September 01, 2006
7 min read

Fungal infections, antibiotic resistance causes for culturing

You've successfully added to your alerts. You will receive an email when new content is published.

Click Here to Manage Email Alerts

We were unable to process your request. Please try again later. If you continue to have this issue please contact

The introduction of the fourth-generation fluoroquinolones has weakened the threat of gram-negative bacteria, and, as a result, practitioners are now concerned with other bacteria that are resistant to these fluoroquinolones.

“The number one reason for culturing is suspicion of resistance,” said Milton M. Hom, OD, FAAO, a Primary Care Optometry News Editorial Board member and diplomate in the Cornea and Contact Lens Section of the American Academy of Optometry. “When the fourth-generation fluoroquinolones first came out, they were clearly superior to other available drugs for the treatment of bacterial keratitis. We are probably in mid-lifecycle with them now, and the more resistance we see, the greater the need for culturing.”

When to culture

Elizabeth D. Muckley, OD, FAAO, a practitioner in Kent, Ohio, discussed the conditions for which she considers culturing to be necessary. “I typically recommend culturing for corneal ulcers that are central in location or that demonstrate an epidefect or infiltrate that is greater than 1.5 mm in size,” she told PCON. “Any ulcer that could be vision threatening needs to be cultured.”

Practitioners should suspect Pseudomonas or other atypical infections in contact lens wearers who have large, rapidly progressive, purulent ulcers, Dr. Muckley said. “Many other small peripheral ulcers are less worrisome and may be treated empirically with a fourth-generation fluoroquinolone without the need for cultures,” she said. “These infiltrates may also be sterile or noninfectious, particularly if they are multiple.”

Pseudomonas: Practitioners should suspect this condition or other atypical infections in contact lens wearers who have large, rapidly progressive, purulent ulcers.
Image: Muckley ED


Dr. Muckley said ulcers that do not respond to fourth-generation fluoroquinolones should also be cultured. “For these, I typically culture and change to fortified antibiotics,” she said. “If the condition doesn’t respond within 12 to 24 hours, then the atypical infections or resistant organisms, such as Acanthamoeba, fungi or mycobacteria, need to be ruled out.”

In these situations, Dr. Muckley said, knowledge of patient history and lifestyle is essential. “Swimming in contacts or corneal trauma with any organic matter may help point to Acanthamoeba or fungal infections,” she said. “Poor contact lens cleaning habits or a dirty contact case is another red flag.”

Dr. Muckley said she typically performs cultures of the conjunctiva only in the setting of a hyperpurulent conjunctivitis or chronic conjunctivitis (more than 3 weeks in duration). “The most worrisome organism in hyperacute conjunctivitis is gonorrhea,” she said. “Prompt diagnosis and treatment is critical, because any delay can progress rapidly to corneal involvement and perforation.”

According to Dr. Muckley, the prototypical organism causing chronic conjunctivitis is Chlamydia. She said special chlamydial cultures or immunofluorescence testing can be performed easily in the office.

“Often, I will empirically start a tetracycline antibiotic at the same time I obtain cultures,” she said. “Herpes simplex can also cause a follicular conjunctivitis, but this rarely needs to be cultured due to other diagnostic clues, such as corneal dendrites or vesicular lesions on the lid.”

Dr. Muckley added that her practice also cultures in suspected cases of endophthalmitis. “We are concerned most about endophthalmitis in the immediate postoperative setting,” she said. “Early postsurgical infections typically are caused by staph or strep organisms. These require immediate referral to a vitreoretinal surgeon for anterior chamber and vitreous tap followed by intraocular injection of antibiotics.”

Finally, Dr. Muckley cultures for any infiltrate in postrefractive surgery patients, corneal transplant patients and glaucoma patients who have had a trabeculectomy and show signs of conjunctivitis or blebitis.

Dr. Hom said, in his practice, nosocomial infections are the number one reason for culturing. “This is due to the higher rates of resistance,” he told PCON.

In terms of culturing, Dr. Hom follows the “1-2-3” rule: 1 mm from the visual axis, two or more infiltrates or 3 mm or more in size.


Culturing techniques

Dr. Muckley said, when culturing, she uses either a 15 blade or Kimura spatula for corneal scrapings. She also uses culture swabs or M4 medium for conjunctival staining. Microscope slides are available for cytology and Gram’s staining.

“When I culture for a corneal ulcer, I routinely use chocolate, blood and Sabouraud’s agar for plating if there are enough corneal scrapings for all three,” she said. “Sabouraud’s is specific for fungal keratitis, blood agar for most aerobes and chocolate agar for Neisseria and Haemophilus.”

She said she also uses the MicroGent swab combo (M4), as this is specific for viral herpes and Chlamydia, and a separate anaerobe collector or thioglycollate broth.

“If you suspect Acanthamoeba, a microscopic slide specimen with Giemsa stain or calcoflur white is recommended,” she said. “You can also use a non-nutrient agar with an overlay of E. coli. Often, Acanthamoeba cultures of the solution in the contact lens case can produce positive results, even when corneal cultures are negative.”

Corneal ulcer: Many clinicians will culture ulcers that do not respond to treatment with next-generation fluoroquinolones.
Image: Muckley ED

Dr. Muckley recommended a Lowenstein-Jensen culture for postrefractive surgery or penetrating keratoplasty patients, to test for atypical mycobacteria. “This may require that the LASIK flap be lifted to gain direct access to the infiltrate,” she said. “These organisms grow slowly, and culture results may take weeks.”

Dr. Muckley stressed the importance of warming the agar plates to room temperature prior to use, and she outlined her culturing technique for the lids/conjunctiva. “I typically do not use anesthetic for the conjunctiva. I use a sterile cotton-tipped swab moistened with sterile saline,” she said. “I rub the swab along the conjunctiva or lids several times and then roll it onto the broths. For a viral transport tube, the swab should have a plastic handle with a Dacron or polyester tip (not cotton), but most now come together. The swab is then broken off and inserted into the tube.”

Dr. Muckley said when culturing the cornea, she first anesthetizes it with proparacaine. “Proparacaine is the least bactericidal of all the anesthetics,” she said. “I typically use a sterile blade or spatula to scrape the edge and bed of the ulcer. Any loose epithelium should be removed and can be placed on a microscopic slide for examination.”

She said she plates out a zig-zag pattern on the media and repeats the scraping process for each plate. “You can also use a multiple ‘C’ pattern to inoculate the plate,” she said.

James Fanelli, OD, FAAO, a practitioner based in Wilmington, N.C., said when culturing, he has found standard culturettes to yield little information. “Bacterial growth from ocular tissue sampling simply did not occur enough to be able to get any growth that could lead to identification of the pathogen and a list of its sensitivities,” he said in an interview. “When I did culture regularly, I did not use standard agar plates. I did culture, however, on blood and chocolate agar. They seemed to afford moderate growth in a short amount of time, and sensitivities were readily established.”

Fungal infections a big concern

Dr. Fanelli said while he once deemed gram-negative bacteria such as Pseudomonas to be the most suspicious, the relative strength of the fourth-generation fluoroquinolones in dealing with gram-negative bacteria has caused his priorities to shift.

“Today, the concern is fungi,” he said. “Fungal infections are difficult to treat and often take a while to manifest. This should always be in the back of the clinician’s mind, especially when there is delayed healing.”

Dr. Fanelli said the fact that fungal keratitis often takes days to clinically manifest can be used to the practitioner’s advantage in the initial management.

“Of course, a corneal insult from an organic origin, such as a tree branch, should raise the distinct possibility that the offending microbe may be fungal rather than bacterial,” he said. “But in my experience, the incidence of fungal keratitis is much lower than bacterial keratitis. So, chances are, in the absence of a vegetative corneal insult, you are probably dealing with bacterial keratitis.”

Choosing empirical treatment

Assuming the existence of a bacterial keratitis, Dr. Fanelli said he usually feels safe in treating the patient with a fourth-generation fluoroquinolone and not initially culturing.

“It can certainly be argued that culturing should still be done, and I can agree with that in theory,” he said. “But in clinical practice, the use of gatifloxacin and moxifloxacin in conjunction with an aminoglycoside, such as tobramycin, will almost certainly result in resolution of the keratitis if the offending pathogen is bacterial.”

When to consider fungus

Dr. Fanelli added, however, that if there is little resolution of the microbial keratitis after 3 to 5 days of aggressive therapy with this regimen on an every-hour basis, the practitioner must consider the possibility of fungal origin.

“The clinician then needs to rethink the possibility of fungal infection,” he said. “Of course, in the initial few days of treating a presumed bacterial keratitis, if the clinical picture shows the development of satellite lesions or branching patterns to the initial lesion, fungal infection should be immediately suspected.”

At this point, a culture for fungi should be done promptly, Dr. Fanelli said. He prefers to refer the patient to a corneal specialist for the culture. “It is simply not cost-effective to maintain fungal culture media in the office, as the incidence of fungal keratitis is relatively low and these patients typically have a long road ahead of them,” he said. “While I am aggressive in what I treat in my office, fungal keratitis is one of those things I simply prefer not to deal with. The incidence of permanent vision loss is too high.”

Dr. Fanelli stipulated that steroids should not be used in the initial stages of any microbial keratitis. “While the anterior segment will become significantly inflamed in these cases, steroids should be avoided, as they cloud the clinical picture significantly as far as resolution of the lesion is concerned,” he said. “While they might make the eye feel better, the risk is simply too high to use them during the phase of active microbial proliferation. Instead, the clinician should use a potent cycloplegic such as 0.25% scopolamine up to four times daily to help quiet the anterior chamber. ”

For more information:
  • Milton M. Hom, OD, FAAO, can be reached at 1131 East Alosta Ave., Azusa, CA 91702-2740; (626) 334-1585; fax: (626) 335-1402; e-mail:
  • Elizabeth D. Muckley, OD, FAAO, can be reached at Northwest Ohio Eye Surgeons, 2013 State Rt. 59, Kent, OH 44240; (330) 678-0201; e-mail:
  • James Fanelli, OD, FAAO, can be reached at 5311 S. College Rd., Wilmington, NC 28412; (919) 452-7225; fax: (910) 452-7229; e-mail: