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Dolosigranulum pigrum in the shadow of Staphylococcus aureus causing nosocomial pneumonia.

Hervé Lécuyer1, Juliette Audibert2,3, Astrid Bobigny1, Catherine Eckert1,3,4, Caroline Jannière-Nartey2, Annie Buu-Hoï1,3, Jean-Luc Mainardi1,3,4 and Isabelle Podglajen1,3,4*

AP-HP, Hôpital Européen Georges Pompidou, Service de Microbiologie1 and Service de Réanimation Médicale2, Paris, France; Université Paris-Descartes, Faculté de Médecine3 , Paris, France; UMR-S 872-Pôle 4-Equipe 124, Laboratoire de Recherche Moléculaire sur les antibiotiques, Centre de Recherche Biomédical des Cordeliers, Paris, France

*Corresponding author. Mailing address: Service de Microbiologie, AP-HP Hôpital Européen Georges Pompidou, 20 rue Leblanc 75908 Paris Cedex 15, France; UMR-S 872-Pôle 4-Equipe 12, LRMA, Centre de Recherche Biomédical des Cordeliers, 15 rue de l’Ecole de Médecine.

Phone 33 1 56 09 39 51. Fax 33 1 56 09 24 46.

E-mail: isabelle.podglajen@hop.egp.ap-hop-paris.fr or ipodgla@ccr.jussieu.fr

Summary

We report a case of nosocomial pneumonia in which Dolosigranulum pigrum, a rare Gram-positive catalase-negative coccus, was isolated together with Staphylococcus aureus from bronchoalveolar fluid and blood. D. pigrum was identified after 16S rRNA gene sequencing.

Case Report

A 73-year-old man was hospitalized, in December 2006, in the Pulmonary Diseases Unit of the European Hospital Georges Pompidou in Paris, for acute respiratory insufficiency. The medical history of this patient, a heavy smoker, included chronic respiratory insufficiency caused by chronic obstructive pulmonary disease and by sequels of a treated pulmonary tuberculosis contracted during childhood. The patient also suffered from essential thrombocythemia treated with hydroxycarbamide and from an abdominal aortic aneurysm.

A bilateral pulmonary embolism was initially diagnosed using chest computed tomography, and the patient was subsequently treated with heparin. Ten days later, his respiratory state worsened, with onset of a septic shock. He was referred to the intensive care unit for supportive care and endotracheal intubation became necessary. Because of suggestive clinical signs and bilateral opacities on the chest X-rays, a bronchoalveolar lavage was performed. The analysis of the fluid showed 250 leukocytes per mm3 (with 86 % polymorphonucleolar neutrophils and 27 % infected cells) and abundant clusters of Gram-positive cocci. An initial treatment with vancomycin and gentamicin was given. At this time, a first set of cultures (using aerobic and anaerobic BacT/Alert media, bioMérieux) from blood collected, because of fever, two days before the septic shock, turned out positive. Staining also revealed Gram-positive cocci. Culture of the bronchoalveolar fluid yielded catalase-negative, Gram-positive cocci at 7x106 colony forming units (CFU)/ml, initially, using the API rapid ID32 STREP test strip (bioMérieux), presumed to be Aerococcus spp., and also Staphylococcus aureus at 2x104 CFU/ml. Subculture of blood samples also yielded Gram-positive cocci, presumed to be Aerococcus spp. using the API technique. Susceptibility was tested in vitro using disk diffusion on Mueller- Hinton agar with 5 % of sheep blood, according to the guidelines concerning Streptococcaceae of the French Society for Microbiology (7). The presumptive Aerococcus isolates were susceptible to amoxicillin and resistant to trimethoprim, while the S. aureus strain was a methicillin-susceptible penicillinase producer. This motivated a switch to treatment with amoxicillin-clavulanic acid (while gentamicin was continued for two more days). At this time, a second set of cultures from blood collected during the septic shock yielded S. aureus with the same phenotype as above. The state of the patient improved slowly and he was extubated after 15 days. Unfortunately, he presented a second episode of nosocomial pneumonia one month later, in this case due to Klebsiella oxytoca and Serratia marcescens, which proved to be fatal.

Because the biochemical techniques used were not sufficiently accurate to identify the Aerococcus spp. to the species level, 16S RNA gene amplification and sequencing was performed. Bacterial DNA was extracted using IntaGene Matrix (Bio-Rad, CA, USA) according to the manufacturer’s instructions. A 475-bp segment of the 16S rDNA was amplified using the universal primers p13B (5’-CGGGATCCCAGGCCCGGGAACGTATTCAC-3’) and p91E (5’-GGAATTCAAA(GT)GAATTGACGGGGGC-3’). The nucleotide sequence was determined with a 3700 DNA analyzer following the BigDye Terminator Cycle Sequencing Ready Reaction (Applied Systems, CA, USA). The search for similarities was carried out against NCBI databases using NCBI Blast and BiBi phylogenetic tools (). Sequence analysis of the fragment led to the identification of Dolosigranulum pigrum in both bronchoalveolar lavage and blood samples. The nucleotide homology was 100% with the corresponding sequence of the reference strain of the

species (X70907).

The species Dolosigranulum pigrum was first reported in 1993 by Aguirre et al. and identified in two specimens, a frozen spinal cord collected in 1988 and an infected eye collected in 1991, which yielded unknown catalase-negative Gram-positive cocci, arranged in pairs, tetrads, and clusters (1). Biochemical tests performed with conventional methods using commercial kits were not conclusive but the isolates appeared to be related to Gemella species. However, they gave a positive reaction in the arginine dehydrogenase test and there was no immunological reaction between polyclonal antisera raised against the unknown isolates and type strains of Gemella. Finally, 16S rRNA gene sequencing revealed no close relation with previously known bacteria, and a new genus and species were established. The most closely related species, based on 16S RNA sequences, are Aerococcus species with 89 % of homology.

Recently, molecular methods have led to the establishment of close to 15 genera of catalase-negative, Gram-positive cocci in addition to Streptococcus and Enterococcus (8). More than a consequence of the growing use of 16S rDNA sequencing in clinical laboratories, the increasing isolation of these unusual bacteria is probably due to their real emergence as novel opportunistic pathogens among patients with compromised immunity.

The phenotypic characteristics of D. pigrum have been described (5, 8) but attempts to identify this species with commercial test strips very often lead to determination of "unacceptable ID" or to misidentification because the profiles generated by D. pigrum are not included in the databases provided by the manufacturers. If they were, identification with the API rapid ID32 STREP test strip system would be possible as a study by LaClaire et al., analyzing 27 strains, has shown (5). Interpreting the test conventionally, before 16S rRNA gene sequencing, we misidentified D. pigrum as Aerococcus sp., which has the profile closest to that of by D. pigrum.

The 27 strains of D. pigrum have been found susceptible to β-lactam antibiotics and resistant to erythromycin in 52%, with one strain resistant to trimethoprim-sulfamethoxazole (4). The strain isolated in our case was susceptible to β-lactams and erythromycin, but resistant to trimethoprim.

Little is known about the habitat of D. pigrum and its pathogenicity. Of the 27 isolates, 12 were from blood cultures, but bacterial sepsis was reported in only 5 cases. One was an isolate from a spinal cord, after autopsy, and one from urine, while the remaining were isolates from the eye (n=6), nasopharynx (n=4), sinus (n=1) sputum (n=1) or stomach (n=1), suggesting that D. pigrum may be a commensal mainly of the upper respiratory tract (4).

There are only three reports of D. pigrum-related infections. One is of multiple synovitis, with two sets of D. pigrum-positive cultures from blood but not from synovial aspirates (2). In this case, before aspiration of the synovial fluid, the patient had received parenteral cefazolin for 24 hours which was continued until after blood culture and susceptibility testing results were obtained. However, the high level of white blood cells in the synovial fluid (more than 80,000/ml with 83 % of neutrophils) suggested septic arthritis. In this case, the patient was immunocompromised since he received prednisolone daily and methotrexate weekly for chronic rheumatoid arthritis. This could have facilitated the bacteriaemia and the secondary localization of D. pigrum into the synovial fluid. The second report is of a case of acute cholecystitis and pancreatitis potentially caused by D. pigrum (6). Here a set of blood cultures was positive, but there was no search of bacteria in the biliary tract. The patient recovered after two weeks of appropriate antimicrobial therapy (empirical ampicillin-sulbactam, switched to an oral cephalosporin after the blood culture results). Neither chronic disease nor immunodepressive treatment was reported that could have explained infection with D. pigrum. The third report is of a case of ventilator-associated pneumonia caused by D. pigrum (3). The patient was hospitalized and intubated for ten days in the ICU for severe subarachnoid hemorrhage with respiratory failure when he developed pneumonia. He received oral decontamination with topical polymyxin E, tobramycin and amphotericin B which could have facilitated pneumonia due to D. pigrum. The bacteria were cultured in abundance from the bronchial aspirate but not from blood. In the three reports, D. pigrum was identified using 16S rRNA gene sequencing.

The case of nosocomial pneumonia reported here is the first in which D. pigrum was isolated simultaneously from blood and bronchoalveolar fluid. Although in the latter S. aureus was also found, D. pigrum was present at a substantially higher concentration, with 7.106 as opposed to 2.104 CFU/ml. Interestingly, D. pigrum was found in blood cultures two days before onset of the septic shock and acute respiratory failure. The patient received hydroxycarbamide for essential thrombocytemia which in itself might have favored the supervening of an opportunistic pathogen such as D. pigrum.

Among the numerous catalase-negative Gram-positive cocci considered as emerging opportunistic pathogens, D. pigrum is among the least frequently described. The use of appropriate molecular methods of identification should help to determine its true rate of involvement in infectious diseases.

References

1. Aguirre, M., D. Morrison, B. D. Cookson, F. W. Gay, and M. D. Collins. 1993. Phenotypic and phylogenetic characterization of some Gemella-like organisms from human infections: description of Dolosigranulum pigrum gen. nov., sp. nov. J Appl Bacteriol 75:608-12.

2. Hall, G. S., S. Gordon, S. Schroeder, K. Smith, K. Anthony, and G. W. Procop. 2001. Case of synovitis potentially caused by Dolosigranulum pigrum. J Clin Microbiol 39:1202-3.

3. Hoedemaekers, A., T. Schulin, B. Tonk, W. J. Melchers, and P. D. Sturm. 2006. Ventilator-associated pneumonia caused by Dolosigranulum pigrum. J Clin Microbiol 44:3461-2.

4. LaClaire, L., and R. R. Facklam. 2000. Antimicrobial susceptibility and clinical sources of Dolosigranulum pigrum cultures. Antimicrob Agents Chemother 44:2001-3.

5. LaClaire, L. L., and R. R. Facklam. 2000. Comparison of three commercial rapid identification systems for the unusual gram-positive cocci Dolosigranulum pigrum, Ignavigranum ruoffiae, and Facklamia species. J Clin Microbiol 38:2037-42.

6. Lin, J. C., S. J. Hou, L. U. Huang, J. R. Sun, W. K. Chang, and J. J. Lu. 2006. Acute cholecystitis accompanied by acute pancreatitis potentially caused by Dolosigranulum pigrum. J Clin Microbiol 44:2298-9.

7. Société Francaise de Microbiologie, 2006. Recommandations du Comité de l'Antibiogramme.

8. Ruoff, K. L. 2002. Miscellaneous catalase-negative, gram-positive cocci: emerging opportunists. J Clin Microbiol 40:1129-33.

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