A STUDYOF THE RELATIONSHIP OF THE NORMALBACTERICIDALACTIVITY OF HUMANSERUMTO BACTERIAL INFECTION *

BY ROBERTJ. ROANTREEt AND LOWELLA. RANTZ

(From the Department of Medical Microbiology and Department of Medicine, Stanford Uni-versity School of Medicine, Stanford, Calif.)

(Submitted for publication July 13, 1959; accepted August 12, 1959)

The potent heat-labile bactericidal property offresh mammalian defibrinated blood or serum invitro has been known since the studies of Nuttall(1) and Buchner (2) over 70 years ago. By1928, it had been shown that serum componentsvery like those of complement were necessary forthis action (3). Although a number of investi-gators in the fields of bacteriology and immunologyhave interested themselves in this bactericidalpower of fresh serum, little notice has been takenof it in clinical medicine, probably, as noted else-where (4), because it has been believed to be ofdoubtful effect in vivo.

Interest has been revived in the bactericidal ac-tivity of serum by the work of Pillemer, Wardlawand associates (5, 6). They described a normallyoccurring serum protein of large molecular weightwhich they named properdin. They attributed thebactericidal property of serum, in part at least,to the presence of properdin, which acted with thefour components of complement in the presenceof magnesium to kill the bacteria.

The findings of Wardlaw and Pillemer (6)confirmed those of Mackie and Finkelstein (7)that the bacterial strains sensitive to the effect ofserum are principally from species of gram-nega-tive bacilli and that the degree of sensitivity is acharacteristic of the strain rather than of the ge-nus. However, whereas Wardlaw and Pillemerhave described the action of the properdin systemas being nonspecific and unrelated to antibody,Mackie and Finkelstein presented data showingthat the absorption of a serum in the cold by asensitive strain resulted in the removal of the kill-ing effect only for that strain, leaving the effect

* This study was supported in part by a grant (No.H700) from the National Heart Institute of the NationalInstitutes of Health, Bethesda, Md.

t Formerly Bank of America-Giannini Foundation Fel-low, and presently recipient of the Lederle MedicalFaculty Award.

against other strains intact. The question as towhether the complement-dependent bactericidaleffect depends wholly upon a system includingproperdin or one including an antibody-like sub-stance, or whether it may depend partially on both,is not as yet decided.

The belief that the properdin system plays a rolein the native resistance of animals to bacterial in-fection is based upon the finding that an induceddecrease in the serum properdin level of labora-tory animals is associated with reduced resistanceto bacterial challenge, while increased properdinlevels correlate with increased resistance (8-10).

What relationship such changes in properdinlevel in vivo bear to the bactericidal effect of serumin vitro is unclear. Nearly all the work on pro-perdin levels and resistance to bacterial challengehas utilized the mouse, an animal whose serum isnonbactericidal in vitro owing to the lack of thesecond component of complement ( 11, 12). Stud-ies using human sera in vitro have indicated thatproperdin levels of 1 to 10 units per ml. of serumshow an equal and optimal activity, while in-creasing the properdin above this range leadsparadoxically to a lesser bacterticidal activity (6).Although properdin levels less than 1 unit per ml.have been reported in human disease, it has notbeen stated whether these levels altered the bac-tericidal power of the serum (13).

The present study was initiated to determinewhether a lessened bactericidal activity of undi-luted serum for gram-negative bacilli could bedemonstrated using sera from patients with serumprotein abnormalities or with diseases commonlyassociated with bacterial infections. It soon be-came clear that such a lack of bactericidal effectrarely, if ever, played a role, and that once thesensitivity of a given bacterial strain was deter-mined to any individual serum, it had a very simi-lar sensitivity to the great majority of other hu-man sera, whether from normal or pathological

72

BACTERICIDAL ACTIVITY OF HUMANSERUM

subjects. Thus all bacterial strains used could beclassified as to their degree of serum sensitivityThe problem as to whether strains resistant to thekilling effect of serum were more often culturedfrom the blood of bacteremic patients than mightbe expected was then investigated.

MATERIALS AND METHODS

Serum. The human blood specimens used in this studywere collected aseptically, allowed to stand at room tem-perature for about an hour, rimmed and centrifuged.The sera were frozen at - 200 C. in 6.5 ml. amounts.The whole procedure was completed within two hoursof the time of bleeding.

Culture medium. Peptic digest of liver broth was em-ployed as a medium for preparing frozen stock culturesand for the 20 hour cultures used for inoculation. Thisbroth was prepared by mixing 1,000 Gm. ground hog'sliver, 1,000 Gm. ground hog's stomach and 100 ml. ofconcentrated HCl in 10 L. of distilled water and digestingat 50° C. for 18 to 24 hours. This mixture was boiledfor five minutes and filtered through cotton. Four Gm.KHPO4, 3 Gm. glucose and 0.1 Gm. p-amino benzoic acid(PABA) were added per L. of this filtrate and the pHwas adjusted to 7.6 by the addition of 20 per cent NaOH.This 0.3 per cent glucose peptone-rich broth with aslightly alkaline pH includes the main requisites of amedium in which a bacterial strain can develop its maxi-mum resistance against the heat-labile bactericidal effectof normal serum (14, 15).

Bacteria. The bacterial strains used in the study, withthe exception of the standard Shigella dysenteriae, wereisolated from various human sources by the InfectiousDisease Laboratory of Stanford University Hospitals.They were taken from single colonies of the originalisolation, grown overnight in peptic digest of liver broth,and then this culture was divided into the desired num-ber of samples and frozen at - 20° C. The standardShigella dysenteriae was a laboratory strain agglutinatingspecifically with antiserum to its species. It was se-lected because previous workers had used this species(6) and because the sera of few patients in the San Fran-cisco area possess agglutinins against it. It was grownfor 24 hours in 150 ml. of peptic digest of liver broth.This culture was divided among 125 test tubes andfrozen at - 200 C. for use as starter culture for eachof the tests to be performed with it.

All strains used were of smooth colonial form andwere able to remain in uniform suspension in 0.85 percent saline at 370 C. for a 24 hour period.

The test for bactericidal activity of serum used through-out this study was set up as follows: A 24 hour brothculture of the chosen test organism was diluted in physio-logic saline solution by serial 10-fold dilutions to 10'.Then 0.1 ml. aliquots of each of the six dilutions, 101 to106, were distributed into three or four 10 X 1 cm. testtubes. This distribution resulted in a pattern of test

TABLE I

Results of two typical bactericidal tests

Panel of fresh active serafrom three human donors

Size of Heatedinoculum* J G B serum

Klebsiella 321.6 X 107 1+ 1+ 1+1.6X106 11 2 01.6 X 105 0 0 01.6 X 104 0 0 0 2+1.6 X 103 0 0 0 261.6X102 0 0 0 2

E. coli 342.8 X 107 4+ 4+ 4+2.8 X 106 1+ 12 102.8X105 5 2 22.8 X 104 0 0 0 2+2.8 X 103 0 0 0 282.8 X 102 0 0 0 0

* Determined bythe 10-61 dilution.

plate counts on duplicate aliquots of

tubes in the rack similar to the pattern of the results oftwo such tests recorded in Table I. Each of the freshlythawed test sera (10 minutes in a 190 C. water bath) wasdistributed in 1 ml. amounts through one row of theserially diluted inocula. In each test, a serum heated to560 C. for 20 minutes was tested with the 10' to 10' di-lutions as shown in Table I to serve as a control to in-dicate whether the killing in the test was indeed due to aheat-labile factor. Pour plate counts were done on dupli-cate 0.1 ml. amounts of the 10' dilution. From thesecounts the number of bacteria in each inoculum could becalculated. The rack of tubes containing the serum andbacteria was shaken well and placed in a 370 C. waterbath for two hours. Loop subcultures were made fromeach tube to a one-sixth section of a standard Petri dishcontaining nutrient agar. The same 2 mm. diameter loopwhich delivered approximately 0.002 ml. of serum wasused throughout. Special care was taken to insert theloop to the center of the test tube bottom and with-draw it without touching the sides. If the section wascompletely covered by growth, the result was recordedas 4 +; if one quarter was covered, as 1 +; and if lessthan 50 colonies appeared, these were counted.

Only the tube with the greatest inoculum giving a ster-ile subculture was taken into account when the sensitivityof a particular bacterial strain was determined or thepotencies of the various sera were compared. For de-termining the sensitivity of a strain, the majority resultfrom the three or four sera tested was taken as the finalreading. For instance, in Table I it is estimated that therate of killing of Klebsiella 32 is 1.6 X 105 organisms perml. of serum per two hours even though one of the threesera shows a sterile subculture for an inoculum of 1.6 X106. As will be seen from the discussion of results, agree-ment among the rates of killing by various sera was goodand little difficulty was experienced in deciding upon the

73

ROBERTJ. ROANTREEANDLOWELLA. RANTZ

end point. For the purpose of classifying the sensitivityof the bacteria in those few tests in which a majority ofsera was not in agreement the test was rerun, and ineach case there was a majority agreement on the secondrun. In each test at least one of the sera includedwas from one of five healthy donors whose sera hadshown good agreement with each other in bactericidaleffect in preliminary tests. This normal serum was neverat more than a one tube variance with the majority.

Blood culture technique. Blood cultures from patientswere obtained by inoculating 10 ml. of freshly drawnblood into 100 ml. of peptic digest of liver broth. Thetime of transfer of blood from the vein to the broth didnot exceed one minute. Tests in vitro indicated thatinocula of from one to three bacteria of a sensitive strainremained viable after such treatment when amounts ofactive serum twice that contained in the blood werepresent.

RESULTS

I. A comparison of the bactericidal rates of varioushuman sera

Sera from 43 patients and 36 healthy adultswere tested by the above method. In every testthe heated serum control failed to kill the 1,000to 4,000 bacteria present in the 10-5 dilution of theseed culture. Often an inoculum of 100 to 400bacteria gave a positive subculture after two hours'incubation in the heated serum.

Except for three instances to be described be-low, all active sera whether from the healthydonors or patients showed a remarkable uniformityin the rate of killing any particular test strain.The clinical states of the 40 patients whose serum

TABLE II

Clinical state of patients from whomtest sera were obtained*

showed no defect in bactericidal power are listedin Table II.

Except for Case 1 (described below), everyserum showed the ability to kill the standard strainof Shigella dysenteriae at a rate of 1 X 107 to 1.5x 108 organisms per ml. of serum in two hours.Since these data were so uniform, they are not in-cluded in the tables.

Forty of the 43 sera from patients and all thesera from healthy donors were tested against atleast one other bacterial strain, and most weretested against more.

TABLE III

Degree of internal agreement in tests using panels of sera fromhealthy donors and in those using sera from healthy donors

and patients

Tests Tests utilizing normalutilizing and patient serum

only samplesnormalserum Nor- Pa-

samples Total mal tient

No. of samples tested 326 162 89 73No. of disagreements 65 36 14 22Disagreements more

bactericidal 29 17 5 12Disagreements less

bactericidal 36 19 9 10%Disagreement 20 22 17 30

No. of two tubedisagreements 6 5 1 4

Two tube disagreementsmore bactericidal 2 0 0 0

Two tube disagreementsless bactericidal 4 5 1 4

%Two tubedisagreements 2 3 1 5.5

Widespread cancer

Lymphomatosis andHodgkin's disease

Myelocytic leukemia

Lymphocyticleukemia

DiabetesPyelonephritis and

bacteremiaUremia, terminalNephrosisCirrhosisInfectious hepatitis

5 Lupus erythematosusWeber Christian's disease

43 Hypogammaglobulinemia,

adult transitory

34

Recurrent bacterialinfections

3 Idiopathic leukopenia2 Infectious mononucleosis2 Congestive heart failure2 Fever, unknown origin2 Anorexia nervosa

* Cases 1, 2 and 3 are excluded.

2 Table I indicates the type of result observed inthe great majority of tests. As may be noted, aone tube disagreement might well be expected asa matter of chance. The degree of internal agree-ment in tests using panels of sera obtained fromhealthy donors and that of tests using panels of

1 both patients' and normal sera is shown in TableIII. Data from the three cases to be describedhave not been included.

Of the 488 serum samples tested, 101 or 20.71 per cent showed a disagreement with the ma-

jority result. Only 11 or 2.3 per cent of the totalwere two tube disagreements; none was greater.None of the serum samples showed a consistent

74

BACTERICIDAL ACTIVITY OF HUMANSERUM

difference from the majority result when testedwith different test strains. When retested againstthe same strain, the difference was not usuallyreproducible.

Although the results from tests using sera frompatients showed a slightly greater percentage oftotal differences from the majority result than didthose for tests in which normal sera were em-ployed, it was not a significant difference. Fur-thermore, the variances that did occur show an al-most equal distribution of those manifesting moreor less bactericidal activity. There are too fewtwo tube variations to attach significance to thegreater number occurring using patient sera.

In three cases the patients' sera were definitelyless bactericidal than normal sera, but only inCase 1 was this deficiency a general one. InCases 2 and 3, it was a defect specific for the or-ganism isolated from the patient's own bloodstream which was resistant to the patient's serumbut not to other human sera.

Case 1. E. C., a 62 year old white man, hadplasma cell leukemia diagnosed at autopsy. Hewas uremic because of leukemic involvement ofthe kidneys. His history was not noteworthyfor frequent infections. Serum electrophoresisshowed a typical myeloma peak in the y-globulinregion and the albumin: globulin ratio was 2.0:7.5. The only infection recognized clinically dur-ing his month long stay was a pneumococcal lobarpneumonia with bacteremia controlled promptlyby penicillin. At death, both lower lobes showedconsolidation. Coccal and bacillary forms werenoted microscopically.

His serum, drawn before the lobar pneumoniaoccurred, was tested three times with the standardstrain of Shigella and once each against one sensi-tive Klebsiella and two moderately sensitive strainsof Proteus. Against none of these strains was abactericidal effect demonstrated. This serum wasfound to be anticomplementary.

Case 2. 0. B., a 67 year old white woman, wasadmitted with a history of low grade fever and arecurring urinary tract infection for four months.Despite antimicrobial therapy, Escherichia coli ofidentical antibiotic sensitivity patterns grew fromthree blood cultures over a span of 23 days. Atlaparotomy six weeks after entry, an adrenal tumorwith an abscess containing E. coli was removed.

E. coli from two of the blood cultures were

SERUMSENSITIVITY OF E. COLI STRAINS

. IXIO 7_

?elx 6 -

4 IX1O5

%;

4.1104

k 1X103

'4'

IIXo 23

1x0O

'/0ood Stoo/

Sensitive

4--

ModeratelySensit ive

Resistant

SOURCEOF BACTERIAL STRAIN

FIG. 1. COMPARISONOF THE DEGREESOF SENSITIVITYTO SERUMBACTERICIDAL EFFECT OF STRAINS OF E. COLIISOLATED FROMTHE BLOOD, STOOL AND URINE

tested with seven normal human sera and were

found to be moderately sensitive, as shown inFigure 1 where the black circle labeled 3 depictsone of the strains. The upper circle representsthe sensitivity of this strain as tested against theseven sera from other donors. The lower circleat the tip of the arrow is the level of bactericidaleffect of the patient's own serum. This latter re-

sult was obtained from three separate tests on

serum samples obtained within several days of thepositive blood cultures. Thus, serum other thanthe patient's could kill this strain at a rate of2 x 105 bacteria per ml. of serum in two hourswhile her serum could kill only 200, or in realitymay have had no bactericidal effect at all sincesuch a low count would not always be detected bythe method used. This patient's serum was, how-ever, fully effective against three other strains ofE. coli, the standard strain of Shigella and one

strain of Proteus.Three months later, the patient was readmitted

because of recurrent carcinoma and a staphylo-coccal subdiaphragmatic abscess. At this time herserum, as tested three times, was just as bacteri-cidal for Strain 3 from her previous bacteremia as

were the sera from the normal donors.

0 0.0 0

0 0.0 0

0l0.0 0.. .

.0 ~~0.

0

.0 00

00 0

-

4

L

75

ROBERTJ. ROANTREEAND LOWELLA. RANTZ

SERUMSENSITIVITY OF VARIOUSGRAM-NEGATIVE ENTERIC BACTERIA

tyln 7-A. IAIV

CW IXIO IL

w~o ~-

N. ~

".4

3 IXlO -~

14J

I. Ixlo5

Ixi

EIX10 I2_

BloodSOURCEOF

Sloo/

4

Sensitive

Modcratel 9Sensitiven

Rcsistan t

BACTERIAL STRAIN

A Proteus 0 SalreorielloaA Pssuckomonas 0 Si63cllo.

x Paracolon

4 KleqbsielIoa0 Acro bcier

FIG. 2. COMPARISONOF THE DEGREESOF SENSITIVITYTO SERUMBACTERICIDAL EFFECT OF STRAINS OF ENTERIcBACILLI OTHERTHANE. COLI ISOLATED FROMTHE BLOOD,STOOLANDURINE

Case 3. A. D., a 47 year old white woman, was

admitted because of cirrhosis and right pyelo-nephritis. At entry a strain of "paracolon" bacil-lus was isolated from the urine and blood. Shewas treated with tetracycline and chloramphenicol.Her fever subsided after 17 days of treatment.Twenty-two days after entry, an antibiotic-freeserum was drawn to test its bactericidal qualities.

The X which is labeled 45 in Figure 2 illustratesa situation analogous to that in Case 2. The serum

of three normal donors killed at the rate of 4 x105 and two at a rate of 4 x 106 organisms per ml.of serum in two hours, whereas the patient's own

serum was able to kill only 400 organisms, a num-

ber barely detectable by this technique. The pa-

tient's serum was fully as effective as normal se-

rum against the test strain of Shigella and sensitivestrains of Klebsiella and E. coli.

In addition to Cases 2 and 3 described above,antibiotic-free sera from five other patients withgram-negative bacillary bacteremia were avail-able for study. Their sera were fully active againstsensitive and moderately sensitive test strains.

TABLE IV

Disease states associated with bacteremia from which bacterialstrains studied were obtained

No. No.of of

cases cases

E. coli SalmonellaPyelonephritis 6 Disseminated lupus

erythematosus 1Carcinoma 2 Lymphocytic leukemia 1Lymphoma 2 Unknown (stool negative) 1Lymphocytic leukemia 1Acute yellow atrophy, 1 Paraolon group

liver Agranulocytosis 1Acute cholecystitis (?) Cirrhosis of liver

KlebsiellaLymphoblastic Pseudomonas

lymphosarcoma 1 Lymphocytic leukemia IAerobacter Proteus

Multiple myeloma 1 Pyelonephritis 2

However, the strains obtained from their bloodwere resistant to normal serum as well as to theirown and thus were not suitable for detecting sucha phenomenon as described in Cases 2 and 3.

Thus, in this investigation of differences in theability of sera from different individuals to killgram-negative bacteria, only three instances ofmajor differences were found. In one of theseit was a lack of killing effect for all test strains andwas probably caused by the anticomplementaryaction of the serum. The other two instancesshowed a defect specific for the organism withwhich they were infected and the possible explana-tion for this phenomenon will be discussed later.

II. A comparison of the sensitivity to serum ofbacteria isolated from the blood with those iso-lated from the stool or urine

Throughout the preliminary stages of the study,it was found that the various test strains evi-denced great differences in their sensitivity to thecomplement-dependent bactericidal effect. Oncethe sensitivity of a particular strain to one serum

was determined, it could be predicted that itwould have nearly the same sensitivity to the greatmajority of other human sera.

It seemed then that a comparison of the sensi-tivities to serum of bacillary strains isolated fromthe blood of bacteremic patients to those of strainsisolated from the stool or urine might throw some

light upon whether the complement-dependentbactericidal effect played any role in native im-munity.

A 00* A

-4;

00

0~ 0

A @

A 0

AA A K

76

I-

BACTERICIDAL ACTIVITY OF HUMANSERUM

Table IV shows the type of disease associatedwith the bacteremias investigated. The majorityof organisms from the stool came from routinecultures and those from the urine were isolatedfrom consecutive positive cultures with no attemptmade to distinguish pyelonephritis from cystitis.Neither stool nor urine cultures were from cases

with known bacteremia and care was taken to besure that each specimen came from a different indi-vidual.

Figures 1 and 2 contain all the data on theserum sensitivities of the strains of gram-negativebacilli investigated. Three arbitrary zones ofsensitivity have been defined, and these form thebasis for the categories presented in Table V.

Figure 1 compares the serum sensitivities ofstrains of E. coli isolated from the blood to thoseof strains from stool and urine cultures. (Strain3 has been discussed with Case 2 and will not be

included in the interpretation of these data.) Itwill be noted that 10 of the 12 strains isolated fromthe blood are in the resistant category. Four of19 strains from the stool, and four of 16 strainsfrom the urine are also resistant. If the Wilcoxon2 sample test (16) is used to analyze the datapresented in Figure 1, the probability of such a

distribution's happening by chance is 0.1 per cent.Figure 2 presents the data on bacterial strains

other than E. coli. Organism 45 has been dis-cussed in Case 3 and will not be included in theinterpretation of these data.

As may be noted from Table V, the various bac-terial genera differ markedly as to the percentageof strains in any particular category of sensitivity.The Shigella and Klebsiella groups show a higherpercentage of sensitive strains than do the otherswhereas there is a higher percentage of resistantstrains of Salmonella.

TABLE V

Comparison of the degree of sensitivity of bacterial strainsisolated from the blood, stool or urine

Blood Stool Urine Blood Stool Urine

E. coli AerobacterSensitive 1 5 4 Sensitive 0 0 0Moderately Moderately

sensitive 1 10 8 sensitive 0 1 0Resistant 10 4 4 Resistant 1 1 0Total 12 19 16 Total 1 2 0

Salmonella ProteusSensitive 0 0 0 Sensitive 0 1 1Moderately Moderately

sensitive 1 4 0 sensitive 0 0 0Resistant 2 8 0 Resistant 2 2 0Total 3 12 0 Total 2 3 1

Shigella PseudomonasSensitive 0 5 0 Sensitive 0 0 0Moderately Moderately

sensitive 0 2 0 sensitive 0 2 1Resistant 0 2 0 Resistant 1 2 1

Total 0 9 0 Total 1 4 2

Klebsiella Paracolon groupSensitive 0 3 5 Sensitive 0 0 0Moderately Moderately

sensitive 1 0 3 sensitive 0 1 1Resistant 0 0 0 Resistant 1 2 2

Total 1 3 8 Total 1 3 3

77

ROBERTJ. ROANTREEANDLOWELLA. RANTZ

No strain of Shigella from a bacteremia wasstudied. The percentage of resistant strains ofSalmonella isolated from the blood compared tothat from the stool was exactly the same. Of theremaining five bacterial groups studied, thosestrains isolated from the blood showed a higherproportion of resistant strains than those from thestool or urine. Such results agree with the find-ings from the more extensive study on the strainsof E. coli.

Considering all the bacteria studied, and ex-cluding the strains of Cases 2 and 3, one finds thatthere were 21 strains of gram-negative bacilli iso-lated from the blood. Of these, four were sensi-tive or moderately sensitive. The two such strainsof E. coli in Figure 1 came from a case of lympho-cytic leukemia and one of diabetes with pyelo-nephritis. Antibiotic-free serum from neither pa-tient was available for testing. Of the strains ofmoderate sensitivity depicted in Figure 2, the Sal-monella came from a case of lymphocytic leukemiawhose serum was not obtained. The strain ofKlebsiella was cultured on two different occasionsfrom a case of lymphoblastic sarcoma. This pa-tient's serum was studied and proved to be just asbactericidal for this strain of Klebsiella as was nor-mal serum. His serum was also normally activeagainst two other test organisms.

DISCUSSION

This study was initiated to determine whethermajor differences in the heat-labile bactericidaleffect of individual human sera can be demon-strated and, if such differences exist, whether theycan be correlated with an altered susceptibility tobacterial infection. The bactericidal effect wasmeasured by adding bacterial inocula containingvarying numbers of bacteria to undiluted serumand noting the surviving organisms after twohours. This method was adopted rather than oneusing a standard inoculum and varying dilutionsof serum because it seemed to simulate moreclosely the natural conditions under which an in-fection, and particularly a bacteremia, might occur.Furthermore, since a deficiency of any one of atleast six different components will limit the ac-tivity of a serum (6, 17), a result from a dilutedserum may not reflect the activity of the wholeserum. Although the technique employed would

not detect minor dissimilarities among sera fromvarious individuals, it seemed evident early thatsmall differences should play little part in affect-ing native resistance when there were such ob-vious, large variations among bacterial strains intheir sensitivity to the serum.

Except for the three cases described, all sera,whether from normal donors or from patients withdiseases in which a serum defect might be sus-pected to be present, killed any given sensitive bac-terial strain at a remarkably uniform rate. Allsera were consistently ineffective against resistantstrains. Whether the anticomplementary effect ofsome hyperglobulinemic sera such as that de-scribed in Case 1 has any relationship to decreasedresistance to infection is unknown. This anti-complementary quality certainly has a profoundeffect on the bactericidal system in vitro, allowingeven the most sensitive strains to survive. Thatthe anticomplementary activity of the sera of pa-tients with multiple myeloma interferes with thebactericidal effect in vitro has been noted before(6).

In both Cases 2 and 3, sera from patients withprolonged bacteremia lacked bactericidal activityagainst their own infecting strains but possessedfull potency against other test strains. If thisfinding were caused by a lack of a specific anti-body, it would seem that such a deficiency wouldhave appeared occasionally among the sera fromthe 76 other individuals tested. It seems morelikely that this phenomenon is caused by antibodyinterfering with the bactericidal system (18) andis related to the Neisser-Wechsberg effect (19).The latter describes the inability of a serum con-taining high concentrations of antibody againsta gram-negative bacillus to kill that strain in thepresence of complement although the bactericidaleffect can be demonstrated if the antiserum is di-luted. In the cases described, the sera from bothpatients were tested at times when antibody inlarge concentrations should have appeared. Theagglutinin titers of their sera were not tested be-cause the possibility of antibody interference hadnot occurred to us. A situation like that describedhas been reproduced in the rabbit. As this ani-mal is immunized with a strain of E. coli, the un-diluted serum loses its normal bactericidal effectfor the immunizing strain but retains its activity

78

BACTERICIDAL ACTIVITY OF HUMANSERUM

for other test organisms (20). What role, if any,

this in vitro finding plays in native immunity isuncertain.

With the exception of the three cases describedabove, the sera from individual patients and nor-

mal donors were very much alike in their abilityto kill any given sensitive test strain. When a

strain was resistant to one serum, it was resistantto all the sera tested. In all, antibiotic-free sera

from six cases of bacteremia other than Cases 2and 3 were available for study. All were effectiveagainst sensitive and moderately sensitive teststrains. In five cases, a lack of effect against theinfecting strains, even if present, could not be dem-onstrated because these strains were resistant toserum. In the other case, the patient's serum was

fully active against the moderately sensitive strainof Klebsiella causing the infection as well as

against other test strains. Such an occurrence

might have several explanations. Perhaps thesensitive organisms were being constantly fed intothe blood stream from a protected infected focus.They may have been sheltered by an intracellularlocation within phagocytes. The bactericidal sys-

tem may not be nearly as effective in vivo as

in vitro.On the basis of the evidence presented, it would

appear that changes in the bactericidal potency ofundiluted serum as measured in vitro rarely takeplace even in the serum of patients with bacteremiaor with diseases often associated with infection.

On the other hand, the finding that the greatmajority of the enteric bacillary strains causingbacteremia were resistant to killing by serum indi-cates that the system may be of importance as a

factor limiting the number of strains capable ofinvading or persisting in the blood stream. Sincethe enteric strains causing bacteremia ordinarilygain entrance from either the intestine or urinarytract, the data showing that the majority ofstrains isolated from these sources are moderatelyor highly sensitive suggest that there is a limita-tion of the number of strains capable of causingbacteremia. The finding of one instance in whichbacteremia of at least two days' duration was

caused by a strain moderately sensitive to the pa-

tient's own serum indicates that the limitation isnot absolute.

The percentages of resistant and of sensitivestrains isolated from the urine were not signifi-cantly different from those isolated from the stool.This finding supports the belief that there is ac-tually a difference between strains isolated fromthese two sources and those from the blood, andis compatible with the belief that the kidney isnot ordinarily infected via the blood stream.

From the results of this study, one might hy-pothesize that one characteristic of a systemicallypathogenic enteric bacillary strain would be its re-sistance to killing by serum. There are few stud-ies to confirm this theory, but those which havebeen reported are in agreement with it. Jensenwas able to derive a strain of Salmonella typhi-murium nonpathogenic for the mouse from a pa-thogenic strain by passing it in vitro at graduallyincreasing temperatures of incubation for 206 pas-sages. This strain had the same antigenic quali-ties and fermentative characteristics as the parentstrain (21). Maaloe showed the parent strain tobe resistant to, and the derived strain sensitive to,the complement-dependent serum factor (14).He showed the two strains to be equally toxic andinfective for the mouse, but the apathogenic variantappeared unable to multiply and spread. Rowleycompared the pathogenicity for mice of strains ofE. coli sensitive to active guinea pig serum andthose resistant. He found that the resistant strainswere in each instance the more pathogenic (22).In a recent study it has been found that 18 of 19strains of gram-negative bacilli isolated from burnswere resistant to the bactericidal action of normal,active serum (23).

Obviously, the property of resistance to the bac-tericidal effect of serum does not alone impartpathogenicity to a bacterial strain. However, oncegiven the opportunity of access to the blood stream,the survival of any particular bacterium and itsability to spread may well depend upon such a qual-ity.

Although, as noted above, a more sensitive strainmay be derived from a resistant one by artificialmethods and sensitivity can vary with the culturemedium used to prepare the inoculum (14, 15), wewere impressed by the stability of the quality ofsensitivity to serum as a characteristic of a strainif the same medium and cultural conditions were

79

ROBERTJ. ROANTREEAND LOWELLA. RANTZ

employed. Our attempts to derive a strain sig-nificantly more resistant to killing by serum byserial passage through active human serum havethus far met with no success.

It may be noted from Table V and Figure 2that a high proportion of the strains of Shigellaare sensitive, whereas a high proportion of strainsof Salmonella are resistant. If, as our results in-dicate, resistance to serum is of importance to astrain's ability to cause bacteremia, this distribu-tion may be related to the frequency of cases ofbacteremia cause by strains of Salmonella and therarity of such cases caused by Shigella.

SUMMARY

An investigation of the heat-labile bactericidalproperties of undiluted sera from 36 healthy do-nors and 43 patients with diseases often associatedwith infection has been conducted. With the ex-ception of the sera from three patients, no majordifference in bactericidal ability was found amongthe various individual sera. Two of these threesera showed a specific inability to kill the orga-nism which was cultured from the patient's ownblood. It is hypothesized that the inability of thepatient's own serum to kill the infecting bacterialstrain is due to interference by antibody with thenormal bactericidal effect. The third serum wasineffective against any test strain of bacteria em-ployed because it was anticomplementary.

In studying the sensitivities of strains of gram-negative enteric bacilli to the heat-labile bacteri-cidal quality of blood, it was found that a muchhigher proportion of bacterial strains isolated fromthe blood, than of those isolated from the stool or

urine, was resistant to killing by serum. The hy-pothesis is presented that the main role of theheat-labile bactericidal system in native resistanceto infection is to prevent many strains from in-vading and persisting in the blood, and thatchanges in this bactericidal property are infrequentand of less importance.

ACKNOWLEDGMENT

The authors thank Dr. Lincoln E. Moses for his aidin statistical analysis.

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