Central venous catheter-related bacteremia due to Tsukamurella species in the immunocompromised host: a case series and review of the literature.

We report 6 cases of bacteremia due to Tsukamurella species, all of which were in immunosuppressed patients with indwelling central venous catheters (CVCs). Fewer than 20 cases of serious illness due to these gram-positive bacilli have been reported in the medical literature; these cases have mostly been ascribed to the species Tsukamurella paurometabola. Tsukamurella species are frequently misidentified as Rhodococcus or Corynebacterium species. We used high-performance liquid chromatography to identify these organisms to the genus level and 16S ribosomal RNA gene sequencing and DNA-DNA dot blots for species identification. Three of our isolates were identified as Tsukamurella pulmonis, 1 was identified as Tsukamurella tyrosinosolvans, and 1 was identified as a unique species. One isolate was not maintained long enough for species identification. All patients were successfully treated with antimicrobial therapy and CVC removal. Infection with this organism should be considered in the immunosuppressed patient with an indwelling CVC and gram-positive bacilli in the blood.

Tsukamurella strandjordae sp. nov., a proposed new species causing sepsis.

We have isolated a gram-positive, weakly acid-alcohol-fast, irregular rod-shaped bacterium from cultures of blood from a 5-year-old girl with acute myelogenous leukemia. This isolate was compared with 14 other strains including reference strains of Tsukamurella species by a polyphasic approach based on physiological and biochemical properties, whole-cell short-chain fatty acid and mycolic acid analyses, DNA-DNA hybridization, and sequencing of the 16S rRNA gene. This isolate represents a new taxon within the genus Tsukamurella for which we propose the name Tsukamurella strandjordae sp. nov. Our study also revealed that Tsukamurella paurometabola ATCC 25938 represents a misnamed Tsukamurella inchonensis isolate and confirms that Tsukamurella wratislaviensis belongs to the genus Rhodococcus.

Diversity within reference strains of Corynebacterium matruchotii includes Corynebacterium durum and a novel organism.

Corynebacterium matruchotii has been the subject of numerous dental pathogenesis studies. The purpose of the present study was to resolve concerns about diversity within the reference strains of C. matruchotii through analysis of seven strains procured from the American Type Culture Collection (ATCC). Analysis of whole-cell fatty acid profiles with the library generation software of Microbial ID Inc. revealed that three types of organisms have been deposited in the ATCC as C. matruchotii. These three groups of organisms were also distinguishable by DNA-DNA dot blot hybridization, by sequences of two hypervariable regions of the 16S rRNA gene, and by the pyrrolidonyl arylamidase test. These studies indicate that two C. matruchotii reference strains, ATCC 33449 and ATCC 33822, are members of the recently proposed species, Corynebacterium durum. The colonial morphology and biochemical reactions of the C. durum strains are more diverse than originally reported. Strain ATCC 43833 is unique and represents a novel species. In addition to the type strain, ATCC 14266, true members of the species C. matruchotii include ATCC strains 14265, 33806, and 43832 plus two reference strains, L2 and Richardson 13, which comprise the vast majority of strains used in dental pathogenesis research with this species.

Standardized BACTEC method to measure clarithromycin susceptibility of Mycobacterium genavense.

A standardized clarithromycin susceptibility test for Mycobacterium genavense is reported. The BACTEC radiometric broth dilution test method recommended for Mycobacterium avium complex was modified to develop a reliable and reproducible procedure. Test development involved optimization of medium pH and inoculum densities for antibiotic vials as well as growth control vials. MIC control organisms included Mycobacterium simiae, Mycobacterium avium, and Mycobacterium xenopi. Growth control vials required two to three inoculum dilutions, which varied for each species. Clarithromycin MICs and MBCs for 12 isolates and 1 colonial variant of M. genavense ranged from < or = 0.06 to 0.25 microg/ml.

A new agent of mycobacterial lymphadenitis in children: Mycobacterium heidelbergense sp. nov.

Nontuberculous mycobacterial lymphadenitis presents an increasing clinical problem in immunocompetent young children. A slowly growing, nonphotochromogenic mycobacterium was recovered twice (isolates 2553/91 and 2554/91) from the lymphatic tissue of a child with recurrent cervical lymphadenitis. It could be differentiated biochemically from described Mycobacterium species, although it most closely resembled Mycobacterium malmoense by thin-layer chromatography and high-performance liquid chromatography of mycolic acids. A striking characteristic of the isolate was its high degree of susceptibility to antituberculous drugs in vitro, including isoniazid. Direct determination of the 16S rRNA gene sequence revealed a unique sequence and positioned the strain phylogenetically on a branch separate from M. malmoense within a group of slowly growing mycobacteria that show a high degree of similarity to M. simiae at the 16S rRNA gene level. Despite 99.6% sequence identity with M. simiae at the 16S rRNA gene level, DNA-DNA hybridization studies (hydroxyapatite method) demonstrated DNA relatedness of less than 40%. We conclude that this organism is a new species for which we propose the name M. heidelbergense. A culture of the type strain, strain 2554/91, has been deposited in the American Type Culture Collection as strain ATCC 51253.

Evaluation of an extended blood culture protocol to isolate fastidious organisms from patients with AIDS.

Recent reports of fastidious pathogens suggest the need for special blood cultures for immunocompromised patients. Blood cultures from 45 human immunodeficiency virus (HIV)-infected patients with unexplained fever (> or = 38.0 degrees C) and CD4 counts of < 125 cells per mm3 were collected into a vacuum tube with sodium polyanetholsulfonate, an Isolator tube, and BACTEC aerobic and anaerobic bottles. Blood from the sodium polyanethosulfonate tube was inoculated into BACTEC 13A bottles, which were read weekly for 16 weeks. Isolator sediment was divided among eight agar media, including four sheep blood agar media: chocolate agar, brain heart infusion blood agar, heart infusion blood agar, and brucella blood agar. Other agar plates included Sabouraud's, buffered charcoal-yeast extract, Middlebrook 7H11 (M7H11) with hemoglobin, and M7H11 with mycobactin J. Incubation conditions included air and CO2-enriched aerobic, microaerophilic, and anaerobic atmospheres. Aerobic BACTEC broths received an acridine orange stain on day 8 and were subcultured at 2, 4, and 8 weeks. Anaerobic BACTEC bottles were subcultured at 4 weeks. All solid media, including subcultures, were incubated for 8 weeks, providing a total of 16 weeks of incubation for each specimen. Clinically significant isolates included eight Mycobacterium avium complex isolates and one each of Bartonella henselae, Bartonella quintana, Shigella flexneri, Klebsiella oxytoca, and Cryptococcus neoformans. All isolates were detected with commercially available media and, with the exception of Bartonella spp., were recovered within incubation times routinely used in most clinical laboratories.

Differentiation of Mycobacterium genavense and Mycobacterium simiae by automated mycolic acid analysis with high-performance liquid chromatography.

Mycobacterium genavense, a fastidious opportunist in patients with AIDS, cannot be identified by conventional biochemical methods. Computerized mycolic acid analysis by high-performance liquid chromatography offers an alternative that distinguishes the mycolic acid profile of M. genavense from those of all other organisms in the database developed at the Centers for Disease Control and Prevention.

Isolation and characterization of a unique group of slowly growing mycobacteria: description of Mycobacterium lentiflavum sp. nov.

A distinct group of slowly growing mycobacteria was identified on the basis of growth characteristics, biochemical and lipid profiles, and nucleic acid analyses. The isolates showed growth at 22 to 37 degrees C, yellow pigmentation, and negative tests for Tween 80 hydrolysis, nicotinic acid, nitrate reductase, and urease; tests for arylsulfatase, pyrazinamidase, and heat-stable catalase were variable. Analysis of cellular fatty acids by gas-liquid chromatography and mycolic acids by thin-layer chromatography and high-performance liquid chromatography indicated a distinctive pattern which was unlike those of other species. Determination of the 16S rRNA gene sequence showed a unique sequence closely related to Mycobacterium simiae and M. genavense. On the basis of DNA homology studies, we suggest that these organisms are representatives of a novel species, for which the name M. lentiflavum sp. nov. is proposed.

Hyperkeratosis in athymic nude mice caused by a coryneform bacterium: microbiology, transmission, clinical signs, and pathology.

The purpose of this study was to characterize a spontaneous disease condition causing hyperkeratosis in nude mice and to explore the etiologic role of a particular species of coryneform bacteria in this disease, colloquially known as "scaly skin disease." The study was divided into two parts. In the first phase, a series of inoculation experiments was conducted with a field isolate of the coryneform species used to study the clinical and histopathologic development of the disease syndrome. Athymic nude mice (4 to 5 weeks old) were inoculated on the skin of the back with a suspension of a pure culture of the coryneform bacterium that had been isolated from a field case. The culture was applied with a sterile cotton swab in concentrations varying from 6.1 x 10(4)/ml to 5.0 x 10(7)/ml. All inoculated mice became persistently infected throughout the 33 days of the experiment. Clinically evident hyperkeratosis in inoculated animals developed more frequently in mice housed in a microisolator cage than in a semi-rigid isolator and more frequently in mice inoculated with higher numbers of organisms. In all animals in which hyperkeratosis developed, it was first noted on day 7 after inoculation. The second series of experiments was designed to determine the success of various housing methods in excluding the infection, mechanisms of transmission, susceptibility of other stocks and strains of mice to the organism, and whether the other strains might serve as a source of the organism. Results of the study in various strains indicated that both immunocompetent and immunodeficient mice, whether glabrous or hirsute, could be infected with the organism, but only glabrous animals developed hyperkeratosis.(ABSTRACT TRUNCATED AT 250 WORDS)

Prosthetic valve endocarditis caused by Corynebacterium afermentans subsp. lipophilum (CDC coryneform group ANF-1).

Corynebacteria are important causes of endocarditis in individuals with valvular prostheses. We report the first published case of prosthetic valve endocarditis caused by the newly defined species Corynebacterium afermentans subsp. lipophilum (former CDC coryneform group ANF-1). The isolate was recovered from a perivalvular abscess specimen and 5 of 15 Bactec blood cultures after 7 to 15 days of incubation. The isolation, identification, and susceptibility testing of Corynebacterium species are discussed.

Bartonella (Rochalimaea) quintana bacteremia in inner-city patients with chronic alcoholism.

BACKGROUND: Bartonella (Rochalimaea) quintana is a fastidious gram-negative bacterium known to cause trench fever, cutaneous bacillary angiomatosis, and endocarditis. Between January and June 1993 in Seattle, we isolated B. quintana from 34 blood cultures obtained from 10 patients not known to be infected with the human immunodeficiency virus (HIV). METHODS: After identifying the isolates as B. quintana by direct immunofluorescence and DNA-hybridization studies, we determined strain hybridization with studies of restriction-fragment-length polymorphisms (RFLPs) of the intergenic spacer (noncoding) region of ribosomal DNA amplified by the polymerase chain reaction (PCR). To characterize the epidemiologic and clinical features of bartonella infections in these patients, we performed a retrospective case-control study using as controls 20 patients with blood cultures obtained at approximately the same time as those obtained from the index patients. RESULTS: B. quintana isolates from the 10 patients were indistinguishable by PCR-RFLP typing. All 10 patients had chronic alcoholism, and 8 were homeless (P = 0.001 for both comparisons with controls). The six patients who underwent HIV testing were seronegative. At the time of their initial presentation, seven patients had temperatures of at least 38.5 degrees C. Six patients had three or more blood cultures that were positive for B. quintana, and in four of these patients B. quintana was isolated from blood cultures obtained 10 or more days apart. Subacute endocarditis developed in two patients and required surgical removal of the infected aortic valve in one of them. Nine patients recovered; one died of sepsis from Streptococcus pneumoniae infection. CONCLUSIONS: B. quintana is a cause of fever, bacteremia, and endocarditis in HIV-seronegative, homeless, inner-city patients with chronic alcoholism.

Native-valve endocarditis due to CDC coryneform group ANF-3: report of a case and review of corynebacterial endocarditis.

Endocarditis due to a bacterium of CDC coryneform group ANF-3 developed in the native aortic valve of a patient without predisposing factors other than valvular calcification. A review of the literature indicates that corynebacteria are an increasingly important cause of endocarditis. Problems in identification and treatment remain. Techniques for the culture and quick identification of these organisms and effective regimens for treatment of the infections they cause are needed.

Detection of Bartonella (Rochalimaea) quintana by routine acridine orange staining of broth blood cultures.

Bartonella quintana was isolated from 34 BACTEC nonradiometric aerobic resin blood cultures for 10 adults. Nine patients were initially diagnosed by routine acridine orange staining of routine cultures that had been incubated for 8 days. All subcultures grew on chocolate agar within 3 to 12 days (median, 6 days). The PLUS 26 high-volume aerobic resin medium, combined with acridine orange stain and subculture, is an effective system for detection and isolation of B. quintana from blood.

Molecular evidence of person-to-person transmission of a pigmented strain of Corynebacterium striatum in intensive care units.

During a 14-month period, a unique strain of Corynebacterium striatum that produces a diffusible brown pigment was isolated from purulent sputa of nine patients and from nonrespiratory 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 of the brown isolates, three of the 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.

Effects of media, atmosphere, and incubation time on colonial morphology of Arcanobacterium haemolyticum.

Arcanobacterium haemolyticum causes pharyngitis as well as skin and other wound infections. Although it is a beta-hemolytic organism, the hemolysis is less well defined than that of beta-hemolytic streptococci and may be overlooked in cultures with heavy growth of commensal throat flora. To determine whether routine throat culture conditions are sufficient to produce recognizable colonies of A. haemolyticum, the morphology of six distinct strains was studied after various combinations of incubation time, medium, and atmosphere. The agar media, containing 5% sheep blood, were Trypticase soy agar, Columbia agar, and heart infusion agar. Cultures were incubated in ambient air, 6 to 8% CO2, or an anaerobic atmosphere. Cultures were compared after 24, 48, and 72 h of incubation for colony size, clarity and size of hemolytic zone, and macroscopic evidence of agar pitting. A minimum of 48 h was needed for expression of beta-hemolysis and pitting. Trypticase soy agar was the superior medium and CO2 was the superior atmosphere for beta-hemolysis. Agar pitting was not significantly affected by variations in medium or atmosphere. Strains differed in their expression of hemolysis and production of pits at 48 h. After 72 h of incubation, beta-hemolysis and pitting were visible in over 96% of culture observations.

Laboratory review of reference strains of Corynebacterium diphtheriae indicates mistyped intermedius strains.

All biotyped strains of Corynebacterium diphtheriae from the American Type Culture Collection (ATCC) were compared for morphology and biochemical reactions. Biotypes of all gravis strains and most mitis strains were confirmed, but intermedius strains were found to be misclassified. New lipid-dependent intermedius strains have been deposited with the ATCC.

Mycobacterium genavense sp. nov.

Strains of a suggested novel type of mycobacterium have been repeatedly isolated from patients with AIDS. We summarize the results of tests performed to determine enzymatic activities and metabolic properties, the results of fatty acid analyses, and the results of a comparative 16S rRNA sequence determination. We propose formally that this organism represents a new species, Mycobacterium genavense. The type strain is strain 2289, a culture of which has been deposited in the American Type Culture Collection as strain ATCC 51234.

Pursuit of the Corynebacterium striatum type strain.

The description of Corynebacterium striatum (Chester 1901) Eberson 1918AL in Bergey's Manual of Systematic Bacteriology has many inconsistencies with the identification scheme from the Centers for Disease Control and Prevention. We have studied the four C. striatum reference strains available from the American Type Culture Collection and the National Collection of Type Cultures and found that they fit the Centers for Disease Control and Prevention description of this species. However, it appears that the wrong strains were deposited for this species, because none of the reference strains fits the descriptions in the original publications. This is a substantial case for declaring it a nomen dubium, but the name could be rescued if a careful search reveals a strain that was used in making the original description. It is hoped that some readers may have the missing strains labeled with the early names Bacillus striatus, Bacillus flavidus, or Corynebacterium flavidum.

Examination gloves as barriers to hand contamination in clinical practice.

OBJECTIVE: To test the effectiveness of vinyl and latex gloves as barriers to hand contamination with gram-negative organisms and enterococci during routine hospital procedures. DESIGN AND INTERVENTIONS: We studied 137 procedures during which a health care worker's gloved hand contacted a patient's mucous membrane and was thus potentially contaminated with gram-negative rods or enterococci. Quantitative hand cultures were obtained from each health care worker before and after the gloved contact using a modified glove juice method, and the exterior glove surface was also quantitatively cultured after patient contact. Used gloves were then tested for leaks using the American Society for Testing and Materials' watertight test. SETTING: Harborview Medical Center, a 330-bed city-county hospital and level I regional trauma and burn center, is both a teaching facility affiliated with the University of Washington and the major provider of care to indigent and uninsured persons in Seattle-King County, Washington. PATIENTS AND OTHER PARTICIPANTS: Respiratory therapists performing endotracheal tube care on intubated intensive care unit patients, registered nurses performing digital rectal stimulation for bowel training on patients with spinal cord injury in the rehabilitation ward, and dentists performing routine dental examinations and procedures on healthy outpatients in the dental clinic. MAIN OUTCOME MEASURE AND RESULTS: Eighty-six of the 135 gloves cultured had gram-negative rods or enterococci on the external surface after use and were thus sources of potential hand contamination. Microbial contamination of the health care worker's hands occurred in 11 (13%; 95% confidence interval, 6% to 20%) of these 86 events, and was more frequent with vinyl (10 of 42) than latex (one of 44) gloves (P < .01). After use, glove leaks were also more frequent in vinyl gloves (26 of 61) than with latex gloves (six of 70) (P < .001). Even when leaks were present, gloves prevented hand contamination in 77% of instances and quantitative counts of microorganisms contaminating hands were 2 to 4 logs less than counts on external glove surfaces. Health care workers reported awareness of the presence of glove leaks in only seven (22%) of the 32 events in which leaks were subsequently demonstrated. CONCLUSIONS: Under conditions of routine use, gloves effectively function as a protective barrier even when leaks are present. Latex gloves were less frequently associated with leaks and hand contamination. Since hand contamination occurred after 13% of exposures and cannot be readily identified by health care workers, routine hand washing should be done after each patient contact.

Evidence of multiple taxa within commercially available reference strains of Corynebacterium xerosis.

Attempts to identify coryneform isolates resembling Corynebacterium xerosis can lead clinical microbiologists to identification schemes with conflicting descriptions which result in confusing C. xerosis with Corynebacterium striatum. For the present study we purchased all available American Type Culture Collection and National Collection of Type Cultures reference cultures of C. xerosis (n = 10) and C. striatum (n = 4) and analyzed them as follows: (i) analysis of biochemical reactions in conventional tests and in the Rapid CORYNE system, (ii) whole-cell fatty acid analysis by using the gas-liquid chromatography research software of Microbial ID, Inc., and (iii) analysis of DNA homology in dot blot hybridizations. Three C. xerosis strains were indistinguishable from the C. striatum strains in whole-cell fatty acid analyses and DNA hybridizations and shared very similar biochemical reactions. The remaining seven strains of C. xerosis clustered into five groups on the basis of fatty acid patterns, DNA hybridizations, and biochemical tests. No reference strain of C. striatum fit the species description in Bergey's Manual of Systematic Bacteriology. The type strains of both C. striatum and C. xerosis fit their respective descriptions by the Centers for Disease Control and Prevention. This study suggests that the 10 commercially available reference strains of C. xerosis represent six different taxa which should be assigned to new species.