First report of Tsukamurella keratitis: association between T. tyrosinosolvens and T. pulmonis and ophthalmologic infections.

We describe the first two cases of Tsukamurella keratitis, presented as eye pain with or without blurring of vision. One case was associated with trichiasis and the other with contact lens wear. The two isolates were identified as T. tyrosinosolvens and T. pulmonis, respectively, by phenotypic characterization and 16S rRNA sequencing.

Development of a multi-locus sequence typing scheme for Laribacter hongkongensis, a novel bacterium associated with freshwater fish-borne gastroenteritis and traveler's diarrhea.

BACKGROUND: Laribacter hongkongensis is a newly discovered, facultative anaerobic, Gram-negative, motile, sea gull-shaped rod associated with freshwater fish borne gastroenteritis and traveler's diarrhea. A highly reproducible and discriminative typing system is essential for better understanding of the epidemiology of L. hongkongensis. In this study, a multilocus sequence typing (MLST) system was developed for L. hongkongensis. The system was used to characterize 146 L. hongkongensis isolates, including 39 from humans and 107 from fish. RESULTS: Fragments (362 to 504 bp) of seven housekeeping genes were amplified and sequenced. Among the 3068 bp of the seven loci, 332 polymorphic sites were observed. The median number of alleles at each locus was 34 [range 22 (ilvC) to 45 (thiC)]. All seven genes showed very low d(n)/d(s) ratios of < 0.04, indicating that no strong positive selective pressure is present. A total of 97 different sequence types (STs) were assigned to the 146 isolates, with 80 STs identified only once. The overall discriminatory power was 0.9861. eBURST grouped the isolates into 12 lineages, with six groups containing only isolates from fish and three groups only isolates from humans. Standardized index of association (I(S)(A)) measurement showed significant linkage disequilibrium in isolates from both humans and fish. The I(S)(A) for the isolates from humans and fish were 0.270 and 0.636, indicating the isolates from fish were more clonal than the isolates from humans. Only one interconnected network (acnB) was detected in the split graphs. The P-value (P = 0) of sum of the squares of condensed fragments in Sawyer's test showed evidence of intragenic recombination in the rho, acnB and thiC loci, but the P-value (P = 1) of maximum condensed fragment in these gene loci did not show evidence of intragenic recombination. Congruence analysis showed that all the pairwise comparisons of the 7 MLST loci were incongruent, indicating that recombination played a substantial role in the evolution of L. hongkongensis. A website for L. hongkongensis MLST was set up and can be accessed at http://mlstdb.hku.hk:14206/MLST_index.html. CONCLUSION: A highly reproducible and discriminative MLST system was developed for L. hongkongensis.

Life-threatening invasive Helcococcus kunzii infections in intravenous-drug users and ermA-mediated erythromycin resistance.

We report the first two cases of life-threatening invasive Helcococcus kunzii infection, with primary bacteremia and empyema thoracis, respectively. Gram smears of both H. kunzii isolates showed a mixture of gram-positive and gram-negative cocci. The isolate from the first patient, resistant to erythromycin and clindamycin, possessed an ermA gene.

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.

Enterococcus cecorum empyema thoracis successfully treated with cefotaxime.

We report the first case of Enterococcus cecorum empyema thoracis and spontaneous bacterial peritonitis in a 44-year-old man with underlying cirrhosis. The patient responded to cefotaxime (MIC, 0.25 microg/ml) treatment and drainage of the empyema. Susceptibility of E. cecorum to expanded-spectrum cephalosporins could be due to its production of types of penicillin-binding proteins similar to those produced by Streptococcus species rather than to those produced by Enterococcus species (as predicted by phylogenetic analysis of the 16S rRNA gene sequences).

"Streptococcus milleri" endocarditis caused by Streptococcus anginosus.

Unlike other viridans streptococci, members of the "Streptococcus milleri group" are often associated with abscess formation, but are only rare causes of infective endocarditis. Although it has been shown that almost all S. intermedius isolates and most S. constellatus isolates, but only 19% of S. anginosus isolates, were associated with abscess formation, no report has addressed the relative importance of the 3 species of the "S. milleri group" in infective endocarditis. During a 5-year period (April 1997 through March 2002), 6 cases of "S. milleri" endocarditis (out of 377 cases of infective endocarditis), that fulfil the Duke's criteria for the diagnosis of infective endocarditis, were encountered. All 6 "S. milleri" isolates were identified as S. anginosus by 16S ribosomal RNA (rRNA) gene sequencing. Three patients had underlying chronic rheumatic heart disease and 1 was an IV drug abuser. Five had monomicrobial bacteremia, and 1 had polymicrobial (S. anginosus, S. mitis, Granulicatella adiacens, and Slackia exigua) bacteremia. Two patients died. None of the 6 isolates were identified by the Vitek system (GPI) or the API system (20 STREP) at >95% confidence. All 6 isolates were sensitive to penicillin G (MIC 0.008-0.064 microg/mL), cefalothin, erythromycin, clindamycin, and vancomycin. Accurate identification to the species level, by 16S rRNA gene sequencing, in cases of bacteremia caused by members of the "S. milleri group", would have direct implication on the underlying disease process, hence guiding diagnosis and treatment. Infective endocarditis should be actively looked for in cases of monomicrobial S. anginosus bacteremia, especially if the organism is recovered in multiple blood cultures.

Usefulness of the MicroSeq 500 16S ribosomal DNA-based bacterial identification system for identification of clinically significant bacterial isolates with ambiguous biochemical profiles.

Due to the inadequate automation in the amplification and sequencing procedures, the use of 16S rRNA gene sequence-based methods in clinical microbiology laboratories is largely limited to identification of strains that are difficult to identify by phenotypic methods. In this study, using conventional full-sequence 16S rRNA gene sequencing as the "gold standard," we evaluated the usefulness of the MicroSeq 500 16S ribosomal DNA (rDNA)-based bacterial identification system, which involves amplification and sequencing of the first 527-bp fragment of the 16S rRNA genes of bacterial strains and analysis of the sequences using the database of the system, for identification of clinically significant bacterial isolates with ambiguous biochemical profiles. Among 37 clinically significant bacterial strains that showed ambiguous biochemical profiles, representing 37 nonduplicating aerobic gram-positive and gram-negative, anaerobic, and Mycobacterium species, the MicroSeq 500 16S rDNA-based bacterial identification system was successful in identifying 30 (81.1%) of them. Five (13.5%) isolates were misidentified at the genus level (Granulicatella adiacens was misidentified as Abiotrophia defectiva, Helcococcus kunzii was misidentified as Clostridium hastiforme, Olsenella uli was misidentified as Atopobium rimae, Leptotrichia buccalis was misidentified as Fusobacterium mortiferum, and Bergeyella zoohelcum was misidentified as Rimerella anatipestifer), and two (5.4%) were misidentified at the species level (Actinomyces odontolyticus was misidentified as Actinomyces meyeri and Arcobacter cryaerophilus was misidentified as Arcobacter butzleri). When the same 527-bp DNA sequences of these seven isolates were compared to the known 16S rRNA gene sequences in the GenBank, five yielded the correct identity, with good discrimination between the best and second best match sequences, meaning that the reason for misidentification in these five isolates was due to a lack of the 16S rRNA gene sequences of these bacteria in the database of the MicroSeq 500 16S rDNA-based bacterial identification system. In conclusion, the MicroSeq 500 16S rDNA-based bacterial identification system is useful for identification of most clinically important bacterial strains with ambiguous biochemical profiles, but the database of the MicroSeq 500 16S rDNA-based bacterial identification system has to be expanded in order to encompass the rarely encountered bacterial species and achieve better accuracy in bacterial identification.

Streptococcus sinensis sp. nov., a novel species isolated from a patient with infective endocarditis.

A bacterium was isolated from the blood culture of a patient with infective endocarditis. The cells were facultative anaerobic, nonsporulating, gram-positive cocci arranged in chains. The bacterium grows on sheep blood agar as alpha-hemolytic, gray colonies of 0.5 to 1 mm in diameter after 24 h of incubation at 37 degrees C in ambient air. Growth also occurs in 10 or 40% bile and on bile esculin agar but not in 6% NaCl. No enhancement of growth is observed in 5% CO(2). It is nongroupable with Lancefield groups A, B, C, D, F, or G antisera and is resistant to optochin and bacitracin. The organism is aflagellated and is nonmotile at both 25 and 37 degrees C. It is Voges-Proskauer test positive. It produces leucine arylamidase and beta-glucosidase but not catalase, urease, lysine decarboxylase, or ornithine decarboxylase. It hydrolyzes esculin and arginine. It utilizes glucose, lactose, salicin, sucrose, pullulan, trehalose, cellobiose, hemicellulase, mannose, maltose, and starch. 16S rRNA gene sequencing showed that there were 3.6, 3.7, 4.3, 4.7, and 5.9% differences between the 16S rRNA gene sequence of the bacterium and those of Streptococcus gordonii, Streptococcus intermedius, Streptococcus constellatus, Streptococcus sanguis, and Streptococcus anginosus, respectively. The G+C content of it (mean plus minus standard deviation) was 53.0% plus minus 2.9%. Based on phylogenetic affiliation, it belongs to the mitis or anginosus group of Streptococcus. For these reasons a new species, Streptococcus sinensis sp. nov., is proposed, for which HKU4 is the type strain. Further studies should be performed to ascertain the potential of this bacterium to become an emerging cause of infective endocarditis.