Micrococcus species, members of the family Micrococcaceae, are usually regarded as contaminants from skin and mucous membranes.1 Nevertheless they have been documented to be causative organisms in cases of bacteremia, endocarditis, ventriculitis, peritonitis, pneumonia, endophthalmitis, keratolysis and septic arthritis.2-9 In these reports micrococci were identified on the basis of their colonial morphology, antibiotic susceptibility and species identity to determine their etiologic relevance. Modern molecular typing of serially isolated micrococcal strains was not performed. We present a patient with rhabdomyosarcoma in whom a single clone of Micrococcus luteus was isolated from blood during septic episodes during a period of 7 weeks despite appropriate therapy. To show the clonal identity of the blood isolates and to separate blood from skin and Broviac catheter M. luteus strains required the development of molecular typing methods.
Case report. A 16-year-old girl with a disseminated embryonal rhabdomyosarcoma received aggressive chemotherapy via a three-lumen Broviac catheter. During the first and, 5 weeks later, during the third chemotherapy cycle, she became neutropenic, toxic and febrile (40°C). Both episodes were treated with a combination of ceftazidime, gentamicin and vancomycin for 10 days after the first febrile episode and for 7 days after the second episode. Each time fever and toxicity resolved within 48 h. M. luteus grew in both aerobic blood cultures drawn before initiation of antimicrobial treatment of each episode. Anaerobic blood cultures were negative. Physical examination, chest roentgenogram, abdominal ultrasound examination, echocardiography and urine culture were normal. After rapid improvement during the therapy of the second febrile episode, antibiotics were stopped for 3 days. Another aerobic blood culture, drawn at this time as a test of cure, grew M. luteus again. Antimicrobial therapy was reinitiated with rifampin plus ampicillin and the Broviac catheter was removed after 5 days of therapy. While she was treated for an additional 5 weeks the patient remained afebrile, and four further blood cultures remained sterile.
Materials and methods. Culture procedure. Blood cultures were drawn through the Broviac catheter. After explantation the vascular, tunneled, the uninserted portions and the cuff of the Broviac catheter were separated and cultured with roll plate10 and both enrichment cultures. Before explanation of the Broviac catheter, swab cultures of hubs and skin surrounding the catheter were taken. Identification as M. luteus was based on typical Gram stain morphology and aerobic growth of catalase-positive, circular, convex and yellowish colonies on furazolidone agar. All isolates were resistant to lysostaphin and susceptible to bacitracin (0.04 U) and lysozyme. The organisms grew strictly aerobically and in the presence of 7.5% NaCl. No acid production was detected from glucose or glycerol, and no growth was recorded on Simmons citrate agar. Gelatin hydrolysis reaction was positive.11 Organisms were further identified with the Api-Staph® system (ATB32 Staph, Marcy-l'Etoile, France).12 Susceptibilities to antibiotics were tested by agar diffusion and agar dilution methods.13, 14
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of whole cell proteins. After incubation micrococci were harvested from a washed autoclaved dialysis membrane that was placed on CDM (chemically defined medium) agar. Bacteria were washed, then resuspended in sample buffer (100 mM Tris-HCl (pH 6.8), 2% SDS, 30% glycerol, 10% beta-mercaptoethanol), boiled and centrifuged; 0.1% bromophenol blue sodium salt was added to the supernatant; and proteins were separated by SDS-PAGE as described.15
SDS-PAGE of extracellular proteins. Cultures were prepared and harvested from the membrane as described for SDS-PAGE of whole cell proteins. After centrifugation supernatant was solubilized with sample buffer (0.0625 M Tris-HCl, pH 6.8, containing 1% SDS, 10% saccharose, 20% beta-mercaptoethanol, 0.005% bromophenol blue sodium salt), boiled and resolved by SDS-PAGE.
Pulsed-field gel electrophoresis. An overnight culture was harvested, washed, and resuspended in EC buffer (6 mM Tris-HCl, 1 M NaCl, 0.1 M EDTA, 0.5% sodium deoxycholate, 1.0% N-lauroylsarcosine, pH 7.5), mixed with a low melting temperature agarose and allowed to solidify in a 100-μl mold.16 The block was incubated overnight at 37°C in a lysis solution (1 ml of EC buffer containing 1 mg lysozyme/l and 0.12 mg RNase/ml). After a washing the block was incubated overnight at 55°C in a second lysis solution (0.5 M EDTA (pH 7.5), 1% N-lauroylsarcosine, supplemented with proteinase K (1 mg/ml)) and then washed four times. Thinly sliced sections of block were digested with 20 U of SspI (New England BioLabs, Beverly, MA) for 4 h at 37°C and then electrophoresed through a 1% agarose gel in 45 mM Tris-base, 45 mM boric acid, 1.25 mM EDTA buffer at 14°C by using the contour-clamped homogeneous electric field (CHEF DR-II®) system.
Results and discussion. M. luteus was cultured in three blood cultures at three separate times during a period of 7 weeks. Additionally, M. luteus was cultured from two skin sites around the catheter and from the catheter at the uninserted portion, the cuff and two hubs with one, four, four, seven, two and three colonies found from each site, respectively. All isolates were susceptible to beta-lactam antibiotics, aminoglycosides, glycopeptides, clindamycin, tetracycline, ofloxacin, ciprofloxacin, trimethoprim-sulfamethoxazole, rifampin and fusidic acid.
Whole cell protein patterns were identical for the three blood isolates (Fig. 1); however, protein patterns showed the distinctive nature of the six micrococcal strains isolated from skin and different portions of the Broviac catheter. Protein profiles of the extracellular proteins were identical for the three blood culture isolates, but different for the other six strains (data not shown). Restriction fragment analysis showed clonality of the strains isolated from the blood, but diversity of the micrococcal strains isolated from skin and Broviac catheter (Fig. 2). The failure to recover the blood culture strain from the catheter was probably the result of the rifampin therapy before the catheter was cultured because an extensive clinical evaluation failed to demonstrate another source and the patient had no further bacteremic episodes once the catheter was removed. This response would be in agreement with our in vitro data14 that rifampin showed the highest activity against micrococci.
A major decision facing clinicians is whether or not to remove a surgically placed catheter, especially when vascular access is difficult. In our patient recurrent sepsis with M. luteus presented a major challenge to distinguish recurrent infection from reinfection. The catheter was not removed initially, because the patient rapidly improved while she was receiving antibiotic therapy. Additionally the micrococci were regarded as contaminants from skin or mucous membranes. Because the antibiogram and biochemical profiles of different strains of M. luteus are most often identical,1, 12, 14 we defined the etiologic relevance of micrococci isolated from multiple blood samples by developing and using molecular typing of the strains through protein pattern and DNA restriction fragment analysis.
These results demonstrate the usefulness of molecular typing methods, e.g. whole cell polypeptide analysis by SDS-PAGE and DNA restriction fragment analysis by pulsed-field gel electrophoresis, for the determination of strain relationship and thus for assessing a possible etiologic relevance of serially isolated micrococcal strains. These methods can also be applied in typing of micrococci for epidemiologic purposes.
Acknowledgments. This work was supported by a grant from the Deutsche Forschungsgemeinschaft, Bonn, Germany. We thank Richard A. Proctor, M.D., University of Wisconsin, for his helpful comments and for reviewing the manuscript.
Christof von Eiff, M.D.; Nana Kuhn, M.D.; Mathias Herrmann, M.D.; Susanne Weber, M.T.; Georg Peters, M.D.
Institute of Medical Microbiology (CvE, MH, SW, GP)
Department of Pediatric Oncology (NK)
Westfälische Wilhelms-Universität Münster,
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Keywords:© Williams & Wilkins 1996. All Rights Reserved.
Micrococcus luteus; bacteremia; molecular typing