Abstract
Purpose:
Ocular bacterial infections are important causes of morbidity and vision loss. Early antimicrobial therapy is necessary to save vision, but their efficacy is increasingly compromised by antimicrobial resistance (AMR). Here we assessed the etiology of ocular bacterial infections seen at Massachusetts Eye and Ear and investigated the molecular epidemiology and AMR profiles of contemporary isolates.
Design:
Laboratory investigation.
Methods:
We used a combination of phenotypic tests and genome sequencing to identify the predominant lineages of leading ocular pathogens and their AMR profiles.
Results:
A total of 1,601 isolates were collected from 2014 to 2021, with S. aureus (n=621), Coagulase-negative staphylococci (CoNS; n=234), P. aeruginosa (n=213), Enterobacteriaceae (n=167) and S. pneumoniae (n=95) being the most common. Resistance was high among staphylococci, with methicillin-resistance (MR) detected in 28% of S. aureus and 39.8% of CoNS isolates. Multidrug resistance (MDR) was frequent among MR staphylococci (MRSA, 60%; MRCoNS, 76.1%). The population of S. aureus isolates consisted mainly of two clonal complexes (CCs): CC8 (26.1%) and CC5 (24.1%). CC5 strains carried a variety of AMR markers resulting in high levels of resistance to first-line therapies. Similarly, the population of ocular S. epidermidis was homogenous with most belonging to CC2 (85%), which were commonly MDR (48%). Conversely, ocular S. pneumoniae, P. aeruginosa and Enterobacteriaceae were often susceptible to first-line therapies and grouped into highly diverse genetic populations.
Conclusion:
Our data showed that ocular bacterial infections in our patient population are disproportionately caused by strains that are resistant to clinically relevant antibiotics and are associated with major epidemic genotypes with both community and hospital associations.
Keywords: ophthalmic infections, bacterial pathogens, genomics, acquired resistance genes, multidrug resistance, methicillin resistance
Table of contents statement
Ocular infectious diseases rank among leading causes of vision loss, with bacteria being the most common etiologies of these infections. With high rates of antimicrobial resistance compromising empiric broad spectrum therapies, it was of interest to report the current microbiology of bacterial ocular infections seen at Massachusetts Eye and Ear combined with genomic epidemiology and antimicrobial resistance profiles. This offers important epidemiological data that can help guide empirical treatments to prevent treatment failures.
Introduction
Ocular infectious diseases rank among leading causes of ocular morbidity and vision loss worldwide, with bacteria being the most common etiologies of these infections 1. Ocular bacterial infections are most often empirically treated with antibiotics, but the emergence of antimicrobial resistance (AMR) is compromising efficacy. AMR within and beyond ophthalmology is a costly pandemic that poses a serious public health threat worldwide 2. Antibiotic resistance among ocular bacteria is rapidly increasing 3,4 resulting in treatment failures with serious consequences, such as progressive visual loss and eye evisceration 5,6.
Early initiation of appropriate therapy can improve clinical efficacy and save vision 7. In an effort to limit pathology, patients are typically treated empirically with broad-spectrum therapies prior to specific diagnosis and determination of antimicrobial susceptibility which are currently performed using time consuming and poorly sensitive methods 7,8. In the absence of culture and susceptibility results, antibiotic resistance data from surveillance studies can guide the choice of initial empirical treatment. The Antibiotic Resistance Monitoring in Ocular Microorganisms (ARMOR) study is a multicenter, nationwide, prospective surveillance study initiated in 2009 and designed to extend on the Ocular Tracking Resistance in US Today (Ocular TRUST) study in surveying antibacterial resistance among clinically relevant isolates of Staphylococcus aureus, Coagulase-Negative Staphylococci (CoNS), Streptococcus pneumoniae, Pseudomonas aeruginosa, and Haemophilus influenzae 3,4. These programs found that antibiotic resistance was high among staphylococci, with 35% of S. aureus and 50% of CoNS resistant to methicillin with a high probability of concurrent resistance to other commonly used antibiotic classes.
With high rates of antimicrobial resistance compromising empiric broad spectrum therapies, it was of interest to understand the population structure of these infections, to identify and track genetic lineages that are most pathogenic or enriched in specific diseases as the result of differential tissue tropism, and to characterize the genomic antimicrobial resistance profile of bacterial isolates. Thus, here we report the current microbiology of bacterial ocular infections seen at Massachusetts Eye and Ear (MEE) combined with an investigation of the genomic epidemiology and antimicrobial resistance profiles of contemporary ocular bacterial isolates. We identified the common bacterial species associated with ocular and periocular infections in our service, assessed the overall phenotypic antibiotic resistance profiles, and examined trends over time for ocular bacterial isolates collected over an 8-year period. For a select subpopulation of relevant species, we determined the sequence types and clonal complexes that are predominant in this population of ocular bacteria, as well as their pool of acquired antimicrobial resistance markers.
Material and methods
This is an experimental laboratory investigation study approved by the Mass General Brigham Institutional Review Board (protocol 2019P001001). Protocols for collection of discarded isolates were approved by the MGB IRB (protocol 2021P000695) for prospective sampling, and the need for informed consent was waived. The study was in adherence to the tenets of the Declaration of Helsinki and is in accordance with HIPAA regulations.
Bacterial isolates.
A total of 1,601 bacterial ocular isolates were collected from patients presenting with eye infections at the Massachusetts Eye and Ear from 2014 to 2021. Primary clinical specimens collected from multiple ocular infection sites were obtained by the attending ophthalmologist or resident following institutional guidelines and were submitted to the clinical microbiology laboratory for processing. Bacterial identification was performed by using the MicroScan WalkAway system (Beckman Coulter, Brea, CA) following the manufacture’s protocol. Isolates were stored at −80°C in Microbank cryopreservative tubes (ProLab Diagnostics). Frozen isolates were cultured on 5% sheep blood agar plates (BD Biosciences, San Jose, CA) and incubated at 37°C.
Antibiotic susceptibility testing.
Routine antimicrobial susceptibility testing was performed using the MicroScan WalkAway system (Beckman Coulter, Brea, CA) following the manufacture’s protocol. Quality assurance was performed by concurrently testing CLSI-recommended strains. Susceptibility breakpoints are not available for topical therapy of ocular surface infections using eye drops or for antibiotics given as intraocular injections to treat endophthalmitis. Because of that, antimicrobial susceptibility data from ocular bacteria are routinely interpreted using CLSI-approved interpretive break point criteria that are developed for non-ocular infections treated with oral or intravenous antibiotics 9, and these breakpoints were applied in our study to classify bacterial isolates into susceptible and non-susceptible, NS (NS= intermediate resistant plus resistant isolates combined). The following antibiotics from 9 different classes were tested as appropriate for each bacterial species: erythromycin (macrolide); clindamycin (lincosamide); ciprofloxacin and levofloxacin (fluoroquinolones); ampicillin/sulbactam, amoxicillin/clavulanic acid, aztreonam, cefepime, ceftazidime, ceftriaxone, imipenem, meropenem, oxacillin, and penicillin (beta-lactams); tetracycline (tetracycline); amikacin, gentamicin and tobramycin (aminoglycosides); trimethoprim/sulfamethoxazole (dihydrofolate reductase inhibitor); linezolid (oxazolidinone) and vancomycin (glycopeptide). Staphylococci were categorized as methicillin-resistant or methicillin-susceptible based on oxacillin susceptibility; the break point for oral penicillin was used to determine susceptibility of S. pneumoniae to penicillin. Non-susceptibility rates were calculated by combining the rates of intermediate and resistant isolates. Multidrug-resistance (MDR) was defined as resistance to 3 or more classes of antibiotics.
Genome sequencing and assembly.
To identify genetic lineages of bacteria causing ocular infections, we performed whole genome sequencing on a subpopulation of isolates (n=563) included in this study. Total DNA was purified using the DNeasy DNA extraction kit (Qiagen, Valencia, CA) from an overnight pure culture in 5 mL Brain Heart Infusion broth (BHI). DNA quality was verified on a Bio-Tek Synergy 2 microplate reader (Winooski, VT) prior to quantification using a Qubit fluorometer and dsDNA High-Sensitivity assay kit (Invitrogen, Carlsbad, CA). Library preparation for Illumina sequencing was carried out using the Nextera XT DNA Library Preparation kit (Illumina, San Diego, CA), according to the manufacturer’s specifications. Quality and quantity of each sample library was measured on a TapeStation instrument (Agilent Technologies, Santa Clara, CA). The genomes were sequenced as 2 x 150 bp or 2 x 250 bp reads on an Illumina HiSeq sequencer, according to the manufacture’s specifications with a minimum depth of coverage of 30X. Sequence reads were assembled de novo using CLC Genomics Workbench (CLC Bio, Cambridge, MA).
Prediction of sequence types and antibiotic resistance genes.
We used the Center for Genomic Epidemiology pipeline to obtain confirmation of species identification and sequence type (ST). ResFinder algorithm10 and CARD (Comprehensive Antibiotic Resistance Database) 11 algorithms were used to identify the pool of acquired antibiotic resistance genes in each genome . Clonal complexes (CC) were determined using the MLST data and the goeBURST algorithm12.
Statistical analysis.
Analyses of differences in antimicrobial resistance rates of staphylococci with distinct methicillin-resistance phenotypes were performed by χ2 test using GraphPad Prism Software (San Diego, CA, US). A value of P < 0.05 was considered statistically significant, and n.s., *, ***, and **** represent P greater than 0.05, < 0.05, = 0.0001 and < 0.0001, respectively.
Results
Species distribution across distinct eye infections.
From January 2014 to December 2021, a total of 1,601 bacterial ocular isolates were collected at MEE from multiple ocular infections including keratitis (n=677), conjunctivitis (n=236), periocular infections (n=264), endophthalmitis (n=229) and lacrimal system infections (n=195) (Table 1). This population of isolates was dominated by Staphylococcus aureus (38.8%), followed by Coagulase-negative staphylococci (CoNS; 14.6%), Pseudomonas aeruginosa (13.3%), various Enterobacteriaceae (10.4%), Streptococcus pneumoniae (5.9%), Haemophilus influenzae (4.0%), viridans group streptococci (3.7%), beta-haemolytic Streptococcus (2.3%), Enterococcus spp. (2.1%), non-fermenting Gram-negative bacteria (NF-GNB; 1.9%), and Moraxella spp. (1.0%). Rarer species isolated in small numbers accounted for 2% when combined (Table 1). Conjunctivitis was mainly caused by S. aureus (48.7%), H. influenzae (16.9%) and S. pneumoniae (13.1%), whereas keratitis was mainly associated with S. aureus (31.6%) and P. aeruginosa (25.0%). Half of the lacrimal system infections (e.g., dacryocystitis and canaliculitis) (50.8%), were associated with S. aureus, and less frequently by enterobacteria (22.1%). Periocular infections (e.g., eyebrow abscess, orbital abscess and cellulitis, subperiosteal abscess and preseptal cellulitis), were also mainly associated with S. aureus (60.6%) followed by Enterobacteriaceae species (9.8%). Endophthalmitis was mostly associated with CoNS (47.2%), with S. aureus (14.4%) and viridans group streptococci (13.1%) being the second and third most common etiologies.
Table 1.
Main causative agents of bacterial ocular infections seen at MEE from 2014 to 2021.
| Organism | No. of isolates (%) for the following eye diseases: | |||||
|---|---|---|---|---|---|---|
| Conjunctivitis | Keratitis | Lacrimal system4 | Periocular infections5 | Endophthalmitis | Total | |
| Staphylococcus aureus | 115 (48.7) | 214 (31.6) | 99 (50.8) | 160 (60.6) | 33 (14.4) | 621 (38.8) |
| Coagulase-negative Staphylococcus1 | 10 (4.2) | 75 (11.1) | 6 (3.1) | 35 (1.3) | 108 (47.2) | 234 (14.6) |
| Pseudomonas aeruginosa | 6 (2.5) | 169 (25.0) | 20 (10.3) | 12 (4.5) | 6 (2.6) | 213 (13.3) |
| Enterobacteriaceae2 | 12 (5.1) | 73 (10.8) | 43 (22.1) | 26 (9.8) | 13 (5.7) | 167 (10.4) |
| Streptococcus pneumoniae | 31 (13.1) | 42 (6.2) | 5 (2.6) | 4 (1.5) | 13 (5.7) | 95 (5.9) |
| Haemophilus influenzae | 40 (16.9) | 11 (1.6) | 7 (3.6) | 4 (1.5) | 2 (0.9) | 64 (4.0) |
| Viridans group streptococci | 3 (1.3) | 22 (3.2) | 2 (1.0) | 2 (0.8) | 30 (13.1) | 59 (3.7) |
| Beta-haemolytic Streptococcus | 6 (2.5) | 13 (1.9) | 2 (1.0) | 8 (3.0) | 8 (3.5) | 37 (2.3) |
| Enterococcus spp. | 5 (2.1) | 14 (2.1) | 2 (1.0) | 4 (1.5) | 8 (3.5) | 33 (2.1) |
| Non-fermenting GNB3 | 6 (2.5) | 11 (1.6) | 5 (2.6) | 4 (1.5) | 4 (1.7) | 30 (1.9) |
| Moraxella spp. | 0 | 15 (2.2) | 0 | 1 (0.4) | 0 | 16 (1.0) |
| Others | 2 (0.8) | 18 (2.7) | 4 (2.1) | 4 (1.5) | 4 (1.7) | 32 (2.0) |
| Total | 236 (100.0) | 677 (100.0) | 195 (100.0) | 264 (100.0) | 229 (100.0) | 1601 (100.0) |
Coagulase-negative Staphylococcus includes: S. epidermidis (n=172), S. lugdunensis (n=26), S. capitis (n=15), S. warneri (n=6), S. haemolyticus (n=5), S. hominis (n=4), S. scheilferi (n=3) and one isolate of each following species: S. pasteuri, S. saprophyticus and S. simulans.
Enterobacteriaceae includes Serratia marcescens (n=56), Klebsiella pneumoniae (n=23), Proteus mirabilis (n=21), Escherichia coli (n=16), Klebsiella oxytoca (n=11), Enterobacter aerogenes (n=10), Morganella morganii (n=8), Enterobacter cloacae (n=6), Citrobacter koseri (n=6), Serratia liquefaciens (n=4), Pantoea agglomerans (n=2) and one isolate each of: Citrobacter freundii, Citrobacter youngae, Enterobacter gergoviae, Kluyvera cryocrescens.
Non-fermeting gram negative bacilli, other than P. aeruginosa. This group is comprised of Stenotrophomonas maltophilia (n=11), Pseudomonas spp. (n=6), Achromobacter xylosoxidans (n=4), Acinetobacter spp. (n=3), Chryseobacterium indologenes (n=3), Elizabethkingia meningoseptica (n=1), Alcaligenes spp. (n=1), and Burkholderia cepacia complex (n=1).
Including dacryocystis and canaliculitis.
Includes eyebrow abscess, orbital abscess and cellulitis, subperiosteal abscess and preseptal cellulitis.
Antimicrobial resistance rates and multidrug resistance (MDR).
To determine the rates of antimicrobial resistance and the antibiotics that might result in better treatment outcomes of bacterial ocular infections based on in vitro susceptibility profiles, we tested the susceptibilities of all isolates against a panel of clinically relevant antimicrobials (macrolide, fluoroquinolones, beta-lactams, aminoglycosides and glycopeptide). These classes include the antibiotics that are most commonly used as empirical therapies for keratitis (e.g., fluoroquinolones or combined therapy with tobramycin/cefazolin plus vancomycin) 13, endophthalmitis (vancomycin, ceftazidime, amikacin or moxifloxacin)14 and periocular infections (amoxicillin-clavulanate, cephalosporins either alone or combined with other antibiotics including clindamycin and vancomycin) 15. The ocular infections included in this study typically occurs non-hospitalized patients in the community setting. Despite largely community origins, we found moderate to high rates of resistance to antibiotics often used for treatment of ocular infections, especially among staphylococcal isolates. Ocular S. aureus were commonly resistant to penicillin (80.5%), erythromycin (53.8%), clindamycin (33.2%), and levofloxacin (21.2%) (Figure 1A). Gentamicin, tetracycline and trimethoprim/sulfamethoxazole (TMP/SMX) possessed good in vitro activity against ocular S. aureus isolates, with resistance rates below 6% (Figure 1A). Methicillin resistance (MRSA) was found in 28% of S. aureus isolates (Figure 1A). The proportion of MRSA was higher in periocular infections (50.6%) when compared to other infections (Figure 1B). Methicillin-resistance status paralleled resistance to other antimicrobials. Co-resistance rates were highest for erythromycin (88.4% vs 40.2%), levofloxacin (53.8% vs 8.6%) and clindamycin (44.8% vs 28.7%) (P < 0.0001) (Figure 1C). MDR was defined as resistance to at least 3 classes of antibiotics based on a retrospective review of the antibiotic susceptibility testing performed by the clinical laboratory. The proportion of MRSA that was MDR was much higher than for MSSA strains (63.1% vs 7.0%) (Figure 1D).
Figure 1.
A) Antibiotic resistance profiles for ocular Staphylococcus aureus isolates. B) Distribution of S. aureus methicillin-phenotype according to the diagnosis. C) Percentage of resistance to other antibiotics by methicillin-resistance and methicillin-susceptible phenotype among ocular Staphylococcal isolates. D) Concurrent resistance to other antibiotics by methicillin-resistant and methicillin-susceptible ocular S. aureus isolates.
Among CoNS species, we found moderate to high resistance rates to antibiotics such as clindamycin (28.9%), levofloxacin (36.2%), erythromycin (54.4%) and penicillin (78%) (Figure 2A). Overall, 39.8% of CoNS isolates were resistant to methicillin (MR-CoNS) (Figure 2A). As found for MRSA, MR-CoNS isolates were also significantly more likely than methicillin-susceptible CoNS (MS-CoNS) isolates to be resistant to erythromycin or clindamycin (P < 0.0001), with resistance rates ≥70% for these antibiotics (Figure 2B). In line with these observations the rate of MDR was higher among MR-CoNS isolates (76.1%) than among MS-CoNS isolates (12.7%) (Figure 2C). All ocular Staphylococcal isolates were susceptible to last-line antibiotics linezolid and vancomycin (Figure 1A and 2A).
Figure 2.
A) Antibiotic resistance profiles for ocular Coagulase-negative staphylococci (CoNS) isolates. B) Percentage of resistance to other antibiotics by methicillin-resistance and methicillin-susceptible phenotype among ocular CoNS isolates. C) Concurrent resistance to other antibiotics by methicillin-resistant and methicillin-susceptible ocular CoNS isolates.
We found low resistance rates (< 10%) among S. pneumoniae isolates for all antibiotics tested except penicillin (16.2%) and erythromycin (48.9%). None of these isolates were resistant to fluoroquinolones (Table 2). Viridans group streptococci (VGS) presented higher resistance rates than S. pneumoniae, with 62.7% being resistant to erythromycin and 22.6% being resistant to penicillin. Resistance to levofloxacin (9.4%) was found among ocular VGS. Most of Enterococcus spp. isolates were susceptible to penicillin (97%) and 3% of them (n=1; E. faecium) were resistant to vancomycin (Table 2). Rates of antibiotic resistance among P. aeruginosa and Enterobacteriaceae isolates were low overall, with 5.2% to 7.4% of isolates resistant to fluoroquinolones, and 2.4% to 6.7% resistant to tobramycin (Table 3). Isolates in the Enterobacteriaceae family were commonly resistant to the beta-lactam/beta-lactamase combination ampicillin-sulbactam (55.4%), and were less resistant to cephalosporins such as ceftriaxone (11.7%), ceftazidime (9.6%) and cefepime (1.8%). Non-susceptibility to imipenem varied from 3.8% among P. aeruginosa isolates and 4.8% among enterobacteria.
Table 2.
Antibiotic resistance profiles for Streptococcus spp. and Enterococcus faecalis. at MEE from 2014 to 2021.
|
|
|
|
||||
|---|---|---|---|---|---|---|
| Antibacterial drug |
S. pneumoniae
|
VGS |
Enterococcus spp. |
|||
| No. tested | % NS | No. tested | % NS | No. tested | % NS | |
| Penicillin | 68 | 16.2 | 53 | 22.6 | 33 | 3.0 |
| Amox/Clav | 85 | 2.4 | - | ND | - | ND |
| Ceftriaxone | 67 | 1.5 | - | ND | - | ND |
| Meropenem | 89 | 7.9 | - | ND | - | ND |
| Clindamycin | - | ND | 53 | 11.3 | - | ND |
| Erythromycin | 92 | 48.9 | 51 | 62.7 | 31 | 80.6 |
| Levofloxacin | 91 | 0 | 53 | 9.4 | 31 | 22.6 |
| Tetracycline | - | ND | 53 | 37.7 | 31 | 97.8 |
| Vancomycin | 89 | 0 | 53 | 0 | 33 | 3.0 |
NS: non-susceptible
ND: not-determined
Table 3.
Antibiotic resistance profiles for Pseudomonas aeruginosa and Enterobacteriaceae at MEE from 2014 to 2021.
|
|
|
|
||||
|---|---|---|---|---|---|---|
| Antibacterial drug |
P. aeruginosa
|
Enterobacteriaceae |
||||
| No. tested | % NS | No. tested | % NS | |||
|
|
|
|
||||
| Aztreonam | 213 | 6.6 | 165 | 7.9 | ||
| Amp/Sul | - | ND | 166 | 55.4 | ||
| Ceftazidime | 213 | 2.8 | 166 | 9.6 | ||
| Ceftriaxone | - | ND | 163 | 11.7 | ||
| Cefepime | 213 | 2.8 | 165 | 1.8 | ||
| Imipenem | 210 | 3.8 | 147 | 4.8 | ||
| Ciprofloxacin | 212 | 5.7 | 163 | 7.4 | ||
| Levofloxacin | 212 | 5.2 | 166 | 6.6 | ||
| Amikacin | 213 | 0.9 | 164 | 0.6 | ||
| Tobramycin | 212 | 2.4 | 165 | 6.7 | ||
| Gentamicin | 211 | 4.7 | 164 | 5.5 | ||
| TMP/SMX | - | ND | 165 | 10.3 | ||
NS: non-susceptible
ND: not-determined
Antibiotic resistance rates over time.
To understand how antimicrobial resistance among ocular bacteria changed over time, we examined resistance trends over an 8-year period (2014-2021) for antimicrobials routinely tested in our clinical microbiology laboratory. Erythromycin resistance rates were high in the beginning of the study period (39.5%, 95.1% and 54.5% for MSSA, MRSA and CoNS respectively) and remained high until the end (40.2%, 89.3% and 43.5% for MSSA, MRSA and CoNS respectively) (Figure 3A-C). MSSA isolates showed moderate rates of resistance to clindamycin over time (between 25-31.5%) and low rates of resistance to levofloxacin (<15.0%) (Figure 3A). On the other hand, MRSA isolates were more commonly resistant to clindamycin during the study period with resistance rates over 40% (Figure 3B). Small decreases over time were noted in resistance to levofloxacin among MRSA isolates even though resistance rates remained high at the end of the study period (40%) (Figure 3B). Among CoNS isolates, levofloxacin and oxacillin resistance rates were similar with a peak of resistance in 2016-2017 (48.7%), followed by a slight decrease. A small increase over time was noted in CoNS resistance to clindamycin, whereas a small decrease was observed to TMP/SMX during the study period (Figure 3C).
Figure 3.
Antibiotic resistance trends over time among A) MSSA, B) MRSA and C) CoNS isolates.
Changes in S. aureus resistance rates over time were also stratified for isolates from keratitis and periocular infections, infections mainly caused by this species in our population. Rates of oxacillin resistance in S. aureus isolates collected from periocular infections were high at the beginning of the study, peaked in 2016-2017, and then decreased slightly, remaining at 43.2% and 55.2% in the subsequent years (Figure 4A). Rates of MRSA among keratitis isolates were 17.4% in 2014-2015 and remained at similar levels in later years (Figure 4A). Rates of resistance to levofloxacin among periocular isolates remained around 20% during the study period, whereas among keratitis isolates, levofloxacin resistance peaked in 2016-2017 (36.0%) and then decreased to a rate of 2.6% in 2020-2021 (Figure 4B). Erythromycin and clindamycin resistance rates in S. aureus causing keratitis and periocular infections were similar to the broader trends observed in all S. aureus isolates of our study (Figure 4C-D).
Figure 4.
Antibiotic resistance trends among ocular S. aureus isolates (n=621) causing keratitis (in pink) (n=214) and periocular infections (in green) (n=160) over time. A) Oxacillin, B) Levofloxacin, C) Erythromycin and D) Clindamycin.
As ocular CoNS isolates were important causes of keratitis and endophthalmitis in our population, we stratified CoNS resistance rates over time for these two infections. Rates of oxacillin resistance in CoNS isolates causing keratitis were high at the beginning of the study, peaked in 2016-2017 (76.9%), and then decreased in the subsequent years to the lowest rate of 27.8% in 2020-2021. Rates of MR-CoNS among endophthalmitis isolates were 47.5% in 2014-2015 and then decreased slightly, reaching 33.3% and 37.5% in the subsequent years (Figure 5A). Rates of levofloxacin resistance in CoNS isolates causing keratitis were lower at the beginning and at the end of the study (between 33.3% and 22.2%) than in the middle of the study with a peak in 2016-2017 (69.7%). For CoNS isolates causing endophthalmitis, a levofloxacin resistance peak was observed in the beginning of the study (48.6%) (Figure 5B). Erythromycin resistance rates in CoNS causing keratitis and endophthalmitis were similar to trends observed for all CoNS isolates of our study (Figure 5C). Clindamycin resistance in CoNS causing keratitis was relatively high (38.9%) at the end of the study period (Figure 5D). Clindamycin resistance in CoNS causing endophthalmitis was low during the study period (≤20%), except for a peak in 2018-2019 (38.9%) (Figure 5D).
Figure 5.
Antibiotic resistance trends among ocular CoNS isolates (n=234) causing keratitis (in pink)(n=75) and endophthalmitis (in blue) (n=108) over time. A) Oxacillin, B) Levofloxacin, C) Erythromycin and D) Clindamycin.
Common clonal complexes and acquired resistance genes.
To define the genetic lineages most commonly associated with ocular bacterial infections and to identify genes likely associated with acquired mechanisms of resistance to antibiotics, we performed whole genome sequencing on a representative subpopulation of our patient isolates (n=563 isolates). As determined by goeBURST analysis, the population of S. aureus isolates (n=291) was largely comprised of 3 major clonal complexes, including CC8 (26.1%), CC5 (24.1%) and CC30 (11.3%) (Table 4). The occurrence of methicillin resistance was enriched among certain lineages, with 55.3% of CC8 being MRSA, and 51.4% of CC5 being MRSA. Interestingly, infections of the wet epithelial tissues of the ocular surface were substantially enriched in the CC5 lineage, which was particularly pronounced for infectious keratitis cases (57.1% for CC5 versus 19.7% for CC8), while CC8 was mainly found in periocular infections (47.4% for CC8 versus 4.3% for CC5) (Table 4). We found that more than half of S. aureus CC5 were MDR (60.0%), whereas 28.9% of S. aureus CC8 were MDR.
Table 4.
Distribution of major and minor CCs, proportion of methicillin-resistant and sites of isolation among ocular staphylococci isolates.
| No. (%) of isolates from: | |||||||
|---|---|---|---|---|---|---|---|
| Clonal complex (CC) | No. of isolates (%) | No. of methicillin-resistant (%) | Conjunctivitis | Keratitis | Lacrimal system | Pericocular infections |
Endophthalmitis |
| S. aureus n=291 | |||||||
| CC8 | 76 (26.1) | 42 (55.3) | 8 (10.5) | 15 (19.7) | 15 (19.7) | 36 (47.4) | 2 (2.6) |
| CC5 | 70 (24.1) | 36 (51.4) | 11 (15.7) | 40 (57.1) | 8 (11.4) | 3 (4.3) | 8 (11.4) |
| CC30 | 33 (11.3) | 1 (3.0) | 6 (18.2) | 18 (54.1) | 4 (12.1) | 3 (9.1) | 2 (6.1) |
| CC45 | 23 (7.9) | - | 3 (13.0) | 14 (60.9) | 4 (17.4) | 2 (8.7) | 0 (0.0) |
| CC15 | 20 (6.9) | 1 (5.0) | 4 (20.0) | 10 (50.0) | 2 (10.0) | 3 (15.0) | 1 (5.0) |
| CC1 | 14 (4.8) | - | 5 (37.5) | 4 (28.6) | 3 (21.4) | 2 (14.3) | - |
| CC97 | 14 (4.8) | - | 7 (50.0) | 1 (7.1) | 3 (21.4) | 3 (21.4) | - |
| CC398 | 13 (4.5) | - | 5 (38.5) | 4 (30.8) | 1 (7.7) | 2 (15.4) | 1 (7.7) |
| CC59 | 11 (3.8) | 5 (45.5) | 3 (27.3) | 5 (45.5) | 1 (9.1) | 2 (18.2) | - |
| CC121 | 3 (1.0) | - | - | - | 2 (66.7) | 1 (33.3) | - |
| CC12 | 2 (0.7) | - | 1 (50.0) | 1 (50.0) | - | - | - |
| CC22 | 2 (0.7) | - | - | 1 (50.0) | 1 (50.0) | - | - |
| CC20 | 1 (0.3) | - | - | 1 (100.0) | - | - | - |
| CC672 | 1 (0.3) | - | - | - | 1 (100.0) | - | - |
| CC96 | 1 (0.3) | 1 (100.0) | - | 1 (100.0) | - | - | - |
| Unknown | 7 (2.4) | 1 (14.3) | 1 (14.3) | 5 (71.4) | - | - | 1 (14.3) |
| S. epidermidis n=100 | |||||||
| CC2-II | 58 (58.0) | 33 (56.9) | - | 17 (29.30) | - | 4 (6.9) | 37 (63.8) |
| CC2-I | 27 (27.0) | 11 (40.7) | 1 (3.7) | 10 (37.0) | 1 (3.7) | 3 (11.1) | 12 (44.4) |
| CC19 | 1 (1.0) | - | - | - | - | - | 1 (100.0) |
| CC290 | 1 (1.0) | 1 (100.0) | - | - | - | - | 1 (100.0) |
| Unknown | 13 (13.0) | 4 (30.8) | - | 6 (46.2) | 1 (7.7) | 1 (7.7) | 5 (38.5) |
CC2 was the main clonal complex identified among S. epidermidis isolates (85.0%) (Table 4) and subclusters within those were identified as previously described 16. The majority of S. epidermidis isolates in our population belonged to CC2 subcluster II (Table 4), which includes lineages that are widespread in the US 17. Isolates within subcluster I, which are commonly found among hospital-associated infections in Europe 18,19 and in about one third of S. epidermidis infections in US 17, were less common in our population. We found that more than half of S. epidermidis CC2-II isolates were MDR (53.4%), while 37.0% of S. epidermidis CC2-I isolates were MDR.
Among S. pneumoniae isolates, the distribution of CCs diverged across different infection sites, with a pattern of lineage predominance in conjunctivitis, which is commonly caused by strains grouped belonging to the Epidemic Conjunctivitis Cluster (ECC) 20, predominantly CC448 (Table 5).
Table 5.
Distribution of major and minor CCs and proportion of sites of isolation among 45 ocular S. pneumoniae isolates.
| No. (%) of isolates from: | ||||||
|---|---|---|---|---|---|---|
| Clonal complex (CC) |
No. of isolates (%) |
Conjunctivitis | Keratitis | Lacrimal system |
Periocular infections |
Endophthalmitis |
| CC448 (9) | 9 (20.0) | 8 (88.9) | - | - | 1 (11.1) | - |
| CC199 (5) | 5 (11.1) | 1 (20.0) | 3 (60.0) | - | - | 1 (20.0) |
| CC558 (5) | 5 (11.1) | 2 (40.0) | 1 (20.0) | 1 (20.0) | - | 1 (20.0) |
| CC180 (3) | 3 (6.7) | 2 (66.7) | 1 (33.3) | - | - | - |
| CC62 (2) | 2 (4.4) | - | 2 (100.0) | - | - | - |
| CC63 (2) | 2 (4.4) | - | 1 (50.0) | 1 (50.0) | - | - |
| CC439 (2) | 2 (4.4) | 1 (50.0) | 1 (50.0) | - | - | - |
| CC100 (2) | 2 (4.4) | - | 1 (50.0) | 1 (50.0) | - | - |
| CC433 (2) | 2 (4.4) | 2 (100.0) | - | - | - | - |
| CC344 (1) | 1 (2.2) | 1 (100.0) | - | - | - | - |
| CC66 (1) | 1 (2.2) | - | 1 (100.0) | - | - | - |
| CC338 (1) | 1 (2.2) | - | 1 (100.0) | - | - | - |
| CC5178 (1) | 1 (2.2) | - | 1 (100.0) | - | - | - |
| CC97 (1) | 1 (2.2) | - | 1 (100.0) | - | - | - |
| CC9355 (1) | 1 (2.2) | - | 1 (100.0) | - | - | - |
| CC1262 (1) | 1 (2.2) | - | 1 (100.0) | - | - | - |
| CC383 (1) | 1 (2.2) | - | 1 (100.0) | - | - | - |
| CC113 (1) | 1 (2.2) | - | - | - | - | 1 (100.0) |
| CC393 (1) | 1 (2.2) | - | - | - | - | 1 (100.0) |
| CC235 (1) | 1 (2.2) | - | - | 1 (100.0) | - | - |
| Unknown CC (2) | 2 (4.4) | - | 2 (100.0) | - | - | - |
Conversely, S. pneumoniae keratitis isolates comprised a highly diverse population representing 15 different CCs (Table 5). The population of P. aeruginosa isolates was also diverse with 44 different CCs or STs observed (Table 6). The distribution of STs among Enterobacteriaceae isolates is shown in Supplementary table 1, with E. coli ST131, a globally disseminated lineage associated with extraintestinal infections, being the most common ST identified for this species.
Table 6.
Distribution of major and minor CCs among 104 ocular P. aeruginosa isolates.
| Clonal complex (CC) |
No. of isolates (%) |
|---|---|
| CC274 | 14 (13.5) |
| CC253 | 7 (6.7) |
| CC390 | 5 (4.8) |
| CC179 | 4 (3.8) |
| CC155 | 3 (2.9) |
| CC252 | 3 (2.9) |
| CC282 | 3 (2.9) |
| CC381 | 3 (2.9) |
| ST2060 | 3 (2.9) |
| ST2613 | 3 (2.9) |
| CC261 | 2 (2.9) |
| CC309 | 2 (2.9) |
| CC395 | 2 (2.9) |
| CC463 | 2 (2.9) |
| CC487 | 2 (2.9) |
| CC532 | 2 (2.9) |
| ST1050 | 2 (2.9) |
| CC1076 | 1 (1.0) |
| CC1097 | 1 (1.0) |
| CC1197 | 1 (1.0) |
| CC260 | 1 (1.0) |
| CC267 | 1 (1.0) |
| CC277 | 1 (1.0) |
| CC314 | 1 (1.0) |
| CC3762 | 1 (1.0) |
| CC389 | 1 (1.0) |
| CC560 | 1 (1.0) |
| CC569 | 1 (1.0) |
| CC611 | 1 (1.0) |
| CC639 | 1 (1.0) |
| ST1094 | 1 (1.0) |
| ST1121 | 1 (1.0) |
| ST1248 | 1 (1.0) |
| ST1285 | 1 (1.0) |
| ST1400 | 1 (1.0) |
| ST1682 | 1 (1.0) |
| ST2475 | 1 (1.0) |
| ST2483 | 1 (1.0) |
| ST2707 | 1 (1.0) |
| ST291 | 1 (1.0) |
| ST296 | 1 (1.0) |
| ST3017 | 1 (1.0) |
| ST386 | 1 (1.0) |
| Unknown | 16 (15.4) |
As revealed by ResFinder and CARD analysis, the presence of the mecA gene that confers resistance to methicillin, was present in our staphylococcal population, with 56.6%, 51.4% and 56.9% and 40.7% of S. aureus CC8, S. aureus CC5, S. epidermidis CC2-II and S. epidermidis CC2-I harboring the mecA gene respectively. These findings correlated with phenotypic resistance to oxacillin (Table 7). Aminoglycoside resistance in S. aureus CC8 isolates was mainly associated with the aminoglycoside phosphotransferase gene aph(3')-lll (63.2%) and the aminoglycoside nucleotidyltransferase gene ant(6)-Ia (59.2%), whereas for S. aureus CC5 isolates, aminoglycosides resistance was mainly caused by aminoglycoside nucleotidyltransferase gene ant(9)-Ia (57.1%) (Table 7)21. Macrolide resistance in S. aureus was frequently associated with mphC/msrA genotype (61.7%) in CC8 strains and with ermA gene (60.0%) in CC5 the group (Table 7)22. However, more than 1 in 4 S. aureus CC5 also carried the mphC/msrA genes. The mphC/msrA genotype confers low levels of resistance to macrolides and streptogramin B only, whereas ermA confers resistance to macrolides, streptogramin B and lincosamides. Our CC5 isolates were more commonly phenotypically resistant to clindamycin (58.0%) than CC8 (16%) (Table 7). Macrolide resistance in S. epidermidis was also frequently associated with mphC/msrA (> 40%), but also with ermC (17.6%) (Table 7). Like ermA, ermC also confers resistance to lincosamides. We found that 22.4% of our S. epidermidis isolates were phenotypically resistant to clindamycin (Table 7). Some S. epidermidis isolates also encoded the tetK gene (10.6%) which confers resistance to tetracycline (Table 7).
Table 7.
Acquired resistance genes in bacteria causing ocular infections according to the species.
|
Antibiotic classes/Genes No. (%) |
Phenotypic resistance, % | |||||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| AS | METHI | MLSb | TET | |||||||||||||||||
| Species No. | aph(3')- lll |
ant(6)- Ia |
ant(9)- Ia |
ant(4') | aac(6')- aph(2") |
mecA | erm(A) | erm(B) | erm(C) | mef(A) | mphC | msr(A) | msr(D) | tet(K) | tet(M) | Oxacillin | Gentamicin | Erythromycin | Clindamycin | Tetracycline |
| S.aureus CC8 n=76 | 48 (63.2) | 45 (59.2) | 2 (2.6) | 1 (1.3) | 1 (1.3) | 43 (56.6) | 4 (5.3) | - | 7 (9.2) | - | 52 (68.4) | 51 (67.1) | - | 1 (1.3) | 1 (1.3) | 56.0 | 1.3 | 68.9 | 16.0 | 4.0 |
| S.aureus CC5 n=70 | 16 (22.9) | 16 (22.9) | 40 (57.1) | 29 (41.4) | 1 (1.4) | 36 (51.4) | 42 (60.0) | - | 3 (4.3) | - | 18 (25.7) | 19 (27.1) | - | 2 (2.9) | 1 (1.4) | 52.2 | 2.9 | 77.9 | 58.0 | 2.9 |
| S. epidermidis CC2 n=85 | 7 (8.2) | 5 (5.9) | - | 29 (34.1) | 6 (7.1) | 43 (50.6) | - | - | 15 (17.6) | - | 35 (41.2) | 43 (50.6) | - | 9 (10.6) | - | 52.4 | 7.1 | 67.9 | 22.4 | 15.5 |
| S.ppeemoniaa n=45 | - | - | - | - | - | - | - | 4 (8.7) | - | 15 (32.6) | - | - | 15 (32.6) | - | 4 (8.7) | NA | NA | 40.0 | NA | NA |
AS: Aminoglycosides; METHI: Methicillin; M: Macrolides; L: Lincosamides; Sb: Streptogramin b TET: Tetracycline
NA: not applicable
Among S. pneumoniae isolates, the common mechanism of resistance to macrolides was the presence of the mefA/msrD genes (32.6%) followed by a smaller percentage of isolates positive for the ermB gene (8.7%) (Table 7). This correlated well with what we observed phenotypically with 40.0% of S. pneumoniae in total being resistant to erythromycin. S. pneumoniae isolates that carried the ermB gene also harbored the tetM gene, which confers resistance to tetracycline. Similar to what has been found in other infectious sites 23, all ocular P. aeruginosa isolates genomes analyzed (n=104) carried one or two β-lactamase genes (blaOXA-50, blaPAO) and 3 additional resistance genes, aph(3')-IIb, fosA and catB7, that confer resistance to aminoglycosides, fosfomycin and chloramphenicol respectively. The most common acquired resistance genes among Enterobacteriaceae isolates were related to β-lactam resistance and included the non-extended-spectrum β-lactamases blaSHV1 (in K. pneumoniae, 85.7%), and blaTEM (in E. coli 33.3%). Only one Enterobacteriaceae isolate in our population (E. coli) carried an extended-spectrum β-lactamase (ESBL; blaCTX-M-15). This was an ST131 multidrug-resistant strain isolated from a recurrent keratitis case 24. A detailed description of each acquired resistance gene, the antibiotics they confer resistance to, and the molecular mechanisms of resistance can be found at https://card.mcmaster.ca/ontology/36006.
Discussion
This study describes the microbiology of eye infections at MEE over an 8-year period. In total, we isolated 1,601 bacterial isolates during this time, with Gram-positive organisms, especially staphylococcal species, being most prevalent. S. aureus was the most common cause of conjunctivitis, keratitis, lacrimal system and orbital and periorbital infections, while CoNS species were the major causes of endophthalmitis. Gram-negative organisms such as P. aeruginosa and Enterobacteriaceae species were common depending on the site of isolation, and were among the top causes of keratitis and lacrimal system infections. Haemophilus influezae ranked second in conjunctivitis cases. These findings are in line with other studies in the US, which demonstrate that S. aureus and P. aeruginosa are leading causes of keratitis and that CoNS are major causes of endophthalmitis 25, whereas in conjunctivitis cases, S. aureus, H. influenzae and S. pneumoniae are the most frequently identified bacteria 25. The distribution of bacterial species causing ocular infections varies somewhat in other parts of the world, especially Asia 26,27. In India, for example, S. pneumoniae isolates are the most commonly identified cause of infectious keratitis 27,28. Among Gram-negative bacteria, P. aeruginosa remains a major causative organism of keratitis worldwide and has been commonly identified as a leading cause of these infections in studies from major centers based in the US 25,29, UK 30 and Asia 27.
Overall, nearly one third of S. aureus isolates and 40% of CoNS isolates were methicillin resistant. We found that the ocular MRSA population was dominated by two major clonal complexes that are also common causes of MRSA infections in other body sites: CC8 and CC5 31. The ocular infections sampled in our study developed mainly in patients within the community setting, thus it was not surprising that we found strains related to the CC8 lineage, the most common community-acquired (CA)-MRSA lineage in the US, among the major MRSA lineages in our population 32. Conversely, the CC5 lineage has been found to be a leading cause of antibiotic-resistant hospital-associated MRSA infections in the US 33. These results are consistent with what we previously observed in our hospital 34 and demonstrate that a heterogeneous population of lineages with both community and hospital origins composes the reservoir of primarily community-associated ocular infections that we sampled. The distribution of lineages within these clonal complexes followed a pattern of enrichment that was split into the main ocular niches tested, with periocular infections being predominantly associated with CC8, while keratitis was frequently caused by CC5 strains. Our results are consistent with other studies 35,36 and suggest an enhanced ability of CC5 to endure selective pressures and colonize the cornea and adjacent tissues. In addition, S. aureus isolates associated with CC5 were more likely to display a MDR phenotype, and are commonly highly resistant to first-line antibiotics used in ophthalmology, such as fluoroquinolones and aminoglycosides 4,35.
The majority of S. epidermidis isolates examined in the present study belonged mainly to CC2 sub-cluster II, a sub-cluster commonly found in US hospitals 17. Lineages within CC2-II were also found to be the most prevalent among ocular S. epidermidis isolates from Brazil 16. In Europe, the most common clones isolated from the hospital environment also belonged to CC2 but within sub-cluster I 19,37,38. In our study, S. epidermidis belonging to CC2-II were more likely to display a methicillin resistance phenotype in comparison to CC2-I (56.9% vs 40.7%) whereas other studies from the US have reported a higher proportion of methicillin resistance in CC2-I isolates (95.2% vs 70.7%) 17. As we previously reported in ocular S. pneumoniae isolates 20,39, our results suggest that ECC tropism is specific to the conjunctiva, whereas keratitis is caused by a highly diverse population. For conjunctivitis, isolates belonging to CC448 were the most common in our study (88.9%). This lineage has been implicated in many outbreaks of pneumococcal conjunctivitis in the US since 198040 and has become established as a persistent lineage that is trophic for, and well adapted to, the eye tissues 20. In our sample, infectious keratitis was caused by a heterogeneous population formed by isolates grouping within a variety of CCs with no apparent clonal dominance. This population structure appears to mirror the current post–PCV-13 strain compositions in invasive pneumococcal diseases 41, where CC180, CC338, CC66, CC62, CC558 and CC199 are commonly found. P. aeruginosa ST274 and ST253 are two of the most prevalent clones in cystic fibrosis patients 42 and were also frequent lineages identified in our P. aeruginosa population. Among ocular E. coli isolates, ST131 was the most common lineage identified. This lineage is widely disseminated worldwide and is a leading cause of extra-intestinal E.coli infections 43.
Nationwide surveillance studies have found remarkably high rates of resistance among ocular bacteria, especially for Gram-positive pathogens with staphylococci playing a major role 4,44. Staphylococci and select other Gram-positive bacteria are notable for their ability to acquire and express various antimicrobial resistance mechanisms leading to MDR phenotypes that further complicate treatment management 45,46. Methicillin-resistant strains pose a major challenge for management of eye infections as they are substantially more resistant to first-line antibiotics used for empirical treatment of ocular infections such as fluoroquinolones and aminoglycosides 4. Prevalence of methicillin resistance in our population (ranging from 28.0% for S. aureus to 39.8% for CoNS) was slightly lower than in the ARMOR study (ranging from 34.9% for S. aureus to 49.3% for CoNS) 4. In agreement with ARMOR and other nationwide multicenter surveillance studies 4,44,47, MRSA and MRSE isolates in our population were significantly more likely than MSSA and MSSE isolates to be concurrently resistant to additional antibacterial drug classes, including first-line antibiotics used for empirical treatment of ocular infections. Clindamycin, erythromycin and levofloxacin resistance rates in MRSA isolates remained high during the study period, meaning that first-line treatments would cover only a fraction of patients infected by MRSA.
Our study highlights the major threat of antimicrobial resistance in ocular infections, with more than 60% of our MRSA isolates and 76.1% of MRCoNS being MDR. In contrast to staphylococcal isolates, comparatively low resistance rates were observed among Enterobacteriaceae and P. aeruginosa isolates in this collection, suggesting that first line therapies should still have good coverage for infections caused by these pathogens. None of our Gram-negative isolates were resistant to carbapenem and only one, a ST131 E. coli isolated from a recurrent keratitis case, was an ESBL producer (blaCTM-X-15). Lower levels of in vitro resistance to penicillin were noted among our S. pneumoniae isolates compared to the ARMOR study (16.2% vs 33.3%) 47. S. pneumoniae isolates in our population were susceptible to fluoroquinolones and vancomycin commonly used to treat Gram-positive keratitis infections, but showed high rates of resistance to macrolides. Resistance to macrolides in S. pneumoniae isolated in US is relatively common, with rates ranging from 20% to 40% 48. Resistome characterization of the most common ocular bacterial species in our study revealed that resistance was achieved through various mechanisms including antibiotic inactivation (aph(3’)-III, ant(6)-Ia, mphC), target alteration (ermA and mecA) and drug efflux (mefA/msrD). Macrolide resistance genes (ermB and mefA/msrD) among S. pneumoniae isolates are easily transferred as they are integrated into mobile genetic elements. mefA/msrD genes were located in the Macrolide Efflux Genetic Assembly cassette and ermB was localized in a Tn-916-like transposon 39. In S. aureus isolates, transfer of ermA and ant(9)-Ia genes between strains is facilitated by horizontal gene transfer as they are found in the transposon Tn554 49.
In summary we describe the genomic epidemiology and rates of resistance to first line empirical treatments of bacterial ocular infections treated at MEE over the last 8 years. Our data demonstrate that ocular bacterial infections are composed of strains that are commonly resistant to clinically relevant antibiotics, especially staphylococcal isolates that are frequently MDR, and are associated with major epidemic lineages of both community and hospital origins. To prevent treatment failures and further increases in antimicrobial resistance among ocular bacterial infections, close and continuing monitoring of AMR rates and tracking of high-risk clones that cause serious and difficult to treat infections are important epidemiological tools that can help guide empirical treatments locally and support the development of novel approaches to control and prevent these infections.
Supplementary Material
Acknowledgments
A. Funding/Support:
This work was supported in part by the New England Corneal Transplant Research Fund and the Massachusetts Lions Eye Research Fund, and by NIH grant EY031600. CA was supported by a scholarship from Fondation pour la Recherche Medicale (FDM202006011203).
B. Financial Disclosures:
Funding agencies had no role in study design, data analysis, decision to publish or preparation of the manuscript.
C. Other Acknowledgments:
The authors thank medical technologists from the Clinical Microbiology Laboratory at MEE, including Lisa Bove and Nancy Sutcliffe for their support in creating a microbial repository of strains isolated from ocular infections.
Footnotes
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