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biochemical profiles by either API (Analytab. Products, Plainview, N.Y.) or Micro-ID (Gen- eral Diagnostics, Morris Plains, N.J.). Of the 50 bacteremic patients, 13 ...

Vol. 13, No. 3

JOURNAL OF CLINICAL MICROBIOLOGY, Mar. 1981, p. 606-608 0095-1137/81/030606-03/02.00/0

Unique Temperature-Sensitive Nutritional Requirements of Bacteremic Escherichia coli Isolates W. D. WELCH,t* L. SPURGEON, D. KITTS, H. S. MOYED, AND L. D. THRUPP Departments of Medicine and Medical Microbiology, University of California-Irvine, Irvine, California 92664

Of 50 strains of Escherichia coli isolated from blood cultures of bacteremic patients, 14 (28%) were unable to grow on minimal medium at 42°C, compared to only 2 of 50 nonbacteremic strains. In 7 of the 14 bacteremic strains, growth at 42°C was restored by adding nicotinic acid. These unique temperature-sensitive auxotrophic patterns warrant evaluation as a marker correlating with clinical pathogenicity in E. coli.

Guze et al. found that the enhanced capacity of several laboratory strains of Escherichia coli to grow in minimal medium (MM) correlated with increased severity and mortality rates of experimental renal infection in mice (4). In a previous study from this laboratory, some 60% of 50 bacteremic E. coli isolates grew better at 30°C than at 37°C on MM compared to enriched media, whereas this reversal of the expected growth pattern was present in only 30% of 60 nonbacteremic isolates (9). These studies supported the hypothesis that some strains which are more pathogenic may have fewer nutritional requirements than less pathogenic isolates, and suggested that such strains may represent auxotrophs with temperature-sensitive metabolic markers. We now report further experiments with bacteremic and nonbacteremic E. coli isolates to determine the presence of temperature-sensitive metabolic markers among such isolates. To develop a method permitting analysis of temperature-sensitive metabolic markers, E. coli isolates were initially screened for their ability to grow at elevated incubation temperatures (42°C) on MM. This approach was based on the hypothesis that a higher growth temperature might reverse the relative growth advantage that these strains showed in MM at low temperatures (9) and instead result in restriction or absence of bacterial growth. The identification of specific nutrients (amino acids, vitamins, purines, pyrimidines) required for growth of such bacteremic strains at elevated temperatures could provide insight into the mechanisms of the metabolic patterns of these strains as possible correlates of pathogenicity. (This work was presented in part at the 16th Interscience Conference on Antimicrobial t Address reprint requests to: Department of Medicine,

UCI Medical Center, Orange, CA 92668.

Agents and Chemotherapy [L. Spurgeon, S. Raza, and L. Thrupp, Program Abstr. 16th ICAAC, abstr. no. 9, 1976] and the 80th Annual Meeting of the American Society for Microbiology [W. D. Welch, L. Spurgeon, D. Kitts, H. Moyed, and L. Thrupp, Abstr. Annu. Meet. Am. Soc. Microbiol. 1980, D49, p. 46].) E. coli blood cultures isolates from 50 septicemic patients were studied in comparison with 50 nonbacteremic strains. All E. coli isolates were identified by standard criteria including biochemical profiles by either API (Analytab Products, Plainview, N.Y.) or Micro-ID (General Diagnostics, Morris Plains, N.J.). Of the 50 bacteremic patients, 13 had acute pyelonephritis, 30 had extra-renal infection, and 7 were neonates with sepsis. Of the 50 nonbacteremic E. coli strains examined, 38 were from patients with urinary tract infections, and 12 were stool isolates (including cystitis and nonbacteremic pyelonephritis) from normal subjects. These E. coli strains, both bacteremic and nonbacteremic, were the subject of preliminary experiments previously reported (9). Each stored isolate was subcultured to a blood agar plate, incubated overnight at 37°C, and then subcultured again to a tryptic soy agar slant and incubated for 4 h. Growth from the slants was suspended in either MM broth or enriched broth (tryptic soy broth; Difco Laboratories, Detroit, Mich.), adjusted to 108 colonyforming units per ml (using an appropriate McFarland standard), and plated onto the desired test medium with a 0.001-ml calibrated platinum loop. The MM agar used had the following composition in grams per liter of distilled water: Noble agar (Difco), 15; Na2HP04, 6; KH2PO4, 3; NH4Cl, 1; NaCl, 0.5; MgSO4.7H20, 0.2; and glucose, 4. MM broth had the same formula without agar. For enriched agar medium, tryptic soy agar (Difco) was used.

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TABLE 1. Amino acids, vitamins, purines, and Growth on MM at 42°C. Ail E. coli isolates pyrimidines used in specific nutrient tested for growth on MM or enriched mesupplementation experiments dium at 37 and 420C. At 37°C, all isolates demConen (mg/ml onstrated confluent growth on either MM or Supplement' of MM) enriched medium. In contrast, at 420C, all strains grew on enriched medium, but on MM Specific amino acidsb ................ 0.1 0.05 14 bacteremic and 2 nonbacteremic strains failed Adenine ... 0.01 .. ... ... to grow. Thus, for the nonbacteremic isolates, 2 Biotin ... of 50 (4%) failed to grow on MM at 420C, Hypoxanthine ....................... 0.05 .... 0.05 whereas among the bacteremic isolates signifi- Folic acid............................ 0.05 Cytosine cantly more strains, 14 of 50 (72%, P < 0.05), Pantothenic acid .................... 0.005 failed to grow on MM at 420C. All 14 bacteremic Guanine .. 0.05 isolates unable to grow on MM at 420C also Pyridoxin ..... 0.005 demonstrated increased growth at 300C on MM Sodium thiosulfate .0.05 0.05 ........... ..... compared with growth on enriched medium at Thymine .. .. 0.01 300C (9), whereas the 2 nonbacteremic isolates Thiamin ................. 0.005 did not show enhanced growth on MM at 300C. para-Aminobenzoic acid ............. There was no specific clinical entity associated Uracil ... 0.05 0.025 .... with this unique temperature-sensitive growth Riboflavin acid .0.005 pattern. Of the two nonbacteremic strains, one Nicotinic 0.01 Choline ....... was a normal stool isolate and the other was a 0.005 Inositol ..... cystitis strain. The 14 bacteremic isolates resolutions were in sterile Stock aseptically prepared a flected a range of clinical diagnoses representawater. tive of and not identifiably different from the distilled bAmino acids tested: phenylalanine, serine, tyroremainder of the 50 bacteremic isolates. sine, alanine, cysteine, threonine, methionine, argiNutritional supplementation experi- nine, ornithine, aspartic acid, proline, glutamic acid, ments. This phenomenon of failure to grow on leucine, glycine, isoleucine, histidine, lysine, valine. MM at 420C was then used as a tool to analyze nutritional requirements among these isolates. of the first enzyme of the methionine pathway, MM agar was supplemented with specific nutri- homoserine transuccylase. The bacteremic E. ents including amino acids, vitamins, purines, or coli isolates we studied could possibly possess a pyriniidines (Sigma Chemical Co., St. Louis, similar temperature-sensitive enzyme. Mo.) at concentrations given in Table 1. Amino The bacterial synthesis of nicotinic acid from acid requirements in the selected E. coli isolates aspartic acid involves several synthetases, transwere screened initially by the addition to MM ferases, and hydrolases (2). Because addition of of nine amino acid pools each containing from aspartic acid did not restore growth at 420C on three to six amino acids (5). By such reconsti- MM in seven of our isolates which could grow tution experiments, certain amino acids could be after supplementation with nicotinic acid, the eliminated due to absence of bacterial growth on temperature-sensitive defect probably resides in particular pools. Determination of growth in all one of these biosynthetic enzymes. The ability experiments was made at 24, 48, and 72 h. of all of our isolates to grow on enriched medium Of the 14 bacteremic isolates which failed to at 420C suggests: (i) that a ribosomal or deoxygrow on MM at 420C, growth was restored on ribonucleic acid defect, such as a deficiency due MM at 420C in 7 of 14 isolates after addition of to inadequate amounts of a specific enzyme or nicotinic acid to the media. Growth of 1 of the production of a defective enzyme, is probably 14 bactergmic isolates was restored by methio- not responsible; and (ii) that in enriched menine supplementation. For the remaining six iso- dium, the temperature-sensitive enzyme at 420C lates, none of the specific single nutrients or the was compensated for by the presence of nicotinic amino acid pools studied restored growth in MM acid in the medium or utilization of other reacat 420C; these strains grew at 420C only on tants needed for nicotinic acid biosynthesis. enriched media such as crude Casamino Acids For an organism to be pathogenic, it must first digest (Difco) or 5% sheep blood in agar base. be able to become established in and supported Nutrient supplementation of MM with methio- by the host. An adequate nutritional environnine has been shown to restore the ability of ment in the host allowing growth of the organism several laboratory E. coli auxotrophs to grow on is therefore required. Potential infectivity of an MM at 420C (8). The temperature-sensitive organism could thus be enhanced in an organism growth rate was related to the decreasing activ- with reduced nutritional requirements. Specific ity (concomitant with increasing temperatures) nutritional requirements necessary for the

were

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expression of virulence have been reported for Salmonella typhi and Klebsiella pneumoniae (1, 3). In vivo studies with these bacteria revealed a lack of mortality in mice unless purines and para-aminobenzoic acid were injected simultaneously with the challenging organism. A high proportion of Neisseria gonorrhoeae strains causing disseminated infection have also been found to be auxotrophs requiring specific nutrient supplementation for growth (6). Interestingly, in a study examining nutritional requirements of 268 clinical isolates of Shigella flexneri, 52% required nicotinic acid for growth on MM at 37°C (7). In a similar respect the requirement of nicotinic acid or of methionine for growth of the bacteremic E. coli isolates examined in this study under "stressed" growth conditions could represent a marker apparently associated with clinical pathogenicity in certain strains of E. coli. However, the mechanisms of any such pathogenic potential remain unknown. LITERATURE CITED 1. Bacon, G. A., T. W. Burrows, and M. Yates. 1951. The effects of biochemical mutation on the virulence of Bacterium typhosum: the loss of virulence in certain

J. CLIN. MICROBIOL. mutants. Br. J. Exp. Pathol. 32:85-96. 2. Foster, J. W., and A. G. Moat. 1980. Nicotinamide adenine dinucleotide biosynthesis and pyridine nucleotide cycle metabolism in microbial systems. Microbiol. Rev. 44:83-105. 3. Garber, E. D., A. J. Hackett, and R. Franklin. 1952. The virulence of biochemical mutants of Klebsiella pneumoniae. Proc. Natl. Acad. Sci. U.S.A. 38:693-697. 4. Guze, L D., J. Z. Montgomerie, C. S. Potter, and G. M. Kalmanson. 1973. Pyelonephritis. XVI. Correlates of parasite virulence in acute ascending Escherichia coli pyelonephritis in mice undergoing diuresis. Yale J. Biol. Med. 46:203-211. 5. Holliday, R. 1956. A new method for the identification of biochemical mutants of micro-organisms. Nature (London) 178:19-87. 6. Knapp, J. S., C. Thornsberry, G. A. Schoolnels, P. J. Wiesner, and K. K. Holmes. 1978. Phenotypic and epidemiologic correlates of auxotype in Neisseria gonorrhoeae. J. Infect. Dis. 138:160-165. 7. Lachowiez, T. M., S. Jankowski, and Z. Lachowicz. 1972. Nutritional requirements of Shigella flexneri bacilli. III. Nicotinic acid as a main growth factor of Sh. flexneri. Acta Microbiol. Pol. 4:9-12. 8. Ron, E. Z., and B. D. Davis. 1971. Growth rate ofEscherichia coli at elevated temperatures: limitation by methionine. J. Bacteriol. 107:391-396. 9. Spurgeon, L., and L. D. Thrupp. 1980. Relation of virulence of Escherichia coli in septicemia and urinary tract infection to temperature sensitive growth in minimal medium. J. Infect. Dis. 142:773.

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