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Journal of Clinical Microbiology, July 2003, p. 3436-3437, Vol. 41, No. 7
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.7.3436-3437.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

CASE REPORT

Utilization of 16S Ribosomal DNA Sequencing for Diagnosis of Septicemia Due to Neisseria elongata subsp. glycolytica in a Neutropenic Patient

Cecile Hombrouck-Alet,1 Isabelle Poilane,1 Claire Janoir-Jouveshomme,2 Olivier Fain,3 Philippe Cruaud,1 Michel Thomas,3 and Anne Collignon1,2*

Service de Microbiologie,1 Service de Médecine Interne, Hôpital Jean Verdier, Bondy,3 Département de Microbiologie, Faculté de Pharmacie, Université Paris-Sud, ChÂtenay-Malabry, France2

Received 15 November 2002/ Returned for modification 7 February 2003/ Accepted 1 April 2003


   ABSTRACT
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 Abstract
Case report
 References
 
Septicemia due to Neisseria elongata subsp. glycolytica occurs infrequently. We report a case of septicemia in a patient undergoing antimitotic chemotherapy. Gram-negative coccobacilli were isolated from blood cultures. The identity of the isolate by phenotypic methods was uncertain. In contrast, identity was confirmed by 16S ribosomal DNA sequencing, which appeared to be very useful for correct identification.


   CASE REPORT
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 Abstract
 Case report
References
 
A 66-year-old retired male was admitted to the internal medicine unit of the hospital for a second antimitotic chemotherapy treatment for a squamous cell carcinoma. Nine days after the beginning of the chemotherapy (cisplatin-vinocelbine), he was leukopenic (leukocyte count of 300/mm3), and he presented with fever (39°C), diarrhea, and pulmonary rhonchi. The chest X-ray film did not show pulmonary infiltrates. Laboratory studies revealed the following values: hemoglobin, 89 g/liter (normal range, 120 to 150 g/liter); platelet count, 21,000/mm3 (normal range 150,000 to 400,000/mm3); and C-reactive protein, 209 mg/liter (normal range, <5 mg/liter). Two sets of blood cultures (Vital; bioMérieux, Marcy l'Etoile, France) were performed. Subsequently, empirical antimicrobial therapy was started with intravenous ceftazidime (1 g/8 h) plus intravenous ciprofloxacin (200 mg/12 h) as recommended in our hospital for neutropenic fever. The fever decreased in 24 h, but the patient suddenly died 8 days after the onset of the antimicrobial therapy.

Gram-negative, aerobic, nonmotile coccobacilli were detected in two blood culture bottles after 2 days of incubation. Subcultures were performed, and growth was observed after 24 h of incubation with 5% CO2 on plates containing chocolate, blood, and Trypticase soy agar (bioMérieux). The colonies were small (0.5 to 1 mm) and nonpigmented. No hemolysis on sheep or horse blood was observed. The organism was strictly aerobic and catalase and oxidase positive. Its growth was enhanced in a 5% CO2 atmosphere. The glucose test result was ambiguous with the API NH assay (bioMérieux), but the oxidation of glucose was negative in medium supplemented with 1% carbohydrate (MEVAG; Bio-Rad, Marnes-la-Coquette, France). All tests were negative with the API NE strip (bioMérieux). The strain was negative for the following characteristics: urease, indole production, o-nitrophenyl-ß-D-galactopyranoside, growth on Simmons citrate, hydrolysis of Tween 80, accumulation of lipids after growth on ß-hydroxybutyrate, and {gamma}-glutamyl transferase. It was asaccharolytic, resistant to the vibriostatic compound O/129, and did not reduce nitrate but did reduce nitrite. The phenylalanine deaminase reaction is dependent on the culture medium used (7). Therefore, to successfully utilize this test, the isolate was grown on various media (Mueller-Hinton, blood, and chocolate agar) prior to inoculation with a heavy inoculum into the phenylalanine deaminase solution. Pseudomonas aeruginosa and Proteus mirabilis were used as negative and positive controls, respectively. The phenylalanine deaminase reaction was positive for the strain tested after incubation for 18 h with all of the media. All of these phenotypic characteristics were close to those of Neisseria weaveri and Neisseria elongata subsp. glycolytica (1, 2, 5, 6, 8). In contrast, N. elongata subsp. elongata and N. elongata subsp. nitroreducens differ from the isolate by two and three phenotypic characteristics, respectively: they were catalase negative, phenylalanine deaminase negative, and, for Neisseria elongata subsp. nitroreducens, nitrate positive (Table 1).


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  		TABLE 1. Phenotypic characteristics of several Neisseria species and the isolate obtained in this study

 
The antibiotic susceptibility of the strain was tested by the disk diffusion method on Mueller-Hinton agar (Bio-Rad, Marnes-la-Coquette, France) incubated at 37°C for 24 h and interpreted according to the CA-SFM criteria (C. J. Soussy et al., Report of the Antibiogram Committee of the French Society for Microbiology, 2002). The isolate was susceptible to amoxicillin, amoxicillin-clavulanic acid, cephalothin, cefotaxime, gentamicin, pefloxacin, and sulfonamides but resistant to trimethoprim.

Since unequivocal identification of the isolate could not be established by phenotypic tests, a molecular approach based on 16S ribosomal DNA (rDNA) sequencing was used. A 1,487-bp fragment of the 16S rDNA gene was amplified by PCR with primers A (5'AGTTTGATCCTGGCTCAG3') and B (5'GGTTACCTTGTTACGACTT3') (Eurobio, Les Ulis, France). Automated sequencing of the purified PCR product was performed on the two strands with the Big Dye terminator kit (Applied Biosystems), and the sequence was analyzed with an ABI PRISM 310 genetic analyzer (Applied Biosystems). The sequence was aligned by the ClustalW multisequence alignment program, giving 99.5% homology with that from N. elongata subsp. glycolytica strain J2 (GenBank accession no. AJ 239278). The homologies were only 96.1% with N. weaveri CDC 8142 and 95.4% with N. flavescens ATCC 13120 and N. canis ATCC 14687 (GenBank accession no. L10738, L06168, and L06170, respectively). The 16S rDNA sequencing demonstrated that this isolate was related to N. elongata subsp. glycolytica and not to N. weaveri.

Only partial comparison of 457 nucleotides over 1,355 total was possible with the two other subspecies of N. elongata: the homologies were 99.8% with N. elongata subsp. elongata (strain LMG 5124) and 100% with N. elongata subsp. nitroreducens (strain ATCC 49377), respectively (GenBank accession no. AJ 247252 and AJ 247254, respectively). The partial sequences of these subspecies were not informative enough to conclusively identify the organism to the subspecies level. However, the combination of genotypic and phenotypic characteristics led unambiguously to the identification of N. elongata subsp. glycolytica.

Among the three subspecies of N. elongata, N. elongata subsp. nitroreducens has been frequently described in human invasive disease, particularly in endocarditis (3, 4). N. elongata subsp. elongata and subsp. glycolytica have been considered as transient colonizers of the human upper respiratory tract and urogenital tract. One case of endocarditis caused by N. elongata subsp. glycolytica has been reported in the literature, suggesting a potential pathogenic role for this bacterium (2).

In conclusion, we report a second case of invasive disease caused by N. elongata subsp. glycolytica. The isolation of the organism from two blood cultures from this patient indicates the clinical significance of this subspecies. Neither the classical phenotypic methods nor the API NH strips allowed us to identify this gram-negative short rod with good agreement. In contrast, 16S rDNA sequencing in combination with biochemical reactions allowed us to correctly identify N. elongata subsp glycolytica. This simple molecular technique is complementary to phenotypic methods in cases of difficult identification.

Nucleotide sequence accession number. The nucleotide sequence data obtained in this study have been deposited in the GenBank nucleotide sequence database under accession no. AY167422.


   FOOTNOTES
 
* Corresponding author. Mailing address: Hôpital Jean Verdier, Assistance Publique-Hôpitaux de Paris, Service de Microbiologie, Avenue du 14 Juillet, F-93143 Bondy Cedex, France. Phone: (33) 1 48 02 65 73. Fax: (33) 1 48 02 68 31. E-mail: anne.collignon{at}jvr.ap-hop-paris.fr. Back


   REFERENCES
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 Abstract
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 References
 

  1. Andersen, B. M., A. G. Steigerwalt, S. P. O'Connor, D. G. Hollis, R. S. Weyant, R. E. Weaver, and D. J. Brenner. 1993. Neisseria weaveri sp. nov., formerly CDC group M-5, a gram-negative bacterium associated with dog bite wounds. J. Clin. Microbiol. 31:2456-2466.[Abstract/Free Full Text]
  2. Andersen, B. M., R. S. Weyant, A. G. Steigerwalt, C. W. Moss, D. G. Hollis, R. E. Weaver, D. Ashford, and D. J. Brenner. 1995. Characterization of Neisseria elongata subsp. glycolytica isolates obtained from human wound specimens and blood cultures. J. Clin. Microbiol. 33:76-78.[Abstract]
  3. Dominguez, E. A., and T. L. Smith. 1998. Endocarditis due to Neisseria elongata subspecies nitroreducens: case report and review. Clin. Infect. Dis. 26:1471-1473.[Medline]
  4. Grant, P. E., D. J. Brenner, A. J. Steigerwalt, D. G. Hollis, and R. E. Weaver. 1990. Neisseria elongata subsp. nitroreducens subsp. nov., formerly CDC group M-6, a gram-negative bacterium associated with endocarditis. J. Clin. Microbiol. 38:2591-2596.
  5. Holmes, B., M. Costas, S. L. W. On, P. Vandamme, E. Falsen, and K. Kersters. 1993. Neisseria weaveri sp. nov. (formerly CDC group M-5), from dog bite wounds of humans. Int. J. Syst. Bacteriol. 43:687-693.[Abstract/Free Full Text]
  6. Knapp, J. S., and E. H. Koumans. 1999. Neisseria and Branhamella, p. 586-603. In P. R. Murray, E. J. Baron, M. A. Pfaller, F. C. Tenover, and R. H. Yolken (ed.), Manual of clinical microbiology, 7th ed. American Society for Microbiology, Washington, D.C.
  7. Richard, C., and M. Kiredjian. 1992. Recherche de la L-phenyl-alanine-désaminase après 30 minutes, 4 heures et 18 heures, p. 144. In Institut Pasteur (ed.), Méthodes de laboratoire pour l'identification des bacilles à Gram négatif aérobies stricts. Institut Pasteur, Paris, France.
  8. Schreckenberger, P. C., and A. von Graevenitz. 1999. Acinetobacter, Achromobacter, Alcaligenes, Moraxella, Methylobacterium, and other nonfermentative gram-negative rods, p. 539-560. In P. R. Murray, E. J. Baron, M. A. Pfaller, F. C. Tenover, and R. H. Yolken (ed.), Manual of clinical microbiology, 7th ed. American Society for Microbiology, Washington, D.C.

Journal of Clinical Microbiology, July 2003, p. 3436-3437, Vol. 41, No. 7
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.7.3436-3437.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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