Bacterial L-form isolation from inflammatory bowel disease patients

GASTROENTEROLOGY

1983:85:364-g

Bacterial L-Form Isolation From Inflammatory Bowel Disease Patients M. R. BELSHEIM, R. Z. DARWISH, B. SCHIEVEN

W. C. WATSON,

and

Departments of Medicine and Divisions of Gastroenterology, Victoria Hospital, St. Joseph’s Hospital, and University of Western Ontario, London, Ontario, Canada

This study was designed to investigate a possible relationship between bacterial L forms and inflammatory bowel disease. Homogenates of intestinal mucosal biopsies from Crohn’s disease, ulcerative colitis, and control patients underwent bacterial culture on hypertonic media designed for the recovery of L-form bacteria and parental organisms. L forms were recovered from 24 of 71 Crohn’s disease, 51 of 121 ulcerative colitis, and 2 of 140 control biopsy specimens. These isolation rates are significantly different when Crohn’s disease biopsy specimens [p < 0.001) and ulcerative colitis biopsy specimens (p < 0.001)are compared with controls. Six diRerent L-form types were recovered, of which the most common were Escherichia coli and Streptococcus fecalis. No marked diferences were observed in L-form recovery rates or L-form types recovered between Crohn’s disease and ulcerative colitis patients. Drug treatment of inflammatory bowel disease patients did not affect L-form recovery rates or the type of L forms recovered. The results suggest either that L forms are involved in the causation of inflammatory bowel disease or that their presence in mucosal biopsy tissues is a result of the disease process. An association between bacterial L forms and inflammatory bowel disease (IBD) has been reported Received March 30, 1982. Accepted February 24, 1983. Address requests for reprints to: M. R. Belsheim, M.D., FRCP(C), Department of Medicine, St. Joseph’s Hospital, 268 Grosvenor Street, London, Ontario, Canada N6A 4V2. This work was partially completed while M. R. Belsheim was the recipient of a Canadian Foundation for Ileitis and Colitis Fellowship. The authors thank Dr. J. L. Whitby (Professor] from the Department of Bacteriology and Immunology, University of Western Ontario, for his assistance in editing. The authors are also grateful to the members of the Gastroenterological Units of Victoria Hospital, St. Joseph’s Hospital, and University Hospital, London, Canada, for their invaluable assistance in the collection of biopsy specimens and patient assessment. 0 1983 by the American Gastroenterological Association 0016-5085/83/$3.00

sporadically since 1971 (l-6).Pseudomonas (2,3) and mycobacterial (4) L forms have been isolated from IBD patients but not from control patients. Antibodies against revertants of pseudomonas L forms have been reported in Crohn’s disease (CD) patients (5). Streptococcus fecalis L forms injected into rabbit ileum have been reported to cause granuloma development, while injection of the parental organism did not (6).L-form studies to this point have reported only small numbers of patients and have employed differing methodologies. L forms are cell-wall-deficient (CWD) variants of conventional bacteria that continue to grow and divide, and which require a hypertonic environment for survival (7,8). Their isolation requires media adapted to their osmotic and metabolic requirements (7,8). L-form pathogenicity in humans is controversial, although L-form isolation has been reported from a wide variety of human illnesses (7-11). If L forms are pathogenic organisms in humans, it .is not clear whether direct pathogenicity is possible or whether the L phase merely permits persistence in the proper tissue environment, with the recrudescence of clinical disease occurring only after L-form reversion to the parental form (7). This paper reports the results of the culture of 332 intestinal mucosal biopsy specimens from Crohn’s disease, ulcerative colitis, and non-IBD control patients on media designed to isolate L-form and parental bacteria.

Materials

and Methods

Population

Studied

Intestinal mucosal biopsy specimens were obtained from three groups of patients seen either as outpatients or during hospital admission. The first two groups consisted of 44 CD and 69 ulcerative colitis (UC) patients, and included newly diagnosed, untreated individuals and previously diagnosed patients with varying disease activi-

August 1983

L FORMS

ty, extent, and treatment status. The diagnosis of IBD patients was confirmed if a combination of clinical, radiologic, and pathological criteria were met. Criteria utilized for diagnosis generally conformed to previously published recommendations (12-l 3). The third group, designated as controls (C), consisted of 75 patients with a heterogeneous group of non-IBD gastrointestinal disorders. The diagnosis included 25 patients with irritable bowel syndrome, 13 with colonic polyps, 12 with carcinoma (colonic or gastric), 5 with infectious diarrhea, 5 with peptic ulcer disease, 4 with celiac disease, 4 with pseudomembranous colitis, 2 with ischemic colitis, and 1 each with Peutz-Jeghers syndrome, anorexia nervosa, postgastrectomy diarrhea, lactase deficiency, and laxative abuse.

Biopsy

Material

All biopsy specimens were taken during routine diagnostic procedures on IBD and control patients. Biopsy procedures and locations varied slightly. Most of the biopsy specimens were taken during colonoscopy or sigmoidoscopy, and 299 of the 332 biopsy specimens studied were from the colon. The largest group of biopsy specimens (113) consisted of single biopsy specimens taken from areas of active disease. To ascertain the effect of the location of biopsy material on L-form recovery, 51 patients (7 CD, 20 UC, 24 C) underwent biopsy from multiple colonic locations (ascending, transverse, descending, rectosigmoid) during a single colonoscopy. In these patients, biopsy specimens were taken from areas remote from disease activity as well as from areas of active disease. An additional 20 patients (5 CD, 12 UC, 3C) had single or multiple biopsies repeated on one or more occasions.

Table BRH

MCK

TSA

M7HlO

1. Media Brain heart infusion agar (Difco)” Yeast extract Sucrose Osmolality I.098 mosmol/kg MacConkey agar (Difco) Cysteine HCl Proteose peptone #3 (Difco) Sucrose Osmolality 996 mosmol/kg Tryptic soy (BBLlb Trypticase peptone (BBL) Agar (special Nobel] Sucrose Osmolality 896 mosmol/kg Middlebrook and Cohn 7HlO Agar (BBL) Glycerol OADC Enrichment (BBL) Sucrose Penicillin G Osmolality 921 mosmol/kg

4% 10% 17% 5% 0.02% 1.5% 17% 2% 0.05% 1.2% 17%

1.8% 0.5% 10% 17%

IN INFLAMMATORY

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DISEASE

365

Thirty-three biopsy specimens were taken from extracoionic sites during upper endoscopy, small bowel biopsy, and from freshly resected surgical specimens. The total number of biopsy specimens studied was 71 CD, 121 UC, and 140 C, and these were obtained from 44 CD, 69 UC, and 75 C patients, respectively. Thirty-two patients had stool samples taken by suction from the bowel lumen adjacent to areas of diseased mucosa during a sigmoidoscopy or colonoscopy. These samples underwent processing and culture in the same manner as tissue biopsy specimens.

Tissue

Preparation

and Culture

All biopsy specimens were immediately placed into a sterile hypertonic transport medium consisting of 2.5% brain heart infusion broth, 15.0% sucrose, 0.6% glucose, and 10.0% horse serum (inactivated). Samples and transport media were stored for a maximum of 3 days at 4°C before processing and culture. All biopsy specimens underwent manual homogenization in 19 x 150-mm tissue grinders (Canlab T4025-3, CANLAB Inc., Canada). One-half milliliter of the homogenates was plated directly onto each of four different freshly prepared media modified to permit L-form growth as well as growth of parental organisms. The media used were brain heart infusion agar (BRH), MacConkey agar (MCK), tryptic soy agar (TSA), and Middlebrook and Cohn’s 7HlO agar (M7HlO). In addition to standard constituents, the media contained 15%-17% sucrose, IO%-20% horse serum, and stabilizing factors consisting of MgS04 . 7H,O, CaC12, cysteine HCL, and glucose (Table 1). One-half milliliter of the homogenate was passed through a 0.2-pm filter (Gelman Acrodisc 0.2pm, Gelman Sciences Inc., Ann Arbor, Mich.), and plated onto each of an identical set of four fresh media to which 5000-10,000 IUiml of penicillin G had been added to maintain L-form growth. Since conventional bacteria cannot pass a filter of this size (14), it was felt that the first set of media could grow stable L forms and parental organisms, while the second set could isolate only L forms. Because of this, it would be impossible for the penicillin in the second set of media to induce L forms from parental organisms, since no parental organisms would be present after filtration. As an additional safeguard, the first 60 samples studied were provided with an additional set of conventional, nonhypertonic media consisting of BRH, MCK, and a pleuropneumonia-like organism (PPLO) medium to ascertain whether there was an appreciable incidence of filter rupture or leakage. As L forms do not grow on nonhypertonic media, only parental bacteria escaping through ruptured or leaking membrane filters should grow on these media, and thus bacterial growth would indicate filter dysfunction.

25,000 IUiml

a Difco Laboratories, Detroit, Mich. L-form versions contain 500010,000 W/ml of Penicillin G (sodium). In addition, these media contain: glucose 0.8%, horse serum (whole) or ascitic fluid 10%. MgS04. 7Hz0 0.05%, and CaCI, 0.15%. b BBL Laboratories, Cockysville, Md.

Incubation

and Identification

Both sets of media were incubated at 37°C until bacterial growth had occurred or for a maximum of 6 days. Media for mycobacterial isolation (Middlebrook and Cohn

366

GASTROENTEROLOGY Vol. 85, No. 2

BELSHEIM ET AL.

7HlO) were incubated for 6 mo. L-form colonies have a similar gross appearance and were identified by their “fried egg” colonial morphology on light microscopy, by gram staining revealing faintly stained gram-negative coccobacillary forms on light microscopy, and by the ability of these cells to revert to their parental counterpart when subcultured on nonhypertonic media. Identification of bacterial type cannot proceed until reversion of the parental organism has occurred. Selected cells also underwent electron microscopy. Culture plates were coded so that investigators who examined the revertant cultures were blinded as to the source of the inocula. Identification of parental and revertant bacteria was done independently by ourselves and by the bacteriology laboratory at Victoria Hospital. Gram staining and standard biochemical testing were the discriminatory procedures used.

Statistical

Analysis

Statistical comparisons were by the x2 test. Probability values were derived from x2 values.

Results The frequency of L-form isolation and the types of L forms isolated from the three patient groups were compared in three main categories: overall or total results, and two subcategories consisting of multiple colonic biopsy specimens and extracolonic biopsy specimens. In some instances, two different L forms were isolated from a single biopsy specimen, and for that reason in some of the tables that follow, the total number of L forms exceeds the number of biopsy specimens. The extra set of nonhypertonic culture media was discontinued when it was evident that non-L-form bacteria were not passing through the filter. By that time trends in the results indicated that if membrane leakage was responsible for the positive L-form cultures, it was selectively affecting only the filters used for CD and UC biopsy specimens. This was regarded as unlikely. Table

2. Incidence

of L-Form

Recovery

Related

to Number

Overall

L-Form

A total of 27 L forms were isolated from CD patients (Table 2). A single L-form type grew from 21 biopsy specimens while three biopsy specimens grew two different L-form types each. L-form growth, therefore, occurred from 24 of 71 biopsy specimens studied. Forty-eight biopsy specimens from UC patients showed growth of a single L-form type, and another three biopsy specimens grew two L forms each for a total of 54 L forms isolated. L-form growth, therefore, occurred from 51 of 121 biopsy specimens studied. Only two control biopsy specimens were positive for L forms. L-form isolation rates were significantly different when CD biopsy specimens (p < 0.001) and UC biopsy specimens (p < 0.001) were compared with control biopsy specimens. To be certain that the study of L-form positive biopsy specimens did not introduce error into our data by including a small number of patients with many positive biopsy specimens, we also studied the number of patients with positive biopsy specimens. The results were strikingly similar (Table 2). Twenty of 44 CD patients and 37 of 69 UC patients had at least one biopsy specimen positive for L-form growth. Statistical analysis revealed significant differences when CD patients (p < 0.001) and UC patients (p < 0.001) were compared with controls. When L-form isolation rates were compared between CD and UC patients, no significant differences were found. Cultures from 8 CD biopsy specimens, 20 UC biopsy specimens, and 12 C biopsy specimens grew CWD bacterial colonies whose cells displayed an inability to revert to a parental type, grow, or divide. On light microscopy, these colonies did not take up vital stains and appeared as ghost cells. On electron microscopy, they lacked any vestige of a cell wall. We designated these isolates as protoplasts, using the terminology defined by McGee et al. (15). We believe they are dying L-form colonies, but because of Biopsy

Specimens

Taken

L form + No.

No.

Recovery

and Patients

Protoplasts %

L form + protoplasts

+

No.

Studied

%

% positive

Crohn’s disease Biopsy specimens Patients Ulcerative colitis

71 44

24 20

Biopsy specimens Patients Controls

121 69

51 37

Biopsy specimens Patients

140 75

2 2

L-form results for Crohn’s disease and ulcerative

33.8

8

11.3

45.1

45.4

6

13.6

59.1

42.1

20

16.5

58.7

53.6

14

20.3

73.9

1.4

12

8.6

10.0

2.7

9

12.0

14.7

colitis are both significantly

different from controls (p <

O.OOI).

L FORMS

August 1983

Table

3. L-Form Recovery: Treatment

Drug Treatment

CD

UC

Treatment L form + L form Protoplast

n = 45 15(33%) 26 4

n = 75 31 (41%) 30 14

No treatment L form + L form Protoplast

n = 26 9 (35%) 13 4

n = 46 20 (43%) 20 6

Controls n=2

0 2 0 n = 138 2 124 12

of morphologic differences, they may be unrelated to L forms. We have, therefore, classified them separately for the time being. L-form recovery did not appear to be affected by the drug treatment status of the population studied [Table 3). Drug treatment was defined as prednisone, sulfasalazine, antibiotics, or combinations of these medications given within 10 days before biopsy. Both treated and untreated groups of CD and UC patients showed the same proportion of L-form positive biopsy specimens. Because most control patients had received no treatment before biopsy, this separation had no relevance. Because antibiotics that act upon bacterial cell walls have been reported to induce L forms, we looked for this relationship closely. Only 2 CD-positive patients (cefazolin and metronidazole), 1 C-positive patient (erythromycin and tobramycin), and 2 C-negative patients (ampicillin and septra, respectively) had received antibiotic therapy within the specified period. There were no statistical differences when L-form isolation was compared between treated and untreated UC and CD groups. Stool samples taken by suction during sigmoidoscopy or colonoscopy from the bowel lumen adjacent to diseased mucosa did not show L-form recovery in any of the three groups of patients studied (8 CD, 14 UC, and 10 C). 4. L-Form Types Isolated:

Comparing

Escherichia

coli

Streptococcus

fecalis

Pseudomonas aeruginosa Aerococcusviridans Proteus mirabilis Enterobacter cloaca Total CD = Crohn’s specimens

DISEASE

367

Treatment

Identification of L-form revertants revealed that six different L-form types had been isolated from the IBD group (Table 4). In all biopsy specimens, the L-form type recovered matched one of the parental bacteria isolated separately on the non-penicillincontaining media. There were no differences in identification of L-form types between the two centers studying revertant cultures. No distinctive pattern of L-form recovery emerged either in the CD or UC groups. Escherichia coli and Streptococcus fecalis were the most common L-form types isolated in both groups. Three biopsy specimens in each of the CD and UC groups grew two different L-form types, which accounts for the difference between total Lform positives and the number of positive biopsy specimens. Only one stable L form was isolated during the study, and because stable L forms do not revert, parental type could not be ascertained. The stable L form was from a control patient. No mycobacterial L forms were isolated in any of the three patient groups at any time during the study. When the L-form positive IBD biopsy results were subdivided into treated and untreated patients, neither CD nor UC biopsy specimens showed distinctive differences in the L-form types recovered or the frequencies with which each type was seen (Table 4). Although the total number in each group was small, a slightly higher proportion of Streptococcus fecalis L forms were recovered from the treated group of UC biopsy specimens. L-Form Recovery From Multiple Biopsy Specimens

CoJonic

A total of 159 biopsy specimens from all groups were taken from four colonic locations during colonoscopy. No biopsy site in any of the three patient groups was predominant in L-form isolation (Table 5). L forms were cultured both from areas of active disease and from uninvolved bowel. More UC multiple biopsy samples were obtained than CD

vs. No Treatment

CD (n = 241 Treated

BOWEL

L-Form Types Isolated

vs. No

CD = Crohn’s disease. UC ulcerative colitis. n = number of biopsy specimens.

Table

IN INFLAMMATORY

UC (n = 51)

Untreated

8 4 2 1 1

5 4 1 0 0 0

17

10

1

disease, UC = ulcerative colitis. n = number each grew two different L-form types.

Treated

Untreated

17 10 4 2 0 0

16 3 0 1 1 0

33 of biopsy

specimens.

21 Three

CD biopsy

Total

%

46 21 7 4 2 1

56.8 25.9 8.6 4.9 2.5 1.2

81 specimens

and three

UC biopsy

368

Table

GASTKOENTEROLOGY

BELSHEIM ET AL.

5. L-Form Biopsy

Recovery

From Multiple

Colonic

Specimens Colon

Ascending CD

+ _

Transverse

Descending

Rectosigmoid

0 3 0

3 1 0

0 3 0

5 3 0

7 8 0

10 7 2

6 3 5

4 15 2

0 15 1

1 12 3

0 16 0

0 21 3

(n = 187 UC

+ _

(n = 697 c

+ -

(n = 725) CD = Crohn’s disease. UC = ulcerative colitis. biopsy specimens. C = controls. p = protoplasts.

samples, and total L-form higher in the UC group. Extracolonic

recovery

n = number

of

was therefore

Biopsies

Culture of biopsy specimens obtained outside the colon did not reveal high rates of L-form recovery at any site except for the ileum in CD patients (Table 6). The number of biopsies at each location, however, was small. Discussion This study shows that L-form colonies can be consistently isolated from gut mucosal homogenates in 40%-50% of IBD patients. A highly significant difference exists between controls and IBD patients. Recovery of L forms on specific media after homogenates have been passed through a O&pm filter rules out the possibility of L-form induction by the penicillin in the media since conventional bacteria cannot pass this filter size (3,14,16), Preliminary studies ruled out artefactually positive results due to a filter rupture or leakage. No difference in the recovery rate or L-form type was observed between treated and untreated groups of patients, which indicates that L forms are not induced by the standard drug treatments for IBD. Although certain antibiotics have been reported to induce L forms, the number of patients in this study who received antibiotics within 10 days of biopsy was small and did not influence the results. The lack of L-form recovery from stool samples taken from areas adjacent to diseased mucosa suggests that it is the tissue environment itself that is important to L-form growth. The observation that Lform growth occurred from both diseased tissue and

Vol. 85. No. 2

apparently uninvolved bowel in IBD patients undergoing multiple biopsy is intriguing. It implies that the entire colonic mucosa of IBD patients is different from that of controls, since it supports L-form growth while that of the controls does not. Whether or not L forms are involved in the causation of IBD, this finding may imply that IBD is a diffuse process, the local expression of which may depend upon decreased tissue resistance in the area of apparent disease. L-form recovery rates showed little variation between CD and UC patients. Several L-form types were recovered from both CD and UC patients, and the pattern of recovery did not separate CD and UC patients. The observation that L forms are common to both diseases may support the idea that CD and UC represent different expressions of a disease spectrum with a common pathogenesis (17). While L forms may be involved in one process that results in clinical disease in susceptible individuals, it is equally possible that L-form growth is promoted by the abnormal tissue found in CD and UC patients. L forms are a possibility as transmissible agents in IBD. They may persist for long periods in the proper hypertonic environment (8), and an intestinal countercurrent multiplier system has been described that creates a hypertonic mucosal environment suitable for L-form survival (18). Because of their relative lack of cell wall antigens, L forms present less of an antigenic challenge to the host (16), and this may aid their persistence in the host tissue. L forms may be able to escape phagocytosis by moving to an intracellular location in the mucosal tissues (3,~). High serum lysozyme levels occur in IBD patients, and lysozyme is important in the experimental induction of L forms (19). The necessity for special handling and media could explain the relative paucity of reports of Lform isolation from IBD patients thus far. The six Lform types isolated from IBD patients in this study

Table

6. L-Form Recovery Specimens

From Extracolonic

CD + Node Fistula Ileum Polyp Esophagus Stomach Duodenum Colostomy

Biopsy C

UC _

_

+

+

_

6 1 5

4

1

3 1 3

4

CD = Crohn’s disease. UC = ulcerative number of biopsy specimens (n = 33).

2 1 1 1

colitis.

C = controls.

n =

August

L FORMS

1983

are neither unusual nor exotic, but form part of the normal gut flora and may be difficult to accept as potentially causative organisms. Because multiple Lform types were isolated from the patient groups, it is unlikely that a single causative L-form organism can be implicated in either disease process. The recovery of L forms from IBD patients does not explain the considerable evidence that genetic (2022) and immunologic (23-28) factors are involved in the causation or perpetuation of these diseases. It is possible that no single cause and effect relationship will be established in IBD. A complex, unpredictable disease process with variable severity and expression such as CD or UC may be caused by the interaction of several mechanisms. L-form infection may be an initiating factor in an individual whose genetic background, environment, or immune system renders him susceptible. The nature of the disease process and its severity may depend upon the nature of the immune response or tissue resistance in the affected individual. The isolation of an organism from a disease process is only the first step in establishing a causal relationship. We do not claim to have established a causal relationship, but believe that the findings reported here warrant further investigation.

11.

12.

13.

14. 15.

16.

17.

18.

19. 20.

21.

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