Listeriosis Due to Infection with a Catalase-Negative Strain of Listeria monocytogenes
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微生物临床杂志 2006年第5期
Department of Medical Microbiology, Barts and The London NHS Trust, London, United Kingdom
Department of Renal Medicine, Barts and The London NHS Trust, London, United Kingdom
Centre for Infectious Disease, Institute of Cell and Molecular Science, Barts and The London, Queen Mary's School of Medicine and Dentistry, London, United Kingdom
ABSTRACT
A strain of Listeria monocytogenes recovered from blood and cerebrospinal fluid had no detectable catalase activity, a characteristic used for primary identification. The sporadic occurrence of pathogenic catalase-negative strains highlights the need for a reconsideration of diagnostic criteria and questions the role of catalase in the pathogenesis of listeria infection.
CASE REPORT
A 28-year-old Caucasian male receiving immunosuppressive therapy with cyclosporine and prednisolone following renal transplantation developed a sudden onset of rigors, high fever (38.9°C), vomiting, and progressive confusion after routine hemodialysis. On arrival in the emergency room, he sustained a generalized tonic-clonic seizure requiring anticonvulsants. Physical examination revealed no obvious source of infection or skin rash, mild photophobia but no other signs of meningeal irritation, and generalized hyperreflexia consistent with a postictal state. A lumbar puncture was performed, and a full septic screen was obtained before the patient was started on intravenous ceftriaxone therapy (2 g every 12 h). Cerebrospinal fluid (CSF) revealed a lymphocyte count of 1,008 cells/mm3; therefore, intravenous acyclovir (10 mg/kg of body weight every 8 h) and ampicillin (2 g every 4 h) were added as per local protocol. The patient's general condition rapidly deteriorated, and he required respiratory and hemodynamic support in the intensive care unit.
Two days later, blood cultures in four bottles became positive for a gram-positive, rod-shaped organism suggestive of a Listeria sp., but catalase testing (performed by observing the generation of bubbles of oxygen formed when a colony was suspended in a drop of hydrogen peroxide on a glass slide) showed consistently negative results. Biochemical identification (esculin positive, glucose positive, and maltose positive) using an API Coryne system (bioMerieux, Basingstoke, United Kingdom) repeatedly failed to identify the organism as Listeria monocytogenes due to negative catalase and xylose reactions. On the following day, an additional set of blood cultures and a CSF sample grew the same organism.
Initial sensitivity testing using the British Society of Antimicrobial Chemotherapy disk diffusion method showed that the sizes of the zones with ampicillin were inconsistent between the blood and CFS isolates. Ampicillin was therefore discontinued, and co-trimoxazole (10 mg/kg every 12 h) commenced. Subsequent susceptibility testing of both isolates by agar dilution at the Health Protection Agency antibiotic reference laboratory confirmed the organism's susceptibility to ampicillin and co-trimoxazole, with MICs of 0.5 mg/liter and 0.064 mg/liter, respectively.
The patient made a slow but progressive recovery, regained consciousness, and experienced no further seizures. The antibiotic therapy with co-trimoxazole was continued for a total of 4 weeks.
The organism was identified as L. monocytogenes by PCR amplification and sequence analysis of 1,156 base pairs of the 16S rRNA genes and confirmed by the Food Safety Microbiology Laboratory at the Health Protection Agency (Colindale, London, United Kingdom) as L. monocytogenes serogroup 4.
The apparent lack of catalase activity was investigated further using crude whole-cell extracts as follows. The isolate was grown at 37°C in 5 ml of Luria-Bertani broth to an optical density at 600 nm of 0.6 to 0.8 (mid-log phase), and cells were harvested by centrifugation. Cell extracts were obtained by resuspending the bacteria in 2.5 ml of phosphate-buffered saline containing 2% (wt/vol) sodium dodecyl sulfate (SDS) and then incubating the suspension at 37°C for 30 min with shaking. Suspensions were centrifuged, and proteins present in the supernatants were quantified by Bradford's method (2a).
Proteins were also extracted from a catalase-positive strain (Listeria monocytogenes NCTC 10357) following growth under similar conditions. Extracts (5-μl volumes; 2 mg protein/ml) were diluted in 1 ml of distilled water, and 0.5 ml of 59 mM H2O2 diluted in 50 mM K2HPO4 was added. The absorbance at 240 nm was measured every 15 s for 1 min with a DU 800 spectrophotometer (Beckman Coulter, High Wycombe, United Kingdom), and the specific catalase activity was calculated using the formula described by Beers and Sizer (2).
The isolate had a catalase-specific activity of 0 compared to an activity of 17.2 ± 0.56 for the catalase-positive control. Catalase activity was also investigated by zymography. Crude cell extracts (25 μl) and purified bovine catalase were mixed with 5 μl of 5x SDS-polyacrylamide gel electrophoresis buffer and electrophoresed on a 7.5% SDS-polyacrylamide gel. Gels were soaked in horseradish peroxidase (50 μg/ml) in 50 mM K2HPO4, pH 7.0, for 45 min, and H2O2 was added to a final concentration of 5 mM. Zymograms were developed by the addition of 0.5 mg/ml diaminobenzadine in a phosphate buffer (5). The 55-kDa band corresponding to L. monocytogenes catalase was absent from the catalase-negative strain (Fig. 1).
The kat gene in the catalase-negative strain was successfully amplified using PCR, and its complete DNA sequence was determined. Analysis of the sequence showed a 4-nucleotide insertion-duplication (GGCC) at position 1044.
Listeria monocytogenes is a well-recognized human pathogen, causing invasive disease in debilitated individuals, children, and pregnant women. Serotypes 1/2 and 4 are the most commonly reported (1) and may cause meningitis in immunocompromised patients or patients receiving immunosuppressive therapy (11). Identification of L. monocytogenes relies on a number of phenotypic criteria, including Gram stain appearance, colonial morphology, hemolysis, tumbling motility, and a number of biochemical reactions. The production of catalase is thought to be an important characteristic and is employed by a number of commercial identification systems (7). Misidentification using the API Coryne test but not the API Rapid ID Strep, which does not rely on catalase production, has been previously reported (3). Strains of L. monocytogenes that do not produce catalase have only rarely been reported in sporadic cases of human infection (12) but can fairly readily be isolated from a number of foods (4; Food Safety Microbiology Laboratory, personal communication). Catalase production is thought to be a key virulence factor contributing to intracellular survival by neutralizing the free radical killing effect of hydroxyl radicals formed within macrophages and other phagocytic cells during infection. Studies in vitro, however, suggest that levels of intracellular survival are similar in both catalase producers and nonproducers (10). Catalase-deficient mutants have also been shown to be fully virulent in mice (8), although such strains may compensate for the lack of catalase production by the overexpression of superoxide dismutase (9). In contrast to previously reported cases of catalase-negative listeriosis, our strain did not regain catalase activity upon subculture (6); in addition, it harbored an insertion within the kat gene which puts a stop codon in frame, leading to the premature termination of translation. The resulting predicted polypeptide was 357 residues in length, 131 residues less than the wild-type protein. Prior to this insertion, point mutations accounted for only four other polymorphisms, I192V, I217V, C243R, and N271S. The truncated polypeptide still retains its heme-binding pocket but lacks many of the residues involved in the tetramer interface. We therefore hypothesize that the lack of catalase activity is due to defective tertiary and quaternary structures in the protein. This case provides further evidence that catalase-negative strains of L. monocytogenes can be pathogenic to humans. The reliance on catalase production as an identification characteristic should be reviewed, as this diagnostic criterion may lead to misidentification and delay appropriate therapy.
Nucleotide sequence accession number. The Listeria sp. catalase sequence has been submitted to GenBank with accession number DQ304650.
REFERENCES
Aouaj, Y., L. Spanjaard, N. van Leeuwen, and J. Dankart. 2002. Listeria monocytogenes meningitis: serotype distribution and patient characteristics in the Netherlands, 1975-95. Epidemiol. Infect. 128:405-409.
Beers, R. F., and I. W. Sizer. 1952. A spectrophotometric method for measuring the breakdown of hydrogen peroxide by catalase. J. Biol. Chem. 195:130-140.
Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248-254.
Bubert, A., J. Riebe, N. Schnitzler, A. Schnberg, W. Goebel, and P. Schubert. 1997. Isolation of catalase-negative Listeria monocytogenes strains from listeriosis patients and their rapid identification by anti-P60 antibodies and/or PCR. J. Clin. Microbiol. 35:179-183.
Chen, T., Y. Feng, J. Dai, and H. Chen. 2000. Identification of Listeria species in 167 kinds of food. Wei Sheng Yan Jiu 29:246-247. (In Chinese.)
Clare, D. A., M. N., Duong, D. Darr, F. Archibald, and I. Fridovich. 1984. Effects of molecular oxygen on detection of superoxide radical with nitroblue tetrazolium and on activity stains for catalase. Anal. Biochem. 140:532-537.
Elsner, H. A., I. Sobottka, A. Bubert, H. Albrecht, R. Laufs, and D. Mack. 1996. Catalase-negative Listeria monocytogenes causing lethal sepsis and meningitis in a hematologics patient. Eur. J. Clin. Microbiol. Infect. Dis. 15:965-967.
Gasanov, U., D. Hughes, and P. M. Hansbro. 2005. Methods for the identification of Listeria spp. and Listeria monocytogenes: a review. FEMS Microbiol. Rev. 29:851-875.
Leblond-Francillard, M., J.-L. Gaillard, and P. Berche. 1989. Loss of catalase activity in Tn1545-induced mutants does not reduce growth of Listeria monocytogenes in vivo. Infect. Immun. 57:2569-2573.
Makino, S., K. Mariko, I. Kawamura, M. Fujita, F. Gejo, and M. Mitsuyama. 2005. Involvement of reactive oxygen intermediate in the enhanced expression of virulence-associated genes of Listeria monocytogenes inside activated macrophages. Microbiol. Immunol. 49:805-811.
Myers, E. R., A. W. Dallmier, and S. E. Martin. 1993. Sodium chloride, potassium chloride, and virulence in Listeria monocytogenes. Appl. Environ. Microbiol. 59:2082-2086.
Skogberg, K., J. Syrjanen, M. Jahkola, O. V. Renkonen, J. Paavonen, J. Ahonen, S. Kontiainen, P. Ruutu, and V. Voltonen. 1992. Clinical presentation and outcome of listeriosis in patients with and without immunosuppressive therapy. Clin. Infect. Dis. 14:815-821.
Swartz, M. A., D. F. Welch, R. P. Narayanan, and R. A. Greenfield. 1991. Catalase-negative Listeria monocytogenes causing meningitis in an adult. Clinical and laboratory features. Am. J. Clin. Pathol. 96:130-133.(J. A. Cepeda, M. Millar, )
Department of Renal Medicine, Barts and The London NHS Trust, London, United Kingdom
Centre for Infectious Disease, Institute of Cell and Molecular Science, Barts and The London, Queen Mary's School of Medicine and Dentistry, London, United Kingdom
ABSTRACT
A strain of Listeria monocytogenes recovered from blood and cerebrospinal fluid had no detectable catalase activity, a characteristic used for primary identification. The sporadic occurrence of pathogenic catalase-negative strains highlights the need for a reconsideration of diagnostic criteria and questions the role of catalase in the pathogenesis of listeria infection.
CASE REPORT
A 28-year-old Caucasian male receiving immunosuppressive therapy with cyclosporine and prednisolone following renal transplantation developed a sudden onset of rigors, high fever (38.9°C), vomiting, and progressive confusion after routine hemodialysis. On arrival in the emergency room, he sustained a generalized tonic-clonic seizure requiring anticonvulsants. Physical examination revealed no obvious source of infection or skin rash, mild photophobia but no other signs of meningeal irritation, and generalized hyperreflexia consistent with a postictal state. A lumbar puncture was performed, and a full septic screen was obtained before the patient was started on intravenous ceftriaxone therapy (2 g every 12 h). Cerebrospinal fluid (CSF) revealed a lymphocyte count of 1,008 cells/mm3; therefore, intravenous acyclovir (10 mg/kg of body weight every 8 h) and ampicillin (2 g every 4 h) were added as per local protocol. The patient's general condition rapidly deteriorated, and he required respiratory and hemodynamic support in the intensive care unit.
Two days later, blood cultures in four bottles became positive for a gram-positive, rod-shaped organism suggestive of a Listeria sp., but catalase testing (performed by observing the generation of bubbles of oxygen formed when a colony was suspended in a drop of hydrogen peroxide on a glass slide) showed consistently negative results. Biochemical identification (esculin positive, glucose positive, and maltose positive) using an API Coryne system (bioMerieux, Basingstoke, United Kingdom) repeatedly failed to identify the organism as Listeria monocytogenes due to negative catalase and xylose reactions. On the following day, an additional set of blood cultures and a CSF sample grew the same organism.
Initial sensitivity testing using the British Society of Antimicrobial Chemotherapy disk diffusion method showed that the sizes of the zones with ampicillin were inconsistent between the blood and CFS isolates. Ampicillin was therefore discontinued, and co-trimoxazole (10 mg/kg every 12 h) commenced. Subsequent susceptibility testing of both isolates by agar dilution at the Health Protection Agency antibiotic reference laboratory confirmed the organism's susceptibility to ampicillin and co-trimoxazole, with MICs of 0.5 mg/liter and 0.064 mg/liter, respectively.
The patient made a slow but progressive recovery, regained consciousness, and experienced no further seizures. The antibiotic therapy with co-trimoxazole was continued for a total of 4 weeks.
The organism was identified as L. monocytogenes by PCR amplification and sequence analysis of 1,156 base pairs of the 16S rRNA genes and confirmed by the Food Safety Microbiology Laboratory at the Health Protection Agency (Colindale, London, United Kingdom) as L. monocytogenes serogroup 4.
The apparent lack of catalase activity was investigated further using crude whole-cell extracts as follows. The isolate was grown at 37°C in 5 ml of Luria-Bertani broth to an optical density at 600 nm of 0.6 to 0.8 (mid-log phase), and cells were harvested by centrifugation. Cell extracts were obtained by resuspending the bacteria in 2.5 ml of phosphate-buffered saline containing 2% (wt/vol) sodium dodecyl sulfate (SDS) and then incubating the suspension at 37°C for 30 min with shaking. Suspensions were centrifuged, and proteins present in the supernatants were quantified by Bradford's method (2a).
Proteins were also extracted from a catalase-positive strain (Listeria monocytogenes NCTC 10357) following growth under similar conditions. Extracts (5-μl volumes; 2 mg protein/ml) were diluted in 1 ml of distilled water, and 0.5 ml of 59 mM H2O2 diluted in 50 mM K2HPO4 was added. The absorbance at 240 nm was measured every 15 s for 1 min with a DU 800 spectrophotometer (Beckman Coulter, High Wycombe, United Kingdom), and the specific catalase activity was calculated using the formula described by Beers and Sizer (2).
The isolate had a catalase-specific activity of 0 compared to an activity of 17.2 ± 0.56 for the catalase-positive control. Catalase activity was also investigated by zymography. Crude cell extracts (25 μl) and purified bovine catalase were mixed with 5 μl of 5x SDS-polyacrylamide gel electrophoresis buffer and electrophoresed on a 7.5% SDS-polyacrylamide gel. Gels were soaked in horseradish peroxidase (50 μg/ml) in 50 mM K2HPO4, pH 7.0, for 45 min, and H2O2 was added to a final concentration of 5 mM. Zymograms were developed by the addition of 0.5 mg/ml diaminobenzadine in a phosphate buffer (5). The 55-kDa band corresponding to L. monocytogenes catalase was absent from the catalase-negative strain (Fig. 1).
The kat gene in the catalase-negative strain was successfully amplified using PCR, and its complete DNA sequence was determined. Analysis of the sequence showed a 4-nucleotide insertion-duplication (GGCC) at position 1044.
Listeria monocytogenes is a well-recognized human pathogen, causing invasive disease in debilitated individuals, children, and pregnant women. Serotypes 1/2 and 4 are the most commonly reported (1) and may cause meningitis in immunocompromised patients or patients receiving immunosuppressive therapy (11). Identification of L. monocytogenes relies on a number of phenotypic criteria, including Gram stain appearance, colonial morphology, hemolysis, tumbling motility, and a number of biochemical reactions. The production of catalase is thought to be an important characteristic and is employed by a number of commercial identification systems (7). Misidentification using the API Coryne test but not the API Rapid ID Strep, which does not rely on catalase production, has been previously reported (3). Strains of L. monocytogenes that do not produce catalase have only rarely been reported in sporadic cases of human infection (12) but can fairly readily be isolated from a number of foods (4; Food Safety Microbiology Laboratory, personal communication). Catalase production is thought to be a key virulence factor contributing to intracellular survival by neutralizing the free radical killing effect of hydroxyl radicals formed within macrophages and other phagocytic cells during infection. Studies in vitro, however, suggest that levels of intracellular survival are similar in both catalase producers and nonproducers (10). Catalase-deficient mutants have also been shown to be fully virulent in mice (8), although such strains may compensate for the lack of catalase production by the overexpression of superoxide dismutase (9). In contrast to previously reported cases of catalase-negative listeriosis, our strain did not regain catalase activity upon subculture (6); in addition, it harbored an insertion within the kat gene which puts a stop codon in frame, leading to the premature termination of translation. The resulting predicted polypeptide was 357 residues in length, 131 residues less than the wild-type protein. Prior to this insertion, point mutations accounted for only four other polymorphisms, I192V, I217V, C243R, and N271S. The truncated polypeptide still retains its heme-binding pocket but lacks many of the residues involved in the tetramer interface. We therefore hypothesize that the lack of catalase activity is due to defective tertiary and quaternary structures in the protein. This case provides further evidence that catalase-negative strains of L. monocytogenes can be pathogenic to humans. The reliance on catalase production as an identification characteristic should be reviewed, as this diagnostic criterion may lead to misidentification and delay appropriate therapy.
Nucleotide sequence accession number. The Listeria sp. catalase sequence has been submitted to GenBank with accession number DQ304650.
REFERENCES
Aouaj, Y., L. Spanjaard, N. van Leeuwen, and J. Dankart. 2002. Listeria monocytogenes meningitis: serotype distribution and patient characteristics in the Netherlands, 1975-95. Epidemiol. Infect. 128:405-409.
Beers, R. F., and I. W. Sizer. 1952. A spectrophotometric method for measuring the breakdown of hydrogen peroxide by catalase. J. Biol. Chem. 195:130-140.
Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248-254.
Bubert, A., J. Riebe, N. Schnitzler, A. Schnberg, W. Goebel, and P. Schubert. 1997. Isolation of catalase-negative Listeria monocytogenes strains from listeriosis patients and their rapid identification by anti-P60 antibodies and/or PCR. J. Clin. Microbiol. 35:179-183.
Chen, T., Y. Feng, J. Dai, and H. Chen. 2000. Identification of Listeria species in 167 kinds of food. Wei Sheng Yan Jiu 29:246-247. (In Chinese.)
Clare, D. A., M. N., Duong, D. Darr, F. Archibald, and I. Fridovich. 1984. Effects of molecular oxygen on detection of superoxide radical with nitroblue tetrazolium and on activity stains for catalase. Anal. Biochem. 140:532-537.
Elsner, H. A., I. Sobottka, A. Bubert, H. Albrecht, R. Laufs, and D. Mack. 1996. Catalase-negative Listeria monocytogenes causing lethal sepsis and meningitis in a hematologics patient. Eur. J. Clin. Microbiol. Infect. Dis. 15:965-967.
Gasanov, U., D. Hughes, and P. M. Hansbro. 2005. Methods for the identification of Listeria spp. and Listeria monocytogenes: a review. FEMS Microbiol. Rev. 29:851-875.
Leblond-Francillard, M., J.-L. Gaillard, and P. Berche. 1989. Loss of catalase activity in Tn1545-induced mutants does not reduce growth of Listeria monocytogenes in vivo. Infect. Immun. 57:2569-2573.
Makino, S., K. Mariko, I. Kawamura, M. Fujita, F. Gejo, and M. Mitsuyama. 2005. Involvement of reactive oxygen intermediate in the enhanced expression of virulence-associated genes of Listeria monocytogenes inside activated macrophages. Microbiol. Immunol. 49:805-811.
Myers, E. R., A. W. Dallmier, and S. E. Martin. 1993. Sodium chloride, potassium chloride, and virulence in Listeria monocytogenes. Appl. Environ. Microbiol. 59:2082-2086.
Skogberg, K., J. Syrjanen, M. Jahkola, O. V. Renkonen, J. Paavonen, J. Ahonen, S. Kontiainen, P. Ruutu, and V. Voltonen. 1992. Clinical presentation and outcome of listeriosis in patients with and without immunosuppressive therapy. Clin. Infect. Dis. 14:815-821.
Swartz, M. A., D. F. Welch, R. P. Narayanan, and R. A. Greenfield. 1991. Catalase-negative Listeria monocytogenes causing meningitis in an adult. Clinical and laboratory features. Am. J. Clin. Pathol. 96:130-133.(J. A. Cepeda, M. Millar, )