ABSTRACT

Purpose: The aims of this study were to characterize alpha-hemolytic streptococci among isolates from cases of infectious endophthalmitis and keratitis and to determine their distributions.
Methods: The sample included 27 and 35 nonduplicated isolates of alpha-hemolytic streptococci recovered from patients with infectious endophthalmitis (2002-2013) and keratitis (2008-2013), respectively. Isolates were identified by the optochin susceptibility and bile solubility tests, using a biochemical identification system. The minimum inhibitory concentration was determined by the broth microdilution method. Molecular identification was performed by analyses of three constitutive genes and the complementary multilocus sequence. The molecular epidemiology of Streptococcus pneumoniae was investigated using multilocus sequence typing, and the presence of the capsular polysaccharide-encoding gene was assessed using conventional polymerase chain reaction. Outcomes were evaluated using the patients’ medical records.
Results: Phenotypic tests differentiated S. pneumoniae from other alpha-hemolytic streptococci, consistent with later molecular identifications. Streptococcus oralis was significantly prevalent among the endophthalmitis isolates, as was S. pneumoniae in the keratitis isolates. High levels of susceptibility to antibiotics were observed, including vancomycin, cephalosporins, and fluoroquinolones. High genetic variability was detected among the 19 S. pneumoniae strains, with 15 predicted to be encapsulated. The medical records of patients with infectious endophthalmitis were reviewed (n=15/27; 56%), and final visual acuity was assessed in 12 cases (44%). Many patients progressed to a final visual acuity state of “no light perception” (6/12; 50%), “light perception” (3/12; 25%), or “hand motion” (1/12; 8%). The medical records of patients with infectious keratitis were also reviewed (n=24/35; 69%), and final visual acuity was assessed in 18 cases (51%). Similarly, most patients progressed to a final visual acuity state of “no light perception” (6/18; 33%), “light perception” (1/18; 6%), or “hand motion” (6/18; 33%). Overall, the majority of patients progressed to a final visual acuity state of “no light perception” (12/30), “light perception” (4/30), or “hand motion” (7/30).
Conclusions: The distribution of alpha-hemolytic streptococci in ocular infections suggested the presence of a species-specific tissue tropism. The prognoses of patients with ocular streptococcal infections were highly unfavorable, and antibiotic resistance did not contribute to the unfavorable clinical progressions and poor outcomes.

Keywords: Endophthalmitis; Keratitis; Eye infections, bacterial; Streptococcal infections; Viridans streptococci/isolation & purification; Drug resistance, microbial; Fluoroquinolones

RESUMO

Objetivo: O objetivo deste estudo foi caracterizar os estreptococos alfa-hemolíticos isolados de endoftalmite infecciosa e ceratite e determinar sua distribuição.
Métodos: A amostra incluiu 27 e 35 isolados não-duplicados de estreptococos alfa-hemolíticos recuperados de pacientes com endoftalmite infecciosa (2002-2013) e ceratite (2008-2013), respectivamente. Os isolados foram identificados pelos testes de suscetibilidade à optoquina e bile solubilidade, utilizando um sistema de identificação bioquímica. A concentração inibitória mínima foi determinada pelo método de microdiluição em caldo. A identificação molecular foi realizada pela análise de três genes constitutivos e análise complementar de sequências multilocus. A epidemiologia molecular do Streptococcus pneumoniae foi investigada por tipagem de sequência multilocus, e a presença do gene codificador do polissacarídeo capsular foi avaliada por reação em cadeia da polymerase convencional. Os resultados foram avaliados utilizando os prontuários médicos dos pacientes.
Resultados: Os testes fenotípicos diferenciaram S. pneumoniae dos outros estreptococos alpha-hemolíticos, consistentes com identificações moleculares posteriores. S. oralis foi significativamente prevalente entre os isolados de endoftalmite, assim como S. pneumoniae nos isolados de ceratite. Foram observados altos níveis de suscetibilidade a antibióticos, incluindo vancomicina, cefalosporinas e fluoroquinolonas. Alta variabilidade genética foi detectada entre as 19 cepas de S. pneumoniae, com 15 previstas para serem encapsuladas. Os prontuários médicos dos pacientes com endoftalmite infecciosa foram revisados (n=15/27; 56%), e a acuidade visual final foi avaliada em 12 casos (44%). Muitos pacientes evoluiram para um estado final de acuidade visual de “sem percepção luminosa” (6/12; 50%), “percepção luminosa” (3/12; 25%) ou “movimentos de mãos” (1/12; 8%). Também foram revisados os prontuários médicos dos pacientes com ceratite infecciosa (n=24/35; 69%), e a acuidade visual final foi avaliada em 18 casos (51%). Da mesma foram, a maioria dos pacientes evoluiu para um estado final de acuidade visual de “sem percepção luminosa” (6/18; 33%), “percepção luminosa” (1/18; 6%) ou “movimentos de mãos” (6/18; 33%). No geral, a maioria dos pacientes evoluiu para um estado final de acuidade visual de “sem percepção luminosa” (12/30), “percepção luminosa” (4/30) ou “movimentos de mãos” (7/30).
Conclusões: A distribuição de estreptococos alfa-hemolíticos nas infecções oculares sugeriu a presença de um tropismo de tecido específico da espécie. Os prognósticos dos pacientes com infeções oculares por estreptococos foram altamente desfavoráveis e a resistência a antibióticos contribuiu não para as progressões clínicas desfavoráveis e os maus resultados.

Descritores: Endoftalmite; Ceratite; Infecções oculares bacterianas; Infecções estreptocócicas; Estreptococos viridans/isolamento & purificação; Resistência antimicrobiana a medicamentos; Fluoroquinolonas

INTRODUCTION

Alpha-hemolytic streptococci comprise a large group of primarily commensal organisms usually found in the mucosae of humans. These streptococci can cause serious infections, such as endocarditis, meningitis, pneumonia, abscesses, and septicemia⁽¹⁾. These organisms have also emerged as important causes of serious and sight-threatening eye infections, such as infectious keratitis and endophthalmitis, especially following intravitreal injections^(2-4). Streptococcal endophthalmitis and keratitis are usually acute, rapid-onset infections that are aggressive and frequently lead to worse outcomes in comparison with other causative bacteria^(5-7). These ocular bacterial infections are associated with risk factors such as contact lenses, trauma, surgery, age, dry eye state, chronic nasolacrimal duct obstruction, and previous ocular infection^(8,9).

In developing countries, most ocular infections are caused by members of the genus Staphylococcus. However, members of the genus Streptococcus have also been involved in ocular infections, resulting in worse outcomes than staphylococcal infections, especially for post-cataract endophthalmitis⁽¹⁰⁾. At our university, bacterial keratitis is caused by members of the genus Staphylococcus (51.7%) (coagulase-negative Staphylococcus followed by Staphylococcus aureus), Corynebacterium spp. (14.1%), Streptococcus spp. (9.9%), Pseudomonas spp. (6.3%), Moraxela spp. (5.5%), Serratia spp. (4.2%), Enterobacter spp. (1.9%), and others⁽¹¹⁾. Coagulase-negative staphylococci were the primary agents responsible for bacterial endophthalmitis (56.5%) in our setting, but alpha-hemolytic streptococci were ranked second (15.2%)⁽¹²⁾. These organisms are routinely separated into viridans group streptococci (VGS) and Streptococcus pneumoniae on the basis of their susceptibility to optochin and bile solubility.

Alpha-hemolytic streptococci comprise several species that are clustered into six major groups: mutans, salivarius, mitis, anginosus, sanguinis, and bovis⁽¹³⁾. S. pneumoniae is phylogenetically placed in the mitis group⁽¹⁴⁾. Phenotypic identification at the Streptococcus species-level is challenging and frequently erroneous⁽¹⁵⁾. The use of only one genetic target for species differentiation is often not reliable, as interspecies recombination events are common in this genus^(16-18).

The present study aimed to determine the species of alpha-hemolytic streptococci causing endophthalmitis and keratitis and their prevalence patterns, along with their antimicrobial resistance profiles, using a combination of phenotypic and genotypic approaches. In addition, we identified potential factors associated with disease pathogenesis and clinical outcomes.

METHODS

Bacterial isolates

We included 62 nonduplicated VGS isolates recovered from patients with endophthalmitis (n=27; 2002-2013) and keratitis (n=35; 2009-2013) seen at the Department of Ophthalmology and Visual Sciences, Federal University of São Paulo, Brazil. After confirmation in the clinical laboratory, when in vitro isolates derived from clinical samples showed substantial and pure growth in different culture media and were identified as part of routine laboratory protocols, the specimens were stored at -80°C in tryptic soy broth (TSB) with 15% glycerol. These isolates were recovered for research purposes from a freezer storage bank for isolates by culturing using commercial 5% sheep blood agar or chocolate agar (PROBAC, São Paulo, Brazil) at 37°C in a 5% CO₂ atmosphere before testing.

Patient data

The Federal University of São Paulo Institutional Review Board approved review of the medical records of the patients from whom the isolates were collected. Patient demographics included age and sex, risk factors associated with infection (e.g., surgery, intravitreal injection, and trauma), antibiotic treatment, and outcomes.

Phenotypic identification

Isolates were initially classified on the basis of biochemical tests regularly used to differentiate S. pneumoniae from other alpha-hemolytic streptococci, such as the optochin susceptibility test and bile solubility test using sodium deoxycholate. All tests were performed twice. The control S. pneumoniae ATCC49619 strain was used as a reference strain in all tests.

Antimicrobial susceptibility testing

Minimum inhibitory concentrations were determined by the broth microdilution method using a commercial panel of antibiotics (STP6F, Sensititre Trek; Thermo Scientific, Waltham, MA, USA). Quality control was performed by testing with the S. pneumoniae ATCC49619 strain. Interpretive criteria published by the Clinical and Laboratory Standards Institute, document M100-S27, were followed.

DNA extraction

DNA was extracted using Chelex 100 Molecular Biology Resin (BioRad, Hercules, CA, USA), as previously reported⁽¹⁹⁾. Briefly, fresh isolates grown on 5% sheep blood agar (PROBAC) were cultured in 5 mL of TSB without shaking overnight at 37°C in a 5% CO₂ atmosphere. An aliquot of 1 mL was centrifuged for 5 min at 13,000 rpm (5415R; Eppendorf, Hamburg, Germany), and the cells were washed twice using 1× phosphate-buffered saline. The pellets were resuspended in 300 µL of 10% Chelex 100 resin and incubated for 30 min at 95°C. After centrifugation for 5 min at 13,000 rpm, a 1 µL aliquot of the supernatant was collected, diluted 1:10 (v/v), and used for polymerase chain reaction (PCR) amplification.

Molecular identification

Molecular identification was initially performed by sequencing three constitutive genes, rpoB, sodA, and tuf, as previously reported⁽²⁰⁾. The PCR products were purified (QIAquick PCR Purification Kit; Qiagen, San Diego, CA, USA), and both strands were sequenced using the BigDye fluorescent terminator with an ABI 3000 genetic analyzer (Applied Biosystems, Foster City, CA, USA). The sequences obtained were edited using SeqMan (DNASTAR, Madison, WI, USA). Sequence identities were searched for in GenBank using the BLAST tool (https://blast.ncbi. nlm.nih.gov). Isolates without identification agreement for these three genes were subjected to multilocus sequence analysis (MLSA), as previously described⁽²⁰⁾. A neighbor-joining tree was constructed on the concatenated MLSA alleles using MEGA software, version 5.0 (https://mega.software.informer.com/5.0/).

Multilocus sequence typing (MLST) scheme for Streptococcus pneumoniae

All S. pneumoniae isolates (n=19) were subjected to MLST, as previously described⁽²¹⁾. The MLST procedure and details of the full analysis are available at the S. pneumoniae MLST website (https://pubmlst.org/spneumoniae/). Sequence types (STs) were assigned using the same database, and clonal complexes (CCs) were determined using the goeBURST algorithm (http://www.phyloviz.net/goeburst/).

Detection of the capsular polysaccharide (cps)-encoding gene

PCR amplification of an internal product of the cpsA gene (654 bp) was performed using the following primer pairs: forward, 5'-TACTAGTTGCCTTGGTAGG-3'; reverse, 5'-CGATTGGTACATAGGCATCA-3'. A 25 µL reaction mix was prepared using 12.5 µL of 2× GoTaq Green Master Mix (2.5 U GoTaq DNA Polymerase, 400 µM of each dNTP, 3.0 mM MgCl₂, and reaction buffer) (Promega, Madison, WI, USA), 0.5 µL of each primer, 1 µL of DNA template, and sterile Milli-Q water. The PCR conditions were as follows: initial denaturation at 95°C for 10 min; 30 cycles of 95°C for 60 s, 51°C for 60 s, and 72°C for 60 s; and a final extension at 72°C for 10 min. The amplicons were separated using agarose gel electrophoresis.

Statistical analyses

Fisher’s exact test was used to analyze the distributions of the infectious species that caused endophthalmitis and keratitis. Simpson’s diversity index (SDI) was used to evaluate S. pneumoniae diversity.

RESULTS

Streptococcus oralis and Streptococcus pneumoniae are the leading causes of ocular streptococcal infections

To determine the most common species of alpha-hemolytic streptococci causing ocular infections in our setting, we used a combination of phenotypic and genotypic tests to identify 62 alpha-hemolytic streptococcal isolates from patients with endophthalmitis or keratitis. On the basis of the optochin susceptibility and bile solubility tests, 30.6% (n=19) of the isolates in our collection were identified as S. pneumoniae, and the other 69.4% (n=43) were placed in the viridans group.

Species-level identifications of the 43 alpha-hemolytic streptococcal isolates were initially performed by analyzing the rpoB, soda, and tuf sequences. Further analysis using a full MLSA scheme⁽²⁰⁾ was then performed on 15 isolates that were not successfully identified by the initial approach (Figure 1). Phenotypic identification of isolates as S. pneumoniae was confirmed molecularly. In total, nine different species were identified in our collection (Table 1). Overall, S. oralis (32.2%) and S. pneumoniae (30.6%) predominated. The distribution of these species was not random across different diseases. S. oralis was significantly prevalent in endophthalmitis (13/27; 48.1%; p=0.0013), whereas S. pneumoniae was the leading cause of keratitis (17/35; 48.6%; p=0.0013).

Figure 1. Phylogenetic tree displaying the clonal relationship between the strains under identification in this study (squares) and reference strains (circles) stored in the eMLSA database (http://viridans.emlsa.net/). The phylogenetic tree was constructed using the neighbor-joining algorithm and concatenated sequences of seven housekeeping gene fragments (map, pfl, ppaC, pyk, rpoB, sodA, and tuf). The outgroup was represented by Streptococcus agalactiae, S. pyogenes, and S. suis (triangles).

Resistance rates of clinically relevant antibiotics

In general, VGS and S. pneumoniae were highly susceptible to commonly used antibiotics (Table 2). Only a small percentage of VGS isolates were resistant to the fluoroquinolones frequently used in ophthalmology, namely, levofloxacin (7.0%) and moxifloxacin (2.3%), whereas all S. pneumoniae isolates were susceptible to these drugs. All isolates were susceptible to vancomycin (MIC₉₀ ≤0.5 ìg/mL), linezolid (MIC₉₀ 2 ìg/mL), and daptomycin (MIC₉₀ 1 ìg/mL for VGS and 0.12 ìg/mL for S. pneumoniae). Using oral penicillin V interpretive breakpoints, the rates of penicillin resistance were 21.1% for S. pneumoniae and 16.3% for VGS. However, these isolates were susceptible to other beta-lactam agents tested, including amoxicillin clavulanate and the third- and fourth-generation cephalosporins ceftriaxone and cefepime. Only 2.3% of VGS isolates were not susceptible to ertapenem and meropenem. The highest rates of resistance among VGS were seen with the macrolides azithromycin (60.5%, MIC₉₀ >2 ìg/mL) and erythromycin (62.8%, MIC₉₀ >2 ìg/mL). By contrast, S. pneumoniae isolates were sensitive to both drugs. Resistance to trimethoprim-sulfamethoxazole (57.9%) and chloramphenicol (5.3%) was more frequently observed in S. pneumoniae than in VGS (39.5% and 0.0%, respectively), whereas resistance to tetracycline (25.6%) and clindamycin (16.3%) was more frequently observed in VGS than in S. pneumoniae (10.5% and 0.0%, respectively).

The population structure of Streptococcus pneumoniae was highly diverse

A deviant and unique clade of unencapsulated S. pneumoniae has been identified as the predominant cause of outbreak-related and outbreak-nonrelated conjunctivitis in the United States⁽²²⁾. To determine whether these conjunctivitis strains were present in our setting, we detected cpsA using a combination of MLST and PCR. We found that most of the S. pneumoniae isolates (15/19; 78.9%) tested positive for the cpsA gene, and these were predicted to be encapsulated strains (Table 3). MLST analysis demonstrated that our collection was highly diverse. In total, 13 different STs were found in our population (SDI=0.982). All STs belonged to different CCs, except for ST66 and ST73, which belonged to CC66. Three isolates (15.8%) were found to have new STs (ST13545, ST13546, and ST13547). Among the isolates lacking the cpsA gene (n=4), only one (ST2315) was found to be part of the previously reported epidemic conjunctivitis cluster of unencapsulated strains. The other cpsA-negative isolates belonged to ST1262 (two isolates) and ST11374 (one isolate), which were not reported as part of the conjunctivitis cluster.

Endophthalmitis and keratitis caused by alpha-hemolytic streptococci resulted in poor outcomes

The clinical features, antibiotic treatments, and visual outcomes of patients with endophthalmitis and keratitis are summarized in tables 4 and 5. Most of the endophthalmitis cases were postoperative following phacoemulsification (n=13; 48.1%). The others were post-traumatic (n=3; 11.1%), post-intravitreal injection (n=2; 7.4%), and endogenous (n=1; 3.7%) or due to corneal perforation (n=1; 3.7%), among other causes. The average age was 53 years (range, 1-89 years). Most of the patients were female (16/27, 59.2%). Most of the patients had vitreous humor collected for culture by means of vitrectomy (n=13) or vitreous tap (n=8), and aqueous humor culture was performed on a smaller number of patients (n=6). In a subset of patients for whom treatment information was available (n=15), the majority (n=14) were treated with intravitreal injections of antibiotics, mainly vancomycin and ceftazidime, as well as topical fluoroquinolones (n=12) and a variety of oral or intravenous antibiotics. Vitrectomy was performed in 10 patients and anterior chamber washing in 2 patients. Corticoid use was reported in 11 cases. Despite prompt clinical and surgical treatment, the patients had poor visual outcomes. Among 12 patients with recorded final visual acuity (VA) outcomes, 1 patient had a final VA of 20/30 and another had a VA of 20/150. The others had final VA scores of “hand motion” (n=1), “light perception” (n=3), and “no light perception” (n=6).

The average age of patients with keratitis was 54 years (range, 7-93 years) for 22 females and 13 males. Ophthalmological procedures prior to keratitis included cataract surgery (n=10; 28.6%), corneal transplant (n=5; 14.3%), and glaucoma surgery (n=3; 8.6%). It was possible to retrieve medical records for some patients (n=22), and we found that most patients were treated with topical fluoroquinolones (n=21) as monotherapy (n=13) or in combination with other antibiotics (n=8). Among 18 patients with recorded final VA outcomes, only a small fraction had a final VA ≥20/200 (n=3), whereas in the others, the final VA scores were “hand motion” (n=6), “light perception” (n=1), or “no light perception” (n=6; including one case with a previously low VA). Among patients with a final VA of “no light perception,” evisceration was performed in three patients, and one patient developed phthisis bulbi.

DISCUSSION

Alpha-hemolytic streptococci are part of a large group of organisms, for which species-level identification is not routinely performed. Generally, clinicians are limited to differentiation between S. pneumoniae and the viridans species as a group⁽²³⁾. This creates a gap in our understanding of ocular infections caused by this group of bacteria. Here we sought to determine the species distribution of alpha-hemolytic streptococci causing ocular infections, their associated antibiotic sensitivity profiles, and clinical outcomes. We found nine different species representing the mitis and sanguinis groups, with S. oralis, S. pneumoniae, Streptococcus sanguinis, and Streptococcus mitis predominating. Differences were present according to the site of infection. A high number of S. oralis isolates were identified in both endophthalmitis and keratitis. S. sanguinis was more common in endophthalmitis, S. pneumoniae was more common in keratitis, and S. mitis was only recovered from keratitis patients.

Microbiological studies have reported that the genus Streptococcus, along with the genera Staphylococcus, Corynebacterium, and Propionibacterium, comprises the commensal bacterial population on the ocular surscies is the upper respiratory tract, which is why it is also speculated that the growth of S. pneumoniae in endophthalmitis that develops post-intravitreal injection may be associated with aerosolization of saliva. This may occur more often when the injection is performed in the physician’s office if regular precautions such as mask-wearing or a no-talking rule are not followed⁽²⁵⁾. It should also be noted that in ophthalmology, the use of preoperative povidone iodine antiseptic in eye preparations is highly recommended, because it is considered effective and economically reasonable and does not induce antibiotic resistance⁽²⁶⁾.

We found that S. oralis was the most prevalent species causing streptococcal endophthalmitis in our population (p=0.0013), followed by S. sanguinis. However, most of the endophthalmitis cases included in our study were associated with phacoemulsification surgery, where the route of contamination was expected to differ from that in-office intravitreal injection^(24,27). When inoculated into the posterior chamber, VGS can cause aggressive and rapidly developing endophthalmitis. In this study, the majority of endophthalmitis infections resulted in very poor final VA, with patients scored as “no light perception” (n=6), “light perception” (n=3), or “hand motion” (n=1). Of the six patients with no light perception, one was subjected to enucleation and another to evisceration, and two resulted in phthisis bulbi. Similar clinical outcomes were described for patients involved in an outbreak of post-intravitreal injection endophthalmitis caused by S. mitis/S. oralis in southern Florida, where 11 of 12 patients were left with minimal vision with a VA ≤ “hand motion”⁽⁶⁾.

In contrast to endophthalmitis, infectious keratitis was predominantly caused by S. pneumoniae (p=0.0013) and less frequently by S. oralis or S. mitis. S. pneumoniae is not only a major cause of conjunctivitis⁽²²⁾ but also a common cause of infectious keratitis. Pneumococcal keratitis is not typically associated with contact lens wear, but predisposing conditions such as ocular trauma or surgery are important risk factors⁽²⁸⁾. In our population, most patients with pneumococcal keratitis had a history of cataract or glaucoma surgery, corneal transplantation, or trauma. Most of these patients were treated with fluoroquinolones as monotherapy and also had poor visual outcomes.

There was high genetic diversity (SDI=0.982) in the S. pneumoniae population studied, with 13 different STs found among 19 isolates (2 isolates from endophthalmitis and 17 from keratitis). All STs belonged to different CCs, except for ST66 and ST73 (CC66). Most of these STs (n=15; 78.9%) tested positive for the cpsA gene and were involved in the biosynthesis of capsular polysaccharide, which is a key virulence factor; the capsule surrounds the bacterial cell and forms a protective barrier to resist the host immune system⁽²⁹⁾. Only four isolates (21.1%) tested negative for the cpsA gene and were therefore predicted to be unencapsulated. These isolates belonged to ST1262 and ST11374 and the conjunctivitis-associated strain ST2315⁽²²⁾. Antibodies against the capsule are the basis of current vaccines that are composed of capsular polysaccharides conjugated to protein (PCV7, PCV10, and PCV13), and epidemiological reports indicate that there is an increasing prevalence of conjunctivitis outbreaks and otitis media infections caused by unencapsulated S. pneumoniae strains^(29,30). In the present study, all but four S. pneumoniae isolates tested positive for the cpsA gene. This may indicate a possible failure of current pneumococcal immunization targeting the common polysaccharide capsular serotypes. Therefore, further studies using a larger population and serotyping information will be necessary to determine if this vaccine fails to prevent ocular pneumococcal infections.

Fluoroquinolones combined or as monotherapy are the most frequently used class of topical antibiotics in the treatment of ocular infections. Although resistance to fluoroquinolones among alpha-hemolytic streptococci may arise after exposure, it is still relatively uncommon. In our population, only 7.0% of the VGS isolates were resistant to levofloxacin and 2.3% were resistant to moxifloxacin. All S. pneumoniae isolates were susceptible to the fluoroquinolones.

High levels of susceptibility were observed for antibiotics frequently used to treat endophthalmitis, such as cephalosporins (cefotaxime, ceftriaxone, and cefepime) and vancomycin (100%). The cases included in our study were mainly treated with intravitreal injections of vancomycin and ceftazidime, along with topical use of fluoroquinolones and corticoids. Despite the sensitivity of the isolates to the antimicrobials used, most of the patients had poor visual outcomes, demonstrating that virulence factors other than resistance to antibiotics played an important role in the course of eye infections caused by alpha-hemolytic streptococci.

In the present study, the majority of keratitis cases were treated with fluoroquinolones (n=21 out of 22 data recoveries) as monotherapy (n=13) or combined with other antibiotics (n=8). VGS exhibited high susceptibility to cephalosporins, although the in vitro susceptibility of VGS recovered from infectious keratitis cases was lower for levofloxacin (93.0%) and moxifloxacin (97.7%).

In conclusion, the distribution of alpha-hemolytic Streptococcus species in ocular infections is not random, suggesting that possible species-specific tissue tropism exists. This finding is consistent with a model in which S. pneumoniae is better able to attach to the corneal epithelium and resist local immune defenses at this site, whereas S. oralis exhibits a greater capacity to invade the posterior chamber and cause endophthalmitis. Additionally, antibiotic resistance does not seem to be an important contributor to the differential selection of these species in different ocular tissues and is also not likely to play a role in poor clinical evolution and outcomes.

ACKNOWLEDGMENTS

This work was supported by FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo, 2012/11094-3). The funding agency had no role in the study design, data analysis, decision to publish, or preparation of the manuscript. Dr. A. Leyva (USA) helped with English editing of the manuscript.

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Submitted for publication: May 23, 2019.
Accepted for publication: November 1, 2019.

Approved by the following research ethics committee: UNIFESP (# 0138/12).

Funding: This study was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo, 2012/11094-3.

Disclosure of potential conflicts of interest: None of the authors have any potential conflicts of interest to disclose.