Nosocomial infection in Auckland Healthcare hospitals

AIM: To determine the prevalence of nosocomial infection in Auckland Healthcare hospitals. BACKGROUND: Nosocomial infections cause patient morbidity and prolong hospital stay. Reporting surveillance results to staff has been shown to reduce nosocomial infection rates. METHOD: Point prevalence study for all patients in Auckland, Green Lane and National Women's hospitals. Standard definitions for nosocomial infections were used. RESULTS: One hundred and ten (12%) of 932 patients had 129 nosocomial infections: 27 (20%) surgical site infections; 25 (19%) lower respiratory tract infections; 23 (18%) skin/ soft tissue infections; 19 (15%) urinary tract infections; 14 (11%) bloodstream infections; and 21 (17%) other infections. Predominant organisms were: Staphylococcus aureus (29%), Escherichia coli (21%), other gram negative bacilli (14%), Pseudomonas aeruginosa (6%), streptococci (6%) and Candida albicans (6%). The prevalence of nosocomial infection was lower in National Women's Hospital (5%) than either Green Lane or Auckland hospitals (15% and 14% respectively), p < 0.01. The prevalence of nosocomial infection was the same in medical and surgical patients, 53 of 394 (14%) and 42 of 297 (14%), respectively. The highest prevalence was in intensive care unit patients, 7 of 31 (23%). The prevalence of nosocomial infection increased with patient age, 17-50 yr (8%) vs > 50 yr (14%), p < 0.01, and duration of hospitalisation 2% for < 2 days, 6% for 2-7 days vs 22% for > 7 days, p < 0.01. Risk factors for nosocomial infection were present in many patients: 339 (36%) had intravenous catheters in place; 268 (29%) patients had undergone surgery during their current admission; 122 (13%) had urinary catheters in place; and 122 (13%) had other invasive devices in situ. CONCLUSION: Our results are comparable with hospitals of similar size overseas. This study provides a base line for future studies which will enable the monitoring of trends over time and the impact of focused infection control initiatives.     

Case presentation in small animal medicine. What is your diagnosis?

Atrial fibrillation and dilated cardiomyopathy were diagnosed in a 9-year old boxer who was examined because of anorexia and ascites. Longterm treatment included a diuretic (furosemide), an ACE-inhibitor (benazepril), digoxin, and a betablocker (atenolol). The prognosis for DCMP is unfavorable--the described dog died 11 months after diagnosis.     

Rapid increase in PKA activity during long-term potentiation in the hippocampal afferent fibre system to the prefrontal cortex in vivo

The purpose of the present study was to examine whether cAMP-dependent protein kinase (PKA) was implicated in the process of long-term potentiation (LTP) in the hippocampal afferent fibre system to the prefrontal cortex in vivo. Using a biochemical approach, we measured PKA activity at different times after induction of LTP. We show that there is an NMDA receptor-dependent increase in PKA activity in the prefrontal cortex, only at five minutes after LTP induction. These data demonstrate a role of PKA in the induction and not the expression of cortical LTP and suggest that if PKA is involved in the late phase of LTP, it does not appear to be a persistent activation.     

Effects of overexpression of the alkyl hydroperoxide reductase AhpC on the virulence and isoniazid resistance of Mycobacterium tuberculosis

Mutations to the regulatory region of the ahpC gene, resulting in overproduction of alkyl hydroperoxide reductase, were encountered frequently in a large collection of isoniazid (INH)-resistant clinical isolates of Mycobacterium tuberculosis but not in INH-susceptible strains. Overexpression of ahpC did not seem to be important for INH resistance, however, as most of these strains were already defective for catalase-peroxidase, KatG, the enzyme required for activation of INH. Transformation of the INH-susceptible reference strain, M. tuberculosis H37Rv, with plasmids bearing the ahpC genes of M. tuberculosis or M. leprae did not result in a significant increase in the MIC. Two highly INH-resistant mutants of H37Rv, BH3 and BH8, were isolated in vitro and shown to produce no or little KatG activity and, in the case of BH3, to overproduce alkyl hydroperoxide reductase as the result of an ahpC regulatory mutation that was also found in some clinical isolates. The virulence of H37Rv, BH3, and BH8 was studied intensively in three mouse models: fully immunocompetent BALB/c and Black 6 mice, BALB/c major histocompatibility complex class II-knockout mice with abnormally low levels of CD4 T cells and athymic mice producing no cellular immune response. The results indicated that M. tuberculosis strains producing catalase-peroxidase were considerably more virulent in immunocompetent mice than the isogenic KatG-deficient mutants but that loss of catalase-peroxidase was less important when immunodeficient mice, unable to produce activated macrophages, were infected. Restoration of virulence was not seen in an INH-resistant M. tuberculosis strain that overexpressed ahpC, and this finding was confirmed by experiments performed with appropriate M. bovis strains in guinea pigs. Thus, in contrast to catalase-peroxidase, alkyl hydroperoxide reductase does not appear to act as a virulence factor in rodent infections or to play a direct role in INH resistance, although it may be important in maintaining peroxide homeostasis of the organism when KatG activity is low or absent.     

Heme packing motifs revealed by the crystal structure of the tetra-heme cytochrome c554 from Nitrosomonas europaea

Cytochrome c554 (cyt c554), a tetra-heme cytochrome from Nitrosomonas europaea, is an essential component in the biological nitrification pathway. In N. europaea, ammonia is converted to hydroxylamine, which is then oxidized to nitrite by hydroxylamine oxidoreductase (HAO). Cyt c554 functions in the latter process by accepting pairs of electrons from HAO and transferring them to a cytochrome acceptor. The crystal structure of cyt c554 at 2.6 A resolution shows a predominantly alpha-helical protein with four covalently attached hemes. The four hemes are arranged in two pairs such that the planes of the porphyrin rings are almost parallel and overlapping at the edge; corresponding heme arrangements are observed in other multi-heme proteins. Striking structural similarities are evident between the tetra-heme core of cyt c554 and hemes 3-6 of HAO, which suggests an evolutionary relationship between these redox partners.     

Hematopoietic cells from -spectrin-deficient mice are sufficient to induce thrombotic events in hematopoietically ablated recipients

Thrombotic events are life-threatening complications of human hemolytic anemias such as paroxysmal nocturnal hemoglobinuria, sickle cell disease, and thalassemia. It is not clear whether these events are solely influenced by aberrant hematopoietic cells or also involve aberrant nonhematopoietic cells. Spherocytosis mutant (Spna1(sph)/Spna1(sph); for simplicity referred to as sph/sph) mice develop a severe hemolytic anemia postnatally due to deficiencies in -spectrin in erythroid and other as yet incompletely defined nonerythroid tissues. Thrombotic lesions occur in all adult sph/sph mice, thus providing a hematopoietically stressed model in which to assess putative causes of thrombus formation. To determine whether hematopoietic cells from sph/sph mice are sufficient to initiate thrombi, bone marrow from sph/sph or +/+ mice was transplanted into mice with no hemolytic anemia. One set of recipients was lethally irradiated; the other set was genetically stem cell deficient. All mice implanted with sph/sph marrow, but not +/+ marrow, developed severe anemia and histopathology typical of sph/sph mice. Histological analyses of marrow recipients showed that thrombi were present in the recipients of sph/sph marrow, but not +/+ marrow. The results indicate that the -spectrin-deficient hematopoietic cells of sph/sph mice are the primary causative agents of the thrombotic events.     

Mycobacterium avium complex augments macrophage HIV-1 production and increases CCR5 expression

Infection with HIV-1 results in pronounced immune suppression and susceptibility to opportunistic infections (OI). Reciprocally, OI augment HIV-1 replication. As we have shown for Mycobacterium avium complex (MAC) and Pneumocystis carinii, macrophages infected with opportunistic pathogens and within lymphoid tissues containing OI, exhibit striking levels of viral replication. To explore potential underlying mechanisms for increased HIV-1 replication associated with coinfection, blood monocytes were exposed to MAC antigens (MAg) or viable MAC and their levels of tumor necrosis factor alpha (TNFalpha) and HIV-1 coreceptors monitored. MAC enhanced TNFalpha production in vitro, consistent with its expression in coinfected lymph nodes. Using a polyclonal antibody to the CCR5 coreceptor that mediates viral entry of macrophage tropic HIV-1, a subset of unstimulated monocytes was shown to be CCR5-positive by fluorescence-activated cell sorter analysis. After stimulation with MAg or infection with MAC, CCR5 expression was increased at both the mRNA level and on the cell surface. Up-regulation of CCR5 by MAC was not paralleled by an increase in the T cell tropic coreceptor, CXCR4. Increases in NF-kappaB, TNFalpha, and CCR5 were consistent with the enhanced production of HIV-1 in MAg-treated adherent macrophage cultures as measured by HIV-1 p24 levels. Increased CCR5 was also detected in coinfected lymph nodes as compared with tissues with only HIV-1. The increased production of TNFalpha, together with elevated expression of CCR5, provide potential mechanisms for enhanced infection and replication of HIV-1 by macrophages in OI-infected cells and tissues. Consequently, treating OI may inhibit not only the OI-induced pathology, but also limit the viral burden.     

Engineering steroid 5 beta-reductase activity into rat liver 3 alpha-hydroxysteroid dehydrogenase

Delta 4-3-Ketosteroid-5 beta-reductase (5 beta-reductase) precedes 3 alpha-hydroxysteroid dehydrogenase (3 alpha-HSD) in steroid hormone metabolism. Both enzymes are members of the aldo-keto reductase (AKR) superfamily and possess catalytic tetrads differing by a single amino acid. In 3 alpha-HSD, the tetrad consists of Tyr55, Lys84, Asp50, and His117, but a glutamic acid replaces His117 in 5 beta-reductase. By introducing the H117E point mutation into 3 alpha-HSD, we engineered 5 beta-reductase activity into the dehydrogenase. Homogeneous H117E 3 alpha-HSD reduced the double bond in testosterone to form 5 beta-dihydrotestosterone with kcat = 0.25 min-1 and Km = 19.0 microM and reduced the double bond in progesterone to generate 5 beta-dihydroprogesterone with kcat = 0.97 min-1 and Km = 33.0 microM. These kinetic parameters were similar to those reported for homogeneous rat liver 5 beta-reductase [Okuda, A., and Okuda, R. (1984) J. Biol. Chem. 259, 7519-7524]. The H117E mutant also reduced 5beta-dihydrosteroids to 5 beta, 3 alpha-tetrahydrosteroids with a 600-1000-fold decrease in kcat/Km versus wild-type 3 alpha-HSD. The ratio of 5 beta-reductase:3 alpha-HSD activity in the H117E mutant was approximately 1:1. Although the H117A mutant reduced Delta 4-3-ketosteroids, the 3 alpha-HSD activity predominated because the 5 beta-dihydrosteroids were rapidly converted to the 5 beta,3 alpha-tetrahydrosteroids. The pH-rate profiles for carbon-carbon double-bond and ketone reduction catalyzed by the H117E mutant were superimposable, suggesting a common titratable group (pKb = 6.3) for both reactions. In wild-type 3 alpha-HSD, the titratable group responsible for 3-ketosteroid reduction has a pKb = 6.9 and is assignable to Tyr55. The pH-rate profiles for 3-ketosteroid reduction by the H117A mutant were pH-independent. Our data indicate that Tyr55 functions as a general acid for both 3 alpha-HSD and 5 beta-reductase activities. We suggest that a protonated Glu117 increases the acidity of Tyr55 to promote acid-catalyzed enolization of the Delta 4-3-ketosteroid substrate. Further, the identity of amino acid 117 determines whether an AKR can function as a 5 beta-reductase by reorienting the substrate relative to the nicotinamide cofactor. This study provides functional evidence that utilization of modified catalytic residues on an identical protein scaffold is important for evolution of enzymatic activities within the same metabolic pathway.