Incidence of Bilophila wadsworthia in appendiceal, peritoneal and fecal samples from children

Authors


In the course of their studies on appendicitis, Baron and coworkers recovered a new anaerobic rod, which was isolated from about 50% of the appendiceal samples from patients with complicated appendicitis and which was described in 1989 as Bilophila wadsworthia [1]. The lower intestinal tract seems to be the natural habitat of B. wadsworthia, where it was recovered from 60% of the subjects, with counts ranging from 3 × 103 to 2 × 108/g stool. Additionally, B. wadsworthia was isolated from 4% of saliva samples and from 3% of vaginal specimens from healthy volunteers [2]. Quite recently, B. wadsworthia has been described also in other infectious processes, such as scrotal abscess, mandibular osteomyelitis, pleural empyema and bacteremia, suggesting that B. wadsworthia is capable of acting as a pathogen [3–5]. There are no reports about the occurrence of B. wadsworthia in clinical specimens or in feces from children.

Included in this study were 229 children aged 3 months to 15 years, admitted to the department of pediatric surgery (University of Tübingen) during a 12-month period (6 January 1994 to 5 January 1995) because of acute abdominal pain in the right hypogastrium. Stool samples were taken from all patients. The main cause of abdominal pain was gastroenteritis (136 cases), but 51 of the children underwent appendectomy; in these patients the appendices were also studied. The appendices were divided lengthwise under sterile conditions for histopathologic and microbiological examination. According to the histopathologic findings and the visual assessment at surgery, seven cases were diagnosed as chronic appendicitis, 26 as acute or phlegmonous appendicitis and 14 as complicated (gangrenous or perforated) appendicitis. In four cases appendectomy was performed in the presence of abdominal pathology other than appendicitis (Table 1). In cases with suspicious intraoperative findings, peritoneal swabs were also examined (38 cases).

Table 1.  Data on patients undergoing surgery
 
 NegativeChronicAcuteComplicated
No.472614
Sex (M/F)2/24/314/127/7
Mean age (years)8.211.710.09.3
Mean WBC count at admission (per μL)1019093851513417820
Mean temperature at admission (°C)37.237.637.838.2

In stool samples the occurrence of B. wadsworthia only was investigated; from the appendix tissue only anaerobic isolates were recovered; and in peritoneal swabs aerobic and anaerobic organisms were isolated. The appendiceal samples were incubated on two Bacteroides–bile–esculin (BBE), brain–heart infusion (BHI) and kanamycin–vancomycin (KV) agar plates each. The peritoneal swab was used to inoculate plates of Columbia blood agar, Endo agar, BBE, BHI and KV agar and an enrichment broth. From each stool sample one loop was inoculated on two BBE plates. Media were incubated anaerobically (Anaerocult A, Merck) at 37°C for at least 7 days. Blood and Endo agar plates were incubated aerobically at 37°C for at least 48 h. The identification of B. wadsworthia was by the following methods: Gram stain; detection of bile resistance; positive catalase reaction (5% H2O2); short-chain fatty acid analysis (detection of acetate) by gas–liquid chromatography; negative reactions for glucose fermentation, esculin hydrolysis and indole production; positive reactions for H2S production and nitrate reductase; and testing for urease activity.

Cultures of the appendices yielded 293 strains of more than 44 different anaerobic species (Table 2). Bacteroides fragilis (62.7%) and B. wadsworthia (50.9%) were the anaerobic species most often isolated from appendiceal samples, followed by Bacteroides ovatus, Peptostreptococcus anaerobius, Peptostreptococcus micros and Prevotella intermedia. The results presented showed marked similarities between non-inflamed and chronically inflamed appendices, in contrast to findings in acute and complicated appendicitis with respect to species and number of species of anaerobes recovered. Non-acutely inflamed appendices harbored on average four to five different anaerobic species, whereas in acute or complicated cases more than six different anaerobic species were identified. B. wadsworthia was recovered from 25% and 28.5%, respectively, of the patients with non-infected and chronically inflamed appendices (mean 27.7%), whereas 53.8% and 64.2%, respectively, of the patients with acute and complicated appendicitis (mean 57.5%) yielded growth of B. wadsworthia. Two recent studies which focus on the bacteriology of appendicitis recovered B. wadsworthia in 46% of the tissue samples, as well as Bacteroides fragilis, Bacteroides thetaiotaomicron and P. micros, with a mean recovery rate of more than seven anaerobes in complicated appendicitis cases [2,6]. Baron and coworkers reported that the recovery rate of B. wadsworthia increased according to the grade of appendicitis [2].

Table 2.  Anaerobic bacterial species isolated from appendices according to pathologic diagnosis
 Type of appendicitis
OrganismNegative (n=4)Chronic (n=7)Acute (n=26)Complicated (n=14)All (n=51)
Bilophila wadsworthia1214926
Bacteroides fragilis13161232
B. thetaiotaomicron2210317
B. ovatus3414223
B. uniformis1311318
B. vulgatus034310
B. distasonis214310
B. caccae11316
B. stercoris00426
B. splanchnicus00156
B. eggerthii00123
B. merdae00202
B. putredinis00022
Dialister pneumosintes00314
Prevotella intermedia138719
P. zoogleoformans00101
P. oris00101
P. buccae01001
Porphyromonas gingivalis00101
P. macacae00101
Fusobacterium nucleatum017210
F. necrophorum00123
F. varium00101
Campylobacter rectus00404
Peptostreptococcus anaerobius3011822
P. micros1010819
P. prevotii00101
Peptostreptococcus spp.00101
Ruminococcus albus00101
Veillonella parvula00101
Clostridium clostridioforme00314
C. ramosum00011
C. innocuum00011
C. difficile10001
Lactobacillus catenaformis02002
L. minutus00101
Eubacterium lentum016411
E. saburreum10001
Eubacterium spp.005510
Actinomyces odontolyticus01001
Bifidobacterium bifidum01102
B. infantis01102
B. angulatum00101
B. adolescentum00101
B. breve01001
B. catenulatum01001

B. wadsworthia was recovered relatively often together with anaerobic species which are considered as pathogens of appendicitis [6–9]: 60% of all Bacteroides fragilis, 76.9% of all Bacteroides thetaiotaomicron and 72.7% of all P. micros isolates were recovered together with B. wadsworthia. Associations with P. intermedia (66.6%) and P. anaerobius (63.1%) were also frequent. In contrast, only 37.5% of Bacteroides ovatus and 35.7% of Bacteroides uniformis isolates were detected together with B. wadsworthia.

From 38 patients a peritoneal swab was available. In cultures from 24 swabs, no growth occurred. Peritoneal swabs taken during complicated appendicitis revealed a higher occurrence of isolates (60%) than during other stages of infection (0%, 25%, 38.3%). B. wadsworthia was recovered in three peritoneal specimens and was only isolated in acute and complicated appendicitis together with Escherichia coli, P. anaerobius and Bacteroides fragilis.

Of the 229 stool samples studied, 135 (58.9%) yielded B. wadsworthia. Although B. wadsworthia was isolated even from the youngest children in the study, an age-dependent increase was observed in the recovery rate from 42% (≦2 years) up to 64–69.2% (>9 years), although this was not statistically significant. A comparison of its presence in feces and appendiceal samples gives an 82.4% concordance.

The frequent occurrence of B. wadsworthia in inflamed appendices from children and adults, its association with other known anaerobic pathogens and its recovery from other infectious processes are suggestive of pathogenic properties in this species.

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