Identification by 16S ribosomal RNA gene sequencing of an Enterobacteriaceae species from a bone marrow transplant recipient

Abstract

Aims: To ascertain the clinical relevance of a strain of Enterobacteriaceae isolated from the stool of a bone marrow transplant recipient with diarrhoea. The isolate could not be identified to the genus level by conventional phenotypic methods and required 16S ribosomal RNA (rRNA) gene sequencing for full identification.

Methods: The isolate was investigated phenotypically by standard biochemical methods using conventional biochemical tests and two commercially available systems, the Vitek (GNI+) and API (20E) systems. Genotypically, the 16S bacterial rRNA gene was amplified by the polymerase chain reaction (PCR) and sequenced. The sequence of the PCR product was compared with known 16S rRNA gene sequences in the GenBank database by multiple sequence alignment.

Results: Conventional biochemical tests did not reveal a pattern resembling any known member of the Enterobacteriaceae family. The isolate was identified as Salmonella arizonae (73%) and Escherichia coli (76%) by the Vitek (GNI+) and API (20E) systems, respectively. 16S rRNA sequencing showed that there was only one base difference between the isolate and E coli K-12, but 48 and 47 base differences between the isolate and S typhimurium (NCTC 8391) and S typhi (St111), respectively, showing that it was an E coli strain. The patient did not require any specific treatment and the diarrhoea subsided spontaneously.

Conclusions: 16S rRNA gene sequencing was useful in ascertaining the clinical relevance of the strain of Enterobacteriaceae isolated from the stool of the bone marrow transplant recipient with diarrhoea.

Figure 1

DNA products from PCR of 16S ribosomal gene. Lane M, molecular marker SPP1 EcoRI digest; lane 1, bacterial isolate from bone marrow transplant recipient; lane 2, negative control containing DNase I treated distilled water.

Figure 2

DNA sequences of the 16S rRNA gene of the isolate from the bone marrow transplant recipient, Escherichia coli K-12, Salmonella typhimurium (NCTC 8391), and Salmonella typhi (St111). The shaded bases represent those in the isolate that are different from the corresponding ones in E coli K-12, S typhimurium (NCTC 8391), or S typhi (St111).