Validation of a novel method to identify healthcare-associated infections.

Despite its potential for use in large-scale analyses, previous attempts to utilise administrative data to identify healthcare-associated infections (HAI) have been shown to be unsuccessful. In this study, we validate the accuracy of a novel method of HAI identification based on antibiotic utilisation patterns derived from administrative data. We contemporaneously and independently identified HAIs using both chart review analysis and our method from four Japanese hospitals (N=584). The accuracy of our method was quantified using sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) relative to chart review analysis. We also analysed the inter-rater agreement between both identification methods using Cohen's kappa coefficient. Our method showed a sensitivity of 0.93 (95% CI: 0.87-0.96), specificity of 0.91 (0.89-0.94), PPV of 0.75 (0.68-0.81) and NPV of 0.98 (0.96-0.99). A kappa coefficient of 0.78 indicated a relatively high level of agreement between the two methods. Our results show that our method has sufficient validity for identification of HAIs in large groups of patients, though the relatively lower PPV may imply limited utilisation in the pinpointing of individual infections. Our method may have applications in large-scale HAI identification, risk-adjusted multicentre studies involving cost of illness, or even as the starting point of future cost-effectiveness analyses of HAI control measures.

Low-intensity pulsed ultrasound accelerates periodontal wound healing after flap surgery.

BACKGROUND AND OBJECTIVE: A study was conducted to evaluate the effects of low-intensity pulsed ultrasound on wound healing in periodontal tissues after mucoperiosteal flap surgery. MATERIAL AND METHODS: Bony defects were surgically produced bilaterally at the mesial roots of the mandibular fourth premolars in four beagle dogs. The flaps were repositioned to cover the defects and sutured after scaling and planing of the root surface to remove cementum. The affected area in the experimental group was exposed to low-intensity pulsed ultrasound, daily for 20 min, for a period of 4 wk from postoperative day 1 using a probe, 13 mm in diameter. On the control side, no ultrasound was emitted from the probe placed contralaterally. After the experiment, tissue samples were dissected out and fixed in 10% formalin for histological and immunohistochemical analyses. RESULTS: The experimental group showed that the processes in regeneration of both cementum and mandibular bone were accelerated by low-intensity pulsed ultrasound compared with the control group. In addition, the expression level of heat shock protein 70 was higher in the gingival epithelial cells of the low-intensity pulsed ultrasound-treated tooth. CONCLUSION: Our results suggest that osteoblasts, as well as cells in periodontal ligament and gingival epithelium, respond to mechanical stress loaded by low-intensity pulsed ultrasound, and that ultrasound accelerates periodontal wound healing and bone repair.

Dipeptidyl peptidase IV on activated T cells as a target molecule for therapy of rheumatoid arthritis.

The extracellular domain of the T cell co-stimulatory molecule CD26 possesses dipeptidyl peptidase IV (DP IV) enzyme activity. Activated T cells are known to increase expression of cell surface DP IV and some specific inhibitors of this enzyme have been reported to suppress T cell function. Previously we have identified a DP IV inhibitor, designated TMC-2, found in culture supernatant of Aspergillus oryzae. Administration of TMC-2 to rats with adjuvant arthritis caused marked suppression of paw swelling. To elucidate the mechanism of TMC-2 antiarthritic activity, we have studied its effects on T cell function. Here we show that TMC-2 inhibited DP IV activity of CD26 immunoprecipitated from T cell lysates, and also inhibited proliferative responses of T cells to specific antigen or anti-CD3 antibody. Suppression of IL-2 production was demonstrated at both the mRNA and protein levels. TMC-2 did not alter the PTPase activity of pure CD45, but when this molecule was co-precipitated from T cell lysates together with associated CD26, its PTPase was virtually completely abolished by TMC-2. These results suggest that modulation of CD45 PTPase activity might be responsible for functional suppression of T cells by TMC-2. Because the effects of TMC-2 on T cells were reversible and it was not toxic at the concentrations used, TMC-2 may be a candidate novel therapeutic agent for rheumatoid arthritis.

Cloning and characterization of the ddc homolog encoding L-2,4-diaminobutyrate decarboxylase in Enterobacter aerogenes.

L-2,4-diaminobutyrate decarboxylase (DABA DC) catalyzes the formation of 1,3-diaminopropane (DAP) from DABA. In the present study, the ddc gene encoding DABA DC from Enterobacter aerogenes ATCC 13048 was cloned and characterized. Determination of the nucleotide sequence revealed an open reading frame of 1470 bp encoding a 53659-Da protein of 490 amino acids, whose deduced NH2-terminal sequence was identical to that of purified DABA DC from E. aerogenes. The deduced amino acid sequence was highly similar to those of Acinetobacter baumannii and Haemophilus influenzae DABA DCs encoded by the ddc genes. The lysine-307 of the E. aerogenes DABA DC was identified as the pyridoxal 5'-phosphate binding residue by site-directed mutagenesis. Furthermore, PCR analysis revealed the distribution of E. aerogenes ddc homologs in some other species of Enterobacteriaceae. Such a relatively wide occurrence of the ddc homologs implies biological significance of DABA DC and its product DAP.

Two genes involved in the 1,3-diaminopropane production pathway in Haemophilus influenzae.

We previously cloned and sequenced the Acinetobacter baumannii dat and ddc genes encoding L-2,4-diaminobutyrate: 2-ketoglutarate 4-aminotransferase (DABA AT) and DABA decarboxylase, respectively, involved in the 1,3-diaminopropane (DAP) production pathway. Homology searches of the gene products provided an indication that a similar gene cluster is present in the genome of Haemophilus influenzae Rd. This was first verified by detection of the corresponding enzyme activities in and the production of DAP by all H. influenzae strains examined. Both of the crude enzymes from the representative strain of H. influenzae showed catalytic properties essentially similar to the A. baumannii DABA AT and DABA DC. An Escherichia coli clone carrying the dat homolog of H. influenzae Rd showed a high level of DABA AT activity, the enzyme protein responsible being detected by immunoblot analysis. These results specify the two H. influenzae genes involved in DAP production.

Identification and analysis of a gene encoding L-2,4-diaminobutyrate:2-ketoglutarate 4-aminotransferase involved in the 1,3-diaminopropane production pathway in Acinetobacter baumannii.

The ca. 2.2-kbp region upstream of the ddc gene encoding L-2,4-diaminobutyrate decarboxylase in Acinetobacter baumannii was sequenced and found to contain another open reading frame of 1,338 nucleotides encoding a protein with a deduced molecular mass of 47,423 Da. Analysis of the homologies observed from the deduced amino acid sequence indicated that the gene product is an enzyme belonging to subgroup II of the aminotransferases. This was first verified when examination of the crude extracts from Escherichia coli transformants led to detection of a novel aminotransferase activity catalyzing the following reversible reactions: L-2,4-diaminobutyric acid + 2-ketoglutaric acid<-->L-glutamic acid + L-aspartic beta-semialdehyde. Further confirmation was obtained when the gene was overexpressed in E. coli and the corresponding protein was purified to homogeneity. It catalyzed the same reactions and its N-terminal amino acid sequence was consistent with that deduced from the nucleotide sequence. Therefore, the gene and its product were named dat and L-2,4-diaminobutyrate:2-ketoglutarate 4-aminotransferase (DABA AT), respectively. Feeding experiments of A. baumannii with L-[U-14C]aspartic acid resulted in the incorporation of the label into 1,3-diaminopropane. Apparent homologs of dat and DABA AT were detected in other Acinetobacter species by PCR amplification and Western blotting. These results indicate that the dat gene (as well as the ddc gene) participates in the synthesis of 1,3-diaminopropane, the only diamine found in this genus. However, the biological role, if one exists, of 1,3-diaminopropane synthesis is unknown.

Cloning and sequencing of the Vibrio parahaemolyticus fur gene.

A ferric uptake regulatory gene (fur) was cloned from Vibrio parahaemolyticus WP1 by a polymerase chain reaction-based technique followed by functional complementation of a fur mutation in Escherichia coli. A sequence analysis showed that, at the amino acid level, the V. parahaemolyticus Fur protein is 81% identical with the Fur protein from E. coli and over 90% identical with those of the Vibrio species.

Occurrence of a novel L-2,4-diaminobutyrate decarboxylase activity in some species of Enterobacteriaceae, and purification and characterization of the enzymes of Enterobacter aerogenes and Serratia marcescens.

L-2,4-Diaminobutyrate decarboxylase (DABA DC) is a novel enzyme yielding 1,3-diaminopropane (DAP) from DABA, which has previously been purified from strains of the genera Vibrio and Acinetobacter. In this study, we also detected DABA DC activity in the species of Enterobacteriaceae: E. aerogenes, E. cloacae, E. agglomerans, Serratia marcescens, S. liquefaciens, Klebsiella pneumoniace, K. oxytoca and Citrobacter freundii, all of which produced DAP in sufficient amounts. Subsequently, the DABA DCs of E. aerogenes and S. marcescens were purified to homogeneity and characterized. Two separate enzymes had similar properties with respect to chromatographic behaviors, and were a dimer with subunits of identical molecular mass of about 51 kDa. The maximal activity of each enzyme was obtained at pH 8.0-8.25. Both enzymes required pyridoxal 5'-phosphate and Mg2+ for full activity, and were highly specific for L-DABA. There was immunological similarity, but not identity between these proteins, as determined by Ouchterlony double diffusion analysis with antiserum against the E. aerogenes DABA DC. They showed the same N-terminal amino acid sequence up to the 8th residue (S-K-L-N-P-I-L-A-). These enzymes were different in molecular mass, N-terminal amino acid sequence and antigenicity from DABA DCs of Acientobacter and Vibrio species.

Sequence analysis of the gene encoding a novel L-2,4-diaminobutyrate decarboxylase of Acinetobacter baumannii: similarity to the group II amino acid decarboxylases.

The gene (ddc) encoding a novel enzyme, l-2,4-diaminobutyrate decarboxylase (DABA-DC; EC 4.1.1.-) in Acinetobacter baumannii was sequenced, and an open reading frame of 1,530 nucleotides was detected. The sequence of 20 N-terminal amino acids of purified DABA-DC and of its proteolytic peptide fragments coincided with those deduced from the nucleotide sequence determined. Comparison of the predicted amino acid sequence of the A. baumannii enzyme with those of other pyridoxal 5'-phosphate-dependent decarboxylases revealed significant similarity to the group II amino acid decarboxylases and conservation of the putative pyridoxal 5'-phosphate-binding domain.

Occurrence and antigenic heterogeneity of L-2,4-diaminobutyrate decarboxylase in Acinetobacter species.

We have previously reported that a novel enzyme, L-2,4-diaminobutyrate decarboxylase (DABA DC), which is responsible for the formation of 1,3-diaminopropane, occurs in two Acinetobacter species. The present study extends this observation to additional Acinetobacter species and strains (6 reference strains and 30 clinical isolates). Furthermore, the DABA DC protein was detected in every strain by Western blot analysis with the antiserum against the enzyme purified from A. baumannii ATCC 19606. However, only the DABA DCs in the A. calcoaceticus and Acinetobacter genospecies 3 in addition to A. baumannii strains strongly cross-reacted with the antiserum, suggesting antigenic heterogeneity among the DABA DC proteins in Acinetobacter species. Therefore, immunological testing of the DABA DC protein may provide an additional method for differentiating and identifying Acinetobacter strains.

Cloning and expression in Escherichia coli of the gene encoding a novel L-2,4-diaminobutyrate decarboxylase of Acinetobacter baumannii.

The gene encoding L-2,4-diaminobutyrate decarboxylase (DABA DC) was cloned from Acinetobacter baumannii ATCC 19606. The gene was evidently under the control of its own promoter. Interestingly, the host carrying this clone also produced an appreciable amount of 1,3-diaminopropane. Restriction mapping and subsequent subcloning of the cloned insert localized the DABA DC gene within a 2.45-kb SphI/EcoRI fragment. For endogenous production of DAP, a 1.75-kb EcoRI/PstI region downstream from the DABA DC gene was further required. Southern blot hybridization revealed some heterogeneity in the DABA DC genes among other Acinetobacter species.