Conserved sequence motifs among bacterial, eukaryotic, and archaeal phosphatases that define a new phosphohydrolase superfamily.

Members of a new molecular family of bacterial nonspecific acid phosphatases (NSAPs), indicated as class C, were found to share significant sequence similarities to bacterial class B NSAPs and to some plant acid phosphatases, representing the first example of a family of bacterial NSAPs that has a relatively close eukaryotic counterpart. Despite the lack of an overall similarity, conserved sequence motifs were also identified among the above enzyme families (class B and class C bacterial NSAPs, and related plant phosphatases) and several other families of phosphohydrolases, including bacterial phosphoglycolate phosphatases, histidinol-phosphatase domains of the bacterial bifunctional enzymes imidazole-glycerolphosphate dehydratases, and bacterial, eukaryotic, and archaeal phosphoserine phosphatases and threalose-6-phosphatases. These conserved motifs are clustered within two domains, separated by a variable spacer region, according to the pattern [FILMAVT]-D-[ILFRMVY]-D-[GSNDE]-[TV]-[ILVAM]-[AT S VILMC]-X-¿YFWHKR)-X-¿YFWHNQ¿-X( 102,191)-¿KRHNQ¿-G-D-¿FYWHILVMC¿-¿QNH¿-¿FWYGP¿-D -¿PSNQYW¿. The dephosphorylating activity common to all these proteins supports the definition of this phosphatase motif and the inclusion of these enzymes into a superfamily of phosphohydrolases that we propose to indicate as "DDDD" after the presence of the four invariant aspartate residues. Database searches retrieved various hypothetical proteins of unknown function containing this or similar motifs, for which a phosphohydrolase activity could be hypothesized.

Identification of an Escherichia coli periplasmic acid phosphatase containing of a 27 kDa-polypeptide component.

An acid phosphatase containing a 27-kDa polypeptide component has been identified in Escherichia coli by means of a zymogram technique. The enzyme is secreted in the periplasmic space and is able to hydrolyze several organic phosphate esters, but not diesters, showing preferential activity on p-nitrophenyl phosphate and other phenolic phosphate esters. Production of the enzyme apparently occurs only in cells growing on carbon sources other than glucose.

Identification of the gene (aphA) encoding the class B acid phosphatase/phosphotransferase of Escherichia coli MG1655 and characterization of its product.

An open reading frame located in the tyrB-uvrA intergenic region of the Escherichia coli MG1655 chromosome was identified as encoding the class B acid phosphatase of this species on the basis of cloning and expression experiments. A protocol for purification of the enzyme (named AphA) was developed, and its properties were analyzed. The enzyme is a 100-kDa homotetrameric protein which apparently requires a metal co-factor for activity. Similarly to other bacterial class B acid phosphatases, it is able to dephosphorylate several organic phosphomonoesters as well as to catalyze the transfer of low-energy phosphate groups from phosphomonoesters to hydroxyl groups of various organic compounds.

Genetic rearrangements in the tyrB-uvrA region of the enterobacterial chromosome: a potential cause for different class B acid phosphatase regulation in Salmonella enterica and Escherichia coli.

Unlike in Escherichia coli, in Salmonella enterica production of class B acid phosphatase (AphA) was detectable also in cells growing in the presence of glucose. Characterization of the aphA locus from a S. enterica ser. typhi strain showed that the aphA determinant is very similar to the E. coli homolog, and that its chromosomal location between the highly conserved tyrB and uvrA genes is retained. However, the aphA flanking regions were found to be markedly different in the two species, either between tyrB and aphA or between aphA and uvrA. The differences in the aphA 5'-flanking region, which in S. enterica is considerably shorter than in E. coli (183 vs. 1121 bp) and includes potential promoter sequences not present in E. coli, could be responsible for the different regulation of class B acid phosphatase observed in the two species.

Bacterial acid phosphatase gene fusions useful as targets for cloning-dependent insertional inactivation.

The Morganella morganii phoC gene, encoding a class A acid phosphatase, was used to generate gene fusions with modified amino-terminal moieties of the Escherichia coli lacZ gene carrying a multiple-cloning site flanked by phage-specific promoters and recognition sites for universal sequencing primers. The corresponding hybrid proteins retained a PhoC-like enzymatic activity which is easily detectable by a plate histochemical assay, rendering similar gene fusions potentially useful as targets for cloning-dependent insertional inactivation. Cloning experiments performed in plasmids carrying similar lacZ-phoC fusions confirmed their usefulness as cloning vectors for direct screening of recombinants. As compared to conventional lacZ alpha-complementation-based vectors, which can only be used in E. coli hosts carrying specific lacZ mutations, the lacZ-phoC fusion-based vectors can be used in combination with any E. coli host and require a less expensive histochemical assay for screening of recombinants, while retaining all the advantageous features that made the former so popular as general purpose cloning vehicles.

Effect of the inoculum size on susceptibility tests performed on sessily growing bacteria.

It has been clearly established that the inoculum size greatly affects the results of antibiotic susceptibility tests performed in both liquid and solid media in standard laboratory growth conditions (i.e. planktonic). Recently methods were developed to perform antibiotic susceptibility tests on bacteria growing in sessile conditions. The present study investigates the effect of the inoculum size on results obtained by these methods. Results show that the inoculum size does not affect tests performed in sessile conditions. A simple and reliable method is proposed to be applied to routine microbiological laboratory procedures.

Effect of slime production on the antibiotic susceptibility of isolates from prosthetic infections.

The antibacterial activity of 6 antibiotics towards 10 gram-positive and 6 gram-negative glycocalyx-producing strains, has been evaluated by employing a method which partially simulates the in vivo colonization of prosthetic devices. The results showed that routine antibiotic sensitivity tests are not predictive about the response of the glycocalyx-embedded bacteria, and that prophylaxis may be useful with ofloxacin and clindamycin, before placing a prosthetic device. Once bacterial colonization had already occurred, however, none of the tested antibiotics was able to eradicate the sessile bacterial form. The minimum bactericidal concentration (MBC) values, indeed, were much higher than those determined on the planktonic form, and were much higher than serum and tissue levels that can be reached in vivo.

Taxonomy and identification of periodontopathogenic bacteria and related species.

The tumultuous evolution of oral and periodontal microbiology has focussed the attention of many researchers on the necessity to clarify the taxonomic position and identification criteria of putative periodontopathogens, in order to elucidate the role played by each species in the pathogenesis of periodontal disease. Many of the most important periodontopathogens recently underwent a radical reclassification process that may create some confusion and certainly deserves an accurate analysis aimed at focussing the actual situation of these bacteria. The taxonomic evolution and identification criteria of species of the genera Bacteroides, Prevotella, Porphyromonas and Actinobacillus is here analyzed in detail to clarify this recent evolutive taxonomic process, and to explain the importance of molecular studies for both taxonomic and identification purposes in this field.

A rapid staining procedure to demonstrate glycocalyx production and bacterial biofilms.

A novel staining procedure to demonstrate glycocalyx production by clinical isolates is presented. The short times required, specificity and sensitivity suggest that the staining could be applied to routine in vitro diagnostic procedures.

[A new diagnostic scheme for the identification of bacterial species belonging to the Enterobacteriaceae family]. / Utilizzazione di un nuovo schema diagnostico per l'identificazione di specie di germi appartenenti alla famiglia delle Enterobacteriaceae.

A new diagnostic two-steps scheme for the species identification of enterobacteria was employed with 703 strains. The diagnoses were compared with those obtained by employing Api 20 E and Enterotube II. On the basis of the reported results this new scheme is shown to be reliable (99.71% right diagnosis) and easy to perform.

[Up-to-date diagnostic method for the diagnosis of environmental species of the Enterobacteriaceae family]. / Schema diagnostico aggiornato per la diagnosi di specie nell'ambito della famiglia delle Enterobacteriaceae.

The Authors propose a new two-steps key for the identification of the species belonging to the Enterobacteriaceae Family. This key can be employed both with standard tube methods and most commercial kits. The proposed method is a simple, easy and rapid one; moreover it considers each genus and species of Enterobacteriaceae Family, associated with human pathology, which has been defined within the month May, 1984.

o-Nitrophenyl-beta-D-galactopyranoside-urease-indole broth, a new composite tube medium for Salmonella screening.

A new composite broth medium combining o-nitrophenyl-beta-D-galactopyranoside (ONPG) and urease and indole tests in a single tube is described. High-level agreement with individual conventional tests was recorded in comparative studies with 2,412 cultures of members of the family Enterobacteriaceae, i.e., 100% agreement with the exception of Hafnia spp. (96.3% agreement) for the ONPG test and Citrobacter, Enterobacter, and Hafnia spp. (75, 86.4, and 98.2% agreement, respectively) for the urease test. The new medium seems especially promising as a screen for Salmonella subgroup I which encompasses most pathogenic Salmonella species other than the Arizona subgroup.

Characterization of yellow-pigmented enterococci from severe human infections.

Four strains of yellow-pigmented enterococci that resembled the species Enterococcus casseliflavus were isolated from patients who had undergone surgical treatment. They were substantially homologous in terms of biochemical properties, antibiotic susceptibilities, and plasmid DNA profiles. Yellow-pigmented enterococci could be another potentially important cause of nosocomial infection in surgical units.

Notes on the appearance of double and triple anomalies in Escherichia coli strains.

The authors report the appearing of Escherichia coli strains differing from typical E. coli patterns as lacking in indole production, lactose fermentation and motility and as some of them are positive for malonate test. On 131 isolates studied, 22 strains carried double or triple anomalies.

A Kluyvera cryocrescens strain from a gall-bladder 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 with acute cholecystitis is described. This is the first reported recovery of a K. cryocrescens strain from such a sample.

Commercial identification systems often fail to identify Providencia stuartii.

We tested 145 clinical isolates in an attempt to evaluate some of the most widely used commercial identification systems in Europe in terms of their ability to identify Providencia strains. Two manual miniaturized systems (API 20E and Enterotube II) and three mechanized-automated systems (Cobas-Bact, Sceptor System, and Titertek-Enterobac-Rapid Automated System) were evaluated. Providencia alcalifaciens and Providencia rettgeri strains were correctly identified by all systems in all cases, and in most cases identification was achieved without the aid of supplementary tube tests. By contrast, Providencia stuartii was identified without the aid of supplementary tube tests for only 42.5% (API 20E), 37.5% (Enterotube), 68.7% (Sceptor), and 71.2% (Cobas-Bact) of the isolates. The overall misidentification rates were 16.3, 11.3, 11.3, and 10%, respectively. The Titertek-Enterobac-Rapid Automated System failed to identify only 1 of 80 strains (1.3%) and required supplementary tests in 2 other cases (2.5%). Since four of the multitest systems examined often failed to correctly identify P. stuartii, we conclude that supplementary conventional tube tests should always be used to distinguish this species from the other taxa of the Proteeae tribe.

Unusual behaviour of Klebsiella rhinoscleromatis strains on API 20E strips.

Seven Klebsiella rhinoscleromatis standard cultures and two wild isolates were examined for their responses to Api 20E (API System, S.A.) strips. Several strains yield incostant results for arabinose fermentation test in Api strips and, when positive, they were not identified. The arabinose positive test indeed, led to the numerical profiles 0004773 (not mentioned in the analytical catalogue), or 0004553 (two strains) corresponding to a Klebsiella ozaenae identification. The mathematical analysis of the biochemical results confirmed the identity of the strains as K. rhinoscleromatis.

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 beta-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.

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 gram-negative 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.