Planctomycetes DNA in Febrile Aplastic Patients with Leukemia, Rash, Diarrhea, and Micronodular Pneumonia

Abstract

We found Planctomycetes DNA in 2 out of 100 blood samples from patients suffering from leukemia with neutropenia induced by chemotherapy, as well as fever, rash, pneumonia, and diarrhea. Antibiotic-resisting Planctomycetes may be pathogenic in these patients.

CASE REPORT

Case 1 was a 45-year-old man treated for acute myeloblastic leukemia by induction chemotherapy (daunomycin and cytarabine). Seven days after induction, the patient entered aplasia and had a 48-h fever, which dissipated after treatment with piperacillin-tazobactam. Ten days after induction, he suffered from mild erythema, diarrhea, abdominal pain, and fever of 38.5°C; a computed tomography (CT) scan confirmed sigmoiditis. The patient's symptoms improved after treatment with imipenem and levofloxacin. On day 18, levofloxacin treatment was stopped, and fever relapsed. Routine investigations, including blood cultures incubated for 5 days, remained negative, as was the molecular detection of Archaea, Mimiviridae, and “broad-spectrum” 16S rRNA PCR. A Planctomycetes-specific PCR amplified directly from blood a 276-bp sequence exhibiting 96.7% similarity to that from an uncultured bacterium detected in the human skin microbiome and mountain lake biofilms (Fig. 1). In this patient, we further amplified directly from blood a 60-bp rpoB sequence that yielded 88% similarity to Gemmata obscuriglobus, but tentative culture of Planctomycetes remained negative. The CT scan was consistent with micronodular pneumonitis. Caspofungin was added, and the patient became apyretic 13 days later. At the last follow-up, the patient was healthy and disease free 15 months following an allogeneic transplantation.

FIG 1.

Multiple sequence alignments carried-out with ClustalX software and phylogenic analysis using the neighbor-joining method (Mega 5 software) were used to construct a phylogenetic tree, including the 16S rRNA gene sequences of the 7 most closely related strains, according to a BLAST search with the case 1 and case 2 sequences. Bootstrap values of >90% are indicated at the nodes. The size bar indicates 2% substitutions.

Case 2 was a 76-year-old woman treated for acute myeloblastic leukemia by induction chemotherapy (daunomycin, cytarabine, and lenalidomide). Nine days after induction, the patient developed the first episode of fever, which dissipated after treatment with piperacillin-tazobactam. On day 18, the patient developed erythema, diarrhea, and a 38.5°C fever in the context of severe sepsis. The patient's treatment was changed to imipenem, amikacin, and levofloxacin. The CT scan showed micronodular pneumonitis. Routine investigations remained negative, including sterile blood cultures after a 5-day incubation, molecular detection of Archaea, Mimiviridae, and “broad-spectrum” 16S rRNA PCR. PCR directly performed on blood detected Planctomycetes 16S rRNA sequence with 100% similarity to uncultured Planctomycetes from a lake water sample closely related to Schlesneria paludicola MPL7T (Fig. 1). However, the rpoB-based detection failed as a tentative culture of Planctomycetes. Antibiotic treatment was changed to ceftazidime and teicoplanin, and the patient became apyretic 10 days later and was discharged. The patient achieved complete remission, but her leukemia relapsed 12 months later.

Planctomycetes are a group of fastidious Gram-negative bacteria related to Verrucomicrobia and Chlamydia in a so-called “PVC (Planctomycetes-Verrucomicrobia-Chlamydiae) superphylum” (1). These organisms are mainly environmental bacteria found in water sources, but recent searches detected DNA sequences specific for Planctomycetes in the digestive tract of healthy individuals (2). These bacteria may exhibit intracellular compartments, including a nucleus, but the cell wall lacks peptidoglycan, and consequently, Planctomycetes are naturally resistant to β-lactams and glycopeptides (3). Furthermore, they generally exhibit broad-spectrum resistance to gentamicin, rifampin, and chloramphenicol (3). Planctomycetes are not detected using common molecular amplification techniques (1, 4). Moreover, Planctomycetes are such fastidious organisms, that no isolate has been cultured from humans, and 9/31 (29%) known Planctomycetes species have no cultured representative. Moreover, specific growth factors are not known, most isolates being progressively enriched from environmental specimens, which also serve as the culture medium.

For patients suffering from aplastic neutropenia, determination of the infectious origins of sepsis remains an unresolved challenge (5, 6). The implementation of new tests such as the SeptiFast test and Ibis technology has not significantly changed how often the causative agent is identified (7). We thought that aplastic patients in whom fever was resistant to antibiotics may have infection due to unusual, naturally antibiotic-resistant bacteria, such as Planctomycetes. Using specifically designed primers, we identified Planctomycetes in human feces (2). Here, we tested these primers on blood samples of febrile patients recruited from the regional center for cancer treatment, meeting the following criteria: (i) chemotherapy for acute leukemia or allogeneic stem cell transplantation, (ii) neutrophil levels below 0.5 g/liter, and (iii) persisting fever 3 days after broad-spectrum antibiotic treatment. Additionally, 100 healthy blood donors were included as controls. Blood DNA was amplified using “broad-spectrum” bacterial 16S rRNA PCR primers (4), and primers targeting the Planctomycetes 16S rRNA gene (2, 3). Positive detections were confirmed by PCR sequencing of the Planctomycetes rpoB gene. One negative control was used for every 8 samples, and no positive controls were used. The study was approved by the local IFR48 Ethic Committee.

This study is the first to report the recovery of Planctomycetes DNA from a normally sterile human tissue. We detected Planctomycetes DNA in the blood of patients suffering from fever, rash, pneumonia, diarrhea, and neutropenia. The association of these signs and symptoms in unusual in such patients—in particular, micronodular pneumonia is rarely seen and is caused by few pathogens, with detection negative in the two patients reported here. The detection of the Planctomycetes 16S rRNA gene remained negative in the 100 controls and was positive in the two patients reported among the 100 specimens collected from 83 patients over 13 months. We believe that there is a link between Planctomycetes and this clinical situation. In particular, the fact that three specific PCRs were positive directly from blood suggests that a high concentration of Planctomycetes DNA was circulating in these patients. It is possible that patients with neutropenia are more likely to have circulating Planctomycetes than other patients, and the presence of the Planctomycetes DNA may be incidental. However, as Planctomycetes is part of the gut flora, it would not be surprising if Planctomycetes species were translocated from the gut and caused bacteremia in immunocompromised hosts receiving broad-spectrum antibiotics. Also, tropism for the lung would be congruent with the phylogenetic proximity of Planctomycetes with Chlamydia, a genus also comprising fastidious organisms responsible for pneumonia (1, 8). Several elements still need to be confirmed to show that Planctomycetes are pathogenic to humans, including their direct isolation from blood.

In conclusion, we report the presence of Planctomycetes DNA in blood samples from two neutropenic patients suffering from fever and rash. Planctomycetes have a different cell wall and, consequently, are usually resistant to all β-lactam antibiotics and vancomycin. They are inconsistently susceptible to fluoroquinolones in vitro (3). This susceptibility may have played a role in the favorable evolution of these cases along with the reversal of the neutropenia. Tetracyclines are active against Planctomycetes in vitro and may be a second-line treatment option for these patients (9).