JOURNAL OF CLINICAL MICROBIOLOGY, Jan. 1994, p. 164-169 Vol. 32, No. 1 0095-1137/94/$04.00+0 Copyright © 1994, American Society for Microbiology Molecular Evidence of Person-to-Person Transmission of a Pigmented Strain of Corynebacterium striatum in Intensive Care Units REBECCA B. LEONARD,1t DAVID J. NOWOWIEJSKI,1 JOHN J. WARREN,2* DONALD J. FINN,2§ AND MARIE B. COYLEl,3* Department ofLaboratory Medicine, Harborview Medical Center, University of Washington, Seattle, Washington, 98104,1 and University Hospital Medical Center2 and Department ofMicrobiology,3 University of Washington, Seattle, Washington, 981953 Received 27 July 1993/Returned for modification 19 August 1993/Accepted 14 October 1993 During a 14-month period, a unique strain of Corynebacteyium striatum that produces a diffusible brown pigment was isolated from purulent sputa of nine patients and from nonrespiratoxy sites of two additional patients. Seven nonpigmented clinical isolates from the same period and three reference strains of C. striatum were compared with the brown isolates. Most patients had multiple sputum cultures with no coryneforms before the brown strain emerged, suggesting that the organism was hospital acquired. DNA restriction fragment patterns and Southern hybridization with the att site probe of Corynebacterium diphtheriae indicated that the brown isolates were a single strain which was distinct from the heterogeneous nonpigmented strains. A common source for the brown C. striatum was not recognized, although all of these patients were located in two adjoining intensive care units. All ofthe brown isolates, three ofthe nonpigmented clinical isolates, and two reference strains had positive CAMP reactions with Staphylococcus aureus, which has not been reported for C. striatum prior to this study. Because many Corynebacterium species and other has not been reported prior to this study. Another previously coryneforms, commonly called diphtheroids, are members unreported characteristic of C striatum is the positive of the normal flora of skin and mucous membranes, oppor- reaction on. the CAMP test which occurred in all of the tunistic infections with these organisms are usually believed brown strains and 5 of the 10 nonpigmented strains, includ- to originate endogenously. The possibility of patient-to- ing two reference strains. DNA fingerprinting indicated that patient transmission of Corynebacterium jeikeium has been all of the brown isolates were a single strain which differed documented in only two (24, 34) of the four studies that used from the diverse group of nonpigmented isolates, suggesting molecular techniques (24, 25, 34, 36). This report presents patient-to-patient transmission. evidence of transmission of a single strain of Cotynebacte- Reports of C. striatum infections are extremely rare (2, 4, rum striatum to 11 hospitalized patients. During a 14-month 28). Although three of nine patients with this organism in period, nine patients from the burn and neurosurgery-neu- their sputum were treated with vancomycin and all were rology intensive care units at Harborview Medical Center noted to have improved in response to the antibiotic cover- (HMC) had respiratory tract colonization with a brown- age, the role of the brown-pigmented C. striatum as a pigmented strain of C. striatum. One additional patient had possible cause of disease in this patient population is doubt- transient bacteremia with this organism, and another patient ful. This unusual experience of a coryneform outbreak raises had an infected burn wound. Although coryneforms are questions about the spread of coryneform organisms in a usually considered normal flora in respiratory secretions, it hospital environment as well as their pathogenic potential. is policy at HMC to identify any organism from purulent material that predominates on Gram stain and in culture. All of these patients had purulent sputa with the brown C. MATERIALS AND METHODS striatum strain recovered in significant quantities, including one case with a pure culture of this organism. Strains. All potentially significant isolates of C striatum For the purpose of comparison, we studied the nonpig- that were recovered from routine cultures of HMC patients mented as well as brown isolates of C striatum recovered at between January 1987 and February 1990 were included in HMC from potentially significant cultures between January this study (see Table 1). The three reference strains of C. 1987 and February 1990. Brown pigmentation in C. striatum striatum were purchased from the American Type Culture Collection. Biochemical and susceptibility tests. Conventional bio- chemical testing was performed by the methods of Krech * Corresponding author. Mailing address: Department of Labora- and Hollis (26), and the identification of a representative tory Medicine, Harborview Medical Center ZA-52, 325 9th Avenue, brown strain was confirmed in R. E. Weaver's laboratory at Seattle, WA 98104. Phone: (206) 223-3311. Fax: (206) 223-3930. Disease Control and Prevention. Isolates Electronic mail address: [email protected]. the Centers for t Present address: Associated Regional and University Patholo- were tested with the Rapid CORYNE identification strip gists, Salt Lake City, UT 84108. (bioMerieux Vitek Systems, Hazelwood, Mo.) as described * Deceased. by Gavin et al. (18). Enhancement of pigment production by § Present address: Department of Pathology, Bay Area Hospital, tyrosine was tested on nutrient agar with and without 0.1% Coos Bay, OR 97420. L-tyrosine. 164 VOL. 32, 1994 HOSPITAL-ACQUIRED BROWN STRAIN OF C. STRLATUM 165 Standardized disk diffusion tests with cefazolin, ceph- Seven of the nine patients had intubated airways, and six alothin, clindamycin, erythromycin, gentamicin, oxacillin, were in the hospital for at least 2 weeks before colonization penicillin, tetracycline, and vancomycin were done on un- with the brown strain was evident. Six of the nine patients supplemented Mueller-Hinton agar by the method recom- with the brown C. striatum organism in their sputa had three mended by the National Committee for Clinical Laboratory or more consecutive sputum samples containing diphthe- Standards (32). Zone diameters were read after 18 to 20 h of roids (Table 1). Two additional patients (HR118 and HR203) incubation. had multiple sputum samples with unidentified diphtheroids Gas-liquid chromatography. Whole-cell fatty acid analysis that we presume were the brown strain of C. striatum was performed by gas-liquid chromatography with the Mi- because this organism was identified in concurrent cultures crobial Identification System (MIS; MIDI, Newark, Del.). from other sites (Table 1). All of the patients with the brown The Microbial Identification System included a model 5890A strain of C striatum were located in the adjoining intensive gas chromatograph with a capillary column and flame ion- care units of either the neurosurgery-neurology service (n = ization detector, an automatic sampler, an integrator, and a 8) or the burn service (n = 3). Six of the neurological patients microcomputer (Hewlett-Packard, Palo Alto, Calif.). Peaks were admitted after closed-head trauma. were automatically integrated, fatty acid identities and per- After routine incubation for 48 h on blood agar, the cent composition were calculated, and organism identifica- creamy gray-white colonies of all the C. striatum strains in tion was determined by the CLIN library data base. The this study were very similar to those of coagulase-negative manufacturer's protocol was followed for all stages of sa- staphylococci, but cultures of the brown isolates exhibited a ponification, methylation, extraction, and chromatography darkening of the surrounding medium (see Fig. 1, isolate procedures. HR111) that was not recognizable as a brown pigment. This DNA hybridization. DNA was prepared from cells incu- pigment was first recognized as a slight darkening of Muel- bated with shaking at 37C overnight in 200 ml of heart ler-Hinton agar after overnight incubation of disk diffusion infusion broth supplemented with Tween 80 (final concen- darker when the plates were tration, 0.2%) as described previously (12). The methods for tests, but it gradually became nick translation, using [a-32P]dATP, and autoradiography held at 35°C or room temperature for another day. The were those described by Buck and Groman (7). Purified brown isolates produced no pigment on nutrient agar after 72 DNA preparations were digested with the restriction en- h of incubation, but the pigment was clearly visible after 24 zymes BamHI, EcoRI, and HincII and analyzed in agarose h on the tyrosine-supplemented nutrient agar. Nonpig- gels. Southern blots with each of the three digests were mented strains were unaffected by growth in the presence of hybridized to the att site of the beta phage of Corynebacte- tyrosine. rium diphtheriae as described previously (12). The results of conventional biochemical tests are shown in CAMP test. Because Munch-Petersen described C. stria- Table 2. All but one of the 21 isolates including reference tum (Corynebacteinum flavidum) as producing "an agent strains were indistinguishable when tested with the standard which protects ruminant erythrocytes from Staphylococcal battery. The nonpigmented strain HR161 was unusual in its beta lysin" (31), we tested our brown isolates for this ability to reduce NO2. In the Rapid CORYNE system, all reaction. A thin line of Staphylococcus aureus (ATCC brown isolates, two reference strains, and four nonpig- 25923)