Journal of Clinical InvestigatioxVol. 44, No. 12, 1965

Significance of Bacterial Variants in Urine of Patients withChronic Bacteriuria *

LAURAT. GUTMAN,MARVINTURCK,t ROBERTG. PETERSDORF,ANDRALPHJ. WEDGWOOD

(From the Departments of Pediatrics and Medicine, University of Washington School of Medi-cine, and the King County Hospital, Seattle, Wash.)

Some persistent and recurrent bacterial infec-tions of the urinary tract may result from theability of organisms to remain viable in the kidneyas bacterial variants, often termed "protoplasts,""L-forms," or "spheroplasts." These variants areinduced in vitro by most antibiotics and by naturalimmune systems such as serum factors (1), lyso-zyme (2), and leukocyte granules (3). L-formshave also been produced in vivo in experimentalanimals (4-6).

Probably every bacterial species is capable offorming a cell wall-free variant. These variantsretain the ability to proliferate and may revert tothe dassical bacterial form, called the parent.Besides lacking a cell wall, bacterial variants dif-fer from the parent because they may be osmoti-cally fragile, reproduce by budding and by produc-tion of viable granules, and are filtrable. Theyalso appear to be resistant to killing by serum (7)and to many antibiotics that attack the parent or-ganism (8).

Bacterial variants may include at least severalforms. Some are probably bizarre forms not nec-essarily related to either protoplasts or L-forms.Protoplasts and L-forms may fall along a contin-uum, and it is often difficult to separate one fromthe other by their morphological or physicochemi-cal properties. For the purposes of this paper,

* Submitted for publication May 3, 1965; acceptedAugust 19, 1965.

This investigation was carried out in part under thesponsorship of the Commission on Immunization of theArmed Forces Epidemiological Board and was supportedin part by U. S. Army Medical Research and Develop-ment Command grant DA-49-193-MD-2308, by traininggrants 5 TI-AI-227-03 and AI-146-05, and by grant Al-06311-01 from the National Institutes of Health, U. S.Public Health Service.

tAddress requests for reprints to Dr. Marvin Turck,Dept. of Medicine, University of Washington Schoolof Medicine, Seattle, Wash. 98105.

"protoplasts" and "L-forms" are used interchange-ably and are defined as filtrable bacterial formsthat appear as granular or budding bodies in broth,grow as distinct colonies on a medium modifiedfor pleuropneumonia-like organisms (PPLO), andmay or may not revert to the parent, classical bac-terial form on repeated subcultures. PPLO aredistinct from L-forms and protoplasts. They areprobably a naturally occurring cell wall-free spe-cies and do not revert to any other form (9).

In previous studies regarding the pathogenesisof L-forms and protoplasts in urinary tract infec-tion, direct microscopy has shown round, bizarreforms in the urine of patients with bacteriuria re-ceiving antimicrobial agents (10, 11). The pres-ent study differs from those previously reportedin that a modified PPLO medium was used thatwill support the growth of bacterial variants in pa-tients with chronic bacteriuria and permit furtherassessment of the role of these forms in the patho-genesis of renal infection. The results on the oc-currence of bacterial variants in renal disease sug-gest that L-fornis and protoplasts may be onecause of persistent or chronic infection of the uri-nary tract.

MethodsCollection of urine specimens. Urine specimens were

carefully collected by a clean-voiding procedure and wereseparated promptly into three portions: 1) 5 ml wasadded to a tube containing 5.0 ml of 20% sucrose solu-tion for osmotic stabilization as suggested by Leclerberg(12); 2) 3.0 ml was used on random samples for deter-mination of osmolality by the freezing point technique;3) 3.0 ml was processed for routine quantitative bacterio-logic culture, serologic grouping of Escherichia coli, andantimicrobial susceptibility testing by methods previouslydescribed (13).

Separation of bacterial variants from classical or-ganisms. The urine-sucrose solution was transferred toa 10-ml glass syringe fitted with a Luer lock and filtered

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GUTMAN,TURCK, PETERSDORF,ANDWEDGWOOD

TABLE I

Total experience in culturing for bacterial variants

No. posi- %Posi-tive for tive for

Study group variants variants

I. Patients with chronic bacteri-uria or pyelonephritisTotal no. patients 57 11 19Total no. urine samples 146 23 16

II. Patients with renal diseaseother than chronic bacteriuriaor pyelonephritisTotal no. patients 59 0 0Total no. urine samples 73 0 0

III. Consecutive admissions formedical reasons other thanrenal diseaseTotal no. patients 15 0 0Total no. urine samples 15 0 0

through a 0.45-IAe Millipore filter in a Swinny-type syringeadapter. Since L-forms and protoplasts, but not classicbacterial forms, will pass through this pore size (14),variants are separated from classical bacteria.

Preparation of L-form medium. L-form agar mediumcontained the following (grams per liter): sucrose 100.0;Phytone1 20.0; NaCl 5.0; MgSO4-4 H20 2.5; yeast ex-tract2 10.0; cholesterol 0.04 dissolved in 10 ml 95%ethanol; Ionagar3 no. 2 8.0. The pH was adjusted to7.8 with normal NaOHand the media autoclaved for 15minutes at 1210 C. L-form broth was. prepared by omit-ting the agar and using 200 g per L sucrose.

Horse serum 1 was heat inactivated before use.Pour plates were made by adding 2 ml horse serum,

8 ml L-form agar, and inoculum. Falcon 12- X 50-mmpetri dishes with tight covers were used to prevent dry-ing of media during prolonged incubation.

A second pour plate contained horse serum, L-formagar, inoculum, and 25 ,ug per ml of tetrazolium redindicator.

Biphasic tube medium contained L-form broth on topof an L-form agar slant. One ml horse serum was addedto 3 ml agar and cooled as a slant. Eight ml L-formbroth, 2 ml horse serum, and inoculum were decantedonto this.

Culture procedures for isolation of variants. The urinefiltrate was processed as follows: 1) 0.25 ml was inocu-lated into an L-form medium pour plate; 2) 0.25 ml wasinoculated into an L-form medium pour plate containing25 ,ug per ml tetrazolium red indicator; 3) 0.1 ml was in-oculated onto a standard sheep blood agar plate; 4) adrop was examined directly by phase contrast microscopy;5) the remainder of the filtrate was inoculated into bi-phasic L-form medium. All plates were incubatedaerobically at 370 C. The blood agar plates served as acheck on contamination by classical bacterial forms, and

1 BBL, Baltimore, Md.2 Difco, Detroit, Mich.3 Oxoid, Chicago Heights, Ill.

the samples were discarded if urine filtrate producedgrowth on the blood plate. The initial cultures wereexamined at 3 to 7 days by direct observation. One weekafter initial inoculation, 1.0 ml of broth from the biphasicmedium was subcultured into an L-form medium pourplate and into a second biphasic broth. One and 2 weekslater, the second biphasic broth was subcultured intoL-form medium pour plates. All broth cultures were ex-amined by phase contrast microscopy for evidence ofvariant growth. All cultures were held for 1 month be-fore being discarded as negative. An uninoculated bi-phasic tube and a pour plate were prepared along withthe study specimens to serve as sterility controls; ifthese showed growth, presumably evidence of contamina-tion, the samples made from that particular urine prepa-ration were discarded.

Criteria for growth of bacterial variants. L-form andprotoplast colonies are small and may appear only afterprolonged incubation. Only those samples that showedgrowth of colonies on L-form medium agar plates wereconsidered positive, including colonies that were macro-scopically visible in the agar and colonies that reducedthe indicator dye to visible pink and red. Althoughslightly inhibitory to variant growth, the tetrazoliumred indicator was useful because the color change of dye,indicative of metabolic activity, allowed for more rapidscreening of plates. Forms that could be identified onlyin the broth, or forms that were visible in the agar onlyby microscopy, were not accepted because of possibleartifacts.

Attempts at reversion of L-form colonies. Variantsmay readily revert to the parent form on the first orsecond subculture. In general, this has occurred in pa-tients receiving treatment with antimicrobial agents.If the culture failed to revert, the variants were seriallyinoculated into media of decreasing horse serum con-centration (20%, 15%o, 10%o, 5%o serum), into brothcontaining 50%o L-form broth and 50% trypticase soybroth,' and on one occasion into an animal, for animalpassage.

50-

'A 40-

t 30-.ci

%- 20-

C t0

7

~7>

0-Urine culture attime of positivevariant culture

O-Urine culture attime of negativevariant culture

O 2 3 4 5 6Log Bacteria / ml urine

FIG. 1. DISTRIBUTION OF 23 URINE CULTURESPOSITIVEFOR VARIANTS IN RELATION TO 146 ROUTINE BACTERIALCOUNTS.

1946

7, 64 777 77,

BACTERIAL VARIANTS IN URINE OF PATIENTS WITH CHRONICBACTERIURIA

TABLE II

Bacteriologic and clinical data of patients with and without variants

Routine urine cultures at time culture forRoutine urine cultures at time culture for variant was positive variant was negative

Concurrent ConcurrentPatient antimicrobial antimicrobial

no. Routine culture Reversion therapy Routine culture therapy

1. Negative None Ampicillin Negative AmpicillinEscherichia coli >103 None Ampicillin Escherichia coli >104 Ampicillin

2. Escherichia coli >105 None Ampicillin Negative (8 samples) AmpicillinKlebsiella <102 None Ampicillin Escherichia coli >106Klebsiella <102 None Ampicillin (5 samples)

3. Escherichia coli >104 Escherichia coli Ampicillin Escherichia coli and >104 Noneand Proteus mirabilis Proteus mirabilis

Proteus mirabilis and >10' NoneEscherichia coli

KMebsiella (2 samples) <102 None- Negative None

4. Escherichia coli >105 None Ampicillin Negative (16 samples) AmpicillinEscherichia coli <102 None Ampicillin Escherichia coli >106Negative None Ampicillin (11 samples) NoneEscherichia coli >105 Escherichia coli AmpicillinEscherichia coli <102 Escherichia coli AmpicillinNegative Escherichia coli Ampicillin

5. Escherichia coti >106 None None

6. Streptococcus >106 Streptococcus Nonefaecalis faecalis

7. Negative None None Staphylococcus and >104 NoneStreptococcus

8. Escherichia coli >105 Escherichia coli CephalothinNegative Escherichia coli Cephalothin

9. Escherichia coli >105 None Chloramphenicol Escherichia coli >106 None

10. Negative None None

11 .* Negative None NoneNegative None NoneNegative None NoneNegative None None

* A renal biopsy from this patient was positive for variants, which subsequently reverted to Proteus mirabilis.

Results

Prevalence of bacterial variants in differentpopulation groups. The data relating to isolationof bacterial variants are summarized in Table I.

These patients were from three study groups.

Group I included patients with chronic bacteriuriaor pyelonephritis. The majority of these patientshad had persistent or repeated episodes of signifi-cant bacteriuria, defined as greater than 105 bac-teria per ml urine. In this group 11 of 57 patientswere found to have bacterial variants in their urineon one or more occasions. Figure 1 depicts thedistribution of cultures positive for variants in re-

lation to routine quantitative bacterial counts.The greatest number of urine samples positive forvariants was obtained at a time when the patientswere receiving antimicrobial therapy and con-

tained between 0 and 10 classical organisms perml urine. Variants were cultured sometime dur-ing the period of observation in 23 of 146 (16%)specimens and 11 of 57 (19%o) patients withchronic infection of the urinary tract.

Group II included 59 patients with renal diseaseother than urinary tract infection. Diagnoses inthis group included chronic glomerulonephritis,collagen vascular disease, diabetic nephropathy,polycystic disease, and familial nephritis. Patientswho showed concomitant bacteriuria were ex-

cluded from the study. No variants were isolatedfrom patients in this group. Variants were alsonot isolated from patients in group III, whichconsisted of 15 consecutive patients admitted tothe hospital for reasons other than renal disease.

Characteristics of cultures containing variants.

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GUTMAN,TURCK, PETERSDORF,ANDWEDGWOOD

FIG. 2. VARIOUS FEATURESOF VARIANTS AS SEENBY PHASEMICROSCOPY. Upper left: A variant colony withinan agar block. Notice the cluster of organisms in the center of the colony. These organisms were obtained fromPatient 4, and reverted to E. coli (agar smash X 300).

Upper right: A stable L-form or protoplast isolated from the urine of Patient 11. Notice the large body sur-rounded by granules and spheres in various stages of development. These mature to become variants (wetmount of broth culture X 3,000).

Lower left: A budding protoplast obtained from Patient 3; these variants reverted to E. coli (wet mount ofbroth culture X 3,000).

Lower right: A variant during the process of reversion to Proteus mirabilis isolated from the kidney biopsyof Patient 11. The long filamentous extensions from the central round body segment to become bacilli (wetmount of broth culture X 3,000).

When variants were found on the L-form me-

dium, they were always present in large numbersand reduced the tetrazolium indicator dye. Theirsmall size, however, precluded precise quantita-tion. When only a few isolated colonies were

cultured on L-form agar, they were found to beeither diphtheroids or staphylococci, presumablycontaminants. In no instance was a colony simi-lar to a variant either morphologically or by phasemicroscopy found on blood agar. The rigid cri-teria employed for definition of positive culturesfor bacterial variants plus the fact that they were

always present in large numbers make it unlikelythat their failure to grow on blood agar was a

chance occurrence. When colonies of typicalvariants were found on L-form agar, budding or

granular forms were also demonstrated in broth.

There were, however, five instances not includedin tabulation of the data, four in patients withchronic bacteriuria and one in a patient with re-nal disease other than chronic pyelonephritis, inwhich microscopic examination of the brothshowed "typical" budding or granular forms, butgrowth failed to occur on solid media; these pa-tients' urines were not considered positive forvariants.

Characteristics of patients wtith bacterial variantsin urine. The bacteriologic and clinical data ofthe 11 patients from whomL-forms or protoplastswere recovered are summarized in Table II. Al-though bacterial variants were usually demon-strated at a time when the patient was receivingconcurrent antimicrobial therapy, cultures forvariants were also positive from five patients at

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BACTERIAL VARIANTS IN URINE OF PATIENTS WITH CHRONICBACTERIURIA

TABLE III

Patients studied for bacterial variants while receiving antimicrobial therapy

Patient no. Infecting organism Variant culture After therapy

1 Streptococcus faecalis and + Streptococcus faecalis andEscherichia coli 04 Escherichia coli 04

2 Escherschsa coli 07 + Escherichia coli 074 Escherichia coli 016-62 + Escherichia coli 016-629 Escherichia coli + Escherichia coli

12 Escherichia coli Klebsiella13 Escherichia coli No relapse14 Intermediate coliform - Proteus mirabilis15 Escherichia coli No relapse16 Escherichia coli 075 - Escherichia coli 075

a time when they had not received therapy for atleast 6 weeks; in one patient no known antimi-crobial treatment had been given.

In Table II are also listed the results of routinequantitative urine cultures and the state of anti-microbial therapy at a time when these 11 patientsdid not have bacterial variants isolated from theurine. A total of 49 specimens was examinedfrom which variants could not be identified, 26during antimicrobial treatment and 23 without con-

current therapy. In all, 23 samples from these 11patients showed growth of variants, and in seven

instances the variant subsequently reverted to a

classical bacterial form, which was the same spe-

cies or serogroup of organism with which the pa-

tient had been infected initially. Urine samplesfrom three patients (no. 7, 10, and 11) were posi-tive for variants at a time when the routine bac-teriologic culture was negative and when the pa-

tient was not receiving chemotherapy. Patient 11was of particular interest. She was admitted tothe hospital because of progressive azotemia of un-

known etiology. She was found to have multiplemyeloma, but in addition, renal biopsy revealedhistologic evidence of pyelonephritis. Variantswere cultured from homogenates of the tissuespecimen and ultimately reverted to classical Pro-teus mirabilis. Urinary variants, which were

demonstrated on four separate occasions, failed torevert to the parent. However, a passage of thesestable variants into rats resulted in isolation offorms that readily reverted to P. mirabilis. Re-nal tissue obtained at post-mortem also grew

variants that failed to revert to classical bacterialforms.

A second patient (No. 4) had been followed forasymptomatic bacteriuria for 5 years and, con-

sistently harbored the same strain of E. coli016-62. During this period of observation she hadremained completely asymptomatic despite per-sistent bacteriuria; she had also remained rela-tively refractory to attempts at eradication of in-fection because of her inability to tolerate oralantimicrobial agents for more than a few days ata time. Because variants were demonstrated inher urine during treatment with ampicillin, shewas admitted to the hospital for further treatmentand study. Percutaneous renal biopsy was per-formed, and dassical E. coli 016-62 was culturedfrom the tissue homogenate although variantscould be identified only in the urine. A course ofparenteral ampicillin, 2 g per day for 10 days, wasadministered with prompt sterilization of the urine,but subsequently the infection relapsed with E. coli016-62, shortly after cessation of therapy. Eryth-romycin had also been added to the treatmentregimen because of apparent in vitro suscepti-bility of the variants to this antibiotic. However,addition of this agent, did not prevent recrudes-cence of bacteriuria with the same serologicalstrain of E. coli.

Figure 2 depicts characteristic examples of anagar smash of a variant colony, stable granularvariant forms, budding, and filamentous formsidentified from patients in this study.

Isolation of bacterial variants during therapy.Nine patients were studied for the presence ofL-forms or protoplasts before, during, and afterantibiotic treatment (Table III). These repre-sented a selected sample because not all patientswere studied serially in relation to chemotherapy.Eight of the patients in this group had receivedseveral courses of antimicrobials for chronic uri-nary tract infection in the past and either were

1949

10;UTMAN, TURCK, PETERSDORF,AND WEDGWOOD

refractory to treatment or had had recurrencesafter cessation of therapy. Although all nine pa-tients became abacteriuric during therapy, onlytwo remained free of significant infection for atleast 6 weeks after therapy. All relapses occurredwithin 1 week of cessation of treatment. Variantswere cultured from the urine of four of these ninepatients, and it is noteworthy that recurrences ofinfection among them were associated with thesame organism or serogroup of E. coli that waspresent before therapy. Although relapse of in-fection with the same strain of E. coli 075 was alsofound in one patient from whom L-forms or pro-toplasts were not grown, it is of particular interestthat this patient had renal lithiasis, and relapse ofinfection was not unexpected.

Discussion

The existence of a filtrable stage of bacterialvariation has been recognized since the early partof this century. In 1931, Hadley, Delves, andKlimek (15) reviewed the field of filtrable bac-teria and suggested that they were associated withchronic infectious disease. In 1935, Klienebergernoticed a small colony variant on a plate of Strep-tobacillus moniliformis (16). She originally be-lieved this to be a symbiont. Subsequently, how-ever, it was determined to be a variant form of theStreptobacillus.

The colony studied by Klieneberger arose spon-taneously from a culture of classical bacteria.Since then, bacterial variants have been producedfrom virtually all species of bacteria (and yeast)lby means of various stimuli. Adverse conditionsin general, such as prolonged incubation, incuba-tion at suboptimal temperatures, and high aminoacid and salt concentrations will predispose toL-form transformation (1). Exposure of bacteriato many antibiotics may also produce variants(17). The transformation from the classical bac-terial to the variant form is probably a generalresponse of the organism rather than the result ofa specific action of the variant-producing agentupon the bacteria. For example, a history of anti-microbial therapy may not be a necessary pre-requisite for bacterial variants in human infection.

Since these studies were concerned primarilywith urinary tract infections, they were limitedmainly to enteric bacterial variants. These gram-

negative variants will pass a 0.45-,u Millipore filter,which restricts classical organisms. Morphologi-cally they are round, vary in size, and are seenmost easily by phase contrast microscopy. Theytake Dienes' methylene blue azure stain (18, 19).On gram stain they break up and are seen only asdebris unless precautions are taken to preventtheir lysis by the hypotonic fixative (11). Theydo not grow in trypticase soy broth or blood agarplates and grow as very small, opaque colonies inthe L-form medium described above. Their smallsize and the extreme difficulty in propagating thesevariants on solid media make precise enumerationof these forms impossible at this time. Protoplastsand L-forms produced from gram-positive organ-isms, on the other hand, tend to be much largerand may be quantitated (8).

In attempting to relate variants to the patho-genesis of infection of the kidney, it may be worth-while to attempt to distinguish protoplasts fromL-forms. There is evidence that L-forms are notso osmotically fragile as are protoplasts (20), andthe greater osmotic stability of the L-form may,in part, explain its survival in the isotonic environ-ment of the body. Distinct from L-forms, proto-plasts may lyse in an isotonic milieu, and the renalmedulla alone may be sufficiently hypertonic tosupport their growth. It is also conceivable thatprotoplasts are formed in the renal medulla duringinfection and subsequently develop into stableL-forms that can be recovered from the urine. Inpursuing this question, urinary osmolality has beendetermined on some urine samples from whichvariants were isolated and from a number ofcontrol specimens. To date the total number ofsamples is too small to draw definitive conclusions,but none with an osmolality below 312 mOsmhaveyielded L-forms or protoplasts. The effect of uri-nary osmolality on isolation of bacterial variantshas not been settled and is under continued study.

Bacterial variants in the urine after antibiotictherapy have been reported previously. In 1951,Voureka observed bizarre bacterial forms in theurine of patients who were treated with chloram-phenicol and also noted their reversion to theparent form after repeated subcultures (10). Tenyears later Braude, Siemienski, and Jacobs re-ported a patient with Proteus pyelonephritis inwhose urine round, osmotically fragile forms were

1950

BACTERIAL VARIANTS IN URINE OF PATIENTS WITH CHRONICBACTERIURIA

found during penicillin therapy ( 11 ) . Whentheseorganisms were injected into the bladder of rats,a Proteus pyelonepritis was produced 7 to 8 dayslater.

Guze and Kalmanson incriminated protoplastsin the persistent infection that characterizes Strep-tococcus faecalis pyelonephritis in rats (21). Ratswere inoculated with S. faecalis and treated withpenicillin. After therapy had been completed, thekidneys were homogenized in salt solution, and thehomogenates were then inoculated into osmoti-cally stabilized and standard media. The numberof classical colonies was enumerated, and the dif-ference between colony counts on the two mediawas taken to be the number of osmotically fragileprotoplasts originally present. In these studiesthe kidneys were often sterile on routine media,whereas a significant number of colonies, presum-ably representing reverted protoplasts, occurredon the osmotically stabilized media.

Alderman and Freedman (22) injected E. coliprotoplasts, produced from the parent by contactwith penicillin, into the renal medulla and intothe skin of rabbits. The animals developedE. coli pyelonephritis presumably from revertedprotoplasts. However, the protoplasts injectedinto the dermis failed to produce evidence of in-fection., It was inferred from these data that thehigher osmolality of the renal medulla favoredsurvival and reversion of protoplasts, whereas theorganisms in the isotonic dermis had lysed.

When bacterial variants are recovered from theurine of patients receiving antimicrobials, espe-cially ampicillin and cephalothin, which act pri-marily on the cell wall, the question can be raisedas to whether these variants are formed in vivo orwhether induction has occurred after voiding hastaken place. There are at least four reasons tobelieve that in urinary tract infection L-forms andprotoplasts are induced in vivo. First, these formshave been observed by direct microscopy offreshly voided urine; second, in the isolation pro-cedures performed in the present study, antibioticsin the urine were probably diluted to a concentra-tion far below that required to produce protoplastsin vitro; third, the urine specimens were promptlyfiltered, excluding classical forms from the filtrate;finally, urine samples from some patients who werenot receiving antibiotics contained variants.

The finding that the antibiotic sensitivity pat-tern of a variant usually differs from that of itsparent (23) has opened up new therapeutic con-siderations in patients with chronic bacteriuria.Guze and Kalmanson (24) have demonstratedthat when rats with S. fecalis pyelonephritis aretreated with penicillin, followed by erythromycin,protoplasts fail to persist. In contrast, when peni-cillin alone is used, protoplasts can be demon-strated many weeks after therapy has ended. Al-though this type of approach has not been triedin patients, it deserves consideration. Whethervariants themselves are pathogenic and are capableof tissue invasion, apart from their ability to revertto a pathogenic classical organism, remains to bedetermined. Some variants of gram-negative rodsexert cytopathogenic action on cells in tissue cul-ture (25) and therefore may cause progressivedisease in vivo. Variants may be pathogenic, andit is important to be able to identify them and todefine their relation to chronic infection.

The identification of bacterial variants in pyelo-nephritis may have wide implications. First,variants cannot be isolated and identified by theusual techniques in use in classical bacteriology,and as was the case with PPLO or viral cultures,techniques for isolation of variants extend the areaof clinical microbiology. The success of this studydepended in part on the preparation of a mediumsuitable for the growth of variants and on a methodfor separating variants from classical bacteriawithout the use of inhibitors. Second, if variantsare causal agents in some cases of chronic pyelo-nephritis, some concepts of the diagnosis and treat-ment of this relatively common disease will haveto be re-evaluated. On the basis of this study,approximately 20%o of patients with chronic bac-teriuria may have protoplast or L-form infectionof the kidney at some time in their course. Third,if L-forms appear as a result of antibiotic therapy,and are in themselves pathogenic, eradication ofbacteriuria can no longer be considered as the solecriterion for successful treatment of urinary tractinfection. Finally, since the antibiotic sensitivitypattern of variants differs from the parent, theidentification of variants as pathogenic agentswould necessitate some changes in concepts ofantibiotic therapy.

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GUTMAN,TURCK, PETERSDORF,AND WEDGWOOD

Summary

A total of 234 urine cultures was processedfrom 131 patients. L-forms or protoplasts were

demonstrated from 11 of 57 (19%) patients withchronic urinary tract infection and pyelonephritis.No filtrable bacterial variants were cultured fromurine of 59 patients with renal disease other thanchronic bacteriuria or from 15 consecutively hos-pitalized patients without renal disease. Methodsof isolating and culturing enteric bacterial variantswere described. Patients from whom variantswere identified during treatment with antibioticswere shown to relapse with the original parent

strain when therapy was completed. These ob-servations suggest that L-forms and protoplastsmay play a role in microbial persistence of chronicrenal infection.

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