Rapid Presumptive Identification of Gram-Negative Rods Directly from Blood Cultures by Simple Enzymatic Tests

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1 JOURNAL OF CLINICAL MICROBIOLOGY, Feb. 1990, p /90/ $02.00/0 Copyright 1990, American Society for Microbiology Vol. 28, No. 2 Rapid Presumptive Identification of Gram-Negative Rods Directly from Blood Cultures by Simple Enzymatic Tests JORGE L. SEPULVEDA, CHARLES E. STAGER, AND JAMES R. DAVIS* Department of Pathology, The Methodist Hospital and Baylor College of Medicine, Houston, Texas Received 26 June 1989/Accepted 6 October 1989 Gram-negative rods were presumptively identified directly from blood cultures within 15 min as Escherichia coli, a member of the Klebsiella-Enterobacter group, or oxidase positive. Samples of artificially seeded blood cultures (193 cultures) and patient blood cultures (78 cultures) were filtered into a Dynadepth test card with the Bac-T-Screen instrument (Vitek, Inc., Hazelwood, Mo.). Triton X-100 was then filtered into the test card to lyse the blood cells but not the entrapped bacteria, and either methylumbelliferone-labeled substrates or oxidase reagent was applied to the filter surface. The oxidase test was read within 30 s, and the methylumbelliferone and indole tests were read after a 10-min incubation at room temperature. Positive IP-galactosidase, P-glucuronidase, and indole test results predicted the identification of E. coli with a 96 to 100% sensitivity and a 99 to 100% specificity. Positive P-xylosidase and P-galactosidase test results and negative oxidase and P-glucuronidase test results were 85 to 93% sensitive and 100% specific for a Klebsiella-Enterobacter organism. A positive oxidase test result and negative jp-glucuronidase, 0-xylosidase, and indole test results were highly predictive of Pseudomonas aeruginosa (sensitivity, 100%; specificity, 99%). The procedures described are rapid and simple and provide a direct presumptive identification of the gram-negative rods most commonly found in blood cultures. The incidence of bacteremia caused by gram-negative rods (GNRs) in the United States is estimated to be between 70,000 and 300,000 cases annually (4, 29), with the associated mortality being from 19 to 50% (5, 12, 22, 29, 32). Early detection, identification, and susceptibility testing are very important in determining the outcome of these infections; however, these procedures usually require 48 h or more. There have been many attempts to reduce the time required to perform these procedures. They have included the use of conventional media (8, 30), commercial biochemical kits (1, 3, 18, 21, 27), automated systems (16, 21, 26), and immunological techniques (13, 31). All of these procedures require extensive processing of the inoculum and at least 4 h of incubation. We describe here a rapid and simple method to separate bacteria from the fluid culture medium by filtration. After the addition of chromogenic or fluorogenic substrates and a 10-min incubation, the test results can presumptively identify the organisms. Our initial studies involved the detection of P-galactosidase (,BGALA), 3-glucuronidase (PGLUR), and indole for the presumptive identification of Escherichia coli (7, 10, 11, 14, 17, 20, 24);,-xylosidase (PXYLO) as a marker for the Klebsiella-Enterobacter group (4, 11, 17); and the oxidase enzyme to detect oxidase-positive GNRs. Since the overwhelming majority of the oxidase-positive GNRs isolated from blood cultures are identified as Pseudomonas aeruginosa (4, 12, 22, 30, 33), a direct oxidase test could be expected to have a high predictive value for this organism. Rapid differentiation of E. coli from the Klebsiella-Enterobacter group or Pseudomonas septicemia may be important because of the higher mortality from (12, 32) and antimicrobial resistance of (15, 23, 25) the latter. * Corresponding author. 177 MATERIALS AND METHODS Simulated blood cultures. Suspensions of 193 clinical isolates of GNRs were prepared in 0.85% saline to a turbidity equivalent to a 0.5 McFarland standard, and 1,ul was inoculated into 5 ml of medium (NR6A medium; BACTEC system; Johnston Laboratories, Inc., Towson, Md.) containing 5% recently collected human blood. Cultures were incubated aerobically at 35 C for 16 to 20 h. These cultures were then treated as unknowns in blind studies. Patient blood cultures. Blood (3 to 5 ml) was obtained from patients at The Methodist Hospital and Ben Taub General Hospital (Houston, Tex.), inoculated into vials containing NR6A medium, and then processed on the BACTEC NR660 system (Johnston Laboratories). Cultures found to be positive by the instrument or by visual inspection were Gram stained, and those showing only GNRs or gram-variable rods were tested in our protocol. Semiquantitative counts were also performed by culturing 1,uI of a 1/100 saline dilution of the culture broth. All GNRs were subcultured onto MacConkey and 5% sheep blood agar plates and identified by the AutoMicrobic System (Vitek, Inc., Hazelwood, Mo.) or the API 20E identification system (Analytab Products, Inc., Plainview, N.Y.). Filtration of blood cultures. For the filtration of blood cultures, we used a modification of a method originally developed by Craig Wallis and Joseph Melnick at the Baylor College of Medicine and Applied Polytechnology, Inc., Houston, Tex. (28; C. Wallis and J. Melnick, personal communication). A Dynadepth card (Vitek) was placed into the Bac-T-Screen (BTS; Vitek) machine, and 0.2 ml of the blood culture fluid was filtered by using the manual control. Five milliliters of a 0.03% solution of Triton X-100 in 0.1 M Sorensen phosphate buffer (ph 8.0) was flushed through the card to lyse the blood cells and wash the filter. jpgala, IIGLUR, and IIXYLO tests. After the filter was treated with 0.2 ml of 70% isopropyl alcohol followed by 5 ml of distilled water, 10,ul of a 0.1 mm solution of 4-methylumbelliferyl-p-galactoside, 4-methylumbelliferyl-,-glucuronide,

2 178 SEPÛLVEDA ET AL. J. CLIN. MICROBIOL. Organism TABLE 1. Results of direct tests performed on 193 simulated blood cultures No. of cultures % of cultures positive for:,gala,bglur [XYLO Indole Oxidase Escherichia coli Escherichia hermanii Shigella sonnei Yersinia ruckeri Salmonella typhimurium Salmonella sp. (group B) Klebsiella pneumoniae Klebsiella oxytoca Klebsiella ozaenae Enterobacter cloacae Enterobacter aerogenes Enterobacter agglomerans Citrobacter freundii Citrobacter diversus Citrobacter amalonaticus Serratia marcescens Serratia odorifera Morganella morganii Proteus mirabilis Proteus vulgaris Providencia rettgeri Providencia stuartii Acinetobacter calcoaceticus subsp. anitratus Pseudomonas aeruginosa Pseudomonas maltophilia or 4-methylumbelliferyl-p-xyloside (Sigma Chemical Co., St. Louis, Mo.) in 0.1 M Sorensen buffer (ph 7.0) was dropped onto the surface of a processed filter. The filters were observed, after 10 min of incubation at room temperature, for a blue fluorescence under a hand-held longwavelength UV lamp (366 nm; Ultra-Violet Products, Inc., San Gabriel, Calif.). If very weak or no fluorescence was detected, 5,ul of 0.1 M sodium carbonate-bicarbonate buffer (ph 10) was added, and if a marked increase in fluorescence developed, the test was considered positive. In order to determine the lowest detectable concentration of free 4- methylumbelliferone, a blood culture which was negative at 7 days on the BACTEC NR660 system was filtered as described above, and 10,ul of serial dilutions of 4-methylumbelliferone (Sigma) in water was added to the filters and immediately observed under the UV light. In addition, we performed the tests on isolates of E. coli and Klebsiella pneumoniae, which were grown for 16 to 20 h at 35 C in Trypticase soy broth (BBL Microbiology Systems, Cockeysville, Md.), serially diluted in the NR6A medium containing 10% blood, and then processed as described above. Oxidase test. A drop of 1% tetramethyl-p-phenylenediamine in 100% dimethyl sulfoxide was placed on Whatman no. 3 filter paper and allowed to absorb for 1 min. The BTS filter was washed with 5 ml of distilled water and then pressed against the Whatman filter paper. The development of a dark blue color in the BTS filter in less than 30 s was considered a positive test result. Serial dilutions of an overnight Trypticase soy broth culture of P. aeruginosa were performed in NR6A medium containing 10% blood and processed on the BTS, and the oxidase test was performed to determine the lowest detectable level of the organism. Indole test. A 1-ml portion of the blood culture was transferred to a glass tube (10 by 75 mm) containing 0.4 ml of a tryptophan solution (L-tryptophan [10 mg/ml], peptone [40 mg/ml], and yeast extract [20 mg/ml] in Sorensen buffer) and incubated at 35 C for 10 min. Xylene (0.4 ml) was then added, the tubes were well shaken; and one drop of Kovacs indole reagent was added after the formation of an upper organic phase. Any development of a pink color in the upper layer was a positive test result. In order to determine the lowest level of indole detectable by this procedure, dilutions of indole (Sigma) were performed in BACTEC NR6A medium containing 10% blood, and the test was performed as described above. RESULTS Levels of detection. Free 4-methylumbelliferone was detected at.1,um (1% of the conjugated substrate concentration), and indole was detected at 21,ug/ml. The,GALA test was positive with a minimal concentration of 1 x 106 CFU of E. coli per ml and 5 x 106 CFU of K. pneumoniae per ml, whereas,glur and,3xylo required 1 x 107 CFU of E. coli per ml and 5 x 107 CFU of K. pneumoniae per ml, respectively. The oxidase test was positive for P. aeruginosa at a minimal concentration of 107 CFU/ml. Simulated blood cultures. The results of the direct tests performed on 193 simulated blood cultures are given in Table 1. The majority of E. coli strains reacted with a pattern of positive,gala, PGLUR, and indole and negative,xylo and oxidase test results. A total of 2 of 57 strains (3.5%) were negative for,glur, and 1 of these was an atypical strain of the Alkalescens-Dispar group. Both strains were negative with a 4-h nitrophenyl-,bglur test performed with pure cultures on sheep blood agar as described by Kilian and

3 VOL RAPID IDENTIFICATION OF GNRs IN BLOOD CULTURES 179 TABLE 2. Results of direct tests performed on 78 patient blood cultures % of cultures positive for: Organism isolated No. of cultures pgala PGLUR PXYLO Indole Oxidase Escherichia coli Klebsiella pneumoniae Klebsiella oxytoca Klebsiella ozaenae Enterobacter aerogenes Enterobacer cloacae Morganella morganii Proteus mirabilis Salmonella sp. (group B) Serratia marcescens Acinetobacter calcoaceticus subsp. anitratus Bacteroides thetaiotaomicron Haemophilus influenza type b Aeromonas hydrophila Pseudomonas aeruginosa il Pseudomonas maltophilia Bacillus sp Diphtheroid Enterobacter cloacae + Serratia marcescens Flavobacterium menigosepticum + Pseudomonas acidovorum Haemophilus influenza type b + Staphylococcus aureus Klebsiella pneumoniae + Proteus mirabilis Bulow (17). Only Escherichia hermanii, a rare isolate from blood cultures (9), produced the direct test profile for E. coli. Two Shigella sonnei isolates and one Yersinia ruckeri isolate were IGALA and PGLUR positive but did not produce detectable indole. None of the remaining 132 isolates was positive for IGLUR. Forty-six isolates of the Klebsiella- Enterobacter group were studied in the simulated blood cultures. Ail of the Klebsiella-Enterobacter group cultures were PGALA positive and PGLUR and oxidase negative. A11 four Klebsiella oxytoca cultures were indole positive, whereas the other Klebsiella-Enterobacter group organisms were indole negative. The PXYLO test served as a marker for Klebsiella-Enterobacter group organisms: 85% of the Klebsiella-Enterobacter cultures were PXYLO positive, and no false-positive results were obtained with non-klebsiella- Enterobacter organisms. The Klebsiella-Enterobacter organisms that did not react with the PXYLO substrate included 2 of 17 isolates (12%) of K. pneumoniae, 1 of 1 isolate of K. oxytoca, 1 of 1 isolate of Klebsiella ozaenae, and 4 of 4 isolates of Enterobacter agglomerans. A total of 50 of 50 (100%) P. aeruginosa and 1 of 1 (100%) Pseudomonas maltophilia isolates were positive for the oxidase test. There were no false-positive oxidase test results. Patient blood cultures. In 78 blood cultures, only GNRs or gram-variable rods were seen (Table 2). In 66 cases an aerobic GNR was isolated in pure culture; one anaerobic organism (Bacteroides thetaiotaomicron) was also isolated. A mixed culture of GNRs was obtained in four cases, and in seven cases, gram-positive rods were isolated. In all cases, -107 organisms per ml were present and more than 10 bacilli per oil immersion field were observed on Gram stain. A total of 25 of 25 (100%) E. coli isolates yielded the direct test profile typical of this organism, and there were no falsepositive results. One diphtheroid organism initially identified as gram-negative was positive for PGLUR but negative for PGALA and indole. The,XYLO test was positive for 12 of 13 Klebsiella-Enterobacter organisms in pure culture and for 1 E. cloacae organism and 1 K. pneumoniae organism that were present in mixed cultures. Only one K. oxytoca isolate was negative for PXYLO. All Klebsiella-Enterobacter organisms reacted positively with PGALA and negatively with the PGLUR, oxidase, and indole tests. Among the non- Klebsiella-Enterobacter organisms, there were no falsepositive results for PXYLO. With organisms present in pure culture, all of the P. aeruginosa, Aeromonas hydrophila, and diphtheroid isolates and 25% of Haemophilus influenza isolates were oxidase positive. There were two mixed cultures that contained at least one oxidase-positive organism (Flavobacterium meningosepticum + Pseudomonas acidovorum and Haemophilus influenzae type b + Staphylococcus aureus), and both specimens yielded a positive oxidase test. All of the 11 cultures of P. aeruginosa yielded the direct test profile of positive oxidase and negative,glur,"xylo, and indole test results, and of the remaining samples, only a mixed culture containing H. influenza and S. aureus yielded this profile. The sensitivity, specificity, and predictive values for specific patterns used for the presumptive identification of E. coli, the Klebsiella-Enterobacter group, and P. aeruginosa directly from blood cultures are shown in Table 3. DISCUSSION Previous studies attempting the direct identification of bacteria in blood cultures have established the requirement for a good separation of the organisms from the other components in the blood culture, especially blood cells, which contain enzymes that may interfere in several biochemical tests (7, 27, 30). Centrifugation of the blood cells at low speed, followed by high-speed sedimentation of the

4 180 SEPÛLVEDA ET AL. J. CLIN. MICROBIOL. TABLE 3. Sensitivity, specificity, and predictive values for typical direct test profiles obtained with simulated and patient blood cultures Pattern of results suggesting: Sensitivity Specificity Positive Negative Incidence of predictive value predictive value organisms Escherichia colia 96/1O0b 99/100 98/100 99/100 30/32 Klebsiella-Enterobacter groups 85/93 100/ /100 96/98 24/19 Pseudomonas aeruginosad 100/100 99/99 98/92 100/100 26/14 a Positive PGALA, IGLUR, and indole test results and negative PXYLO and oxidase test results. b The first number represents percentages calculated from simulated blood culture results, and the second number represents percentages obtained from patient blood cultures. c Positive PGALA and PXYLO test results and negative IGLUR and oxidase test results. d Positive oxidase test results and negative PGLUR, PXYLO, and indole test results. bacteria, has been effectively used for separation in most of these studies. However, this is a lengthy procedure involving some labor-intensive manipulations. The filtration technique described in this report provides a faster and simpler alternative. The BTS system has been widely used to screen urine samples for urinary tract infections. The filters are composed of a fiber glass paper that is able to electrostatically bind the bacteria (28). Interfering substances and blood cells in the blood culture fluid can then be efficiently removed by drawing a solution, containing Triton X-100 as a lysing agent, through the filter. When the same amount of a chromogenic or fluorogenic substrate was added to either fluid suspensions or filterretained organisms, reactions seemed to take place faster in the latter (data not shown). While the colored or fluorescent products of the reactions are diluted by the suspension fluid, they are concentrated in a small area in the filter. The filter also provides a flat surface and a white, nonfluorescent background against which any development of color or fluorescence can be easily interpreted. Preliminary experiments determined that reactions with 4-methylumbelliferyl-labeled glucoside substrates require previous treatment of the filtered bacteria with 50 to 70% isopropyl alcohol for optimal sensitivity. This may be related to the intracellular location of the enzymes and to a permeating effect of the alcohol on the cell wall. A certain degree of hydration of the alcohol seemed to be required, since alcohol concentrations outside the 50 to 70% range were not as effective. The addition of alkaline buffer to the filter after incubation (20) improved the sensitivity of the reaction by flushing the free 4-methylumbelliferone into a concentric ring and increasing its fluorescence because of the alkaline ph of the buffer. The positivity of an enzymatic reaction depends on the intracellular concentration, availability and activity of the enzyme, and the number of cells involved in the reaction. When successive dilutions of E. coli and K. pneumoniae were tested for PGALA, PGLUR, and PXYLO activities, concentrations of 107 CFU/ml were required for a positive reaction. When a culture was detected as positive by either the BACTEC NR660 system or visual inspection, bacteria usually reached concentrations above 107 CFU/ml. However, in rare cases, lower bacterial concentrations were detected as positive blood cultures and may have caused false-negative direct test results. The,GALA reaction was strongly positive with all strains of E. coli and the Klebsiella-Enterobacter group, whereas P. aeruginosa strains were usually negative or weakly positive PGALA can thus be used as an initial test, and for this test. the result can be used to choose further direct tests. The,GLUR substrate was hydrolyzed by members of the genera Escherichia, Shigella, and Yersinia. Previous studies on PGLUR activity in pure culture suspensions of aerobic gram-negative bacilli revealed a limited number of species that reacted positively (E. coli, 89 to 100%; Salmonella sp., 17 to 30%; Shigella sp., 40 to 50%) (7, 10, 14, 17, 19). These results are in accordance with our data. The rapid identification of E. coli by positive PGLUR, IGALA, and indole test results has been successful with commercial kits (7, 24). Because of the absence of false-positive direct identifications of E. coli in our series of patient blood cultures and the very low incidence of E. hermanii in patients with bacteremia (9), this pattern of results can be used to presumptively report the presence of E. coli in blood cultures. It is important to perform the indole and PGALA tests, in addition to the PGLUR test, because of the possibility of bacteremia caused by Salmonella sp., which can be IGLUR positive (17, 19), but are easily differentiated from E. coli by negative indole and PGALA tests. Strains of Shigella spp. or Yersinia spp. can occasionally be rglur, PGALA, or indole positive; but they are unlikely to be found as blood culture isolates, and in our limited series they were never simultaneously positive for these three direct tests. E. coli septicemia has been associated with lower mortality and higher susceptibility to antibiotic therapy compared with other enteric gramnegative bacilli (12, 32). The PXYLO enzyme was present in a high percentage of Klebsiella-Enterobacter organisms, with the exceptions of Enterobacter agglomerans, K. ozaenae, and Klebsiella rhinoscleromatis (4, 17), which was in complete agreement with our findings. A test that can rapidly predict the presence of Klebsiella-Enterobacter organisms in a blood culture may be of some importance, since these organisms are frequently of nosocomial origin and are associated with a high mortality and a high frequency of multiple antimicrobial resistance (15, 23, 25, 32). P. aeruginosa is the gram-negative organism most likely to cause death directly related to septicemia (32). A positive direct oxidase test with negative indole, PGLUR, and PXYLO test results had a strong predictive value for P. aeruginosa, because of the very high prevalence of this organism among the oxidase-positive bacteria in our series. Other oxidase-positive organisms were isolated; but they were weakly oxidase positive, produced detectable amounts of indole, or were PGLUR positive, whereas P. aeruginosa was always strongly oxidase positive and indole negative, but other organisms that behave similarly may be more prevalent in a larger series. However, with the possible exception of H. influenza, all other oxidase-positive GNRs in our series are also well known for being opportunistic, multiresistant organisms that are more frequently found in patients with nosocomial infections. A positive direct oxidase test result should be useful for earlier correct management of gram-negative bacteremia. Fluorescent-antibody identification of P. aeruginosa has

5 VOL. 28, 1990 RAPID IDENTIFICATION OF GNRs IN BLOOD CULTURES 181 been successfully applied to the direct identification of this organism in blood cultures (6), and antibodies reacting against other GNRs may become available for direct examination of blood cultures. The techniques described here may be useful as a screen for groups of organisms and, hence, limit the number of specific antibodies to be tested. In conclusion, this filtration procedure, combined with the described substrates, is a fast, efficient, and effective method to presumptively identify the GNRs that are most frequently isolated from patients with bacteremia. A correct presumptive identification could be made directly from 49 of 78 (63%) patient blood cultures within 10 to 15 min. The sooner that results are available, the greater the likelihood that decisions about antimicrobial therapy can be influenced. 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