Ten-Minute Test for Differentiating Between Klebsiella and Enterobacter Isolates

A 10-minute test, utilizing a urease paper-reagent strip (PATHO-TEC), for differentiating Klebsiella and Enterobacter species is described. By using a heavy suspension of organisms and 50 C temperature for incubation, 93% of Klebsiella strains (186/200) were positive and 95% of Enterobacter strains (190/200) were negative with this testing system. The rapid nature of the test (10 min), the facility with which it can be carried out, and the ease with which the strips can be stored and handled may make this a useful aid for the clinical microbiologist.     

Klebsiella, Enterobacter, and Serratia: Biochemical Differentiation and Susceptibility to Ampicillin and Three Cephalosporin Derivatives

Three hundred twenty-nine strains of the tribe Klebsielleae were compared by several biochemical tests and by susceptibility to selected antibiotics. Biochemical tests included urease, amino acid decarboxylase, and hydrogen sulfide production; fermentation of lactose and dextrose; motility; and tests in the IMViC (indole, methyl red, Voges-Proskauer, citrate) series. The isolates were: Klebsiella species, 67.5%; Enterobacter species, 28%, and Serratia species, 4.5%. Minimal inhibitory concentrations of cephaloridine, cephalothin, and a new cephalosporin, cephalexin, and of ampicillin were determined by the agar dilution procedure. Cephalosporins at 20 mug/ml or less inhibited 90% of the Klebsiella strains but only 15% of the Enterobacter strains. Ampicillin inhibited 27% of Enterobacter strains and 17% of Klebsiella strains. Serratia isolates were insensitive to the cephalosporins and ampicillin. The results suggest that precise identification of this group to the generic level can be accomplished readily in the clinical laboratory and that such information is helpful in the preliminary selection of an antibiotic for treatment of clinical infections.     

Catalase Test as an Aid to the Identification of Enterobacteriaceae

Although the catalase test has been used for many years for rapid differentiation of the genera of gram-positive organisms, little has been said about its use in the family Enterobacteriaceae. It was further noted that a wide variety of methods exist for the execution of the catalase test, that there is no universally accepted strength specified for the hydrogen peroxide, and that no gradations for the vigor and speed of the reaction have been mentioned. Under the conditions of the clinical laboratory, we have developed a simple, rapid, and accurate method for the catalase test that has been of great value as an aid in the identification of the Enterobacteriaceae. With 3% H(2)O(2), it was observed that Serratia, Proteus, and Providencia were vigorous catalase reactors. Only Salmonella and rare Escherichia, Enterobacter, and Klebsiella isolates were moderate catalase reactors. Escherichia and Shigella strains were mostly nonreactive, with less than one-third weekly (+) reactive, whereas most Enterobacter strains tended to be weakly reactive. Klebsiella strains were divided equally between nonreactive and weakly reactive. In practice, this test was also of great value in discerning nonpigmented Serratia cultured from the hospital environment and in detecting mixed flora containing nonspreading Proteus.     

Comparison of EC broth and medium A-1 for the recovery of Escherichia coli from frozen shucked snow crab.

Two variations of the multiple-tube fermentation technique were used to enumerate fecal coliforms in commercially processed, frozen crab meat. These were the EC confirmation test and a more rapid method that requires medium A-1. The method with medium A-1 was more specific than the EC confirmation test for detecting Escherichia coli type 1. E. coli was isolated from 84% of the positive medium A-1 tubes, whereas it was isolated from only 64% of the positive tubes of EC broth. When samples of crab meat were inoculated with known amounts of E. coli, better estimates of the known numbers were obtained by the medium A-1 method. Several species of nonfecal coliforms were isolated from cultures in EC broth. These belonged to the genera Klebsiella, Citrobacter, Enterobacter, and Serratia. Apparently these strains were naturally adapted to growth at an elevated temperature because the majority were able to grow at 44.5 degrees C when retested in EC broth. Fewer species of nonfecal coliforms were isolated from medium A-1. Those that were isolated belonged to the genera Citrobacter and Enterobacter.     

Comparison of EC broth and medium A-1 for the recovery of Escherichia coli from frozen shucked snow crab.

Two variations of the multiple-tube fermentation technique were used to enumerate fecal coliforms in commercially processed, frozen crab meat. These were the EC confirmation test and a more rapid method that requires medium A-1. The method with medium A-1 was more specific than the EC confirmation test for detecting Escherichia coli type 1. E. coli was isolated from 84% of the positive medium A-1 tubes, whereas it was isolated from only 64% of the positive tubes of EC broth. When samples of crab meat were inoculated with known amounts of E. coli, better estimates of the known numbers were obtained by the medium A-1 method. Several species of nonfecal coliforms were isolated from cultures in EC broth. These belonged to the genera Klebsiella, Citrobacter, Enterobacter, and Serratia. Apparently these strains were naturally adapted to growth at an elevated temperature because the majority were able to grow at 44.5 degrees C when retested in EC broth. Fewer species of nonfecal coliforms were isolated from medium A-1. Those that were isolated belonged to the genera Citrobacter and Enterobacter.     

A specific property of bacteria in the genus Klebsiella--a color reaction with 5-aminosalicylic acid in the nutrient medium

For the first time bacteria of the genus Klebsiella have been found to possess a specific property, characteristic of this genus only, i.e. the capacity of giving color reaction with 5-aminosalicylic acid. This reaction can be observed in all Klebsiella species under study: K. pneumoniae (94.4 +/- 2.3%), K. ozaenae (93.3 +/- 4.5%), K. rhinoscleromatis (100%), K. oxytoca (88.0 +/- 4.9%), K. mobilis (92-5 +/- 4.3% of the strains). In all other bacteria under study (40 species, 22 genera and 7 families) the reaction is negative. The test for the color reaction with 5-aminosalicylic acid confirms the belonging of K. mobilis (Enterobacter aerogenes) to the genus Klebsiella, thus making it possible to simplify and accelerate the identification of Klebsiella.     

STAPHYOGRAM, a new rapid identification kit for the aerobic, gram-positive, catalase-positive cocci--application of fluorometric microplate hybridization for the pre-identification of 386 isolates used

A new simplified test kit, STAPHYOGRAM plate, was developed for 4-hr identification of aerobic, Gram-positive and catalase-positive cocci. The plate has 18 wells, in which different dehydrated substrates and nutrients are fixed. An 18-hr agar-culture suspension of a test strain with a turbidity of McFarland No. 4 was distributed into all wells in 50-microliters quantities. After 4-hr incubation at 37C, the profile number was obtained by summarizing positive reactions. The ability of the plate to differentiate the type strains of the 30 species of the three genera in the family Micrococcaceae was confirmed. These three genera are Staphylococcus, Micrococcus and Stomatococcus. The applicability of the fluorometric microplate hybridization technique to identification of aerobic, Gram-positive and catalase-positive cocci was confirmed by homologous hybridization among the type strains of the 30 species. Thus, 386 isolates of human and animal origin were pre-identified by microplate hybridization and used for evaluating the STAPHYOGRAM plate. Of the 236 profile numbers thus obtained with the 386 isolates, 218 (92.4%) were species-proper each and all for the 15 species of Staphylococcus and Stomatococcus mucilaginosus. A total of 342 (88.6%) of the 386 isolates were given such profile numbers, and were identified without any additional test. Among the 15 species identified primarily by the results of STAPHYOGRAM plate culture, S. caprae, S. lugdunensis, S. gallinarum and S. delphini were validly published after Approved Lists of Bacterial Names. The identified strains of S. caprae (48), S. haemolyticus (46), S. capitis (35) numbered between those of S. epidermidis (67) and S. saprophyticus (31). Profile numbers common to two species were seven (27 strains) and that to four species was one (17 strains). These 44 strains were identified with one to three additional tests. From these results, we were convinced that the STAPHYOGRAM test plate is useful for the rapid identification of members of family Micrococcaceae. By compiling STAPHYOGRAM plate data on genetically identified strains, an exclusive list of profile numbers will soon be prepared for perfection of the kit.     

Phylogenetical relationship based on groE genes among phenotypically related Enterobacter,Pantoea, Klebsiella,Serratia and Erwinia species

In an attempt to define the phylogenetical relationship among 17 phenotypically related species of genera Enterobacter, Pantoea, Serratia, Klebsiella and Erwinia, we determined almost all of their groE operon sequences using the polymerase chain reaction direct sequencing method. The number of nucleotide substitutions per site was 0.12+/-0.030. The value was 3.6-fold higher than that of 16S rDNA. As a result, we were successful in constructing molecular phylogenetic trees which had a finer resolution than that based on the 16S rDNA sequences. The phylogenetic trees based on the nucleotide sequences and deduced amino acid sequences of groE operons indicated that the members of genera Enterobacter, Pantoea and Klebsiella were closely related to each other, while Serratia and Erwinia species except Erwinia carotovora, made distinct clades. The close relationship between Enterobacter aerogenes and Klebsiella pneumoniae, that had been suggested by biochemical tests and DNA hybridization, was also supported by our molecular phylogenetic trees.     

Bacterial antagonists of Shigellae

Bacterial antagonism between a microorganisms and Shigella sonnei strains was studied in model experiments simulating conditions of the natural aquatic environment. In these studies surface waste samples from the river Vltava served as the experimental environment. To ensure bacteriologically defined conditions all water samples were heat-sterilized prior to antagonism testing. Consistently with the literature data and author's own observations the following bacterial species and genera were chosen as test organisms to be tested for antagonism against Shigella sonnei strains in water; E. coli, Citrobacter, Enterobacter, Klebsiella pneumoniae, Proteus, Pseudomonas aeruginosa and the fecal streptococci S. fecalis and S. faecium. Presence or absence of microbial antagonism against shigellae was determined in the experimental water medium contaminated with shigella-test organism mixtures of density ratios within the range 1 : 1 through 1 : 10(4). The highest degree of antagonism was observed with Pseudomonas aeruginosa that at density ratio 1 : 1 inhibited the Shigella sonnei growth in water within 42 hours of incubation. A similar degree of antagonism was also observed with Klebsiella pneumoniae at the density ratio 1 : 10(1) and with Enterobacter aerogenes at 1 : 10(2). At lower density ratios the antagonism exhibited by these two species was also present, but occurred much later, i.e. after 72 hours up to 5 days. The remaining test organisms used showed no antagonistic action Shigella sonnei strain in the model aquatic environment.     

Genetic analysis of Escherichia coli urease genes: evidence for two distinct loci.

Studies with two uropathogenic urease-producing Escherichia coli strains, 1021 and 1440, indicated that the urease genes of each are distinct. Recombinant plasmids encoding urease activity from E. coli 1021 and 1440 differed in their restriction endonuclease cleavage sites and showed minimal DNA hybridization under stringent conditions. The polypeptides encoded by the DNA fragments containing the 1021 and 1440 urease loci differed in electrophoretic mobility under reducing conditions. Regulation of urease gene expression differed in the two ureolytic E. coli. The E. coli 1021 locus is probably chromosomally encoded and has DNA homology to Klebsiella, Citrobacter, Enterobacter, and Serratia species and to about one-half of the urease-producing E. coli tested. The E. coli 1440 locus is plasmid encoded; plasmids with DNA homology to the 1440 locus probe were found in urease-producing Salmonella spp., Providencia stuartii, and two E. coli isolates. In addition, the 1440 urease probe was homologous to Proteus mirabilis DNA.     

TreeSeq,a Fast and Intuitive Tool for Analysis of Whole Genome and Metagenomic Sequence Data

Next-generation sequencing is not yet commonly used in clinical laboratories because of a lack of simple and intuitive tools. We developed a software tool (TreeSeq) with a quaternary tree search structure for the analysis of sequence data. This permits rapid searches for sequences of interest in large datasets. We used TreeSeq to screen a gut microbiota metagenomic dataset and a whole genome sequencing (WGS) dataset of a strain of Klebsiella pneumoniae for antibiotic resistance genes and compared the results with BLAST and phenotypic resistance determination. TreeSeq was more than thirty times faster than BLAST and accurately detected resistance gene sequences in complex metagenomic data and resistance genes corresponding with the phenotypic resistance pattern of the Klebsiella strain. Resistance genes found by TreeSeq were visualized as a gene coverage heat map, aiding in the interpretation of results. TreeSeq brings analysis of metagenomic and WGS data within reach of clinical diagnostics.     

Epiphytic bacteria biodiversity in Brazilian Cerrado fruit and their cellulolytic activity potential

The Cerrado is the second largest Brazilian biome, yet little is known about its wild fauna, flora and microbiota. This work aimed to identify epiphytic bacteria present in fruits native to three different regions of the Cerrado and to select cellulase-producing bacteria. Culture-dependent and culture-independent (PCR-DGGE) methods were used to characterize the microbiota from 32 native Cerrado fruits, and the selection of cellulase-producing bacteria was performed by a semi-quantitative test on carboxymethylcellulose agar medium. Analysis of the 16S rRNA gene sequences of 69 profile representatives showed that the isolates belonged to 29 bacterial genera (Arthrobacter, Bacillus, Paenibacillus, Pseudomonas, Serratia, Staphylococcus, Streptomyces, Enterobacter, Microbacterium, Aerococcus, Bradyrhizobium, Methylobacterium, Erwinia, Pantoea, Acidithiobacillus, Ochrobactrum, Stenotrophomonas, Curtobacterium, Clostridium, Lactobacillus, Xanthomonas, Delftia, Klebsiella, Enterococcus, Burkholderia, Escherichia, Streptococcus, Citrobacter and Achromobacter). Species in the genera Methylobacterium, Stenotrophomonas, Clostridium, Pantoea and Enterobacter were detected by both culture-dependent and culture-independent methods. The species Lactobacillus fermentum, Acinetobacter sp. and Methylomonas methanica were detected only by PCR-DGGE. Additionally, 30 % (178 isolates) of the bacteria tested were able to produce cellulase. The best producers belonged to the genera Bacillus, Streptomyces, Paenibacillus, Enterobacter and Burkholderia, indicating that this ecosystem could be an attractive source for the study of novel enzymes.     

FT-IR microspectroscopy for early identification of some clinically relevant pathogens

AIMS: To investigate the potentials and limitations of Fourier transform-infrared (FT-IR) microspectroscopy as a tool to identify, at the level of microcolonies, pathogenic bacteria frequently isolated in the clinical environment. METHODS AND RESULTS: A total of 1570 FT-IR spectra from 164 gram-positive and gram-negative bacteria isolated from patients were recorded from 6 to 10-h old microcolonies of 50-150 microm size. A classification of 100% was obtained for the most frequent gram-positive bacteria, such as Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, and Enterococcus faecium at the species level. An average accuracy of about 80% was reached with Gram negative bacteria from the Enterobacteriaceae and Pseudomonaceae families; Enterobacter aerogenes, Enterobacter cloacae, Klebsiella spp., and Citrobacter koseri; and Proteus mirabilis and Escherichia coli. Results were comparable with FT-IR measurements on dried suspensions from 18-h cultures. CONCLUSIONS: Early identification of young microcolonies is feasible with FT-IR microscopy with a very high accuracy for gram-positive bacteria. Some improvement in the transfer of microcolonies is necessary to increase the accuracy for gram-negative bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: Combination of FT-IR microscopy and multivariate data analysis could be a complementary, rapid, and reliable tool for screening and discriminating, at species and subspecies level, micro-organisms of clinical, food-borne, or environmental origins.     

The influence of Helicobacter pylori infection on the prevalence of endoscopic erosive esophagitis

OBJECTIVES: This study aimed to determine the frequency of endoscopic esophagitis and Helicobacter pylori infection in a large Turkish population over a 6-year period. METHODS: We studied a consecutive series of 14,380 patients who had been newly referred for diagnostic esophagogastroduodenoscopy from 2000 to 2006. The mean age value was 45 +/- 10 (18-89) years. All endoscopic findings were retrospectively evaluated. Two antral and two corpus biopsies were taken from patients for rapid urease test. Endoscopic esophagitis was defined as the presence of erosions and/or ulceration. The relationship between erosive esophagitis and various relevant factors was analyzed. RESULTS: The overall prevalence of endoscopic esophagitis was 7.8% (95% CI, 6.9-8.1). The prevalence of positive rapid urease test was 49% (95% CI, 38-53) in patients with esophagitis and 85% (95% CI, 70-96) in patients without esophagitis (p < .001). From 2000 to 2006, the frequency of endoscopic esophagitis and the rate of positive rapid urease test remained unchanged. After adjusting for the effects of mean age, male gender, and percentage of hiatal hernia, there was a 0.785% risk reduction in esophagitis with every 1% increase in the rate of positive rapid urease test result. CONCLUSIONS: The frequency of endoscopic esophagitis is significantly lower in patients with a positive rapid urease test result. This negative correlation with H. pylori infection reflects a protective effect of H. pylori from endoscopic esophagitis in a Turkish population and deserves further investigation.     

Evaluation of a commercial beta-glucuronidase test for the rapid and economical identification of Escherichia coli

A commercial beta-glucuronidase (beta-GUR) test for the rapid and economical identification of Escherichia coli was evaluated. A total of 762 clinical strains and 228 environmental isolates were studied. More than 95% of the E. coli strains were found to be beta-GUR positive. Thirty-one clinical isolates of Shigella sonnei, 10 of Enterobacter cloacae, eight of Enterobacter aerogenes, nine of Citrobacter freundii and one of Salmonella enteritidis also gave positive results. The enzyme beta-GUR was also detected in two environmental strains of E. cloacae and one C. freundii. A comparative study between the beta-GUR test and the conventional identification system was carried out in 233 consecutive isolates of lactose positive enterobacteria. Agreement was observed in 223 cases and 190 E. coli strains were correctly identified using this test. Discrepancies were found in 10 cases: nine E. coli were beta-GUR negative and one C. freundii was beta-GUR positive. Escherichia coli was the only species positive for both beta-GUR and indole tests. This procedure permits a rapid, easy, precise and inexpensive identification of E. coli. beta-GUR positive Enterobacter strains have not previously been described.     

Pollution indicator bacteria associated with municipal raw and drinking water supplies.

A total of 3819 bacterial cultures isolated from municipal water samples were identified using a combination of Enterotubules and confirmatory media. Frequency distributions for the different genera or groups of bacteria were similar for raw water and drinking water isolations, except for Escherichia organisms which doubled their frequency in raw water. Differences between the membrane filter (MF) and presence-absence (P-A) test with regard to types of organisms isolated were limited to Klebsiella organisms which were preferentially cultured from MF plates. Members of the genus Enterobacter were isolated more than twice as frequently as any of the other coliform genera dealt with in this study. Aeromonas organisms were detected almost as often as such individual genera as Escherichia, Citrobacter, or Klebsiella. Although non-lactose fermenting colonies (false-negatives) of the coliform genera would not be detected by the MF technique, their lack of detection would likely be offset by the Aeromonas colonies (false-positives). At least 25% of the coliform isolates were either anaerogenic or non-lactose fermenters and would therefore go undetected by the most probably number (MPN) technique.     

Profiles of the utilization of 20 amino acids as the only source of nitrogen and carbon in bacteria of the genera Klebsiella, Enterobacter, Serratia, Escherichia

The profiles of the utilization of 20 protein amino acids in 118 Klebsiella pneumoniae sub- sp. pneumoniae, K. oxytoca, K. planticola, K. mobilis, Enterobacter cloacae, Serratia marscescens, S. liquefaciens, Escherichia coli strains isolated from clinical material were studied. The utilization of amino acids was determined on minimal saline agar containing amino acid as the only source of nitrogen and carbon; the results were evaluated after 72-hour incubation at 37 degrees C. 17 profiles of amino-acid utilization were thus determined, most of them genus-specific in enterobacteria: Klebsiella (profiles No. 1--6, 9, 10), Enterobacter (No. 11--13), Serratia (No. 14--16), Escherichia (No. 17). The full coincidence of amino-acid utilization profiles in bacteria of K. mobilis (No. 1, 6) and K. pneumoniae subsp. pneumoniae with out of such profiles in bacteria of the genera Enterobacter, Serratia, Escherichia was established, which confirmed that K. mobilis (formerly Enterobacter aerogenes) belonged to the genus Klebsiella.     

Micro-Complement Fixation in Klebsiella Classification

The alkaline phosphatases of 29 strains of bacteria assigned by various authors to the genera Aerobacter, Klebsiella and Enterobacter were compared by the micro-complement fixation technique. On the basis of phosphatase resemblance, we recommend that all strains hitherto assigned to Aerobacter aerogenes and Enterobacter aerogenes be assigned to the genus Klebsiella.     

Characterization of Ornithine Decarboxylase-Positive, Nonmotile Strains of the Klebsiella-Enterobacter Group

Thirty-seven strains of ornithine decarboxylase-positive, nonmotile Klebsiella-Enterobacter organisms isolated from 36 patients were studied by biochemical and serological testing. Five strains gave biochemical reactions which conformed closely to those of Escherichia coli; three strains gave positive Quellung reactions to specific Klebsiella antisera. (Two of these were thought to be Enterobacter in spite of this typing reaction.) The remaining 29 strains were classified as Enterobacter. These results demonstrate the necessity of doing both an ornithine decarboxylase test and a motility test to differentiate Klebsiella from Enterobacter. Had only a motility test been done, they all would have been called Klebsiella.     

Membrane Filter Procedure for Enumerating the Component Genera of the Coliform Group in Seawater

A facile, quantitative, membrane filter procedure (mC) for defining the distribution of coliform populations in seawater according to the component genera was developed. The procedure, which utilizes a series of in situ substrate tests to obviate the picking of colonies for identification, also provides an estimate of the total coliform density. When pure cultures of Escherichia coli, Klebsiella pneumoniae, and Enterobacter cloacae were suspended in seawater and held at 4 C for 24 h, between 56 and 100% of the cells which grew on nutrient agar spread plates at 35 C could be recovered by the mC procedure. Confirmation as coliforms of typical colonies from natural samples was about 95%. Assay variability was found to be insignificant. The recovery of coliforms from marine waters by the mC procedure was comparable to those obtainable by current methods. Klebsiella was differentiated by the urease reaction and E. coli by its ability to form indole. The confirmation frequencies for colonies designated as Klebsiella and E. coli by the in situ tests approached 95% for the former and 98% for the latter.