Novel Phagocytosis-Resistant Extended-Spectrum β-Lactamase–Producing Escherichia coli From Keratitis

Importance  Extended-spectrum β-lactamase (ESBL)-producing Escherichia coli are highly antibiotic resistant, and primary ocular infection by ESBL E coli has rarely been reported. A novel mutation conferring phagocytosis resistance would position a strain well to infect the cornea.

Observations  A woman with recurrent keratitis presented with a corneal ulcer, which was culture positive for ESBL E coli. Resistant to nearly all other antimicrobials, the infection was treated with amikacin and polymyxin B–trimethoprim, and the ulcer resolved over 3 weeks. Analysis of the E coli genome showed it to belong to multilocus sequence type 131 (ST131). This isolate was found to possess a novel deletion in yrfF, an essential regulator of bacterial capsule synthesis. Disruption of yrfF, which confers mucoidy and increased virulence, has not been previously observed in ESBL E coli from any infection site. This novel variant was experimentally proven to cause the mucoid phenotype, and corresponding resistance to phagocytic killing.

Conclusions and Relevance  Increased resistance to immune clearance in an ESBL E coli lineage already known for its virulence is an unsettling development. This phenotype, which likely positioned it as an unusual cause of corneal ulcer, can be easily recognized in the laboratory, which should help limit its spread.

Bacterial keratitis occurs when the integrity of the corneal epithelium is breached, allowing bacteria access to the stroma and sometimes resulting in sight-threatening ulceration. The most common bacterial agents are Streptococcus, Staphylococcus, Pseudomonas, and other Enterobacteriaceae (Klebsiella, Enterobacter, Serratia, and Proteus) species. Escherichia coli is a rare cause.¹

E coli producing an extended-spectrum β-lactamase (ESBL) recently emerged and are usually resistant to most other antibiotics, leaving few therapeutic options. Multilocus sequence type 131 (ST131) ESBL strains are now globally disseminated.^2,3 ST131 E coli is now a leading cause of extraintestinal infection, including bacteremia and pneumonia, as well as more common urinary tract, intra-abdominal, and wound infections. Although found in both clinical and community settings, ST131 infection is most common in the elderly and those residing in long-term care facilities.⁴

Because of its exposure to the environment, host defenses of the ocular surface are robust.⁵ Regarding ESBL-producing E coli as a cause of eye infection, rare cases have been reported.⁶ We present a case of keratitis caused by ESBL-producing E coli in a long-term care facility resident and show that the strain possessed a novel mutation that conferred mucoidy and increased virulence. We report the genome sequence, as well as observable phenotypes for this strain to aid in its recognition.

A female nursing home resident in her 60s with a history of glaucoma, blepharitis, and recurrent corneal ulcers in the right eye presented to the Massachusetts Eye and Ear Infirmary (MEEI) with a central corneal ulcer in the same eye. Her previous ulcers were exacerbated by exposure keratopathy and led to perforation, requiring 2 penetrating keratoplasties. Previous culture samples had grown Pseudomonas aeruginosa and Bacteroides fragilis. Because of her history, she had continued to receive treatment with topical prednisolone acetate, 1%, erythromycin ointment, and moxifloxacin hydrochloride. She had been hospitalized elsewhere 2.5 months prior for severe pneumonia. A sputum culture sample grew ESBL-producing E coli, which was not retained.

Hourly administration of fortified vancomycin (25 mg/mL) and tobramycin (14 mg/mL) was initiated following presentation, which was later changed to topical amikacin, 2.5%, and polymyxin B–trimethoprim (polymyxin B sulfate, 10 000 units/mL; trimethoprim, 1 mg/mL) every 3 hours after abundant growth of extensively antibiotic-resistant E coli was reported (Table 1). Subsequent cultures 6 days later had negative results, and the ulcer resolved after 18 days.

Because of its extensive antibiotic resistance profile and unusual occurrence in corneal infection, the ESBL E coli genome was sequenced and analyzed by the MEEI Infectious Disease Institute (eMethods in the Supplement). The genome of the strain, designated MEEI01, showed that it belonged to ST131, a widely disseminated ESBL E coli,^7-9 and was replete with antibiotic resistance determinants, including OXA-1 and CTX-M-15 β-lactamases (Table 1). Basic Local Alignment Search Tool (BLAST) comparison of the fimH adhesin showed it to be of the FimH30 sublineage.² Sequence identification of virulence genes identified 2 closely related copies of iss (which confer complement resistance and contribute to increased serum survival¹⁰), autotransporter secreted toxin sat, and adhesin genes iha and nfaE.

To gain insight into its unusual occurrence in a corneal ulcer, we compared MEEI01 to sequenced ST131 E coli, and found that it was very closely related to strain EC958¹¹—so close that no single-nucleotide polymorphisms were detected, implying rapid dissemination. The MEEI01 genome did harbor 7 insertion/deletion variations (indels) that distinguished it from EC958 (Table 2). The sequences surrounding and including each indel were compared to the National Center for Biotechnology Information database by nucleotide BLAST,¹² which revealed that 6 of the indels were shared with other ST131 strains, while 1 was unique. The unique indel occurred within yrfF, an essential gene in E coli.¹³ The 12-nucleotide deletion results in an in-frame variant eliminating 4 amino acids from the most C-terminal membrane-spanning helix domain (655delYLST). Although essential, a recent study found that repeated rounds of selection in vitro for resistance to macrophage phagocytosis and killing selected for yrfF mutations conferring increased capsule production, a mucoid phenotype, and enhanced virulence in a non-ESBL, laboratory strain.¹⁴

To determine whether the in-frame yrfF deletion observed in MEEI01 conferred the same virulence-associated phenotypes as those obtained by in vitro selection for increased resistance to phagocytic killing,¹⁴ we complemented MEEI01 with a wild-type copy of yrfF (Figure). Comparison of colony morphologies confirmed that the yrfF mutation in MEEI01 confers the mucoid phenotype, which was eliminated on complementation with the wild-type gene but not with empty vector (Figure, A-C). To determine whether the yrfF mutation also conferred increased phagocytosis resistance, RAW264.7 murine macrophages were exposed to each bacterial strain.¹⁴ As predicted, MEEI01 was more resistant to phagocytic elimination than the yrfF-complemented strain possessing the wild-type colony morphology (Figure, D). These data show that phagocytosis-resistant variants of E coli ST131 arise in vivo, effected by a novel yrfF mutation.

E coli is a rare cause of microbial keratitis,¹ and cases due to multidrug-resistant, ESBL-producing strains have been rarely reported.⁶ Among possible forces that selected for the outgrowth of a phagocytosis-resistant ST131 strain, the patient from whom MEEI01 was isolated had been treated with a protracted course of erythromycin and moxifloxacin in the affected eye for months. These antibiotics likely vacated the ocular surface, providing an opportunity for the outgrowth of MEEI01, which both possesses high-level fluoroquinolone resistance and is intrinsically resistant to erythromycin. Moreover, the innate mucosal defenses of the ocular surface, including complement as well as diurnal neutrophil surveillance,⁵ likely selected for outgrowth of this strain, which possesses multiple complement resistance genes, in addition to the novel yrfF mutation conferring mucoidy and phagocytosis resistance.

The recent global spread of the ST131 lineage has been attributed to its extensive antibiotic resistance, increased transmissibility, and enhanced virulence.³ Although known to confer mucoidy and enhanced virulence in E coli,¹⁴ to our knowledge, mutations in yrfF have not been reported previously in ESBL-producing strains. In MEEI01, an in-frame deletion of 12 bases in this essential gene results in loss of 4 amino acids from a predicted membrane-spanning helix of YrfF, which likely affects protein folding in the bacterial cell membrane and alters function. YrfF complementation did not alter the drug resistance profile or the growth rate of MEEI01 (data not shown), indicating that other forces, such as phagocytosis resistance, selected for its occurrence.

We postulate that the yrfF mutation in MEEI01 was selected for by its protracted occurrence on an ocular surface treated prophylactically with moxifloxacin and erythromycin, combined with cyclic exposure to phagocytic clearance by neutrophils. The latter may have allowed the microbe to persist and accumulate at a site predisposed to infection. Consistent with the known behavior of yrfF reduced-function mutants,¹⁴ MEEI01 exhibited a mucoid phenotype that is readily identifiable on specific growth media. Awareness of the phenotype of this virulent, highly antibiotic-resistant variant may help limit its spread.

A novel mucoid variant of the extensively drug-resistant ST131 lineage of ESBL-producing E coli was isolated from a corneal ulcer. The recent rapid global spread of the highly antibiotic-resistant and pathogenic ST131 ESBL E coli lineage is an alarming development.^3,15 Here we find that ST131 is also capable of developing resistance to immune clearance via increased capsule production, showing the adaptability and continued evolution of a leading cause of extraintestinal pathogenic E coli infection, now extending its range to the ocular surface.

Accepted for Publication: July 18, 2016.

Corresponding Author: Michael S. Gilmore, PhD, Department of Ophthalmology, Harvard Medical School, Massachusetts Eye and Ear Infirmary, 243 Charles St, Boston, MA 02114 (michael_gilmore@meei.harvard.edu).

Published Online: September 15, 2016. doi:10.1001/jamaophthalmol.2016.3283

Author Contributions: Dr Gilmore had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Study concept and design: Van Tyne, Ciolino, Durand, Gilmore.

Acquisition, analysis, or interpretation of data: All authors.

Drafting of the manuscript: Van Tyne, Ciolino, Wang, Gilmore.

Critical revision of the manuscript for important intellectual content: Van Tyne, Ciolino, Durand, Gilmore.

Statistical analysis: Van Tyne.

Obtained funding: Gilmore.

Administrative, technical, or material support: Van Tyne, Ciolino, Wang, Gilmore.

Study supervision: Van Tyne, Ciolino, Gilmore.

Conflict of Interest Disclosures: All authors have completed and submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Dr Gilmore reports grants from the National Eye Institute and the National Institute of Allergy and Infectious Diseases of the National Institutes of Health during the conduct of the study. No other disclosures are reported.

Funding/Support: This work was supported by Public Health Service grant EY024285, as well as support provided by the Harvard Medical School Department of Ophthalmology for the Massachusetts Eye and Ear Infirmary Infectious Disease Institute. Additional funding was provided by the Harvard University–wide Program on Antibiotic Resistance (AI083214). Dr Van Tyne receives support from grant AI109855, and Dr Ciolino is supported by a Career Development Award from Research to Prevent Blindness, Inc.

Role of the Funder/Sponsor: The funders had no role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; preparation, review, or approval of the manuscript; and decision to submit the manuscript for publication.

Additional Contributions: James J. Cadorette, M(ASCP), and Rick P. Boody, (MT)MLT, Henry Whittier Porter Bacteriology Laboratory at the Massachusetts Eye and Ear Infirmary, isolated, processed, and drug tested the ESBL E coli strain. Paulo J. M. Bispo, PhD, Jenna I. Wurster, BA, and Wolfgang Haas, PhD, MEEI Infectious Disease Institute, played a role in developing, managing, and curating the microbial strain collection. José Saavedra, BS, MEEI, provided assistance in preparing the MEEI01 genomic DNA library for sequencing and photographic assistance. Elizabeth Selleck Fiore, PhD, MEEI, provided help with macrophage phagocytosis assays. We fondly acknowledge the memory of Kim Gaither, PhD, Oklahoma Christian University, for the creation of the pKIM2 vector. The collaborators listed here received no additional compensation for their contributions to this study.