Coagulase-Negative Staphylococci

Transcription

1 JOURNAL OF CLINICAL MICROBIOLOGY, Apr. 1987, p /87/ $02.00/0 Copyright 1987, American Society for Microbiology Vol. 25, No. 4 Antimicrobic Susceptibility and Plasmid Profile Analysis as Identity Tests for Multiple Blood Isolates of Coagulase-Negative Staphylococci ALAN I. HARTSTEIN, 23* MIGUEL A. VALVANO,4 VIRGINIA H. MORTHLAND,2 PETER C. FUCHS,5 SUSAN A. POTTER,4 AND JORGE H. CROSA4 Department of Hospital Infection Control, University Hospitals,' and Departments of Medicine,2 Clinical Pathology,3 and Microbiology and Immunology,4 Oregon Health Sciences University, Portland, Oregon 97201, and Department of Pathology, St. Vincent Hospital and Medical Center, Portland, Oregon Received 19 June 1986/Accepted 15 December 1986 We compared a disk diffusion antimicrobic susceptibility panel with plasmid DNA profiles as tests for identity of 106 isolates of coagulase-negative staphylococci cultured from the blood of 45 patients on multiple occasions. The antimicrobic panel included penicillin, oxacillin, clindamycin, trimethoprim-sulfamethoxazole, chloramphenicol, tetracycline, tobramycin, kanamycin, and gentamicin. Nineteen patterns of antimicrobic susceptibility were found. The most common pattern was present in 25% of the isolates, and at least one isolate from 31% of the patients had this pattern. Forty-seven distinct plasmid DNA profiles were found. The most common plasmid profile was present in 8.5% of the isolates, and at least one isolate from 15% of the patients had this profile. Twenty-eight patients had multiple isolates that were identical by plasmid profile analysis. Twenty-seven (96%) of these patients had isolates that were also identical by antimicrobic susceptibility. Nineteen patients had multiple isolates that were different by plasmid profile analysis. In 18 (95%) of these patients, the isolates were also different by antimicrobic susceptibility. Although plasmid DNA profile analysis is a more discriminating tool, these data confirm that a selected disk diffusion antimicrobic susceptibility panel may be used to screen multiple blood isolates of coagulase-negative staphylococci for identity or differences. Coagulase-negative staphylococcal bacteremia is recognized as a frequent and morbid medical illness (4-6, 8, 11, 24, 27, 30). However, coagulase-negative staphylococci are also the most frequent contaminants of blood cultures (17, 28, 29). Many authors, therefore, recommend that multiple blood cultures should be positive for the organism when presuming that true bacteremia is present (6, 8, 11, 24, 27, 30). When more than one culture grows coagulase-negative staphylococci, this still may be indicative of multiple episodes of culture contamination rather than true bacteremia (14). The phenotypic comparison of multiple isolates of coagulase-negative staphylococci has been suggested to support their clinical significance, to trace the epidemiology and pathogenesis of infections, and to confirm relapse (1, 2, 7-9, 13, 20, 23, 26). Bacteriophage typing, biotyping, and antimicrobic susceptibility patterns have all been used to compare isolates with one another, but with limited success (2, 8, 9, 13, 20, 22, 23, 26). Plasmid profile analysis is considered to be a precise method for comparing one isolate with another (1, 2, 20). Antimicrobic susceptibility patterns have been criticized as a comparison test for two reasons: (i) a tendency for isolates from different patients to have similar antimicrobic susceptibilities and (ii) an inability to obtain reproducible results on retesting the same isolate (1, 9, 20). However, tested antimicrobics have not been specifically selected for the phenotypic comparison of one isolate with another. Furthermore, antimicrobic susceptibility testing has not been compared with plasmid profile analysis of isolates cultured on multiple occasions from a large number of patients. * Corresponding author. 589 The present study was directed at the selection of an antimicrobic susceptibility panel with the discriminating capability necessary to differentiate isolates of coagulasenegative staphylococci. Our results suggest that the selected antimicrobic susceptibility panel may be used as an alternative method for isolate comparison when plasmid DNA profile analysis is not readily available. (This study was presented in part at the 86th Annual Meeting of the American Society for Microbiology, Washington, D.C., 23 to 29 March 1986 [V. H. Morthland, A. I. Hartstein, and M. A. Valvano, Abstr. Annu. Meet. Am. Soc. Microbiol. 1986, C157, p. 354].) MATERIALS AND METHODS Bacteria. A total of 140 clinical blood isolates of coagulasenegative staphylococci were used for this study; 106 isolates were from 45 patients who had more than one positive blood culture over less that a 1-week interval, and 34 isolates were from patients who had single positive blood cultures. All were derived from specimens submitted to the Clinical Microbiology Laboratory of Oregon Health Sciences University, grown in enriched tryptic soy broth (BACTEC; Johnston Laboratories, Inc., Towson, Md.), and subcultured on 5% sheep blood agar (Prepared Media Laboratories, Tualatin, Ore.). Isolates were identified as coagulasenegative staphylococci by conventional means, including an inability to coagulate rabbit plasma (BBL Microbiology Systems, Cockeysville, Md.) after overnight incubation at 35 C (16). Three to five colonies were then inoculated into 10% skim milk (Difco Laboratories, Detroit, Mich.) and frozen at -70 C until tested. Testing included species determination by the scheme of Kloos and Schliefer (15). Antimicrobic susceptibility. Susceptibility to antimicrobial agents was determined by the disk diffusion method on

2 590 HARTSTEIN ET AL. Mueller-Hinton medium (BBL) as advocated by the National Committee for Clinical Laboratory Standards (19). Disks were obtained from BBL (penicillin, 10,ug; oxacillin, 1 p.g; cephalothin, 30 p.g; cefoperazone, 75,ug; cefuroxime, 30 p.g; cefotaxime, 30 p.g; cefoxitin, 30 jxg; tobramycin, 10 p.g; gentamicin, 10,ug; kanamycin, 30 ptg; netilmicin, 30 ptg; erythromycin, 15,ug; clindamycin, 2,ug; tetracycline, 30 p.g; chloramphenicol, 30 jxg; trimethoprim, 5 Fpg; and trimethoprim-sulfamethoxazole, 1.25 ptg ptg), Eli Lilly and Co., Indianapolis, Ind. (cefazolin, 30,ug; cefamandole, 30 Ftg; moxalactam, 30 p.g), Roche Laboratories, Nutley, N.J. (ceftriaxone, 30,ug), Miles Pharmaceuticals, West Haven, Conn. (ciprofloxacin, 5 p.g), and Smith Kline and French Laboratories, Philadelphia, Pa. (ceftizoxime, 30,ug). Recommended zone size cutoffs were used to define isolates as susceptible, intermediately susceptible, or resistant to the antimicrobial agents (3, 19). Each isolate was tested against all antimicrobial agents on the same day during each part of the study. Once the antimicrobic susceptibility panel was selected, all isolates from the same patient were tested against all of the antimicrobial agents in the panel on the same day. For determining reproducibility, isolates derived from patients on multiple occasions were retested once at a later date without reference to prior results. Plasmid profile analysis. Plasmid DNA of isolates from patients with multiple positive cultures was obtained by a modification of the method of Parisi and Hecht (20). All centrifugations were performed in Eppendorf tubes utilizing the same Eppendorf microfuge (Sarstadt, Princeton, N.J.) at 14,000 x g. After overnight incubation in brain heart infusion broth at 37 C with shaking, bacteria contained in 3-ml samples were collected by a brief centrifugation. Sedimented cells suspended in 200,ul of NaCl-EDTA buffer (5 mm EDTA, 2.5 M NaCI, ph 7.5) and 3 tld of a stock solution containing 7.5 mg of lysostaphin (Sigma Chemical Co., St. Louis, Mo.) per ml were incubated at 37 C for 30 min. Protoplasts were lysed by adding 300,ul of a solution containing Brij 58, deoxycholate, and EDTA (20). These mixtures were inverted several times, incubated for 10 min at room temperature, heated at 60 C for 30 min, and then centrifuged for 30 min. Supernatants were then incubated at 37 C with 3 of pancreatic RNase (Sigma) and extracted once with 300 pl of phenol-chloroform (1:1). The aqueous upper layer was transferred to a fresh tube, and DNA was precipitated with 95% ethanol as described previously (21). Each sample yielded enough material for approximately five agarose gels. Samples were electrophoresed in vertical 0.9% agarose slabs, and plasmid DNA bands were visualized under UV lamp after staining with ethidium bromide (10). Samples derived from each patient's multiple isolates were electrophoresed on the same gel, and plasmid profiles were compared. RESULTS Selection of the antimicrobic susceptibility panel. Since our goal was to find reproducible differences between isolates, we sought antimicrobics to which bacteria were frequently susceptible and resistant and rarely intermediately susceptible. Twenty-four antimicrobial agents were used to screen the susceptibility of up to 58 isolates derived from 58 patients (34 from patients with one positive blood culture and randomly selected single isolates from 24 patients with more than one positive blood culture) (Table 1). All 10 of the cephalosporins and imipenem were considered unsatisfactory. Only one isolate was resistant to cephalothin, cefamandole, and cefoperazone, and this isolate and three others were resistant to cefazolin. Seven to thirty-three percent of isolates showed intermediate susceptibility to six of the seven other antimicrobial agents in these groups. Although ceftizoxime testing did not lead to intermediate results (Table 1), all seven of the susceptible isolates were among the eight that were susceptible to oxacillin. Except for netilmicin, the tested aminoglycosides were good agents. No isolates were intermediately susceptible, and 10 to 28% were susceptible to tobramycin, gentamicin, and kanamycin. Only one isolate was resistant to netilmicin. The miscellaneous antimicrobial agents yielded results that would likely demonstrate differences between isolates. Except for ciprofloxacin, to which all isolates were susceptible, these agents never demonstrated intermediate susceptibility and frequently identified isolates as susceptible (10 to 79%) and resistant (21 to 90%). All isolates were identically susceptible to trimethoprim and trimethoprim-sulfamethoxazole, and all six isolates that were susceptible to erythromycin were among the nine that were susceptible to clindamycin. Following these studies, we chose penicillin, oxacillin, clindamycin, trimethoprim-sulfamethoxazole, chloramphenicol, tetracycline, tobramycin, kanamycin, and gentamicin as our antimicrobic susceptibility test panel, and we developed a coding system to record results (Fig. 1). In this TABLE 1. agentmcb J. CLIN. MICROBIOL. Antimicrobic susceptibility of coagulase-negative staphylococci Antimicrobial No. of No. (%) of isolates: agent isolates tested Susceptible Intermediate Resistant Penicillins Penicillin 57 4 (7) 53 (93) Oxacillin 57 8 (14) 49 (86) Highly active cephalosporins Cephalothin (98) 1 (2) Cefamandole (98) 1 (2) Cefoperazone (98) 1 (2) Cefazolin (86) 4 (14) Less active cephalosporins Ceftriaxone 57 8 (14) 16 (28) 33 (58) Cefuroxime (40) 9 (16) 25 (44) Cefotaxime 58 7 (12) 19 (33) 32 (55) Cefoxitin (28) 13 (22) 29 (50) Moxalactam 58 2 (3) 7 (12) 49 (84) Ceftizoxime 58 7 (12) 51 (88) Carbapenem Imipenem (29) 4 (7) 37 (64) Aminoglycosides Tobramycin 57 9 (16) 48 (84) Gentamicin (28) 41 (72) Kanamycin 57 6 (10) 51 (89) Netilmicin (97) 1 (3) Miscellaneous Ciprofloxacin (100) Erythromycin 57 6 (10) 51 (89) Clindamycin 57 9 (16) 48 (84) Tetracycline (79) 12 (21) Chloramphenicol (58) 24 (42) Trimethoprim (37) 36 (63) Trimethoprim (37) 36 (63) sulfamethoxazole

3 VOL. 25, 1987 IDENTITY TESTING OF COAGULASE-NEGATIVE STAPHYLOCOCCI 591 Antimicrobic abbreviation Susceptible (S) or Resistant (R) Number If susceptible Code for group of antimicroblcs Possible codes for group PEN OX CLIND T-S TET N GENT R R S R S S R Antimicroblc susceptibility code for example strain = 467 FIG. 1. Sample assignment of the antimicrobic susceptibility code. Abbreviations for antimicrobial agents: PEN, penicillin; OX, oxacillin; CLIND, clindamycin; T-S, trimethoprim-sulfamethoxazole; CHLOR, chloramphenicol; TET, tetracycline; TOB, tobramycin; KAN, kanamycin; GENT, gentamicin. system, the nine antimicrobial agents were arranged in three groups, and isolates found susceptible to an antimicrobial agent were given a numerical designation of 1, 2, or 4. Numbers in each group were added, leading to a three-digit code representing the antimicrobic susceptibility of each isolate. Distribution of isolates by antimicrobic susceptibility codes and plasmid profiles. All 106 isolates from 45 patients with more than one positive blood culture were analyzed with the panel for antimicrobic susceptibility and for their plasmid DNA content (Table 2). Nineteen different antimicrobic susceptibility codes were found. Nine percent or more of the isolates fell into one of four codes (040, 060, 070, and 777), and isolates with these codes were cultured from 18% or more of the patients. The frequency of finding isolates with given codes was not predictive of whether a patient had more than one isolate with a given code. Isolates with low-frequency codes were cultured from patients on multiple occasions (000, 034, 075, 440, 460, and 737) as well as single occasions (014, 050, 624, and 637). Alternatively, isolates with high-frequency codes TABLE 2. Antimicrobic susceptibility codes and plasmid profiles of isolates from patients with multiple positive blood cultures Antimicrobic No. (%) No. (%) No. of distinct code of Isolates of patients plasmid profiles (2) 1 (2) (1) 1 (2) (3) 1 (2) (25) 14 (31) (1) 1 (2) (9) 8 (18) (2) 2 (4) (14) 8 (18) (7) 5 (11) (2) 1 (2) (7) 2 (4) 2a (3) 2 (4) (3) 2 (4) (2) 2 (4) (1) 1 (2) (1) 1 (2) lb (6) 6 (13) 5c (3) 2(4) il (10) 8 (18) 5 a One of these plasmid profiles was also found in one isolate with antimicrobic code 074. b This plasmid profile was also found in one isolate with antimnicrobic code 737. c A sixth isolate in this antimicrobic code did not have plasmids as assessed by our methodology. (040, 060, 070, 074, and 777) were cultured once as well as multiple times from patients. Isolates with the most prevalent code, 040 (susceptible only to tetracycline), were cultured at least once from 31% of the patients and accounted for 25% of the total isolates studied. Forty-seven distinct plasmid profiles were found. The number (one to six) and molecular mass (1.0 to 40 megadaltons) of plasmids varied considerably. Isolates from different patients within the same antimicrobic code frequently had different plasmid profiles (Table 2). Figure 2 shows 24 of the 30 different plasmid profiles of isolates within commonly found antimicrobic codes (040, 060, 070, 074, 677, and 777); these isolates had different plasmid profiles. In addition, isolates with different antimicrobic codes had different plasmid profiles except on two occasions. The isolate with code 637 was identical to an isolate with code 737, and one isolate with code 074 was identical to five isolates from a different patient with code 424 (Table 2). The most common plasmid profile (Fig. 2A, lane c) was present in 8.5% of the isolates, and at least one isolate from 15% of the patients had this profile. Comparison of antimicrobic susceptibility pattern and plasmid profiles for isolate identification. Twenty-six patients had from two to five (mean 2.5) isolates that were identical by plasmid profile analysis. Seventeen patients had from two to four (mean, 2.2) isolates that were different from each other by plasmid profile analysis. Two patients had three isolates, two of which were identical and one of which was different by plasmid profile analysis. For the purpose of this comparison, these two patients with isolates that were identical and different from each other by plasmid profile analysis are counted in both groups. A total of 27 of 28 patients (96%) with isolates that were identical by plasmid profile analysis had isolates that were identical by antimicrobic susceptibility, and 18 of 19 patients (95%) with isolates that were different by plasmid profile analysis had isolates that were different by antimicrobic susceptibility. The predictive values of the antimicrobic susceptibility patterns for corroborating plasmid profile findings were 0.96 for patients with isolates that were identical and 0.95 for patients with isolates that were different, respectively. Reproducibility of antimicrobic susceptibility tests. Of the 954 susceptibility tests of isolates from patients with multiple positive blood cultures, 950 (99.6%) were repeatable on blinded retesting. Two isolates demonstrated variable penicillin susceptibility. Both of these isolates were shown to contain strains with variability in only penicillin susceptibility when each of 10 colonies subcultured from the frozen inoculum was retested. Two isolates demonstrated variability in gentamicin susceptibility. These variable results could

4 592 HARTSTEIN ET AL. J. CLIN. MICROBIOL. Md 35.8 A a b c d e f :-, u u u u u 1 B Md 35.8 c.. I..: h c d p g h M> AC040 AC060 AC677 AC777 FIG. 2. Agarose gel electrophoresis of plasmid DNA obtained from isolates with commonly found antimicrobic codes. MW, Molecular mass markers, plasmids from Escherichia coli V517 (18). Md, Megadaltons; AC, antimicrobic codes. Each lane shows the plasmid profile of a specific isolate within a given antimicrobic code. not be explained by further retesting. Separate colonies derived from the frozen inocula did not demonstrate differences in antimicrobic susceptibility, and further testing revealed consistent susceptibility test results. Both of these isolates were shown to be resistant to gentamicin on only one occasion. These discrepancies did not have a major effect on the value of the antimicrobic susceptibility pattern as a method for comparison of isolates. Three of the four isolates with nonreproducible results were different from comparison strains regardless of which susceptibility pattern was used. Species determination as an identity test. Only five patients had isolates that were not Staphylococcus epidermidis. One patient had Staphylococcus capitis in one culture and S. epidermidis in another. These isolates were different from each other by plasmid profile analysis and antimicrobic susceptibility pattern. Two patients had two isolates that were Staphylococcus haemolyticus, and one patient had two isolates that were Staphylococcus warneri. Isolates from all three of these patients were identical by plasmid profile analysis as well as antimicrobic susceptibility pattern. The last patient had three isolates, two of which were S. capitis and one of which was Staphylococcus simulans. All three isolates were different from each other by plasmid profile analysis and antimicrobic susceptibility pattern. DISCUSSION The results reported in this study strongly suggest that our selected disk diffusion panel of nine antimicrobial agents is capable of identifying similarities and differences among isolates of coagulase-negative staphylococci cultured on multiple occasions from blood. This conclusion is supported by the comparison of the antimicrobic susceptibility patterns with plasmid DNA profiles of multiple blood isolates from each of 45 patients. Multiple isolates that were identical according to their plasmid profiles had identical antimicrobic susceptibility codes. Similarly, multiple isolates with different plasmid profiles had different antimicrobic susceptibilities. A correlation between the plasmid profiles and the antimicrobic susceptibility codes was not evident in only 2 of 47 comparisons. The discriminating capability of the antimicrobic susceptibility panel was achieved by the inclusion of agents that frequently found isolates to be susceptible and resistant and the exclusion of agents which yielded results that were similar to those of other antimicrobial agents tested. Despite these efforts, we found 19 susceptibility patterns compared with 47 plasmid DNA profiles when testing isolates from our 45 patients. Therefore, although not a problem in this study, identical susceptibility patterns with different plasmid profiles may occur when testing serial isolates from a patient. More importantly, isolates from different patients commonly have identical susceptibility patterns and different plasmid profiles (Table 2). We advise consideration of confirmatory secondary testing by plasmid profile analysis in the former circumstance, and we strongly advocate such secondary testing whenever an outbreak of coagulase-negative staphylococcal infections caused by identically susceptible isolates is being investigated. Alternatively, discrimination may be further improved by the addition of other antimicrobial agents to this panel. Among those partially evaluated in this study, cefazolin and erythromycin seem the most promising. Streptomycin, neomycin, and fusidin are other agents which deserve further evaluation (22). In our study, species determination was marginally useful in comparing isolates. This is chiefly related to the fact that the vast majority of blood isolates of coagulase-negative staphylococci are S. epidermidis (9, 12, 25). Reproducibility of the antimicrobic susceptibility tests (99.6%) was achieved by the exclusion of antimicrobial agents which demonstrated intermediate susceptibility on initial screening. Variability in penicillin susceptibility (2 of 106 isolates) was probably due to the heterogeneous nature of susceptibility in the isolates. The reason for the variability in gentamicin susceptibility (2 of 106 isolates) was not identified. These minor problems with reproducibility did not confound the interpretation of susceptibility testing in 44 of the 45 patients. The validity of our antimicrobial susceptibility panel in comparing isolates of coagulase-negative staphylococci depended upon the variable susceptibility of isolates that have been cultured at our hospital laboratory. Whether our panel will work as well at other laboratories remains to be demonstrated. We suggest that each laboratory examine its own cumulative susceptibility data as the initial step in selecting the potentially best antimicrobial susceptibility panel for use or evaluation. Finally, the question of whether isolates of coagulasenegative staphylococci derived from the same clone could have identical antimicrobic susceptibilities but different plasmid profiles has been raised (21). Care must be taken in the

5 VOL. 25, 1987 IDENTITY TESTING OF COAGULASE-NEGATIVE STAPHYLOCOCCI 593 interpretation of results when the plasmid profiles of isolates are close to identical, particularly when the difference is limited to the presence or absence of plasmid bands located above the chromosomal bands. Our experience suggests that this is not a common finding (Fig. 2). Wider availability and application of an extended phage typing system for these infrequent problem isolates may be useful for solving this dilemma (21). In summary, disk diffusion susceptibility testing with carefully selected antimicrobial agents may be used to screen multiple blood isolates of coagulase-negative staphylococci for phenotypic identity and differences. Such testing may also be used as an alternative method for isolate differentiation in those laboratories without the ready availability of plasmid DNA analysis. ACKNOWLEDGMENTS This study was supported in part by a grant from the Medical Research Foundation of Oregon to J.H.C. M.A.V. was supported by a fellowship from the Consejo Nacional de Investigaciones Cientificas y Tecnicas of Argentina. We thank Tellelyn Wellner for her help in preparing the manuscript. LITERATURE CITED 1. Archer, G. L., A. W. Karchmer, N. Vischniavsky, and J. L. Johnston Plasmid-pattern analysis for the differentiation of infecting from noninfecting Staphylococcus epidermidis. J. Infect. Dis. 149: Archer, G. L., N. Vishniavsky, and H. G. Stiver Plasmid pattern analysis of Staphylococcus epidermidis isolates from patients with prosthetic valve endocarditis. Infect. Immun. 35: Barry, A. L., R. J. Fass, J. P. Anhalt, H. C. Neu, C. Thornsberry, R. C. Tilton, B. G. Painter, and J. A. Washington H Ciprofloxacin disk susceptibility tests: interpretive zone size standards for 5 ug discs. J. Clin. Microbiol. 21: Battisti, O., R. Michison, and P. A. 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