Biochemical characteristics and identification of Enterobacteriaceae isolated from meats.

The isolation and identification of 2,220 Enterobacteriaceae from meats indicated that Escherichia coli biotype I, Enterobacter agglomerans, and Serratia liquefaciens were the principal types to be differentiated in meats. Citrobacter freundii, Klebsiella pneumoniae, Enterobacter cloacae, and Enterobacter hafniae were also commonly identified. Identification of isolates by the Encise II (Roche Diagnostics Inc., Nutley, N.J.) and Minitek (BBL Microbiology Systems, Cockeysville, Md.) coding systems gave similar results with only 255 (11.5%) discrepancies in identity, but both systems required large numbers of supplementary tests for identification of the isolates. Not only the distribution of Enterobacteriaceae types isolated from meats but also some of the biochemical reactions of the isolates differed from those of clinical isolates. The Minitek technique is recommended because of its versatility. However, with the addition of cellobiose and salicin disks and the inclusion of methyl red to the Minitek test and the use of the Voges-Proskauer test and gas production in EC medium at elevated temperature as standard tests, the identification of these Enterobacteriaceae from meats would be greatly facilitated. The inclusion of the motility test, for example, using nitrate motility agar, would also be of value to Enterobacteriaceae identification.     

Differentiation of Klebsiella-Enterobacter (Aerobacter)-Serratia by biochemical tests and antibiotic susceptibility

Studies were undertaken for the differentiation of members of the Klebsiella-Enterobacter (Aerobacter)-Serratia division by biochemical tests and antibiotic susceptibility. A total of 67 cultures were tested. Strain identification was readily accomplished with the use of motility tests and arabinose fermentation. In addition, a practical schema, based on sensitivity pattern, proved valuable in the classification of the different strains. Most (if not all) Klebsiella strains were susceptible to cephalothin, and all were sensitive to colistin. Enterobacter strains were resistant to cephalothin but susceptible to colistin. In contrast to those other members of the group, all Serratia strains were resistant to both cephalothin and colistin. The combination of a limited number of biochemical reactions and single-disc sensitivity tests appears to be a logical approach for the tentative identification of Klebsiella-Enterobacter (Aerobacter)-Serratia strains.     

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.     

Limitations of the Moeller lysine and ornithine decarboxylase tests.

A total of 40 fecal and environmental isolates, including 26 Escherichia coli strains, 9 members of the genus Klebsiella, and 5 members of the genus Enterobacter, were tested by enzyme assay for their endogenous and induced levels of lysine decarboxylase and ornithine decarboxylase when grown in Moeller decarboxylase medium. All of the coliforms examined had measurable lysine decarboxylase and ornithine decarboxylase activities whether or not they were positive in the Moeller test. In general, the Moeller lysine decarboxylase test reflected the inducibility of lysine decarboxylase whereas the Moeller ornithine decarboxylase test did not relect the inducibility of ornithine decarboxylase. Neither test measured the amount of intracellular enzyme; rather, they indicated whether the amount of polyamine liberated was sufficient to raise the pH of the culture medium above 7. Changing the growth conditions (i.e., the concentrations of glucose, lysine, and amino acids other than lysine) greatly influenced the lysine decarboxylase activity in coliforms. The limitations on the interpretation of the Moeller test results are discussed.     

Limitations of the Moeller lysine and ornithine decarboxylase tests.

A total of 40 fecal and environmental isolates, including 26 Escherichia coli strains, 9 members of the genus Klebsiella, and 5 members of the genus Enterobacter, were tested by enzyme assay for their endogenous and induced levels of lysine decarboxylase and ornithine decarboxylase when grown in Moeller decarboxylase medium. All of the coliforms examined had measurable lysine decarboxylase and ornithine decarboxylase activities whether or not they were positive in the Moeller test. In general, the Moeller lysine decarboxylase test reflected the inducibility of lysine decarboxylase whereas the Moeller ornithine decarboxylase test did not relect the inducibility of ornithine decarboxylase. Neither test measured the amount of intracellular enzyme; rather, they indicated whether the amount of polyamine liberated was sufficient to raise the pH of the culture medium above 7. Changing the growth conditions (i.e., the concentrations of glucose, lysine, and amino acids other than lysine) greatly influenced the lysine decarboxylase activity in coliforms. The limitations on the interpretation of the Moeller test results are discussed.     

In vitro adhesion of n(2)-fixing enteric bacteria to roots of grasses and cereals

Nitrogen-fixing Klebsiella and Enterobacter strains isolated from several plants were assayed for fimbriae and for adhesion to plant roots in vitro. All eight Klebsiella strains formed type 3 fimbriae, and five strains also formed type 1 fimbriae; all 21 Enterobacter strains had type 1 fimbriae. Three strains of Klebsiella carrying either type 1, type 3, or no fimbriae were used as model organisms in developing an in vitro adhesion test. Adhesion was assayed with bacterial cells labeled with [H]leucine. Fifteen N(2)-fixing strains and the three model strains were compared for adhesion to the roots of seven grasses and five cereals. Type 3-fimbriated Klebsiella strains adhered better than the other strains, and type 3 fimbriae appeared to be major adhesins for the Klebsiella strains. Although variations between plants were observed, no host specificity for bacterial adhesion was found.     

Differentiation of Aerobacter-Klebsiella isolated from sugarcane

Three hundred and eighty-four isolates were obtained in the completed test portion of the most probable number determinations of coliforms in sugarcane sources. Of these isolates, 88% were of the (- - + +) indole, methyl red, Voges-Proskauer, citrate (IMViC) type and were identified as Aerobacter aerogenes according to the protocol of the American Public Health Association (1). Employing 359 of these cultures, a comparative biochemical, serological, and pathogenicity study was carried out with Klebsiella pneumoniae CDC no. 2211-66 type 9. More than 86% of the organisms tested gave biochemical reactions typical of K. pneumoniae. Of the other isolates, 2% were Enterobacter aerogenes, and the remaining 12% were identified as atypical, nonmotile IMViC types. Comparable agglutination titers were also observed between A. aerogenes and the CDC strain of K. pneumoniae when several randomly selected sugarcane strains were reacted with prepared K. pneumoniae whole cell antiserum. Neither the K. pneumoniae reference organism nor selected sugarcane isolates displayed pathogenicity for mice. On the basis of all the analyses performed, it was suggested that such organisms be classified as K. pneumoniae.     

Four-Hour Urease Test for Distinguishing Between Klebsiella and Enterobacter

Infections with Klebsiella and Enterobacter have increased among hospitalized patients. To study such infections, relatively simple but precise methods are needed for clinical laboratories to identify the two genera accurately. Moreover, a rapid identification is essential for assisting with the therapy of the patients. For this purpose, a new 4-hr urease test was developed so that colonies could be tested directly from blood-agar plates which have been inoculated with clinical material and allowed to incubate overnight. This 4-hr test was positive with 98.5% of 202 Klebsiella species and negative with 80 Enterobacter species. As a single criterion for distinguishing between the two major genera, the new 4-hr urease test was just as accurate as a motility test (99% of the 282 isolates were accurately identified with either). The 4-hr urease test represents a simple, rapid, and reliable technique which is ideally suited for use in clinical microbiology laboratories.     

Coliforms from hides and meat.

Coliform tests were performed on 85 hide and 75 meat samples. IMViC reactions were determined on isolates from positive confirmed and fecal tests, and strains other than Escherichia coli were identified. Strains typed as Aerobacter aerogenes types I and II were identified as Enterobacter cloacae (51.4%), Klebsiella pneumoniae (21.5%), Enterobacter aerogenes (15%), and Enterobacter liquefaciens, Serratia, and unidentified coliforms (12.1%). K. pneumoniae appeared to be responsible for less than 1% positive fecal tests.     

Coliforms from hides and meat.

Coliform tests were performed on 85 hide and 75 meat samples. IMViC reactions were determined on isolates from positive confirmed and fecal tests, and strains other than Escherichia coli were identified. Strains typed as Aerobacter aerogenes types I and II were identified as Enterobacter cloacae (51.4%), Klebsiella pneumoniae (21.5%), Enterobacter aerogenes (15%), and Enterobacter liquefaciens, Serratia, and unidentified coliforms (12.1%). K. pneumoniae appeared to be responsible for less than 1% positive fecal tests.     

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.     

Evaluation of Modified R-B System for Identification of Members of the Family Enterobacteriaceae

In a paired, double-blind study, the modified ("Beckford tube") R-B system was compared with conventional bacteriological procedures for the identification of members of the family Enterobacteriaceae from clinical isolates and stock cultures. The tests in the R-B system yielding positive reactions comparable to those predicted by Ewing's taxonomic classification of Enterobacteriaceae were production of hydrogen sulfide and presence of lysine and ornithine decarboxylasè activities. The test reactions in the R-B system found to be comparable to those in the conventional method were fermentation of glucose, hydrogen sulfide production, and lysine and ornithine decarboxylase activities. The production of gas from glucose was positive in the R-B system more often than in the conventional method; however, the motility test and the production of indole were positive less often in the R-B system. Adequate preliminary identification of the Enterobacteriaceae with the R-B system is enhanced if Simmons' citrate and Christensen's urea tests are used concomitantly. These findings emphasize the manufacturer's instructions that, in interpretation of results, colonial morphology and biochemical reactions must be used concurrently to make an accurate identification.     

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.     

Differentiation of Serratia from Enterobacter on the Basis of Nucleoside Phosphotransferase Production

Precoated cellulose thin-layer chromatograms were used to detect the production of guanosine 5'-phosphate from guanosine and p-nitrophenylphosphate by whole-cell preparations. One-hundred per cent of Serratia (163 strains) and 84% of E. liquefaciens (15 of 18 strains) produced the nucleotide. All other Enterobacter (23 strains), Klebsiella (10 strains), and E. coli (10 strains) were negative for the production of this nucleotide. The entire test procedure could be carried out in 4 hr. It is proposed that E. liquefaciens is more closely related to Serratia than Enterobacter and that reclassification of these organisms should be investigated.     

Epidemiological Differentiation of Serratia marcescens: Typing by Bacteriocin Sensitivity

Strains of Serratia marcescens were compared and differentiated by a new method. Bacteriocin lysates were prepared from mitomycin-induced S. marcescens and added to lawns of test strains. From 100 bacteriocin producers, 12 were chosen with the aid of computer analysis as the most useful in differentiation. Uniform drops of the 12 standard bacteriocins were added simultaneously with a bacteriocin-bacteriophage dropper to each strain to be typed. All 93 strains of S. marcescens tested were typable and were differentiated into 79 different sensitivity patterns. One pattern had three strains, 12 patterns had two strains each, and 66 patterns had only one strain. The bacteriocins also inhibited Shigella, Klebsiella, and Enterobacter, but no other Enterobacteriaceae. Bacteriocin sensitivity was less stable as an epidemiological marker than bacteriocin production. Several colonial mutants had sensitivity patterns different from the wild types, but most mutants were identical. In three different instances when cross-infection had been shown by other methods, bacteriocin sensitivity also gave the correct epidemiological results. Until the significance and frequency of genetic variations are known, a more stable epidemiological technique should be used in conjunction with bacteriocin sensitivity.     

Taxonomy of Citrobacter rods found in human specimens

The aim of this study was the identification of 181 Citrobacter strains on the basis of the recently proposed taxonomic changes of Brenner. All strains were isolated from diarrhoeic patients; 124 strains were originally sent for identification to Laboratory of Enterobacteriaceae DB NIH, 57 strains was isolated in Czech Republic. Citrobacter isolates were initially identified as C. koseri (3 strains), C. amalonaticus (1 strain) or as members of the C. freundii complex (177 strains). Additionally some biochemical tests were performed. The ability to grow in medium containing KCN, lysine decarboxylase production, lactose fermentation and PYR test were examined. Strains belonging to the C. freundii complex were identified to the species level by biochemical methods on the basis of the results of Brenner, who found some tests to be useful in separating Citrobacter species. These test included citrate and acetate utilization, arginine dihydrolase and ornithine decarboxylase activities, motility, urease production, esculin hydrolysis, and acid production from sucrose, dulcitol, melibiose, raffinose and salicin. On the basis of the criteria described above, 96.6% of the strains tested could be assigned to one of the recently named species of C. freundii complex. Using biochemical tests suggested by Brenner we were able to identify Citrobacter strains members of newly recognised species. A five-test system is proposed to identify the most frequently encountered species currently residing in the C. freundii complex.     

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.     

The use of bile — esculin agar for the taxonomic classification of the family Enterobacteriaceae

Bile-esculin medium has been used for many years for the presumptive identification of group D Streptococcus. The test is based on the ability of a bacterium to grow in the presence of 40% bile and produce esculinase. 2935 strains of Enterobacteriaceae were inoculated onto bile—esculin agar slants and incubated at 35 C. Esculin hydrolysis was determined after 24 and 48 hours. At 24 hours of incubation esculin hydrolysis was limited to the generaKlebsiella, Enterobacter, Serratia, and the speciesP. vulgaris, P. rettgeri, andC. diversus. Not all strains of these species were positive, however. All other members of the family were negative. At 48 hours of incubation 37% ofE. coli gave a positive reaction; all other Enterobacteriaceae which were negative at 24 hours remained negative. Esculin hydrolysis is a valuable test for the taxonomic classification of the family Enterobacteriaceae.     

Substrate specificity of the beta-lactamases found in a number of clinical strains of gram-negative bacteria

The substrate profiles of beta-lactamases defected in 46 clinical polyresistant strains of gram-negative bacteria were determined. By the substrate profile and sensitivity to inhibitors (dicloxacillin and p-CMB) beta-lactamases were considered to belong to classes I, II, III, IV and V of the Richmond classification. The molecular weights of the enzymes were measured. Enterobacter aerogenes 6803, Enterobacter aerogenes 11030 and Klebsiella pneumoniae 970 produced simultaneously two beta-lactamases belonging to different classes. beta-Lactamases of classes I and III were detected in the cells of Enterobacter aerogenes 6803. The cells of Enterobacter aerogenes 11030 contained beta-lactamases of classes V and III and the cells of Klebsiella pneumonia 970 beta-lactamases of classes II and III. Therefore, in all the cases one of beta-lactamases belonged to the class III enzyme close to TEM beta-lactamases by its substrate profile, molecular weight and sensitivity to the inhibitors. Cephalexin and dicloxacillin were most frequently stable to the effect of the above beta-lactamases. The enzymes from 26 strains did not hydrolyse or hydrolysed slightly cephalexin and the enzymes from 19 strains did not hydrolyse of hydrolysed slightly dicloxacillin.