Identification of intracellular bacteria in adenoid and tonsil tissue specimens: the efficiency of culture versus fluorescent in situ hybridization (FISH).
Monocyte/macrophage cells from human nasopharyngeal lymphoid tissue can be a source of bacteria responsible for human chronic and recurrent upper respiratory tract infection. Detection and characterization of pathogens surviving intracellularly could be a key element in bacteriological diagnosis of the infections as well as in the study on interactions between bacteria and their host. The present study was undertaken to assess the possibility of isolation of viable bacteria from the cells expressing monocyte/macrophage marker CD14 in nasopharyngeal lymphoid tissue. Overall, 74 adenotonsillectomy specimens (adenoids and tonsils) from 37 children with adenoid hypertrophy and recurrent infections as well as 15 specimens from nine children with adenoid hypertrophy, which do not suffer from upper respiratory tract infections (the control group), were studied. The suitability of immunomagnetic separation for extraction of CD14(+) cells from lymphoid tissue and for further isolation of the intracellular pathogens has been shown. The coexistence of living pathogens including Haemophilus influenzae, Staphylococcus aureus, and Streptococcus pyogenes with the bacteria representing normal nasopharyngeal microbiota inside CD14(+) cells was demonstrated. Twenty-four strains of these pathogens from 32.4 % of the lysates of CD14(+) cells were isolated. Concurrently, the fluorescent in situ hybridization (FISH) with a universal EUB388, and the species-specific probes demonstrated twice more often the persistence of these bacterial species in the lysates of CD14(+) cells than conventional culture. Although the FISH technique appears to be more sensitive than traditional culture in the intracellular bacteria identification, the doubts on whether the bacteria are alive, and therefore, pathogenic would still exist without the strain cultivation.
Department of Microbiology, Military Institute of Hygiene and Epidemiology, Kozielska 4, 01-163, Warsaw, Poland.
M
M
Stępińska
O
O
Olszewska-Sosińska
M
M
Lau-Dworak
B
B
Zielnik-Jurkiewicz
E A
EA
Trafny
D000234:Adenoids / Q000166:cytology / Q000382:microbiology*; D001419:Bacteria / Q000737:chemistry / Q000302:isolation & purification*; D001431:Bacteriological Techniques / Q000379:methods*; D018929:Cell Culture Techniques; D002648:Child; D002675:Child, Preschool; D006801:Humans; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D042541:Intracellular Space / Q000382:microbiology*; D018950:Lipopolysaccharide Receptors; D008264:Macrophages / Q000382:microbiology; D009000:Monocytes / Q000382:microbiology; D014066:Palatine Tonsil / Q000166:cytology / Q000382:microbiology*; D010784:Photomicrography
D018950:Lipopolysaccharide Receptors