Two cases of bacteremia due to an unusual pathogen , Comamonas testosteroni in Iran and a review literature

Here we describe two cases of bacteremia caused by Comamonas testosteroni in two malignant patients, a 10-year-old boy with brain medulloblastoma and a 19-year-old girl with osteosarcoma admitted in the same hospital at short intervals. This is the first report in Iran on this low inherent virulence organism as a human pathogen.


Introduction
Comamonas testosteroni is characterized as a Gram-negative, aerobic, motile and non-spore forming bacterium belonging to the Comamonadacae family.Their arrangements on Gram staining usually are in singles or pairs [1].The organism has been isolated from a wide range of natural habitats such as water, soil, and plants, so it is recognized as an environmental organism.In spite of its uncommon pathogenesis, there are a few reports on the aggressive manner of it as an opportunistic pathogen.
We isolated this organism in a short time from the blood cultures of two patients with malignancy that presented with fever and neutropenia.The first one was diagnosed with brain medulloblastoma and the other with osteosarcoma.

Case reports
The first case was a 10-year-old boy who was hospitalized in the hematological ward of Amir Oncology Hospital, Shiraz, Iran, in September 2010.He had been diagnosed one year previously with brain medulloblastoma and had received six courses of chemotherapy after surgery.He was in good health without any serious condition until November 2010, when he was found febrile (38.5ºC) on examination.
Subsequently, blood and urine cultures were taken.Wide-spectrum antibiotics, ciprofloxacin (10 mg/kg/day for 21 days) and amikacin (15 mg/kg/day for 21 days), were also received intravenously.Laboratory findings were as follows: white blood cell 3700 cell/mm3; hemoglobin 9.1g/dl; platelets 114000 cell/mm3; erythrocyte sedimentation rate 91/h; and creactive protein three plus.Lactate dehydrogenase, creatine phosphokinase, calcium, alkaline phosphatase and urine analysis were within normal range.Aspartate aminotransferase was 78 u/l, phosphorous 7.2 mg/dl, and urine culture showed no growth but blood culture was positive.During this hospital stay (i.e., September to November) he was placed in different wards.
The second patient was a 19-year-old girl who had been diagnosed with osteosarcoma at the age of 15.She had since then relapsed following surgery and five courses of chemotherapy, and for the past three months she had developed dry cough.Evaluation showed tumor metastasis to the lungs.She was admitted to the emergency ward of Amir Oncology Hospital in November 2010 with septic shock and after two days she was placed in the same room where the first case was hospitalized.On examination she had drowsiness, a bedsore on the buttock, low blood pressure (60 systolic mm Hg), and high-grade fever (39.8ºC).Blood and urine samples were taken and cultured.Broad-spectrum antibiotics, vancomycin (60 mg/kg/day for 14 days) and imipenem (100 mg/kg/day for 14 days), were taken intravenously, and oral ciprofloxacin (30 mg/kg/day for three weeks) was prescribed upon discharge.She also received granulocyte colony stimulating factor for severe neutropenia.The laboratory data were as follows: white blood cell 300 cell /mm3; hemoglobin 9.1g/dl; platelets 82000 cell/mm3; blood urea nitrogen 13mg/dl; uric acid 5.4mg/dl; lactate dehydrogenase 753u/l.Calcium, phosphorous, sodium, potassium and glucose were in normal range.Both blood cultures were positive for Comamonas testosteroni.
The two patients presented here were cured of the bacteremia after appropriate treatment and discharged from hospital with normal white blood cell counts.

Sampling
Blood samples (1-3 ml from a 10-year-old boy and 8-10 ml blood from a 19-year-old girl) were inoculated into blood culture bottles (BD BACTEC peds plus/F, BD BACTEC plus + Aerobic/F, Shannon, Ireland).One droplet of the positive growth blood cultures (BD BACTEC 9240 Blood Culture System 02390, Becton, Dickinson and Company Franklin Lakes, NJ, USA) was placed on sheep blood agar, chocolate blood agar and MacConkey agar plates and pricked by a sterile needle.Sheep and chocolate blood agar plates were incubated in micro aerobic conditions prepared by a candle jar at 37ºC.MacConkey agar plates were incubated in aerobic conditions at 37ºC for 24 to 48 hours.Gram staining was performed for both single colonies and patients' blood samples.

Biochemical methods
The isolated bacteria were tested by catalase and oxidase reactions.Motility and potency of the bacterium for indole and hydrogen sulfate production were checked on sulfur-indole-motility agar.Because no acid production occurred in the OF-glucose medium, complementary identical tests were performed using an api 20 NE identical kit (Biomérieux, Marcy l'Etoile, France), according to the manufacturer's instructions.

Antimicrobial tests
Antimicrobial susceptibility tests were performed based on the Kirby Bauer disk-diffusion method on Mueller Hinton agar plates (Merck, Darmstadt, Germany).Antibiotic disks (Mast Diagnostics Co, Merseyside, United Kingdom) were selected on the basis of United States Federal Drug Administration approved antimicrobial agents that should be considered for routine testing and reporting on nonfastidious organisms.

Results
For both cases pink pigmented colonies with a mucoid and bulgy surface and without hemolysis appeared on the blood and chocolate blood agar plates after 48 hours of incubation at 37ºC.Bacteria also grew on MacConkey agar plates.
Gram staining indicated dumpy Gram negative bacilli mostly revealed in pair arrangement.Catalase and oxidase reactions were negative and positive, respectively.This bacterium was motile and able to reduce nitrate.Due to its disability for glucose fermentation OF-glucose media, complementary diagnostic tests were done using an api 20 NE identical kit (Biomérieux, Marcy l'Etoile, France), according to the manufacture's protocol.Finally, this strain was identified as Comamonas testosteroni with high certainty.

Discussion
In 1987, Tamaoka et al. replaced the name of Pseudomonas testosteroni with a new genus known as Comamonas testosteroni [2].This genus is a motile bacterium by a polar tuft of up to six distinctive and long wavelength flagella [3].Its name is derived due to its ability to decompose testosterone as the carbon source [4,5].Comamonas testosteroni, referred to as an environmental organism, is a commensal microorganism that prefers niches, including soil and water.There are some reports indicating its isolation from activated sludge [6].In spite of its wide environmental distributions, there are a few reports on its involvement in human infections.Copper et al. (2005) suggested that the organism survives for extended periods of time with diverse nosocominal environments, and most of the reported infections by this organism are community acquired [7].It has also been isolated from some hospital devices such as intravenous lines and water contained in the  [25] humidifier reservoirs used in respiratory treatment [8].
There was only one report of the infective role of Pseudomonas testosteroni in a human until 1987 [9].
Here we collected all the sites of infection related to Comamonas testosteroni from 1987 up to date (Table 1).The collected data are divided on the basis of the patient's age, sex, and sites of infection, predisposing factors, antibiotic treatment, and outcome.Considering the low virulence potency of Comamonas spp., the majority of the reported literature focused on two species, Comamonas acidovorans and Comamonas testosteroni.Most Comamonas acidovorans strains were isolated from ocular sites [10][11][12] , followed by urinary track, central venous catheter, indwelling catheter, tricuspid valve in a case of endocarditis, and acute suppurative otitis draining pus [13][14][15][16][17].However, in 33 reported cases of Comamonas testosteroni including the two present cases, the most common isolation sites were the bloodstream in 13 cases, followed by the abdominal cavity in 10 cases mainly due to secondary dissemination after perforated appendix or other anatomic abnormalities in the gastrointestinal tract; three cases from cerebrospinal fluids; one case of the organism isolated from the embryonic cord of a stillborn infant of an intravenous drug abuse mother; one isolation from an ocular site; one from urine; one from a patient with immunodeficiency syndrome as an agent of pneumonia; and one from the infection site of an animal bite.The infection site of one last case is missing (Table 1).All were responsive to antibacterial treatment except for the three cases consisting of two newborn and stillborn infants and a case of meningitis in a homeless man, [18,19].Taking into account the reported studies, it seems that the strains of Comamonas testosteroni show more sensitivity to common antibiotics than Comamonas acidovorans.
The two present cases are the first reports of the role of Comamonas testosteroni in bacteremia in Iran.Therefore, our two cases are the 32nd and 33rd reported cases on the infection since then.Having detected two positive blood cultures in a two-day interval with an uncommon pathogen microorganism, we became suspicious of a probable common source and consequently investigations found a direct contact between the two patients who were admitted for a few hours at the same time in the same room.On the basis of the two patients' histories and presentation of infections, it seems that the possible source and predisposing factor in the osteosarcoma case might be acquired Comamonas bacteremia from the environment with unknown origin, due to neutropenia.As for the case with brain medulloblastoma, probably inhalation in the same room where the other case with dry cough was hospitalized was responsible.Unfortunately, we could not find any source, and air contact such as respiratory inhalation or through air conditioners, which might have been the source of the infections.Further investigations should be conducted to confirm and consolidate the findings.Finally, we informed the Infection Control Committee of the hospital to take proper disinfecting measures, including isolation of such cases, educating the health staff, and using effective antiseptic methods.

Table 1 .
Summary of reported cases of infection by Comamonas testosteroni