New Diagnostic System for the Identification of Lactose-fermenting Gram-negative Rods

The identification of prompt lactose-fermenting gram-negative rods has generally relied heavily upon colonial morphology coupled with one or more indole, methyl red, Voges-Proskauer, citrate (IMViC) parameters, hydrogen sulfide, and motility. Studies were undertaken to compare diagnoses dependent solely upon the more orthodox criteria to a system for identification based upon hydrogen sulfide, ornithine decarboxylase, and citrate utilization (HOC). The results suggest that the IMViC scheme of identification is neither consistent nor applicable when applied to the current nomenclature of the above group of organisms and should be discarded, whereas the HOC system may prove to be of significant value to clinical microbiologists.     

Practical Substitution for the Indole, Methyl Red, Voges-Proskauer, Citrate System

A substitution for the indole, methyl red, Voges-Proskauer, citrate system is proposed. The scheme employs indole and ampicillin susceptibility tests routinely and uses selectively three other media for six reactions.     

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.     

Simplified 48-hour IMVic test: an agar plate method.

An agar plate method was developed for the performance of the IMVic (indole, methyl red, Voges-Proskauer, and citrate) tests in lieu of the conventional tubed liquid media. By modifying the composition of the media and adding agar, a single "X"-compartmented petri dish was prepared containing all four IMVic test media. Ease of performance and simplification of the test were achieved by inoculating all four media simultaneously from a single colony (single inoculum) on eosin-methylene blue agar. Tests with 87 cultures, representing 7 genera in the family Enterobacteriaceae, were completed with typical (correct) IMVic patterns for all cultures within 48 h. Parallel tests with conventional media showed that the agar plate method was superior, more sensitive, faster, and simpler to perform, and less time was required to identify Escherichia coli by 72 h.     

Simplified 48-hour IMVic test: an agar plate method.

An agar plate method was developed for the performance of the IMVic (indole, methyl red, Voges-Proskauer, and citrate) tests in lieu of the conventional tubed liquid media. By modifying the composition of the media and adding agar, a single "X"-compartmented petri dish was prepared containing all four IMVic test media. Ease of performance and simplification of the test were achieved by inoculating all four media simultaneously from a single colony (single inoculum) on eosin-methylene blue agar. Tests with 87 cultures, representing 7 genera in the family Enterobacteriaceae, were completed with typical (correct) IMVic patterns for all cultures within 48 h. Parallel tests with conventional media showed that the agar plate method was superior, more sensitive, faster, and simpler to perform, and less time was required to identify Escherichia coli by 72 h.     

Characterization of the coliform and enteric bacilli in the environment of calves with colibacillosis.

In the first part of the present study the coliform and enteric bacilli in the environment of calves with colibacillosis were examined. The occurrence, number, and pathogenic properties of Escherichia coli in barnyard soils were obtained from six cattle ranches. The O and K serogroups of E. coli isolates obtained from the feces of calves with colibacillosis born at these cattle ranches were determined, and their serotypes were compared with the E. coli O and K serotypes found in soils. The results showed a reservoir of potentially pathogenic E. coli in barnyard soils contaminated with bovine feces. For the second part of this study, 6 healthy calves and 51 calves with colibacillosis were studied. The numbers of total aerobic heterotrophic bacteria, total streptococci, fecal streptococci, total coliforms, and fecal coliforms in the feces of calves were determined. In addition, coliform and enteric bacilli from the feces of both healthy and diseased calves were identified, and their indole, methyl red, Voges-Proskauer, citrate (IMViC) types were described. In parallel, the IMViC types of coliform and enteric bacilli isolated from barnyard soils previously contaminated with bovine feces were compared with those isolated from uncontaminated soils. All fecal specimens were also examined for the presence of rotavirus. No significant effect on the numbers of the bacterial types was found. The results suggest that the predominant IMViC types found in the feces of calves with colibacillosis originate from the soil. From this study it is apparent that the occurrence, number, and survival of E. coli in barnyard soils is related to ranch husbandry and sanitary practices.     

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.     

Characterization of the coliform and enteric bacilli in the environment of calves with colibacillosis

In the first part of the present study the coliform and enteric bacilli in the environment of calves with colibacillosis were examined. The occurrence, number, and pathogenic properties of Escherichia coli in barnyard soils were obtained from six cattle ranches. The O and K serogroups of E. coli isolates obtained from the feces of calves with colibacillosis born at these cattle ranches were determined, and their serotypes were compared with the E. coli O and K serotypes found in soils. The results showed a reservoir of potentially pathogenic E. coli in barnyard soils contaminated with bovine feces. For the second part of this study, 6 healthy calves and 51 calves with colibacillosis were studied. The numbers of total aerobic heterotrophic bacteria, total streptococci, fecal streptococci, total coliforms, and fecal coliforms in the feces of calves were determined. In addition, coliform and enteric bacilli from the feces of both healthy and diseased calves were identified, and their indole, methyl red, Voges-Proskauer, citrate (IMViC) types were described. In parallel, the IMViC types of coliform and enteric bacilli isolated from barnyard soils previously contaminated with bovine feces were compared with those isolated from uncontaminated soils. All fecal specimens were also examined for the presence of rotavirus. No significant effect on the numbers of the bacterial types was found. The results suggest that the predominant IMViC types found in the feces of calves with colibacillosis originate from the soil. From this study it is apparent that the occurrence, number, and survival of E. coli in barnyard soils is related to ranch husbandry and sanitary practices.     

Comparison of m-Endo LES, MacConkey, and Teepol media for membrane filtration counting of total coliform bacteria in water.

Total coliform counts obtained by means of standard membrane filtration techniques, using MacConkey agar, m-Endo LES agar, Teepol agar, and pads saturated with Teepol broth as growth media, were compared. Various combinations of these media were used in tests on 490 samples of river water and city wastewater after different stages of conventional purification and reclamation processes including lime treatment, and filtration, active carbon treatment, ozonation, and chlorination. Endo agar yielded the highest average counts for all these samples. Teepol agar generally had higher counts then Teepol broth, whereas MacConkey agar had the lowest average counts. Identification of 871 positive isolates showed that Aeromonas hydrophila was the species most commonly detected. Species of Escherichia, Citrobacter, Klebsiella, and Enterobacter represented 55% of isolates which conformed to the definition of total coliforms on Endo agar, 54% on Teepol agar, and 45% on MacConkey agar. Selection for species on the media differed considerably. Evaluation of these data and literature on alternative tests, including most probable number methods, indicated that the technique of choice for routine analysis of total coliform bacteria in drinking water is membrane filtration using m-Endo LES agar as growth medium without enrichment procedures or a cytochrome oxidase restriction.     

Comparison of m-Endo LES, MacConkey, and Teepol media for membrane filtration counting of total coliform bacteria in water.

Total coliform counts obtained by means of standard membrane filtration techniques, using MacConkey agar, m-Endo LES agar, Teepol agar, and pads saturated with Teepol broth as growth media, were compared. Various combinations of these media were used in tests on 490 samples of river water and city wastewater after different stages of conventional purification and reclamation processes including lime treatment, and filtration, active carbon treatment, ozonation, and chlorination. Endo agar yielded the highest average counts for all these samples. Teepol agar generally had higher counts then Teepol broth, whereas MacConkey agar had the lowest average counts. Identification of 871 positive isolates showed that Aeromonas hydrophila was the species most commonly detected. Species of Escherichia, Citrobacter, Klebsiella, and Enterobacter represented 55% of isolates which conformed to the definition of total coliforms on Endo agar, 54% on Teepol agar, and 45% on MacConkey agar. Selection for species on the media differed considerably. Evaluation of these data and literature on alternative tests, including most probable number methods, indicated that the technique of choice for routine analysis of total coliform bacteria in drinking water is membrane filtration using m-Endo LES agar as growth medium without enrichment procedures or a cytochrome oxidase restriction.     

一株耐高温纤维素酶产生菌的分离和鉴定

为获得耐高温的纤维素酶产生菌,从农田旁稻草堆底取样,采用液体滤纸条试管法和纤维素刚果红平板法,分离到6株能够在45℃生长良好且降解纤维素的耐高温菌株,分别标为A1-A6,其中菌株A1透明圈直径和菌落直径比值为4,DNS法测定还原糖浓度较高,确定为实验菌株。菌体呈短杆状,G+,易褪色,具有运动性,过氧化氢酶、淀粉水解、明胶液化、甲基红试验、硫化氢、葡萄糖氧化发酵、P HB类脂粒、异染粒、纤维素分解、反硝化试验呈阳性,乙酰甲基甲醇试验、吲哚试验、硝酸盐还原试验、卵磷脂酶实验呈阴性,以FP A酶活为主,羧甲基纤维素酶活为辅。根据细菌形态、生理生化特征,参照《伯杰氏细菌系统鉴定手册》初步鉴定为纤维单胞菌属,即Cellulomonas。     

Comparison of Enterococci and Coliform Microorganisms in Commercially Produced Pecan Nut Meats

Pecan nut meats in the unbroken shell are sterile for enteric microorganisms. Recovery of coliform microorganisms or enterococci from finished pecan nut meats indicated contact contamination, assuming the tempering procedures to be satisfactory. Results of specific studies, designed toward developing background data on the sanitary significance of enterococci and coliform microorganisms in the production of pecan meats are reported. Unbroken pecan nuts or nut meats from various stages of shelling operations were diluted with a phosphate-buffered diluent. Serial dilutions were inoculated into Lactose Broth and Azide Dextrose Broth. The lactose fermentors were carried through indole, methyl red, Voges-Proskauer, and citrate reactions; the positive Azide Dextrose cultures were confirmed in Ethyl Violet Azide Broth and microscopically. Viable plate counts were obtained. Enterococci were found resistant to many deterrent factors affecting coliforms. Recoveries of enterococci were detected long after pollution had occurred. Little correlation was found between enterococcal recovery and observed insanitary practices in commercial shelling operations. Using the coliaerogenes group and, specifically, Escherichia coli as a sanitation index, microorganisms allowed accurate appraisal of tempering, personnel practices, and contact surface contaminating factors. It is felt this was due, in part, to the more delicate growth characteristics of E. coli. The fact that other pathogenic microorganisms, capable of causing gastrointestinal upsets, are associated with the presence of E. coli introduces a health factor which is important to regulatory agencies concerned with consumer protection.     

Comparative Study of the Efficacy of Seven Paper-Reagent Strips and Conventional Biochemical Tests in Identifying Gram-negative Organisms

Gram-nagative organisms were tested with commercially available reagentimpregnated strips (PATHO-TEC). Of the 291 strains, all were tested by using seven paper tests and their conventional counterparts. Excellent correlation was obtained with the oxidase, phenylalanine-deaminase, and Voges-Proskauer tests. Indole tests made on liquid medium cultures also gave complete correlation, but some false-negative results with indole-positive Proteus strains were obtained when growth from solid medium was tested by the strip method. Paper strip urease tests were positive within 2 hr with all Klebsiella and some Serratia, Herellea, and Citrobacter strains as well as with Proteus strains. Approximately 15% of citrate strip test results differed from those of the conventional tests, and reproducibility was poor on retest. The lysine decarboxylase strip test showed a number of discrepancies and posed problems of interpretation and readability. Paper reagent strip methods are simple and convenient and merit further development to increase the specificity of those which depend on pH change up to that achieved with the Voges-Proskauer, oxidase, phenylalanine, and indole methods.     

The Occurrence of Aeromonads in Activated Sludge: Isolation of Aeromonas sobria and its Possible Confusion with Escherichia coli

Strains of Aeromonas have been isolated in high numbers from several activated sludge samples. Immediately after isolation, some displayed only weak or negative oxidase activity, produced both acid and gas from lactose, formed 'metallic'colonies on Endo agar, and had IMViC reactions which were identical with those of Escherichia coli . Although such strains could have been readily confused with E. coli , further biochemical characterization showed that these belonged to the recently-described A. sobria . Nitrogenase activity was absent in all strains of both A. hydrophila and A. sobria . Almost all strains of both taxa were resistant to ampicillin and penicillin V and 67% of both were bete -hemolytic.     

Evaluation of methods for the isolation and detection of Escherichia coli O157 in minced beef

This study has evaluated enrichment and detection procedures for the isolation and detection of Escherichia coli O157 inoculated into minced beef. The use of a 24 h enrichment in modified EC broth containing novobiocin allowed low numbers of contaminating cells to multiply to levels detectable on culture media and by ELISA test kits. Total analysis time was reduced by the use of the Dynabead^TM immunomagnetic separation system. The use of the Petrifilm^TM Test Kit-HEC for E. coli O157: H7 and Organon Teknika EHEC-TEK system detected low numbers of contaminating cells following enrichment and reduced analysis time by 1 d. The incorporation of cefixime and tellurite into Sorbitol MacConkey Agar increased the rate and ease of isolation of E. coli O157 and its use is therefore recommended.     

Quantitative evaluation of growth-promoting properties of selected culture media used for isolation of Salmonella and Shigella strains

Growth promoting properties and selectivity of 11 commercially produced media recommended for Salmonella and Shigella isolation were evaluated. The following media were tested: EMB (Eosin methylene blue agar), Endo, P?oskiriew, MacConkey, DC (Deoxycholate citrate agar), SS (Salmonella-Shigella agar), BS (Bismuth sulfite agar) and Mueller-Hinton as a medium with no selective properties. The media were produced in Czechoslovakia, East Germany, West Germany, Poland, and Soviet Union. Quantitative studies were performed on 71 strains representing 8 genera of Enterobacteriaceae family; both reference and wild newly + isolated from clinical material strains were included. It was found that none of DC and BS media provided suitable growth conditions for Shigella strains and in particular for S. dysenteriae, S. boydii, and S. flexneri. It was also found that the same medium (name and content) but derived from different producer can vary significantly in respect to growth promotion and selectivity especially for Shigella strains. All media with selective, differentiating properties for Salmonella and Shigella isolation should not be used without previous quantitative control test for their selective and growth promoting properties checked by user. The need for such a control performed both on reference and freshly isolated strains was shown in this study. In the set of control strains all species of Shigella should be represented.     

Leclercia adecarboxylata Gen. Nov., Comb. Nov., formerly known asEscherichia adecarboxylata.

The nameLeclercia adecarboxylata is proposed for a group of the family Enterobacteriacae previously known asEscherichia adecarboxylata. Leclercia adecarboxylata can be phenotypically differentiated from all other species of Enterobacteriaceae. The members of this species are positive for motility, indole production, methyl red, growth in the presence of KCN, malonate, beta-galactosidase, beta-xylosidase, esculin hydrolysis, gas production fromd-glucose, and acid production fromd-cellobiose,d-lactose, melibiose,l-rhamnose, adonitol,d-arabitol, dulcitol, and salicin; the strains were negative for Voges-Proskauer, citrate (Simmons), H₂S (Kligler), lysine and ornithine decarboxylases, arginine dihydrolase, phenylalanine deaminase, gelatinase, DNase, Tween-80 hydrolysis, and acid production from myoinositol and alpha-methyl-d-glucoside. Fermentation ofd-raffinose,d-sucrose, andd-sorbitol is variable with strains. DNA relatedness of 11 strains ofL. adecarboxylata to three strains including the type strain of this species averaged 80% in reactions at 65°C. DNA relatedness to other species in Enterobacteriaceae was 2%–32%, indicating that this species was placed in a new genusLeclercia gen. nov. The type strain ofL. adecarboxylata is ATCC 23216.     

Rapid Methods for the Determination of Faecal Contamination in Oysters

S ummary . Two methods for the rapid detection and estimation of numbers of faecal coliforms and Escherichia coli type I in oysters have been developed. That for faecal coliforms involves incubation of tubes of MacConkey broth for 2 h at 37° and then for 22–24 h at 44°. The second method, a modification of MacKenzie, Taylor & Gilbert's (1948) specific method for E. coli type I, makes use of the same system of incubation, but requires the inoculation of tubes of peptone water as well as MacConkey broth, the former tubes being used for subsequent testing for indole formation. Both methods take only 24–26 h and are as sensitive and accurate as the Most Probable Number methods which are in common use and which take upwards of 72–96 h to complete.     

Effect of Storage at 1° and 4°C on Viability and Injury of Staphylococcus aureus, Escherichia coli and Streptococcus faecalis

Staphylococcus aureus and Escherichia coli incubated at 1° or 4°C became increasingly sensitive to Mannitol Salt Agar and Violet Red Bile Agar respectively. The increased sensitivity was most marked with exponential phase cultures. Streptococcus faecalis was refractory to similar treatments. The viability of all 3 organisms on selective and non-selective media is reported.     

Arsenic-resistant proteobacterium from the phyllosphere of arsenic-hyperaccumulating fern (Pteris vittata L.) reduces arsenate to arsenite

An arsenic-resistant bacterium, AsRB1, was isolated from the fronds of Pteris vittata grown in a site contaminated with copper chromium arsenate. The bacterium exhibited resistance to arsenate, arsenite, and antimony in the culture medium. AsRB1, like Pseudomonas putida, grew on MacConkey and xylose-lactose-desoxycholate agars and utilized citrate but, unlike P. putida, was positive for indole test and negative for oxidase test. A phylogenetic analysis of the 16S rRNA gene showed that AsRB1 is a proteobacterium of the beta subclass, related to Pseudomonas saccharophila and Variovorax paradoxus. Following an exogenous supply of arsenate, most arsenic occurred as arsenite in the medium and the cell extracts, suggesting reduction and extrusion of arsenic as the mechanism for arsenic resistance in AsRB1.