Inquilinus limosus and Cystic Fibrosis

To the Editor: Inquilinus limosus, a new multidrug-resistant species, was reported in 1999 as an unidentified gram-negative bacterium in a lung transplant patient with cystic fibrosis (CF) (1). This species was later characterized by the description of 7 new isolates of I. limosus and 1 isolate of Inquilinus sp (2). Infections and colonizations by I. limosus have been documented mainly in adolescent or adult patients with CF. To date, 8 clinical cases have been described in Germany (3,4), 1 case in the United States (1), 5 cases in France (5), and 1 case in the United Kingdom (6) (Table). Only 1 isolate of Inquilinus sp. has been recovered from blood samples of a patient without CF who had prosthetic valve endocarditis (7). 
 
 
 
Table 
 
Clinical and epidemiologic features of cystic fibrosis (CF) patients with Inquilinus limosus* 
 
 
 
Because this bacterium is not recorded in all commercial identification system databases currently available, a longitudinal study for I. limosus detection with a new real-time PCR assay with a Taqman probe (Applied Biosystems, Foster City, CA, USA), that targets the 16S rRNA gene, has been developed and compared with the culture isolation. Primers il1d (5′-TAATACGAAGGGGGCAAGCGT-3′) and il1r (5′-CACCCTCTCTTGGATTCAAGC-3′) and probe ilProbe (6FAM-GGTTCGTTGCGTCAGATGTGAAAG-TAMRA), which were used in this study, were designed on the basis of multisequence alignment of all I. limosus 16S rDNA sequences available in the GenBank database. 
 
To confirm specificity, the primers and probe were checked by using the BLAST program (www.ncbi.nlm.nih.gov/blast/Blast.cgi) and also by using suspension of several bacteria recovered habitually in patients with CF. For sensitivity of the Taqman PCR assay (Applied Biosystems), the minimal CFU detectable was 2 CFU/PCR. From January 2006 through June 2007, 365 sputum samples recovered from 84 children and 61 adults with CF and 71 sputum samples recovered from 54 patients without CF were screened blindly for I. limosus. By using our real-time PCR, we detected 9 I. limosus-positive samples from 4 patients with CF (Table); 8 of these samples were also culture positive. However, all sputum samples from patients without CF were negative. In 1 patient (Table, case 17), I. limosus was detected by using real-time PCR 3 months before the culture was positive. Retrospectively, the patient’s medical file was rechecked and his clinical respiratory condition worsened briefly at that stage, which indicates an infection by this bacterium. Thus, in our study, the incidence of I. limosus was 2.8% (4.9% for adults with CF and 1.2% for children with CF). The incidence of Burkholderia cepacia complex during the same period and in the same patients was 2.1% (3 adults with CF were positive, data not shown). 
 
The genus Inquilinus belongs to the α-Proteobacteria; the genus Azospirillum is the most closely related bacteria (2). This cluster of bacteria contains several strains that are able to grow under saline conditions and in biofilms (8,9). The mucoid phenotype of I. limosus may contribute to its colonization and resistance to many antimicrobial drugs. Recently, the exopolysaccharides (EPS) produced by I. limosus were studied. The authors indicated that I. limosus produces mainly 2 EPSs that exhibit the same charge per sugar residue present in alginate, the EPS produced by Pseudomonas aeruginosa in patients with CF. This similarity may be related to common features of the EPS produced by these 2 opportunistic pathogens that are related to lung infections (10). Transmission of I. limosus between patients with CF is not known, but in the report from Chiron et al., 1 of the 5 patients with I. limosus had a brother who had never been colonized with this bacterium despite living in the same home (5). Schmoldt et al. reported that 3 patients were treated in the same outpatient CF clinic during overlapping time periods and each patient was infected/colonized by an individual I. limosus clone, which suggests that there was no transmission among these patients (4). This bacterium has been recovered mainly from sputum of adolescents (mean age 17 ± 6.47 years, range 8–35), except in our study with a 2-year-old boy, which suggests that this emerging bacterium may be hospital acquired, as recently suggested (7). Because this bacterium is multiresistant to several antimicrobial drugs, particularly colistin, which is widely used for treatment for P. aeruginosa colonization (as was the case for our 4 patients), we hypothesize that this bacterium is selected during the evolution of the disease. 
 
We have developed a real-time PCR molecular method that is faster and easier than amplification-sequencing for prompt detection and accurate identification of I. limosus with good specificity and sensitivity. By using this screening assay, we identified 4 additional cases of patients with CF who were also infected with this bacterium, including a 2-year-old child. In addition, by using this technique, we were able to detect I. limosus in a patient with deteriorated respiratory function 3 months before the culture-based isolation, indicating that a low bacterial load, insufficient for being isolated in culture, can be detected by PCR in the lower respiratory tract of patients with CF.


Inquilinus limosus and Cystic Fibrosis
To the Editor: Inquilinus limosus, a new multidrug-resistant species, was reported in 1999 as an unidentifi ed gram-negative bacterium in a lung transplant patient with cystic fi brosis (CF) (1). This species was later characterized by the description of 7 new isolates of I. limosus and 1 isolate of Inquilinus sp. (2). Infections and colonizations by I. limosus have been documented mainly in adolescent or adult patients with CF. To date, 8 clinical cases have been described in Germany (3,4), 1 case in the United States (1), 5 cases in France (5), and 1 case in the United Kingdom (6) (Table). Only 1 isolate of Inquilinus sp. has been recovered from blood samples of a patient without CF who had prosthetic valve endocarditis (7).
Because this bacterium is not recorded in all commercial identifi cation system databases currently available, a longitudinal study for I. limosus detection with a new real-time PCR assay with a Taqman probe (Applied Biosystems, Foster City, CA, USA), that targets the 16S rRNA gene, has been developed and compared with the culture isolation. Primers il1d (5′-TAATACGAAGGGGGCAAGCGT-3′) and il1r (5′-CACCCTCTCTTGGA TT CAAGC-3′) and probe ilProbe (6FAM-GGTTCGTTGCGTCAGAT GTGAAAG-TAMRA), which were used in this study, were designed on the basis of multisequence alignment of all I. limosus 16S rDNA sequences available in the GenBank database.
To confi rm specifi city, the primers and probe were checked by using the BLAST program (www.ncbi.nlm. nih.gov/blast/Blast.cgi) and also by using suspension of several bacteria recovered habitually in patients with CF. For sensitivity of the Taqman PCR assay (Applied Biosystems), the minimal CFU detectable was 2 CFU/PCR. From January 2006 through June 2007, 365 sputum samples recovered from 84 children and 61 adults with CF and 71 sputum samples recovered from 54 patients without CF were screened blindly for I. limosus. By using our real-time PCR, we detected 9 I. limosus-positive samples from 4 patients with CF (Table); 8 of these samples were also culture positive. However, all sputum samples from patients without CF were negative. In 1 patient (Table, case 17), I. limosus was detected by using real-time PCR 3 months before the culture was positive. Retrospectively, the patient's medical fi le was rechecked and his clinical respiratory condition worsened briefl y at that stage, which indicates an infection by this bacterium. Thus, in our study, the incidence of I. limosus was 2.8% (4.9% for adults with CF and 1.2% for children with CF). The incidence of Burkholderia cepacia complex during the same period and in the same patients was 2.1% (3 adults with CF were positive, data not shown).
The genus Inquilinus belongs to the α-Proteobacteria; the genus Azospirillum is the most closely related bacteria (2). This cluster of bacteria contains several strains that are able to grow under saline conditions and in biofi lms (8,9). The mucoid phenotype of I. limosus may contribute to its colonization and resistance to many antimicrobial drugs. Recently, the exopolysaccharides (EPS) produced by I. limosus were studied. The authors indicated that I. limosus produces mainly 2 EPSs that exhibit the same charge per sugar residue present in alginate, the EPS produced by Pseudomonas aeruginosa in patients with CF. This similarity may be related to common features of the EPS produced by these 2 opportunistic pathogens that are related to lung infections (10). Transmission of I. limosus between patients with CF is not known, but in the report from Chiron et al., 1 of the 5 patients with I. limosus had a brother who had never been colonized with this bacterium despite living in the same home (5). Schmoldt et al. reported that 3 patients were treated in the same outpatient CF clinic during overlapping time periods and each patient was infected/colonized by an individual I. limosus clone, which suggests that there was no transmission among these patients (4). This bacterium has been recovered mainly from sputum of adolescents (mean age 17 ± 6.47 years, range 8-35), except in our study with a 2-year-old boy, which suggests that this emerging bacterium may be hospital acquired, as recently suggested (7). Because this bacterium is multiresistant to several antimicrobial drugs, particularly colistin, which is widely used for treatment for P. aeruginosa colonization (as was the case for our 4 patients), we hypothesize that this bacterium is selected during the evolution of the disease.
We have developed a real-time PCR molecular method that is faster and easier than amplifi cation-sequencing for prompt detection and accurate identifi cation of I. limosus with good specifi city and sensitivity. By using this screening assay, we identifi ed 4 additional cases of patients with CF who were also infected with this bacterium, including a 2-year-old child. In addition, by using this technique, we were able to detect I. limosus in a patient with deteriorated respiratory function 3 months before the culture-based isolation, indicating that a low bacterial load, insuffi cient for being isolated in culture, can be detected by PCR in the lower respiratory tract of patients with CF.

Splenic Rupture and Malignant Mediterranean Spotted Fever
To the Editor: Mediterranean spotted fever (MSF) is a Rickettsia conorii infection endemic to the Mediterranean. In this case, a 55-year-old man was referred to the Necker-Enfants Malades Hospital, Paris, France, for fever, myalgia, and hypotensive shock. The patient had been in Southern France (Montpellier) 6 days before symptom onset and had been bitten by a tick on the left hand. Four days later, he reported fatigue, fever (39°C), and myalgia. His medical history showed polycystic kidney disease, which had necessitated hemodialysis and a kidney transplant. He was receiving ongoing treatment with an immunosuppressive regimen of cyclosporine, prednisolone, and tacrolimus; his baseline hemoglobin level was 15 g/dL, and creatinine level was 230 μmol/L.
At admission, the patient's temperature was 39.5°C, blood pressure 55/40 mm Hg, and heart rate 104 beats/min. Physical examination showed a diffusely tender abdomen with guarding, no hepatosplenomegaly, a nontender renal transplant, and no lymphadenopathy. Results of cardiovascular, respiratory, and neurologic examinations were unremarkable. A diffuse maculopapular cutaneous eruption was noted on the lower limbs; no eschar was detected.
A computed tomographic scan showed hemoperitoneum secondary to a ruptured subcapsular splenic hematoma (online Appendix Figure, available from www.cdc.gov/EID/content/ 14/6/995-appG.htm), and an emergency splenectomy was performed. Histopathologic evaluation of the spleen showed white pulp atrophy; the red pulp indicated congestion and illdefi ned nodules, varying in size and comprising macrophages, polymorphonuclear neutrophils, and necrotic cells (Figure, panels A, B). Skin biopsy of the macular eruption on day 2 demonstrated a leukocytoclastic vasculitis with nonocclusive luminal thrombi in the dermal capillaries (Figure, panel C).
Universal 16S rRNA gene PCR amplifi cation on spleen and skin tissue samples and direct sequencing identifi ed an R. conorii-specifi c 16S rRNA sequence match. We confi rmed this by using primers for gltA and ompA specifi c for R. conorii. Immunohistochemical staining demonstrated Rickettsia in endothelial cells and macrophages in the spleen and skin (Figure, panels D-F). Blood culture, skin biopsy specimens, and splenic tissue cultures were subsequently R. conorii positive. Doxycycline therapy (100 mg intravenously twice a day) was instituted at day 2 because rickettsiosis was suspected. The patient dramatically improved within 72 hours and remained well 36 months after diagnosis.
MSF is a rickettsiosis belonging to the tick-borne SFG caused by R. conorii, an obligate intracellular bacteria transmitted by the dog tick Rhipicephalus sanguineus. Endemic to Mediterranean countries, MSF generally results in a benign febrile illness accompanied by a maculopapular rash, myalgia, and local black eschar at a tick bite inoculation site. A minority of persons seeking treatment display a malignant form, which results from disseminated vasculitis associated with increased vascular permeability, thrombus-mediated vascular occlusion, and visceral perivascular lymphohistiocytic infi ltrates (1). Focal thrombi have been identifi ed in almost all organs of patients with fatal cases. Manifestations of MSF include neurologic involvement, multi-organ failure, gastric hemorrhage, and acute respiratory distress syndrome; the case-fatality rate is 1.4%-5.6%.