Identification and Antibiotic Susceptibility of Coagulase Negative

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Br J Ophthalmol 1999;83:771–773 771 Identiﬁcation and antibiotic susceptibility of coagulase negative staphylococci isolated in Br J Ophthalmol: first published as 10.1136/bjo.83.7.771 on 1 July 1999. Downloaded from corneal/external infections

Antonio Pinna, Stefania Zanetti, Mario Sotgiu, Leonardo A Sechi, Giovanni Fadda, Francesco Carta

Abstract microbiology laboratory, identification of sta- Aims—To identify and determine anti- phylococci is often limited to a rapid screening biotic susceptibility of coagulase negative test for S aureus, while non-S aureus isolates are staphylococci (CoNS) isolated from pa- simply reported as CoNS species. Apart from tients with chronic blepharitis, purulent being a common component of the normal conjunctivitis, and suppurative keratitis. ocular flora, these bacteria have also been Methods—A retrospective review of all reported to cause chronic blepharitis,23 culture positive cases of chronic blephari- conjunctivitis,45and keratitis.67 tis, purulent conjunctivitis, and suppura- Despite being important ocular pathogens, tive keratitis between July 1995 and CoNS have so far received little attention in December 1996 was performed. Cases in ophthalmology. The purpose of this study was which CoNS were the sole isolates were to identify and determine antibiotic suscepti- analysed. Species identification was per- bility of CoNS isolated from patients with formed by using a commercially available chronic blepharitis, purulent conjunctivitis, standardised biochemical test system. and suppurative keratitis. Antibiotic susceptibility to penicillin, gentamicin, tetracycline, erythromycin, ciprofloxacin, and teicoplanin was deter- Materials and methods mined by agar disc diVusion (Kirby– A retrospective review of all culture positive Bauer method). Teicoplanin resistance cases of chronic blepharitis, purulent conjunc- was confirmed by agar dilution. tivitis, and suppurative keratitis between July Results—42 Staphylococcus epidermidis, 1995 and December 1996 was performed. All four S warneri, three S capitis,twoS case in which CoNS were the sole isolates were hominis, one each of S xylosus, S simu- selected. In total 55 clinically significant strains lans, S equorum, and S lugdunensis were of CoNS from 45 outpatients were analysed. Thirty one CoNS were from eyes with chronic identified. 37 CoNS were penicillin resist- http://bjo.bmj.com/ ant, 12 gentamicin resistant, 28 tetracy- blepharitis, 12 from eyes with purulent con- cline resistant, 18 erythromycin resistant, junctivitis, and 12 from eyes with suppurative keratitis. Eight patients had bilateral chronic Institute of four ciprofloxacin resistant, and one te- Ophthalmology, icoplanin resistant (MIC, 32 µg/ml). In blepharitis and two bilateral acute conjunctivi- University of Sassari, total, 16 strains were resistant to three or tis; in the remaining 35, ocular infection Sassari, Italy more antibiotics. involved one eye only. A Pinna Conclusion—Species of CoNS apart from Specimens were collected from the inferior M Sotgiu fornix of the palpebral conjunctiva, lid mar- on October 3, 2021 by guest. Protected copyright. F Carta S epidermidis may be isolated from patients with corneal and external infec- gins, and corneal ulcers using Dacron swabs. Department of tion. Antibiotic susceptibility of CoNS is Three swabs were used for each eye. The first Biomedical Sciences, unpredictable and multiresistant strains swab was inoculated into thioglycolate broth Section of are common. As a result, antibiotic sus- supplemented with sterile fetal calf serum Experimental and ceptibility testing should be performed in (10% vol/vol) for the detection of aerobic and Clinical Microbiology, all cases of clinically significant ocular anaerobic bacteria. Tubes were incubated at University of Sassari, 37°C for at least 72 hours. Subcultures were Sassari, Italy infections caused by CoNS. S Zanetti (Br J Ophthalmol 1999;83:771–773) performed by using chocolate, mannitol salt, L A Sechi and McConkey agar plates. The second swab was rolled on a Sabouraud dextrose agar plate Institute of Members of the genus Staphylococcus are non- for the detection of fungi. The third swab was Microbiology, motile, non-spore forming, usually unencapsu- inoculated into 4SP transport medium for Università Cattolica lated Gram positive cocci; most species are . Cultures for the detec- del Sacro Cuore, Chlamydia trachomatis Rome, Italy facultative anaerobes. They are indigenous tion of bacteria, fungi, and C trachomatis were G Fadda microflora of the skin and mucous membranes performed according to a previously recom- of humans and are a common cause of eyelid, mended protocol.9 CoNS were identified by Correspondence to: Dr conjunctival, or corneal infection.1–7 The pres- Gram staining, colony morphology on agar Antonio Pinna, Istituto di 8 Clinica Oculistica, Università ence or absence of coagulase, an enzyme that plates, and slide test for coagulase. degli Studi di Sassari, Viale clots plasma, divides the Staphylococcus species Over the 18 month period, as they were iso- San Pietro 43 A, 07100 into two broad groups: the coagulase positive lated, CoNS were immediately speciated. Spe- Sassari, Italy. staphylococci and coagulase negative staphylo- cies identification was performed by using the Accepted for publication cocci (CoNS). Fourteen species of CoNS are API ID 32 Staph (bioMérieux, La Balme les 4 February 1999 currently recognised as human isolates.8 In the Grottes, Montalieu Vercieu, France), a stand- 772 Pinna, Zanetti, Sotgiu, et al

Table 1 Species identiﬁcation of coagulase negative Table 3 Multiple antibiotic resistance of coagulase staphylococci isolated from patients with chronic blepharitis, negative staphylococci

purulent conjunctivitis, and suppurative keratitis Br J Ophthalmol: first published as 10.1136/bjo.83.7.771 on 1 July 1999. Downloaded from Resistance to Number (%) of isolates No of cases 2 antibiotics 12 (22) Species (No of Chronic Purulent Suppurative 3 antibiotics 11 (20) isolates) blepharitis conjunctivitis keratitis 4 antibiotics 3 (5) 5 antibiotics 1 (2) S epidermidis (42) 21 12 9 6 antibiotics 1 (2) S warneri (4) 4 — — S capitis (3) 2 — 1 S hominis (2) 2 — — Strains which showed multiple antibiotic S xylosus (1) — — 1 S simulans (1) 1 — — resistance were stored in nutrient broth with S equorum (1) 1 — — 15% glycerol at −80°C for further reference. S lugdunensis (1) — — 1

Results ardised and miniaturised biochemical test sys- Forty two S epidermidis, four S warneri, three S tem with a specially adapted database. The capitis,twoS hominis, one each of S xylosus, S API ID 32 Staph system strip consists of 32 simulans, S equorum, and S lugdunensis were cupules, 26 of which contain dehydrated identified (Table 1). The results of antibiotic biochemical media for colorimetric tests. The susceptibility testing are given in Tables 2 and tests included acid production from glucose, 3. Overall, 37 (67%) isolates were resistant to fructose, mannose, maltose, lactose, trehalose, penicillin, 12 (22%) to gentamicin, 18 (33%) mannitol, raYnose, sucrose, N-acetyl- to erythromycin, four (7%) to ciprofloxacin, glucosamine, turanose, ribose, arabinose, and one (2%) to teicoplanin. Susceptibility to cellobiose; decarboxylation of arginine and tetracycline was tested on 49 isolates; 28 (57%) ornithine; production of urease, â glucuroni- were found to be tetracycline resistant. On the dase, â galactosidase, acetoin, alkaline phos- whole, eight (15%) strains were sensitive to all phatase, arginine arylamidase, and antibiotics, whereas 16 (29%) were resistant to pyrrolidonyl-arylamidase; hydrolysis of aescu- three or more antibiotics. One strain of S lin; reduction of nitrate; and susceptibility to epidermidis was found to be resistant to all anti- novobiocin. The manufacturer’s recom- biotics. Teicoplanin resistance determined by agar disc di usion was confirmed by the agar mended procedures were followed. Strain pro- V dilution method (MIC 32 µg/ml). files were read and identified with automatic testing bacteriology equipment and were inter- preted with APILAB software. Discussion Antibiotic susceptibility tests were deter- Methods for the identification of CoNS mined by agar disc diVusion (Kirby–Bauer include conventional as well as commercial 10 method). The following antibiotics were systems. The conventional method8 is relatively http://bjo.bmj.com/ tested: penicillin, gentamicin, tetracycline, cumbersome for routine laboratory use and erythromycin, ciprofloxacin, and teicoplanin. A may take up to 5 days of incubation to obtain a standard agar dilution method11 was used to result. Commercial biochemical systems can confirm teicoplanin resistance determined by identify a number of CoNS with accuracy of agar disc diVusion. In brief, overnight cultures more than 95% with relative speed and of bacteria (≥109 colony forming units/ml) simplicity.13 In this study, species identification were plated at a series of dilutions on tryptic of CoNS was performed by using API ID 32 soy agar plates containing antibiotic-free me- Staph, a commercially available biochemical on October 3, 2021 by guest. Protected copyright. dium or twofold dilutions of teicoplanin within test system which can identify 24 species. This the drug concentration range of 0.1–128 system has been reported to be able to provide µg/ml. Plates were incubated at 37°C for 48 a correct species identification for 95.2% of hours and the number of bacterial colonies was CoNS analysed, with an accuracy of 98%– counted. Teicoplanin minimal inhibitory con- 100% for S epidermidis, S lugdunensis, S capitis, centration (MIC) was defined as the lowest S warneri, and S xylosus.13 In our study, eight concentration of the antibiotic causing 99.9% species of CoNS were identified, the most loss of the bacterial inoculum. Teicoplanin common being S epidermidis (76% of all MICs for most clinical isolates of CoNS have isolates). Other species of CoNS were identi- been reported to be ≤8 µg/ml or ≤16 µg/ml.12 fied in 10 patients with chronic blepharitis and in three with suppurative keratitis. Interest- Table 2 Antibiotic susceptibility testing of coagulase negative staphylococci ingly, all four isolates of S warneri were from patients with chronic blepharitis. Strains of S Species Penicillin Gentamicin Tetracycline Erythromycin Ciprofloxacin Teicoplanin capitis, S xylosus, and S lugdunensis were S epidermidis 30/42* 9/42 20/36† 14/42 3/42 1/42 isolated from patients with suppurative kerati- S warneri 3/4 1/4 3/4 0/4 0/4 0/4 tis. S capitis 1/3 1/3 2/3 2/3 1/3 0/3 S hominis 1/2 0/2 2/2 1/2 0/2 0/2 Because of their ubiquitous nature and rela- S xylosus 1/1 1/1 1/1 1/1 0/1 0/1 tively low virulence, staphylococci other than S S simulans 0/1 0/1 0/1 0/1 0/1 0/1 are often simply reported as CoNS in S equorum 1/1 0/1 0/1 0/1 0/1 0/1 aureus S lugdunensis 0/1 0/1 0/1 0/1 0/1 0/1 the microbiology laboratory. However, over the Total 37/55 12/55 28/49† 18/55 4/55 1/55 past 15 years, there has been an increase in the *Numbers indicate resistant isolates/total isolates. documentation of ocular infections caused by †Susceptibility to tetracycline was not performed in 6 isolates. CoNS. Identification and antibiotic susceptibility of coagulase negative staphylococci isolated in corneal/external infections 773

Evidence indicates that these bacteria play a aims of this study. Other authors reported that primary role in the production of disease in treatment with ciprofloxacin in ciprofloxacin clinical staphylococcal and mixed seborrhoeic/ resistant S epidermidis keratitis was Br J Ophthalmol: first published as 10.1136/bjo.83.7.771 on 1 July 1999. Downloaded from staphylococcal blepharitis.3 In a relatively unsuccessful.18 Similar results have been re- recent study, CoNS have been reported to be ported in the treatment of ciprofloxacin resist- the most frequently isolated pathogens from ant Pseudomonas keratitis (Garg P, presented at patients with acute bacterial conjunctivitis.5 the AAO Annual Meeting, New Orleans, Two recent studies performed by the Bacterial 1998). Keratitis Study Research Group6 and the Cip- In conclusion, the need for species identifi- rofloxacin Bacterial Keratitis Study Group7 cation of clinically significant CoNS must be have shown that the most frequent isolates emphasised. With the availability of highly spe- from patients with bacterial keratitis were cific commercial identification systems, it is CoNS. Moreover, CoNS are currently the likely that species of CoNS apart from S epider- most common organisms isolated from cases of midis will be increasingly implicated as causa- postoperative endophthalmitis.14 15 tive organisms in corneal and external infec- Because of their increasing importance, tion. Since the antibiotic susceptibility of clinically significant CoNS should be identified CoNS is unpredictable and multiresistant to the species level. Species identification will isolates are common, we recommend to improve our knowledge about the role played perform antibiotic susceptibility testing in all in clinical diseases by this group of bacteria. cases of clinically significant ocular infections In this study, CoNS isolated from patients caused by these organisms. with corneal and external infection showed highly variable patterns of antibiotic suscepti- Presented in part as a poster at the American Academy Annual bility. Eight (15%) strains only were sensitive Meeting, San Francisco, California, 26–29 October 1997. to all antibiotics. Most isolates were penicillin 1 McCulley JP, Dougherty JM, Denau DG. Classification of and tetracycline resistant, whereas resistance to chronic blepharitis. Ophthalmology 1982;89:1173–80. ciprofloxacin and teicoplanin was found in four 2 Dougherty JM, McCulley JP. Comparative bacteriology of chronic blepharitis. Br J Ophthalmol 1984;68:524–8. (7%) and one (2%), respectively. Given the 3 McCulley JP, Dougherty JM. Bacterial aspects of chronic high broad spectrum activity against most blepharitis. Trans Ophthalmol Soc UK 1986;105:314–8. 16 4 Soukiasian SH, Baum J. Bacterial conjunctivitis. In: Krach- bacteria and the reduced frequency of ocular mer JH, Mannis MJ, Holland EJ, eds. Cornea. St Louis, toxic eVects,7 ciprofloxacin is currently consid- Mosby-Year Book, 1996; vol 2: chap 63. 5 Leibowitz HM. Antibacterial eVectiveness of ciprofloxacin ered the drug of choice in the therapy for bac- 0.3% ophthalmic solution in the treatment of bacterial terial keratitis. However, ophthalmologists conjunctivitis. Am J Ophthalmol 1991;112:29S–33S. 6 O’Brien TP, Maguire MG, Fink NE, et al. EYcacy of should be aware that there is growing evidence ofloxacin vs cefazolin and tobramycin in the therapy for for ciprofloxacin resistant strains of CoNS and bacterial keratitis. Arch Ophthalmol 1995;113:1257–65. 7 Hyndiuk RA, Eiferman RA, Caldwell DR, et al. Comparison should be prepared to modify treatment of ciprofloxacin ophthalmic solution 0.3% to fortified accordingly. tobramycin-cefazolin in treating bacterial corneal ulcers. Ophthalmology 1996;103:1854–63. Teicoplanin, as well as vancomycin, is an 8 Kloos WE, Bannerman TL. Staphylococcus and Micrococ- anti-staphylococcal antibiotic to which resist- cus. In: Murray PR, Baron EJ, Pfaller MA, Tenover FC, Yolken RH, eds. Manual of clinical microbiology. 6th ed. http://bjo.bmj.com/ ance is rarely seen; their use should be reserved Washington, DC: American Society for Microbiology, for the treatment of staphylococcal infections 1995: chap 22. 17 9 Jones DB, Liesegang TJ, Robinson NM. Cumitech 13: labo- resistant to all other antibiotics. ratory diagnosis of ocular infections. Washington, DC: Ameri- Multiple antibiotic resistance of CoNS is a can Society for Microbiology, 1981. 10 National Committee for Clinical Laboratory Standards. well recognised problem, especially in nosoco- Performance standards for antimicrobial disk susceptibility tests. mial infections.12 17 In this study, 16 (29%) iso- Approved standards M2-A5. Villanova, PA: National Committee for Clinical Laboratory Standards, 1993. lates were resistant to three or more antibiotics. 11 National Committee for Clinical Laboratory Standards. Other authors2 reported resistance to two or Methods for dilution antimicrobial susceptibility tests for bacteria on October 3, 2021 by guest. Protected copyright. that grow aerobically. Approved standard M7-A4. Villanova, more antibiotics in 25% of the staphylococcal PA: National Committee for Clinical Laboratory Stand- isolates from patients with chronic blepharitis. ards, 1997. 12 Archer GL, Climo MW. 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