Klebsiella, Enterobacter, and Serratia:

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1 APPLIED MICROBIOLOGY, Aug. 1969, p Copyright 1969 American Society for Microbiology Klebsiella, Enterobacter, and Serratia: Vol. 18, No. 2 Printed in U.S.A. Biochemical Differentiation and Susceptibility to Ampicillin and Three Cephalosporin Derivatives RONALD J. ZABRANSKY, JACK W. HALL, FRED E. DAY, AND GERALD M. NEEDHAM Mayo Clinic and Mayo Foundation, Rochester, Minnesota Received for publication 3 April 1969 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 ug/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 Kiebsiella 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. The recent change in nomenclature of organisms in the tribe Klebsielleae (10) [Klebsiella, The introduction of new antibiotics and chemotion is not warranted, are no longer acceptable. Enterobacter, and Serratia; 5, 11, 13], the increasing importance of gram-negative bacteria ness against certain gram-negative bacteria, therapeutic agents with a wide range of effective- as causes of clinically important infections, and particularly ampicillin and the cephalosporins, the conflicting reports pertaining to the susceptibility of organisms of this group (2, 8, 16, organisms as precisely as possible. For example, has made it necessary to identify infecting 17) to certain antibiotics prompted us to evaluate ampicillin is usually effective against E. coil and the biochemical characteristics and the antibiotic susceptibility of members of the tribe siella, Enterobacter, Serratia, or Providencia Proteus mirabilis strains but not against Kleb- Klebsielleae isolated in the Mayo Clinic laboratories. associates (15), that Klebsiella species are sensi- species (1). Similarly, the report by Fleming and For many years the appearance of the colonies tive to cephalothin but Aerobacter (Enterobacter) of the lactose-fermenting Enterobacteriaceae species are not (because of the production of a on eosin-methylene blue-agar has been a primary cephalosporinase by the latter), suggests that it is basis for identifying and separating the "colonaerogenes" group. This method readily dis- accurate bacterial identification. important for the clinical laboratory to provide tinguishes Escherichia coil from the Klebsiella- Our study was undertaken to determine (i) Enterobacter-Serratia group of bacteria with a the frequency of isolation and (ii) the susceptibility to selected antibiotics of members of the high degree of accuracy, but it provides no means of identifying atypical strains and it fosters the tribe Klebsielleae in material presented to the lumping of organisms into a broad, ill-defined clinical laboratory of the Mayo Clinic. In addition, biochemical tests used for the classification group. The suggestions of Lampe (18) and Thaler (22), that these organisms cause similar were evaluated in the clinical laboratory setting. infections requiring similar treatment and therefore the use of complicated and lengthy labora- study were the cephalosporins, of which one has The primary group of antibiotics chosen for this tory procedures for separation and differentia- given conflicting results (2, 4, 6, 8, 16, 17, 20). 198

2 VOL. 18, 1969 KLEBSIELLA, ENTEROBACTER, AND SERRATIA 199 The new orally administered derivative, cephalexin, and cephaloridine were included to determine whether they exhibit any differences in activity against these organisms. Ampicillin was added to determine whether its effectiveness was uniform for this group when tested under the same circumstances as the cephalosporins. Other antibiotics which may be more effective in the treatment of clinical infections were not included because this was not the purpose of the study. MATERIALS AND METHODS Isolates (352) from clinical material (urine, sputum, blood, wound swabs, and swabs, tissue, or fluids from miscellaneous infections) were selected initially by gross colonial morphology on eosin-methylene blue and blood-agar plates. The reactions of the isolates in selected fermentative, biochemical, and motility tests were then determined. Basic tests were: fermentation of lactose and dextrose (triple sugar-iron-agar or peptone water with 1% carbohydrate); production of indole (nutrient broth with 0.5% additional peptone and tested with Kovacs' reagent); acetoin (methyl red-voges-proskauer medium); hydrogen sulfide (triple sugar-iron-agar); urease (Christensen's ureaagar); ornithine and lysine decarboxylase production (Moeller's decarboxylase medium with 0.3% agar and 2% amino acid); acidity with methyl red (methyl red- Voges-Proskauer medium); motility (motility agar, 0.3%); and citrate utilization (Simmons' citrate-agar). Tubes were incubated overnight at 37 C (motility medium was incubated at 22 C), and all reactions were read at 18 to 24 hr, except the methyl red-voges- Proskauer test and the amino acid decarboxylase media, which were read at 48 and again at 72 hr. If the reactions in motility agar and citrate-agar were not decisive at the end of 24 hr, these tests were reincubated and read again at 48 hr. When atypical results were obtained, the tests were repeated and certain alternative procedures were attempted, particularly motility studies at different incubation temperatures and in liquid media (hangingdrop preparation). Determination of lysine decarboxylase or deaminase and hydrogen sulfide production was done on lysine-iron-agar in a few instances. Organisms that remained difficult to classify were classified "atypical" and were tested for fermentation of sucrose, rhamnose, arabinose, raffinose, and sorbitol and for production of arginine dihydrolase and phenylalanine deaminase. TABLE 1. The minimal inhibitory concentration (MIC) of ampicillin and three cephalosporin antibiotics (cephalothin, cephaloridine, and cephalexin) for the isolates was determined by the agar dilution procedure (9) with the Steers et al. (21) replicator. Dilutions of each antibiotic at 5, 10, 20, 50, 100, and 200,ug/ml were prepared in Trypticase Soy Agar. Six-hour nutrient broth cultures were used as inocula and the plates were examined after 18 hr of incubation at 37 C. The MIC was recorded as that concentration producing complete inhibition of growth. RESULTS Biochemical tests. Quick and accurate identification of groups of organisms by colonial morphology has long been recognized by experienced bacteriologists (3, 14, 19) as having certain practical advantages. In our experience, the selection of isolates solely on the basis of gross colonial morphology in eosin-methylene blue and blood-agar was accurate 94% of the time in identifying members of the Klebsiella- Enterobacter-Serratia group. Only 23 of the original 352 isolates were found not to be KMbsiella, Enterobacter, or Serratia. These 23 strains were subsequently identified as E. coli (17 strains), Providencia (2 strains), and Citrobacter (4 strains) and were excluded from this study. The descriptions of the species of Klebsiella, Enterobacter, and Serratia by Edwards and Ewing (7), Ewing (11), and Fife et al. (13) were used for classification. The distribution and source of the 329 strains of the tribe Klebsielleae in our study are presented in Table 1. Klebsiella isolates were found more than twice as often as Enterobacter. Serratia isolates were found infrequently, in agreement with the results of Lerner and Weinstein (20). Table 2 shows the distribution of the positive reactions in the basic biochemical tests. Twentyeight Klebsiella strains (13%) varied from the typical or definitive reactions of the genus (indole-negative, citrate-positive, ornithine-decarboxylase-negative, lysine-decarboxylase-positive, nonmotile). The significant variances were in the results for indole production (3%), citrate Distribution and source of 329 strains of tribe Klebsielleae Source Genus Total no. Per cent of total Abscess Urine Sputum wounds, Blood Other etc. Klebsiella Enterobacter Serratia

3 200 ZABRANSKY ET AL. APPL. MICROBIOL. utilization (6%), and lysine-decarboxylase production (7%), but these variances are within the limits reported by Fife et al. (13). Twenty of the 92 strains of Enterobacter (22 %) varied from typical or definitive reactions of the genus (indole-negative, citrate-positive, ornithinedecarboxylase-positive, motile). The significant variances were with strains that were either ornithine-decarboxylase-negative (8 %) or nonmotile (12%). The majority of these strains were identified as either E. liquefaciens or E. hafniae by use of additional tests. Atypical results are common among these two species, and comparison of the results of reactions at 22 and 37 C was frequently helpful in separation and identification of these organisms. The results of the biochemical tests of the 15 strains of Serratia are similar to those reported in the literature, even though only a small number of strains were examined. All strains were incubated for 3 days at 22 C on nutrient agar for pigment production, but only two strains produced prodigiosin. None of these strains fermented rhamnose, raffinose, or arabinose. Antibiotic sensitivity. Table 3 shows the distribution of MIC for the four antibiotics tested. Of the Klebsiella strains, 57% or greater were inhibited by each of the three cephalosporins at 5,g/ml. Most of the remaining strains were inhibited by either 10 or 20 jig/ml of these same antibiotics. Higher concentrations of ampicillin were needed to inhibit the Klebsiella strains, and greater variability in antibiotic susceptibility was noted. Only 37 strains (17%) TABLE 3. Antibiotic were inhibited by 20 Ag/ml or less, and 64 strains (29%) were not inhibited by 200,ug/ml. The Enterobacter strains exhibited the same susceptibility pattern to ampicillin as did the Klebsiella strains, 27% of the strains being inhibited by concentrations of 20 Ag/ml or less. However, these strains differed markedly from the Klebsiella strains in susceptibility to the cephalosporins. Only six (7%) Enterobacter strains were inhibited by any of the three cephalosporin antibiotics at 5,ug/ml, and large numbers TABLE 2. Biochemical reactions of 329 strains of tribe Klebsielleae Positive reactions Test Klebsiella Enterobacter Serratia (222 strains) (92 strains) (15 strains) No. % No. % No. % Lactose Dextrose Indole Methyl red Voges-Proskauer Citrate Lysine decarboxylase Ornithine decarboxylase Motility H2S Urease Numbers of strains inhibited at various concentrations of antibiotics Antibiotic concn (jug/ml) >200 Klebsiella strains (total 222) Ampicillin Cephalothin Cephaloridine Cephalexin Enterobacter strains (total 92) Ampicillin Cephalothin Cephaloridine Cephalexin Serratia strains (total 15) Ampicillin Cephalothin Cephaloridine Cephalexin

4 VOL. 18, 1969 KLEBSIELLA, ENTEROBACTER, AND SERRATIA of the strains (47 to 83 %) were not inhibited by concentrations of 200,ug/ml or more. The Serratia strains gave a pattern of antibiotic susceptibility similar to that of the Enterobacter strains, with almost all the strains resistant to the cephalosporins. The terms "resistant" and "sensitive" as used here are interpretations of in vitro tests. No implication is made as to the actual clinical response of these organisms to the antibiotics tested. A "sensitive" organism was arbitrarily defined as one inhibited by any one of the antibiotics at a concentration of 20 jig/ml or less. An average of 90% of the Klebsiella strains were sensitive to the cephalosporins, compared to only 15% of the Ezterobacter strains (Fig. 1). Ampicillin appeared to be slightly more effective against Enterobacter strains than against Klebsiella strains. The sensitivity of the Klebsiella and Enterobacter strains to the cephalosporin antibiotics was fairly uniform. If a strain was inhibited by one of these antibiotics, it was usually inhibited by the other two as well. However, a few strains were inhibited by one of the cephalcsporins but not the other two, or by two but not by the third. At the 20 Ag/ml level, only 22 strains exhibited this peculiarity (Table 4). Consequently, there was a 90% chance that, if an organism was sensitive to one of the cephalosporins, it would be sensitive to the other two as well. To avoid bias that may have been introduced by our interpretation of the results of the several biochemical tests, the atypical strains were ex- 17% AMPICILLIN 3[27% CEPHALOTH IN CEPHALORI DINE CEPHALEXIN 12% 16% 15% KLEBSIELLA ENTEROBACTER SERRATIA FIG. 1. Inhibition of Klebsiella, Enterobacter, and Serratia strains by antibiotics at 20 jg/ml or less. Shown as percentages of total tested. TABLE 4. Klebsiella, Enterobacter, and Serratia strains sensitivea to only one or two cephalosporin antibiotics Antibiotic No. of strains Klebsiela oenater Serratia Cephalexin Cephaloridine Cephalothin Cephalothin + cephaloridine Cephalothin + cephalexin Cephaloridine + cephalexin a By definition, "sensitive" means inhibited at a concentration of 20,ug/ml or less. amined with particular emphasis on their antibiotic sensitivity patterns. The occasional atypical Enterobacter strain that was nonmotile or was ornithine-decarboxylase-negative exhibited about the same susceptibility to the cephalosporins as did the motile, ornithine-decarboxylase-positive strains. Similarly, the indole-positive or citratenegative strains of Enterobacter or Klebsiella were no more susceptible to ampicillin than were the typical strains. This comparison suggested that our original classification of the atypical strains was correct and that the pattern of sensitivity to selected antibiotics might be a useful additional aid in identification of hard-to-classify members of the Klebsiella-Enterobacter-Serratia group. DISCUSSION By using several easily performed biochemical tests (indole production, citrate utilization, urease production, and reactions on triple sugariron-agar and motility agar), 88% of our isolates could be readily identified as Klebsiella, Enterobacter, or Serratia species. These same tests provide the additional benefit of allowing recognition of other organisms such as E. coli, Citrobacter, Salmonella, Shigella, Providencia, and Proteus. In our study, further biochemical testing for classification was needed for only 42 strains. When determined in a semisolid medium and not confused with growth down the side of the tube (6), motility is an adequate procedure for differentiating Klebsiella from Enterobacter or Serratia. Twelve per cent of our Enterobacter strains were nonmotile. The ornithine-decarboxylase test was slightly more reliable in that

5 202 ZABRANSKY ET AL. APPL. MICROBIOL. only 8% of the Enterobacter strains were negative. Routine use of both tests would eliminate errors in classification. Although identification to the level of species could be achieved in the majority of instances by the tests used or by addition of one or two other tests, this information is not included since it was our purpose to indicate the ease of identification of the genera and to test antibiotic susceptibility differences inherent in these genera to the selected antibiotics. Speciation or serological typing would, of course, add to the epidemiological value of the information obtained, but such an undertaking was beyond the intent of the study. The frequency of isolation and the antibiotic sensitivity of members of the tribe Klebsielleae from clinical material vary among reported series (2, 4, 6, 8, 16, 20). Klebisella isolates outnumber Enterobacter isolates. Lerner and Weinstein (20) reported a ratio of 2.5:1, Benner et al. (2) reported 4:1, and Eickhoff et al. (8) reported 5:1; in our material the ratio was 2.5:1. Almost as variable is the reported antibiotic sensitivity of the different strains-for example, the percentage of strains of Klebsiella sensitive to cephalothin varies from 22 to 100% (2, 8). The disparities in isolation rate and antibiotic sensitivity of Klebsiella and Enterobacter strains are difficult to explain but might be the result of differences in methods used in isolation and identification or in determination of antibiotic sensitivity of the organisms, or they might be a reflection of a bona fide difference in geographic distribution. It has also been suggested that organisms isolated from different specimens may have different antibiotic susceptibilities, at least in terms of the sensitivity of Klebsiella to cephalothin (16). Consequently, comparisons between studies not specifying the source of isolation may not be completely valid. Only two of our Serratia strains produced pigment, and reliance on the production of pigment for identification of Serratia is not recommended. The combination of motility and production of ornithine and lysine decarboxylases permitted the classification of Serratia strains without difficulty in most instances, but confirmation was provided by appropriate fermentative reactions in arabinose, rhamnose, and raffinose. Edwards and Ewing (7) reported pigmentation in only 26% of their strains of Serratia. More recently, Fields et al. (12) described pigmentation in less than 10% of their clinical isolates of Serratia and also pointed out the resistance of these organisms to most antibiotics, findings corroborated in our study. Before the availability of ampicillin there was little reason to differentiate organisms in the "colon-aerogenes" group because of the similarity in antibiotic susceptibility among members of this large heterogeneous group. Until cephalothin was made available, there was little reason to differentiate between Klebsiella and Enterobacter species except for epidemiological purposes. However, our study suggests that a distinct difference in antibiotic sensitivity does exist for members of this ubiquitous and genetically related group and that classification into genera by the clinical laboratory can be readily accomplished, providing useful information to the clinicians. Of the Klebsiella species defined by our system, 90% were inhibited by low concentrations of the cephalosporin antibiotics, compared to only 15% of the Enterobacter strains. The differences in sensitivity between the various cephalosporins for the Klebsiella were small. Prior results (9) with the agar dilution procedure for antibiotic sensitivity testing indicate the reliability of the method, and repeated testing of the same or more strains would probably further minimize the differences. Our results most closely parallel those of Lerner and Weinstein (20), both in the degree of sensitivity of Klebsiella isolates to cephalothin and in the frequency of isolation of Klebsiella strains from clinical material. The results of our study contrast sharply with those of Bulger (4) and Eickhoff and et al. (8). By using a tube dilution procedure for determining antibiotic susceptibility, Bulger found that 53% of the Klebsiella strains were inhibited by cephalothin at 15 Ag/ml or less; Eickhoff et al. reported that 22% of Klebsiella strains were inhibited by cephalothin at 25,g/ml. This lack of agreement could be the result of differences in methods of antibiotic sensitivity testing or in identification of the organisms. More likely, it represents one or a combination of several other factors. Benner et al. (2) suggested that the adaptive production of cephalosporinase by some strains of Klebsiella may be responsible for differences in antibiotic susceptibility patterns. The medical importance of infective drug resistance or resistance transfer factors was realized early by the Japanese (23), but until recently only the genetic aspects of this phenomenon have been of interest in this country. As a consequence of the use of cephalosporin antibiotics, cephalosporinase production or resistance transfer factors may direct the development of resistant strains in a hopsital, in isolated areas of a hospital, or in patients under treatment. The predominance of such strains would then vary from institution to institution. In addition, it is

6 VOL. 18, 1969 KLEBSIELLA, ENTEROBACTER, AND SERRATIA 203 well recognized that multiple isolations from the same patient could influence the statistics. With the wider clinical application of cephalothin and, to a lesser extent, cephaloridine, increasing numbers of cephalosporin-resistant Klebsiella may be seen in the future. Although the cephalosporin-resistant strains in this study were not examined for the presence of resistance transfer factors or cephalosporinase activity, future studies could combine the efforts of the clinical microbiologist, the geneticist, the clinician, and the epidemiologist to evaluate the significance of these factors. Proper bacteriological classification, as accomplished in the clinical laboratory, provides information for a better understanding of the taxonomic relationship of organisms and for epidemiological studies in tracing sources of infection. It can serve also as an aid to the clinician in the preliminary selection of an antibiotic for treatment. An understanding of the genetic capabilities of these organisms will also contribute to the taxonomy, epidemiology, and evaluation of antibiotic susceptibility. The actual treatment of the patient, however, should be based upon the accurate in vitro determination of the antibiotic susceptibility of the organism isolated in the clinical laboratory. LITERATURE CITED 1. Anderson, K. N., R. P. Kennedy, J. J. Plorde, J. A. Shulman, and R. G. Petersdorf Effectiveness of ampicillin against gram-negative bacteria: In vitro and in vivo studies of a new antibiotic. J. Amer. Med. Ass. 187: Benner, E. J., J. S. Micklewait, J. L. Brodie, and W. M. M. Kirby Natural and acquired resistance of Klebsiella- Aerobacter to cephalothin and cephaloridine. Proc. Soc. Exp. Biol. Med. 119: Braun, W Dissociation in Brucella abortus: a demonstration of the role of inherent and environmental factors in bacterial variation. J. Bacteriol. 51: Bulger, R. J In vitro effectiveness of kanamycin and kanamycin/cephalothin against Klebsiella: comparison with other antibiotics. Ann. Intern. Med. 67: Editorial Board Opinion 28, rejection of the bacterial generic name Cloaca Castellani and Chalmers and acceptance of Enterobacter Hormaeche and Edwards as a bacterial generic name with type species Enterobacter cloacae (Jordan) Hormaeche and Edwards. Int. Bull. Bacteriol. Nomencl. Taxon. 13: Edmundson, E. B., and J. P. Sanford The Klebsiella- Enterobacter (Aerobacter)-Serratia group: a clinical and bacteriological evaluation. Medicine 46: Edwards, P. R., and W. H. Ewing Identification of Enterobacteriaceae, 2nd ed. Burgess Publishing Co., Minneapolis. 8. Eickhoff, T. C., B. W. Steinhauer, and M. 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