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PubMed1982

Evaluation of Gram-stain screen and Micro-ID methods for direct identification of Enterobacteriaceae from urines.

A rapid method of urine screening and enterobacterial identification was evaluated. Results indicated that an average of 13.5 bacteria/oil immersion field (threshold value greater than or equal to 1) was observed in unsedimented urine of patients with significant bacteriuria, with an average of less than 1 bacterium/field in urines of patients without significant bacteriuria. In centrifuged urines, numbers of bacteria divided by amount of urine sedimented yielded similar results. Of 1758 urines studied, 136 yielded greater than or equal to 10(5) bacteria/ml, and 58 greater than 10(4) but less than 10(5) bacteria/ml, by conventional techniques. Gram-screening of unsedimented specimens gave sensitivity rates of 94.1%, specificity of 97.7%, and predictive positive and negative values of 78.5%, 99.5%, respectively; similar values were obtained with sedimented urines. Sensitivity rates of both screening methods for the 58 urines with greater than 10(4) but less than 10(5) bacteria/ml were 9.0%, 10.0%, respectively. Total correct enteric identification in 113 urines with positive screens and significant bacteriuria (greater than or equal to 10(5)/ml) was 82.3% and 90.3% with direct saline and broth Micro-ID methods, respectively. In 99 urines yielding pure or predominantly pure growth of 1 species of Enterobacteriaceae identification by direct saline and broth Micro-ID corresponded with isolated colony identification in 85.9%, 94.9% of cases, respectively. Gram-stain screening (together with back-up conventional plating in certain patient categories) and enterobacterial identification by direct broth Micro-ID, of urines with pure stains suggestive of greater than or equal to 10(5) Gram-negative rods/ml has been shown to be useful in laboratories without automated equipment for urine screening.

Bacteriological Techniques↗
PubMed1982

Differential production of acid by Enterobacteriaceae in OF media and in fermentation broth.

Several species of Enterobacteriaceae were checked for acid formation from some carbohydrates or alcohols in OF medium [OF Basal Medium, Baltimore Biological Laboratories] and in a traditional "fermentation broth". After selection of those species that formed acid only oxidatively or fermentatively in OF, it was found that acid formation in the fermentation broth could reflect fermentation or oxidation, but that it was not necessarily related to either. In oxidative species, it occurred less frequently than oxidation in OF and was usually delayed. Fermentative species (in OF) either yielded acid in fermentation broth or did not, depending on the species. Thus, OF media cannot be substituted for traditional broths, although they may be of value in differentiating certain species.

Culture Media↗
PubMed1997

3-Methylaspartate ammonia-lyase as a marker enzyme of the mesaconate pathway for (S)-glutamate fermentation in Enterobacteriaceae.

The enzyme 3-methylaspartase (3-methylaspartate ammonia-lyase, EC 4. 3.1.2) was found in the cells of enteric bacteria, especially in the genera Citrobacter and Morganella, that were grown under anoxic and oxygen-limited conditions. The enzymes were purified to homogeneity from the cell-free extracts of 18 active strains and had similar enzymological properties such as action on columns, specific activity, molecular weight, subunit structure, and N-terminal amino acid sequence similarity. The production of the enzyme was dependent on the limitation of oxygen during growth and was arrested by aeration. The addition of external electron acceptors such as dimethylsulfoxide could support cell growth and production of the enzyme. Activities of glutamate mutase (EC 5.4.99.1) and (S)-citramalate hydrolyase (EC 4.2.1.34), key enzymes of the mesaconate pathway of (S)-glutamate fermentation in the genus Clostridium, were detected in the cells of the active strains grown under oxygen-limited conditions. Based on the results, the mesaconate pathway is proposed to explain the (S)-glutamate fermentation process observed in Enterobacteriaceae, and 3-methylaspartase could be a marker enzyme for this pathway.

Aerobiosis↗
PubMed2005

Screening of antibiotics resistance to Enterobacteriaceae, Pseudomonas aeruginosa, and Acinetobacter baumannii by an advanced expert system.

The VITEK2 advanced expert system (AES) gives information about the antibiotics-resistance mechanisms based on the biological validation derived from the VITEK2 susceptibility result. In this study, we investigated whether or not this system correctly categorized the beta-lactamase resistance mechanism data derived from the VITEK2 susceptibility result using the testing card, AST-N025, with Enterobacteriaceae, Pseudomonas aeruginosa, and Acinetobacter baumannii. We used 131 strains, and their phenotypes were determined according to the biological and genetic screening. The AES analysis result matched the phenotype testing in 120 (91.6%) of the 131 strains. Incorrect findings were found in six strains, including three strains of Serratia marcescens. The resistance mechanism could not be determined in five strains, including three strains of Providencia rettgeri. The analysis of those phenotypes agreed in 34 (97.1%) among 35 strains with extended spectrum beta-lactamase (ESBL), and in 27 (96.4%) among 28 strains with high-level cephalosporinase. The agreement ratio in the phenotype was very high as we expected. The incorrect and nondeterminable samples were strains with relatively high cephalosporinase that has variation of outer membrane protein. The AES was able to detect the phenotype for carbapenemase. The AES is a clinically useful system that allows taking prompt measures to treat patients because it can provide information about the resistance mechanism in less than half a day after starting the analysis.

Acinetobacter baumannii↗
PubMed1993

Pseudomonas aeruginosa and Enterobacteriaceae bacteremia after biliary endoscopy: an outbreak investigation using DNA macrorestriction analysis.

PURPOSE: An outbreak of gram-negative bacteremia in patients undergoing endoscopic retrograde cholangiopancreatography (ERCP) was investigated to determine the sources of infection and to control transmission. PATIENTS, METHODS, AND RESULTS: The incidence of post-ERCP bacteremia increased from 1.6% (60 of 3,696) procedures to 3.6% (53 of 1,454) procedures (relative risk 2.3, p < 0.0001) after endoscopes were processed in a new automated disinfector. Bacteremia involved nine species of Pseudomonas and Enterobacteriaceae, which were also isolated from processed endoscopes. Seven epidemic strains with highly related genomic macrorestriction profiles each infected 2 or more patients, accounting for 29 (55%) episodes of post-ERCP bacteremia. Strains recovered from endoscopes and from the disinfector were associated with 22 (42%) and 5 (9%) bacteremic episodes respectively. Effective endoscope disinfection was achieved by cleansing and disinfection of a blind channel not processed in the disinfector, additional isopropanol-air flush of all channels, and auto-disinfection of the disinfector. In the following period, the incidence of post-ERCP bacteremia returned to the pre-epidemic rate (1.7%, p = 0.0001). CONCLUSION: Bacterial genome fingerprinting by macrorestriction analysis enabled delineation of a multi-strain outbreak of post-ERCP bacteremia. Cross-contamination, and to a lesser extent, common-source contamination, appeared related to inadequate disinfection of endoscopes processed in an automated disinfector.

Bacteremia↗
PubMed1989

Characterization of the structural genes for the DNA-binding protein H-NS in Enterobacteriaceae.

The promoter region of Escherichia coli hns, the structural gene for the DNA-binding protein H-NS, has been identified by use of a promoter search vector and the in vivo transcriptional start point by primer extension analysis. The homologous hns genes of two other Enterobacteriaceae, Proteus vulgaris and Serratia marcescens, were identified by heterologous hybridization with a DNA probe derived from E. coli hns, cloned and sequenced. Taking into account only the invariant nucleotides and amino acids, the homology of H-NS among the three organisms was found to be greater than 70% at the DNA level and greater than 75% at the protein level. The three hns genes were also found to have nearly identical transcriptional and translational signals.

Amino Acid Sequence↗
PubMed1995

Pilins of fimbrial adhesins of different member species of Enterobacteriaceae are structurally similar to the C-terminal half of adhesin proteins.

The structural relatedness of pilins and the C-terminal half of adhesin proteins in different member species of Enterobacteriaceae was deduced from their two-dimensional sequence analysis using the hydrophobic cluster analysis (HCA) and secondary structure predictions from the profile network Hei-Delberg program (PHD). Despite a large evolutionary distance between the two protein families, we show that pilins and the C-terminal domain of adhesins have a similar folding that can serve as modules for pilus assembly.

Adhesins, Bacterial↗
PubMed1985

Evolution of the lipoprotein gene in the enterobacteriaceae. Cloning and DNA sequence of the lpp gene from Proteus mirabilis.

We cloned the lipoprotein gene from Proteus mirabilis and determined its DNA sequence. Comparison with the lpp genes from Escherichia coli, Serratia marcescens, Erwinia amylovora and Morganella morganii revealed several unique features of the evolution of the lpp gene in the Enterobacteriaceae and enabled us to establish phylogenetic relationships between these bacteria.

Base Composition↗
PubMed1988

Functional and structural map of pLST1000: a multiresistance plasmid widely distributed in Enterobacteriaceae.

pLST1000, an 80-kb plasmid found in Enterobacteriaceae in North and South America, harbors the aadB and several other resistance genes. We suggested earlier that, because of its widespread distribution, pLST1000 could act as a carrier plasmid, bringing the aadB gene to new locations. This paper presents the restriction enzyme recognition site and functional map of the plasmid. The resistance genes lie in a discreet region. The aadB and aadA genes form an operon with the aadB gene promoter proximal. This operon is flanked by bla-TEM and bla-OXA2 genes, the former located in a functional Tn3-like transposon. This arrangement is similar to that of relatives of the transposon TN21, where additional resistance genes are precisely inserted in recombinational "hot spot" sequences that flank the aadA gene. We were not able to demonstrate transposition of the aadB gene in Escherichia coli. A sul gene and mer operon lie beyond the bla-OXA2 gene. The transfer genes form a single region, defined by insertions of Tn5-132 that give the Tra- phenotype.

Drug Resistance, Microbial↗
PubMed1993

Contamination of beef carcasses by psychrotrophic Pseudomonas and Enterobacteriaceae at different stages along the processing line.

The extent of the contamination of beef carcasses with psychrotrophic Pseudomonas spp. and Enterobacteriaceae during slaughter, chilling and cutting was estimated by introducing a new analytical procedure; the contamination index. Comparisons were made between the initial viable counts and the contamination index. The contamination index was calculated as the sum of the bacterial counts obtained during aerobic cold storage of excised meat samples. The presence and composition of spoilage bacteria in the slaughter environment and on the carcasses was also determined at one plant. Rapid chilling was identified as a critical processing step by the contamination index. In addition to this, the dehiding and the chilling in cold storage rooms were implicated as critical operations, with respect to aerosol contamination and surface cross-contamination. Comparison of the composition of spoilage bacteria in the slaughter environment and the bacteria proliferating on the carcass surface samples taken at the corresponding steps showed similar distributions of the identified Pseudomonas spp. In five surveys at two plants, the contamination of beef carcasses along the processing line was estimated. Statistically significant variations between different processing steps were more pronounced for the contamination index than for the conventional counts. It was concluded that the contamination index could be used for identifying critical processing steps, with respect to the extent of contamination of carcasses by psychrotrophic spoilage bacteria.

Acinetobacter↗
PubMed1995

Media for 'total' Enterobacteriaceae, coliforms and Escherichia coli.

Selective and enrichment media for the isolation and enumeration of 'total' Enterobacteriaceae, coliforms, faecal coliforms and E. coli from foods are described. The effects of time and temperature of incubation are discussed. Reports of some comparative studies of these media and factors to be considered in the selection of media for this group of organisms are noted and the difficulties associated with the isolation and enumeration of pathogenic serotypes of E. coli considered.

Bacteriological Techniques↗
PubMed1995

Decreased transmission of Enterobacteriaceae with extended-spectrum beta-lactamases in an intensive care unit by nursing reorganization.

In our gastrointestinal surgical intensive care unit (SICU), the large number of patients with multiple enterostomies enhances the risk of nosocomial transmission of gut extended-spectrum beta-lactamase-producing Enterobacteriaceae (ESBLE) by health care workers. A control study performed in our SICU from June-August 1992 showed an ESBLE gut colonization rate of 70%. To reduce this rate, nursing procedures were intensified or modified, particularly handwashing, single-use equipment and waste control. To test the efficiency of these procedures, 64 patients hospitalized for more than two days from September 1992-March 1993 were screened for gut acquisition of ESBLE. Rectal samples were taken within 48 h after admission and then weekly. After nursing reorganization, the ESBLE colonization rate dropped significantly to 40% (P < 0.001). Twenty patients (31.7%) acquired a gut ESBLE, after a mean of 24.3 +/- 13.7 days. Each patient was colonized with one, two or three ESBLE (Klebsiella pneumoniae, Escherichia coli and Enterobacter aerogenes). Baseline characteristics of the 20 colonized and 39 non-colonized patients showed no significant difference (Student's t-test, P > 0.05). The nursing workload, estimated as a omega index, was greater in the colonized group (P < 0.001). These findings show that strict observance of nursing procedures can significantly reduce ESBLE acquisition in a high-risk surgical unit.

Aged↗
PubMed1990

Biotinylated oligonucleotide probes for the detection and the characterization of TEM-type extended broad spectrum beta-lactamases in Enterobacteriaceae.

Point mutations in the nucleotide sequence of the structural genes for the TEM-type penicillinases can broaded their substrate spectrum towards all beta-lactams except cephamycins and imipenem. The presence of such variants on self-transferable plasmids accounts for the dissemination of this new type of resistance to numerous species of Enterobacteriaceae in various countries. We have synthetized biotinylated oligonucleotide probes for the detection and the discrimination of parental and mutated nucleotide sequences of TEM enzymes. Seven clinical isolates belonging to four species and harbouring TEM-1, TEM-3 or TEM-6 were studied. The results obtained indicate that detection of TEM-derived broad spectrum beta-lactamases in clinical isolates of Entero-bacteriaceae is possible with biotinylated oligonucleotide probes.

Base Sequence↗
PubMed1991

Demonstration of formaldehyde dehydrogenase activity in formaldehyde-resistant Enterobacteriaceae.

Clinical isolates of different Enterobacteriaceae strains and genetically modified variants which were resistant to the disinfectant formaldehyde were investigated. In cell-free extracts of all formaldehyde-resistant strains a glutathione-dependent formaldehyde dehydrogenase activity was demonstrated. In contrast cell extracts from formaldehyde sensitive strains did not show any formaldehyde dehydrogenase activity. The enzymatic degradation of formaldehyde seems to play an important role in formaldehyde resistance.

Aldehyde Oxidoreductases↗
PubMed1985

Differentiation of Pseudomonas aeruginosa and Enterobacteriaceae in direct smears based on measurements by scanning electron microscopy.

We believe that experienced observers can often distinguish between Enterobacteriaceae and Pseudomonas aeruginosa, by differences in dimensions in Gram-stained direct smears. Many microbiologists question whether this should be attempted because of overlap in dimensions. We have found that culture results confirm our observations about 80% of the time and that such reporting is helpful in diagnosis and treatment. We decided to try to verify the differences in dimensions objectively. Because of the limitations of making measurements by light microscopy, exudate from clinical specimens and from experimental mouse infections were examined by scanning electron microscopy. Discriminant function analysis was applied to the dimensions of length and width, and this showed that 83% of the organisms in both groups could be correctly classified on the basis of the dimensions. This supports our premise that experienced observers should be able to differentiate between these organisms in Gram-stained direct smears, using light microscopy with sufficient confidence to provide clinically useful information.

Animals↗
PubMedDate not supplied

Ceftibuten and bactericidal kinetics. Comparative in vitro activity against Enterobacteriaceae producing extended spectrum beta-lactamases.

Ceftibuten, compared to cefixime, cefetamet, cefpodoxime, loracarbef, cefprozil, cefuroxime, cefaclor, and cefadroxil, was the most active oral cephalosporin derivative against Enterobacteriaceae producing plasmid-encoded broad spectrum beta-lactamases. In a pharmacodynamic model, ceftibuten was bactericidal for Haemophilus influenzae and Streptococcus pneumoniae at concentrations simulating human serum levels following 200 mg, p.o., b.i.d.

Administration, Oral↗
PubMed1994

Prediction of piperacillin-tazobactam susceptibility among Enterobacteriaceae, Pseudomonas aeruginosa, and other bacteria using ticarcillin-clavulanic acid, ceftazidime, and other broad-spectrum antimicrobial in vitro test results.

The ability of various in vitro beta-lactam susceptibility test results to predict the susceptibility of piperacillin-tazobactam (a new beta-lactam-beta-lactamase inhibitor combination) was assessed using more than 46,000 recent clinical isolates. The organisms were tested by reference-quality National Committee for Clinical Laboratory Standards (NCCLS) broth microdilution procedures and interpreted by the currently published NCCLS criteria. The recommended antimicrobial tests that would accurately predict the piperacillin-tazobactam in vitro efficacy had an overall very major, false-susceptible rate of only 0.6% (< or = 1.5% is acceptable). The following drug tests can be used to judge piperacillin-tazobactam activity and spectrum (low patient risk) conservatively: for Enterobacteriaceae use ticarcillin-clavulanic acid results (0.6% very major error); for Pseudomonas aeruginosa use piperacillin (0.1%) results; for enterococci use ampicillin or ampicillin-sulbactam (1.8%) results; for Haemophilus influenzae and Moraxella catarrhalis use cefotaxime or cefuroxime or ceftriaxone (1.5%); and for staphylococci use oxacillin by NCCLS recommendations. When the piperacillin-tazobactam testing reagents become available, the direct testing of this combination should be applied to relevant clinical isolates. The piperacillin-tazobactam break points should be reassessed as indicated by the cited minimum inhibitory concentration population analysis to improve predictive accuracy; H. influenzae susceptibility modified to < or = 2/4 micrograms/ml and Enterococcus species susceptibility tested at < or = 16/4 micrograms.

Anti-Bacterial Agents↗