Objectives: To characterize isolates of Klebsiella pneumoniae producing KPC carbapenemase (KPCKp) associated with an outbreak in a long-term acute care hospital (LTACH) in South Florida.Methods: During 21 March to 20 April 2008, 241 K. pneumoniae isolates detected at Integrated Regional Laboratories (Ft. Lauderdale, FL) for which the ertapenem MICs were 4 mg/L were studied. PCR, cloning and sequence analysis were used to detect bla KPC and to characterize the b-lactamase and outer membrane proteins (Omps). The expression level of KPC enzymes was studied by immunoblotting. Genetic relatedness of isolates was investigated with rep-PCR and PFGE. Clinical records of patients were investigated.Results: Seven KPC-Kp strains were isolated from different patients located at a single LTACH, with a further three isolates being recovered from patients at different hospitals. All KPC-Kp isolates in patients from the LTACH and from one hospital patient were genetically related and shared PFGE patterns that clustered with known sequence type (ST) 258 strains. These strains were highly resistant to carbapenems (MICs32 mg/L) due to an increased level of KPC expression and loss of Omps. Rectal colonization was documented in all LTACH patients with KPC-Kp isolates. Treatment failures were common (crude mortality rate of 69%). Active surveillance and enhanced infection control practices terminated the KPC-Kp outbreak.
Conclusions:The detection of KPC-Kp in an LTACH represents a serious infection control and therapeutic challenge in a new clinical setting. The speed at which the epidemic of KPC-Kp is spreading in our healthcare system mandates urgent action.
Pseudomonas pseudoalcaligenes JS52 grows on nitrobenzene via partial reduction of the nitro group and enzymatic rearrangement of the resultant hydroxylamine. Cells and cell extracts of nitrobenzene-grown JS52 catalyzed the transient formation of 4-hydroxylamino-2,6-dinitrotoluene (4HADNT), 4-amino-2,6-dinitrotoluene (4ADNT), and four previously unidentified metabolites from 2,4,6-trinitrotoluene (TNT). Two of the novel metabolites were identified by liquid chromatography/mass spectrometry and 1 H-nuclear magnetic resonance spectroscopy as 2,4-dihydroxylamino-6-nitrotoluene (DHANT) and 2-hydroxylamino-4-amino-6-nitrotoluene (2HA4ANT). A polar yellow metabolite also accumulated during transformation of TNT by cells and cell extracts. Under anaerobic conditions, extracts of strain JS52 did not catalyze the production of the yellow metabolite or release nitrite from TNT; moreover, DHANT and 2HA4ANT accumulated under anaerobic conditions, which indicated that their further metabolism was oxygen dependent. Small amounts of nitrite were released during transformation of TNT by strain JS52. Sustained transformation of TNT by cells required nitrobenzene, which indicated that TNT transformation does not provide energy. Transformation of TNT catalyzed by enzymes in cell extracts required NADPH. Transformation experiments with 14 C-TNT indicated that TNT was not mineralized; however, carbon derived from TNT became associated with cells. Nitrobenzene nitroreductase purified from strain JS52 transformed TNT to DHANT via 4HADNT, which indicated that the nitroreductase could catalyze the first two steps in the transformation of TNT. The unusual ability of the nitrobenzene nitroreductase to catalyze the stoichiometric reduction of aromatic nitro compounds to the corresponding hydroxylamine provides the basis for the novel pathway for metabolism of TNT.
In the course of measuring the concentrations of retinoic acids (RA) in bovine plasma, a major peak was observed which comigrated with 9-cis-RA on normal-phase high-performance liquid chromatography. Rechromatography of this retinoic on reverse-phase high-performance liquid chromatography showed that it was distinct from 9-cis-,13-cis-, and all-trans-RA, but comigrated with 9-cis,13-cis-retinoic acid (9,13-di-cis-RA). This retinoid was identified as 9,13-di-cis-RA based on its chemical, spectral, and chromatographic properties. Plasma concentrations of 9,13-di-cis-RA increased from < or = 0.5 ng/mL at birth to 5-6 ng/mL by 48 h of age in the calf. The 9,13-di-cis-RA was also a major circulating product of 9-cis-RA dosed intramuscularly to rats; conversely, intravenous administration of 9,13-di-cis-RA produced circulating 9-cis-RA in the rat. 9,13-Di-cis-RA had little or no affinity for cellular retinoic acid binding protein types I and II. This study establishes 9,13-di-cis-RA as a naturally-occurring retinoid under physiological conditions, shows that it undergoes interconversion with 9-cis-RA, and emphasizes a need for careful chromatography to resolve 9-cis-RA and 9,13-di-cis-RA. This is consistent with in vivo 13-cis isomerization operating to modify the concentration and perhaps the activity of 9-cis-RA in vivo.
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