Malignancy-Associated Microorganisms from Patients with

JOURNAL OF CLINICAL MICROBIOLOGY, Dec. 1982, p. 1102-1105 0095-1137/82/121 102-04$02.00/0 Copyright (© 1982, American Society for Microbiology

Vol. 16, No. 6

Failure to Recover Alpha-Hemolytic Streptococci or Malignancy-Associated Microorganisms from Patients with Kidney Disease and from Healthy Humans CHARLES H. ZIERDT,L* JAMES HASBARGEN,2 AND JOHN B. COPLEY2t Microbiology Service, Clinical Pathology Department, Clinical Center, National Institutes of Health, Bethesda, Maryland 20205,1 and Department of Nephrology, Walter Reed Army Medical Center, Washington, DC 200122

Received 12 May 1982/Accepted 14 September 1982 We duplicated blood culture techniques and media used by workers reporting alpha-hemolytic streptococci from nephropathy patients as well as from healthy persons. We studied 33 kidney patients presenting 12 diagnosed kidney diseases. The study was expanded to duplicate the experimental blood culture techniques of workers who reported Bacillus licheniformis and Cryptocides tltnefaciens from patients with malignancies and, to a lesser degree, from healthy people. Seven culture media and 21 conditions of growth were used. There were no streptococcal isolates, and the few random isolates of other bacteria indicated that they were contaminants. Thus, our study did not corroborate the previous reports.

Domingue and his co-workers (7-12) and Other reports of this group of microorganisms Green et al. (13) have reported recovery by were those of Livingston et al. (14), and Alexanspecial culture techniques of aberrant, intracel- der-Jackson (1). By phase optics, they demonlular forms of alpha-hemolytic streptococci from strated parasitized erythrocytes of healthy and blood, kidney tissue, other tissues, and urinary cancerous humans. Their most common isolate sediments of patients with glomerulopathies. was a gram-positive rod that bore resemblance These organisms were isolated from 71% of the to mycobacteria, mycoplasmas, and Listeria patients and 7% of healthy humans in the study spp. Certain phases of its growth had acid-fast of Domingue and Schlegel (9). It was hypothe- cellular extensions. They classified the organism sized that antigens derived from these bacteria as Order Actinomyc etales, Family Progenitoragave rise to immune reactions destructive to ceae, Genus Cryptocides, Species Cryptocides various tissues. Cures were effected after 2 to 3 tumefaciens. Bisset (4), Bisset and Bartlett (5), months of antibiotic treatment. Because of the and Bartlett and Bisset (2) reported the recovery obvious potential significance of these reports, a of a gram-positive rod, which they named Bacilstudy was undertaken to repeat the techniques lus licheniformis subsp. endoparasiticus (Benethat led to the cultural evidence. No attempts dek), from blood and tissues of arthritic patients were made to corroborate the reported phase- particularly, but also from healthy humans. In optical examination of fresh samples, including their study, some of the pleomorphic isolates of cultural development of aberrant forms in and Alexander-Jackson and Wuerthle-Caspe Livingon cells in urine sediments. Also, no attempts ston were classified also as B. licheniformis were made to identify aberrant, wall-less spheri- subsp. endoparasiticuis (Benedek). The orgacal forms in electron micrographs of clinical nisms recovered from blood or tissue by Dominsamples such as blood, tissue, and urinary sedi- gue et al. (9, 12), by Pease (15-17), by Wuerthlements. We believe that these latter studies were Caspe Livingston (20), by Alexander-Jackson to a degree subjective, with no assurance that (1), by Bisset and Bartlett (5), and by Diller and the bodies viewed as microbial were in fact Donnelly (6) were in general robust in growth on ordinary culture media, especially after initial microbial. There are reports by Tedeschi et al. (18, 19) of subculture, as they assumed definitive form bacteria isolated from platelets and erythro- growth characteristics. The major purpose of this study was to duplicytes. Wuerthle-Caspe Livingston and Alexander-Jackson (20) isolated a number of pleomor- cate the culture techniques of Domingue and cophic organisms from patients with malignancies. workers. There was an obvious relationship between these techniques and those used in the Fitzsimons Army isolation of cryptic bacteria from the blood of t Present address: Nephrology Service, cancer patients and of healthy humans. DominMedical Center, Aurora, CO 80045. 1102

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TABLE 1. Media used, duplicating media from other studies, and inoculum Authors of previous study

Letter designation

Domingue et al. (11)

A B C D E F G H I J K L M N 0

Bisset and Bartlett (5)

p Q R R' S S' T U

Inoculum

Size (ml) BHI blood culture bottle (BBL)-aerobic 5 BHI blood culture bottle (BBL)-anaerobic 5 VB agar 0.5 VB broth 0.5 Thioglycollate medium 1.0 VB agar 1.0 VB broth 1.0 0.1 Sheep blood agar plate-aerobic Sheep blood agar plate-anaerobic 0.1 Thioglycollate medium 0.1 VB agar 1.0 VB broth 1.0 Sheep blood agar plate-aerobic 0.1 Sheep blood agar plate-anaerobic 0.1 1.0 Thioglycollate medium

Whole blood Whole blood Whole blood Whole blood Whole blood Lysed in 0.25% NaCl Lysed in 0.25% NaCl Lysed in 0.25% NaCl Lysed in 0.25% NaCl Lysed in 0.25% NaCl Lysed blood filtrate Lysed blood filtrate Lysed blood filtrate Lysed blood filtrate Lysed blood filtrate

PPLO broth BHI plus adjuvantsb PPLO broth PPLO broth BHI plus adjuvantsb BHI plus adjuvantsb PPLO broth BHI plus adjuvantsb

Whole blood Whole blood Packed blood Packed blood Packed blood Packed blood Plasma Plasma

Mediuma

a BHI, Brain heart infusion. Adjuvants consisted of 0.5% IsoVitaleX

b

1.0 1.0 0.5 0.5 0.5 0.5 1.0 1.0

Type or treatment

cells cells cells cells

(BBL)-1.0% Fildes enrichment (Difco)-0.1% agar.

gue et al. (12) fully appreciated this and related their findings to those from the cancer studies. Therefore, a minor study was included, using media and techniques of both groups of authors, with the exception that no blood cultures were done on cancer patients. MATERIALS AND METHODS Patient population. A total of 33 patients between 15 and 75 years of age were studied. Five were female. Nine were being treated with hemodialysis. One had a successful renal transplant. Serum creatinine ranged from normal to 10.0 mg/100 ml. Nine had been treated with antibiotics in the 8 weeks preceding blood culture. Blood cultures were also done according to the protocol on 50 healthy blood donors. Of the 33 patients, 12 were diagnosed as having immunoglobulin A nephropathy. Nine were diagnosed as having primary renal hematuria; two were diagnosed as having systemic lupus erythematosus, and the remaining 10 were diagnosed as having Goodpasture's syndrome, membranous glomerulonephritis, membranoproliferative glomerulonephritis type I, focal segmental sclerosing glomerulonephritis, Wegener's granulomatosis, myeloma kidney, arteriolar nephrosclerosis, hemolytic uremic syndrome, rhabdomyolysis-associated acute renal failure, and glomerulonephritis of unknown etiology. All diagnoses included biopsy data. Media and culture techniques. Seven media were used (Table 1)-five to repeat the culture techniques of

Domingue et al. (11), and two to repeat the culture techniques of Bisset and Bartlett (5) and of Pease (1517). Media containers used by these workers were duplicated, such as the 30-ml prescription bottle (McCartney bottle) used by Bisset and Bartlett. Brain heart infusion broth with 1% IsoVitaleX (BBL Microbiology Systems) and 0.5% Fildes Enrichment (Difco Laboratories) was used in place of bovine lung "sensitive broth" of Pease (15). Thioglycollate medium (BBL) was used in place of Brewer's thioglycollate medium. The seven base media were used to provide 22 conditions for growth of each blood sample, including lysis, filtration, and centrifugation, as well as aerobic and anaerobic culture conditions. Blood (35 ml) was drawn into 0.2 ml of heparin and then was distributed and processed (Table 2). Lysis was in 10 ml and 0.25% NaCl for 1 h. Filtration was done through 0.2-p.m cellulosic membrane filters (Millipore Corp.). Examination of cultures. Plate media were examined at 2, 7, and 14 days and then were discarded. Bottle and tube media were examined at 2, 7, and 14 days and then at monthly intervals for 2 years. Subcultures from whole blood and blood cell-inoculated media were done at 1 week, 1 month, 3 months, and thereafter at 3month intervals for 2 years. Clear media were examined visually at these intervals, and a final subculture was made before the cultures were discarded. Subcultures followed the report of Domingue and Schlegel (8). They were made to variant bacterial (VB) medium agar, VB broth, tryptose broth, Todd-Hewitt broth, VB broth plus 0.2% trypsin, VB broth plus 0.4%

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ZIERDT, HASBARGEN, AND COPLEY

J. CLIN. MICROBIOL.

TABLE 2. Positive cultures, showing organism recovered from culture Kidney disease (33 patients) no. B51 to b A50bB83

Healthy humans (50) humans no. Al to A5Ob no. (50) Healthy Sourcea

A B

Staphylococcus epidermidis

Bacillus

Corynebacterium

licheniformis A13

species

A6 A36

C D E F

G H I J K L

A12 A39 A10 A8

A29 A29

Staphylococcus

epidermidis B59

Corynebacterium species

B59 B69

A29 B52

A9 A8

M N

0 p

Q

R R'

A29

S

St B52 T A29 U B59 a The letter designation indicates the medium and culture condition as listed in Table 1. b The number indicates the subject from whom the blood sample was obtained.

trypsin, thioglycollate medium, and sheep blood agar. Inoculated sheep blood agar plates were incubated under 10% CO2 anaerobically and aerobically at 35°C and at room temperature.

RESULTS Bacterial isolations from the 50 healthy blood donors and from 33 patients with kidney disease are shown in Table 2. In the group of 50 blood samples from healthy donors, there were 6 positive for Staphylococcus epidermidis, but only 1 sample was positive under more than one culture condition. One grew a Bacillus sp. under 1 of 21 culture conditions. One sample grew a Corynebacterium sp. under five conditions of growth, and one grew a Corynebacterium sp. under one condition of growth. No sample grew more than one bacterial species. In the group of 33 blood samples from patients with kidney disease, 3 grew S. epidermidis. One of these grew it under three conditions of growth, one grew it under two conditions, and one grew it under one condition of growth. The blood of one patient grew a Corynebacterium sp. under one condition of growth. Thus, there were seven isolations of S. epidermidis, two of Corynebacterium sp., and one of

B51

B. licheniformis among blood samples from 50 healthy humans. There were three S. epidermidis and one Corynebacterium sp. isolations among the 33 patients. All of these isolates were considered contaminants. There were 13 organisms isolated from 83 blood samples (16%). This is a high rate of contamination, but it is based on the 21 growth conditions and seven media. If only the conventional blood culture bottles are considered, there were four organisms isolated (5% contamination). No streptococcal isolates were made.

DISCUSSION Since the etiology of many glomerulonephritic diseases remains an enigma, the implication of the reports of Domingue and others is far reaching. It is for this reason that the present work was undertaken in an attempt to duplicate reported results in patients with well-defined renal disease. Results of the present study were uniformly negative. There were no isolations of alphahemolytic streptococci or any other streptococci in the present study. The reasons for the discrepancy in the two studies are uncertain. This

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study required bacterial growth for evidence of etiological relationship. Domingue et al. also presented light microscopic and electron microscopic evidence. Although Domingue et al. did not describe the patients studied other than to state the clinical diagnoses, it is unlikely that population differences can explain the difference in results. The patients in this study represent a spectrum of renal disease, with various degrees of renal function and various treatment modalities. Thus, at least some of the patients would have had positive cultures had population differences been responsible. Finally, many but not all of Domingue and Schlegel's patients were treated with antibiotics during the culture period. Antimicrobial agents could have been initiating factors for the variant bacteria. However, several of their patients had positive cultures but no antimicrobial therapy. Diller and Donnelly (6) isolated acid-fast pleomorphic organisms from blood and tissues of lymphomatous mice and from humans with malignancies. They believed that the organisms they recovered from about 75% of tumor-bearing mice were the same as the organisms described by other workers who studied human

malignancies. Pease (15, 16) described L forms in and on the erythrocytes of 170 of 176 arthritic humans. Pease later identified the parent form of these organisms as B. licheniformis (17) and related it to organisms reported by Alexander-Jackson, Benedek (3), Bisset, and others. She at first believed the parent bacterium to be Listeria sp., but this was shown to be antigenically related to B. licheniformis. Pease, as others had done, found the same organisms in a smaller percentage of apparently healthy persons. There was one B. licheniformis isolate from the aerobic conventional blood culture bottle of one of the healthy donors in this study. The other six media and 20 growth conditions were negative. These included the lysed blood cultures that would be expected to increase the recovery of B. licheniformis. The individual from whom this blood sample was obtained had a subsequent negative blood culture. LITERATURE CITED 1. Alexander-Jackson, E. 1954. A specific type of microorganism isolated from animal and human cancer: bacteriology of the organism. Growth 18:137-151.

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2. Bartlett, R., and K. A. Bisset. 1979. Induction of reversion from the L-form to the sporogenous phase of Bacillus licheniformis var. endoparasiticus (Benedek). J. Med. Microbiol. 12:239-243. 3. Benedek, T. 1955. Rheumatoid arthritis and psoriasis vulgaris. Edwards Bros.. Inc.. Ann Arbor. Mich. 4. Bisset, K. A. 1977. Isolations of micro-organisms from arthritic patients and healthy normals. Ann. Rheum. Dis. 36:96-97. 5. Bisset, K. A., and R. Bartlett. 1978. The isolation and characters of L-forms and reversions of Bacillus licheniformis var. endoparasiticus (Benedek) associated with the erythrocytes of clinically normal persons. J. Med. Microbiol. 11:335-349. 6. Diller, I. C., and A. J. Donnelly. 1970. Experiments with mammalian tumor isolates. Ann. N.Y. Acad. Sci. 174:655-674. 7. Domingue, G. J. 1980. Filterable cell-associated cryptic bacterial forms in immunologic renal diseases. Urol. Surv. 30:1-4. 8. Domingue, G. J., and J. U. Schlegel. 1977. Novel bacterial structures in human blood: cultural isolation. Infect. Immun. 15:621-627. 9. Domingue, G. J., and J. U. Schlegel. 1978. Novel bacterial structures in human blood. II. Bacterial variants as etiologic agents in idiopathic hematuria. J. Urol. 120:708-711. 10. Domingue, G. J., J. U. Schlegel, P. M. Heidger, Jr., and M. Ehrich. 1976. Novel bacterial structures in human blood: cultural isolation, ultrastructural and biochemical evaluation. Colloq. INSERM 65:279-298. 11. Domingue, G. J., J. U. Schlegel, and H. B. Woody. 1976. Naked bacteria in human blood. Microbia 2:3-31. 12. Domingue, G. J., H. B. Woody, K. B. Farris, and J. U. Schlegel. 1979. Bacterial variants in urinary casts and renal epithelial cells. Arch. Intern. Med. 139:1355-1360. 13. Green, M. T., P. M. Heidger, Jr., and G. Domingue. 1974. Demonstration of the phenomena of microbial persistence and reversion with bacterial L-forms in human embryonic kidney cells. Infect. Immun. 10:889-914. 14. Livingston, A. M., V. Wuerthle-Caspe Livingston, E. Alexander-Jackson, and G. H. Wolter. 1970. Toxic fractions obtained from tumor isolates and related clinical implications. Ann. N.Y. Acad. Sci. 174:675-689. 15. Pease, P. 1969. Bacterial L-forms in the blood and joint fluids of arthritic subjects. Ann. Rheum. Dis. 28:270-274. 16. Pease, P. 1970. Morphological appearances of a bacterial L-form growing in association with the erythrocytes of arthritic subjects. Ann. Rheum. Dis. 29:439-444. 17. Pease, P. 1974. Identification of bacteria from blood and joint fluids of human subjects as Bacillus licheniformis. Ann. Rheum. Dis. 33:67-69. 18. Tedeschi, G. G., 1). Amici, I. Santarelll, M. Paparelli, and C. Vitali. 1976. Unstable L-forms of micrococci in human platelets, p. 325-330. In R. Fuller and D. W. Lovelock (ed.), Microbial ultrastructure: the use of the electron microscope. Academic Press, Inc., New York. 19. Tedeschi, G. G., A. Bondi, M. Papareill, and G. Sprovieri. 1978. Electron microscopical evidence of the evaluation of corynebacteria-like microorganisms within human erythrocytes. Experientia 34:458-460. 20. Wuerthle-Caspe Livingston, V., and E. Alexander-Jackson. 1970. A specific type of organism cultivated from malignancy: bacteriology and proposed classification. Ann. N.Y. Acad. Sci. 174:636-654.