Application of 16S rRNA gene sequencing to identify Bordetella hinzii as the causative agent of fatal septicemia

We report on the first case of fatal septicemia caused by Bordetella hinzii. The causative organism exhibited a biochemical profile identical to that of Bordetella avium with three commercial identification systems (API 20E, API 20 NE, and Vitek GNI+ card). However, its cellular fatty acid profile was not typical for either B. avium or previously reported strains of B. hinzii. Presumptive identification of the patient's isolate was accomplished by traditional biochemical testing, and definitive identification was achieved by 16S rRNA gene sequence analysis. Phenotypic features useful in distinguishing B. hinzii from B. avium were production of alkali from malonate and resistance to several antimicrobial agents.     

Specimen volume versus yield in the BACTEC blood culture system.

During a 24-month period, 5,625 blood culture specimens were collected at the Seattle Veterans Administration Medical Center in 20-ml volumes and divided into separate 10-ml aliquots. The two aliquots were processed as duplicate sets (set 1, set 2) by the BACTEC system (Johnston Laboratories, Inc., Towson, Md.). Specimens (5 ml) from each set were inoculated into aerobic (6B) and anaerobic (7C/7D) vials. A total of 434 significantly positive blood cultures were found. In 342 of these positive cultures, yielding 379 isolates (112 members of the family Enterobacteriaceae, 104 staphylococci, 87 streptococci, 27 anaerobes, 20 yeasts, 14 pseudomonads, and 15 miscellaneous organisms), there was adequate specimen volume to fill all four vials. The utilization of set 1 would have resulted only in the failure to detect 65 of 379 (17.2%) significant isolates, 52 of 342 (15.2%) positive cultures, and 20 of 198 (10.1%) bacteremic episodes. There were no significant differences in the recovery of individual species in sets 1 and 2. Although the range of isolates recovered by the aerobic and anaerobic vials of each set differed, the percent yield of total isolates was similar, indicating total isolate yield was predominantly a function of specimen volume. The addition of set 2 most dramatically increased the recovery of Escherichia coli (30%), yeasts (33%), and anaerobes (42%).     

Inhibition of phospholipase A2 (PLA2) activity by nifedipine and nisoldipine is independent of their calcium-channel-blocking activity

The effects of several calcium antagonists on phospholipase A₂ (PLA₂) activity were examined. Nifedipine and nisoldipine inhibited a cell-free preparation of PLA₂ in a dose-dependent manner with maximal inhibition of 71–77% observed at 100M. More potent or equipotent dihydropyridine calcium antagonists such as nitrendipine and felodipine did not inhibit PLA₂ activity. In addition, nondihydropyridine calcium antagonists such as diltiazem, verapamil, and cinnarazine failed to reduce PLA₂ activity markedly. Nifedipine and nisoldipine also reduced PLA₂ activity in intact mouse peritoneal macrophages where PLA₂ activity was monitored by free [¹⁴C]arachidonic acid release from [¹⁴C]arachidonic acid-prelabeled cells. When levels of PGE₂ and LTC₄ were measured by radioimmunoassay, it was found that the synthesis of these two metabolites was concomitantly inhibited by nifedipine and nisoldipine. In vivo, nifedipine and nisoldipine inhibited tetradecanoylphorbol acetate (TPA) induced ear edema. UV irradiation of nifedipine and nisoldipine (which destroys the slow caicium-channel-blocking activity of these compounds) did not result in a loss of PLA₂ inhibitory activity. In fact, in both instances the UV-irradiated forms of nifedipine and nisoldipine were slightly more potent PLA₂ inhibitors than the parent compound alone. We therefore conclude that the ability of nifedipine and nisoldipine to inhibit PLA₂ was direct and unrelated to their actions on slow calcium channels.     

The (α2→8)-Linked Polysialic Acid Capsule and Lipooligosaccharide Structure Both Contribute to the Ability of Serogroup B Neisseria meningitidis To Resist the Bactericidal Activity of Normal Human Serum

The molecular basis for the resistance of serogroup B Neisseria meningitidis to the bactericidal activity of normal human sera (NHS) was examined with a NHS-resistant, invasive serogroup B meningococcal isolate and genetically and structurally defined capsule-, lipooligosaccharide (LOS)-, and sialylation-altered mutants of the wild-type strain. Expression of the (α2→8)-linked polysialic acid serogroup B capsule was essential for meningococcal resistance to NHS. The very NHS-sensitive phenotype of acapsular mutants (99.9 to 100% killed in 10, 25, and 50% NHS) was not rescued by complete LOS sialylation or changes in LOS structure. However, expression of the capsule was necessary but not sufficient for a fully NHS-resistant phenotype. In an encapsulated background, loss of LOS sialylation by interrupting the α2,3 sialyltransferase gene, lst, increased sensitivity to 50% NHS. In contrast, replacement of the lacto-N-neotetraose α-chain (Galβ1-4GlcNAcβ1-3Galβ1-4Glc) with glucose extensions (Glc_N) in a galE mutant resulted in a strain resistant to killing by 50% NHS at all time points. Encapsulated meningococci expressing a Hep₂(GlcNAc)→KDO₂→lipid A LOS without an α-chain demonstrated enhanced sensitivity to 50% NHS (98% killed at 30 min) mediated through the antibody-dependent classical complement pathway. Encapsulated LOS mutants expressing truncated Hep₂→KDO₂→lipid A and KDO₂→lipid A structures were also sensitive to 50% NHS (98 to 100% killed at 30 min) but, unlike the wild-type strain and mutants with larger oligosaccharide structures, they were killed by hypogammaglobulinemic sera. These data indicate that encapsulation is essential but that the LOS structure contributes to the ability of serogroup B N. meningitidis to resist the bactericidal activity of NHS.Serogroup B Neisseria meningitidis (the meningococcus) is an obligate human pathogen and remains a leading cause of fulminant septicemia and meningitis. In addition to sporadic outbreaks, large epidemics of serogroup B meningococcal disease continue to occur in many parts of the world, including South America, the United States Pacific Northwest, Western Europe, and New Zealand (4, 22). After penetrating upper respiratory tract mucosal surfaces, N. meningitidis must survive and multiply in the bloodstream to cause sepsis, meningitis, and other manifestations of invasive meningococcal disease. A major mechanism inhibiting or preventing the multiplication of meningococci in the blood is the complement-mediated bactericidal activity of human sera (17, 39). The importance of this activity in the prevention of systemic meningococcal disease is reinforced by host factors that alter bactericidal activity and increase the risk for development of invasive disease. These factors include the absence of bactericidal antibodies against meningococci (17, 18, 45), deficiencies in the complement cascade (13), and the presence of blocking immunoglobulin A antibodies that inhibit the bactericidal activity of human sera (19). The bactericidal activity of human sera against meningococci is also used as a surrogate marker for assessing meningococcal vaccine efficacy.Meningococci have evolved mechanisms that protect them from the bactericidal activity of human sera. Invasive serogroup B meningococcal strains recovered from blood and cerebrospinal fluid often resist being killed by human sera (48). The molecular basis for resistance has been attributed to the expression by this organism of an (α2→8)-linked polysialic acid capsule and a short-chained lipooligosaccharide (LOS) with terminal sialic acid residues (23, 34, 35). Meningococci isolated from the bloodstream in invasive disease, in contrast to nasopharyngeal isolates, are heavily encapsulated (9) and express the L3,7,9 LOS immunotypes (28). These immunotypes have a lacto-N-neotetraose originating from HepI of the inner core, which may be terminally sialylated (34, 62). However, the experimental data defining the precise contributions of the capsule, LOS sialylation, and LOS structure to the ability of serogroup B meningococci to resist the bactericidal activity of human sera is conflicting (11, 15, 20, 21, 27, 37, 63–65).LOS epitopes are immunogenic in infants and children and induce protective bactericidal antibodies in convalescent sera (10, 12). These bactericidal LOS antibodies appear to be directed at conserved low-molecular-weight LOS epitopes (10, 12). LOS is also a component of new serogroup B outer membrane vesicle (OMV) vaccines and is proposed as a basis for other new meningococcal vaccines (1–3, 50). Although changes in the structure of LOS are known to influence the amount and epitopes of bactericidal and other functional antibodies elicited by OMV vaccines (2), the precise LOS structure(s) to include in these and other LOS-containing meningococcal vaccines is uncertain.To help understand the basis for meningococcal survival following mucosal invasion and to facilitate development of meningococcal vaccines which may contain LOS, we created a series of genetically and structurally defined capsule-, sialylation-, and LOS-altered mutants of the serogroup B meningococcal strain NMB. We used these mutants to study the contributions of the capsule, LOS sialylation, and changes in LOS structure to meningococcal resistance to the bactericidal activity of normal human sera (NHS).     

Ultrastructural morphometry of capillary basement membrane thickness in normal and transgenic diabetic mice

Capillary basement membrane (CBM) thickening is an ultrastructural hallmark in diabetic patients and in animal models of diabetes. However, the wide variety of tissues sampled and diverse methods employed have made the interpretation of thickness data difficult. We showed previously that acellular glomerular BMs in OVE26 transgenic diabetic mice were thickened beyond normal age-related thickening, and in the current study we hypothesized that other microvascular BMs likewise would show increased widths relative to age-matched controls. Accordingly, a series of tissues, including skeletal and cardiac muscle, ocular retina and choriod, peripheral nerve, lung, pancreas, and renal glomerulus was collected from 300-350-day-old normal and transgenic mice. Transmission electron micrographs of cross sections through capillary walls were prepared, and CBM thickness (CBMT) was determined by the "orthogonal intercept" method. Morphometric analyses showed highly variable transgene-related BMT increases in the sampled tissues, with glomerular BM showing by far the greatest increase (+87%). Significant thickness increases were also seen in the retina, pulmonary alveolus, and thoracoabdominal diaphragm. BMT increases were not universal; however, most were modestly widened, and those that were thickest in controls generally showed the greatest increase. Although the pathogenesis of diabetes-related increases in CBM is poorly understood, data in the current study showed that in OVE26 transgenic mice increased BMT was a frequent concomitant of hyperglycemia. Accordingly, it seems likely that hyperglycemia-induced microvascular damage may be a contributing factor in diabetic BM disease, and that microvessel cellular and extracellular heterogeneity may limit the extent of CBM thickening in diverse tissues.     

Paradoxical esterification of plasma cholesterol in fish eye disease

The activity of lecithin: cholesterol acyl transferase (LCAT), the enzyme which catalyses the esterification of human plasma cholesterol, has been measured by two independent methods in plasma from the two known living Swedish patients with fish eye disease. The enzyme activity was in both cases about 15% of that of normal plasma. Paradoxically, however, the percentage of plasma cholesterol which was esterified was almost normal in both patients. In addition, a normal spectrum of the fatty acids of the cholesteryl esters was present indicating a normal cholesterol esterification pathway in vivo. Incubation experiments in vitro of plasma from the two patients also yielded normal cholesterol esterification rates when measured by two different methods. These paradoxical results for cholesterol esterification are discussed on the basis of the present biochemical knowledge of fish eye disease and LCAT deficiency.     

Nonspecific cytotoxic cells in fish (Ictalurus punctatus). IV. Target cell binding and recycling capacity

The morphology of nonspecific cytotoxic cells (NCC) was identified. NCC were purified by target cell conjugate formation and density gradient separation. NCC are monocyte-like. They have reniform nuclei and a low nucleus/cytoplasm ratio. Cytoplasmic granules were not seen after giemsa staining. Scanning electron microscopy demonstrated moderate surface villi and target cell attachment occurred via long membraneous filament-like surface projections extending to the target cell membranes. Transmission electron microscopy of effector:target cell conjugates revealed membrane contact areas without fusion or fragmentation. The nucleus of the NCC had accentuated peripheral chromation and a prominent Golgi apparatus; the cytoplasm contained osmiophilic granules. Michaelis-Menten and Lineweaver-Burk transformation of target cell binding revealed a Vmax of 11-15,000 and a Km of 40,000. The percentage of NCC bound to target cells was 16-18%. Results of these studies were combined with the conjugate experiment to obtain an estimated percentage of active NCC (5-7%). A maximum recycling capacity of .16-.30 indicated that once attachment by NCC to the target cell occurred (and a lethal signal delivered by an effector cell), either the NCC did not recycle or a long lag period was required to restore its cytotoxic capability.