JOURNAL OF CLINICAL MICROBIOLOGY, JUlY 1987, p. 1258-1261 Vol. 25, No. 7 0095-1137/87/071258-04$02.00/0 Copyright © 1987, American Society for Microbiology Prevalence of Thymidine-Dependent Staphylococcus aureus in Patients with Cystic Fibrosis PETER H. GILLIGAN,l2* PATTI A. GAGE,' DAVID F. WELCH,3'4 MICHAEL J. MUSZYNSKI,4 AND KIMBERLEY R. WAIT' Clinical Microbiology-Immunology Laboratories, North Carolina Memorial Hospital,' and Departments ofMicrobiology- Immunology and Pathology, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27514, and Clinical Microbiology Laboratories, Oklahoma Teaching Hospitals,3 and Department ofPediatrics, Division of Infectious Disease, University of Oklahoma Health Sciences Center,4 Oklahoma City, Oklahoma 73104 Received 6 November 1986/Accepted 13 April 1987 During a 1-year period, the prevalence of thymidine-dependent (TD) Staphylococcus aureus in patients at two geographically distinct cystic fibrosis (CF) centers was determined. Of 200 CF patients who had their respiratory secretions cultured, 95 harbored S. aureus, and 20 (21%) had TD S. aureus as their predominant staphylococcal isolate. All 20 TD S. aureus-positive patients had received trimethoprim-sulfamethoxazole for an average of 30.9 months. It was also observed that TD S. aureus exhibited aberrant colony morphologies or did not grow on media commonly used in CF centers for S. aureus isolation, suggesting that this organism could be missed by routine culture methods. In contrast, all 20 isolates had typical staphylococcal morphology on mannitol salt agar after 48 h of incubation. Mannitol salt agar is recommended for primary isolation of TD S. aureus. Staphylococcus aureus continues to be an important res- 1985 and 15 May 1986. The patients ranged in age from 0.5 to piratory tract pathogen in patients with cystic fibrosis (CF). 37 years. Charts were available for 90 of 95 patients from Hoiby (2) recently reported that S. aureus was involved in whom S. aureus was recovered. They were reviewed to 20% of all pulmonary exacerbations in CF patients. These determine the history of previous antimicrobial therapy with patients are often treated with prophylactic antimicrobial TMP-SMX. agents, in part, to prevent pulmonary complications associ- Culture techniques. Respiratory secretions, including spu- ated with S. aureus infection. In a recent survey of 114 CF tum, bronchial washings, deep pharyngeal swabs, tracheal centers in the United States, 0. Tablan (personal communi- aspirates, and Bartlett bronchial brushings were routinely cation) found that in 58 centers (51%), trimethoprim- cultured to ensure the specific recovery of Haemophilus sulfamethoxazole (TMP-SMX) was a frequently used pro- influenza, Pseudomonas aeruginosa, Pseudomonas phylactic agent. Although most strains of S. aureus are cepacia, enteric and glucose-nonfermenting gram-negative exquisitely susceptible to TMP-SMX (7), several investiga- rods, Streptococcus pneumoniae, and S. aureus. At the tors in Great Britain (3, 8; R. George and R. E. Hewling, University of North Carolina, cultures were done with horse Letter, Lancet ii:1081, 1977; R. W. Lacey and E. Lewis, blood agar, colistin-nalidixic acid (CNA) agar, MacConkey Letter, Br. Med. J. 4:165, 1973) have described the emer- agar (Difco Laboratories, Detroit, Mich.), PC medium, and gence of TMP-SMX-resistant S. aureus isolates in CF pa- mannitol salt agar. Plates were incubated at 35°C and were tients during or after therapy with this agent. Resistance of examined daily for 3 days for the pathogens already listed. these organisms is presumably due to their ability to bypass At Children's Memorial Hospital, the cultures were per- the folic acid synthetic pathway, which is blocked by TMP- formed with horse blood agar (heart infusion base; Difco) SMX, and directly procure thymidine, a major end product containing 100 ,ug of bacitracin (The Upjohn Co., Kalama- of this pathway, from their environment (11). Because a zoo, Mich.); 5% sheep Columbia blood agar (Columbia base; medium with a low thymidine content will not support the Difco); MacConkey agar; OF base with polymyxin, growth of these S. aureus strains, these organisms are bacitracin, and lactose (OFPBL) agar (Remel, Lenexa, referred to as thymidine dependent (TD). We present data on Kans.); and mannitol salt agar. PC medium and OFPBL agar the prevalence of TD S. aureus in two CF centers over a are selective and differential media used for the isolation of period of 1 year. We also examined the ability of TD S. P. cepacia (1). aureus to grow on laboratory media commonly used for the S. aureus isolates were recognized to be TD if they grew isolation of S. aureus in CF centers. on mannitol salt agar or Columbia blood agar but failed to grow for susceptibility testing on Mueller-Hinton agar or MATERIALS AND METHODS broth. Thymidine dependence was confirmed by the growth of these isolates on Mueller-Hinton agar which was first studied. with CF seen at the CF Patients All individuals swabbed with a 1% solution of thymidine. All TD S. aureus centers of either the University of North Carolina, Chapel organisms were confirmed as S. aureus on the basis of a Hill, or the University of Oklahoma Children's Memorial positive catalase and coagulase test and fermentation on Hospital, Oklahoma City, were included in this study if they mannitol salt agar. Antimicrobial susceptibility testing was had cultures of respiratory secretions done between 15 May done by disk diffusion (6). Growth response studies. To study the growth response on * Corresponding author. media commonly used to isolate S. aureus from respiratory 1258 VOL. 25, 1987 PREVALENCE OF TD S. AUREUS IN CF PATIENTS 1259 TABLE 1. Comparison of characteristics of TMP-SMX therapy RESULTS Age of No. of patients Duration of patient at receiving TMP-SMX We cultured respiratory secretions from 200 CF patients at Species isolation TMP-SMX/total therapy various times over a 1-year period. S. aureus was recovered (yr)a no. in group (mo)a from 95 individuals (48%). Of these 95 patients, 20 (21%) had TD S. aureus 14.2 (20) 20/20 30.9 (18) TD S. aureus as their predominant isolate on either mannitol Non-TD S. aureus 10.5 (75)b 19/70c 11.0 (19)d salt agar or Columbia blood agar. TD S. aureus was consis- tently recovered from several patients from whom multiple a Values in parentheses are the total number of individuals. b p O.l. cultures were Qbtained. The isolates from these 20 patients c P 0.0001. were, by definition, all resistant to TMP-SMX. Otherwise, d p = 0.0067. their in vitro susceptibility was typical for S. aureus, in that the isolates were resistant to penicillin and susceptible to beta-lactamase-stable penicillins, gentamicin, chloramphen- secretions of CF patients, 15 isolates of TD S. aureus were icol, clindamycin, erythromycin, cephalosporins, and van- selected. The media chosen for study were determined in comycin. Charts were available for review on 90 of 95 part by reviewing the results of a survey of 114 CF centers in patients. A summary of these data is found in Table 1. which media used to culture respiratory secretions of CF Patients from whom TD S. aureus was recovered were not patients were outlined (O. Tablan, personal communica- significantly older than patients harboring non-TD S. aureus tion). The media studied were Mueller-Hinton agar, choco- (P = 0.1). All 20 TD S. aureus-positive patients had received late agar, phenylethyl alcohol (PEA) agar, CNA agar, 5% TMP-SMX. The duration of TMP-SMX therapy in this horse blood agar, mannitol salt agar, 5% sheep blood agar, population ranged from 3 months to over 8 years. Many of and 5% sheep blood agar supplemented with 1 or 100 ,ug of these patients received TMP-SMX intermittently, usually thymidine (Calbiochem-Behring, La Jolla, Calif.) per ml. during the winter months or when respiratory symptoms Mueller-Hinton, mannitol salt, and CNA agars were pur- worsened. In contrast, only 19 of 70 (27%) CF patients with chased from Difco Laboratories. PEA agar and basal media non-TD S. aureus were receiving TMP-SMX. The average for chocolate agar (GC base) and for horse and sheep blood duration of TMP-SMX therapy, 10.6 months, in the patients agars (Trypticase soy agar) were purchased from BBL harboring non-TD S. aureus was approximately one-third Microbiology Systems, Cockeysville, Md. the duration of therapy for the TD S. aureus-positive pa- Isolates were grown overnight in Trypticase soy broth tients. (BBL), adjusted to a 0.5 McFarland standard with Mueller- We next examined the ability of nine different media to Hinton broth (BBL), and diluted 1:10,000 in Mueller-Hinton support the growth of 15 isolates of TD S. aureus. Six of the broth. One hundred microliters was then inoculated onto media, 5% sheep blood agar, PEA agar, CNA agar, choco- each medium and spread with a glass rod. The plates were late agar, horse blood agar, and mannitol salt agar, have incubated at 35°C and were examined daily for up to 3 days. been reported to be used for the primary isolation of patho- The colony morphology of each isolate on each medium was gens, especially gram-positive ones, from the respiratory compared with that of the isolate on 5% sheep blood agar secretions of CF patients in the United States (O. Tablan, supplemented with 100 ,ug of thymidine per ml and with that personal communication). The results demonstrate the ca- of S. aureus ATCC 25923 growing on the same medium. pacity of the nine different media to support growth of the 15 Isolates showing typical S. aureus morphology when com- TD S. aureus strains (Table 2). Typical S. aureus colonies pared with the two controls were said to have normal were seen on both 5% sheep blood agar supplemented with growth.