Prevalence and Antibiotic Resistance Patterns of Ocular Bacterial Strains Isolated from Pediatric Patients in University Hospital of Campania “Luigi Vanvitelli,” Naples, Italy

Eye infections caused by bacteria are a serious public health problem among pediatric patients. These diseases, if not properly treated, can cause blindness and impaired vision. The study aimed to evaluate the antimicrobial resistance profiles of the main pathogens involved in eye infections. This study involved pediatric patients enrolled at the “Luigi Vanvitelli” University Hospital of Campania in Naples, Italy, between 2017 and 2019. Of a total of 228 pediatric patients, 73 (32%) tested positive for bacterial infection. In terms of strain distribution, 85% were Gram-positive bacteria, while 15% were Gram-negative bacteria. The most frequently isolated strains were coagulase-negative Staphylococci (60.4%), followed by Staphylococcus aureus (16.4%). The isolated bacteria showed a significant percentage of resistance to multiple antibiotics. Therefore, the identification of the causal bacteria and antimicrobial sensitivity tests are mandatory to select the effective drug for the treatment of eye infections and prevent the development of antibiotic-resistant bacteria.


Introduction
Ocular infections and their complications represent an important public health problem [1]. ese diseases are associated with a high degree of visual morbidity and blindness worldwide [2]. e ocular infections distribution in the population is conditional on many factors: (i) the use of contact lenses; (ii) surgery; (iii) trauma; (iv) previous eye infections; (v) obstruction of the nasolacrimal duct; (vi) age; and (vii) dry eye [3,4]. ese infections are commonly observed in pediatric patients, affecting infants and preschool-aged children of both genders [5]. Bacteria are the main cause of ocular infection, although viruses, fungi, and parasites may be involved in the origin of this infection [6,7]. ese microorganisms contribute to 32-74% of eye infections, globally [8]. Bacteria are associated with different types of eye surface infections including keratitis, dacryocystitis, blepharokeratoconjunctivitis, and conjunctivitis [9,10]. e most common bacterial pathogens, involved in pediatric ocular infection are coagulase-negative Staphylococci (CoNS), Staphylococcus aureus (S. aureus), Streptococcus pneumoniae, Pseudomonas aeruginosa (P. aeruginosa), and Haemophilus influenzae [11]. Gram-positive bacteria are primarily responsible for pediatric ocular infection [12]. A prospective study conducted in the United States has revealed that 65% of children have ocular infections caused by Gram-positive bacteria [13]. According to the guidelines, the diagnosis of ocular bacterial infection is based on the examination of the patient's clinical symptoms and laboratory testing [14]. Cultural analysis and antibiotic susceptibility testing are ideal for guiding therapy. Although the guidelines for the treatment of these infections recommend the laboratory procedures, empirical broadspectrum antibiotics treatment is initially used [15]. is contributes to the development of antimicrobial resistance (AMR) among ocular pathogens, which has increased dramatically in recent decades [16]. A national surveillance study started in 2009 (ARMOR) has monitored the resistance profiles among bacterial species that most commonly cause eye infections: Staphylococci species, S. pneumoniae, P. aeruginosa, and H. influenzae. e study reported high rates of AMR, particularly among the Staphylococci species [17]. Ocular diseases, if not treated properly, can cause irreversible damage to the structures of the eye, leading to visual impairment and blindness [18]. Drug-resistant bacteria and the high prevalence of ocular bacterial infections in pediatric patients stress the importance of knowing the causative microorganisms and antimicrobials susceptibility profile. erefore, the goal of our study was to evaluate the etiology and antimicrobial resistance profiles of ocular infection pathogens isolated from pediatric patients in the University Hospital of Campania "Luigi Vanvitelli," Naples.

Sample Collection.
Our retrospective study was conducted on 228 pediatric patients with clinical diagnoses of ocular infections, at the University Hospital of Campania "Luigi Vanvitelli" (UOC) in Naples, Italy, between July 2017 and November 2019. Each patient had undergone a conjunctival sampling. is procedure consisted of rolling a thin cotton swab over the lower fornix of the conjunctival sac. e eye swab was inserted into the transport media and delivered to the bacteriology laboratory and processed.

Bacterial Culture and Identification.
e samples were transferred into 5 ml of Brain-Heart Infusion broth (Oxoid, Hampshire, UK) and incubated overnight at 37°C. e broth was inoculated on blood agar, chocolate agar, MacConkey agar, mannitol salted agar, modified ayer-Martin agar, and Sabouraud glucose agar (Oxoid, Hampshire, United Kingdom). All plates were incubated overnight at 37°C. e chocolate and ayer-Martin agar were maintained in the presence of CO 2 . After 24 hours of incubation, each plate was examined, and negative plates were incubated for an additional 24 hours. Bacterial identification was obtained via Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) (Bruker Dal-tonics, Germany). Identifications were performed according to the manufacturer's instructions. A score higher than 2 allowed a reliable identification of the species [19].

Data Analysis.
Data were analyzed using IBM SPSS software (version 22.0; IBM SPSS Inc., New York, USA). Descriptive statistics were computerized for the study, and variables such as sex and pathogenic bacteria were isolated from the study population. e tables show the frequency of isolated ocular bacteria and also compare the resistance percentage of different antibiotics. For the categorical variables, the chi-square test values <0.05 were considered significant [21].

Ethical Consideration Statement.
Ethical approval by the Human Research Ethics Committee was not requested for this study. e resignation was given as our study used laboratory management data and clinical information on patients, collected from databases.
is is a retrospective study and not directly associated with patients. is study was consistent with the principles of the Helsinki Declaration.

Incidence of Ocular Infections in Pediatric Patients.
In this study, 228 ocular samples, obtained from pediatric patients, were processed. Ocular infections were diagnosed based on the patient's clinical symptoms, redness with mucopurulent discharge. As reported in Table 1, 32% of patients were positive for bacterial growth, while 68% were negative (Table 1). Among the isolated strains, 85% was Gram-positive bacteria, while 15% was Gram-negative bacteria (Table 1). Our study showed a high frequency of ocular infections in males compared to females (31.5%) ( Table 1). In addition, we observed most cases over a 12month group (Figure 1).
Bacterial species, which appertain to 9 genera, were identified by 73 positive cultures. For Gram-positive bacteria, isolates of CoNS (Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus warneri, Staphylococcus hominis, and Staphylococcus lugdunensis) were the most commonly isolated bacteria, followed by S. aureus, Enterococcus species, Streptococcus salivarius, and Bacillus megatherium ( Table 2). For Gram-negative bacteria, Enterobacter cloacae and Serratia marcescens have represented the bacteria frequently encountered, followed by Klebsiella pneumoniae, Escherichia coli, and Pseudomonas aeruginosa (Table 3).
Resistance profile of the most represented bacterial isolates for common drugs of choice for empirical therapy is shown in Table 6. Bacterial strains most resistant to the common treatments of ocular infections were Enterococcus spp. for Gram-positive and K. pneumoniae for Gram-negative.

Discussion
e bacterial ocular infections are commonly diagnosed in pediatric patients, affecting infants and preschool-aged children of both genders [22]. ese diseases affect about 1 in every 8 children every year [23]. Although most cases are self-limiting, in others, about three weeks may be needed to remove the infection [24]. e gold standard for the treatment of bacterial ocular infection should be the identification of the agent and antibiotic susceptibility testing. In order to reduce antibiotic resistance, surveillance data of resistance profiles can guide the choice of appropriate empirical therapy in the absence of culture and sensitivity data [25]. e current analysis shows the incidence of ocular infections among pediatric patients, evaluating the pathogens involved in the infection and the related resistance profiles. In this study, 228 pediatric patients with supposed ocular surface infection were enrolled. Of these, 32% were suffering from an ocular infection. Similar proportions had been observed in India (34.5%), Japan (32.2%), and Iran (37.5%) [26][27][28]. A higher incidence was recorded in Ethiopia (74.7%) and Jordan (54.2%) [29,30].
Sociodemographic and geographic aspects could explain these differences [31]. e high frequency of ocular infections was observed in the 12-month group. e elevated prevalence in this age group is mainly due to poor hand hygiene [32]. It was interesting to note that males (68.5%) were more susceptible to ocular infections, contrary to the study of Teweldemedhin et al. (Ethiopia) [8], where the females represented 55.9%. In another study conducted in Iran, there was no significant difference in the incidence of ocular infection among male and female patients [33]. is variation in the gender rate can differ from country to country. As reported in India, Iran, Ethiopia, and Jordan, Gram-positive bacteria were mainly responsible for bacterial ocular infections among pediatric patients [34]. In our study, CoNS were the most isolated strains (60.4%), and according to the study of Muluye et al., although CoNS constitute the normal flora of the skin and their presence could be due to contamination during sampling, we believe that they represent a source of infection as they are associated with clinical symptoms [35]. In a retrospective study conducted in India, CoNS had caused 45.4% of ocular infections [36]. Similar data were reported in Iran with a prevalence of 40%.  However, fluoroquinolones represent the most used antibiotics in ophthalmic practice, and they remain effective against the strains responsible for ocular infections [38]. e Gramnegative and -positive isolated bacteria in our University Hospital had shown a low rate of resistance to the fluoroquinolones tested (ciprofloxacin, moxifloxacin, and levofloxacin). Antibiotics, belonging to this class, inhibit DNA gyrase and topoisomerase IV, enzymes that are involved in DNA replication [39]. ese drugs are broad-spectrum antibiotics, providing excellent coverage against most ocular pathogens [40]. It is well tolerated on the ocular surface, and the topical use reduces the development of bacterial resistance [41]. ese data can be the starting point for outlining the guideline in the treatment of the pediatric patient's ocular infection. In conclusion, the main goal of our study was to report the bacterial profile and antibiotic susceptibility pattern of ocular infection in pediatric patients in order to know the epidemiology of our hospital, reducing the antibiotic resistance and improving the empirical treatment with factual and statistical information.

Data Availability
Epidemiological data used to support the results of this study are included in the article.

Conflicts of Interest
None of the authors have any conflicts of interest related to this submission.