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

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 [1Baron EJ Summanen P Downes J Roberts MC Wexler H Finegold SM Bilophila wadsworthia, gen. nov. and sp. nov., a unique Gram-negative anaerobic rod recovered from appendicitis specimens and human feces.J Gen Microbiol. 1989; 135: 3405-3411PubMed Google Scholar]. 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 [2Baron EJ Bennion R Thompson J et al.A microbiological comparison between acute and complicated appendicitis.Clin Infect Dis. 1992; 14: 227-231Crossref PubMed Scopus (70) Google Scholar]. 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 [3Baron EJ Curren M Henderson G et al.Bilophila wadsworthia isolates from clinical specimens.J Clin Microbiol. 1992; 30: 1882-1884PubMed Google Scholar, 4Finegold SM Summanen P Hunt Gerardo S Baron E Clinical importance of Bilophila wadsworthia.Eur J Clin Microbiol Infect Dis. 1992; 11: 1058-1063Crossref PubMed Scopus (45) Google Scholar, 5Kasten MJ Rosenblatt JE Gustafson DR Bilophila wadsworthia bacteremia in two patients with hepatic abscesses.J Clin Microbiol. 1992; 30: 2502-2503PubMed Google Scholar]. 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 surgeryType of appendicitisNegativeChronicAcuteComplicatedNo.472614Sex (M/F)2/24/314/127/7Mean age (years)8.211.710.09.3Mean WBC countat admission (per μL)1019093851513417820Mean temperatureat admission (°C)37.237.637.838.2 Open table in a new tab 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 [2Baron EJ Bennion R Thompson J et al.A microbiological comparison between acute and complicated appendicitis.Clin Infect Dis. 1992; 14: 227-231Crossref PubMed Scopus (70) Google Scholar,6Bennion RS Baron EJ Thompson JE et al.The bacteriology of gangrenous and perforated appendicitis—revisited.Ann Surg. 1989; 211: 165-171Crossref Scopus (148) Google Scholar]. Baron and coworkers reported that the recovery rate of B. wadsworthia increased according to the grade of appendicitis [2Baron EJ Bennion R Thompson J et al.A microbiological comparison between acute and complicated appendicitis.Clin Infect Dis. 1992; 14: 227-231Crossref PubMed Scopus (70) Google Scholar].Table 2Anaerobic bacterial species isolated from appendices according to pathologic diagnosisType of appendicitisOrganismNegative (n=4)Chronic (n=7)Acute (n=26)Complicated (n=14)All (n=51)Bilophila wadsworthia1214926Bacteroides fiagilis13161232B. thetaiotaomicron2210317B. ovatus3414223B. uniformis1311318B. vulgatus034310B. distasonis214310B. caccae11316B. stercoris00426B. splanchnicus00156B. eggerthii00123B. merdae00202B. putredinis00022Dialister pneumosintes00314Prevotella intermedia138719P zoogleoformans00101P. oris00101P. buccae01001Porphyromonas gingivalis00101P macacae00101Fusobacterium nucleatum017210F. necrophorum00123F. varium00101Campylobacter rectus00404Peptostreptococcus anaerobius3011822P. micros1010819P. prevotii00101Peptostreptococcus spp.00101Ruminococcus albus00101Veillonella parvula00101Clostridium clostridioforme00314C. ramosum00011C. innocuum00011C. difficile10001Lactobacillus catenaformis02002L. minutus00101Eubacterium lentum016411E. saburreum10001Eubacterium spp.005510Actinomyces odontolyticus01001Bifidobacterium bifidum01102B. infantis01102B. angulatum00101B. adolescentum00101B. breve01001B. catenulatum01001 Open table in a new tab B. wadsworthia was recovered relatively often together with anaerobic species which are considered as pathogens of appendicitis [6Bennion RS Baron EJ Thompson JE et al.The bacteriology of gangrenous and perforated appendicitis—revisited.Ann Surg. 1989; 211: 165-171Crossref Scopus (148) Google Scholar, 7Werner H Kunstek-Santos H Schockemöhle C Gündürewa M Bacteroides und Appendizitis.Path Microbiol. 1975; 42: 110-118PubMed Google Scholar, 8Pieper R Kager L Weintraub A Lindberg AA Nord CE The role of Bacteroides fragilis in the pathogenesis of acute appendicitis.Acta Chir Scand. 1982; 148: 39-44PubMed Google Scholar, 9EIhag KM Alwan MH Al-Adnani MS Sherif RA Bacteroides fragilis is a silent pathogen in acute appendicitis.J Med Microbiol. 1986; 21: 245-249Crossref PubMed Scopus (15) Google Scholar]: 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.