A KLUYVERA-CRYOCRESCENS STRAIN FROM A GALLBLADDER INFECTION

The isolation and the identification of a pure-culture Kluyvera cryocrescens strain in a gall.bladder pus specimen from a 76-year-old woman vcith acute cholecystitis is described. This is the first Teported recovery of a K. cryocrescens strain from such a sample

T-MOD PATHWAY, A REDUCED SEQUENCE FOR IDENTIFICATION OF GRAM-NEGATIVE URINARY-TRACT PATHOGENS

In this paper, we describe a reduced sequence of identification that includes T-mod medium, a selective and differential isolation medium which allows accurate presumptive identification of the most common gramnegative bacteria encountered in urine samples. The present study, performed on bacteria isolated from 1,762 independent urine samples, has shown that a few selected tests (lysine and ornithine decarboxylase, urease and trehalose fermentation tests) improve the identification accuracy of T-mod, making it possible both to identify the less frequent species and to prevent some misidentifications of Klebsiella pneumoniae and Proteus mirabilis. The proposed work flow agreed with conventional identification protocols to a 99.3% extent and allowed identification of 87.4% of the isolates directly from the primary plate, 11.4% after 1 to 3 additional tests, and 1.2% after an identification gallery

NEW PLATE MEDIUM FOR SCREENING AND PRESUMPTIVE IDENTIFICATION OF GRAM-NEGATIVE URINARY-TRACT PATHOGENS

A new selective, differential plating medium to screen the common gram-negative urinary tract pathogens is described. The medium combines adonitol fermentation, phenylalanine deaminase, and P-glucuronidase tests and allows the indole and cytochrome oxidase tests to be performed directly from the plates. High-level agreement with individual conventional tests was recorded in comparative studies with 504 cultures of gram-negative rods. There was 100% agreement, except for the Providencia spp. indole spot test (61.6% agreement). Adonitol fermentation by Providencia species could not be determined. Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, and Pseudomonas aeruginosa were identified with a high efficiency (100, 85.7, 83.5, and 100% agreement, respectively) without further testing. There was 96% overall agreement for the 267 infected urine samples tested

Characterization and sequence of PhoC, the principal phosphate-irrepressible acid phosphatase of Morganella morganii.

Phosphatase activities were investigated in Morganella morganii, which is one of the few enterobacterial species producing high-level phosphateirrepressible acid phosphatase activity (HPAP phenotype), and the gene encoding the major phosphate-irrepressible acid phosphatase was cloned, sequenced, and its product characterized. Using p-nitrophenyl phosphate as substrate, Morganella produced a major phosphate-irrepressible acid phosphatase (named PhoC) which is associated with the HPAP phenotype, a minor phosphate-irrepressible acid phosphatase, and a phosphate-repressible alkaline phosphatase. The presence of the PhoC activity prevented induction of alkaline phosphatase when a PhoC-hydrolysable organic phosphate ester, such as glycerol &phosphate, was the sole phosphate source. PhoC is a secreted nonspecific acid phosphatase apparently composed of four 25 kDa polypeptide subunits. The enzyme is resistant to EDTA, Pi# fluoride and tartrate. The M. morganii PhoC showed 84.6% amino acid sequence identity to the PhoN nonspecific acid phosphatase of Providencia stuartii, 45.3 O/O to the PhoN nonspecific acid phosphatase of Salmonella typhimurium, and 3708% to the principal acid phosphatase (PhoC) of Zymomonas mobilis. Comparison of sequence data and of regulation of these enzymes suggested a different phylogeny of members of this gene family within the Enterobacteriaceae

Species diversity, spatial distribution, and virulence associated genes of culturable vibrios in a brackish coastal Mediterranean environment

The Vibrio genus is widespread in marine and brackish environments, and several species are human and animal pathogens of global importance. Vibrios adapt rapidly to many environmental stresses, so that brackish environments can be both a suitable niche and a possible reservoir for them. To test the occurrence of culturable vibrios and their possible correlation with environmental factors in a temperate brackish environment, a 1year sampling study was performed in three brackish ponds located along the Central Thyrrenian coast in the Macchiatonda Nature Reserve (Santa Marinella, district of Rome, Italy). Molecular methods were used to detect Vibrio cholerae, Vibrio arahaemolyticus, and Vibrio vulnificus pathogenicity associated genes among the Vibrio isolates. Out of 130 Vibrio isolates identified by sequencing a recA fragment, 70 harbored virulence associated genes including ctx, ace, tcpA, tdh, trh, vvhA, vllY, and toxRS, so confirming the spread of virulence determinants across the environmental isolates. Ecological analysis showed that, although the water temperature is known to be a strong predictor of abundance and distribution of vibrios, its influence accounts for 27 % of the observed variance in the Macchiatonda samples, increasing to 40 % when combined with salinity

The molecular class C acid phosphatase of Chryseobacterium meningosepticum (OlpA) is a broad-spectrum nucleotidase with preferential activity on 5'-nucleotides

The olpA gene of Chryseobacterium meningosepticum, encoding a molecular class C phosphatase, was cloned and expressed in Escherichia coli. The gene encodes a 29-kDa polypeptide containing an amino-terminal signal peptide typical of bacterial membrane lipoproteins. Expression in E. coli results in a functional product that mostly partitions in the outer membrane. A secreted soluble OlpA derivative (sOlpA) lacking the N-terminal cysteine residue for lipid anchoring was produced in E. coli and purified by means of two steps of ion exchange chromatography. Analysis of the kinetic parameters of sOlpA with several organic phosphoesters revealed that the enzyme was able to efficiently hydrolyze nucleotide monophosphates, with a strong preference for 5'-nucleotides and for 3'-AMP. The enzyme was also able to hydrolyze sugar phosphates and beta-glycerol phosphate, although with a lower efficiency, whereas it was apparently inactive against nucleotide di- and triphosphates, diesters, and phytate. OlpA, therefore, can be considered a broad-spectrum nucleotidase with preference for 5'-nucleotides. Its functional behaviour exhibits differences from that of the Haemophilus influenzae OMP P4 lipoprotein, revealing functional heterogeneity among phosphatases of molecular class C

A structure-based proposal for the catalytic mechanism of the bacterial acid phosphatase AphA belonging to the DDDD superfamily of phosphohydrolases

The Escherichia coli gene aphA codes for a periplasmic acid phosphatase called AphA, belonging to class B bacterial phosphatases, which is part of the DDDD superfamily of phosphohydrolases. After our first report about its crystal structure, we have started a series of crystallographic studies aimed at understanding of the catalytic mechanism of the enzyme. Here, we report three crystal structures of the AphA enzyme in complex with the hydrolysis products of nucleoside monophosphate substrates and a fourth with a proposed intermediate analogue that appears to be covalently bound to the enzyme. Comparison with the native enzyme structure and with the available X-ray structures of different phosphatases provides clues about the enzyme chemistry and allows us to propose a catalytic mechanism for AphA, and to discuss it with respect to the mechanism of other bacterial and human phosphatases. (c) 2005 Elsevier Ltd. All rights reserved

Uneven frequency of Vibrio alginolyticus-group isolates among different populations of Galápagos marine iguana (Amblyrhynchus cristatus)

The presence of Vibrio isolates was investigated in cloacal swabs from the Galápagos marine iguana (Amblyrhyncus cristatus). Such unique iguana is endemic to the Galápagos Archipelago, it is listed as vulnerable in the IUCN Red List (2009), and is strictly protected by CITES and Ecuador laws. Our results revealed an uneven isolation frequency of vibrios from animals living in different settings: maximal among the Santa Fe population, scarce at Bahía Tortuga but practically absent in the samples from Puerto Ayora and Plaza Sur. A 16S sequencing confirmed that the isolates belonged to the genus Vibrio, placing them within the V. alginolyticus group; the biochemical identification was, indeed, consistent with V. alginolyticus features. The reason of the observed discrepancy is not clear, but could be either linked to a higher pollution in the inhabited or more touristic places or to differential influence of chemical and physical parameters at a local scale. As V. alginolyticus is an opportunistic pathogen for man and it is known to cause disease in sea-living animals, the ability of these vibrios to enter and persist to a certain extent in the marine iguana gut should be regarded as a risk for health of both the animals and the human personnel involved in monitoring activities

Biochemical characterization of the POM-1 metallo-β-lactamase from Pseudomonas otitidis

The POM-1 metallo--lactamase is a subclass B3 resident enzyme produced by Pseudomonas otitidis, a pathogen causing otic infections. The enzyme was overproduced in Escherichia coli BL21(DE3), purified by chromatography, and subjected to structural and functional analysis. The purified POM-1 is a tetrameric enzyme of broad substrate specificity with higher catalytic activities with penicillins and carbapenems than with cephalosporins