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Cellulomonas denverensis is a small and thin gram-positive rod-shaped bacterium that was proposed as a new species in 2005. Here we report a female case of acute cholecystitis and sepsis in which C. denverensis was determined to be causative.
On 13 April 2008, a previously healthy female patient, 82 years old, presented in our hospital with a sudden upper abdominal pain. Pancreas swelling and increased adipose tissue density around the pancreas were observed by abdominal computed tomography (CT). Blood tests showed an elevated amylase level of 2,255 IU/liter. A diagnosis of acute pancreatitis was made and its grade was judged as 1 out of 4 points (2 points for vital signs plus 1 point for CT grade plus 1 point for age). She was admitted in the high care unit as a severe case of acute pancreatitis. After receiving medical treatment for acute pancreatitis, her abdominal pain disappeared, her serum amylase level was restored, and the CT findings improved. A bile duct stone was not detected, and the pancreatic duct was normal as determined by magnetic resonance cholangiopancreatography, so liquid food was started on 24 April 2008. However, after 1 week, she had a fever of 38°C and right hypochondralgia. In addition, gallbladder swelling with a gallstone at the cervix was found via ultrasonography and abdominal CT, giving a diagnosis of acute cholecystitis. Laboratory tests showed leukocytosis with 94% neutrophils and elevated C-reactive protein (Table (Table1).1). Percutaneous transhepatic gallbladder puncture was carried out, and 255 ml of green bile fluid was drained. Two days later, abdominal pain, a fever of 38°C, and even rebound tenderness appeared. The diagnosis of peritonitis was made, and the patient was subjected to an emergency cholecystectomy.
To examine pathogenic bacteria, blood and bile cultures were performed. Ten milliliters of peripheral blood was injected into FA and FN bottles (Sysmex bioMerieux, Tokyo, Japan), cultured by BacT/Alert 3D (Sysmex bioMerieux) for up to 1 week, and the supernatant was collected from a BacT/Alert 3D-positive sample. Bile and supernatant from the blood culture were then cultured at 37°C on sheep blood agar (Nissui Pharmaceutical, Tokyo, Japan), chocolate agar (Eiken Kagaku, Tokyo, Japan), and ABHK agar (Nissui Pharmaceutical) under aerobic or anaerobic conditions. In aerobic cultures of the bile sample, bacterial growth was observed on sheep blood agar and chocolate agar at 24 h. In blood cultures, when an inflammatory symptom existed, bacterial growth was observed in the blood culture bottle at 2 days after culturing. After culture supernatants were cultured on the above-mentioned plate under the above-mentioned conditions, bacterial growth was observed at 24 h. The growing bacterial colony showed white and smooth microcolonies after 24 h and pale yellow and smooth colonies after 48 h (Fig. (Fig.1).1). In the anaerobic culture, bacterial growth was observed in neither the blood sample nor the bile sample. The isolated bacterium was stained with the Gram-staining solution neo-B&M Wako (Wako Pure Chemical Industries, Osaka, Japan) and examined by a light microscope, which showed a small and thin gram-positive rod (Fig. (Fig.1).1). However, excess decolorization in the staining process tended to change it to gram negative. The isolated bacterium examined by API Coryne (Sysmex bioMerieux) was identified as a Cellulomonas species (99.8% identical). In addition, PCR amplification and sequencing analyses of the 16S rRNA gene were performed. Sequencing analysis was carried out using a GenBank BLAST search (National Center for Biotechnology, Bethesda, MD). Sequence editing and phylogenetic analyses were performed with ClustalW. The sequence of the 16S rRNA gene differed by 10 bp (1,412 bp over the entire 1,422-bp fragment; 99.3% identical) from that of Cellulomonas denverensis type strain W6929 (ATCC BAA-788 or DSM 15764). Since bacteria with a concordance rate of the base sequence of more than 98.7% are regarded as identical (11), the isolated bacterium was identified as C. denverensis (GenBank accession no. AB491158). In comparison with type strain W6929, the colony formation rate was similar to that of the isolated bacterium at 24- and 48-h cultures, but the colony protruded more markedly than that of the isolated bacterium at the 48-h culture. The bacterial cells of type strain W6929 were morphologically smaller than those of the isolated bacterium (Fig. (Fig.11).
Antimicrobial sensitivities were determined by a previously described broth microdilution method with cation-supplemented Mueller-Hinton broth (Eiken Kagaku, Tokyo, Japan). The antimicrobial agents tested were piperacillin, cefazolin, cefotiam, cefotaxime, ceftazidime (CAZ), cefepime, cefpodoxime (CPD), flomoxef (FMO), sulbactam-ampicillin (SAM), aztreonam (ATM), imipenem (IPM), meropenem (MEM), gentamicin, amikacin, minocycline, fosfomycin (FOM), sulfamethoxazole-trimethoprim (SXT), and levofloxacin (LVX). The plates were incubated at 35°C for 48 h. Currently, there are no interpretative MIC breakpoints for isolates of this genus. Therefore, only the MIC of each antibiotic was determined. Penicillin and cephem antibiotics comparatively inhibited bacterial growth in low concentrations except for CAZ and CPD. Carbapenem antibiotics more effectively inhibited bacterial growth in low concentrations. ATM, FOM, SXT, and LVX showed no inhibitory effect in high concentrations of these drugs. Type strain W6929 had a tendency to grow in high concentrations of cephem and carbapenem antibiotics compared with the isolated bacterium. The others showed almost the same drug sensitivities (Table (Table22).
Because the patient was affected with sepsis and was predicted to have a progression of further systemic infection, SAM was administrated (Unasyn; Pfizer Inc., New York, NY) by intravenous infusion at 6 g/day for 6 days from 3 May 2008. Thereafter, laboratory data improved and she satisfactorily recovered.
This is the first case report of acute cholecystitis with sepsis caused by C. denverensis. Cellulomonas species, which are known as serious opportunistic pathogens of humans, sometimes exist in various places in the environment, including trees and the atmosphere (4, 5, 10). Until now, only clinical cases of infectious disease caused by C. denverensis have been reported (2, 7). Moreover, the detection of Cellulomonas species from a clinical sample is extremely rare. In this case of severe cholecystitis with a gallstone, only C. denverensis was demonstrated in the bile and blood samples. Consequently, the inflammatory response is thought to have been caused by a C. denverensis infection.
Acute cholecystitis is commonly caused by bile duct obstruction and subsequent bacterial infection (3). Acute cholecystitis with a gallstone is generally accompanied by bacterial infection in many cases. Responsible bacteria are detected by a bile culture in 40 to 75% of patients with acute cholecystitis (6, 8), and in some cases the infection progresses to sepsis. In this case, since only C. denverensis was detected from the bile and blood samples, we concluded that the inflammation of the gallbladder and sepsis were caused by infection with C. denverensis. Because Cellulomonas species are reported to exist in the environment, we speculated that the patient contracted them from the environment and held them in the intestinal tract. In the treatment of acute cholecystitis, antibiotics are generally used against enteric bacteria. However, as in this case, the possibility of disease due to unpredictable bacteria should always be taken into consideration. In the antimicrobial sensitivity results, carbapenem antibiotics (IPM, MEM), which are frequently used in empirical treatment, inhibited the bacterial growth at low concentrations, but the bacterium grew at high concentrations of CAZ and CPD. Since it is also reported that MICs of cephem and quinolone antibiotics for C. denverensis are higher than those for other Cellulomonas species (2), we should carefully monitor the data of antimicrobial sensitivity in this bacterial infection.
Although the colony forms and bacterial cells had some differences, the colony formation rate was similar to that of the isolated bacterium in 24- and 48-h cultures in comparison with type strain W6929. It is therefore thought that the colony formation rate in C. denverensis is an important property for identification. There were no reports of detection of this bacterium before 2005, but it may have been misidentified as another gram-positive or gram-negative bacterium, because it can present with variable Gram stain results. Although enteric bacteria, such as Escherichia coli, Klebsiella pneumoniae, Enterococcus species, and anaerobes, are frequently detected in biliary tract infections in many cases (1, 9), clinical microbiologists should keep in mind the possibility of acute cholecystitis caused by unusual organisms, such as Cellulomonas species.
Published ahead of print on 5 August 2009.