Nosocomial infections caused by antibiotic-resistant Klebsiella pneumoniae are emerging as a major health problem worldwide, while community-acquired K. pneumoniae infections present with a range of diverse clinical pictures in different geographic areas. In particular, an invasive form of K. pneumoniae that causes liver abscesses was first observed in Asia and then was found worldwide. We are interested in how differences in gene content of the same species result in different diseases. Thus, we sequenced the whole genome of K. pneumoniae NTUH-K2044, which was isolated from a patient with liver abscess and meningitis, and analyzed differences compared to strain MGH 78578, which was isolated from a patient with pneumonia. Six major types of differences were found in gene clusters that included an integrative and conjugative element, clusters involved in citrate fermentation, lipopolysaccharide synthesis, and capsular polysaccharide synthesis, phage-related insertions, and a cluster containing fimbria-related genes. We also conducted comparative genomic hybridization with 15 K. pneumoniae isolates obtained from community-acquired or nosocomial infections using tiling probes for the NTUH-K2044 genome. Hierarchical clustering revealed three major groups of genomic insertion-deletion patterns that correlate with the strains' clinical features, antimicrobial susceptibilities, and virulence phenotypes with mice. Here we report the whole-genome sequence of K. pneumoniae NTUH-K2044 and describe evidence showing significant genomic diversity and sequence acquisition among K. pneumoniae pathogenic strains. Our findings support the hypothesis that these factors are responsible for the changes that have occurred in the disease profile over time.

A plasmid containing the qnrS quinolone resistance determinant and the gene encoding the SHV-2 ␤-lactamase has been discovered from a clinical Klebsiella pneumoniae strain isolated in Taiwan. The complete 98-kb sequence of this plasmid, designated pK245, was determined by using a whole-genome shotgun approach. Transfer of pK245 conferred low-level resistance to fluoroquinolones in electroporant Escherichia coli epi300. The sequence of the immediate region surrounding qnrS in pK245 is nearly identical (>99% identity) to those of pAH0376 from Shigella flexneri and pINF5 from Salmonella enterica serovar Infantis, the two other qnrS-carrying plasmids reported to date, indicating a potential common origin. Other genes conferring resistance to aminoglycosides (aacC2, strA, and strB), chloramphenicol (catA2), sulfonamides (sul2), tetracycline (tetD), and trimethoprim (dfrA14) were also detected in pK245. The dfrA14 gene is carried on a class I integron. Several features of this plasmid, including three separate regions containing putative replicons, a partitioning-control system, and a type II restriction modification system, suggest that it may be able to replicate and adapt in a variety of hosts. Although no critical conjugative genes were detected, multiple insertion sequence elements were found scattered throughout pK245, and these may facilitate the dissemination of the antimicrobial resistance determinants. We conclude that pK245 is a chimera which acquired its multiple antimicrobial resistance determinants horizontally from different sources. The identification of pK245 plasmid expands the repertoire of the coexistence of quinolone and extended-spectrum-␤-lactam resistance determinants in plasmids carried by various species of the family Enterobacteriaceae in different countries.Plasmid-mediated quinolone resistance was first identified in a clinical Klebsiella pneumoniae strain in the United States in 1998 (29). The gene responsible for this mechanism, qnrA, was later characterized (43). Several QnrA-and QnrB-like proteins have since been identified in different species of the family Enterobacteriaceae, including Escherichia coli, Enterobacter cloacae, and K. pneumoniae from the United States, Europe, and Asia (6,21,22,27,32,36,37,44,45). Another qnr gene, qnrS, was recently found on a transferable plasmid from a clinical Shigella flexneri isolate in Japan (19) and has subsequently been found in a Salmonella enterica serovar Infantis isolate of avian origin (23). Although many qnrA-and qnrB-positive plasmids were also found to carry genes encoding extended-spectrum ␤-lactamases (ESBLs) (21, 32, 34), no ESBLs were found on the plasmids that carried qnrS.We recently identified a multidrug resistance plasmid from K. pneumoniae strain NK245. NK245 was isolated in January 2002 from a patient with hospital-acquired urinary tract infection. The plasmid, designated pK245, conferred resistance to several classes of antimicrobials in the E. coli electroporant, including low-level resistance to fluoroquinolones (FQs). The...

Copper ions switch the oxidation of methane by soluble methane monooxygenase to particulate methane monooxygenase in Methylococcus capsulatus (Bath). Toward understanding the change in cellular metabolism related to this transcriptional and metabolic switch, we have undertaken genomic sequencing and quantitative comparative analysis of the proteome in M. capsulatus (Bath) grown under different copper-to-biomass ratios by cleavable isotope-coded affinity tag technology. Of the 682 proteins identified, the expressions of 60 proteins were stimulated by at least 2-fold by copper ions; 68 proteins were down-regulated by 2-fold or more. The 60 proteins overexpressed included the methane and carbohydrate metabolic enzymes, while the 68 proteins suppressed were mainly responsible for cellular signaling processes, indicating a role of copper ions in the expression of the genes associated with the metabolism of the organism downstream of methane oxidation. The study has also provided a complete map of the C 1 metabolism pathways in this methanotroph and clarified the interrelationships between them.Methanotrophs are a unique group of Gram-negative bacteria that grow aerobically on methane and utilize methane as the sole source of carbon and energy. There has been considerable interest in methanotrophs over the past 30 years, since they can be used to produce single-cell bulk chemicals such as propylene oxide. The ability of these bacteria to co-oxidize a wide range of alkanes, alkenes, and substituted aliphatic compounds has been exploited in bioremediation processes, for example, in the degradation of key pollutants such as trichloroethylene in soil and groundwater. Methanotrophs also play an important role in the global methane cycle (1, 2).Methanotrophs are classified into three distinct types. Type I methanotrophs show disk-like intracellular membranes and use the RuMP (ribulose 5-phosphate) pathway for carbon assimilation. In contrast, Type II methanotrophs possess intracellular membranes in the cell periphery and use the serine pathway to degrade the formaldehyde produced. Methylococcus capsulatus (Bath) is classified as a Type X methanotroph (1), since it shows the physiological properties of both Type I and II methanotrophs, but it develops Type I intracytoplasmic membranes.Two methane monooxygenases (MMO) 1 catalyze the methane oxidation process in methanotrophs, converting methane to methanol. All methanotrophs express a membrane-bound copper-containing particulate MMO (pMMO) (1, 3), while Type X and a few Type II methanotrophic bacteria are capable of producing a second, soluble form (soluble methane monooxygenase, sMMO). Copper ions are known to switch the methane oxidation from sMMO to pMMO. At low copper-to-biomass ratios, the cytoplasmic sMMO is the dominant MMO. When the cells are grown at high copper-to-biomass ratios, pMMO is expressed and produced instead in the plasma membrane.Because of the unique role of copper ions in regulating the switch between sMMO and pMMO, we have applied cICAT (cleavable isot...

SummaryElevated levels of shear stress that occur in stenotic arteries may induce platelet aggregation and initiate thrombosis. Shear-induced platelet aggregation (SIPA) was studied in groups of ischemic stroke patients and normal subjects using a viscometric-flow cytometric technique. Twenty-three patients who sustained an ischemic stroke that was not of cardiac origin were included in this study, and were classified either as atherosclerotic (n = 15) or as lacunar (n = 8) stroke patients. The results show that shear stresses at the levels which occur in arteries partially occluded by atherosclerosis or vascular spasm strongly activate and aggregate platelets, and this response is much more pronounced in non-lacunar stroke patients who had documented atherosclerotic disease of their cerebral vessels. SIPA is not affected by the time of blood drawing after the onset of stroke suggesting that these platelet abnormalities are not transient but chronic. Furthermore, the extent of platelet activation detected by an anti-P-selectin monoclonal antibody and the proportion of neutrophil-platelet aggregates circulating in vivo are significantly higher in the atherosclerotic stroke patients studied at least one month after the onset of stroke. The results indicate that the enhanced platelet responses observed in atherosclerotic stroke patients are not consequences of ischemia, and therefore both platelet activation and elevated SIPA may be considered as important risk factors for stroke. The methodology developed in this work may be useful for characterization of platelet reactivity, and may contribute to our understanding of thrombotic mechanisms.

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