjunctival sac culture (147 males and 118 females); the gender difference was not statistically significant (P > 0.05). There were 285 (52.86%) positive binocular conjunctival sac cultures observed (146 males and 140 females); the gender difference was not statistically significant (P > 0.05). Additionally, 174 chindren (32.16%) with consistent binocular conjunctiva sac flora were reported (84 males and 90 males), and the difference was not statistically significant (P > 0.05).

Microbial flora distribution

42 bacterial species were detected among 827 samples: 91.54% Gram-positive cocci (757 cases), 3.99% Gram-positive bacillus (33 cases), 2.06% Gram-negative bacillus, and 2.42% fungi (Table 1). Staphylococcus (77.51%) was the highest observed microbial flora; S. epidermidis (52.2%) and S. aureus (10.76%) were identified as the predominant staphylococcal species (Fig. 1A). The second was Streptococcus (12.09%), of which S. mitis (5.20%) accounted for the highest proportion (Fig. 1B).

Table 1 Microbial flora distribution in normal children
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Fig. 1
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A Microbial flora distribution in Staphylococcus. B Microbial flora distribution in Streptococcus. C Microbial flora distribution in male and female,Only the top three strains are listed. D Characteristics of main flora at different ages. E Age group characteristics in Streptococcus. F Correlation analysis between S. epidermidis(%) and age

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Positive conjunctival sac flora in single and both eyes

A total of 32 bacterial species were detected in children with positive conjunctival sac flora in a single eye. S. epidermidis (54.90%), S. aureus (9.41%), S. mitis (3.14%), and S. pneumoniae (2.75%) were the most common floras. Children with positive conjunctival sac flora in both eyes reported 34 bacterial species, S. epidermidis (51.92%), S. aureus (11.19%), S. mitis (6.12%), and S. pneumoniae (2.87%). There was no statistically significant difference in the number of S. epidermidis,S. aureus and S. pneumoniae detected between the two groups (P > 0.05). Howere,the prevalence of Streptococcus was found to be higher in children with positive conjunctival sac flora in both eyes compared to those with positive flora in a single eye.

Gender characteristics

Thirty-five bacterial species were detected in male children; S. epidermidis (55.66%), S. aureus (8.60%), and S. mitis (4.52%) accounted for a higher proportion. There were 33 bacterial species detected in female children, and S. epidermidis (49.61%), S. aureus (12.99%), and S. mitis (5.97%) were quite common. There were no significant differences in the number of bacteria detected between different genders (P > 0.05)(Fig. 1C).

Age group characteristics

The proportion of S. epidermitis was the highest in all age groups and increased gradually with age(Fig. 1D). However, in children aged 0–6 (infancy, early childhood, and preschool), Streptococcus(mainly S. mitis) accounted for a higher proportion than S. aureus. Over the age of 6, the proportion of Streptococcus decreased and was lower than that of S. aureus, among which S. pneumoniae became the main strain (Figs. 1D and E).

Children with consistent conjunctival sac flora in both eyes

One hundred seventy-four chindren (32.16%) were presented with consistent binocular conjunctiva sac flora.The average age was 6.16 years. S. epidermidis (63.49%), S. aureus (11.49%), and S. mitis (6.90%) were the main floras.

Correlation analysis between S. epidermidis(%) and age

Correlation analysis identified a significant association between S. epidermidis and age(r = 0.89,P =  = 0.03)(Fig. 1F).

Drug susceptibility analysis of bacterial flora

Bacterial flora

S. epidermidis was sensitive to quinolones, especially gatifloxacin (98.61%) and moxifloxacin (97.22%), and their resistance rate to erythromycin was the highest (87.94%). S. aureus was most sensitive to moxifloxacin and gatifloxacin, whereas Streptococcus was most sensitive to moxifloxacin (96.97%) with the highest resistance rate to tobramycin (92.93%) (Table 2).

Table 2 Drug susceptibility analysis of bacterial flora in children
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Gender analysis

The drug susceptibility trends were almost similar for both genders; Staphylococcus and Streptococcus were both sensitive to quinolones, particularly gatifloxacin and moxifloxacin. However, the Streptococcus resistance rate to tobramycin was higher in female children (Tables 3 and 4).

Table 3 Drug susceptibility analysis of bacterial flora in male children
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Table 4 Drug susceptibility analysis of bacterial flora in female children
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Discussion

Staphylococcus and Propionibacterium were the predominant conjunctival sac floras in normal adults; Corynebacterium and Streptococcus were secondary, while other bacterial species were relatively lesser [6]. Previous studies have reported that the normal flora of the conjunctival sac is established during infancy [7] and that the structural composition of this flora remains constant throughout adulthood [8]. This study observed that S. epidermidis (52.12%), Streptococcus (12.79%), and S. aureus (10.76%) were the significant microbial floras in children, which was consistent with earlier studies [4, 9]. However, our study reported the proportion of S. epidermidis in children was much lower than that in adults. The conjunctival sac flora analysis in different age groups showed that S. epidermidis had the highest proportion in all age groups; the proportion increased gradually with age, similar to earlier research results [4, 10]. The results suggest that while the conjunctival sac flora is established during childhood, the proportion and distribution of the flora are still distinct from those of adults.

Earlier studies showed that Staphylococcus (37.4%) was the main conjunctival sac flora in healthy children, followed by Corynebacterium (30%) and S. pneumoniae (21.4%) [11]; the lower proportion of Staphylococcus could be attributed to the age of the object. Our study sample had a wider age range compared to Ke et al.’s study [11], which only included children aged 0 to 6 years. Additionally, our study found a negative correlation between age and the proportion of Staphylococcus, indicating that the younger the age, the lower the proportion of Staphylococcus. As age increased, the proportion of S. epidermidis gradually increased, approaching the levels observed in adults.

In addition to Staphylococcus, it was found that Streptococcus also had a high detection rate (12.9%) in the conjunctival sac of children, including S. pneumoniae, S. midis, and S. midis/S. sanguis, similar to an earlier study of Zeng et al. [10]. Streptococcus, initially colonizing the conjunctival sac, invaded the ocular tissue causing eye infection and even endophthalmitis under low immunity, trauma, surgery, and other conditions [1]. Streptococcus was the most common endophthalmitis pathogen responsible for ocular trauma in children aged 3–10 years after Staphylococcus [12]. However, Streptococcus endophthalmitis has a worse treatment outcome and prognosis than Staphylococcus endophthalmitis [13]. In contrast to previous studies, this research not only identified the age distribution and change trend of S. epidermidis, S. aureus, and Streptococcus in children but also highlighted the distribution characteristics of Streptococcus in the conjunctival sac of children of different ages. S. mitis was the main bacteria in preschool children (0–6 years old). As the age increased, the proportion of S. mitis decreased, while the proportion of S. pneumoniae increased gradually. It is hypothesized that the differences in proportion may be related to factors such as immune status and hormone levels of children at different ages [14]. These findings have significant reference value for the prevention and treatment of endophthalmitis, particularly in children under 6 years old.

This study detected 35 bacterial species in male and 33 in female children, and the difference between them was not statistically significant. S. epidermidis accounted for the highest proportion of bacterial flora, followed by S. aureus and Streptococcus. No significant differences in the conjunctival sac flora distribution were found in normal children based on gender. However, there were differences in normal conjunctival sac flora between the eyes. The study found that only 32.16% of normal children had the same conjunctival sac flora in both eyes, indicating that most children had an asymmetric distribution of conjunctival sac flora. This may result from multiple factors. Such as the age of the children, the difference of culture condition, the error of sampling, and so on. However, we also speculate that differences in the ocular surface microenvironment may likewise play an important role. The presence of certain bacterial flora in the normal kerato-conjunctival epithelium is believed to be necessary to maintain a stable relationship with the ocular surface epithelium and stimulate the expression of IgA antibodies in the ocular surface mucosa [15]. The IgA antibodies on the ocular surface mucosa play a crucial role in neutralizing antigens and bacterial toxins, as well as in the formation of dendritic cells through interleukin-10. Therefore, it is important to take into account the individual ocular micro-environmental factors when analyzing the conjunctival sac flora. Although dry eyes have a limited effect on the distribution of conjunctival sac flora, they can increase the bacterial population [16], ultimately affecting the detection rate. Unfortunately, the study did not include individuals with dry eye signs, and a quantitative analysis of tear quality was not conducted, which may be another factor contributing to the low consistency rate of the conjunctival sac flora in both eyes.

The drug susceptibility analysis in the study found that S. epidermidis, S. aureus, and Streptococcus were highly sensitive to quinolones, especially to gatifloxacin and moxifloxacin, which were consistent with the previous studies [17,18,19]. The development of resistance to moxifloxacin is a slow process that requires a double mutation [18]. However, the widespread use of moxifloxacin in children requires close monitoring to avoid the development of resistance and to select antibiotics rationally. The study found that fusidic acid, tobramycin, and erythromycin drugs showed a high resistance rate. Fusidic acid, a narrow-spectrum and high-efficiency antibiotic, has been the first-line drug for children’s ocular inflammation in Europe, with good antimicrobial activity and low drug resistance to most Staphylococcus [18]. However, in recent years, there has been an increase in resistance to fusidic acid at all levels, which is attributed to the high incidence of Haemophilus in local children [18]. Nevertheless, this study showed an increased resistance of Gram-positive bacteria Streptococcus and even some S. aureus to fusidic acid; the fusF gene may be responsible for the high resistance of Streptococcus [20]. Additionally, we observed that Streptococcus had a higher resistance to fusidic acid in female children than the male children (female 89.13% > male 72.92%), and whether the expression level of the fusF gene differs between genders requires further investigation. Our findings can provide guidance for empirical drug use in remote and underprivileged areas, where drug sensitivity testing may not be feasible. The study also has implications for developing public health policies and strategies for children.

Recent studies have reported that conjunctival sac flora abnormalities are not limited to infectious eye diseases, but also observed in non-infectious eye diseases, such as allergic conjunctivitis [21] and dry eye [22]. This finding indicates that the conjunctival sac resident flora not only has a close association with infectious ophthalmopathy but also plays a crucial role in non-infectious ophthalmopathy and maintaining the normal microenvironment of the ocular surface. Therefore, understanding the normal distribution of conjunctival sac flora is crucial for comprehending the role of ocular surface microenvironment in maintaining ocular surface health and causing ocular surface diseases.

In conclusion, this study provides insights into the distribution characteristics of conjunctival sac flora in normal children aged 0–18 years and the drug susceptibility of commonly detected strains. It provides instructional guidance on ocular antibiotics selection and ocular infection treatment in children. However, the study has limitations, including a relatively small sample size and the lack of multi-center research across different regions. Further research is needed to explore the characteristics of flora drug susceptibility in children of different ages and regions.