BackgroundNosocomial infections due to vancomycin-resistant enterococci (VRE) have become a major problem during the last years. The purpose of this study was to investigate the economic burden of nosocomial VRE infections in a European university hospital.MethodsA retrospective matched case-control study was performed including patients who acquired nosocomial infection with either VRE or vancomycin-susceptible enterococci (VSE) within a time period of 3 years. 42 cases with VRE infections and 42 controls with VSE infections were matched for age, gender, admission and discharge within the same year, time at risk for infection, Charlson comorbidity index (±1), stay on intensive care units and non-intensive care units as well as for the type of infection, using criteria of the Centers for Disease Control and Prevention.ResultsThe median overall costs per case were significantly higher than for controls (EUR 57,675 vs. EUR 38,344; p = 0.030). Costs were similar between cases and controls before onset of infection (EUR 17,893 vs. EUR 16,600; p = 0.386), but higher after onset of infection (EUR 37,971 vs. EUR 23,025; p = 0.049). The median attributable costs per case for vancomycin-resistance were EUR 13,157 (p = 0.036). The most significant differences in costs between cases and controls turned out to be for pharmaceuticals (EUR 6030 vs. EUR 2801; p = 0.008) followed by nursing staff (EUR 8956 vs. EUR 4621; p = 0.032), medical products (EUR 3312 vs. EUR 1838; p = 0.020), and for assistant medical technicians (EUR 3766 vs. EUR 2474; p = 0.023). Furthermore, multivariate analysis revealed that costs were driven independently by vancomycin-resistance (1.4 fold; p = 0.034).ConclusionsThis analysis suggested that nosocomial VRE infections significantly increases hospital costs compared with VSE infections. Therefore, hospital personal should implement control measures to prevent VRE transmission.
Mycobacterium tuberculosis can utilize various nutrients including nitrate as a source of nitrogen. Assimilation of nitrate requires the reduction of nitrate via nitrite to ammonium, which is then incorporated into metabolic pathways. This study was undertaken to define the molecular mechanism of nitrate assimilation in M. tuberculosis. Homologues to a narGHJI-encoded nitrate reductase and a nirBD-encoded nitrite reductase have been found on the chromosome of M. tuberculosis. Previous studies have implied a role for NarGHJI in nitrate respiration rather than nitrate assimilation. Here, we show that a narG mutant of M. tuberculosis failed to grow on nitrate. A nirB mutant of M. tuberculosis failed to grow on both nitrate and nitrite. Mutant strains of Mycobacterium smegmatis mc 2 155 that are unable to grow on nitrate were isolated. The mutants were rescued by screening a cosmid library from M. tuberculosis, and a gene with homology to the response regulator gene glnR of Streptomyces coelicolor was identified. A DglnR mutant of M. tuberculosis was generated, which also failed to grow on nitrate, but regained its ability to utilize nitrate when nirBD was expressed from a plasmid, suggesting a role of GlnR in regulating nirBD expression. A specific binding site for GlnR within the nirB promoter was identified and confirmed by electrophoretic mobility shift assay using purified recombinant GlnR. Semiquantitative reverse transcription PCR, as well as microarray analysis, demonstrated upregulation of nirBD expression in response to GlnR under nitrogen-limiting conditions. In summary, we conclude that NarGHJI and NirBD of M. tuberculosis mediate the assimilatory reduction of nitrate and nitrite, respectively, and that GlnR acts as a transcriptional activator of nirBD.
Mycobacterium tuberculosis and Mycobacterium bovis cause tuberculosis, which is responsible for the deaths of more people each year than any other bacterial infectious disease. Disseminated disease with Mycobacterium bovis BCG, the only currently available vaccine against tuberculosis, occurs in immunocompetent and immunodeficient individuals. Although mycobacteria are obligate aerobes, they are thought to face an anaerobic environment during infection, notably inside abscesses and granulomas. The purpose of this study was to define a metabolic pathway that could allow mycobacteria to exist under these conditions. Recently, the complete genome of M. tuberculosis has been sequenced, and genes homologous to an anaerobic nitrate reductase (narGHJI), an enzyme allowing nitrate respiration when oxygen is absent, were found. Here, we show that the narGHJI cluster of M. tuberculosis is functional as it conferred anaerobic nitrate reductase activity to Mycobacterium smegmatis. A narG mutant of M. bovis BCG was generated by targeted gene deletion. The mutant lacked the ability to reduce nitrate under anaerobic conditions. Both mutant and M. bovis BCG wild type grew equally well under aerobic conditions in vitro. Histology of immunodeficient mice (SCID) infected with M. bovis BCG wild type revealed large granulomas teeming with acid‐fast bacilli; all mice showed signs of clinical disease after 50 days and succumbed after 80 days. In contrast, mice infected with the mutant had smaller granulomas containing fewer bacteria; these mice showed no signs of clinical disease after more than 200 days. Thus, it seems that nitrate respiration contributes significantly to virulence of M. bovis BCG in immunodeficient SCID mice.
Mycobacterium tuberculosis (Mtb) uses alveolar macrophages as primary host cells during infection. In response to an infection, macrophages switch from pyruvate oxidation to reduction of pyruvate into lactate. Lactate might present an additional carbon substrate for Mtb. Here, we demonstrate that Mtb can utilize L-lactate as sole carbon source for in vitro growth. Lactate conversion is strictly dependent on one of two potential L-lactate dehydrogenases. A knock-out mutant lacking lldD2 (Rv1872c) was unable to utilize L-lactate. In contrast, the lldD1 (Rv0694) knock-out strain was not affected in growth on lactate and retained full enzymatic activity. On the basis of labelling experiments using [U-13C3]-L-lactate as a tracer the efficient uptake of lactate by Mtb and its conversion into pyruvate could be demonstrated. Moreover, carbon flux from lactate into the TCA cycle, and through gluconeogenesis was observed. Gluconeogenesis during lactate consumption depended on the phosphoenolpyruvate carboxykinase, a key enzyme for intracellular survival, showing that lactate utilization requires essential metabolic pathways. We observed that the ΔlldD2 mutant was impaired in replication in human macrophages, indicating a critical role for lactate oxidation during intracellular growth.
Mycobacterium bovis BCG, the only presently available vaccine against tuberculosis, was obtained from virulent M. bovis after serial passages in vitro. The vaccine strain retained at least some of its original virulence, as it persists in immune-competent hosts and occasionally may cause fatal disease in immunedeficient hosts. Mycobacterial persistence in vivo is thought to depend on anaerobic metabolism, an apparent paradox since all mycobacteria are obligate aerobes. Here we report that M. bovis BCG lacking anaerobic nitrate reductase (NarGHJI), an enzyme essential for nitrate respiration, failed to persist in the lungs, liver, and kidneys of immune-competent (BALB/c) mice. In immune-deficient (SCID) mice, however, bacilli caused chronic infection despite disruption of narG, even if growth of the mutant was severely impaired in lungs, liver, and kidneys. Persistence and growth of BCG in the spleens of either mouse strain appeared largely unaffected by lack of anaerobic nitrate reductase, indicating that the role of the enzyme in pathogenesis is tissue specific. These data suggest first that anaerobic nitrate reduction is essential for metabolism of M. bovis BCG in immune-competent but not immune-deficient mice and second that its role in mycobacterial disease is tissue specific, both of which are observations with important implications for pathogenesis of mycobacteria and vaccine development.Mycobacterium tuberculosis claims more human lives each year than any other bacterial pathogen. Mycobacterium bovis BCG, the only presently available vaccine against tuberculosis, belongs phylogenetically to the M. tuberculosis complex. In humans, M. bovis BCG, like M. tuberculosis, forms granulomas and abscesses in various tissues. Following vaccination in immune-competent individuals, M. bovis BCG may persist for extended periods (34). In immune-compromised individuals the vaccine strain may even lead to fatal disease (2,3,10,11,14,21,27,30,36,39).Mycobacteria become firmly established within host tissues, adapting their metabolism to the available source of carbohydrates, nitrogen, and energy (4). Although the acquisition of essential nutrients by mycobacteria is an area of considerable interest, our knowledge of bacterial metabolism throughout the course of infection remains rudimentary. A recent study revealed that metabolism of fatty acids serves as a source of carbohydrates and is required for persistence of M. tuberculosis in mice and activated macrophages (25). Nitrate, through nitrate respiration, could provide energy for bacterial metabolism in an anaerobic environment, because anaerobic nitrate reductase (NarGHJI) couples the reduction of nitrate (NO 3 ) to the generation of ATP by replacing oxygen as a terminal electron acceptor (29). Anaerobic nitrate reductase coding sequences (narGHJI) have been identified in both obligate aerobes such as Bacillus and Pseudomonas and facultative anaerobes such as Escherichia coli (5, 17, 28). However, a role of this enzyme in virulence was not established. In mycobacteria,...
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