Microbiological profile and antibiotic resistance trends of preoperative conjunctival swabs: An 8-year retrospective analysis from a North Indian tertiary care ophthalmic center

Abstract

Purpose:

To assess the spectrum of bacterial flora from preoperative conjunctival swabs and analyze their antimicrobial susceptibility patterns.

Design:

This is a retrospective observational clinical study.

Methods:

This retrospective, observational study analyzed conjunctival swab cultures collected 24–48 hours prior to elective ophthalmic surgeries at a tertiary eye care center in North India from January 2015 to December 2022. Aerobic bacterial cultures were performed using standard microbiological techniques. Isolates were identified and tested for antibiotic susceptibility using the Kirby–Bauer disk diffusion method following CLSI guidelines. Data were stratified by patient age groups and analyzed using descriptive statistics and Chi-square testing.

Results:

Of the 8671 conjunctival swabs analyzed, 1287 (14.8%) showed significant bacterial growth. The most prevalent isolates were Coagulase-negative Staphylococci (CoNS, 53.3%), Corynebacterium species (32.9%), and Staphylococcus aureus (10.7%). Methicillin resistance was observed in 40% of Staphylococcal isolates, with significant coresistance to fluoroquinolones, macrolides, and tetracyclines. No Vancomycin resistance was observed. Gram-negative bacteria, though infrequent (1.7%), were predominantly isolated from adult and elderly patients. Resistance trends showed a rising pattern over the 8-year period, particularly against fluoroquinolones and cefoxitin. Agewise, bacterial positivity was similar across pediatric (12.0%), adult (15.1%), and elderly (15.1%) groups (P = 0.1462).

Conclusion:

This large-scale study highlights the predominance of CoNS and Corynebacterium in the conjunctival flora and the rising resistance among Staphylococcal isolates, particularly to fluoroquinolones. These findings underscore the importance of region-specific surveillance and antimicrobial stewardship in guiding perioperative prophylaxis in ophthalmology. Vancomycin and aminoglycosides remain effective options, while empirical use of fluoroquinolones warrants cautious re-evaluation.

The conjunctival sac hosts a diverse array of commensal microorganisms, which constitutes the ocular microbiome, and in usual circumstances offers a protective defense against ocular infections.[1] However, during intraocular and periocular surgeries, these resident bacteria can gain access to sterile intraocular compartments and become pathogens, leading to postoperative infections such as endophthalmitis.[1,2] While the overall incidence of such infections remains low, their clinical consequences are often severe and sight-threatening. In this context, the role of perioperative antibiotic prophylaxis becomes pivotal.[2,3]

The selection of effective prophylactic agents, however, depends on a sound understanding of the commensal bacteria and their evolving antimicrobial susceptibility patterns. Empirical protocols need to be based on regional variations in colonizing flora and resistance trends, leading to optimal prophylaxis and rational antibiotic use. Inappropriate or blanket use of antibiotics not only risks therapeutic failure but also contributes to antimicrobial resistance, a concern increasingly highlighted in national and global stewardship initiatives.[4] Preoperative assessment of the conjunctival microbiota has thus gained importance in guiding targeted perioperative prophylaxis and minimizing the risk of postsurgical infections such as endophthalmitis.

This study investigates the spectrum of bacterial flora isolated from preoperative conjunctival swabs and analyzes their antimicrobial susceptibility patterns. The findings aim to guide rational selection of perioperative prophylactic antibiotics and contribute to strategies that mitigate the risk of postsurgical ocular infections in an evidence-based manner.

Methods

Study setting

Prior to commencing the study, approval of the Institutional Ethics Committee was taken. The study adhered to the Declaration of Helsinki and is a retrospective observational analysis of demographics, and the bacterial culture reports of all the patients whose conjunctival swabs were sent for preoperative screening. Cases were identified using the medical records system.

Clinical evaluation

Evaluation of the patient included clinical and ophthalmological examination. The anterior segment was assessed using a slit lamp, while the posterior segment was evaluated with ultrasound and an indirect ophthalmoscope. Patients with pre-existing infections were excluded from the study.

Microbiological evaluation

Aerobic bacterial culture of preoperative conjunctival swabs was done using standard ocular microbiology techniques. Briefly, conjunctival swabs were collected 24–48 hours before elective surgery using sterile cotton swabs premoistened in sterile saline. Bilateral swabs were collected from all patients except for patients who were one-eyed. These samples were cultured on blood agar and chocolate agar plates and incubated at 37°C, with bacterial growth being assessed at 24 and 48 hours. Growth was considered significant if it was confluent (more than 10 colonies) on both the culture plates.[5] Bacterial isolates were identified using conventional microbiological methods; and antimicrobial susceptibility was tested using the Kirby-Bauer disk diffusion method or finding the Minimum Inhibitory Concentration, as per the latest Clinical and Laboratory Standards Institute (CLSI) guidelines for each bacterial group.[6] The results were documented in reports, detailing swab positivity, identified organisms, and their susceptibility to various antimicrobials.

Preoperative antisepsis

Preoperative antisepsis was performed as per the center’s protocol. Bilateral eye cleansing was done using 10% Povidone Iodine (Betadine) solution prior to surgery, followed by additional cleaning of the eye to be operated with 10% Betadine in the operation room. After draping, a single wash with 5% Betadine was performed just before the surgery.

Statistical analysis

The patients’ demographic and clinical details, bacterial culture reports- identified pathogens and their antimicrobial susceptibility patterns from preoperative conjunctival swabs, all were reviewed and analyzed on a yearly basis. Data were recorded in a Microsoft Excel spreadsheet, including age, sex, conjunctival swab results (positive or negative), identified organisms, and their antibiotic susceptibility and resistance patterns. Statistical analysis was conducted using the Chi-squared test, with a significance level set at a P value of <0.05.

Results

Demographic profile

A total of 8671 conjunctival swab samples collected from January 2015 to December 2022 were analyzed. Among the 8671 samples, 5726 (66%) were from male patients; 105 (1.2%) were from one-eyed patients, and the rest 8566 were from both eyes of 4283 patients.

Among the 8671 samples, 1287 (14.8%) exhibited significant bacterial growth; 1274 isolates were from conjunctival swabs of both-eyed patients (14.9% of 8566), while 13 (12.4% of 105) were from one-eyed patients. The difference between positivity of one-eyed and both-eyed patients was not statistically significant (P value = 0.4754).

In case of samples collected from both eyes, 3454 patients (6908 swab samples) showed bilateral ‘No Significant Growth.” Out of the rest 829 patients, 384 showed significant growth in only one of the eyes, 231 showed the same bacteria in both eyes (462 isolates), and 214 showed different bacteria in different eyes (428 isolates). For further analysis, only ‘bacterial isolates’ and ‘conjunctival swabs’ are taken into consideration irrespective of whether they were from one eyed or both eyed patients; or from one eye or both eyes where both eyes’ samples were taken.

Coagulase Negative Staphylococci (CoNS) were the most prevalent organisms, identified in 686 samples (53.3% of 1287) across all age groups. This was followed by Corynebacterium species (423, 32.9%); Staphylococcus aureus (138, 10.7%); Streptococcus species (14, 1.1%); Pseudomonas aeruginosa (10, 0.8%); Enterobacterales including Klebsiella species, Proteus species, and Citrobacter species (12, 0.9%); and others like nil-fermenting Gram-negative bacilli, Acinetobacter species, and Moraxella species (4, 0.3%). Fig. 1 illustrates the spectrum of organisms isolated from the conjunctival swab cultures.

Figure 1.

Bacteria isolated from the preoperative conjunctival swab cultures

Agewise analysis was done, in which the study subjects were divided into three groups – pediatric age group (<18 years of age), adults (18–60 years), and elderly (>60 years of age). The positivity in these age groups was 87 out of 726 (12.0%), 752 out of 4981 (15.1%), and 448 out of 2964 (15.1%), respectively. The difference in different age groups was not statistically significant (P value = 0.1462). The most common isolate in all age groups were CoNS, followed by Corynebacterium species and Staphylococcus aureus. Colonization by Gram-negative bacteria was seen in adult and elderly subjects, except for one, Pseudomonas aeruginosa, which was isolated from a 16-year-old patient [Table 1].

Table 1.

Bacterial isolates from different age groups of the study

Age/Bacteria Isolated		<18 years		18–60 years		>60 years
Coagulase Negative Staphylococci		48 (55.2)		395 (52.5)		243 (54.2)
Corynebacterium species		24 (27.6)		253 (33.6)		146 (32.6)
Staphylococcus aureus		13 (14.9)		81 (10.8)		44 (9.8)
Streptococcus species		01 (1.1)		09 (1.2)		04 (0.9)
Enterobacterales		00 (0.0)		09 (1.2)		03 (0.7)
Pseudomonas aeruginosa		01 (1.1)		05 (0.7)		04 (0.9)
Others		00 (0.0)		00 (0.0)		04 (0.9)
Total positives		87 (12.0)		752 (15.1)		448 (15.1)
Total samples		726		4981		2964

Figures in parentheses indicate percentage out of total samples

Resistance of isolated organisms to commonly prescribed antibiotics [Fig. 2]

Figure 2.

Graphical representation of resistance patterns of the study isolates

Resistance pattern of Staphylococcus isolates

Methicillin resistance, as indicated by the surrogate marker, Cefoxitin, was seen in 330 out of 824 (40.0%) of the isolates of Staphylococci; it was 40.5% among CoNS isolates (278 out of 686) and 37.6% among S. aureus isolates (52 out of 138). Maximum resistance was seen against gatifloxacin (73.35%), followed by ciprofloxacin (66.75%), moxifloxacin (49.4%), erythromycin (49.2%), tetracyclines (30.5%), clindamycin (22.15%), and chloramphenicol (21.3%). None of the isolates was resistant to vancomycin.

Analysis of antimicrobial resistance pattern over the years from 2015 to 2022 was done for the most common isolates- CoNS and S.aureus. In CoNS, there have been dips and rises in the graph showing antimicrobial resistance over eight years, and a rise in resistance to cefoxitin and gatifloxacin was observed in the year 2022 [Fig. 3]. In the case of S. aureus, there was a rising trend in the proportions of strains that were resistant to fluoroquinolones; and the proportion of S. aureus strains that was resistant to cefoxitin was higher in 2022 than 211 in other years [Fig. 4].

Figure 3.

Resistance pattern of Coagulase Negative Staphylococci over the 8 years

Figure 4.

Resistance pattern of Staphylococcus aureus over the 8 years

Resistance to other group of antibiotics was compared between methicillin-resistant and susceptible isolates. It was found that resistance against aminoglycosides, fluroquinolones, tetracycline, and erythromycin was higher in methicillin-resistant isolates as compared to methicillin-susceptible isolates, and the differences were statistically significant (except in case of amikacin) [Table 2].

Table 2.

Resistance to other groups of antibiotics in methicillin-resistant and susceptible isolates

		Total Resistant Isolates		% of total Staphylococci		Resistant Isolates in MRS		% of MRS		Resistant Isolates in MSS		% of MSS		P
Gentamycin		82		10.0%		46		13.9%		36		7.3%		0.0018*
Tobramycin		91		11.0%		53		16.1%		38		7.7%		0.0002*
Amikacin		8		1.0%		5		1.5%		3		0.6%		0.1931
Ciprofloxacin		552		67.0%		244		73.9%		308		62.3%		0.0005*
Moxifloxacin		404		49.0%		186		56.4%		218		44.1%		0.0008*
Gatifloxacin		602		73.1%		256		77.6%		346		70.0%		0.017*
Tetracycline		255		30.9%		119		36.1%		136		27.5%		0.0095*
Chloramphenicol		173		21.0%		64		19.4%		109		22.1%		0.3566
Erythromycin		404		49.0%		180		54.5%		224		45.3%		0.0097*
Clindamycin		181		22.0%		67		20.3%		114		23.1%		0.3463

MRS=Methicillin-Resistant Staphylococci, MSS=Methicillin-Susceptible Staphylococci. *P is significant

Resistance pattern of Streptococcus isolates

Isolates showed resistance ranging from 14% to 43% to various cefotaxime was the antibacterial to which the highest proportion of Streptococcus strains was resistant (43%). None of the isolates showed resistance to vancomycin, ampicillin, ceftriaxone, or gentamicin. Yearly susceptibility patterns showed rising resistance to tetracyclines, gatifloxacin, and erythromycin.

Resistance pattern of Pseudomonas aeruginosa isolates

Resistance patterns in the range of 10–22% were noted against the anti-pseudomonal antibiotics. None of the isolates showed resistance to polymixin-B, piperacillin, or piperacillin with tazobactam.

Resistance pattern of enterobacterales

Organisms included in this group are Gram-negative bacilli like Escherichia coli, Klebsiella species, Proteus species, and Citrobacter species. These bacteria were isolated from very few samples and showed varied resistance ranging from 5% to 63% against commonly prescribed antibiotics. Maximum resistance was found against ceftazidime, followed by ciprofloxacin and cefuroxime. In the Enterobacterales identified in the study samples, no resistance was observed against gentamicin.

Corynebacterium species: Routine antibiotic susceptibility testing of Corynebacterium species is not done; hence, no data on its resistance pattern are included in the study.

Discussion

Postoperative infections, including endophthalmitis, are severe complications following ophthalmic surgeries, potentially leading to vision loss and significant psychological distress for patients. There are studies and excellent reviews in literature understanding the predispositions, triggers, pathogens and their virulence factors for understanding the dynamics of these infections for better management of patients.[2,3,7,8]

Previous research has consistently demonstrated that organisms isolated from vitreous samples are often similar to the patient’s ocular flora. Speaker et al. reported that in 80% of post-cataract surgery endophthalmitis cases, the source of infection was indistinguishable from bacteria obtained from the eyelid, conjunctiva, or nose.[3,8] Similarly, the Endophthalmitis Vitrectomy Study (EVS) group found that 68% of postoperative endophthalmitis cases involved isolates indistinguishable from the ocular flora.[9] These findings underscore the importance of targeting adnexal structures and their microbial flora for effective prophylaxis and prevention of bacterial endophthalmitis.

We have done a retrospective analysis of data pertaining to pre-operative conjunctival swabs collected over an eight-year period; the study provides valuable insights into the spectrum of ocular surface colonizers and their antimicrobial resistance patterns in patients undergoing elective ocular surgeries at a tertiary eye care center in northern India. The study revealed an aerobic bacterial culture positivity of 14.8% from the conjunctival swabs. There is a variation in the microbial isolation rates reported in similar cohorts across India and Asia.[10,11,12,13,14,15,16] This likely reflects the effects of regional differences in microbial flora, patient demographics, and variations in sampling and culture methodologies in different study settings. Our findings reaffirm that CoNS remain the predominant colonizers, accounting for over 50% of all isolates, followed by Corynebacterium species and S. aureus. These findings are consistent with previous Indian studies, including those by Srivastava et al.[10] and Sharma et al.,[11] which reported CoNS and Corynebacterium as the most frequent commensals in the conjunctival sac, and Jacob et al.,[12] which identified CoNS as the most commonly isolated organism. Studies by authors from Asia and Europe further emphasize the consistency of these findings across different settings.[13,14,15,16]

The identification of Gram-negative bacteria, such as Pseudomonas aeruginosa and Enterobacterales, though infrequent, underscores the need for vigilance, especially in adult and elderly patients where colonization by Gram negatives was prominent. The detection of these organisms, often implicated in aggressive infections, reinforces the utility of regular surveillance in high-risk surgical populations.

Our findings suggest that the composition of the ocular microbiome remains relatively stable across age groups, with no statistically significant differences in positivity rates. However, the presence of Gram-negative isolates in adults and elderly individuals may reflect age-related changes in local immunity or increased prior exposure to antibiotics or hospital environments. Gupta et al.[17] observed the highest microbial positivity rates in the age group of 51–65 years. These findings suggest that demographic factors may influence conjunctival flora composition and, potentially, susceptibility to infection.

The prevalence of methicillin resistance among Staphylococcus isolates was 40% overall, including 40.5% in CoNS and 37.6% in S. aureus. Methicillin resistance was significantly associated with coresistance to fluoroquinolones, macrolides, and tetracyclines, a trend also observed by previous studies from northern India and the broader South Asian region.[10,11,12,13,14] The rising resistance against fluoroquinolones which are commonly used in ophthalmic prophylaxis has also been reported by Jacob et al.[12] and Das et al.[18] and raises concerns about the continued empirical use of these agents.

No resistance to vancomycin was observed among Gram-positive isolates, affirming its continued efficacy and supporting its use as part of intravitreal therapy in endophthalmitis management, as advocated by the Endophthalmitis Vitrectomy Study and the recent Endophthalmitis Management Study (EMS) report.[9,18] Among Streptococcus and Pseudomonas isolates, overall resistance was moderate, except for the resistance against cephalosporins and fluoroquinolones, which was high. This supports the need for culture-based adjustments in empirical protocols in select patient subsets or when high-risk organisms are suspected.

Our findings of antibiotic resistance are in alignment with those of Jacob et al.[12] from India, who have demonstrated a higher sensitivity of conjunctival isolates to chloramphenicol compared to moxifloxacin. Other authors have demonstrated good susceptibility against fluroquinolones. Srivastava et al.[10] and Fenjan et al.[14] have also observed low resistance against aminoglycosides like amikacin.

The multicentric study on antibiotic susceptibility in EMS highlighted the susceptibility of Gram-positive cocci (GPC) to vancomycin and cefazolin, with no statistically significant difference between the susceptibility for the two antibiotics (P = 0.999). In the same study, Gram-negative bacilli (GNB) showed high susceptibility to amikacin, ceftazidime, colistin, and imipenem, with colistin being superior to ceftazidime against GNB (P = 0.047). The preference for vancomycin and ceftazidime combinations by ophthalmologists in India, as observed in this study, underscores the importance of these agents in managing bacterial infections.[18] The results of antibiotic susceptibility of our study corroborate the recommendations of the EMS.

Another recent large study- Antibiotic Resistance Monitoring in Ocular micRoorganisms (ARMOR) surveillance study (2009–2021) from United States, has reported in vitro antibiotic susceptibility rates and trends among conjunctival-sourced isolates. A total of 2214 conjunctival isolates over a period of 13 years have been analyzed. Amongst Staphylococci, 54.8% and 29.3% isolates have been reported resistant to azithromycin and methicillin respectively. Resistance among S. pneumoniae isolates has been reported to be notable for azithromycin (34.0%) and penicillin (28.9%); while P. aeruginosa and H. influenzae isolates have been found to be susceptible to most tested antibiotics. Methicillin-resistant staphylococci have demonstrated greater concurrent resistance to other antibiotics than methicillin-susceptible isolates and exhibited high rates of multidrug resistance (≥74.0%).[19]

A smaller single centre cross sectional study from Tanzania on bacteria colonizing the external ocular surfaces of 224 patients undergoing ocular surgeries have reported Staphylococcus epidermidis (43.4%), Staphylococcus aureus (21.9%), and Pseudomonas aeruginosa (14.3%) to be the most common bacteria. They have observed low proportions of resistance among predominant Gram-positive and Gram-negative bacteria to gentamicin (≤ 25.0%), and low resistance among Gram negative bacteria to 3rd generation cephalosporins (≤ 25.0%) and piperacillin-tazobactam (0.0%).[20]

Findings of the present study have multifaceted implications. Preoperative screening for conjunctival colonization and tailoring antibiotic prophylaxis to local resistance patterns are critical. Resistance trends against fluoroquinolones highlight the need for cautious and judicious use of these antibiotics. The study emphasizes the importance of periodic susceptibility testing to guide prophylactic strategies effectively. Furthermore, the high sensitivity of CoNS and other isolates to vancomycin and aminoglycosides suggests these agents remain reliable management options.

Strengths and limitations

The current study has several strengths and limitations. Conducted at a tertiary care center in Delhi, it provides insights into the spectrum of organisms and antimicrobial resistance patterns primarily reflective of North India. This study is notable for being one of the very few to focus on preoperative conjunctival swabs and for analyzing a large number of samples over a prolonged 8-year period, enabling the detection of microbiological trends and resistance evolution with statistical robustness. Antibiotic susceptibility testing was performed and interpreted according to updated antimicrobial guidelines recommended by CLSI. It is also one of the few Indian studies providing age-stratified analysis and methicillin resistance-associated multidrug resistance in ocular isolates.

However, there were some limitations due to the retrospective nature of our study. The postoperative course of all the study participants could not be assessed. Also, understanding of the variables such as prior antibiotic use, comorbidities, or seasonal influences was limited to the parameters mentioned in the case records. Sharma et al. and Bruttini et al. highlight the role of systemic comorbidities in shaping conjunctival flora and influencing infection risk, suggesting avenues for future research.[11,16] Moreover, cultures for anaerobic bacteria, which are known ocular commensals, were not done, and results of culture of fungi, especially Candida, which are known to be colonizers in some patient subsets, were not included. These again represent opportunities for further prospective, comprehensive studies.

Conclusion

The study contributes to the growing evidence highlighting the critical role of preoperative conjunctival flora in postophthalmic surgery outcomes. The resistance trends underscore the need for targeted antibiotic prophylaxis based on regional resistance patterns and advocates for ongoing surveillance to mitigate the risk of postoperative infections. Integrating findings from a diverse array of studies enhances the robustness of our conclusions, offering a comprehensive understanding of conjunctival flora and its implications for ophthalmic surgery prophylaxis. The findings also support a cautious, evidence-based approach to perioperative antibiotic use in Ophthalmology, in line with antimicrobial stewardship principles.

Conflicts of interest:

There are no conflicts of interest.

Funding Statement

The study was supported by internal funding of Dr. R. P. Centre for Ophthalmic Sciences, All India Institute of Medical Sciences, New Delhi.