We investigated the antimicrobial resistance profile and the occurrence of Klebsiella pneumoniae carbapenemase (KPC)-producing Gram-negative rods in sewage samples obtained from a Brazilian teaching hospital and from the wastewater treatment plant (WWTP) that receives it for treatment. We identified multidrug-resistant bacteria as well as KPC-2-producing Aeromonas spp. and several Enterobacteriaceae species, including Kluyvera spp., in the hospital effluent and in different sites of the WWTP. Most isolates showed the blaKPC-2 gene harbored on a transposon that was carried by conjugative plasmids. The presence of KPC production among Aeromonas spp., Kluyvera spp., and other Enterobacteriaceae indicates the adaptability of such isolates to aquatic environments, not only in the hospital effluent but also throughout the WWTP. Although secondary treatment seems to decrease the amount of KPC producers in sewage, multidrug-resistant isolates are continually disposed in the urban river. Thus, sewage treatment regulations are urgently needed to decelerate the evolution of antimicrobial resistance beyond hospitals.

X-linked hypophosphatemia (XLH/HYP)-with renal phosphate wasting, hypophosphatemia, osteomalacia, and tooth abscesses-is caused by mutations in the zinc-metallopeptidase PHEX gene (phosphate-regulating gene with homologies to endopeptidase on the X chromosome). PHEX is highly expressed by mineralized tissue cells. Inactivating mutations in PHEX lead to distal renal effects (implying accumulation of a secreted, circulating phosphaturic factor) and accumulation in bone and teeth of mineralization-inhibiting, acidic serine-and aspartate-rich motif (ASARM)-containing peptides, which are proteolytically derived from the mineral-binding matrix proteins of the SIBLING family (small, integrin-binding ligand N-linked glycoproteins). Although the latter observation suggests a local, direct matrix effect for PHEX, its physiologically relevant substrate protein(s) have not been identified. Here, we investigated two SIBLING proteins containing the ASARM motif-osteopontin (OPN) and bone sialoprotein (BSP)-as potential substrates for PHEX. Using cleavage assays, gel electrophoresis, and mass spectrometry, we report that OPN is a full-length protein substrate for PHEX. Degradation of OPN was essentially complete, including hydrolysis of the ASARM motif, resulting in only very small residual fragments. Western blotting of Hyp (the murine homolog of human XLH) mouse bone extracts having no PHEX activity clearly showed accumulation of an $35 kDa OPN fragment that was not present in wild-type mouse bone. Immunohistochemistry and immunogold labeling (electron microscopy) for OPN in Hyp bone likewise showed an accumulation of OPN and/or its fragments compared with normal wild-type bone. Incubation of Hyp mouse bone extracts with PHEX resulted in the complete degradation of these fragments. In conclusion, these results identify full-length OPN and its fragments as novel, physiologically relevant substrates for PHEX, suggesting that accumulation of mineralization-inhibiting OPN fragments may contribute to the mineralization defect seen in the osteomalacic bone characteristic of XLH/HYP. ß

We explored furin substrate requirements in addition to the motif R-X-K/R-R using synthetic fluorescent resonance energy transfer (FRET) decapeptides. These decapeptides were derived from furin cleavage sites in viral coat glycoproteins and human and bacterial protein precursors. The hydrolysis by furin of most substrate was activated by K+ ion, whereas kosmotropic anions of the Hofmeister series were inhibitors. The analysis of furin hydrolytic activity showed that its efficiency is highly dependent on the particular combinations of amino acids at different substrate positions. There is a clear interdependence of furin subsites that must be taken in account in determining its specificity and also for the design of inhibitors. However, clear preferences were detected for substrates with S at P′1, and V at P′2, at P′3 the amino acids D, S, L and A are almost equally frequent. In the non-prime subsites the best substrates presented S and H at P6; basic amino acids at P5; and no clear tendency at P3. Interestingly, two amino acid substitutions on the prime side of the peptide derived from H5N1 influenza hemagglutinin furin processing site highly improved its hydrolysis. These modifications are possible by single point mutations, suggesting a potential yield of a more infectious virus.

This study evaluates the accuracy of liquid chromatography-mass spectrometry (LC-MS) and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF) for detecting carbapenem hydrolytic activity among SPM-1-, GIM-1-, and GES-5-producing Pseudomonas aeruginosa isolates and OXA-143-, IMP-10-, and OXA-58-producing Acinetobacter baumannii isolates. Class A and B carbapenemase activities were rapidly detected by MALDI-TOF in a 2-h assay. However, an extended incubation time was necessary for detection of carbapenem-hydrolyzing class D ␤-lactamase (CHDL) activity in Acinetobacter spp. Over the last two decades, the therapeutic options to treat infections caused by Gram-negative rods have been narrowed by bacterial acquisition of carbapenemase-encoding genes (1). The rapid detection of clinically significant carbapenemases is important for establishing adequate therapy and controlling their spread. Recently, the detection of class A or B carbapenemase activity has been accomplished by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) (2, 3). Although MALDI-TOF MS seems to be a promising tool, there is a lack of evidence showing its usefulness in testing a broader range of carbapenemases. To date, the published studies have mainly focused on KPC-2-and NDM-1-producing Enterobacteriaceae, IMP-1-, VIM-1-, and VIM-2-producing Pseudomonas aeruginosa, and OXA-23-and OXA-24-producing Acinetobacter baumannii isolates (2-4). In the current study, we evaluated the performance of the MALDI-TOF MS assay in detecting carbapenemase activity of GES-5-, GIM-1-, or SPM-1-producing P. aerugi- 128-Ͼ256 64-Ͼ256 64 (7/11) 100 (11/11) 100 (11/11) 100 (11/11) 100 (11/11) 100 (11/11) IMP-1 1 Ͼ256 32 Ͼ256 100 (1/1) 100 (1/1) 100 (1/1) 100 (1/1) 100 (1/1) 100 (1/1) VIM-1 2 256-Ͼ256 256 256 0 (0/2) 100 (2/2) 100 (2/2) 100 (2/2) 100 (2/2) 100 (2/2) GIM-1 4 Ͼ256 128-256 256-Ͼ256 100 (4/4) 100 (4/4) 100 (4/4) 100 (4/4) 100 (4/4) 100 (4/4) Negative control 6 4-Ͼ256 0.5-16 Ͻ0.06-32 0 (0/6) 0 (0/6) 0 (0/6) 0 (0/6) 0 (0/6) 0 (0/6) a ETP, ertapenem; IPM, imipenem; MEM, meropenem; MHT, modified Hodge test; MALDI-TOF, matrix-assisted laser desorption ionization-time of flight mass spectrometry; LC-MS, liquid chromatography-mass spectrometry.

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The MALDI-TOF MS carbapenemase assay is a feasible and rapid test to identify carbapenemase activity directly from blood culture vials. It may contribute to faster readjustment of empirical antimicrobial therapy and implementation of infection control measures.

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