Specific and Rapid Detection by Fluorescent In Situ Hybridization of Bacteria in Clinical Samples Obtained from Cystic Fibrosis Patients

ABSTRACT We report on the rapid and specific detection of bacteria commonly isolated from clinical specimens from cystic fibrosis (CF) patients by fluorescent in situ hybridization (FISH). On the basis of comparative sequence analysis, we designed oligonucleotide probes complementary to species-specific 16S rRNA regions of these microorganisms and demonstrated the specificities of the probes by hybridization of different remotely related as well as closely related reference strains. Furthermore, in a pilot project we investigated 75 sputum samples and 10 throat swab specimens from CF patients by FISH and detected Pseudomonas aeruginosa, Burkholderia cepacia, Stenotrophomonas maltophilia,Haemophilus influenzae, and Staphylococcus aureus within these specimens. The specificity of FISH was 100% in comparison to the results of conventional microbial culture. In contrast, the sensitivity of standard laboratory cultivation was moderately higher, since the limit for microscopic detection of bacteria within sputum samples by FISH was approximately 4 × 105 CFU/ml of sputum (resulting in a 90% sensitivity for FISH). Moreover, we demonstrated that FISH will be useful for the rapid detection of bacteria that cause acute pulmonary exacerbations in CF patients, as demonstrated in patients with H. influenzae, S. aureus, andP. aeruginosa exacerbations. Therefore, FISH is a valuable additional method for the rapid and specific detection of bacteria in clinical samples from CF patients, in particular, patients with pulmonary exacerbations.

[1]  N. Høiby.  MICROBIOLOGY OF LUNG INFECTIONS IN CYSTIC FIBROSIS PATIENTS , 1982 .

[2]  M. Corey,et al.  Pseudomonas cepacia infection in cystic fibrosis: an emerging problem. , 1984, The Journal of pediatrics.

[3]  L. Tsui,et al.  Cystic Fibrosis Locus Defined by a Genetically Linked Polymorphic Dna Marker Author(s): Lap , 2022 .

[4]  P. Gilligan,et al.  Pseudomonas cepacia colonization in patients with cystic fibrosis: risk factors and clinical outcome. , 1985, The Journal of pediatrics.

[5]  R. Stern,et al.  Pseudomonas cepacia colonization among patients with cystic fibrosis. A new opportunist. , 1985, The American review of respiratory disease.

[6]  R. Stern,et al.  Pseudomonas cepacia: decrease in colonization in patients with cystic fibrosis. , 2015, The American review of respiratory disease.

[7]  E. Delong,et al.  Phylogenetic stains: ribosomal RNA-based probes for the identification of single cells. , 1989, Science.

[8]  D A Stahl,et al.  Fluorescent-oligonucleotide probing of whole cells for determinative, phylogenetic, and environmental studies in microbiology , 1990, Journal of bacteriology.

[9]  M. Zach.  Lung disease in cystic fibrosis—an updated concept , 1990 .

[10]  N. Høiby,et al.  Prevention of chronic Pseudomonas aeruginosa colonisation in cystic fibrosis by early treatment , 1991, The Lancet.

[11]  K. Schleifer,et al.  Phylogenetic Oligodeoxynucleotide Probes for the Major Subclasses of Proteobacteria: Problems and Solutions , 1992 .

[12]  N. Høiby,et al.  Role of alginate in infection with mucoid Pseudomonas aeruginosa in cystic fibrosis. , 1992, Thorax.

[13]  C. Vázquez,et al.  Early treatment of Pseudomonas aeruginosa colonization in cystic fibrosis , 1993, Acta paediatrica.

[14]  R Amann,et al.  In situ probing of gram-positive bacteria with high DNA G + C content using 23S rRNA-targeted oligonucleotides. , 1994, Microbiology.

[15]  K. Schleifer,et al.  Phylogenetic identification and in situ detection of individual microbial cells without cultivation. , 1995, Microbiological reviews.

[16]  R Amann,et al.  In situ probing of gram-positive bacteria with high DNA G+C content using 23S rRNA-targeted oligonucleotides. , 1995, Microbiology.

[17]  V. Deretic,et al.  Microbial pathogenesis in cystic fibrosis: mucoid Pseudomonas aeruginosa and Burkholderia cepacia. , 1996, Microbiological reviews.

[18]  P. Vandamme,et al.  Burkholderia cepacia: medical, taxonomic and ecological issues. , 1996, Journal of medical microbiology.

[19]  P. Vandamme,et al.  Occurrence of multiple genomovars of Burkholderia cepacia in cystic fibrosis patients and proposal of Burkholderia multivorans sp. nov. , 1997, International journal of systematic bacteriology.

[20]  J. Heesemann,et al.  Development of rRNA-Targeted PCR and In Situ Hybridization with Fluorescently Labelled Oligonucleotides for Detection of Yersinia Species , 1998, Journal of Clinical Microbiology.

[21]  P. Hawkey,et al.  Molecular Epidemiology of Stenotrophomonas maltophilia Isolated from Clinical Specimens from Patients with Cystic Fibrosis and Associated Environmental Samples , 1998, Journal of Clinical Microbiology.

[22]  Rudolf Amann,et al.  Bacterial Community Dynamics during Start-Up of a Trickle-Bed Bioreactor Degrading Aromatic Compounds , 1998, Applied and Environmental Microbiology.