Studies on the Virulence of Neisseria gonorrhoeae I. Relation of Colonial Morphology and Resistance to Phagocytosis by Polymorphonuclear Leukocytes

Colonial varieties of Neisseria gonorrhoeae that are associated with virulence, types 1 and 2, were more resistant to phagocytosis by rabbit exudative polymorphonuclear leukocytes than colonial types of lesser virulence, types 3 and 4. Type 1 bacteria were resistant and type 4 gonococci were susceptible to phagocytosis by human polymorphonuclear leukocytes. Recent local type 1 isolates were similar in resistance to type 1 organisms of a standard laboratory strain (F62). Living and Formalin-treated, heat-killed, type 1 gonococci were equally resistant to phagocytosis. The antiphagocytic property of virulent colonial types was independent of leukotoxic action. Phagocytosis of both type 1 and type 4 gonococci by rabbit and human leukocytes was bactericidal. Rabbit leukocytes were superior to human leukocytes in killing gonococci. The results suggest that N. gonorrhoeae has virulence properties similar to those of extracellular bacterial pathogens, i.e., virulence is associated with antiphagocytic properties.     

Phagocytic killing of Neisseria gonorrhoeae by human monocytes

The ability of human monocytes to phagocytize and kill nonpiliated opaque (T3) and transparent (T4) gonococci was investigated in a tumbling tube suspension assay. A serum-sensitive strain, F62, and a serum-resistant strain, FA19, were studied. CFU remaining after incubation with monocytes were used to assess the extent of killing. The data show that 50% of T3 and T4 gonococci of both strains were killed by monocytes over a 2-h period. Serum was necessary for the killing of transparent gonococci of both strains as well as for FA19 T3. Concentrations of serum ranging from 0.5 to 10% were equally effective, and heat-labile components were required. Killing of F62 T3, however, occurred in the absence of serum. An increased ratio of bacteria to monocytes decreased the rate of killing. A 30-min preopsonization of gonococci in 10% serum resulted in an enhanced rate of killing. Monocytes were able to kill plate-grown, but not log-phase, organisms. Disruption of the monocytes by sonication to release internalized bacteria did not increase the number of viable organisms. The addition of 10 micrograms of cytochalasin B per ml completely inhibited the reduction in colony numbers over time. These data indicate that freshly isolated human monocytes are capable of phagocytizing and killing nonpiliated gonococci.     

Gonococci-human polymorphonuclear leukocyte interactions: metabolic studies associated with attachment and ingestion

Utilizing monolayers of human polymorphonuclear leukocytes, optimal conditions for attachment and ingestion of Neisseria gonorrhoeae were determined. Both attachment and ingestion were optimal at 36 degrees C when a bacteria-leukocyte ratio of 100:1 was employed. After 30 min of incubation, log-phase viable type 2 gonococci were attached to 90% of leukocytes, whereas log-phase viable type 4 gonococci were ingested by 80 to 90% of cells. Respiratory inhibitors had no effect on attachment or ingestion, whereas glycolytic inhibitors blocked ingestion but did not affect attachment of gonocci to the leukocyte surface. Inhibition was dose dependent and partially reversible. The oxidative metabolism of leukocytes with gonococci attached or ingested was also examined. Attachment of log-phase type 2 gonococci stimulated a minimal increase in glucose oxidation and oxygen consumption by leukocytes in contrast to marked increases by leukocytes that had ingested viable type 4 or heat-killed typed 2 organisms. These results demonstrate that attachment of log-phase type 2 gonococci to the surface membrane does not stimulate significant leukocyte oxidative metabolism nor initiate the phagocytic process.     

Interactions of Neisseria gonorrhoeae with Human Neutrophils: Effects of Serum and Gonococcal Opacity on Phagocyte Killing and Chemiluminescence

Serum-sensitive strains of Neisseria gonorrhoeae were incubated with suspensions of normal or chronic granulomatous disease human neutrophils in the absence or presence of fresh or heat-inactivated human serum; phagocytosis, gonococcal viability, and chemiluminescence were measured. Nonpiliated opaque or transparent gonococci (colony types 3 and 4, respectively) were used for phagocytic bactericidal assays. In the presence of 2.0% fresh human serum, normal neutrophils killed >90% of types 3 and 4 gonococci by 135 min. Serum alone at this concentration was not bactericidal. In the absence of serum, type 4 gonococci were not killed, whereas type 3 gonococci were killed to the same degree as in the presence of serum. Interestingly, heat-inactivated normal serum slightly inhibited phagocytic killing of type 3 gonococci. Results almost identical to those above were obtained when 5% fresh human serum deficient in complement component 7 was substituted for 2% normal autologous serum. This indicated that the later components of complement were not involved in the observed results. To investigate the mechanisms responsible for the intracellular killing of the gonococci, we used neutrophils from patients with chronic granulomatous disease. These neutrophils are deficient in an activable NADPH oxidase and do not produce bactericidal oxygen products upon phagocytic stimulation. Neutrophils from two unrelated boys with chronic granulomatous disease killed type 3 and 4 gonococci to the same degree as did normal neutrophils. As with normal neutrophils, serum was needed for killing type 4 organisms. As expected, neutrophils from these patients showed absolutely no increased chemiluminescence in the presence of type 3 or 4 gonococci, with or without serum. The effects of serum on gonococcus-induced chemiluminescence by normal neutrophils was also investigated. For these studies, in addition to type 3 and 4 gonococci, we also used transparent colony types of lightly (type 1) and heavily (type 2) piliated organisms. Chemiluminescence induced by type 1, 2, or 3 gonococci (i.e., gonococci possessing either pili or opacity-associated proteins, but not both) was augmented only slightly by serum and then only at low ratios of gonococci to neutrophils. On the other hand, chemiluminescence induced by type 4 gonococci (i.e., gonococci possessing neither pili nor opacity-associated proteins) was substantially increased in the presence of serum. Stimulation of chemiluminescence by type 1, 2, 3, or 4 gonococci was dose dependent in the absence or presence of serum. Heat-killed type 3 gonococci induced chemiluminescence to the same degree as did viable organisms. Since the gonococci used in this research was strongly catalase positive, as are gonococci in general, and since it was killed by chronic granulomatous disease neutrophils, the results indicate that gonococci can be effectively killed within neutrophils, i.e., within phagolysosomes, by nonoxidative bactericidal mechanisms. Whereas type 3 gonococci were phagocytized and killed by neutrophils equally well with or without serum, serum was obligatory for phagocytic killing of type 4 gonococci, i.e., gonococci lacking opacity-associated proteins. In addition, either pili or opacity-associated proteins were apparently necessary for maximal stimulation of neutrophil chemiluminescence. The submaximal stimulation of chemiluminescence by gonococci lacking both pili and opacity-associated proteins, i.e., type 4 gonococci was augmented by low concentrations of nonimmune serum.     

Functional characterization of a sialyltransferase-deficient mutant of Neisseria gonorrhoeae.

Previous studies indicate that sialylation of lipopolysaccharide (LPS) by host CMP-N-acetylneuraminic acid (CMP-NANA) catalyzed by bacterial sialyltransferase rendered gonococci resistant to killing by phagocytes, to entry into epithelial cell lines, to killing by immune serum and complement, and to absorption of complement component C3. These results have been confirmed by comparing a sialyltransferase-deficient mutant (strain JB1) with its parent (strain F62) in appropriate tests. In contrast to F62, JB1 was very susceptible to killing by human polymorphonuclear phagocytes in opsonophagocytosis tests and incubation with CMP-NANA did not decrease the level of killing. The inherent resistance of F62 in these tests was probably due to LPS sialylation by CMP-NANA and lactate present in the phagocytes. A JB1 variant expressing the invasion-associated Opa protein was as able to enter Chang human conjunctiva epithelial cells as an Opa-positive variant of F62, suggesting that the sialyltransferase is not required for Opa-mediated entry. After incubation with CMP-NANA, the number of F62 variant gonococci entering cells but not that of JB1 variant gonococci was drastically reduced. Both JB1 and F62 were killed by incubation with rabbit antibody to gonococcal major outer membrane protein, protein I, and human complement, but only F62 was rendered resistant to the killing by incubation with CMP-NANA. Finally, both JB1 and F62 absorbed similar amounts of complement component C3 and the binding was decreased by incubation with CMP-NANA only for the wild type, F62.     

Sialylation of lipopolysaccharide and loss of absorption of bactericidal antibody during conversion of gonococci to serum resistance by cytidine 5'-monophospho-N-acetyl neuraminic acid

Changes in lipopolysaccharide (LPS) which occur when serum susceptible gonococci are converted to resistance by incubation with cytidine 5'-monophospho-N-acetyl neuraminic acid (CMP-NANA) have been investigated. Transfer of radioactivity to bacterial LPS from CMP-NANA labelled with 14C in the NANA moiety was detected by fluorography following lysis, proteinase K digestion and SDS-PAGE. Incorporation of radioactivity was inhibited by cytidine 5'-monophosphate (CMP). Both the radioactivity of the LPS and the resistance of gonococci to fresh human serum were largely lost after incubation with neuraminidase. No evidence was obtained to suggest that CMP-NANA is an inducer of new protein synthesis as well as a substrate for the sialylation of LPS. Little radioactivity was incorporated into components other than LPS. Sialylated, serum resistant gonococci were less able than serum susceptible gonococci to absorb the bactericidal activity of fresh human serum. Hence, we conclude that serum resistance conferred on gonococci by CMP-NANA is due to transfer of sialyl groups to surface LPS sites and this inhibits their reaction with bactericidal antibody in human serum.     

Studies on gonococcus infection. VII. In vitro killing of gonococci by human leukocytes.

The comparative killing of pilated and nonpilated forms of Neisseria gonorrhoeae by human peripheral blood leukocytes was studied in vitro. Some nonpilated gonococci (T2) were killed to a lesser extent than were pilated, T2 organisms, which were killed less readily than another nonpilated (T4*) form of gonococcus. Thus, the relative order of killing of gonococci by human peripheral blood leukocytes appears to be: T4 less than T2 less than T4*. These data suggest that pilation, though correlated with virulence of gonococci, has little influence on the survival or killing of these organisms by human leukocytes.     

Effects of human neutrophil granule extracts on macromolecular synthesis in Neisseria gonorrhoeae.

Neisseria gonorrhoeae were exponentially killed for 120 min (i.e., they were prevented from forming colonies on agar) by extracts of human neutrophil granules; however, macromolecular synthesis, indicated by incorporation of radiolabeled precursors in trichloroacetic acid-precipitable material, continued at or above zero time control values for 45 min. Protein, deoxyribonucleic acid, and ribonucleic acid synthesis appeared to decrease simultaneously after 45 min. Little or no lysis gonococci occurred during the first 60 min of incubation. The ions K+, Na+, Ca2+, Cl1-, SO4(2-) and PO4(3-) at concentrations of less than or equal to 100 mM did not affect granule extract bactericidal activity. On the other hand, 20 mM Mg2+ completely inhibited killing when initially present along with granule extract or when added within 2 to 5 min after granule extract was added to a suspension of gonococci. Gonococci treated with granule extract, washed, and then incubated in gonococci. Gonococci treated with granule extract, washed, and then incubated in the absence of extract died as if extract were still present. The ability of subinhibitory concentrations of actinomycin D or erythromycin to inhibit growth and protein and nucleic acid synthesis was synergistically increased in the presence of granule extract. The above information suggests that a bactericidal component(s) of human neutrophil granules sticks to gonococci, altering their outer membrane permeability and their ability to divide.     

Pathogenesis and Immunology of Experimental Gonococcal Infection: Virulence of Colony Types of Neisseria gonorrhoeae for Chicken Embryos

The virulence of gonococcal strains and colony types was evaluated in embryonated hen eggs of various ages inoculated by different routes. Striking differences in virulence of colony types were revealed by intravenous inoculation of 11-day embryos. T1 and T2 colony types were found to have high virulence for embryos, whereas T3 and T4 colony types were relatively avirulent. These observations are in accord with previous studies in volunteers. The differences in virulence were not related to differences in toxicity of killed gonococci, sonic lysates, or to the susceptibility of gonococci to cidal effects of chicken embryo blood. Rather, they appeared to involve differences in clearance of gonococci from the blood stream and subsequent multiplication of the virulent colony types. Infection with virulent colony types appears to be primarily bacteremic in this model. Preliminary experiments indicated that chicken embryos may be protected against the lethal infection by prior treatment of the inoculum with normal and immune rabbit serum. This protective effect was not associated with bactericidal activity. The chicken embryo model is potentially useful as a means of investigating attributes of virulence of gonococci and factors in immunity against gonorrhea.     

Phase Transition of Gonococci in Mammalian Cell Cultures

Neisseria gonorrhoeae was cultivated in mammalian cell cultures in an effort to determine if this environment will elicit a T4 --> T1 transition. Of four avirulent (T4) isolates tested, only one, H4, yielded T1 colonies. This change was consistently obtained in HeLa, WI-38, and MK2 cells, even when the multiplicity of the gonococcal infection was less than 1 per culture. Growth of the gonococci took place primarily on the surface of the cells, as demonstrated by light and electron microscopy, but occasional bacteria were undoubtedly intracellular. T1 colonies were seen at 24 h and were the major population at 48 h. This shift was favored by the presence of viable cells, since smaller yields of T1 were obtained when the cells were irradiated or heat inactivated. It was also favored by low pH, since T1 recovery was reduced when the buffering capacity of the medium was increased. Although the results suggest that T1 gonococci derived from H4 have a selective advantage over T4 in cell cultures, this is not true of all T1 and T4 colony types. F62 T4, which does not undergo a T4 --> T1 shift, propagated as well as T1 in HeLa cell cultures. The change in colony type of strain H4 to T1 was accompanied by formation of pili and by gain in capacity for deoxyribonucleic acid-mediated transformation. It is concluded that gonococci can undergo T4 --> T1 phase transition in mammalian cell cultures, but this property is not retained by all strains.     

Chemotaxis of human polymorphonuclear leukocytes toward Neisseria gonorrhoeae

Chemoattractive properties of Neisseria gonorrhoeae were studied by measuring leukocyte migration in agarose gel. Human serum albumin (0.5%) was present in the gel and normal human serum was excluded from all components of the assay. Viable cell populations and lysates of colonial types F62T1, F62T2 and F62T3 induced migration of polymorphonuclear leukocytes. Chemotactic activity of the lysate was not altered by heating at 100 degrees C for 10 min and was retained in the 12 100 g supernatant fraction of the heated lysate. Fractionation of the supernate by Sephadex G-100 chromatography showed that the chemotactic activity was associated primarily with an absorbance peak at 280 nm of relatively low mol. wt. The chemotactic activity of this fraction was lost after dialysis and the peak was no longer present in the Sephadex G-100 elution profile of the dialysed supernate. The gonococcal leukotaxins were sensitive to digestion by trypsin, pronase and amyloglucosidase, but insensitive to treatment with RNAase, DNAase or lipase at pH 5.7-7.1.     

Role of urinary solutes in natural immunity to gonorrhea.

Natural resistance of the male urethra to gonococci has not been explained by classical immune mechanisms but could result from antibacterial properties of urine. Accordingly, we measured survival in midmorning urine of 10(7) F-62 T2 gonococci per ml by serial dilutions and plate counts. Fifteen killer urines from eight people all killed greater than 3 logs (average, 5.3), and 13 of 15 were sterilized. Fourteen nonkiller (inhibitor) urines from seven subjects allowed no growth. Killer urines were more acidic (pH 5.4 versus 6.4) and more concentrated (861 versus 717 mosmol/kg) than nonkillers. Upon addition of hydrogen ion, urea, and sodium chloride to urines and broth, pH proved to be the major killing factor, but urea and NaCl were also bactericidal. Susceptibility to urine bactericidal power did not vary with colony type (T2 versus T4) or strain (F-62 versus two fresh isolates). Killing was rapid (0.5 to 3 h) and not bacteriolytic. Escherichia coli multiplied 10-fold in urines that inhibited growth of gonococci. Thus, the bacteriostatic effect of urine may explain why gonococci do not infect the bladder and kidney during gonorrhea. The bactericidal properties of urine may contribute to resistance against gonococcal urethritis.     

Neisseria gonorrhoeae LPS variation, serum resistance and its induction by cytidine 5'-monophospho-N-acetylneuraminic acid.

Inherent serum resistance and the effect of the serum resistance inducing factor cytidine 5'-monophospho-N-acetylneuraminic acid (CMP-NANA) were studied on Neisseria gonorrhoeae with different lipopolysaccharides (LPS). Strain M01 and LPS variants of strain Gc40 (variants D1, D2, D4 and D5) were examined after incubation in the presence or absence of CMP-NANA by bactericidal assays using normal human or immune sera and by SDS-PAGE followed by silver staining or Western blotting. The blots were probed with monoclonal antibody CC1, specific to epitope C of the LPS. Variants D1 and D5 were inherently serum resistant, variants D2 and D4 and strain M01 were susceptible. CMP-NANA induced marked changes in the LPS of all gonococci. However, only some gonococci were converted to serum resistance. Gonococci which were converted to serum resistance had LPS with components of relatively large molecular mass, expressing epitope C. Variants which did not convert to serum resistance had LPS with low molecular mass components only, without epitope C. Conversion to serum resistance increased the size of the large LPS components without affecting the expression of epitope C. The results indicate that conversion to serum resistance by CMP-NANA is not a general occurrence but depends on the quality of the LPS.     

Anaerobic growth of gonococci does not alter their Opa-mediated interactions with human neutrophils.

Gonococci grown anaerobically (anaerobic gonococci) in the presence of nitrite induce the expression of at least three novel outer membrane proteins (PANs 1 to 3). Although PAN 1 is expressed by gonococci during gonorrhea, the function of the PAN proteins remains unknown. In the absence of serum, gonococci possessing opacity-associated (Opa, formerly PII) outer membrane proteins adhere to, stimulate, and are phagocytically killed by human neutrophils. Gonococci lacking Opa proteins demonstrate none of these activities. We investigated whether the PAN proteins, or any other characteristics of anaerobic gonococci, altered the ability of nonpiliated, Opa+ or Opa- gonococci to adhere to, stimulate, or be phagocytically killed by neutrophils. The expression of Opa4 by strain F62, as determined by its relative mobility on sodium dodecyl sulfate-polyacrylamide gels, appeared to be unaltered by anaerobic growth, as seen previously (V. L. Clark, L. A. Campbell, D. A. Palermo, T. M. Evans, and K. W. Klimpel, Infect Immun. 55:1359-1364, 1987). Anaerobic and aerobic Opa+ gonococci adhered to and stimulated neutrophils to the same extent. Similarly, anaerobic and aerobic Opa- gonococci adhered to and stimulated neutrophils equally poorly. Finally, anaerobic and aerobic Opa+ gonococci were equally sensitive to phagocytic killing by neutrophils, while anaerobic and aerobic Opa- gonococci were equally resistant to killing. Thus, the role of Opa proteins in mediating the interactions of gonococci with human neutrophils appears unaltered by anaerobic growth, and the PAN proteins, or other cryptic properties of anaerobic gonococci, do not seem to modulate or mediate these phenomena.     

Opa (protein II) influences gonococcal organization in colonies, surface appearance, size and attachment to human fallopian tube tissues

Opa-expressing variants of Neisseria gonorrhoeae strain F62-SF and an Opa- variant, all non-piliated, were examined for differences in the interaction of the bacteria within colonies and in attachment to and damage of human fallopian tube mucosa. Expression of certain Opas was associated with the formation of transparent colonies where the bacteria were tightly packed and evenly spaced within the colonies. Expression of other Opas was associated with the formation of opaque colonies where the gonococci were less tightly packed and were unevenly spaced. Distinct differences in the size of the gonococci and in their surface characteristics were dependent upon the Opa being expressed. Certain Opas were associated with gonococci that had significantly larger cross-sectional areas and bigger perimeters. Scanning electron microscopy showed that OpaC- and OpaD-containing variants yielded greater mucosal damage than OpaB-containing and Opa- variants with the least damage caused by the OpaA-containing variant (clumped bacteria from dark opaque friable colonies). The mucosal damage after 60 min incubation included shortening and decreased numbers of microvilli on non-ciliated cells and invagination and sloughing of ciliated cells. Differences in the interactions of gonococci within colonies and in attachment to fallopian tube mucosa and damage to the mucosal cells occurred with different Opa-expressing variants of N. gonorrhoeae strain F62-SF.     

Gonococcal lipooligosaccharide sialylation prevents complement-dependent killing by immune sera.

Previous investigators have demonstrated that a sialic acid residue is added to the terminal galactose moiety of gonococcal lipooligosaccharide (LOS) when incubated with 5'-CMP-N-acetylneuraminic acid. When this in vitro sialylation occurs, gonococci become resistant to the bactericidal activity of normal human serum. This is believed to result because the added sialic acid residue blocks the binding of bactericidal anti-LOS antibodies present in normal human serum. We extend these studies by demonstrating that sialylated gonococci also become resistant to the bactericidal effect of immune sera containing antibodies that recognize exposed components of the outer membrane besides LOS. Prevention of antibody binding to the organism was not the cause, since the same percentage of bactericidal antibodies to the major outer membrane protein, Protein I, can be absorbed with sialylated organisms as with wild-type organisms. In addition, gonococcal sialylation prevents opsonophagocytosis by antigonococcal antisera. The negative effect of sialic acid on the complement pathway might be the reason for the findings in this study.     

Studies on Gonococcus Infection V. Observations on In Vitro Interactions of Gonococci and Human Neutrophils

The association of in vitro human leukocytes with pilated, type 2 Neisseria gonorrhoeae exceeds that for nonpilated, type 4 organisms but is less than that for nonpilated, type 4(*) gonococci. The two nonpilated forms of gonococci (types 4 and 4(*)) attach to tissue culture cells to a much lesser extent than do pilated, type 2 organisms of the same strain. Trypsin treatment of either pilated (type 2) or nonpilated (type 4(*)) gonococci markedly reduces the attachment-ingestion of these organisms with leukocytes, but the same trypsin treatment does not depilate the type 2 organisms nor visibly alter the morphology of their pili. Similar reductions in association with leukocytes are found if the gonococci are pretreated with chymotrypsin, heat, or glutaraldehyde. High levels of association between gonococci and leukocytes are reestablished if the trypsin or chymotrypsin-treated organisms are reincubated in protease-free medium. These data suggest that interactions between gonococci and human neutrophils are mediated through surface characteristics of the bacteria, different from those which influence attachment of the organisms to tissue culture cells. In the latter instance, pili appear to positively influence gonococcal attachment, whereas in the former a nonpilus bacterial cell wall nonpilus protein is probably the major determiner in the interaction between leukocytes and gonococci.     

Distinct proinflammatory host responses to Neisseria gonorrhoeae infection in immortalized human cervical and vaginal epithelial cells

In this study we utilized immortalized morphologically and functionally distinct epithelial cell lines from normal human endocervix, ectocervix, and vagina to characterize gonococcal epithelial interactions pertinent to the lower female genital tract. Piliated, but not nonpiliated, N. gonorrhoeae strain F62 variants actively invaded these epithelial cell lines, as demonstrated by an antibiotic protection assay and confocal microscopy. Invasion of these cells by green fluorescent protein-expressing gonococci was characterized by colocalization of gonococci with F actin, which were initially detected 30 min postinfection. In all three cell lines, upregulation of interleukin 8 (IL-8) and IL-6, intercellular adhesion molecule 1 (CD54), and the nonspecific cross-reacting antigen (CD66c) were detected 4 h after infection with piliated and nonpiliated gonococci. Furthermore, stimulation of all three cell lines with gonococcal whole-cell lysates resulted in a similar upregulation of IL-6 and IL-8, confirming that bacterial uptake is not essential for this response. Increased levels of IL-1 were first detected 8 h after infection with gonococci, suggesting that the earlier IL-8 and IL-6 responses were not mediated through the IL-1 signaling pathway. The IL-1 response was limited to cultures infected with piliated gonococci and was more vigorous in the endocervical epithelial cells. The ability of gonococci to stimulate distinct proinflammatory host responses in these morphologically and functionally different compartments of the lower female genital tract may contribute directly to the inflammatory signs and symptoms characteristic of disease caused by N. gonorrhoeae.     

The effect of antistaphylococcal agents used alone and in combinations on the survival of Staphylococcus aureus ingested by human polymorphonuclear leukocytes

The intracellular activity of a number of drugs used alone and in combinations against Staphylococcus aureus was investigated using an experimental design which imitates the clinical situation and differs from other published methods. Staphylococci were phagocytosed by human polymorphonuclear leukocytes and, after differential centrifugation and washing, the granulocytes were incubated in 90% pooled human serum with clinically relevant drug concentrations. When exposed to antibiotics, more than 40-50% of the bacteria were located intracellularly. Fusidic acid (100 mg/l), erythromycin (20 mg/l), and clindamycin (20 mg/l) all had a bacteriostatic effect during the first 6 h of incubation, whereas rifampicin (1 and 5 mg/l), vancomycin (5 and 20 mg/l), and ciprofloxacin (2 mg/l) all acted bactericidally with decreases in viable counts between 1.3-1.9 log10. The greatest bactericidal effect was achieved with tobramycin (10 mg/l), which produced more than a 4 log10 decrease in viable counts at 6 h. Combinations of fusidic acid with other antibiotics all resulted in killing kinetics different from those achieved with the drugs used individually. The bactericidal effect of ciprofloxacin and dicloxacillin during the first 6 h was abolished when these drugs were combined with fusidic acid. However, at 24 h no significant difference was found between the effect of dicloxacillin alone versus the combination dicloxacillin and fusidic acid. The combination of fusidic acid and rifampicin resulted in a killing identical to that achieved with rifampicin used alone during the first 6 h, but at 24 h the killing by the combination was significantly greater. The bactericidal effect of the combination dicloxacillin (20 mg/l) and tobramycin (10 mg/l) equalled that obtained with tobramycin (10 mg/l) used alone. Rifampicin (5 mg/l) antagonized the bactericidal effect of ciprofloxacin (2 mg/l) during the first 6 h of incubation but at 24 h the combination acted synergistically. The results obtained are partly in agreement and partly in conflict with previous results.     

Extraction and partial characterization of a leukotoxin from a plaque-derived Gram-negative microorganism.

The plaque-derived gram-negative microorganism Y4 identified as a member of the genus Actinobacillus, was tested for a soluble cytotoxic factor(s). Sonication or incubation of viable Y4 microorganisms in distilled water or normal human serum resulted in liberation of a soluble material which was cytotoxic in vitro for human polymorphonuclear leukocytes (PMNs). The Y4 soluble sonic extract was also cytotoxic to human peripheral blood monocytes. However, human lymphocytes, platelets, and fibroblasts, as well as rabbit, rat, and mouse leukocytes and chicken embryo fibroblasts, were not killed by exposure to the Y4 sonic extract. No hemolytic activity was detected in the Y4 sonic extract. No hemolytic activity was detected in the Y4 sonic extract. Consequently, the factor(s) in the Y4 sonic extract was referred to as Y4 leukotoxin. The Y4 leukotoxin was inactive at 4 degrees C, heat sensitive (56 degrees C, 30 min), and inactivated by proteases. The cytotoxic effect of Y4 leukotoxin on PMNs was dose, time, and temperature dependent. The leukotoxin did not bind to viable PMNs at 4 degrees C but did bind to dead PMN membrane components at both 4 and 37 degrees C. The addition of bovine serum albumin (51 mg/ml) to PMN-Y4 leukotoxin cultures inhibited the release of lactate dehydrogenase from the PMNs, but did not prevent the death of the cells as indicated by electron microscopy. Lysosomal markers were released in parallel to the cytoplasmic enzyme lactate dehydrogenase from Y4 leukotoxin-treated PMNs. The addition of 0.02 M ethylenedinitrilotetraacetic acid to these cultures inhibited release of lysosomal markers but enhanced the release of lactate dehydrogenase. These results suggested that a soluble leukotoxin with specificity for only human PMNs and monocytes can be liberated from viable Y4. What role this leukotoxin plays in the pathogenicity of the Y4 microorganism is not yet known. However, this leukotoxin is one of the first materials from a plaque-derived microorganism with a potential role in the pathogenesis of juvenile periodontitis.