Primary infective spondylodiscitis caused by Lactococcus garvieae and a review of human L. garvieae infections.

We report the first case of primary infective spondylodiscitis due to Lactococcus garvieae, confirmed by 16S rRNA gene sequencing, in the absence of concomitant endocarditis in a patient with long-standing gastritis on famotidine. He responded to a 6-week course of ampicillin. The gastrointestinal tract is probably the source of infection.

Then and now: use of 16S rDNA gene sequencing for bacterial identification and discovery of novel bacteria in clinical microbiology laboratories.

In the last decade, as a result of the widespread use of PCR and DNA sequencing, 16S rDNA sequencing has played a pivotal role in the accurate identification of bacterial isolates and the discovery of novel bacteria in clinical microbiology laboratories. For bacterial identification, 16S rDNA sequencing is particularly important in the case of bacteria with unusual phenotypic profiles, rare bacteria, slow-growing bacteria, uncultivable bacteria and culture-negative infections. Not only has it provided insights into aetiologies of infectious disease, but it also helps clinicians in choosing antibiotics and in determining the duration of treatment and infection control procedures. With the use of 16S rDNA sequencing, 215 novel bacterial species, 29 of which belong to novel genera, have been discovered from human specimens in the past 7 years of the 21st century (2001-2007). One hundred of the 215 novel species, 15 belonging to novel genera, have been found in four or more subjects. The largest number of novel species discovered were of the genera Mycobacterium (n = 12) and Nocardia (n = 6). The oral cavity/dental-related specimens (n = 19) and the gastrointestinal tract (n = 26) were the most important sites for discovery and/or reservoirs of novel species. Among the 100 novel species, Streptococcus sinensis, Laribacter hongkongensis, Clostridium hathewayi and Borrelia spielmanii have been most thoroughly characterized, with the reservoirs and routes of transmission documented, and S. sinensis, L. hongkongensis and C. hathewayi have been found globally. One of the greatest hurdles in putting 16S rDNA sequencing into routine use in clinical microbiology laboratories is automation of the technology. The only step that can be automated at the moment is input of the 16S rDNA sequence of the bacterial isolate for identification into one of the software packages that will generate the result of the identity of the isolate on the basis of its sequence database. However, studies on the accuracy of the software packages have given highly varied results, and interpretation of results remains difficult for most technicians, and even for clinical microbiologists. To fully utilize 16S rDNA sequencing in clinical microbiology, better guidelines are needed for interpretation of the identification results, and additional/supplementary methods are necessary for bacterial species that cannot be identified confidently by 16S rDNA sequencing alone.

The oral cavity as a natural reservoir for Streptococcus sinensis.

Using Streptococcus sinensis 16S rRNA-specific and groEL gene-specific primers, a 128-bp fragment of the 16S rRNA gene and a 433-bp fragment of the groEL gene were amplified from bacterial DNA recovered from 22 of 100 saliva samples from healthy volunteers. There was no nucleotide difference between the 88-bp 16S rRNA gene fragments from the 22 saliva samples and that of S. sinensis strain HKU4(T), but there were zero to eight nucleotide differences between the 311-bpgroEL gene fragments from the 22 samples and that of S. sinensis strain HKU4(T). The oral cavity is the natural reservoir of S. sinensis.

Globicatella bacteraemia identified by 16S ribosomal RNA gene sequencing.

BACKGROUND: Globicatella are streptococcus-like organisms that have been rarely isolated from clinical specimens. Their epidemiology and clinical significance remain largely unknown. AIMS: To describe two cases of Globicatella bacteraemia identified by 16S ribosomal RNA (rRNA) gene sequencing. METHODS: Two unidentified streptococcus-like bacteria isolated from blood cultures of patients were subject to 16S rRNA gene sequencing. RESULTS: Two cases of Globicatella bacteraemia were identified by 16S rRNA gene sequencing. In the first case, a gram positive coccus was isolated from the blood culture of an 80 year old woman with diabetes mellitus and nosocomial sepsis, who died the day after developing the bacteraemia. The bacterium was unidentified by conventional phenotypic tests, the Vitek (gram positive identification) and the ATB expression (ID32 Strep) systems. In the second case, a similar bacterium was isolated from the blood culture of a 92 year old woman with polymicrobial acute pyelonephritis complicated by septic shock, who subsequently recovered after antibiotic treatment. 16S rRNA gene sequencing of the two isolates showed 0.5% nucleotide difference from that of G. sulfidifaciens and 0.7% nucleotide difference from that of G. sanguinis, indicating that they were Globicatella species. CONCLUSIONS: Because Globicatella is rarely encountered in clinical microbiology laboratories, it may have been overlooked or misidentified in these cases. 16S rRNA gene sequencing is a useful tool to better characterise the epidemiology and clinical significance of Globicatella.

Pseudobacteraemia in a patient with neutropenic fever caused by a novel paenibacillus species: Paenibacillus hongkongensis sp. nov.

AIMS: To characterise a strain of Gram negative aerobic straight or slightly curved rods (HKU3) isolated from the blood culture of a 9 year old Chinese boy with neutropenic fever and pseudobacteraemia. METHODS: The isolate was phenotypically investigated by standard biochemical methods using conventional biochemical tests, scanning electron microscopy, and transmission electron microscopy. Genotypically, the 16S rRNA gene of the bacterium 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 by multiple sequence alignment. The G + C content was determined by thermal denaturation. A phylogenetic tree was constructed by the PileUp method. RESULTS: The cells of the bacterial strain were aerobic, sporulating, Gram negative straight or slight curved rods. The bacterium grew on horse blood agar as non-haemolytic, grey colonies of 1 mm in diameter after 24 hours of incubation at 37 degrees C in ambient air. No enhancement of growth was seen in 5% CO(2). It grew at 50 degrees C as pinpoint colonies after 72 hours of incubation, but did not grow at 65 degrees C or on MacConkey agar. It was non-motile. It produced catalase (weakly positive) and cytochrome oxidase. It reduced nitrate, produced beta galactosidase, hydrolysed esculin, and utilised sodium acetate. A scanning electron micrograph of the bacterium showed straight or slightly curved rods. A transmission electron micrograph of the cell wall of the bacterium revealed multiple electron dense layers, including the outer membrane, middle murein layer, and inner cytoplasmic membrane, compatible with its Gram smear appearance. 16S rRNA gene sequencing showed that there were 7.7%, 8.0%, 8.2%, and 8.6% differences between the 16S rRNA gene sequence of the bacterium and those of Paenibacillus macerans, Paenibacillus borealis, Bacillus ehimensis, and Paenibacillus amylolyticus, respectively. The mean (SD) G + C content of the bacterium was 47.6 (2.1) mol%. Phylogenetically, it belongs to the genus paenibacillus (previously called group 3 bacillus). CONCLUSIONS: A bacterium that exhibited phenotypic and genotypic characteristics that are very different from closely related members of paenibacillus was the cause of pseudobacteraemia in a patient with neutropenic fever. A new species, Paenibacillus hongkongensis sp. nov. is proposed, for which HKU3 is the type strain.

Identification by 16S ribosomal RNA gene sequencing of Arcobacter butzleri bacteraemia in a patient with acute gangrenous appendicitis.

AIMS: To identify a strain of Gram negative facultative anaerobic curved bacillus, concomitantly isolated with Escherichia coli and Streptococcus milleri, from the blood culture of a 69 year old woman with acute gangrenous appendicitis. The literature on arcobacter bacteraemia and arcobacter infections associated with appendicitis was reviewed. METHODS: The isolate was phenotypically investigated by standard biochemical methods using conventional biochemical tests. Genotypically, the 16S ribosomal RNA (rRNA) gene of the bacterium 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 by multiple sequence alignment. Literature review was performed by MEDLINE search (1966-2000). RESULTS: The bacterium grew on blood agar, chocolate agar, and MacConkey agar to sizes of 1 mm in diameter after 24 hours of incubation at 37 degrees C in 5% CO2. It grew at 15 degrees C, 25 degrees C, and 37 degrees C; it also grew in a microaerophilic environment, and was cytochrome oxidase positive and motile, typically a member of the genus arcobacter. Furthermore, phenotypic testing showed that the biochemical profile of the isolate did not fit into the pattern of any of the known arcobacter species. 16S rRNA gene sequencing showed one to two base differences between the isolate and A butzleri, but 35 to 39 base differences between the isolate and A cryaerophilus, indicating that the isolate was a strain of A butzleri. Only three cases of arcobacter bacteraemia with detailed clinical characteristics were found in the English literature. The sources of the arcobacter species in the three cases were largely unknown, although the gastrointestinal tract is probably the portal of entry of the A butzleri isolated from the present case because the two concomitant isolates (E coli and S milleri) in the blood culture were common flora of the gastrointestinal tract. In addition, A butzleri has previously been isolated from the abdominal contents or peritoneal fluid of three patients with acute appendicitis. CONCLUSIONS: 16S rRNA gene sequencing was useful in the identification of the strain of A butzleri isolated from the blood culture of a patient with acute gangrenous appendicitis. Arcobacter bacteraemia is rare. Further studies using selective medium for the delineation of the association between A butzleri and acute appendicitis are warranted.