Automated Broad-Range Molecular Detection of Bacteria in Clinical Samples

Abstract

Molecular detection methods, such as quantitative PCR (qPCR), have found their way into clinical microbiology laboratories for the detection of an array of pathogens. Most routinely used methods, however, are directed at specific species. Thus, anything that is not explicitly searched for will be missed. This greatly limits the flexibility and universal application of these techniques. We investigated the application of a rapid universal bacterial molecular identification method, IS-pro, to routine patient samples received in a clinical microbiology laboratory. IS-pro is a eubacterial technique based on the detection and categorization of 16S-23S rRNA gene interspace regions with lengths that are specific for each microbial species. As this is an open technique, clinicians do not need to decide in advance what to look for. We compared routine culture to IS-pro using 66 samples sent in for routine bacterial diagnostic testing. The samples were obtained from patients with infections in normally sterile sites (without a resident microbiota). The results were identical in 20 (30%) samples, IS-pro detected more bacterial species than culture in 31 (47%) samples, and five of the 10 culture-negative samples were positive with IS-pro. The case histories of the five patients from whom these culture-negative/IS-pro-positive samples were obtained suggest that the IS-pro findings are highly clinically relevant. Our findings indicate that an open molecular approach, such as IS-pro, may have a high added value for clinical practice.

FIG 1

Schematic representation of the concept behind the IS-pro procedure. (A) All bacterial species contain at least one IS region in their chromosome. However, many species contain multiple alleles of the IS region. These regions may vary between different alleles. Depicted here is the schematic situation in E. faecalis that contains four alleles of the IS region. Two alleles have a length of 275 nt, and the other two have a length of 377 nt. When amplified, a profile specific for E. faecalis is obtained. (B) IS profiles are highly diverse between different species. The fact that species commonly have multiple alleles with different lengths dramatically increases the differential potential between species. S. typhimurium, Salmonella enterica serovar Typhimurium; B. longum, Bifidobacterium longum; B. subtilis, Bacillus subtilis; C. jejuni, Campylobacter jejuni; S. mutans, Streptococcus mutans; L. lactis, Lactococcus lactis; L. sakei, Lactobacillus sakei; P. gingivalis; Porphyromonas gingivalis; V. parahaemolyticus; Vibrio parahaemolyticus; S. coelicolor; Streptomyces coelicolor; L. monocytogenes, Listeria monocytogenes. (C) By amplifying IS fragments with phylum-specific fluorescently labeled primers, a next layer of information is added. (D) When an IS profile is made of a sample containing multiple species from different phyla, peaks with different lengths, height, and color are found. These correspond to species, abundance, and phylum. A peak profile may be translated to a list of bacterial species by a software algorithm linked to a database of IS profiles of known bacterial species. The whole IS-pro procedure from unprocessed sample to analyzed data can be performed in 5 h. nc, nucleotides.

FIG 2

Schematic representation of the PacBio sequencing approach. A bacterial chromosome always contains one or more ribosomal regions (top left, red bars). These regions each consist of a 16S, 23S, and 5S region and two interspace regions (IS1 and IS2). The identity of the bacterial species can be assessed either by the sequence of the 16S fragment or by the length of the IS1 region (top right). Amplification primers were chosen to cover a large part (∼700 nt) of the 16S region, containing four variable regions (V6 to V9) for sequence-based species identification. Each amplicon thus consists of both the 16S region and the IS1 region, enabling a comparison of identifications. Forward primers had sequence tags for multiplexing in the PacBio run. Padding sequences were added to prevent damage to the sequence tags and for optimal SMRTbell ligation (bottom). By performing a virtual IS-pro on full-length sequences, best-matching (labeled) IS-pro primers and predicted length of resulting IS fragments was determined. The resulting virtual IS fragments were matched to actual IS fragments. In this fashion, bacterial identifications by 16S sequence analysis could be directly matched to corresponding IS fragments. ID, identification; refpos, E. coli reference position.

FIG 3

IS-pro results of all positive samples depicted as a clustered heat map. Common pathogens known from culture clearly play an important role when considering all species found by IS-pro. In intraperitoneal abscesses and abscesses in the urogenital region, gut microbiota clearly play an important role, as can be seen by the high presence of Bacteroides and other anaerobic species in the rightmost cluster.