Rapid Identification of Enterobacteriaceae from Blood

Transcription

1 JOURNAL OF CLINICAL MICROBIOLOGY, Nov. 1979, p /79/ /05$02.00/0 Vol. 10, No. 5 Rapid Identification of Enterobacteriaceae from Blood Cultures with the Micro-ID System STEPHEN C. EDBERG,* DAPHNE CLARE, M. HELEN MOORE, AND JACQUES M. SINGER Department of Pathology, Division of Microbiology and Immunology, Montefiore Hospital and Medical Center, The Albert Einstein College of Medicine, Bronx, New York Received for publication 7 August 1979 Micro-ID is a new test system designed to identify members of the family Enterobacteriaceae in 4 h. It consists of 15 biochemical tests on reagent-irnpregnated paper disks; each test is in its own compartment in a molded plastic tray. Based on the pattern of positive and biochemical reactions, a five-digit octal code number is calculated. A computer-generated identification manual accompanies the product, and for each octal code listed there is a numerical value that represents the unknown isolate's degree of fit to a typical organism (LFR), a second numerical value that represents its separation from other organisms (PNOR), and a verbal description of the quality of identification. Only one reagent is added to the system. Manufacturer's directions were modified in this laboratory to allow identification from a turbid blood culture bottle within 4 h. Based on 330 routine clinical cultures tested, there was a 96.1% agreement with conventional identification to the genus and species level; 1.2% yielded first two choices possible, with one being the correct choice; 1.2% provided a correct genus, but no species identification; and 1.5% produced an incorrect identification. The Micro-ID is an accurate, facile system for the rapid identification of Enterobacteriaceae from blood cultures. There has been considerable interest in recent years in the rapid identification of septicemiacausing bacteria. The first rapid identification system available to the routine clinical microbiology laboratory was presented by Wasilauskas and Ellner (8) and utilized a mixture of conventional and commercial reagents. With the widespread use of commercial kit systems for the identification of Enterobacteriaceae, it was found that some performed well for the rapid identification from blood cultures. Kocka and Morello (7) were able to identify Enterobacteriaceae from blood cultures within 7 h with the Indolex Enteric I system, and Edberg et al. (4), using a combination of Pathotec strips and some conventional media, were able to accurately identify most genera and species of medically important bacteria within 4 h. Blazevic et al. (2) have reported a modified inoculation procedure with the API strip to allow an identification 18 to 24 h after the observation of a turbid blood culture bottle. Micro-ID (General Diagnostics Division, Warner-Lambert Co., Morris Plains, N.J.) is a new system for the identification of Enterobacteriaceae. It consists of 15 biochemical tests on reagent-impregnated paper disks; each test is in its own compartment in a molded plastic tray. The battery of tests includes Voges-Proskauer, nitrate reductase, phenylalanine deaminase, hydrogen sulfide, indole, ornithine decarboxylase, lysine decarboxylase, malonate utilization, urease, esculinase, o-nitrophenyl-,8-d-galactopyranoside, and the sugar fermentation tests arabinose, adonitol, inositol, and sorbitol. Only one reagent, 20% KOH, is added to the system. No compartment is overlaid with oil. The inoculation procedure recommended by the manufacturer for the identification of Enterobacteriaceae from primary plates was modified in this laboratory to allow the 4-h identification of members of this family directly from blood culture bottles. The method is designed to allow its use in the routine laboratory with a minimum change in the processing of blood culture specimens. 693 MATERIALS AND METHODS Blood culture technique. Blood cultures were drawn aseptically by the attending physician; 5 ml of blood was inoculated directly into a 45-ml Vacutainer (Becton, Dickinson and Co., Rutherford, N.J.). For each venipuncture, two 45-ml Vacutainers were used: Trypticase soy broth (BBL Microbiology Systems, Cockeysville, Md.) and supplemented peptone broth II. The Trypticase soy broth was vented with filtered air immediately; the anaerobic bottle was unvented but had a cap to allow the release of gas produced in the bottle. Appropriate accessory factors were in-

2 694 EDBERG ET AL. cluded in each bottle. Incubation was at 36 C. All bottles were visually observed each day; blind subcultures were routinely performed after 24 to 48 h. Inoculum preparation. When growth was observed, a Gram stain was made. Smears demonstrating gram- bacilli morphologically consistent with Enterobacteriaceae and appearing to be axenic were processed by the rapid identification protocol. A 10- to 15-ml amount of medium was aseptically removed above the blood cell layer with a 22-gauge needle attached to a 20-ml syringe (Fig. 1). The medium was centrifuged for 15 min at 4,000 rpm. The supernatant was discarded. A small portion of the pellet was used to perform a cytochrome oxidase (Pathotec) test (General Diagnostics, Division of Warner-Lambert Co., Morris Plains, N.J.), after which 4 ml of sterile physiological saline was added to the tube. If the suspension visually appeared to be more dense than a 0.5 Mc- Farland turbidity standard, additional saline was added to dilute the suspension to approximate this degree of turbidity; 0.2 ml of the suspension was added to each of the 15 micro-id biochemical tests. Another 0.5 ml was used to perform a direct antibiotic susceptibility test, and the remaining inoculum was used to inoculate a variety of primary isolation and growth media to determine whether the isolate was a mixed, facultative anaerobic, or aerobic culture. After 4 h of incubation at 36 C, 2 drops of 20% KOH was added to the Voges-Proskauer well. Biochemical reactions were then read according to manufacturer's directions, an octal code number was calculated, and an identification was established by using the Micro-ID computergenerated identification manual. If the identification manual yielded more than one choice, possible additional biochemical or serological tests recommended by the manual were done. Each isolate was given one of the following designations: A, isolate correctly identified to genus and species; B, one or more choices as the possible identification; C, organism identified to the genus level only; D, octal number not found in the identification manual; and E, isolate misidentified. Conventional identification. All blood cultures were processed in parallel with the rapid identification method by conventional procedures (1). Enterobacteriaceae were identified from colonies by conventional media from their reactions on triple sugar and motility agars and their ability to produce indole, acetoin, urease, H2S, esculinase, and deoxyribonuclease; to utilize malonate and citrate; to decarboxylate ornithine and lysine; to deaminate phenylalanine; to hydrolyze o-nitrophenyl-,8-d-galactopyranoside; and to ferment sorbitol. When necessary, additional biochemical and appropriate serological tests were performed (5, 6). During the course of this investigation, we used the nomenclature of Edwards and Ewing (5, 6) rather than that of Bergey's Manual of Determinative Bacteriology. Recently, proposals for some taxonomic changes have been made by the Center for Disease Control (CDC) (3). Although some of the changes are of nomenclature only, others involve a biochemical test(s) which was not routinely used in this laboratory when this study was performed. Where, by our system, Edwards and Ewing's and the new CDC nomenclature are the same, we so indicate (Table 1). Three hundred thirty sequential strains of Enterobacteriaceae isolated from blood were studied (Table 1). RESULTS Micro-ID and conventional methodology yielded the same genus and species name 317 of 330 times (96.1%, Table 2). In four cases (1.2%) the Micro-ID produced two or more choices as the possible identification. In each case, the correct genus and species was listed among the choices. Where the Micro-ID identification manual yielded two or more species as a possible identification, it listed a series of biochemical reactions to separate the choices; in each instance, by using the recommended tests, the correct identification was achieved. Micro-ID yielded a correct genus diagnosis four times but gave no species identification (1.2%). The Micro- ID misidentified five isolates (1.5%). It called one Enterobacter cloacae an Enterobacter agglomerans, one Citrobacter diversus a lysine Escherichia coli, one Proteus morganii a Proteus mirabilis, and one Proteus mirabilis a Proteus vulgaris (Tables 2 and 3). DISCUSSION Identification agreement percentages between two different systems are influenced by many factors. Prominent among these are the distri- MICRO-ID J. CLIN. MICROBIOL. BLOOD CULTURE " BOTTLES \ VIDENCE OF GROWTH GRAM STAIN BLIND SUBCULTURE FIG. 1. Flow chart of the study. DIRECT SENSITIVITY DIRECT \ u BIOCHEMICALS 10ML SUBCULTURE (with standardized CENTRIFUGATION identification and antibiotic susceptibility) AND RESUSPENSION

3 VOL. 10, 1979 bution of organisms used to test the system, the system one establishes as the reference, and the method of data analysis. Because the isolates in this study were sequential, the data contained a preponderance of E. coli, Klebsiella pneumoniae, and P. mirabilis, the most common members of the family Enterobacteriaceae isolated from blood cultures. In a companion study, with colonies from primary plates of a variety of clinical specimens (S. C. Edberg, B. Atkinson, C. Chambers, H. Moore, C. Zorzon, and J. M. Singer, Abstr. Annu. Meet. Am. Soc. Microbiol. 1978, C167, p. 305), in which the members of the family Enterobacteriaceae were more evenly represented, the percentage of agreement between the Micro-ID and a conventional identification system was somewhat lower (94.5%) than that reported here. Whether this was due to excess representation by the more common isolates, effects of the growth medium on the organism in a blood culture bottle, or normal statistical variation is not known. However, this distribution is normal for isolates from blood cultures. Since the percentage of agreement is influenced by the distribution of bacterial strains tested, we have presented the data in a way (Table 2) that enables all comparisons to be made readily. We chose to do this by comparison tables in which the user may calculate percentage agreement between the two systems in a variety of dimensions. A conventional media system, comparable to and tested against the one used at the CDC, served as the standard for this study rather than another commercial kit. Each commercial kit system consists of its own biochemical formulations and its own biochemical test series. The number and type of test formulations of each product are designed to work best within its own system rather than being identical to the CDC battery. Most commercial kits are accompanied by a computer identification manual which identifies the isolates based on the biochemical test reaction pattern generated by the kit. The comparison of commercial kit systems is best made, therefore, on the basis of organism identification rather than test-by-test comparisons. Since all commercial kit systems disagree at some level with the CDC conventional battery, a direct comparison of two commercial kit systems merely results in percentage of agreement figures that represent the sum of the innate errors of both (Edberg et al., Abstr. Annu. Meet. Am. Soc. Microbiol. 1978, C167, p. 305). In the course of this study, bacterial isolates other than those belonging to the family Enterobacteriaceae were encountered. The oxidase, glucose nonfermenters included two strains of Pseudomonas maltophilia, four MICRO-ID BLOOD CULTURE IDENTIFICATION 695 TABLE 1. Blood culture isolates testeda No. of Percentage Organism iso- of total lates isolatesb E. coli K. pneumoniae Klebsiella ozaenae E. cloacae Enterobacter aerogenes E. agglomerans P. mirabilis P. morganii Proteus rettgeri P. vulgaris Providencia stuartii Providencia alcalifa ciens Serratia liquefaciens Serratia marcescens Serratia rubideae Citrobacter freundii C. diversus Salmonella groups B, C, and D a Conventional identifications were made according to Edwards and Ewing (5,6). According to recent CDC nomenclature (3) and based on the biochemical tests performed in this study, the following names are interchangeable on the above list: K. pneumoniae, indole positive (K. oxytoca); Enterobacter hafniae (Hafnia alvei); and P. morganii (Morganella morganii). One of the twelve P. rettgeri was urease positive and would correspond to P. stuartii; 11 of the remaining 12 would correspond to P. rettgeri. E. gergoviae, E. sakazakii, and C. amalonaticus, if isolated, would, in most cases, be considered E. aerogenes, E. cloacae, and C. freundii, respectively. 'To the nearest 0.1%. strains of Acinetobacter calcoaceticus subsp. anitratus, and two strains of Acinetobacter calcoacticus subsp. lwoffi. These organisms yielded octal numbers which were either not listed in the identification manual or were biochemically consistent Shigella spp. All showed Shigella serological reactions and were identified by conventional procedures for nonfermenters. Of four strains of Haemophilus influenzae recovered, three grew only chocolate agar in a CO2 atmosphere, and, in addition, all four yielded octal numbers which were found to be unique in this species (J. C. Edberg, E. Melton, and J. Singer, Abstr. Annu. Meet. Am. Soc. Microbiol. 1977, C94, p. 51). The gram- anaerobes included 15 strains of Bacteroides fragilis and 4 strains of Fusobacterium nucleatum. They yielded octal numbers which alerted us to their presence and were identified by gasliquid chromatography. Mixed cultures, whether within or outside the family Enterobacteriaceae, produced octal numbers which indicated

4 696 EDBERG ET AL. J. CLIN. MICROBIOL. TABLE 2. Comparison of the conventional and Micro-ID identification for all isolatesa CONVENTIONAL IDENTIFICATION / TOTAL z 0 (-3 LLz wc E cob ~ S enteritidis 3 3 C freundl 4 4 CcdJversus 2 2 K pneumoniae K ozaenae 4 3 E aerogenes 6 6 E cloacae E agglomerans 3 2. S marcescens 3 3 S liquetaclens 6-6 S rubidaea P vulgarns 2 - P mirabdhs P morganil P rettgerl P alca/faciens 4 4 P stuarti 2- Proteus sp 2 1--I Enterobacter sp _ Klebsiella sp Not in book 0 Not separated 4 2 a This table is used much in the same way a standard chart is used for determining the distance between two cities. For example, if one wished to determine the Micro-ID efficacy for the identification of P. mirabilis, one would determine, from the "conventional identification" column, that 46 strains were tested. Following a parallel line to the "Micro-ID identification," one would find the Micro-ID identified 45 of these as P. mirabilis and one as P. vulgaris. Each - represents a combination not encountered, or zero. TABLE 3. Misidentifications by Micro-ID Conventional Micro-ID identifi- Biochemical Octal identification cation test responsible no. E. cloacae E. agglomerans Ornithine C. diversus E. coli (lysine None ) P. mor- P. mirabilis Indole ganii P. mirabi- P. vulgaris Ornithine lis K. pneumo- K ozaenae Malonate niae and esculin the presence of multiple organisms. One of them yielded a highly atypical interpretation in which the choices were not separated. The methods described in this paper required a minimal extension of procedures routinely used in the laboratory. By relating the identity of the organism with known patterns of antimicrobial sensitivity, valuable information is available to the physician on the same day that a blood culture becomes positive. Conversely, in those laboratories using a rapid antimicrobial susceptibility technique, a more complete report and an added dimension of quality control are obtained. Micro-ID proved efficacious for the 4- h identification of Enterobacteriaceae from blood cultures. LITERATURE CITED 1. Bartlett, R. C., P. D. Ellner, and J. A. Washington Cumitech 1, Blood cultures. Coordinating ed., John C. Sherris. American Society for Microbiology, Washington, D.C. 2. Blazevic, D. J., C. M. Trombley, and M. E. Lund Inoculation of API-20E from positive blood cultures. J. Clin. Microbiol. 4: Brenner, D. K., J. J. Farmer, F. W. Hickman, M. A. Asbury, and A. G. Steigerwalt Taxonomic and nomenclature changes in Enterobacteriaceae. Depart- 2

5 VOL. 10, 1979 MICRO-ID BLOOD CULTURE IDENTIFICATION 697 ment of Health, Education, and Welfare publication number (CDC) Center for Disease Control, Atlanta, Ga. 4. Edberg, S. C., M. Novak, H. Slater, and J. M. Singer Direct inoculation procedure for the rapid classification of bacteria from blood culture. J. Clin. Microbiol. 2: Edwards, P. R., and W. H. Ewing. (ed) Identification of Enterobacteriaceae, 3rd ed. Burgess Publishing Co., Minneapolis, Minn. 6. Ewing, W. H Differentiation of Enterobacteriaceae by biochemical reactions. Department of Health, Education, and Welfare publication number (CDC) , revised. Center for Disease Control, Atlanta, Ga. 7. Kocka, F. E., and J. A. Morello Rapid detection and identification of enteric bacteria from blood cultures. J. Infect. Dis. 121: Wasilauskas, L. L., and P. D. Ellner Presumptive identification of bacteria from blood cultures. J. Infect. Dis. 124: