Single gene target bacterial identification: GroEL gene sequencing for discriminating clinical isolates of Burkholderia pseudomallei and Burkholderia thailandensis

Proper identification of Burkholderia pseudomallei and Burkholderia thailandensis is crucial in guiding clinical management of patients with suspected melioidosis, as more than 99% of cases of melioidosis are caused by B. pseudomallei, whereas B. thailandensis is only responsible for causing less than 1% of the cases. However, the difference between the 16S ribosomal RNA gene sequences of B. pseudomallei and that of B. thailandensis is only 1%, and is therefore not discriminative enough for distinguishing the 2 species confidently. In this study, we amplified and sequenced the groEL genes of 7 strains of B. thailandensis and 6 strains of B. pseudomallei, and compared the sequences with 7 other groEL gene sequences of Burkholderia species. BLAST analysis revealed that the putative protein encoded by the groEL gene of B. thailandensis has 99.6%, 99.5%, 98.4%, 98.5%, and 96.5% amino acid identity with the groEL of B. pseudomallei, B. mallei, B. cepacia, B. vietnamiensis, and B. fungorum respectively. The amino acid sequences of GroEL of the strains of B. thailandensis and B. pseudomallei all showed >99.5% amino acid identity with each other. The nucleotide sequence of the groEL gene of any of the strains of B. thailandensis showed >99.8% nucleotide identity with that of any of the other strains of B. thailandensis, and the nucleotide sequence of the groEL gene of any of the strains of B. pseudomallei showed >99.5% nucleotide identity with that of any of the other strains of B. pseudomallei. However, the nucleotide sequence of any of the strains of B. thailandensis showed 96% amino acid identity with each other. Furthermore, the nucleotide sequence of the groEL genes of the 2 strains of B. cepacia showed >99.5% nucleotide identity with each other, and the nucleotide sequence of the groEL gene of B. mallei showed >99.5% nucleotide identity with any of the strains of B. pseudomallei. The groEL gene sequence is therefore good for distinguishing between B. thailandensis and B. pseudomallei, and the GroEL amino acid and groEL nucleotide sequences of this single gene locus may potentially be useful for a 2-tier hierarchical identification of medically important Burkholderia at the genus and species levels respectively. © 2002 Elsevier Science Inc. All rights reserved.link_to_subscribed_fulltex

Anaerobic, non-sporulating, Gram-positive bacilli bacteraemia characterized by 16S rRNA gene sequencing

Owing to the difficulties in identifying anaerobic, non-sporulating, Gram-positive bacilli in clinical microbiology laboratories, the epidemiology and clinical spectrum of disease of many of these bacteria have been poorly understood. The application of 16S rRNA gene sequencing in characterizing bacteraemia due to anaerobic, non-sporulating Gram-positive bacilli during a 4-year period is described. The first case of Olsenella uli bacteraemia, in a patient with acute cholangitis, is also reported. Among 165 blood culture isolates of anaerobic, Gram-positive bacilli, 75 were identified as Propionibacterium acnes by phenotypic tests and 21 as members of other anaerobic, non-sporulating Gram-positive bacilli by 16S rRNA gene sequencing. Of these 96 isolates, 16 (17 %) were associated with cases of clinically significant bacteraemia, among which 10 (63 %) were caused by Eggerthella, four (25 %) by Lactobacillus and one (6 %) by each of Eubacterium tenue and O. uli. Five of the 10 Eggerthella isolates were Eggerthella lenta, whereas the other five belonged to two novel Eggerthella species, with Eggerthella hongkongensis being almost as prevalent as Eggerthella lenta. Underlying disease in the gastrointestinal tract, isolation of Eggerthella and Lactobacillus, and monomicrobial bacteraemia were associated with clinically significant bacteraemia, whereas isolation of P. acnes and polymicrobial bacteraemia were associated with pseudobacteraemia. Most patients with clinically significant bacteraemia had underlying diseases, with diseases in the gastrointestinal tract being most common. The overall mortality rate was 31 %. Immunocompromised patients with clinically significant bacteraemia due to anaerobic, non-sporulating, Gram-positive bacilli other than P. acnes should be treated with appropriate antibiotics. The unexpected frequency of isolation of Eggerthella from blood cultures and its association with clinically significant disease suggest that this genus is probably of high pathogenicity. Further studies to look for specific virulence factors are warranted.link_to_subscribed_fulltex

Eggerthella hongkongensis sp. nov. and eggerthella sinensis sp. nov., two novel Eggerthella species, account for half of the cases of Eggerthella bacteremia

Eggerthella, one of the human gut flora, was rarely reported to cause bacteremia in the literature. We describe the application of 16S ribosomal RNA gene sequencing in defining the epidemiology and clinical significance of Eggerthella bacteremia during a 4-year period. Among 55 clinically significant blood culture isolates of anaerobic Gram-positive bacilli, 5 were identified as E. lenta and 5 belonged to 2 novel Eggerthella species, proposed as E. hongkongensis and E. sinensis, respectively. The 10 patients with Eggerthella bacteremia were adults, and 9 had underlying diseases. In all cases, the source of the bacteremia was likely from endogenous flora. Septic shock was a complication in 4 patients, and 3 patients died. The present study suggests that Eggerthella bacteremia is much more common than expected and is associated with significant morbidity and mortality. Moreover, the 2 novel species account for half of the cases of Eggerthella bacteremia. © 2004 Elsevier Inc. All rights reserved.link_to_subscribed_fulltex

Alkanindiges hongkongensis sp. nov. A novel Alkanindiges species isolated from a patient with parotid abscess

A bacterium was isolated from the abscess pus of a 72-year-old patient with Warthin's tumor and parotid abscess. The cells were aerobic, non-motile, Gram-negative but difficult to be destained, non-sporulating, coccobacillus. The bacterium grew poorly on sheep blood agar and MacConkey agar as non-hemolytic colonies of 0.5 mm in diameter after 24 h of incubation at 37°C in ambient air. Growth was enhanced by Tween 80. It produces catalase but not cytochrome oxidase. Sequencing of the cloned 16S rRNA PCR products of the bacterium revealed three different 16S rRNA gene sequences, with 12-31 bp differences among them. Phylogenetic analysis showed that the bacterium is closely related to Alkanindiges illinoisensis, with 5.0-5.9% differences between the 16S rRNA gene sequence of the bacterium and that of A. illinoisensis. Tryptophan auxotrophic strain of Acinetobacter trpE27 transformed with DNA extracted from the bacterium was unable to grow on tryptophan deficient medium, indicating that the bacterium was not a strain of Acinetobacter. The G+C content of the bacterium (mean±SD) was 46.9±4.3%. A new species, Alkanindiges hongkongensis sp. nov., is proposed, for which HKU9T is the type strain. Isolates with "small colonies" that are apparently Acinetobacter-like species should be carefully identified. Growth enhancement with aliphatic hydrocarbons should be looked for and 16S rRNA gene sequencing performed in order to find more potential cases of Alkanindiges infections, as well as to define the epidemiology, clinical spectrum, and outcome of infections associated with this genus. © 2005 Elsevier GmbH. All rights reserved.link_to_subscribed_fulltex

Anaerospora hongkongensis gen. nov. sp. nov., a novel genus and species with ribosomal DNA operon heterogeneity isolated from an intravenous drug abuser with pseudobacteremia

A bacterium was isolated from the blood culture of an intravenous drug abuser with pseudobacteremia. The cells were strictly anaerobic, straight or slightly curved, sporulating, Gram-negative rods. It grew on sheep blood agar as non-hemolytic, pinpoint colonies after 48 hr of incubation at 37 C in an anaerobic environment. It was motile but did not produce catalase or cytochrome oxidase. 16S ribosomal DNA (rDNA) sequencing revealed three different copies of 16S rDNA sequences. More than 90% of the differences among them were due to differences in the lengths of the sequences. Phylogenetically, the bacterium is clustered with Dendrosporobacter, Sporomusa, and Propionispora, the other three genera of anaerobic, sporulating, Gram-negative rods. There were 8.6-11.1% differences between the 16S rDNA sequences of the bacterium and that of D. quercicolus, 4.7-15.1% differences between the 16S rDNA sequences of it and those of S. acidovorans, S. aerivorans, S, malonica, S. ovata, S. paucivorans, S. silvacetica, S. spaeroides, and S. termitida, and 7.6-13.1% differences between the 16S rDNA sequences of it and those of P. hippei and P. vibrioides. The G+C content of the bacterium (mean±SD) was 46.8±3.2%. For these reasons, a new genus and species, Anaerospora hongkongensis gen. nov. sp. nov., is proposed, for which HKU15 T is the type strain.link_to_OA_fulltex