Rapid and Economical Identification and Antimicrobial Susceptibility Test Methodology for Urinary Tract Pathogens

STEPHEN C. EDBERG* AND RICHARD W. TREPETA Clinical Microbiology Laboratory, Department of Laboratory Medicine, Yale-New Haven Hospital, Yale University School ofMedicine, New Haven, Connecticut 06504

Received 5 May 1983/Accepted 2 September 1983

To decrease the time and cost of processing urine cultures, we devised a critical pathway to identify and perform antibiotic susceptibility tests on commonly isolated microbial pathogens within 6 h of growth detection. The strategy was based on eliminating expensive kits and automated procedures when not required. A pathway utilizing a statistical matrix and three rapid biochemical tests required to identify the most common pathogen, Escherichia coli, was developed. This species, which represented 82% of urinary isolates, was identified in 1 h for less than 10% the cost of a commercial kit. The specificity of the 1-h E. coli identification battery was .99.9% with a sensitivity of 93%. In addition, this critical pathway, adapting published methods, permitted the identification of other enteric pathogens, the group D streptococci, and Pseudomonas aeruginosa within 4 to 6 h. Furthermore, it accounted for other microbes that required longer periods of incubation. The pathway also included a rapid disk diffusion sensitivity test. Utilizing the critical pathway strategy, 76% (E. coli frequency of 0.82 x E. coli sensitivity of 0.93) of all urinary pathogens were identified within 1 h, and 98% were identified within 4 h with an antibiotic sensitivity test available within 6 h after the observation of growth. Costs were reduced from 2.5 to 5.0 times. This methodology is applicable to other specimen types.

In the last decade we have witnessed a signifi- the largest number of culture requests received cant growth in the number of laboratory tests by the Clinical Microbiology Laboratory of the carried out per hospitalized patient and an in- Yale-New Haven Hospital. The distribution of crease in the sophistication and complexity of microbiology specimens was as follows: urine each individual test. Too often this growth was cultures, 35.9%; blood cultures, 23.2%; stool given impetus by the tendency to axiomatically specimens, 11.9%; respiratory specimens, presume that more was better than less. Costs 10.2%; wound specimens, 9.4%; mycology-tu- have increased concomitantly (10). To address berculosis specimens, 6.4%; and sterile body this situation, we devised a critical pathway fluids (other then blood cultures), 3.0%. Al- strategy to reduce costs, increase accuracy, and though the ratios of some specimen types, par- decrease the time required for identification and ticularly stool and respiratory, can vary by sea- antimicrobial susceptibility testing of microbes son, this division approximated that seen isolated from urinary tract infections. The meth- nationally at primary teaching hospitals. od can also be applied to other specimen types. At Yale-New Haven Hospital and nationally, The strategy was based on developing a critical 95% of urinary tract infections are caused by a pathway of work flow that would permit the single infecting microbe, with Escherichia coli laboratory to specifically identify the most com- being responsible for approximately 80% of all mon isolates with the fewest number of tests and nosocomial and more than 90% of those commu- in the most rapid time span. Complex, time- nity-acquired infections. After E. coli, other consuming, and expensive procedures required members of the family Enterobacteriaceae, the for only a small percentage of laboratory isolates group D streptococci, and Pseudomonas aeru- would not be routinely used for all microbes, as ginosa comprise all but 2% of isolates. The is now common practice. A requirement of the majority of hospital microbiology laboratories strategy was the generation of a completed re- devote a significant portion of their budgets to port for over 95% of isolates within 6 h of the purchasing commercial products to identify E. time significant growth was detected. coli and other enteric pathogens. Single kits The urine specimen was chosen as the critical range in price from $1.90 to $2.50 and can cost pathway strategy model because it represented up to $4.00 if instrumentation is utilized. Be- 1287 1288 EDBERG AND TREPETA J. CLIN. MICROBIOL. cause these kits are designed to identify all petri plate (5, 12). Sodim ascorbate (0.1% final concen- enteric pathogens, they contain a large number tration) was added to this reagent to prevent autoxida- of individual biochemical tests, many of which tion. A commercial reagent (Marion Scientific, Kansas was also to Like are extraneous to the identification of a City, Mo.) found be satisfactory. the particu- indole test, a portion of the colony was rubbed onto lar species such as E. coli. Yet, we pay for the the filter paper; if the reagent turned purple in 30 s, the additional tests and must wait for their reactions test result was positive; if it remained unchanged, it to develop. Most commercial kits require 24 h of was negative. At least 50 tests can be carried out on incubation after inoculation before an identifica- each half of the filter paper. tion is established. The group D streptococci P-Glucuronidase reagent consisted of 0.5 mg of p- and P. aeruginosa can also be identified rapidly nitrophenyl-p-D-glucuronide (Sigma) per ml in 0.05 M and with a much smaller number of tests than Sorenson phosphate buffer (pH 7.5). Portions (0.25 ml) are required for other microbes in their respec- were transferred into test tubes (12 by 75 mm). Several tive families. colonies from MacConkey agar were inoculated into a tube and incubated for 1 h at 35°C. An unchanged, To decrease the cost and time of identification colorless solutionn was regarded as a negative result. of E. coli, a statistical matrix was devised to A yellow hue indicated a positive result as color determine the minimum number of tests re- developed when the p-nitrophenyl was released from quired (1, 6, 15, 16). The tests were selected by p-nitrophenyl-p-D-glucuronide by P-glucosidase. their ability to separate E. coli from other mi- The group D streptococci and P. aeruginosa were crobes and to identify this species with 99.9% also rapidly identified (2, 7, 8). Candida albicans was specificity. These tests were formulated to yield identified by the germ tube test (3). Other pathogenic results within 1 h after inoculation and were microbes were identified as directed by the critical integrated into the routine identification system pathway by conventional means. Processing urine cultures. Agar plates inoculated for urinary tract pathogens. To achieve maximal with patient urine were examined after 18 to 24 h of benefit from this system, the work flow pathway incubation. If growth was not observed, plates were was further designed to yield an identification reincubated and examined again at 48 h. All plates and antimicrobial susceptibility test from 98% of showing significant growth (2104 microbes per ml) human urinary isolates within 6 h. were batch processed. Identification and antibiotic susceptibility proceeded in the following sequence MATERIALS AND METHODS (Fig. 1): (i) The rapid spot indole and oxidase tests were performed and recorded. (ii) The test tubes Analysis of microbial growth. All specimens were containing p-nitrophenyl-f3-D-glucuronide reagent human urine samples submitted for analysis of micro- were inoculated and placed in the incubator. (iii) A 6-h bial content and antibiotic susceptibility to the Clinical plate antibiotic disk susceptibility test (13) was inocu- Microbiology Laboratory of the Yale-New Haven lated and placed in the incubator. (iv) The P-glucuroni- Hospital. Microbes were quantified by means of a dase test was read 1 h after inoculation, and the results standard calibrated loop technique (3). A total of 1,200 were recorded. Those microbes that were indole posi- patient samples were tested, of which 465 showed tive, oxidase negative, lactose positive, and ,3-gluc- significant growth. E. coli was the single pathogen in uronidase positive were identified as E. coli. Those 381 (82%) of these positive cultures; E. coli with organisms that were oxidase negative and negative on another microbe was present in an additional 5%. A any other test were considered an enteric pathogen non-E. coli enteric pathogen, a group D streptococcus, and inoculated into a commercial, 4-h identification or P. aeruginosa represented 16% of the remaining system (Micro-ID; General Diagnostics, Warner-Lam- isolates encountered in this study. Therefore, mi- bert Co., Morris Plains, N.J.). Those organisms that crobes other than members of the family Enterobacte- were oxidase positive were considered nonfermenters riaceae, group D streptococci, or P. aeruginosa were and were identified as P. aeruginosa by a rapid test (2) encountered in 2% of cases during the course of this or as another species by conventional means (3, 6). study. Group D streptococci were identified by rapid tests (7, Identification of E. coli. By applying an analytical 8), and other gram-positive bacteria were identified by matrix (4, 9), the number of tests required to identify conventional tests (7). C. albicans was identified by E. coli specifically were reduced to the following: the 2-h germ tube test, and other yeasts were identified growth and lactose fermentation on MacConkey agar, by a commercial product (Analytab Products, Plain- indole positivity, cytochrome c oxidase negativity, view, N.Y.). (v) After 4 h, the Micro-ID tests were and ,-glucuronidase positivity. read and the enteric pathogens were identified. (vi) The indole test was carried out as a spot procedure The antibiotic susceptibility plates were examined and on filter paper and required 30 s (17). Kovacs reagent recorded. If quality control criteria were fulfilled, final was added to standard laboratory filter paper in one reports containing an identification and antibiotic sen- half of a petri plate; a portion of a bacterial colony was sitivity were available for 98% of isolates 6 h after rubbed onto the filter paper, and if within 30 s the initiation of batch processing. colony became red, the test result was indole positive. If the reagent remained unchanged, it was negative. RESULTS The oxidase test was likewise a spot test, with 1% N,N,N',N'-tetramethyl-p-phenylenediamine dihy- Specificity and sensitivity of the identification of drochloride reagent (Sigma Chemical Co., St. Louis, E. coli. By comparison with the methods statisti- Mo.) added to filter paper in the other one half of the cally the same as the methods used by the VOL. 18, 1983 RAPID METHODOLOGY FROM URINE 1289

7 Final

FIG. 1. Sequence of identification and antibiotic susceptibility determination. Y, Yes; N, no; ,-gluc, 1- glucuronidase.

Centers for Disease Control (1, 6), 354 of 354 and oxidase negative were also identified by microbes that grew on MacConkey agar were ,B- conventional means as E. coli. Theoretically, glucuronidase, indole, and lactose positive, and 8% of Shigella sp. may be 3-glucuronidase, oxidase negative and were identified as E. coli indole, and lactose positive; however, none (specificity of .99.9%). We found that E. coli were encountered from urine during the course was 100% positive for growth on MacConkey of this study, and only two had been identified agar, 95% indole positive, 94% 1-glucuronidase from the previous 200,000 urine cultures at this positive, 88% lactose positive, and 0% oxidase hospital. positive. All of the 12% lactose-negative isolates Seven percent (27 of 381) of E. coli showed that were indole and ,B-glucuronidase positive either a negative indole or negative 3-glucuroni- 1290 EDBERG AND TREPETA J. CLIN. MICROBIOL.

TABLE 1. Comparison of test systems to identify E. coli and other members of the family Enterobacteriaceae

Test system Avg. cost/unita Specificity Sensitivity Incubation time Technologist Testsystem ~($) (% (% (h) labor time (min) Automated 3.60 97 91 4 7 Commercial kit 2.25 98 91 4-24 8 Commercial test tubes 2.60 94 90 24 7 Laboratory-made test 1.82 94 90 24 7 tubes 1-h E. coli tests 0.20 99.9 93 1 2 " Cost of the commercial 4-h system to identify the 18% of urinary isolates not E. coli average $0.45 per total positive culture. All costs are based on prices effective 1 January 1983 at maximum volume discount. dase test (sensitivity of 93%). These atypical E. the group D streptococci and P. aeruginosa coli isolates and other enteric pathogens were were identified by a small number of rapid tests, identified in 4 h by a commercial system, and the with the remaining microbes identified by a large nonenteric pathogens were identified by either series of biochemical tests. A critical pathway rapid or standard Centers for Disease Control for processing specimens automatically directed formulation biochemical tests (Fig. 1). the work flow to higher levels of complexity Cost analysis. Table 1 demonstrates the costs when required. incurred to identify enteric pathogens from urine First, we have demonstrated that by choosing by an average automated system and manual the tests to be used with a statistical matrix, E. commercial kits, laboratory-prepared and com- coli can be identified specifically by the follow- mercially purchased test tube biochemical sys- ing characteristics: growth and lactose fermenta- tems, and the 1-h identification system de- tion on MacConkey agar, production of ,B-gluc- scribed here. For purposes of calculation, the uronidase, production of indole, and lack of commercial systems were considered to yield a production of oxidase. By using these character- definitive diagnosis with no other procedures istics, there is less than a 0.1% chance that it required; in practice, approximately 5 to 10% of may be confused with another organism (99.9% commercial identification tests must be repeat- specificity). Approximately 7% of E. coli will ed. The Micro-ID 4-h commercial system was demonstrate an atypical reaction in any one of calculated as the adjunct identification method these tests (93% sensitivity). However, these to the 1-h E. coli test battery. isolates are captured by inoculation into the 4-h The data showed that the 1-h identification commercial system, as are all other enteric system reduced the expenses of identifying E. bacilli (4). Rapid systems have been reported coli in the urine by a factor of at least 5.0 from that utilize only growth on MacConkey agar, the automated commercial, 3.0 from the manual indole positivity, and oxidase negativity (14) for commercial, and 2.5 from the conventional identification. Without the 3-glucuronidase test, homemade biochemical test tube systems. Over- however, there is a sacrifice of specificity for all, total laboratory kit usage was reduced 65%. sensitivity. For example, Citrobacter diversus We calculated the actual savings in our labora- and Klebsiella oxytica may be confused with E. tory to be from $30,000 to $32,000 in 1982. coli (11). Second, after designing the minimum number DISCUSSION of tests required to identify E. coli and other E. coli is the most common microbe isolated commonly isolated pathogens, we analyzed cost in the Clinical Microbiology Laboratory of the parameters. Our thesis was that by determining Yale-New Haven hospital. It belongs to the whether an organism was or was not E. coli, a family Enterobacteriaceae, of which there are at critical pathway could be developed that would least 14 genera and over 80 species. Commercial obviate the need for using large numbers of kits are designed to identify all these genera with commercial, expensive kits. Our data indicated equal facility. The trend over the last several that we eliminated 93% of E. coli kit identifica- years has been to perform an increasing number tions and reduced kit usage in the laboratory of tests on each isolate to avoid misidentifying a overall by 65%. Because the difference in cost rare or unusual microbe. For the preponderance between a commercial or homemade identifica- of clinical laboratory isolates, these tests are tion system is from 2.5 to 5.0 times more than unnecessary. The approach taken here was to the 1-h test, substantial savings occurred. identify E. coli specifically, rapidly, and inex- After establishing a specific and inexpensive pensively and only to use commercial systems means of identifying E. coli and other frequent for the minority of enteric pathogens. Likewise, urinary isolates, our third and final goal was to VOL. 18, 1983 RAPID METHODOLOGY FROM URINE 1291 perform it in a time frame that would be rapid eases. Addison-Wesley Publishing Co., Menlo Park, Cal- and useful for the Initiation of is if. patient. therapy 4. Edberg, S. C., and L. Konowe. 1982. Statistical and based on two factors: the name of the organism applied meaning of a clinical microbiology product: bacte- and the antibiotic susceptibility pattern. There is rial identification systems, p. 268-299. In V. Lorian (ed.), an association between the species name and its Clinical microbiology and patient care. 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