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

Efficacy of acoustic waves in preventing Streptococcus mutans adhesion on dental unit water line

Background: nei riuniti odontoiatrici, la qualità dell'acqua utilizzata per la refrigerazione e il risciacquo di manipoli, siringhe e altri componenti è un aspetto di notevole importanza sanitaria. L'acqua attraversa questi dispositivi mediante un circuito interconnesso di tubi di piccole dimensioni (circa 2 mm di diametro), denominato “dental unit water line” (DUWL). I DUWL possono essere fortemente colonizzati da varie specie batteriche sia in fase planctonica, che adesi o organizzati in biofilm, rappresentando una potenziale causa di infezione, non solo per i professionisti che usano abitualmente questi dispositivi, ma anche per pazienti occasionali, in particolare per i pazienti immunocompromessi. La contaminazione dei DUWL può essere prevenuta o ridotta con l'uso dei disinfettanti, ma l'eradicazione dei microrganismi adesi alle superfici interne dei DUWL o organizzati in forma di biofilm, è una sfida assai più complessa e spesso i normali metodi di disinfezione non sono pienamente efficaci. Inoltre, in ambito odontoiatrico, i disinfettanti utilizzati abitualmente per disinfettare i DUWL possono alterare la capacità adesiva del materiale utilizzato nella pratica restaurativa. Obiettivi: individuare una strategia innovativa, in grado di contrastare l'adesione batterica alle superfici dei DUWL mediante un approccio di tipo fisico, che sia più efficace nel superare il problema della contaminazione dei DUWL e ridurre il rischio di infezione rispetto ai normali metodi già in uso. A tal fine, fra le molte specie batteriche potenzialmente riscontrabili nei circuiti idrici odontoiatrici, si è deciso di avviare questo studio pilota utilizzando la specie batterica patogena S. mutans, per il suo indubbio interesse in ambito odontoiatrico e per la sua spiccata capacità di aderire e persistere su superfici inerti. Metodi: utilizzo di onde acustiche elastiche ad alta energia nel contrastare l'adesione di Streptococcus mutans alle pareti interne di un circuito idrico sperimentale riproducente un DUWL. Per evidenziare l’efficacia delle onde acustiche anche in condizioni estreme, è stata utilizzata un’elevata carica contaminante di S. mutans. Risultati: Si osserva una significativa riduzione dei batteri adesi soggetti a trattamento con onde acustiche rispetto al controllo (P = 0,003)

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

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

Biotimer assay: A reliable and rapid method for the evaluation of central venous catheter microbial colonization

Adherent bacteria and biofilm frequently colonize central venous catheters (CVCs). CVC colonization is correlated to infections and particularly to bloodstream ones. The classical microbiological methods to determine of CVC colonization are not fully reliable and are time-consuming. BioTimer Assay (BTA) is a biological method already used to count bacteria adherent to abiotic surfaces and biofilm without sample manipulation. BTA employs specific reagents whose color changed according to bacterial metabolism. BTA is based on the principle that a metabolic reaction will be faster when more bacteria are present in the sample. Therefore, the time required for color changes of BTA reagents determines the number of bacteria present in the sample through a correlation line. Here, for the first time, we applied BTA and a specifically developed laboratory procedure to evaluate CVC colonization in comparison with the routine microbiological method (RMM). 125 CVCs removed from patients for suspected catheter-related bloodstream infection (CRBSI) or at hospital discharge were examined. BTA was reliable in assessing sterility and CVC colonization (100% agreement with RMM) and in recognizing the presence of fermenting or non-fermenting bacteria (97.1% agreement with RMM) shortening the analytical time by between 2- and 3-fold. Moreover, the reliability of BTA as early alert of CRBSI was evaluated. The sensitivity, specificity, positive, and negative predictive values for BTA as an early alert of CRBSI were 100, 40.0, 88.8 and 100%, respectively. In conclusion, BTA and the related laboratory procedure should be incorporated into routine microbiological methods since it can be considered a reliable tool to evaluate CVC colonization in a very short time and a rapid alert for CRBSIs

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

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

Combined use of x-ray fluorescence microscopy, phase contrast imaging for high resolution quantitative iron mapping in inflamed cells

X-ray fluorescence microscopy (XRFM) is a powerful technique to detect and localize elements in cells. To derive information useful for biology and medicine, it is essential not only to localize, but also to map quantitatively the element concentration. Here we applied quantitative XRFM to iron in phagocytic cells. Iron, a primary component of living cells, can become toxic when present in excess. In human fluids, free iron is maintained at 10-18 M concentration thanks to iron binding proteins as lactoferrin (Lf). The iron homeostasis, involving the physiological ratio of iron between tissues/secretions and blood, is strictly regulated by ferroportin, the sole protein able to export iron from cells to blood. Inflammatory processes induced by lipopolysaccharide (LPS) or bacterial pathoge inhibit ferroportin synthesis in epithelial and phagocytic cells thus hindering iron export, increasing intracellular iron and bacterial multiplication. In this respect, Lf is emerging as an important regulator of both iron and inflammatory homeostasis. Here we studied phagocytic cells inflamed by bacterial LPS and untreated or treated with milk derived bovine Lf. Quantitative mapping of iron concentration and mass fraction at high spatial resolution is obtained combining X-ray fluorescence microscopy, atomic force microscopy and synchrotron phase contrast imaging