JOURNAL OF CLINICAL MICROBIOLOGY, May 1990, p. 1047-1048 0095-1137/90/051047-02$02.00/0 Copyright © 1990, American Society for Microbiology
Vol. 28, No. 5
NOTES
Septicemia Caused by Cysteine-Dependent Escherichia coli K. Y. YUEN,* W. H. SETO, K. H. TSUI, AND W. T. HUI Department of Microbiology, University of Hong Kong, Hong Kong Received 18 September 1989/Accepted 9 January 1990
A case of septicemia and urinary tract infection caused by cysteine-dependent Escherichia coli in a 70-year-old woman with bilateral staghorn calculi is described. This is the second report of a cysteinedependent E. coli bacteremia. The bacterium was falsely susceptible to ampicillin and co-trimoxazole when tested on a medium without cysteine supplement.
Cysteine-dependent Escherichia coli is a known cause of chronic urinary tract infection, but the isolation of this organism from blood is rare (2, 5). In fact, only one study of five cases is reported in the literature (14). We report in this paper on a cysteine-dependent E. coli with Ki antigen that caused septicemia in a patient with bilateral renal stones. Clinical information. The patient was a 70-year-old female who had been suffering from bilateral renal staghorn calculi for 10 years and had had an extended pyelolithotomy for hydronephrosis of the left kidney 9 years earlier. After the operation, her renal function had remained normal, and the hydronephrotic changes in her kidney were improved with radiology despite the presence of residual stones. One year before admission, she had dysuria and hematuria, which were treated with a 10-day course of co-trimoxazole (two tablets twice a day). Her past health was otherwise unremarkable. On admission, she was febrile (38.5°C) with rigors. Her blood pressure was 90/60 mm Hg, and her pulse rate was 100/min. She had bilateral loin tenderness maximal over the renal angles. Proteinuria (1 g/liter), microscopic hematuria, and pyuria were found by urinalysis. Routine blood tests showed leukocytosis (11.9 x 109 cells per liter) and a slightly raised urea level (7.6 x 10-3 mol/liter). A presumptive diagnosis of septicemia due to acute pyelonephritis was made, and blood cultures were taken. A urine culture was not obtained before the initiation of an antibiotic because she was oliguric on admission. She was started on intravenous cefuroxime (0.75 g every 8 h). After 24 h of antimicrobial therapy, she became afebrile, and the bilateral loin pain and tenderness improved. Two weeks later, intravenous urography showed that the left kidney was obstructed by residual stones. A second operation was however refused by the patient. Microbiological results. Brain heart infusion broth (Mast Diagnostics Ltd., United Kingdom) with 0.05% sodium polyanetholesulfonate was used for conventional aerobic blood culture, and thioglycolate medium (Oxoid Ltd., London, England) supplemented with hemin, vitamin K, and sodium citrate was used for anaerobic blood culture. After 24 h of incubation, a gram-negative bacillus was found by Gram staining in both the aerobic and anaerobic blood culture broths. The broth was subcultured onto blood and chocolate agar, but no growth was noted after 24 h of *
Corresponding author.
incubation. However, after 48 h of incubation, pinpoint translucent colonies could be seen with a magnifying glass. At 72 h, the colonies attained maximal size but were still less than 0.5 mm in diameter. The bacterium was facultative and also grew on MacConkey agar as a non-lactose fermenter. The bacterium was oxidase negative, catalase positive, and motile. The organism was identified as E. coli by both conventional tests and the API 20E system (API, La Balme les Grottes, France) at 48 h of incubation. However, conventional tests for lysine decarboxylase and urea hydrolysis could not be interpreted because of lack of growth, unless cysteine was supplemented. The bacteria were then inoculated onto a cysteine-lactoseelectrolyte-deficient agar plate and a MacConkey agar containing a paper disk soaked with cysteine solution (5 g/liter). A blood agar plate and another MacConkey agar plate were inoculated as controls. After 18 h of incubation, the colonies had attained a diameter of 3 mm, and there was evidence of lactose fermentation on the cysteine-lactose-electrolyte-deficient plate. There were also satellitism and lactose-fermenting activity around the cysteine disk on the MacConkey agar. However, no visible growth was noted on the control plates. The organism was therefore identified as cysteinedependent E. coli. By using the Wellcogen Bacterial Antigen Kit (Wellcome Diagnostics, Research Triangle Park, N.C.), Ki antigen was detected on this bacterium by latex agglutination and counterimmunoelectrophoresis. Antimicrobial susceptibility was tested by the KirbyBauer disk diffusion and agar dilution method, using cysteine-supplemented Mueller-Hinton agar (0.1 g of cysteine per liter). The organism was susceptible to cephalothin, cefuroxime, gentamicin, and chloramphenicol but was resistant to co-trimoxazole and ampicillin by both methods. However, the bacterium appeared susceptible to all six antibiotics when tested on an unsupplemented medium. No organism was found on the urine culture that was collected after the initiation of antimicrobial therapy. Discussion. Nutritionally deficient or auxotrophic bacteria are known to cause difficulty in isolation, identification, or antimicrobial-susceptibility testing as a result of their dependence on various growth factors to achieve normal colony morphology and growth rate. These bacteria include pyridoxal-dependent streptococci (Editorial, Lancet ii:1164, 1977); hemin-, thiamine-, pentothenate-, or menaquinonedependent Staphylococcus aureus (9, 11, 12, 16); thymine1047
1048
NOTES
dependent E. coli (15); Proteus mirabilis (1); Salmonella typhimurium (7); and Streptococcus pneumoniae (4). Cysteine-dependent organisms, such as E. coli (2, 5) and Klebsiella pneumoniae (K. aerogenes) (6), have been reported as occasional urinary tract pathogens. Twenty-one strains of cysteine-dependent E. coli were isolated from about 1,000 specimens of infected urine over a period of 2 years (5), while none were detected in 120 specimens of infected urine from a general-practice population (8). In addition, seven cysteine-requiring strains of K. pneumoniae (6), which constitutes 1.2% of all urinary isolates of Klebsiella species, were found in a hospitalized population. Although E. coli from the urinary tract is the most common source of gram-negative septicemia, only five cysteinedependent isolates have been reported (14). The infrequent reports associated with this organism may not be a reflection of the true incidence. As pointed out above, these organisms may not grow in the most commonly used culture media because of the low cysteine content of these media. If the cysteine content is too low for such nutritionally deficient strains, the strains could be missed on a blind subculture of the blood broth. Furthermore, the API 20E still correctly identified the organism after 48 h of incubation despite atypical colony morphology. Thus, unless a specific test for cysteine dependence is performed, the true nature and the clinical significance of such nutritionally deficient forms of E. coli may simply be ignored. The diagnosis of cysteine-dependent E. coli was considered in this case because there was a discrepancy between growth in the blood broth and in the subculture plates after 48 h of incubation. A differential diagnosis of HB-5, a rare genitourinary pathogen, was also entertained, but this possibility was excluded by the negative oxidase test (3). The clinical data for this patient are rather similar to those from cases of cysteine-dependent E. coli infection reported in the literature. All five reported cases of septicemia were in females over 50 years of age suffering from chronic urinary
problems or debilitating illness (including diabetic papillary necrosis causing obstructive uropathy, post-renal transplantation immunosuppression, indwelling urinary catheters, and myelomatosis). Of the 17 patients with urinary tract infection by cysteine-dependent E. coli (2, 5), 13 were over 50 years of age, and 9 had chronic renal diseases (including nephrolithiasis, hydronephrosis, renal tuberculosis, diabetic nephropathy, benign prostatic hypertrophy, prostatic malignancy, diverticula, and papillomatosis of the bladder). A chronic renal condition was also found in our patient, and the urinary tract was the most likely source of the septicemia in view of her typical uropathic symptoms. Unfortunately, the urine culture was negative because a specimen could be collected only after antimicrobial therapy. As pointed out by Tapsall and McIver (14), the presence of cysteine-dependent E. coli in the blood or urine should prompt a search for an underlying renal condition. This case, involving cysteine-dependent E. coli, is an example of how precise microbiological identification of a bacterial isolate from blood can help in the diagnosis of a medical condition. Other known examples include the association of Streptococcus bovis bacteremia with colonic carcinoma and the association of pyridoxal-dependent streptococci with endocarditis (5, 13). Although Ki antigen is known to be highly associated with
J. CLIN. MICROBIOL.
uropathic E. coli, the prevalence of Ki antigen among cysteine-dependent E. coli is not known, since serotyping was not mentioned in previously reported cases. Since Ki antigen is known to endow the bacteria with invasive potential, probably by blocking the complement-dependent uptake by phagocytosis (10), it would be interesting to know whether the degree of Ki antigenic prevalence within this group of auxotrophic mutants could explain the infrequent detection in blood cultures. The mechanism for the development of most auxotrophic bacteria is largely unknown. For thymidine-dependent E. coli, there is evidence that the mechanism might be induced by chemotherapy. These organisms were found in the urine of patients after at least 8 days of treatment with cotrimoxazole (15). Further studies are needed to identify the mechanism that induces cysteine dependence. LITERATURE CITED
1. Barker, J., D. Healing, and J. G. P. Hutchison. 1972. Characteristics of some cotrimoxazole-resistant Enterobacteriaceae from infected patients. J. Clin. Pathol. 25:1086-1088. 2. Borderon, E., and T. Horodniceanu. 1978. Metabolically deficient dwarf-colony mutants of Escherichia coli: deficiency and resistance to antibiotics of strains isolated from urine culture. J. Clin. Microbiol. 8:629-634. 3. Finegold, S. M., and E. J. Baron. 1988. Gram-negative facultatively anaerobic bacilli and aerobic coccobacilli, p. 438-455. In Bailey & Scott's diagnostic microbiology, 7th ed. C. V. Mosby Co., St. Louis. 4. Friedman, L. R., and A. W. Ravin. 1972. Genetic and biochemical properties of thymidine-dependent mutants of pneumococcus. J. Bacteriol. 109:459-461. 5. Gillespie, W. A. 1952. Biochemical mutants of coliform bacilli in infections of the urinary tract. J. Pathol. Bacteriol. 64:551-557. 6. McIver, C. J., and J. W. Tapsall. 1988. Characteristics of cysteine-requiring strains of Klebsiella isolated from urinary tract infection. J. Med. Microbiol. 26:211-215. 7. Okado, T., J. Homma, and H. Sonohara. 1962. Improved method for obtaining thymineless mutants of Escherichia coli and Salmonella typhimurium. J. Bacteriol. 84:602-603. 8. Sanyal, D. 1987. Incidence of cysteine dependent Escherichia coli in a general practice population. J. Clin. Pathol. 40:930-933. 9. Sasarman, A., M. Surdeanco, J. Szabados, V. Greceanu, and T. Horodniceanu. 1968. Menaphtone-requiring mutants of Staphylococcus aureus. Rev. Can. Biol. 27:333-339. 10. Silver, R. P., W. Aaronson, and W. F. Vann. 1988. The Ki capsular polysaccharide of Escherichia coli. Rev. Infect. Dis.
10(Suppl. 2):S282-S286. 11. Sompolinski, D., Z. Ernst-Geller, and S. Segal. 1967. Metabolic disorders in thiaminless dwarf strains of Staphylococcus aureus. J. Gen. Microbiol. 48:205-213. 12. Sompolinski, D., I. Gluskin, and G. Ziv. 1969. Pantothenaterequiring dwarf colony variants of Staphylococcus aureus as the etiologic agent in bovine mastitis. J. Hyg. 67:511-516. 13. Steinberg, D., and C. Z. Naggar. 1977. Streptococcus bovis endocarditis with carcinoma of colon. N. Engl. J. Med. 297: 1354. 14. Tapsall, J. W., and C. J. McIver. 1968. Septicemia caused by cysteine-requiring isolates of Escherichia coli. J. Med. Microbiol. 22:379-382. 15. Tapsali, J. W., E. Wilson, and J. Harper. 1974. Thymine dependent strains of Escherichia coli selected by trimethoprim-
sulphamethoxazole therapy. Pathology 6:161-167. 16. Yegian, D., G. Gallo, and M. W. Toll. 1959. Kanamycin resistant Staphylococcus mutant requiring heme for growth. J. Bacteriol. 78:10-12.
Vol. 28, No. 5
NOTES
Septicemia Caused by Cysteine-Dependent Escherichia coli K. Y. YUEN,* W. H. SETO, K. H. TSUI, AND W. T. HUI Department of Microbiology, University of Hong Kong, Hong Kong Received 18 September 1989/Accepted 9 January 1990
A case of septicemia and urinary tract infection caused by cysteine-dependent Escherichia coli in a 70-year-old woman with bilateral staghorn calculi is described. This is the second report of a cysteinedependent E. coli bacteremia. The bacterium was falsely susceptible to ampicillin and co-trimoxazole when tested on a medium without cysteine supplement.
Cysteine-dependent Escherichia coli is a known cause of chronic urinary tract infection, but the isolation of this organism from blood is rare (2, 5). In fact, only one study of five cases is reported in the literature (14). We report in this paper on a cysteine-dependent E. coli with Ki antigen that caused septicemia in a patient with bilateral renal stones. Clinical information. The patient was a 70-year-old female who had been suffering from bilateral renal staghorn calculi for 10 years and had had an extended pyelolithotomy for hydronephrosis of the left kidney 9 years earlier. After the operation, her renal function had remained normal, and the hydronephrotic changes in her kidney were improved with radiology despite the presence of residual stones. One year before admission, she had dysuria and hematuria, which were treated with a 10-day course of co-trimoxazole (two tablets twice a day). Her past health was otherwise unremarkable. On admission, she was febrile (38.5°C) with rigors. Her blood pressure was 90/60 mm Hg, and her pulse rate was 100/min. She had bilateral loin tenderness maximal over the renal angles. Proteinuria (1 g/liter), microscopic hematuria, and pyuria were found by urinalysis. Routine blood tests showed leukocytosis (11.9 x 109 cells per liter) and a slightly raised urea level (7.6 x 10-3 mol/liter). A presumptive diagnosis of septicemia due to acute pyelonephritis was made, and blood cultures were taken. A urine culture was not obtained before the initiation of an antibiotic because she was oliguric on admission. She was started on intravenous cefuroxime (0.75 g every 8 h). After 24 h of antimicrobial therapy, she became afebrile, and the bilateral loin pain and tenderness improved. Two weeks later, intravenous urography showed that the left kidney was obstructed by residual stones. A second operation was however refused by the patient. Microbiological results. Brain heart infusion broth (Mast Diagnostics Ltd., United Kingdom) with 0.05% sodium polyanetholesulfonate was used for conventional aerobic blood culture, and thioglycolate medium (Oxoid Ltd., London, England) supplemented with hemin, vitamin K, and sodium citrate was used for anaerobic blood culture. After 24 h of incubation, a gram-negative bacillus was found by Gram staining in both the aerobic and anaerobic blood culture broths. The broth was subcultured onto blood and chocolate agar, but no growth was noted after 24 h of *
Corresponding author.
incubation. However, after 48 h of incubation, pinpoint translucent colonies could be seen with a magnifying glass. At 72 h, the colonies attained maximal size but were still less than 0.5 mm in diameter. The bacterium was facultative and also grew on MacConkey agar as a non-lactose fermenter. The bacterium was oxidase negative, catalase positive, and motile. The organism was identified as E. coli by both conventional tests and the API 20E system (API, La Balme les Grottes, France) at 48 h of incubation. However, conventional tests for lysine decarboxylase and urea hydrolysis could not be interpreted because of lack of growth, unless cysteine was supplemented. The bacteria were then inoculated onto a cysteine-lactoseelectrolyte-deficient agar plate and a MacConkey agar containing a paper disk soaked with cysteine solution (5 g/liter). A blood agar plate and another MacConkey agar plate were inoculated as controls. After 18 h of incubation, the colonies had attained a diameter of 3 mm, and there was evidence of lactose fermentation on the cysteine-lactose-electrolyte-deficient plate. There were also satellitism and lactose-fermenting activity around the cysteine disk on the MacConkey agar. However, no visible growth was noted on the control plates. The organism was therefore identified as cysteinedependent E. coli. By using the Wellcogen Bacterial Antigen Kit (Wellcome Diagnostics, Research Triangle Park, N.C.), Ki antigen was detected on this bacterium by latex agglutination and counterimmunoelectrophoresis. Antimicrobial susceptibility was tested by the KirbyBauer disk diffusion and agar dilution method, using cysteine-supplemented Mueller-Hinton agar (0.1 g of cysteine per liter). The organism was susceptible to cephalothin, cefuroxime, gentamicin, and chloramphenicol but was resistant to co-trimoxazole and ampicillin by both methods. However, the bacterium appeared susceptible to all six antibiotics when tested on an unsupplemented medium. No organism was found on the urine culture that was collected after the initiation of antimicrobial therapy. Discussion. Nutritionally deficient or auxotrophic bacteria are known to cause difficulty in isolation, identification, or antimicrobial-susceptibility testing as a result of their dependence on various growth factors to achieve normal colony morphology and growth rate. These bacteria include pyridoxal-dependent streptococci (Editorial, Lancet ii:1164, 1977); hemin-, thiamine-, pentothenate-, or menaquinonedependent Staphylococcus aureus (9, 11, 12, 16); thymine1047
1048
NOTES
dependent E. coli (15); Proteus mirabilis (1); Salmonella typhimurium (7); and Streptococcus pneumoniae (4). Cysteine-dependent organisms, such as E. coli (2, 5) and Klebsiella pneumoniae (K. aerogenes) (6), have been reported as occasional urinary tract pathogens. Twenty-one strains of cysteine-dependent E. coli were isolated from about 1,000 specimens of infected urine over a period of 2 years (5), while none were detected in 120 specimens of infected urine from a general-practice population (8). In addition, seven cysteine-requiring strains of K. pneumoniae (6), which constitutes 1.2% of all urinary isolates of Klebsiella species, were found in a hospitalized population. Although E. coli from the urinary tract is the most common source of gram-negative septicemia, only five cysteinedependent isolates have been reported (14). The infrequent reports associated with this organism may not be a reflection of the true incidence. As pointed out above, these organisms may not grow in the most commonly used culture media because of the low cysteine content of these media. If the cysteine content is too low for such nutritionally deficient strains, the strains could be missed on a blind subculture of the blood broth. Furthermore, the API 20E still correctly identified the organism after 48 h of incubation despite atypical colony morphology. Thus, unless a specific test for cysteine dependence is performed, the true nature and the clinical significance of such nutritionally deficient forms of E. coli may simply be ignored. The diagnosis of cysteine-dependent E. coli was considered in this case because there was a discrepancy between growth in the blood broth and in the subculture plates after 48 h of incubation. A differential diagnosis of HB-5, a rare genitourinary pathogen, was also entertained, but this possibility was excluded by the negative oxidase test (3). The clinical data for this patient are rather similar to those from cases of cysteine-dependent E. coli infection reported in the literature. All five reported cases of septicemia were in females over 50 years of age suffering from chronic urinary
problems or debilitating illness (including diabetic papillary necrosis causing obstructive uropathy, post-renal transplantation immunosuppression, indwelling urinary catheters, and myelomatosis). Of the 17 patients with urinary tract infection by cysteine-dependent E. coli (2, 5), 13 were over 50 years of age, and 9 had chronic renal diseases (including nephrolithiasis, hydronephrosis, renal tuberculosis, diabetic nephropathy, benign prostatic hypertrophy, prostatic malignancy, diverticula, and papillomatosis of the bladder). A chronic renal condition was also found in our patient, and the urinary tract was the most likely source of the septicemia in view of her typical uropathic symptoms. Unfortunately, the urine culture was negative because a specimen could be collected only after antimicrobial therapy. As pointed out by Tapsall and McIver (14), the presence of cysteine-dependent E. coli in the blood or urine should prompt a search for an underlying renal condition. This case, involving cysteine-dependent E. coli, is an example of how precise microbiological identification of a bacterial isolate from blood can help in the diagnosis of a medical condition. Other known examples include the association of Streptococcus bovis bacteremia with colonic carcinoma and the association of pyridoxal-dependent streptococci with endocarditis (5, 13). Although Ki antigen is known to be highly associated with
J. CLIN. MICROBIOL.
uropathic E. coli, the prevalence of Ki antigen among cysteine-dependent E. coli is not known, since serotyping was not mentioned in previously reported cases. Since Ki antigen is known to endow the bacteria with invasive potential, probably by blocking the complement-dependent uptake by phagocytosis (10), it would be interesting to know whether the degree of Ki antigenic prevalence within this group of auxotrophic mutants could explain the infrequent detection in blood cultures. The mechanism for the development of most auxotrophic bacteria is largely unknown. For thymidine-dependent E. coli, there is evidence that the mechanism might be induced by chemotherapy. These organisms were found in the urine of patients after at least 8 days of treatment with cotrimoxazole (15). Further studies are needed to identify the mechanism that induces cysteine dependence. LITERATURE CITED
1. Barker, J., D. Healing, and J. G. P. Hutchison. 1972. Characteristics of some cotrimoxazole-resistant Enterobacteriaceae from infected patients. J. Clin. Pathol. 25:1086-1088. 2. Borderon, E., and T. Horodniceanu. 1978. Metabolically deficient dwarf-colony mutants of Escherichia coli: deficiency and resistance to antibiotics of strains isolated from urine culture. J. Clin. Microbiol. 8:629-634. 3. Finegold, S. M., and E. J. Baron. 1988. Gram-negative facultatively anaerobic bacilli and aerobic coccobacilli, p. 438-455. In Bailey & Scott's diagnostic microbiology, 7th ed. C. V. Mosby Co., St. Louis. 4. Friedman, L. R., and A. W. Ravin. 1972. Genetic and biochemical properties of thymidine-dependent mutants of pneumococcus. J. Bacteriol. 109:459-461. 5. Gillespie, W. A. 1952. Biochemical mutants of coliform bacilli in infections of the urinary tract. J. Pathol. Bacteriol. 64:551-557. 6. McIver, C. J., and J. W. Tapsall. 1988. Characteristics of cysteine-requiring strains of Klebsiella isolated from urinary tract infection. J. Med. Microbiol. 26:211-215. 7. Okado, T., J. Homma, and H. Sonohara. 1962. Improved method for obtaining thymineless mutants of Escherichia coli and Salmonella typhimurium. J. Bacteriol. 84:602-603. 8. Sanyal, D. 1987. Incidence of cysteine dependent Escherichia coli in a general practice population. J. Clin. Pathol. 40:930-933. 9. Sasarman, A., M. Surdeanco, J. Szabados, V. Greceanu, and T. Horodniceanu. 1968. Menaphtone-requiring mutants of Staphylococcus aureus. Rev. Can. Biol. 27:333-339. 10. Silver, R. P., W. Aaronson, and W. F. Vann. 1988. The Ki capsular polysaccharide of Escherichia coli. Rev. Infect. Dis.
10(Suppl. 2):S282-S286. 11. Sompolinski, D., Z. Ernst-Geller, and S. Segal. 1967. Metabolic disorders in thiaminless dwarf strains of Staphylococcus aureus. J. Gen. Microbiol. 48:205-213. 12. Sompolinski, D., I. Gluskin, and G. Ziv. 1969. Pantothenaterequiring dwarf colony variants of Staphylococcus aureus as the etiologic agent in bovine mastitis. J. Hyg. 67:511-516. 13. Steinberg, D., and C. Z. Naggar. 1977. Streptococcus bovis endocarditis with carcinoma of colon. N. Engl. J. Med. 297: 1354. 14. Tapsall, J. W., and C. J. McIver. 1968. Septicemia caused by cysteine-requiring isolates of Escherichia coli. J. Med. Microbiol. 22:379-382. 15. Tapsali, J. W., E. Wilson, and J. Harper. 1974. Thymine dependent strains of Escherichia coli selected by trimethoprim-
sulphamethoxazole therapy. Pathology 6:161-167. 16. Yegian, D., G. Gallo, and M. W. Toll. 1959. Kanamycin resistant Staphylococcus mutant requiring heme for growth. J. Bacteriol. 78:10-12.
