Bacterial identification based on 16S rRNA gene analysis

Bacterial identification was conducted according to previously published methods of 16S rRNA gene sequencing analysis [10]. Total DNA of the bacteria that showed positive reactions to both of the HoPAT test and HZ were extracted using InstaGene^TM Matrix (Bio-Rad Japan, Tokyo, Japan) according to the manufacturer’s instructions. Full-length16S rRNA gene fragments from the extracted DNAs were amplified by polymerase chain reaction (PCR) with universal primers (forward 27f: 5′-AGA GTT TGA TCC TGG CTC AG-3′, reverse 1492r: 5′-GGT TAC CTT GTT ACG ACT T-3′) using commercial master mix (SapphireAmp Fast PCR Master Mix, Takara BIO Inc., Shiga, Japan). These amplified PCR products were purified with a QIAquick PCR Purification Kit (Qiagen Co., Ltd., Tokyo, Japan). After confirming molecular sizes of the obtained amplicons by means of routine procedures using agarose gel electrophoresis, sequencing analysis using synthesized universal primer sets, the forward and reverse primers of which were f1L, f2L, 926f, and f3L and r1L, r2L, r3L, and r4L, respectively [11, 24], was performed by a commercial service (FASMAC Co., Ltd., Atsugi, Japan). Homology analysis was performed using a Basic Local Alignment Search Tool (BLASTN) search (http://blast.ncbi.nlm.nih.gov/) of the published 16S rRNA gene sequences in GenBank. The species or genus of the isolates was defined according to the criteria of the Clinical and Laboratory Standards Institute (CLSI). In other words, we identified a bacterium with ≥99.0% identity to the type strain as one corresponding to the species level, one with ≥97.0% to <99.0% identity to the type strain as one corresponding to the genus level, and one with <97.0% identity to the type strain as one corresponding to a closely related genus or possibly to a novel bacterium.