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The a 1a Approved EXTRACTION OF LBUCOCIDBT FROM STAPHYLOCOCCI AND ITS PRACTICAL APPLICATION 3N THE DIAGNOSIS OF SEPTICEMIA BY WILLIAM A. SAUSER A THESIS Submitted to the Faculty of the Graduate School of the Creighton University in Partial Fulfillment of the Requirements for the Degree of Master of Science in the Department of Microbiology OMAHA, 1958 TAHUS 0 F CONTENTS Page LIST OF ILLUSTRATIONS......................................................... iv I . INTRODUCTION............................................................ 1 II . REVIEW OF LITERATURE............................................ 3 P h a g o c y t o s is ......................................................... 3 Leucocidin ............................................................. 7 I I I . TECHNIQUE.................................................................... 14 Selection o f O rga n ism s..................................... 14 Extraction of Leucocidin Titre ...................... 15 Determination of a Leucocidin Titre .... 19 IV. RESULTS........................................................................ 25 V. CONCLUSION................................................................. 28 VI. SUMMARY......................................................................... 30 REFERENCES............................................................................. 31 i i i LIST OF ILLUSTRATIONS Figure Page 1. F illin g Jar with Gas M ix tu re............................. 17 2. Average Leucoeidin Titre ..................................... 18 3. Leucoeidin Titre per Organisms ........................ 21 4. Percentage of Positive and Negative Blood C u ltu r e s ..................................................................... 25 5. Genua of Organisms I s o l a t e d ............................ 26 iv I HTRODUCTIOH Since the c la s s ic a l work o f Metclmikoff ( l ) in 1895 the importance of the cellular defense mechanism has been recognized. The great number of investigations on the various aspects of the subject of phagocytosis that appear in the worlds literature serve as convincing evi­ dence for recognition of the necessity for a thorough un­ derstanding of this phenomenon. It is the purpose of this paper to investigate a small but interesting facet of this process of phagocytosis, that is the inhibition or depres­ sion of the mechanism by destruction of polymorphonuclear leukocytes; destruction due to a specific exotoxin elabo­ rated by certain bacteria. In addition an attempt has been made to utilize this process of phagocytic inhibition in a practical manner in the diagnosis of septicemia and bac­ teremia. The problem of continuing phagocytosis after asep­ tic transfer of whole blood to a suitable liquid media has been a probable source of false negative blood culture re­ sults. This idea is due to the fairly high percentage of suspected septicemia patients who have given negative re­ sults with many blood cultures. It also brings to mind 2 the possibility that many so-called 11 normal'• individuals may possess a temporary undetected bacteremia. II REVIEW OF LITERATURE Phagocytosis The mechanism of phagocytosis itself has never "been clearly understood. Mudd (2) following the formula­ tion of Fenn (3) states that phagocytosis is the result of an interplay of surface forces which may be considered as surface tension or surface energy. Phagocytosis may be defined as the ingestion of a particle by a living cell. The particle may be any part of non-living matter, or may be a bacteria or other cell. This mechanism of phagocytosis is known to continue »in vitro" as well as »in vivo." This is especially useful in studies on phagocytic ability of various individuals in determination of opsonic index. More important from our point of view is the in vitro ingestion of organisms after placing a volume of blood in a broth media in an at­ tempt to cultivate bacterial growth. J. de Haan (4) found that polymorphonuclear leuko­ cytes maintain their viability for as long as seven days in undiluted homologous blood serum. In Ringerfe solution and similar physiological fluids the leukocytes lose their phagocytic activities and degenerate within one to two days. Cross (5) observed that polymorphonuclear leukocytes 4 may preserve their phagocytic activity in the body for as long ae eleven days. One must take into consideration also the phago­ cytosis-promoting substances in the serum generally known as opsonins. The ability found in opsonins to increase phagocytic activity is well known and is considered to be most efficient. These substances are known to affect pri­ marily the cells to be phagocytized in such a way as to favor ingestion. The experiments of Mudd (6) have shown the effects of opsonins upon the particles to be ingested and the correlated increase in phagocytosis are both due to the deposition of serum proteins upon the particle. Thus the relationships at the interface between bacterial cell and phagocyte and cell and suspending medium are more favorable fo r the spreading of the phagocyte over the c e l l . A ll of the review above, taken together with many other considerations cited in the original papers, lead to the conclusion that specific precipitation, agglutination, and the surface phagocytosis-promoting effects of serum sen sitization are a ll consequences of one and the same antibody. The method of antibody reaction is governed ac­ cording to the state it is in. This concept is known as the “Unitarian hypotheses.“ These sensitizing opsonic properties are contained in the globulin fractions of im­ mune sera and appear to be globulins with specific 5 differences from normal serum globulin. The tissues and blood stream are from time to time invaded by bacteria of the most diversified character. The difficulty of dealing with such dissimilar parasites finds at least partially adequate solution by the mechan­ ism which makes them sim ilar with respect to the surface properties upon which phagocytosis depends. This simi­ larity is approached in the normal blood stream by the nonspecific absorption of serum proteins. It is more ade­ quately approached in the blood stream containing homolo­ gous antibodies through the specific combination of these antibodies with substances on the bacterial surfaces. The adequacy of this phagocytosis-promoting mecha­ nism depends obviously upon the concentration of phagocy­ tes is -promot in g substances in the serum and upon their af­ fin it ie s fo r the components of the bacterial su rfaces. It also depends, however, upon the intrinsic properties of the bacterial surfaces themselves. Thus, certain bacterium are phagocytized even in absence of serum; the partial coating with serum proteins brought about by nonaal serum suffices to cause phagocytosis of others ; whereas certain virulent and esp ecially encapsulated organisms are phago­ cytized only after interaction with specific immune serum. The next step after initial ingestion of an organ­ ism in the continuing process of phagocytosis can be either 6 destruction of the bacteria by the phagocyte, or continued m ultiplication of the bacteria inside the phagocyte with eventual disruption of the same, thereby releasing viable bacterial cells. It is with the first alternative that we are concerned. It must be kept in mind that any organism capable of producing a diseased state will most likely be more re­ sistant to in vitro phagocytosis. However, by destroying the phagocytes without harming the bacteria i t s e lf nor changing the surrounding growth conditions it would seem that the organism would have a much better opportunity for survival and more rapid growth rate. There are many substances to be considered fo r phagocytic inhibition. Mudd (2) reports cells exposed to sodium iodide were rendered practically incapable of pha­ gocytosis although they became normally phagocytic when placed in sodium bromide. This is an especially interest­ ing result as bromide is not normally present in high con­ centrations in the c e l l . It would appear that the depress ing effect of the iodine anion on phagocytosis does not de pend on loss of ions proper to the cell and that its ef­ fect is reversible. A number of other observers have reported on the e ffe cts of various s a lt s . Hektoen and Ruediger (7) added salts to mixtures of defibrinated blood and bacteria. 7 Phagocytosis was depressed by a ll salts other than sodium chloride in the concentration used. Preliminary treatment o f the leukocytes or the bacteria with the sa lts did not produce this inhibition of phagocytosis; addition of the salts to the defibrinated blood before adding the bacteria did inhibit subsequent phagocytosis. The conclusion drawn from these works was that the salts interfered with the opsonization of the bacteria. Despite the results obtained by Hektoen and Ruediger the use o f sa lts was not considered in this work because first of all the addition of these salts may have an adverse effect on the bacteria itself; and secondly, be­ cause the sa lts had to be added to the blood prior to the addition of the bacteria. Leucocidin Leucocidin, a naturally occurring substance, ful­ fills the two criteria of destroying the phagocyte itself and not altering the surrounding growth conditions. Leu­ cocidin was the first toxic substance of bacteria to be identified and studied. It contributes to pathogenicity by its destructive action on leukocytes. Leucocidin is produced in varying amounts by the majority of pathogenic staphylococci, particularly those which are actively ca­ pable of

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