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Keywords:

  • 16S rDNA gene;
  • bacteria;
  • discovery;
  • identification;
  • review;
  • sequencing

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Use of 16s rDNA sequencing in clinical microbiology laboratories
  6. Usefulness and limitations of using 16s rDNA sequencing in clinical microbiology laboratories
  7. Concluding Remarks
  8. Transparency Declaration
  9. References

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 (= 12) and Nocardia (= 6). The oral cavity/dental-related specimens (= 19) and the gastrointestinal tract (= 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.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Use of 16s rDNA sequencing in clinical microbiology laboratories
  6. Usefulness and limitations of using 16s rDNA sequencing in clinical microbiology laboratories
  7. Concluding Remarks
  8. Transparency Declaration
  9. References

One and a half centuries after the 22-year-old Charles Darwin’s 5-year voyage on HMS Beagle, the analysis of small rDNA gene sequences is another important landmark in the study of the evolution and classification of living organisms. Traditionally, living organisms were classified, according to similarities and differences in their phenotypic characteristics, into prokaryotes and eukaryotes, and these were in turn further classified into various kingdoms, phyla, classes, orders, families, genera and species. However, objective taxonomic classification by these methods can be difficult because of variations in phenotypic characteristics. Three decades ago, Carl Woese and others started to analyse and sequence the 16S rDNA genes of various bacteria, using DNA sequencing, a state-of-the-art technology at that time, and used the sequences for phylogenetic studies [1,2]. The invention of PCR and automated DNA sequencing two decades ago, and subsequent work on 16S rDNA sequencing of bacteria, as well as 18S rDNA sequencing of eukaryotes, has led to the accumulation of a vast amount of sequence data on the rDNA genes of the smaller subunit of the ribosomes in a large number of living organisms. Comparison of these sequences has shown that the rDNA gene sequences are highly conserved within living organisms of the same genus and species, but that they differ between organisms of other genera and species. Using these rDNA gene sequences for phylogenetic studies, three domains of life, Archaea, Bacteria and Eukarya, as opposed to the traditional classification of living organisms into prokaryotes and eukaryotes only, were described [3].

Among the three domains of life, the largest amount of rDNA gene sequencing work concerns bacteria. Using 16S rDNA sequences, numerous bacterial genera and species have been reclassified and renamed, classification of uncultivable bacteria has been made possible, phylogenetic relationships have been determined, and the discovery and classification of novel bacterial species has been facilitated. In the last decade, sequencing of various bacterial genomes and comparison between genome and 16S rDNA gene phylogeny has confirmed the representativeness of the 16S rDNA gene in bacterial phylogeny [4].

As a result of the increasing availability of PCR and DNA sequencing facilities, the use of 16S rDNA sequencing has not been limited to research purposes, but has also been exploited in clinical microbiology laboratories. Accurate identification of bacterial isolates is one of the most important functions of clinical microbiology laboratories. On a patient-to-patient basis, accurate identification is crucial in determining whether the isolate is causing genuine infection or is simply a colonizer or contaminant, the choice and duration of antibiotic treatment, and the appropriate infection control procedures. On a population scale, accurate identification is important in analysing the epidemiology, antibiotic resistance patterns, treatment plans and outcomes of infections associated with a particular bacterium. Traditionally, identification of bacteria in clinical microbiology laboratories was performed using phenotypic tests, including Gram smear and biochemical tests, taking into account culture requirements and growth characteristics. However, these methods of bacterial identification have major limitations. First, organisms with biochemical characteristics that do not fit into the patterns of any known genus and species are occasionally encountered. Second, they cannot be used for uncultivable organisms. Third, identification of some particular groups of bacteria, such as anaerobes and mycobacteria, would require additional equipment and expertise, which are not available in most clinical microbiology laboratories. Using 16S rDNA sequencing, these problems can be overcome by a single technology, which also facilitates the discovery of novel genera and species.

In this article, the use of 16S rDNA sequencing in clinical microbiology laboratories for bacterial identification, the discovery of novel bacterial genera and species, the detection of uncultivable bacteria and the diagnosis of culture-negative infections are reviewed. Automation of 16S rDNA sequencing and the usefulness and limitations of 16S rDNA sequencing in clinical microbiology laboratories are also discussed. The technological aspects of 16S rDNA sequencing are not included in this review.

Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Use of 16s rDNA sequencing in clinical microbiology laboratories
  6. Usefulness and limitations of using 16s rDNA sequencing in clinical microbiology laboratories
  7. Concluding Remarks
  8. Transparency Declaration
  9. References

In the section on ‘Novel bacterial genus and species discovery’, novel species discovered from human specimens that were reported during the first 7 years of the 21st century (2001–2007) in the English literature are reviewed. For initial screening, ‘gen. nov.’ and ‘sp. nov.’ were used as the key words for a Medline search. The results were manually screened for novel bacterial species isolated from human specimens, regardless of the criteria used for their definition. Bacteria that were discovered only in non-human specimens were excluded. Bacteria that were discovered, characterized and named before 2001, but were renamed after 2001 (e.g. Streptococcus lutetiensis and S. pasteurianus [5]), and those that were further subclassified (e.g. S. pseudopneumoniae [6]) because of acquisition of new information such as 16S rDNA sequences or phenotypic test results, were also not included. Additional reports on infections associated with each of the novel species isolated from human specimens, as well as those concerning their isolation from non-human specimens, were searched. All novel species that have been found in four or more subjects are presented.

Use of 16s rDNA sequencing in clinical microbiology laboratories

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Use of 16s rDNA sequencing in clinical microbiology laboratories
  6. Usefulness and limitations of using 16s rDNA sequencing in clinical microbiology laboratories
  7. Concluding Remarks
  8. Transparency Declaration
  9. References

Bacterial identification

Rapid and accurate identification of bacterial isolates is a fundamental task in clinical microbiology, and provides insights into aetiologies of infectious disease and appropriate antibiotic treatment. Although conventional phenotypic methods are relatively inexpensive and allow identification of most commonly encountered bacteria, certain groups of bacteria are difficult to identify, and special equipment and expertise may be required, e.g. gas chromatography–mass spectrometry for anaerobes. These methods also fail in cases of rare bacteria or bacteria with ambiguous profiles. Moreover, phenotypic methods rely on the availability of pure culture and are dependent on subsequent growth characteristics and biochemical profiling. Therefore, considerable time is required for slow-growing bacteria to be identified. 16S rDNA sequencing represents a universal technology that, theoretically, provides solutions to these problems, yielding unambiguous data, even for unusual and slow-growing isolates, often within 48 h, which are reproducible among laboratories.

Identification of rare bacteria and bacteria with unusual phenotypic profiles.  16S rDNA sequencing is particularly useful in identifying unusual bacteria that are difficult to identify by conventional methods, providing genus identification in >90% of cases, and identification of 65–83% of these at the species level [7,8]. The MicroSeq  500 16S rDNA-based identification system was also able to identify 81% of clinically significant bacterial isolates with ambiguous biochemical profiles and 89.2% of unusual aerobic Gram-negative bacilli to the species level [9,10].

In many situations, 16S rDNA sequencing is the ultimate solution to identification of aetiological agents of infectious diseases caused by rarely encountered bacteria [11–22]. This not only allows correct identification and selection of appropriate treatment, but also contributes to a better understanding of the epidemiology and pathogenic role of these bacteria, which has not been possible in the past. For example, using 16S rDNA sequencing, cases of invasive Streptococcus iniae infections, which had previously been reported only in North America, have been recognized in Asia [23–27].

Unlike phenotypic identification, which can be affected by the presence or absence of non-housekeeping genes or by variability in expression of characters, 16S rDNA sequencing provides accurate identification of isolates with atypical phenotypic characteristics. Using the technique, it has been possible to identify thermotolerant Campylobacter fetus strains as important causes of bacteraemia in immunocompromised patients [28]. Similar applications have also been shown to have significant impacts on the decision of whether to prescribe antibiotic treatment [29–31] and on the choice of specific antibiotic regimen [32–34], which could lead to improved clinical outcomes [10].

Mistakes in identifying rarely encountered or phenotypically aberrant isolates are probably quite common in clinical microbiology laboratories. Sometimes, it is even difficult to know whether a bacterium has been incorrectly identified. For example, using phenotypic methods, Francisella tularensis subsp. novicida was consistently misidentified twice, as Neisseria meningitidis or Actinobacillus actinomycetemcomitans [35]. 16S rDNA sequencing will continue to play a major role in the identification of rare bacteria and bacteria with ambiguous characteristics in clinical microbiology laboratories.

Identification of slow-growing bacteria.  16S rDNA sequencing and similar molecular identification methods have the additional advantage of reducing the time required to identify slow-growing bacteria such as mycobacteria, which may take 6–8 weeks to grow in culture sufficiently for phenotypic tests to be performed. Even for rapidly growing mycobacteria, some biochemical reactions may take up to 28 days to complete. As for whole cell fatty acid analysis by gas chromatography, this requires special equipment and expertise that are often not available in clinical microbiology laboratories. Therefore, 16S rDNA sequencing has been used for identification of Mycobacterium species isolated from clinical specimens, making clinical diagnosis more rapid and guiding prompt antibiotic treatment [29,36]. Using this technique, we were able to describe a novel clinical syndrome, acupuncture mycobacteriosis, caused by relatively alcohol-resistant mycobacteria in patients receiving acupuncture [37,38]. However, a limited number of mycobacterial species could not be differentiated from one another by 16S rDNA sequencing, e.g. Mycobacterium avium intracellulare and M. paratuberculosis, M. chelonae and M. abscessus, and members of the M. tuberculosis complex. Therefore, other gene targets, sometimes supplemented by phenotypic results, have to be used for differentiation of these species, e.g. hsp65 and rpoB for those that grow rapidly, and 16S–23S rRNA internal transcribed spacer or gyrB for the mycobacteria that grow slowly [39–42].

Routine identification.  Several studies have been conducted to compare the usefulness of 16S rDNA sequencing with conventional or commercial methods for the identification of various groups of medically important bacteria. In general, 16S rDNA sequencing results in a higher percentage of species identification than conventional or commercial methods. The success rate of species identification by 16S rDNA sequencing ranged from 62% to 92%, depending on the group of bacteria and the criteria used for species definition [43–49]. However, there are ‘blind spots’ within some major genera, in which 16S rDNA sequences are not sufficiently discriminative for the identification of certain species. In these circumstances, alternative targets have to be investigated. For example, groEL is a commonly used essential gene other than 16S rDNA which is useful for classification and identification of many groups of bacteria, e.g. staphylococci and Burkholderia species [50,51]. In particular, it has been shown to be useful in differentiating Burkholderia pseudomallei from B. thailandensis, the 16S rDNA sequences of which are indistinguishable [52,53]. Similarly, 16S rDNA sequencing has limited discriminatory power for closely related Staphylococcus species. Therefore, sequencing of groEL and tuf has been proposed as being more reliable for identification of coagulase-negative staphylococci [47,54].

As simple conventional methods are often available for commonly encountered bacteria, 16S rDNA sequencing for identification of ‘routine’ bacterial strains is most useful in the context of bacterial species that are often difficult to identify with phenotypic tests. This has led to a better understanding of the epidemiology and pathogenicity of these clinically ‘unidentifiable’ bacteria. One remarkable example is provided by anaerobic Gram-positive rods, where conventional methods are simply not reliable, even for genus identification; with the use of 16S rDNA sequencing, many previously undescribed or ignored anaerobic bacterial species were found to contribute to cases of bacteraemia [55–59]. With regard to streptococci, cases of β-haemolytic Lancefield group G streptococcus bacteraemia have been found to be almost exclusively caused by S. dysgalactiae subsp. equisimilis using 16S rDNA sequencing, although S. canis infections in dog owners have been reported rarely [60,61]. In another study, six cases of ‘S. milleri’ endocarditis were attributed to S. anginosus, suggesting that S. anginosus had the highest propensity to cause infective endocarditis among the three species of the ‘S. milleri group’ [62]. Using this ‘state-of-the-art’ technique, it was also found that Haemophilus segnus is an important cause of non-H. influenzae bacteraemia [19,63,64]. In addition, the use of 16S rDNA sequencing for clinically ‘unidentifiable’ bacteria could have clinical significance regarding the choice of antibiotic regimen as well as the duration of treatment. For example, we have previously reported a case of empyema thoracis caused by Enterococcus cecorum that was unidentified by conventional methods and three commercial identification systems. Unlike other Enterococcus species, the organism is known to be susceptible to cefotaxime and ceftriaxone. In this case, the use of 16S rDNA sequencing not only allowed continuation of cefotaxime as treatment for the patient, who responded very well, but also suggested that the bacterium’s cephalosporin susceptibility could well be explained by its unique phylogenetic position, it being the ancestor of other Enterococcus species and more closely related to Streptococcus species [65]. In other instances, 16S rDNA sequencing has been particularly useful in differentiating between Actinomyces and non-Actinomyces anaerobic Gram-positive bacilli, which is often difficult in clinical microbiology laboratories [66–68]. A definitive diagnosis of actinomycosis is important, as a prolonged course of appropriate antibiotic treatment (weeks to months) is indicated to prevent relapse.

Novel bacterial genus and species discovery

During the process of bacterial identification, potential novel species will be encountered when there is a significant difference between the phenotypic characteristics and/or 16S rDNA sequences of the unknown bacterium and those of the most closely related ones. As no single test or gene sequence is ideal for the definition of new species in all groups of bacteria, a polyphasic approach is usually used when a novel species is defined. Depending on the group of bacteria, this involves various combinations of phenotypic characteristics, 16S rDNA sequences, sequences of other housekeeping genes, and DNA–DNA hybridization results. In this section, we review the novel species discovered from human specimens in the first 7 years of the 21st century (2001–2007) in the English literature.

According to the inclusion and exclusion criteria used in the present article, 215 novel species, 29 of which belonged to novel genera, have been discovered from human specimens in the past 7 years. Among these 215 novel species, 100 (15 of novel genera) have been found in four or more subjects (Table 1). Among these 100 novel species, there were four (4%) aerobic Gram-positive cocci, 30 (30%) aerobic Gram-positive rods, three (3%) aerobic Gram-negative cocci, 24 (24%) aerobic Gram-negative rods, four (4%) anaerobic Gram-positive cocci, ten (10%) anaerobic Gram-positive rods, one (1%) anaerobic Gram-negative coccus, 22 (22%) anaerobic Gram-negative rods, and two (2%) spirochaetes. The largest numbers of novel species discovered and isolated from four or more subjects were of the genera Mycobacterium (= 12) and Nocardia (= 6). The oral cavity/dental-related specimens (= 19) and the gastrointestinal tract specimens (= 26) were the most important for discovery and/or the most important reservoirs of novel species. This is in line with the huge diversity of potential novel bacterial species in the human oral cavity and gastrointestinal tract [69,70]. Among the 100 novel species, S. 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 [71–89], although most Nocardia and Mycobacterium species were probably from the environment, and most anaerobes were probably from the oral cavity and/or gastrointestinal tract.

Table 1.   Novel bacterial species discovered in four or more human subjects in the 21st century
BacteriaYear of first publicationReferencesAssociated diseasesSource of isolationReservoir/ source of infection
HumanNon-human
PatientsAsymptomatic individuals 
  1. aAlso a novel genus.

  2. bThe isolates discovered by Steer et al. were Gram-positive, but those found in the subsequent studies were Gram-negative.

  3. BAL, bronchoalveolar lavage; IUCD, intrauterine contraceptive device; CSF, cerebrospinal fluid; UTI, urinary tract infection; CGD, chronic granulomatous disease; CF, cystic fibrosis; CAPD, continuous ambulatory peritoneal dialysis; IPD, intermittent peritoneal dialysis.

Aerobic and facultative anaerobic Gram-positive cocci
 Kytococcus schroeteri2002[159–162]Infective endocarditis, pneumoniaBlood, BAL, prosthetic valve, abscess pus
 Streptococcus oligofermentans2003[163]Dental plaque, saliva
 S. pseudoporcinus2006[164]Female genitourinary tract
 S. sinensis2002[71–75] Infective endocarditisBloodSalivaOral cavity
Aerobic and facultative anaerobic Gram-positive rods
 Actinomyces cardiffensis2002[165]Postmastoidectomy brain and ear abscesses, jaw abscess, pericolic abscess, sinusitis, pelvic actinomycosisAbscess pus, pleural fluid, antral washout, IUCD
 A. funkei2002[166,167]Blood, sternum, abdominal incision, neck–face, thorax, pelvis, IUCD, vagina/penis, brain/CSF, superficial soft tissue lesion
 Bifidobacterium scardovii2002[168]Blood, urine, hip
 Brevibacterium lutescens2003[169]PeritonitisPeritoneal fluid, infected ear discharge, peritoneal dialysate fluidPeptone preparation
 Brevibacterium paucivorans2001[170]CSF, groin abscess, blood, ear discharge, intravascular catheter, wound, groin swab
 Corynebacterium aurimucosum (= Corynebacterium nigricans)2002[171–173] Meningitis, uterine infection, UTI, vaginitis, placental infection, vulval ulcer, female urogenital tract infectionCervix, bartholin gland, vagina, CSF, endometrium, placenta, urine, vulva, amniotic fluid, blood, vaginal swab, vaginal ulcer, vaginal–rectal swab
 Corynebacterium freneyi2001[174,175]BacteraemiaPus from toe, varicose ulcer, subcutaneous abscess fistula, blood
 Corynebacterium resistens2005[176]Bacteraemia, nosocomial pneumonia, cellulitisBlood, bronchial aspirate, cellulitis aspirate
 Lactobacillus coleohominis2001[177]Vagina, urine, cervix
 Microbacterium paraoxydans2003[178]Catheter-related bacteraemiaBlood, bronchial aspirateVegetable
 Mycobacterium arupense2006[179]Respiratory specimen, gastrointestinal tract, various sterile sites, e.g. tendon, lymph node, lung biopsy, pleural fluid, surgical tissue, urinePossibly environmental
 M. bolletii2006[42]Chronic pneumoniaSputum, stomach aspirate
 M. canariasense2004[180]Catheter-related bacteraemiaBlood
 M. colombiense2006[181]Sputum, blood
 M. florentinum2005[182]Lymphadenopathy, pneumoniaSputum, stool, lymph node, gastric aspirate, BAL
 M. holsaticum2002[183,184]Sputum, urine, gastric fluidLymph node of cattle with evidence of bovine tuberculosis
 M. immunogenum2001[185–190]Disseminated cutaneous infection, keratitis, catheter-related infection, septic arthritis, chronic pneumonia, pacemaker-related sepsis, hypersensitivity pneumonitis, chronic leg ulcer, peritonitisSkin, cornea, urine, intravenous catheter site, joint fluid, BAL, blood, broviac site, leg biopsy, corneal scraping, peritoneal fluid of patients receiving IPDHospital environment, metalworking fluid, environment, tap water, water in IPD machinesProbably tap water
 M. massiliense2004[191–193]Pneumonia, post-intramuscular injection abscess, pacemaker pocket infectionSputum, BAL, pus, tissue debris, specimen from surgical debridement, blood
 M. monacense2006[194]Post-traumatic wound infectionBiopsy, sputum, BALPossibly environmental
 M. nebraskense2004[195,196]Respiratory infectionSputum, BAL
 M. palustre2002[184,197]LymphadenitisSubmandibular lymph node biopsySubmandibular lymph node of pig, stream water, lymph node of cattle with evidence of bovine tuberculosis
 M. saskatchewanense2004[198]Pneumonia in bronchiectasis patientsSputum, pleural fluid, respiratory sample
 Nocardia africana2001[199,200]Pulmonary nocardiosisSputumSubcutaneous nodule of cat
 N. aobensis2004[201]
 N.  arthritidis2004[202,203]Thigh abscess, pulmonary nocardiosis, keratitisSputum, thigh inflammatory swelling discharge, cornea
 N. asiatica2004[203–205]Pulmonary nocardiosis, skin infection, keratitis, ocular nocardiosisSputum, granuloma, transtracheal aspirate, cutaneous ulcer, cornea, vitreous bodyPossibly environmental
 N. ignorata2001[206–208]Pulmonary nocardiosisSputum, BAL, blood, tracheal aspirateSoilProbably soil
 N. veterana2001[209–215]Pulmonary nocardiosis, mycetoma, bacteraemia, peritonitisBAL, subcutaneous nodule biopsy, sputum, open lung biopsy tissue, fine-needle transthoracic biopsy and aspirate, blood, peritoneal fluid
 Olsenella profusaa2001[216–218]Periodontitis, acute periradicular abscessSubgingival plaque, carious dental lesion, root canal, pus
 Weissella cibaria (= Weissella kimchii)2002[219–225]GallStoolEar of dog with otitis, cheese whey, sugar cane, canary liver, chili bo, tapai, wheat sourdough, blood sausage, faecal swab of healthy dog, plaa-som (fermented fish product from Thailand), fermented kimchii, whole crop paddy rice silage
Aerobic and facultative anaerobic Gram-negative cocci
 Achromobacter insolitus2003[226]Urine, woundLaboratory sink
 Achromobacter spanius2003[226]Blood
 Kerstersia gyioruma2003[227]Stool, leg and ankle wounds, sputum
Aerobic and facultative anaerobic Gram-negative rods
 Acinetobacter parvus2003[228]Catheter-related bacteraemiaEar, eye, forehead, skin, bloodEar of dog with refractory otitis media
 Acinetobacter schindleri2001[229,230]Cystitis, bacteraemiaUrine, vagina, throat, ear, nasal swab, conjunctiva, skin, pleural effusion, liquor, blood, central catheter
 Acinetobacter ursingii2001[229–231]Infective endocarditis, bacteraemia, UTI, postneurosurgical meningitis, cholangitis, cervical adenopathyBlood, intravenous line, pus, ulcer, eye, wound, urine, hairy skin, toe, abscess, central catheter, ganglion biopsy, CSF
 Advenella incenataa2005[232]Sputum, bloodHorse blood
 Averyella dalhousiensisa2005[233]Catheter-related bacteraemia, cellulitis, wound infectionWound, stool, blood, intestinal fluid
 Burkholderia ambifaria2001[234,235]KeratitisSputum and respiratory tract of CF patients, corneal cultureBiocontrol strains; corn roots; corn, pea, snap bean and sedge rhizosphere; forest soil, leaves of Sesbania exaltata, commercial soil
 B. fungorum2001[236,237]Septic arthritisVaginal secretion of pregnant woman, CSF, bloodWhite-rot fungus Phanerochaete chrysosporium, mouse nose, haemoglobin solution
 Campylobacter hominis2001[88,238,239]BacteraemiaBloodStoolGastrointestinal tract
 Cardiobacterium valvarum2004[240–244]Infective endocarditis, prosthetic valve endocarditisBlood, noma lesionProbably oral cavity
 Cupriavidus respiraculi (= Wautersia respiraculi = Ralstonia respiraculi)2003[245–247]Sputum and respiratory tract of CF patients
 Enterobacter ludwigii2005[248]Nosocomial UTIUrine, trachea, fat tissue of left thigh, venous line, sputum, blood, stool, BAL, biopsy, throat, skin, swab
 Escherichia albertii2003[249]DiarrhoeaStoolProbably gastrointestinal tract
 Granulibacter bethesdensisa2006[250,251]Lymphadenitis in CGD patientsLymph node
 Haematobacter massiliensisa (= Rhodobacter massiliensis)2003[252,253]Bacteraemia, wound infectionNose, blood, wound
 Halomonas phocaeensis2007[254]Fresh frozen plasma transfusion-associated bacteraemia/pseudobacteraemiaBloodProbably environmental, e.g. water
 Helicobacter winghamensis2001[255,256]Stool of patients with gastroenteritisStool of gerbilsProbably gastrointestinal tract
 Inquilinus limosusa2002[257–261]Acute respiratory exacerbation and progressive loss of pulmonary function in CF patients?Respiratory secretion and sputum of CF patientsRoots of grassPossibly environmental
 Laribacter hongkongensisa2001[76–84,262–265]Bacteraemia, empyema thoracis, community-acquired gastroenteritis, travellers’ diarrhoeaBlood, empyema pus, stoolFreshwater fish, drinking water reservoirFreshwater fish
 Neisseria bacilliformis2006[266]Wound infection, bronchitis, lung abscessEar, blood, oral fistula, submandibular wound, sputum, lung abscessPossibly oral cavity
 Paracoccus yeei2003[267]CAPD peritonitis, wound infection, biliary tract infection, bacteraemiaAbdominal dialysate, wound, bile, CSF, blood
 Pseudomonas mosselii2002[268,269]Tracheal aspirate, bronchial aspirate, blood, venous catheter, stool, drainage liquidMineral water
 P. otitidis2006[270]Acute otitis externa, acute otitis media, chronic suppurative otitis mediaEar of patients with acute otitis externa, acute otitis media, chronic suppurative otitis media
 Ralstonia taiwanensis2001[271]Sputum of CF patientsRoot nodule of Mimosa species
 Wautersiella falseniia2006[272]Blood, wound, pus, respiratory tract, ear discharge, vaginal swab, pleural fluid, oral cavity
Anaerobic Gram-positive cocci
 Anaerococcus murdochii2007[273]Infected foot ulcer, infected sternal wound, soft tissue neck infection, post-traumatic thumb abscessWound, abscess
 Peptoniphilus gorbachii2007[273]Infected dry gangrene, cellulitis, infected diabetic foot ulcerWound
 Peptoniphilus olsenii2007[273]Osteomyelitis, infected dry gangrene, infected diabetic foot ulcer, toe infectionBone, wound
 Peptostreptococcus stomatis2006[274,275]Dento-alveolar abscess, endodontic infection, pericoronal infection, bacteraemiaSpecimen from infected structure of oral cavity, oropharyngeal specimen, appendix, stool, bloodPossibly oral cavity and gastrointestinal tract
Anaerobic Gram-positive rods
 Alloscardovia omnicolensa2007[276]Urine, blood, urethra, oral cavity, tonsil, abscesses of lung and aortic valve
 Anaerostipes caccaea2002[277,278]StoolProbably gastrointestinal tract
 Catabacter hongkongensisa2007[59]BacteraemiaBloodProbably gastrointestinal tract
 Clostridium bolteae2003[279–281]Bacteraemia, intra-abdominal abscess, necrotizing fasciitis, pelvic abscess, wound infectionBlood, intra-abdominal abscess, abscess, woundStoolProbably gastrointestinal tract
 C. hiranonis2001[282,283]StoolProbably gastrointestinal tract
 Eggerthella hongkongensis2004[55]Perianal abscess, infected rectal tumour, liver abscess, appendicitisBloodProbably gastrointestinal tract
 Lactobacillus ultunensis2005[284–286]Carious dentineGastric mucosa biopsyStool of pigs
 Roseburia intestinalis2002[287]StoolProbably gastrointestinal tract
 Shuttleworthia satellesa2002[288]PeriodontitisPeriodontal pocket, subgingival plaqueProbably oral cavity
 Varibaculum cambriensisa2003[289]Facial abscess, otogenic cerebral abscessAbscess, IUCDPossibly oral cavity
Anaerobic Gram-negative cocci
 Veillonella montpellierensis2004[290,291]Infective endocarditisGastric fluid, amniotic fluid, blood
Anaerobic Gram-negative rods
 Alistipes onderdonkii2006[292]Intra-abdominal infectionAppendix, abdominal abscess, stool, urineStoolProbably gastrointestinal tract
 Alistipes shahii2006[292]Intra-abdominal infectionIntra-abdominal fluid and appendixStoolProbably gastrointestinal tract
 Bacteroides intestinalis2006[293]StoolProbably gastrointestinal tract
 Bacteroides plebeius2005[294]StoolProbably gastrointestinal tract
 Cetobacterium somerae2003[295]StoolProbably gastrointestinal tract
 Clostridium hathewayib2001[85–88]Bacteraemic acute cholecystitis and liver abscess, bacteraemic acute gangrenous appendicitis, bacteraemiaBlood, liver abscessStoolGastrointestina tract
 Desulfomicrobium  orale2001[296]PeriodontitisSubgingival plaqueProbably oral cavity
 Dialister invisus2003[128,297–299]Endodontic and periodontal infections, UTI, acute periradicular abscess, acute periradicular periodontitis, acute apical abscessDental root canal, deep periodontal pocket, urine of renal transplant recipients, abscess pus, abscess aspirate
 Dialister micraerophilus2005[300,301]Anal, buttock, breast, perinephric abscess, bacteraemia, wound infectionAmniotic fluid, blood, pilonidal cyst, abscess pus, bone, bartholin gland, wound, phlegmon, skin and soft tissues, vagina
 Dialister propionicifaciens2005[300]Groin abscess, wound infectionPressure ulcer, semen, abscess pus, wound
 Dysgonomonas mossii2002[302,303]Biliary drainage, abdominal drain, intestinal juice
 Leptotrichia amnionii2002[304–307]Septic arthritis, chorioamnionitis, renal abscessAmniotic fluid, joint fluid, blood, renal abscess pusPossibly female genital tract
 Parabacteroides goldsteinii (= Bacteroides goldsteinii)2005[308,309]Peritonitis, appendicitis, intra-abdominal abscessPeritoneal fluid, appendix tissue, intra-abdominal abscessProbably gastrointestinal tract
 Porphyromonas somerae2005[310]Chronic skin and soft tissue infections, osteomyelitisSkin and soft tissues, bone
 Porphyromonas uenonis2004[311]Appendicitis, peritonitis, pilonidal abscess, infected sacral decubitus ulcerStoolProbably gastrointestinal tract
 Prevotella baroniae2005[312]Endodontic and periodontal infections, dentoalveolar abscessOral cavityDental plaque
 Prevotella bergensis2006[313]Skin and soft tissue abscesses, wound infectionWound, decubitus ulcer
 Prevotella copri2007[314]StoolProbably gastrointestinal tract
 Prevotella marshii2005[312]Endodontic and periodontal infectionsOral cavitySubgingival dental plaque
 Prevotella multiformis2005[315]Chronic periodontitisSubgingival plaque
 Prevotella multisaccharivorax2005[316]Chronic periodontitisSubgingival plaque
 Sneathia sanguinegensa2001[317,318]Postpartum bacteraemiaBlood, amniotic fluidProbably female genital tract
Spirochetes
 Borrelia spielmanii (= Borrelia spielmani)2004[89]Lyme diseaseErythema migrans lesionTicksGarden dormice
 Treponema parvum2001[319]Periodontitis, acute necrotizing ulcerative gingivitis, acute and chronic apical periodontitisSubgingival plaque, acute necrotizing ulcerative gingivitis lesion, root canal

In our opinion, novel bacterial species recovered from human specimens should be reported, even if only one well-characterized strain is available. Despite extensive investigations, a microbiological cause cannot be determined in approximately half of the patients with infectious disease. For some clinical syndromes, such as neutropenic fever, no microbiological cause can be found in up to 80% of patients who are believed to be suffering from infective causes [90,91]. For some other syndromes, e.g. acute gastroenteritis and community-acquired pneumonia, the cause was undetermined in c. 40% of patients [92,93]. Over the years, tremendous efforts have been made to determine the microorganisms associated with these ‘unexplained infectious disease syndromes’. The discovery of novel aetiological agents responsible for ‘unexplained infectious disease syndromes’ relies quite heavily on the description of novel microbes. Although standard or unusual forms of known microbes are sometimes considered to be novel causes of ‘unexplained infectious disease syndromes’ [94,95], most of the novel causes are indeed previously undescribed microbes. There has been much debate on the number of strains of a particular bacterium required for description of a novel bacterial species, but our experience with the discovery and characterization of L. hongkongensis and S. sinensis suggests that any well-characterized strain of a novel bacterial species recovered from human specimens should be reported. Concerning L. hongkongensis, when the bacterium was first discovered, only one strain was obtained [76]. The description of the novel bacterium and the wide availability of molecular techniques, sophisticated databases and bioinformatics tools have made discovery of additional strains from other countries and rapid sharing of information possible [77]. This has led to rapid confirmation of its association with gastroenteritis and the determination of its reservoir [79]. In retrospect, if the first strain of L. hongkongensis had not been described, the demonstration of its association with gastroenteritis would have been hampered. Therefore, in the context of pathogenic microbes, we think that even one strain of a novel species should be described, so that global, concerted efforts can be made to identify more cases associated with that pathogen.

Detection of uncultivable bacteria and diagnosis of culture-negative infections

Bacterial culture had been the most important technique for diagnosing bacterial infections since the first days of microbiology. Indeed, Koch’s original postulates explicitly required that the pathogen be grown in a pure culture. This specific requirement has since been updated, and the clinical importance of ‘uncultivable’ bacteria, e.g. Treponema pallidum, is now well recognized. Nonetheless, given the central role of bacterial culture in most clinical laboratories, the diagnosis of culture-negative infections and those caused by uncultivable bacteria remains a somewhat arduous task. Direct microscopy and immunology-based assays have been the culture-independent methods traditionally used. The sensitivities and specificities of these methods can vary greatly, depending on the implementation and the organisms concerned. Direct microscopic examination demands a significant microbial presence in the specimen for positive identification, and serological tests suffer from cross-reactivities and may be affected by a patient’s immunocompromised status. The introduction of molecular diagnostics significantly enhanced the ability to diagnose culture-negative infections. Among the various molecular assays available, 16S rDNA sequencing stands out as a useful technique for detecting uncultivable bacteria. The universal presence and sequence conservation of the gene allows broad-range PCR primers to be readily designed. This characteristic is of great importance, as it would be impractical to perform multiple specific assays to rule out individual bacteria associated with the disease.

An important use of 16S rDNA sequencing is the identification of clinical syndromes caused by uncultivable bacteria. These relate to infections caused by bacteria that could not be cultured reliably by known methods at the time of discovery. For example, Whipple’s disease was known only as a systemic disorder of unknown aetiology when first described in 1907. Since then, gradual progress towards better diagnosis and treatment of the disease has been made, although the classic method of periodic acid–Schiff staining for diagnosis lacks both sensitivity and specificity when used alone. The first breakthrough in the understanding of Whipple’s disease arrived with the identification of Tropheryma whippeli as the causative agent. This was accomplished by the PCR amplification and sequencing of its 16S rDNA gene [96,97]. The 16S rDNA sequence allowed the phylogenetic position of T. whippeli to be defined immediately, and enabled the later development of molecular diagnostic tests [97–100]. The definitive identification of the aetiological agent greatly accelerated research into the pathophysiology of the disease. In 2003, the genome sequences of two T. whipplei strains were completed [101,102], marking another milestone in medical history. Similar successes were seen in the context of bacillary angiomatosis (caused by Bartonella henselae and Bartonella quintana) [103,104] and human ehrlichiosis (caused by bacteria of the genera Ehrlichia and Anaplasma) [105–107].

16S rDNA sequencing has also been used for diagnosing culture-negative infections, the best example being culture-negative endocarditis. Up to one-third of all cases of infective endocarditis are culture-negative [108], and diagnosis has relied mainly upon clinical and ultrasonographic findings. Goldenberger et al. reported one of the earliest situations in which 16S rDNA PCR amplification and sequencing were performed on DNA extracted from infected valves [109]. Many subsequent studies confirmed the usefulness of the method [110–119]. In general, the sensitivity was found to be comparable to that of serological testing and histopathological examination. An interesting and important result of these studies is the recognition that many culture-negative cases were caused by cultivable Gram-positive bacteria, but the blood culture results might have been negative due to previous antibiotic usage. Other factors that may contribute to the false-negative blood culture results include inadequate microbiological techniques, e.g. use of an insufficient volume of blood, or infection by a fastidious organism [120].

Diagnosis of other culture-negative infections, including meningitis [121–125], brain abscess [126], keratitis [127], urinary tract infections [128], empyema [129,130], septic arthritis [131,132] and septicaemia [120,133,134], also benefits from the use of 16S rDNA sequencing. The encouraging results from these studies reinforced the idea that 16S rDNA sequencing is broadly applicable as a diagnostic technique in the context of clinical microbiology. Future developments should involve further optimization of universal primer sets and attempts to discriminate between positive results and contamination or true pathogens. Recent progress in these areas has been made through the use of a broad-range real-time PCR design [124,135,136]. With continued improvements in performance and cost profile, 16S rDNA sequencing is expected to play an increasingly significant role in the diagnosis of culture-negative bacterial infections.

Automation of 16S rDNA sequencing

One of the greatest hurdles in putting 16S rDNA sequencing into routine use in clinical microbiology laboratories is automation of the technology. At present, complete automation of 16S rDNA sequencing, i.e. input of the bacterial isolate/clinical sample for identification or its extracted DNA into a machine that will generate the result of the identity of the isolate, is impossible. Most of the steps, including DNA extraction, PCR amplification, purification of PCR products, DNA sequencing and sequence editing, have to be performed manually, although user-friendly commercial kits for some of these steps are available. With the advances in high-throughput technologies, these steps may be incorporated into a robotic system, making complete automation of 16S rDNA sequencing possible in the future.

At present, the only steps that can be automated are the input of the finalized 16S rDNA sequence concerned into one of the software packages that contains a database with 16S rDNA sequences of selected bacterial species and the output of the result of the identity of the isolate. The most well known of these software packages include Ribosomal Database Project (RDP-II) [137–141], MicroSeq [9,48,142,143], Ribosomal Differentiation of Medical Microorganisms (RIDOM) [144–146] and the SmartGene Intergrated Database Network System (SmartGene  IDNS) [147] (Table 2). The databases of RDP-II and SmartGene  IDNS contain sequences downloaded from GenBank, whereas all sequences in the databases of RIDOM and MicroSeq were obtained by sequencing the 16S rDNA genes of bacterial strains of culture collections.

Table 2.   Commonly used software packages for bacterial identification using 16S rDNA sequencing
SoftwareYear of first description Company/OrganizationWebsitePartial/full 16S rDNA sequence includedDatabase sizeSource of sequencesCostQuality controlUpdates
  1. aIt contains two databases, MicroSeq ID 16S rDNA 500 Library v2.0, which contains sequences from the 5′- end 527 bp of 16S rDNA genes, and MicroSeq ID 16S rDNA Full Gene Library v2.0, which contains full 16S rDNA sequences.

  2. bNumber counted from http://rdna2.ridom.de/ridom2/servlet/link?page=list (18 January 2008).

  3. cStaphylococcus, Nocardia and Bacillus species were also studied [149,151,320] but were not found in the software database.

  4. dNumber obtained from reference [147].

RDP-II1991Michigan State University, USAhttp://rdp.cme.msu.edu/Partial and full>450 000 (release 9.56)GenBankNoPartialMonthly
MicroSeq microbial identification systema1998Applied Biosystems, Foster City, CA, USAhttp://www.microseq.comPartial and fulla1716 and 1261 in the two databases (version 2) respectivelyaSequence of 16S rDNA gene of one strain from each speciesYesAll type strains from culture collectionsPeriodically
RIDOM1999Ridom GmbH, Würzburg, Germanyhttp://rdna2.ridom.de/Partial240bSequence of 16S rDNA gene of bacteria in Neisseriaceae, Moraxellaceae and MycobacteriumcNoAll strains from culture collectionsPeriodically
SmartGene IDNS2006SmartGene GmbH, Switzerlandhttp://www.smartgene.ch/index.shtmlPartial and full∼112 000dGenBankYesPartialWeekly

The major studies that have evaluated the usefulness of the various software packages for different groups of bacteria are summarized in Table 3. However, it is not possible to compare accuracy among the different studies, because bacterial isolates from different sources (reference strains vs. clinical strains, identifiable strains vs. unidentified strains), different reference standards and different criteria for ‘correct’ identification were used in the different studies. In some studies, the identities of the isolates were determined by a combination of phenotypic and genotypic methods [43,142,147–151]. In others, such as a recent one on a heterogeneous group of clinical isolates, no reference standard was mentioned, except whether the isolates were ‘identified’ by MicroSeq [152]. As there are intrinsic problems with MicroSeq, e.g. inclusion of bacterial species with 16S rDNA sequences with minimal differences from those of some other species, it would be difficult for readers to assess the usefulness of MicroSeq from this study. As the intrinsic problems of the software packages may not be fully addressed in the publications, some of the reported accuracies of the software packages described may be overestimated.

Table 3.   Studies on the usefulness of commonly used software pachages for bacterial identification using 16S rDNA sequencing
ReferencesBacterial isolates testedMethods for determination of real identity of the isolatesAccuracy of software
  1. hsp65, heat shock protein 65; ATCC, American Type Culture Collection; BLAST, Basic Local Alignment Search Tool; EMBL, European Molecular Biology Laboratory; JCM, Japan Collection of Microorganisms; PRA, PCR-restriction endonuclease analysis.

  2. aFour of the 52 isolates not identified by conventional phenotypic methods were not included.

  3. bTwo of the 113 isolates not identified by phenotypic methods were not included.

  4. cIdentification was considered correct if it was the best matched species, although not a perfect match. Over 1400 bp of the 16S gene was aligned in BLAST and RDP-II, whereas Escherchia  coli bp  54–510 was used in RIDOM.

  5. dSequences resulting in distance score of <0.8%.

  6. eSimilarity ≥99.12% reported by either RIDOM or MicroSeq.

  7. fAn expanded portion of the database was developed from partial 5′ 16S rDNA sequences derived from 28 reference strains (from ATCC and JCM); samples with 99–100% similarity to a species in the expanded MicroSeq  500 library were assigned that species designation, provided that the colony morphology and growth characteristics were consistent.

  8. gPartial 16S rDNA fragment (corresponding to E. coli positions 54–510); classification is based on percentage similarity ≥98.5 to the 16S rDNA sequence of the type and reference strain.

  9. hEight of the 99 isolates not identified by phenotypic methods were not included. Eighty per cent of 30 Actinomadura, Gordona, Rhodococcus, Streptomyces and Tsukamurella species, and 65.6% of 61 Nocardia species.

  10. iEight of the 99 isolates not identified by phenotypic methods were not included. Fifty per cent of 30 Actinomadura, Gordona, Rhodococcus, Streptomyces and Tsukamurella species, and 80.3% of 61 Nocardia species.

  11. jGenus-level and species-level identifications were assigned using the following criteria: ≥99% identity to a reference entry identified a bacterium to the species level, 97.0–98.9% identity identified a bacterium to the genus level, and <97% identity to any sequence was considered to be unable to provide a definite identification.

Tang et al. [10]65 unusual aerobic Gram-negative bacilli from clinical specimensConventional phenotypic methodsMicroSeq  500: 89.2% (72% of 25 fermenters and 100% of 40 non-fermenters; 97.2% to genus level)
Tang et al. [143]52 coryneform Gram-positive bacilli (42 Corynebacterium species and 10 Corynebacterium-related species) from clinical specimensConventional phenotypic methodsaMicroSeq  500: 66.7%a (100% to genus level)
Patel et al. [142]113 Mycobacterium clinical isolates (18 different species)Combination of phenotypic methods, 16S rDNA sequencing using the MicroSeq database, and other methods, e.g. standard biochemical assays, hsp65 gene sequencing, AccuProbe rRNA hybridization, HPLC of mycolic acids. Identifications were counted as correct if two methods provided the same answerMicroSeq   500 (version 1.36): 92.8%b
Turenne et al. [321]79 non-tuberculous mycobacterial isolates (ATCC type strains)Not described, but all were ATCC strainsBLAST (GenBank and EMBL databases, dated 30 March 2001): 38%c RDP-II (dated 30 March 2001): 41%c RIDOM (dated 8 March 2001): 100%c
Cloud et al. [148]119 Mycobacterium isolates (94 clinical isolates and 25 ATCC strains)Combination of conventional phenotypic methods, 16S rDNA sequencing using MicroSeq and RIDOM databases, and additional tests on problematic isolatesMicroSeq   500 (version 1.36) in conjunction with RIDOM database: 96.6%d (100% of 25 ATCC strains and 95.7% of 94 clinical isolates)
Mellmann et al. [149]81 Nocardia clinical isolatesCombination of conventional phenotypic methods, 16S rDNA sequencing using MicroSeq and RIDOM databasesRIDOM (version 1.1): 100%e MicroSeq  500 (version 1.4.3, library version 500-0125): 44.4%e
Woo et al. [9]37 (15 aerobic or facultative anaerobic Gram-positive; 11 aerobic, micro-aerophilic or facultative anaerobic Gram-negative, seven anaerobic; three mycobacterial; and one mycoplasma) clinical isolates with ambiguous biochemical profilesCombination of phenotypic methods and full 16S rDNA sequencingMicroSeq  500 (version 1.0): 81.1% (86.5% to genus level)
Hall et al. [43]387 Mycobacterium isolates (59 ATCC strains and 328 clinical isolates)Combination of conventional phenotypic methods, HPLC, PCR restriction analysis of 65-kDa heat shock protein region, 16S rDNA sequencing using MicroSeq  500 databaseMicroSeq  500 (version 1.4.2): 68% (98.3% of 59 ATCC strains and 62.5% of 328 clinical isolates)
Cloud et al. [150]94 Nocardia clinical isolates (ten separate species)Combination of conventional phenotypic methods, 16S rDNA sequencing using expanded MicroSeq  500 database, restriction endonuclease assay for portions of 16S rDNA and hsp65 genes, sequencing of a 999-bp fragment of the 16S rDNA geneExpanded MicroSeq  500 (version 1.4.3, library version 500-0125)f: 82%
Becker et al. [151]55 clinical Staphylococcus isolatesCombination of phenotypic methods, 16S rDNA sequencing using RIDOM and NCBI databases and additional tests (chemotaxonomy and riboprinting) on isolates with ambiguous or below the-threshold RIDOM resultsRIDOM (version 1.2): 90.9%g GenBank (dated 12 January 2004): 65.5%g
Lau et al. [48]20 anaerobic Gram-positive bacilli isolated from blood culturesPreliminary phenotypic methods and full 16S rDNA sequencingMicroSeq  500 (version 1.0): 65% (80% to genus level)
Patel et al. [322]99 aerobic actinomycetes (28 reference strains and 71 clinical isolates, including members of the genera Streptomyces, Gordonia, and Tsukamurella, and ten taxa of Nocardia)Phenotyping, PRA, drug susceptibility testingMicroseq  500: 70.3%h BLAST (GenBank): 70.3%i
Fontana et al. [152]83 (25 Gram-positive and 58 Gram-negative) clinical isolates not identifiable by conventional systemsNot mentionedNot determined
Simmon et al. [147]300 (158 Gram-positive and 142 Gram-negative) clinical isolatesCombination of conventional phenotypic methods and 16S rDNA sequencing using SmartGene IDNS and MicroSeq   500 databasesSmartGene IDNS (version 3.2.3r8): 87% (98% to genus level)j MicroSeq  500 (version 1.4.3): 74% (90% to genus level)j

Usefulness and limitations of using 16s rDNA sequencing in clinical microbiology laboratories

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Use of 16s rDNA sequencing in clinical microbiology laboratories
  6. Usefulness and limitations of using 16s rDNA sequencing in clinical microbiology laboratories
  7. Concluding Remarks
  8. Transparency Declaration
  9. References

Although 16S rDNA sequencing is being used increasingly for bacterial identification in clinical microbiology laboratories, there are no widely accepted guidelines for using the technique or for the interpretation of sequence data. Given the limitations of the technique for some taxa, the ever-expanding sequence databases and taxonomic complexity, and the inaccuracies in some databases, some recommendations have been suggested for the use of 16S rDNA sequencing for bacterial identification [7,153]. For indications for the use of 16S rDNA sequencing, the strains most often chosen are those that cannot be accurately identified with phenotypic tests in clinical microbiology laboratories. However, as certain groups of bacteria are known to present difficulties in identification by 16S rDNA sequencing, these bacteria should be excluded, and other housekeeping gene targets, e.g. rpoB, if available, are required [39–42,47,50–54,153]. Regarding the interpretation of sequence data, this involves requirements concerning the length and quality of sequences, the choice of appropriate programs for analysis, and the final species assignment based on similarity search results. Although a minimum of 500–525 bp that includes the more variable 5′-region may be adequate for identification of some groups of bacteria, Drancourt et al., according to their several recommendations concerning the criteria for 16S rDNA sequencing as a reference method for bacterial identification, proposed that full 16S rDNA sequences with <1% ambiguities should be used [7]. The comparison should be made using at least 1500 positions in all sequences, of the same length, included in the similarity search, and an ungapped program should be used. In a recent review by Janda and Abbott, the use of full 16S rDNA sequences, whenever possible, and in particular for groups such as Campylobacter species [153], was confirmed as a viable technique.

The major difficulties and controversies in the interpretation of sequence data concern the assignment of bacterial species according to similarity search results, as no threshold values are available, as in the case of DNA–DNA hybridization [154]. Although a 97% similarity level has been proposed for bacterial speciation using 16S rDNA sequences [155], a >0.5% difference may be indicative of a new species [156]. In fact, as different bacterial species are likely to evolve at different rates, it is impossible to determine a universal cut-off for bacterial genus and species delineation. Although, in the study by Drancourt et al., >99% and >97% sequence similarities were used as the cut-offs for species and genus identification, respectively, the authors indicated that these sharp values were set mainly for practical purposes, for interpretation of their large sequence dataset, and it may be necessary to use different cut-off values, depending on the bacterial genus under investigation [7]. On the other hand, Janda and Abbott, in their recommended guidelines, suggested that a minimum of >99%, and ideally >99.5%, sequence similarity be used as the criteria for species identification [153]. They also proposed that for matches with distance scores <0.5% to the next closest species, other properties, e.g. phenotype, should be considered in final species identification. However, using these criteria, it is difficult to determine the species identity with sequences that have very similar distances to the closest and next closest matches, especially those within 0.5–1%. Although different studies have identified groups of bacteria for which 16S rDNA sequences are not sufficiently discriminative and for which other gene targets have to be used, very few studies have addressed and attempted to solve this problem in a systematic way.

The use of 16S rDNA sequencing for bacterial identification depends on significant interspecies differences and small intraspecies differences in 16S rDNA sequences. Therefore, one of the major limitations is that when two different bacterial species share almost the same 16S rDNA sequence, this technique would not be useful for distinguishing between them. In our experience, clinical microbiologists and technicians with limited experience in 16S rDNA sequencing often find interpretation of 16S rDNA similarity search results difficult. As a result of the large number of unvalidated 16S rDNA sequences in GenBank, it is not a straightforward task for inexperienced users to decide whether the ‘first hit’ or ‘closest match’ represents the actual identity of a bacterial isolate.

Regarding the software packages described above, the usefulness is further limited by the choice of bacterial species in the database. If a bacterial species is not included in the database, it would never be given as the identity of an isolate, and if the database also includes bacterial species with minimal differences in their 16S rDNA sequences and which, therefore, cannot be identified confidently by 16S rDNA sequencing, this may also give rise to incorrect identification.

In view of these limitations, we initiated a systematic evaluation of the potential usefulness of full and 527-bp 16S rDNA sequencing and the existing MicroSeq databases for identification of medically important bacteria 2 years ago. The species of medically important bacteria included in this analysis comprise all species of medically important bacteria listed in the Manual of Clinical Microbiology [157]. The most representative 16S rDNA sequence for each species was chosen from the GenBank database. The percentage differences of the 16S rDNA sequences among the different species in the same group/genus were determined by pairwise alignment.

The study first involved medically important anaerobic bacteria, and the results were published in 2007 [158]. Each medically important bacterial species is classified as one of the following: (i) the bacterium can be confidently identified by 16S rDNA sequencing (√ in Table 4 and Supplementary Tables 2–4 of reference [158]), meaning that there is a >3% difference between the 16S rDNA sequence of the species and those of other medically important bacteria; (ii) the bacterium cannot be confidently identified by 16S rDNA sequencing (× in Table 4 and Supplementary Tables 2–4 of reference [158]), meaning that there is a <2% difference between the 16S rDNA sequence of the species and that of a closely related medically important bacterium; and (iii) the bacterium can only be doubtfully identified by 16S rDNA sequencing (? in Table 4 and Supplementary Tables 2–4 of reference [158]), meaning that there is a 2–3% difference between the 16S rDNA sequence of the species and that of a closely related medically important bacterium. According to our guidelines for 16S rDNA sequence analysis, if the bacterium belongs to the ‘?’ or ‘×’ category (e.g. Actinomyces meyeri; Table 4 and Supplementary Tables 2–4 of reference [158]), the bacterial species with similar 16S rDNA sequences will also be known (i.e. A. georgiae, A. odontolyticus and A. turicensis; Table 4 and Supplementary Tables 2–4 of reference [158]). If further species differentiation is necessary, one can look for additional/supplementary methods, which may be key phenotypic tests or sequencing of additional gene loci, to distinguish among these species with similar 16S rDNA sequences. It should be noted that the cut-offs mentioned are not meant to be used as criteria for defining new species, but to provide a clearer meaning for the search results.

Table 4.   An example of guidelines for interpretation of usefulness of full 16S rDNA sequencing and MicroSeq full 16S rDNA bacterial identification system database for identification of medically important Actinomyces species (adapted from Supplementary Table 2 of reference [158] with permission)
Actinomyces speciesUsefulness for species identificationMedically important bacteria with similar (<3% difference) full 16S rDNA gene sequencesUsefulness of existing MicroSeq full 16S rDNA bacterial identification system database
  1. √, >3% difference between the 16S rDNA gene sequence of the species and those of other medically important bacteria; ?, 2–3% difference between the 16S rDNA gene sequence of the species and that of a closely related medically important bacterium; ×, <2% difference between the 16S rDNA gene sequence of the species and that of a closely related medically important bacterium.

  2. aThe species is not included in the existing MicroSeq full 16S rDNA bacterial identification system database.

  3. bAlthough the species is included in the existing MicroSeq full 16S rDNA bacterial identification system database, the high 16S rDNA gene sequence similarity (>98% nucleotide identity) between the species and a closely related species does not allow them to be distinguished confidently.

  4. cAlthough the species is included in the existing MicroSeq full 16S rDNA bacterial identification system database, a 2–3% difference is observed between the 16S rDNA gene of the species and a closely related species.

A. bovis?A. urogenitalis?c
A. bowdeni ?A. viscosus×a
A. canis ×a
A. catuli ×a
A. denticolens ×a
A. europaeu ×a
A. funkei?A. hyovaginalis×a
A. georgiae?A. meyeri×a
A. gerencseriae ×a
A. graevenitzii  ×a
A. hordeovulneris ×a
A. howellii 
A. hyovaginalis?A. funkei×a
A. israelii ×a
A. meyeri?A. georgiae, A. odontolyticus, A. turicensis?c
A. naeslundii×A. viscosus×a
A. neuii 
A. odontolyticus×A. meyeri, A. turicensis×a
A. radicidentis ×a
A. radingae 
A. slackii 
A. turicensis×A. meyeri, A. odontolyticus×a
A. urogenitalis?A. bovis×a
A. viscosus×A. bowdenii, A. naeslundii×b

Concerning MicroSeq database analysis, a supplementary note is given to indicate whether the reason for the inability of the database to identify the bacterium is due to the species being not included in the existing database (xa in Table 4 and Supplementary Tables 2–4 of reference [158]), or high 16S rDNA sequence similarity (>98% nucleotide identity) to a closely related species (xb in Table 4 and Supplementary Tables 2–4 of reference [158]). In the study on anaerobes, full and 527-bp 16S rDNA sequencing were capable of identifying 52–63% of 130 anaerobic Gram-positive rods, 72–73% of 86 anaerobic Gram-negative rods, and 78% of 23 anaerobic cocci. Surprisingly, the MicroSeq databases (version 1.0) were able to identify only 19–25% of 130 Gram-positive anaerobic rods, 38% of 86 Gram-negative anaerobic rods, and 39% of 23 anaerobic cocci. This represents only 45–46% of those that should be confidently identified by full-length and 527-bp 16S rDNA sequencing, indicating that, in order to improve the usefulness of MicroSeq, bacterial species that should be confidently identified by full-length and 527-bp 16S rDNA sequencing but are not included in the MicroSeq databases (version 1.0) should be included. Recently, there has been an expansion of the MicroSeq databases (version 2.0) to include more bacterial species, which has led to some improvement. At present, we are performing further studies of aerobic and facultative anaerobic Gram-positive and Gram-negative bacteria, and the results will be available in the near future for the development of similar guidelines.

Despite the extensive analysis, there are still limitations in these guidelines. First, intraspecies variation in 16S rDNA sequences was not taken into account in the analysis. In the evaluation, only whether there was a significant difference between the 16S rDNA sequence of a particular species and those of other species was determined, and a 3% difference was chosen as the cut-off. Whether there is a high similarity among the 16S rDNA sequences in different strains of the same species is difficult to study, because: (i) 16S rDNA sequence information is available for multiple strains of the same species in only a minority of species; and (ii) even when 16S rDNA sequence information is available for multiple strains of the same species, the strains are often not well characterized phenotypically, so the reliability of the sequence information is difficult to judge. However, despite this limitation, a high degree of conservation of 16S rDNA sequences for the same species is often assumed, because most existing results do not indicate otherwise. On the other hand, for some genera, the intraspecies variation in 16S rDNA sequences may be so small that a difference of 3% in the 16S rDNA sequences between two species may not be necessary for confident identification. Second, the present study included only the bacterial species that are known to be associated with infections. This was deliberate, because, if those bacterial species that have never been reported to cause infections had also been included, some species may have been classified as ‘not confidently identified by 16S rDNA sequencing’ because of the sequence similarity between a bacterium and another that has never been reported to cause infection. One must bear in mind that those species that have never been reported to be associated with infections may still have the potential to do so. Owing to these limitations, we suggest that, in order to increase the accuracy of 16S rDNA sequencing for identification of pathogenic bacteria, it would be necessary to interpret the results of 16S rDNA sequencing with preliminary phenotypic test results. Nevertheless, our data not only facilitate interpretation of sequence data by inexperienced users in the clinical microbiology laboratories, but also provide clues to the potential usefulness of 16S rDNA sequencing for different groups of bacteria before they are chosen for such analysis.

Concluding Remarks

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Use of 16s rDNA sequencing in clinical microbiology laboratories
  6. Usefulness and limitations of using 16s rDNA sequencing in clinical microbiology laboratories
  7. Concluding Remarks
  8. Transparency Declaration
  9. References

Not only has 16S rDNA sequencing contributed to answering some of the most fundamental questions in biology, but during the last decade this technology has evolved beyond the research realm and matured into clinical applications. For the management of individual patients, accurate and objective identification of clinical isolates, especially ‘unidentifiable bacteria’, rarely encountered bacteria, slow-growing bacteria and uncultivable bacteria, has assisted clinicians in the choice of antibiotics and in determining the duration of treatment, as well as in infection control measures. On a population scale, accurate identification has greatly improved our understanding of the epidemiology of clinical syndromes, and hence has improved empirical treatment of these. Our understanding of all these aspects is further improved by the discovery of novel genera and species of bacteria, which has been greatly facilitated by using 16S rDNA sequencing. Nevertheless, automation of 16S rDNA sequencing is not yet available and, despite the wide range of software packages and databases available, interpretation of results is often difficult for inexperienced users, due to their limitations. Hopefully, the generation of guidelines for the interpretation of 16S rDNA sequences can make this technology an even more user-friendly tool in clinical microbiology laboratories in the coming years.

Transparency Declaration

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Use of 16s rDNA sequencing in clinical microbiology laboratories
  6. Usefulness and limitations of using 16s rDNA sequencing in clinical microbiology laboratories
  7. Concluding Remarks
  8. Transparency Declaration
  9. References

This work was partly supported by the Research Grant Council Grant, University Development Fund, Outstanding Young Researcher Award, HKU Special Research Achievement Award and The Croucher Senior Medical Research Fellowship, The University of Hong Kong. The authors declare no competing interests.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Use of 16s rDNA sequencing in clinical microbiology laboratories
  6. Usefulness and limitations of using 16s rDNA sequencing in clinical microbiology laboratories
  7. Concluding Remarks
  8. Transparency Declaration
  9. References
  • 1
    Fox GE, Magrum LJ, Balch WE, Wolfe RS, Woese CR. Classification of methanogenic bacteria by 16S ribosomal RNA characterization. Proc Natl Acad Sci USA 1977; 74: 45374541.
  • 2
    Gupta R, Lanter JM, Woese CR. Sequence of the 16S ribosomal RNA from Halobacterium volcanii, an Archaebacterium. Science 1983; 221: 656659.
  • 3
    Woese CR, Kandler O, Wheelis ML. Towards a natural system of organisms: proposal for the domains Archaea, Bacteria, and Eucarya. Proc Natl Acad Sci USA 1990; 87: 45764579.
  • 4
    Snel B, Bork P, Huynen MA. Genome phylogeny based on gene content. Nat Genet 1999; 21: 108110.
  • 5
    Poyart C, Quesne G, Trieu-Cuot P. Taxonomic dissection of the Streptococcus bovis group by analysis of manganese-dependent superoxide dismutase gene (sodA) sequences: reclassification of ‘Streptococcus infantarius subsp. coli’ as Streptococcus lutetiensis sp. nov. and of Streptococcus bovis biotype 11.2 as Streptococcus pasteurianus sp. nov. Int J Syst Evol Microbiol 2002; 52: 12471255.
  • 6
    Arbique JC, Poyart C, Trieu-Cuot P et al. Accuracy of phenotypic and genotypic testing for identification of Streptococcus pneumoniae and description of Streptococcus pseudopneumoniae sp. nov. J Clin Microbiol 2004; 42: 46864696.
  • 7
    Drancourt M, Bollet C, Carlioz A, Martelin R, Gayral JP, Raoult D. 16S ribosomal DNA sequence analysis of a large collection of environmental and clinical unidentifiable bacterial isolates. J Clin Microbiol 2000; 38: 36233630.
  • 8
    Mignard S, Flandrois JP. 16S rRNA sequencing in routine bacterial identification: a 30-month experiment. J Microbiol Methods 2006; 67: 574581.
  • 9
    Woo PC, Ng KH, Lau SK et al. Usefulness of the MicroSeq  500 16S ribosomal DNA-based bacterial identification system for identification of clinically significant bacterial isolates with ambiguous biochemical profiles. J Clin Microbiol 2003; 41: 19962001.
  • 10
    Tang YW, Ellis NM, Hopkins MK, Smith DH, Dodge DE, Persing DH. Comparison of phenotypic and genotypic techniques for identification of unusual aerobic pathogenic gram-negative bacilli. J Clin Microbiol 1998; 36: 36743679.
  • 11
    Woo PC, Chong KT, Leung K, Que T, Yuen K. Identification of Arcobacter cryaerophilus isolated from a traffic accident victim with bacteremia by 16S ribosomal RNA gene sequencing. Diagn Microbiol Infect Dis 2001; 40: 125127.
  • 12
    Lau SK, Woo PC, Teng JL, Leung KW, Yuen KY. Identification by 16S ribosomal RNA gene sequencing of Arcobacter butzleri bacteraemia in a patient with acute gangrenous appendicitis. Mol Pathol 2002; 55: 182185.
  • 13
    Lau SK, Woo PC, Woo GK, Yuen KY. Catheter-related Microbacterium bacteremia identified by 16S rRNA gene sequencing. J Clin Microbiol 2002; 40: 26812685.
  • 14
    Elshibly S, Xu J, McClurg RB et al. Central line-related bacteremia due to Roseomonas mucosa in a patient with diffuse large B-cell non-Hodgkin’s lymphoma. Leuk Lymphoma 2005; 46: 611614.
  • 15
    Woo PC, Fung AM, Lau SK et al. Granulicatella adiacens and Abiotrophia defectiva bacteraemia characterized by 16S rRNA gene sequencing. J Med Microbiol 2003; 52: 137140.
  • 16
    Woo PC, Lau SK, Fung AM, Chiu SK, Yung RW, Yuen KY. Gemella bacteraemia characterised by 16S ribosomal RNA gene sequencing. J Clin Pathol 2003; 56: 690693.
  • 17
    Woo PC, Tse H, Wong SS et al. Life-threatening invasive Helcococcus kunzii infections in intravenous-drug users and ermA-mediated erythromycin resistance. J Clin Microbiol 2005; 43: 62056208.
  • 18
    Lau SK, Woo PC, Woo GK et al. Bacteraemia caused by Anaerotruncus colihominis and emended description of the species. J Clin Pathol 2006; 59: 748752.
  • 19
    Somers CJ, Millar BC, Xu J et al. Haemophilus segnis: a rare cause of endocarditis. Clin Microbiol Infect 2003; 9: 10481050.
  • 20
    Lau SK, Woo PC, Li NK et al. Globicatella bacteraemia identified by 16S ribosomal RNA gene sequencing. J Clin Pathol 2006; 59: 303307.
  • 21
    Lau SK, Teng JL, Leung KW et al. Bacteremia caused by Solobacterium moorei in a patient with acute proctitis and carcinoma of the cervix. J Clin Microbiol 2006; 44: 30313034.
  • 22
    Woo PC, Lau SK, Lin AW, Curreem SO, Fung AM, Yuen KY. Surgical site abscess caused by Lactobacillus fermentum identified by 16S ribosomal RNA gene sequencing. Diagn Microbiol Infect Dis 2007; 58: 251254.
  • 23
    Weinstein MR, Litt M, Kertesz DA et al. Invasive infections due to a fish pathogen, Streptococcus iniae. N Engl J Med 1997; 337: 589594.
  • 24
    Lau SK, Woo PC, Tse H, Leung KW, Wong SS, Yuen KY. Invasive Streptococcus iniae infections outside North America. J Clin Microbiol 2003; 41: 10041009.
  • 25
    Koh TH, Kurup A, Chen J. Streptococcus iniae discitis in Singapore. Emerg Infect Dis 2004; 10: 16941696.
  • 26
    Lau SK, Woo PC, Luk WK et al. Clinical isolates of Streptococcus iniae from Asia are more mucoid and beta-hemolytic than those from North America. Diagn Microbiol Infect Dis 2006; 54: 177181.
  • 27
    Sun JR, Yan JC, Yeh CY, Lee SY, Lu JJ. Invasive infection with Streptococcus iniae in Taiwan. J Med Microbiol 2007; 56: 12461249.
  • 28
    Woo PC, Leung KW, Tsoi HW, Wong SS, Teng JL, Yuen KY. Thermo-tolerant Campylobacter fetus bacteraemia identified by 16S ribosomal RNA gene sequencing: an emerging pathogen in immunocompromised patients. J Med Microbiol 2002; 51: 740746.
  • 29
    Woo PC, Tsoi HW, Leung KW et al. Identification of Mycobacterium neoaurum isolated from a neutropenic patient with catheter-related bacteremia by 16S rRNA sequencing. J Clin Microbiol 2000; 38: 35153517.
  • 30
    Woo PC, Leung PK, Leung KW, Yuen KY. Identification by 16S ribosomal RNA gene sequencing of an Enterobacteriaceae species from a bone marrow transplant recipient. Mol Pathol 2000; 53: 211215.
  • 31
    Woo PC, Leung AS, Leung KW, Yuen KY. Identification of slide coagulase positive, tube coagulase negative Staphylococcus aureus by 16S ribosomal RNA gene sequencing. Mol Pathol 2001; 54: 244247.
  • 32
    Lefevre P, Gilot P, Godiscal H, Content J, Fauville-Dufaux M. Mycobacterium intracellulare as a cause of a recurrent granulomatous tenosynovitis of the hand. Diagn Microbiol Infect Dis 2000; 38: 127129.
  • 33
    Chen PL, Lee NY, Yan JJ et al. Prosthetic valve endocarditis caused by Streptobacillus moniliformis: a case of rat bite fever. J Clin Microbiol 2007; 45: 31253126.
  • 34
    Woo PC, Cheung EY, Leung K, Yuen K. Identification by 16S ribosomal RNA gene sequencing of an Enterobacteriaceae species with ambiguous biochemical profile from a renal transplant recipient. Diagn Microbiol Infect Dis 2001; 39: 8593.
  • 35
    Clarridge JE 3rd, Raich TJ, Sjosted A et al. Characterization of two unusual clinically significant Francisella strains. J Clin Microbiol 1996; 34: 19952000.
  • 36
    Heller R, Jaulhac B, Charles P et al. Identification of Mycobacterium shimoidei in a tuberculosis-like cavity by 16S ribosomal DNA direct sequencing. Eur J Clin Microbiol Infect Dis 1996; 15: 172175.
  • 37
    Woo PC, Li JH, Tang W, Yuen K. Acupuncture mycobacteriosis. N Engl J Med 2001; 345: 842843.
  • 38
    Woo PC, Leung KW, Wong SS, Chong KT, Cheung EY, Yuen KY. Relatively alcohol-resistant mycobacteria are emerging pathogens in patients receiving acupuncture treatment. J Clin Microbiol 2002; 40: 12191224.
  • 39
    Roth A, Fischer M, Hamid ME, Michalke S, Ludwig W, Mauch H. Differentiation of phylogenetically related slowly growing mycobacteria based on 16S–23S rRNA gene internal transcribed spacer sequences. J Clin Microbiol 1998; 36: 139147.
  • 40
    Ringuet H, Akoua-Koffi C, Honore S et al. hsp65 sequencing for identification of rapidly growing mycobacteria. J Clin Microbiol 1999; 37: 852857.
  • 41
    Kasai H, Ezaki T, Harayama S. Differentiation of phylogenetically related slowly growing mycobacteria by their gyrB sequences. J Clin Microbiol 2000; 38: 301308.
  • 42
    Adekambi T, Berger P, Raoult D, Drancourt M. rpoB gene sequence-based characterization of emerging non-tuberculous mycobacteria with descriptions of Mycobacterium bolletii sp. nov., Mycobacterium phocaicum sp. nov. and Mycobacterium aubagnense sp. nov. Int J Syst Evol Microbiol 2006; 56: 133143.
  • 43
    Hall L, Doerr KA, Wohlfiel SL, Roberts GD. Evaluation of the MicroSeq system for identification of mycobacteria by 16S ribosomal DNA sequencing and its integration into a routine clinical mycobacteriology laboratory. J Clin Microbiol 2003; 41: 14471453.
  • 44
    Bosshard PP, Abels S, Zbinden R, Bottger EC, Altwegg M. Ribosomal DNA sequencing for identification of aerobic gram-positive rods in the clinical laboratory (an 18-month evaluation). J Clin Microbiol 2003; 41: 41344140.
  • 45
    Bosshard PP, Abels S, Altwegg M, Bottger EC, Zbinden R. Comparison of conventional and molecular methods for identification of aerobic catalase-negative gram-positive cocci in the clinical laboratory. J Clin Microbiol 2004; 42: 20652073.
  • 46
    Song Y, Liu C, Bolanos M, Lee J, McTeague M, Finegold SM. Evaluation of 16S rRNA sequencing and reevaluation of a short biochemical scheme for identification of clinically significant Bacteroides species. J Clin Microbiol 2005; 43: 15311537.
  • 47
    Heikens E, Fleer A, Paauw A, Florijn A, Fluit AC. Comparison of genotypic and phenotypic methods for species-level identification of clinical isolates of coagulase-negative staphylococci. J Clin Microbiol 2005; 43: 22862290.
  • 48
    Lau SK, Ng KH, Woo PC et al. Usefulness of the MicroSeq  500 16S rDNA bacterial identification system for identification of anaerobic Gram positive bacilli isolated from blood cultures. J Clin Pathol 2006; 59: 219222.
  • 49
    Bosshard PP, Zbinden R, Abels S, Boddinghaus B, Altwegg M, Bottger EC. 16S rRNA gene sequencing versus the API  20 NE system and the VITEK  2 ID-GNB card for identification of nonfermenting Gram-negative bacteria in the clinical laboratory. J Clin Microbiol 2006; 44: 13591366.
  • 50
    Kwok AY, Chow AW. Phylogenetic study of Staphylococcus and Macrococcus species based on partial hsp60 gene sequences. Int J Syst Evol Microbiol 2003; 53: 8792.
  • 51
    Kwok AY, Su SC, Reynolds RP et al. Species identification and phylogenetic relationships based on partial HSP60 gene sequences within the genus Staphylococcus. Int J Syst Bacteriol 1999; 49 (Pt 3): 11811192.
  • 52
    Woo PC, Woo GK, Lau SK, Wong SS, Yuen K. Single gene target bacterial identification. groEL gene sequencing for discriminating clinical isolates of Burkholderia pseudomallei and Burkholderia thailandensis. Diagn Microbiol Infect Dis 2002; 44: 143149.
  • 53
    Woo PC, Lau SK, Woo GK et al. Seronegative bacteremic melioidosis caused by Burkholderia pseudomallei with ambiguous biochemical profile: clinical importance of accurate identification by 16S rRNA gene and groEL gene sequencing. J Clin Microbiol 2003; 41: 39733977.
  • 54
    Goh SH, Potter S, Wood JO, Hemmingsen SM, Reynolds RP, Chow AW. HSP60 gene sequences as universal targets for microbial species identification: studies with coagulase-negative staphylococci. J Clin Microbiol 1996; 34: 818823.
  • 55
    Lau SK, Woo PC, Woo GK et al. Eggerthella hongkongensis sp. nov. and Eggerthella sinensis sp. nov., two novel Eggerthella species, account for half of the cases of Eggerthella bacteremia. Diagn Microbiol Infect Dis 2004; 49: 255263.
  • 56
    Lau SK, Woo PC, Fung AM, Chan KM, Woo GK, Yuen KY. Anaerobic, non-sporulating, Gram-positive bacilli bacteraemia characterized by 16S rRNA gene sequencing. J Med Microbiol 2004; 53: 12471253.
  • 57
    Bosshard PP, Zbinden R, Altwegg M. Turicibacter sanguinis gen. nov., sp. nov., a novel anaerobic, Gram-positive bacterium. Int J Syst Evol Microbiol 2002; 52: 12631266.
  • 58
    Woo PC, Lau SK, Chan KM, Fung AM, Tang BS, Yuen KY. Clostridium bacteraemia characterised by 16S ribosomal RNA gene sequencing. J Clin Pathol 2005; 58: 301307.
  • 59
    Lau SK, McNabb A, Woo GK et al. Catabacter hongkongensis gen. nov., sp. nov., isolated from blood cultures of patients from Hong Kong and Canada. J Clin Microbiol 2007; 45: 395401.
  • 60
    Woo PC, Fung AM, Lau SK, Wong SS, Yuen KY. Group G beta-hemolytic streptococcal bacteremia characterized by 16S ribosomal RNA gene sequencing. J Clin Microbiol 2001; 39: 31473155.
  • 61
    Lam MM, Clarridge JE 3rd, Young EJ, Mizuki S. The other group G Streptococcus: increased detection of Streptococcus canis ulcer infections in dog owners. J Clin Microbiol 2007; 45: 23272329.
  • 62
    Woo PC, Tse H, Chan KM et al. Streptococcus milleri’ endocarditis caused by Streptococcus anginosus. Diagn Microbiol Infect Dis 2004; 48: 8188.
  • 63
    Lau SK, Woo PC, Mok MY et al. Characterization of Haemophilus segnis, an important cause of bacteremia, by 16S rRNA gene sequencing. J Clin Microbiol 2004; 42: 877880.
  • 64
    Lau SK, Woo PC, Chan BY, Fung AM, Que TL, Yuen KY. Haemophilus segnis polymicrobial and monomicrobial bacteraemia identified by 16S ribosomal RNA gene sequencing. J Med Microbiol 2002; 51: 635640.
  • 65
    Woo PC, Tam DM, Lau SK, Fung AM, Yuen KY. Enterococcus cecorum empyema thoracis successfully treated with cefotaxime. J Clin Microbiol 2004; 42: 919922.
  • 66
    Colmegna I, Rodriguez-Barradas M, Rauch R, Clarridge J, Young EJ. Disseminated Actinomyces meyeri infection resembling lung cancer with brain metastases. Am J Med Sci 2003; 326: 152155.
  • 67
    Woo PC, Fung AM, Lau SK, Yuen KY. Identification by 16S rRNA gene sequencing of Lactobacillus salivarius bacteremic cholecystitis. J Clin Microbiol 2002; 40: 265267.
  • 68
    Woo PC, Fung AM, Lau SK, Hon E, Yuen KY. Diagnosis of pelvic actinomycosis by 16S ribosomal RNA gene sequencing and its clinical significance. Diagn Microbiol Infect Dis 2002; 43: 113118.
  • 69
    Bik EM, Eckburg PB, Gill SR et al. Molecular analysis of the bacterial microbiota in the human stomach. Proc Natl Acad Sci USA 2006; 103: 732737.
  • 70
    Eckburg PB, Bik EM, Bernstein CN et al. Diversity of the human intestinal microbial flora. Science 2005; 308: 16351638.
  • 71
    Woo PC, Tam DM, Leung KW et al. Streptococcus sinensis sp. nov., a novel species isolated from a patient with infective endocarditis. J Clin Microbiol 2002; 40: 805810.
  • 72
    Woo PC, Teng JL, Leung KW et al. Streptococcus sinensis may react with Lancefield group F antiserum. J Med Microbiol 2004; 53: 10831088.
  • 73
    Woo PC, Teng JL, Lau SK, Yuen KY. Clinical, phenotypic, and genotypic evidence for Streptococcus sinensis as the common ancestor of anginosus and mitis groups of streptococci. Med Hypotheses 2006; 66: 345351.
  • 74
    Uckay I, Rohner P, Bolivar I et al. Streptococcus sinensis endocarditis outside Hong Kong. Emerg Infect Dis 2007; 13: 12501252.
  • 75
    Faibis F, Mihaila L, Perna S et al. Streptococcus sinensis: an emerging agent of infective endocarditis. J Med Microbiol 2008; 57: 528531.
  • 76
    Yuen KY, Woo PC, Teng JL, Leung KW, Wong MK, Lau SK. Laribacter hongkongensis gen. nov., sp. nov., a novel gram-negative bacterium isolated from a cirrhotic patient with bacteremia and empyema. J Clin Microbiol 2001; 39: 42274232.
  • 77
    Woo PC, Kuhnert P, Burnens AP et al. Laribacter hongkongensis: a potential cause of infectious diarrhea. Diagn Microbiol Infect Dis 2003; 47: 551556.
  • 78
    Lau SK, Woo PC, Hui WT et al. Use of cefoperazone MacConkey agar for selective isolation of Laribacter hongkongensis. J Clin Microbiol 2003; 41: 48394841.
  • 79
    Woo PC, Lau SK, Teng JL et al. Association of Laribacter hongkongensis in community-acquired gastroenteritis with travel and eating fish: a multicentre case-control study. Lancet 2004; 363: 19411947.
  • 80
    Teng JL, Woo PC, Ma SS et al. Ecoepidemiology of Laribacter hongkongensis, a novel bacterium associated with gastroenteritis. J Clin Microbiol 2005; 43: 919922.
  • 81
    Woo PC, Lau SK, Teng JL, Yuen KY. Current status and future directions for Laribacter hongkongensis, a novel bacterium associated with gastroenteritis and traveller’s diarrhoea. Curr Opin Infect Dis 2005; 18: 413419.
  • 82
    Lau SK, Woo PC, Fan RY, Lee RC, Teng JL, Yuen KY. Seasonal and tissue distribution of Laribacter hongkongensis, a novel bacterium associated with gastroenteritis, in retail freshwater fish in Hong Kong. Int J Food Microbiol 2007; 113: 6266.
  • 83
    Ni XP, Ren SH, Sun JR et al. Laribacter hongkongensis isolated from a patient with community-acquired gastroenteritis in Hangzhou City. J Clin Microbiol 2007; 45: 255256.
  • 84
    Lau SK, Woo PC, Fan RY et al. Isolation of Laribacter hongkongensis, a novel bacterium associated with gastroenteritis, from drinking water reservoirs in Hong Kong. J Appl Microbiol 2007; 103: 507515.
  • 85
    Steer T, Collins MD, Gibson GR, Hippe H, Lawson PA. Clostridium hathewayi sp. nov., from human faeces. Syst Appl Microbiol 2001; 24: 353357.
  • 86
    Elsayed S, Zhang K. Human infection caused by Clostridium hathewayi. Emerg Infect Dis 2004; 10: 19501952.
  • 87
    Woo PC, Lau SK, Woo GK, Fung AM, Yiu VP, Yuen KY. Bacteremia due to Clostridium hathewayi in a patient with acute appendicitis. J Clin Microbiol 2004; 42: 59475949.
  • 88
    Linscott AJ, Flamholtz RB, Shukla D, Song Y, Liu C, Finegold SM. Fatal septicemia due to Clostridium hathewayi and Campylobacter hominis. Anaerobe 2005; 11: 9798.
  • 89
    Richter D, Schlee DB, Allgower R, Matuschka FR. Relationships of a novel Lyme disease spirochete, Borrelia spielmani sp. nov., with its hosts in Central Europe. Appl Environ Microbiol 2004; 70: 64146419.
  • 90
    Pizzo PA. Management of fever in patients with cancer and treatment-induced neutropenia. N Engl J Med 1993; 328: 13231332.
  • 91
    Yuen KY, Woo PC, Hui CH et al. Unique risk factors for bacteraemia in allogeneic bone marrow transplant recipients before and after engraftment. Bone Marrow Transplant 1998; 21: 11371143.
  • 92
    Uhnoo I, Wadell G, Svensson L, Olding-Stenkvist E, Ekwall E, Molby R. Aetiology and epidemiology of acute gastro-enteritis in Swedish children. J Infect 1986; 13: 7389.
  • 93
    Macfarlane JT, Colville A, Guion A, Macfarlane RM, Rose DH. Prospective study of aetiology and outcome of adult lower-respiratory-tract infections in the community. Lancet 1993; 341: 511514.
  • 94
    Woo PC, Wong SS, Lum PN, Hui WT, Yuen KY. Cell-wall-deficient bacteria and culture-negative febrile episodes in bone-marrow-transplant recipients. Lancet 2001; 357: 675679.
  • 95
    Woo PC, Ngan AH, Lau SK, Yuen KY. Tsukamurella conjunctivitis: a novel clinical syndrome. J Clin Microbiol 2003; 41: 33683371.
  • 96
    Wilson KH, Blitchington R, Frothingham R, Wilson JA. Phylogeny of the Whipple’s-disease-associated bacterium. Lancet 1991; 338: 474475.
  • 97
    Relman DA, Schmidt TM, MacDermott RP, Falkow S. Identification of the uncultured bacillus of Whipple’s disease. N Engl J Med 1992; 327: 293301.
  • 98
    Fenollar F, Raoult D. Molecular techniques in Whipple’s disease. Expert Rev Mol Diagn 2001; 1: 299309.
  • 99
    Fenollar F, Fournier PE, Raoult D, Gerolami R, Lepidi H, Poyart C. Quantitative detection of Tropheryma whipplei DNA by real-time PCR. J Clin Microbiol 2002; 40: 11191120.
  • 100
    Fenollar F, Fournier PE, Robert C, Raoult D. Use of genome selected repeated sequences increases the sensitivity of PCR detection of Tropheryma whipplei. J Clin Microbiol 2004; 42: 401403.
  • 101
    Bentley SD, Maiwald M, Murphy LD et al. Sequencing and analysis of the genome of the Whipple’s disease bacterium Tropheryma whipplei. Lancet 2003; 361: 637644.
  • 102
    Raoult D, Ogata H, Audic S et al. Tropheryma whipplei Twist: a human pathogenic Actinobacteria with a reduced genome. Genome Res 2003; 13: 18001809.
  • 103
    Relman DA, Loutit JS, Schmidt TM, Falkow S, Tompkins LS. The agent of bacillary angiomatosis. An approach to the identification of uncultured pathogens. N Engl J Med 1990; 323: 15731580.
  • 104
    Regnery RL, Anderson BE, Clarridge JE 3rd, Rodriguez-Barradas MC, Jones DC, Carr JH. Characterization of a novel Rochalimaea species, R. henselae sp. nov., isolated from blood of a febrile, human immunodeficiency virus-positive patient. J Clin Microbiol 1992; 30: 265274.
  • 105
    Maeda K, Markowitz N, Hawley RC, Ristic M, Cox D, McDade JE. Human infection with Ehrlichia canis, a leukocytic rickettsia. N Engl J Med 1987; 316: 853856.
  • 106
    Anderson BE, Dawson JE, Jones DC, Wilson KH. Ehrlichia chaffeensis, a new species associated with human ehrlichiosis. J Clin Microbiol 1991; 29: 28382842.
  • 107
    Chen SM, Dumler JS, Bakken JS, Walker DH. Identification of a granulocytotropic Ehrlichia species as the etiologic agent of human disease. J Clin Microbiol 1994; 32: 589595.
  • 108
    Kupferwasser LI, Darius H, Muller AM et al. Diagnosis of culture-negative endocarditis: the role of the Duke criteria and the impact of transesophageal echocardiography. Am Heart J 2001; 142: 146152.
  • 109
    Goldenberger D, Kunzli A, Vogt P, Zbinden R, Altwegg M. Molecular diagnosis of bacterial endocarditis by broad-range PCR amplification and direct sequencing. J Clin Microbiol 1997; 35: 27332739.
  • 110
    Wilck MB, Wu Y, Howe JG, Crouch JY, Edberg SC. Endocarditis caused by culture-negative organisms visible by Brown and Brenn staining: utility of PCR and DNA sequencing for diagnosis. J Clin Microbiol 2001; 39: 20252027.
  • 111
    Millar B, Moore J, Mallon P et al. Molecular diagnosis of infective endocarditis—a new Duke’s criterion. Scand J Infect Dis 2001; 33: 673680.
  • 112
    Gauduchon V, Chalabreysse L, Etienne J et al. Molecular diagnosis of infective endocarditis by PCR amplification and direct sequencing of DNA from valve tissue. J Clin Microbiol 2003; 41: 763766.
  • 113
    Grijalva M, Horvath R, Dendis M, Erny J, Benedik J. Molecular diagnosis of culture negative infective endocarditis: clinical validation in a group of surgically treated patients. Heart 2003; 89: 263268.
  • 114
    Bosshard PP, Kronenberg A, Zbinden R, Ruef C, Bottger EC, Altwegg M. Etiologic diagnosis of infective endocarditis by broad-range polymerase chain reaction: a 3-year experience. Clin Infect Dis 2003; 37: 167172.
  • 115
    Lang S, Watkin RW, Lambert PA, Bonser RS, Littler WA, Elliott TS. Evaluation of PCR in the molecular diagnosis of endocarditis. J Infect 2004; 48: 269275.
  • 116
    Lang S, Watkin RW, Lambert PA, Littler WA, Elliott TS. Detection of bacterial DNA in cardiac vegetations by PCR after the completion of antimicrobial treatment for endocarditis. Clin Microbiol Infect 2004; 10: 579581.
  • 117
    Breitkopf C, Hammel D, Scheld HH, Peters G, Becker K. Impact of a molecular approach to improve the microbiological diagnosis of infective heart valve endocarditis. Circulation 2005; 111: 14151421.
  • 118
    Houpikian P, Raoult D. Blood culture-negative endocarditis in a reference center: etiologic diagnosis of 348 cases. Medicine (Baltimore) 2005; 84: 162173.
  • 119
    Kotilainen P, Heiro M, Jalava J et al. Aetiological diagnosis of infective endocarditis by direct amplification of rRNA genes from surgically removed valve tissue. An 11-year experience in a Finnish teaching hospital. Ann Med 2006; 38: 263273.
  • 120
    Cursons RT, Jeyerajah E, Sleigh JW. The use of polymerase chain reaction to detect septicemia in critically ill patients. Crit Care Med 1999; 27: 937940.
  • 121
    Xu J, Millar BC, Moore JE et al. Employment of broad-range 16S rRNA PCR to detect aetiological agents of infection from clinical specimens in patients with acute meningitis—rapid separation of 16S rRNA PCR amplicons without the need for cloning. J Appl Microbiol 2003; 94: 197206.
  • 122
    Schuurman T, De Boer RF, Kooistra-Smid AM, Van Zwet AA. Prospective study of use of PCR amplification and sequencing of 16S ribosomal DNA from cerebrospinal fluid for diagnosis of bacterial meningitis in a clinical setting. J Clin Microbiol 2004; 42: 734740.
  • 123
    Xu J, Moore JE, Millar BC, Webb H, Shields MD, Goldsmith CE. Employment of broad range 16S rDNA PCR and sequencing in the detection of aetiological agents of meningitis. New Microbiol 2005; 28: 135143.
  • 124
    Deutch S, Pedersen LN, Podenphant L et al. Broad-range real time PCR and DNA sequencing for the diagnosis of bacterial meningitis. Scand J Infect Dis 2006; 38: 2735.
  • 125
    Welinder-Olsson C, Dotevall L, Hogevik H et al. Comparison of broad-range bacterial PCR and culture of cerebrospinal fluid for diagnosis of community-acquired bacterial meningitis. Clin Microbiol Infect 2007; 13: 879886.
  • 126
    Tsai JC, Teng LJ, Hsueh PR. Direct detection of bacterial pathogens in brain abscesses by polymerase chain reaction amplification and sequencing of partial 16S ribosomal deoxyribonucleic acid fragments. Neurosurgery 2004; 55: 11541162.
  • 127
    Knox CM, Cevellos V, Dean D. 16S ribosomal DNA typing for identification of pathogens in patients with bacterial keratitis. J Clin Microbiol 1998; 36: 34923496.
  • 128
    Domann E, Hong G, Imirzalioglu C et al. Culture-independent identification of pathogenic bacteria and polymicrobial infections in the genitourinary tract of renal transplant recipients. J Clin Microbiol 2003; 41: 55005510.
  • 129
    Saglani S, Harris KA, Wallis C, Hartley JC. Empyema: the use of broad range 16S rDNA PCR for pathogen detection. Arch Dis Child 2005; 90: 7073.
  • 130
    Le Monnier A, Carbonnelle E, Zahar JR et al. Microbiological diagnosis of empyema in children: comparative evaluations by culture, polymerase chain reaction, and pneumococcal antigen detection in pleural fluids. Clin Infect Dis 2006; 42: 11351140.
  • 131
    Rosey AL, Abachin E, Quesnes G et al. Development of a broad-range 16S rDNA real-time PCR for the diagnosis of septic arthritis in children. J Microbiol Methods 2007; 68: 8893.
  • 132
    Fihman V, Hannouche D, Bousson V et al. Improved diagnosis specificity in bone and joint infections using molecular techniques. J Infect 2007; 55: 510517.
  • 133
    Sleigh J, Cursons R, La Pine M. Detection of bacteraemia in critically ill patients using 16S rDNA polymerase chain reaction and DNA sequencing. Intensive Care Med 2001; 27: 12691273.
  • 134
    Xu J, Moore JE, Millar BC et al. Improved laboratory diagnosis of bacterial and fungal infections in patients with hematological malignancies using PCR and ribosomal RNA sequence analysis. Leuk Lymphoma 2004; 45: 16371641.
  • 135
    Marin M, Munoz P, Sanchez M et al. Molecular diagnosis of infective endocarditis by real-time broad-range polymerase chain reaction (PCR) and sequencing directly from heart valve tissue. Medicine (Baltimore) 2007; 86: 195202.
  • 136
    Schabereiter-Gurtner C, Nehr M, Apfalter P, Makristathis A, Rotter ML, Hirschl AM. Evaluation of a protocol for molecular broad-range diagnosis of culture-negative bacterial infections in clinical routine diagnosis. J Appl Microbiol. 2008; 104: 12281237.
  • 137
    Maidak BL, Cole JR, Parker CT Jr et al. A new version of the RDP (Ribosomal Database Project). Nucleic Acids Res 1999; 27: 171173.
  • 138
    Maidak BL, Cole JR, Lilburn TG et al. The RDP (Ribosomal Database Project) continues. Nucleic Acids Res 2000; 28: 173174.
  • 139
    Maidak BL, Cole JR, Lilburn TG et al. The RDP-II (Ribosomal Database Project). Nucleic Acids Res 2001; 29: 173174.
  • 140
    Cole JR, Chai B, Farris RJ et al. The Ribosomal Database Project (RDP-II): sequences and tools for high-throughput rRNA analysis. Nucleic Acids Res 2005; 33: D294D296.
  • 141
    Cole JR, Chai B, Farris RJ et al. The ribosomal database project (RDP-II): introducing myRDP space and quality controlled public data. Nucleic Acids Res 2007; 35: D169D172.
  • 142
    Patel JB, Leonard DG, Pan X, Musser JM, Berman RE, Nachamkin I. Sequence-based identification of Mycobacterium species using the MicroSeq  500 16S rDNA bacterial identification system. J Clin Microbiol 2000; 38: 246251.
  • 143
    Tang YW, Von Graevenitz A, Waddington MG et al. Identification of coryneform bacterial isolates by ribosomal DNA sequence analysis. J Clin Microbiol 2000; 38: 16761678.
  • 144
    Harmsen D, Rothganger J, Singer C, Albert J, Frosch M. Intuitive hypertext-based molecular identification of micro-organisms. Lancet 1999; 353: 291.
  • 145
    Harmsen D, Rothganger J, Frosch M, Albert J. RIDOM: ribosomal differentiation of medical micro-organisms database. Nucleic Acids Res 2002; 30: 416417.
  • 146
    Harmsen D, Dostal S, Roth A et al. RIDOM: comprehensive and public sequence database for identification of Mycobacterium species. BMC Infect Dis 2003; 3: 26.
  • 147
    Simmon KE, Croft AC, Petti CA. Application of SmartGene IDNS software to partial 16S rRNA gene sequences for a diverse group of bacteria in a clinical laboratory. J Clin Microbiol 2006; 44: 44004406.
  • 148
    Cloud JL, Neal H, Rosenberry R et al. Identification of Mycobacterium spp. by using a commercial 16S ribosomal DNA sequencing kit and additional sequencing libraries. J Clin Microbiol 2002; 40: 400406.
  • 149
    Mellmann A, Cloud JL, Andrees S et al. Evaluation of RIDOM, MicroSeq, and Genbank services in the molecular identification of Nocardia species. Int J Med Microbiol 2003; 293: 359370.
  • 150
    Cloud JL, Conville PS, Croft A, Harmsen D, Witebsky FG, Carroll KC. Evaluation of partial 16S ribosomal DNA sequencing for identification of nocardia species by using the MicroSeq  500 system with an expanded database. J Clin Microbiol 2004; 42: 578584.
  • 151
    Becker K, Harmsen D, Mellmann A et al. Development and evaluation of a quality-controlled ribosomal sequence database for 16S ribosomal DNA-based identification of Staphylococcus species. J Clin Microbiol 2004; 42: 49884995.
  • 152
    Fontana C, Favaro M, Pelliccioni M, Pistoia ES, Favalli C. Use of the MicroSeq  500 16S rRNA gene-based sequencing for identification of bacterial isolates that commercial automated systems failed to identify correctly. J Clin Microbiol 2005; 43: 615619.
  • 153
    Janda JM, Abbott SL. 16S rRNA gene sequencing for bacterial identification in the diagnostic laboratory: pluses, perils, and pitfalls. J Clin Microbiol 2007; 45: 27612764.
  • 154
    Petti CA. Detection and identification of microorganisms by gene amplification and sequencing. Clin Infect Dis 2007; 44: 11081114.
  • 155
    Stackebrandt E, Goebel BM. Taxonomic note: a place for DNA–DNA reassociation and 16S rRNA sequence analysis in the present species definition in bacteriology. Int J Syst Bacteriol 1994; 44: 846849.
  • 156
    Palys T, Nakamura LK, Cohan FM. Discovery and classification of ecological diversity in the bacterial world: the role of DNA sequence data. Int J Syst Bacteriol 1997; 47: 11451156.
  • 157
    Murray PR, Baron EJ, Jorgensen JH, Pfaller MA, Yolken RH. Manual of clinical microbiology, 8th edn. Washington, DC: American Society for Microbiology, 2003.
  • 158
    Woo PC, Chung LM, Teng JL et al. In silico analysis of 16S ribosomal RNA gene sequencing-based methods for identification of medically important anaerobic bacteria. J Clin Pathol 2007; 60: 576579.
  • 159
    Becker K, Schumann P, Wullenweber J et al. Kytococcus schroeteri sp. nov., a novel Gram-positive actinobacterium isolated from a human clinical source. Int J Syst Evol Microbiol 2002; 52: 16091614.
  • 160
    Le Brun C, Bouet J, Gautier P, Avril JL, Gaillot O. Kytococcus schroeteri endocarditis. Emerg Infect Dis 2005; 11: 179180.
  • 161
    Mohammedi I, Berchiche C, Becker K et al. Fatal Kytococcus schroeteri bacteremic pneumonia. J Infect 2005; 51: E11E13.
  • 162
    Mnif B, Boujelbene I, Mahjoubi F et al. Endocarditis due to Kytococcus schroeteri: case report and review of the literature. J Clin Microbiol 2006; 44: 11871189.
  • 163
    Tong H, Gao X, Dong X. Streptococcus oligofermentans sp. nov., a novel oral isolate from caries-free humans. Int J Syst Evol Microbiol 2003; 53: 11011104.
  • 164
    Bekal S, Gaudreau C, Laurence RA, Simoneau E, Raynal L. Streptococcus pseudoporcinus sp. nov., a novel species isolated from the genitourinary tract of women. J Clin Microbiol 2006; 44: 25842586.
  • 165
    Hall V, Collins MD, Hutson R, Falsen E, Duerden BI. Actinomyces cardiffensis sp. nov. from human clinical sources. J Clin Microbiol 2002; 40: 34273431.
  • 166
    Westling K, Lidman C, Thalme A. Tricuspid valve endocarditis caused by a new species of actinomyces: Actinomyces funkei. Scand J Infect Dis 2002; 34: 206207.
  • 167
    Smith AJ, Hall V, Thakker B, Gemmell CG. Antimicrobial susceptibility testing of Actinomyces species with 12 antimicrobial agents. J Antimicrob Chemother 2005; 56: 407409.
  • 168
    Hoyles L, Inganas E, Falsen E et al. Bifidobacterium scardovii sp. nov., from human sources. Int J Syst Evol Microbiol 2002; 52: 995999.
  • 169
    Wauters G, Avesani V, Laffineur K et al. Brevibacterium lutescens sp. nov., from human and environmental samples. Int J Syst Evol Microbiol 2003; 53: 13211325.
  • 170
    Wauters G, Charlier J, Janssens M, Delmee M. Brevibacterium paucivorans sp. nov., from human clinical specimens. Int J Syst Evol Microbiol 2001; 51: 17031707.
  • 171
    Yassin AF, Steiner U, Ludwig W. Corynebacterium aurimucosum sp. nov. and emended description of Corynebacterium minutissimum Collins and Jones (1983). Int J Syst Evol Microbiol 2002; 52: 10011005.
  • 172
    Shukla SK, Bernard KA, Harney M, Frank DN, Reed KD. Corynebacterium nigricans sp. nov.: proposed name for a black-pigmented Corynebacterium species recovered from the human female urogenital tract. J Clin Microbiol 2003; 41: 43534358.
  • 173
    Daneshvar MI, Hollis DG, Weyant RS et al. Identification of some charcoal-black-pigmented CDC fermentative coryneform group 4 isolates as Rothia dentocariosa and some as Corynebacterium aurimucosum: proposal of Rothia dentocariosa emend. Georg and Brown 1967, Corynebacterium aurimucosum emend. Yassin et al. 2002, and Corynebacterium nigricans Shukla et al. 2003 pro synon. Corynebacterium aurimucosum. J Clin Microbiol 2004; 42: 41894198.
  • 174
    Renaud FN, Aubel D, Riegel P, Meugnier H, Bollet C. Corynebacterium freneyi sp. nov., alpha-glucosidase-positive strains related to Corynebacterium xerosis. Int J Syst Evol Microbiol 2001; 51: 17231728.
  • 175
    Auzias A, Bollet C, Ayari R, Drancourt M, Raoult D. Corynebacterium freneyi bacteremia. J Clin Microbiol 2003; 41: 27772778.
  • 176
    Otsuka Y, Kawamura Y, Koyama T, Iihara H, Ohkusu K, Ezaki T. Corynebacterium resistens sp. nov., a new multidrug-resistant coryneform bacterium isolated from human infections. J Clin Microbiol 2005; 43: 37133717.
  • 177
    Nikolaitchouk N, Wacher C, Falsen E, Andersch B, Collins MD, Lawson PA. Lactobacillus coleohominis sp. nov., isolated from human sources. Int J Syst Evol Microbiol 2001; 51: 20812085.
  • 178
    Laffineur K, Avesani V, Cornu G et al. Bacteremia due to a novel Microbacterium species in a patient with leukemia and description of Microbacterium paraoxydans sp. nov. J Clin Microbiol 2003; 41: 22422246.
  • 179
    Cloud JL, Meyer JJ, Pounder JI et al. Mycobacterium arupense sp. nov., a non-chromogenic bacterium isolated from clinical specimens. Int J Syst Evol Microbiol 2006; 56: 14131418.
  • 180
    Jimenez MS, Campos-Herrero MI, Garcia D, Luquin M, Herrera L, Garcia MJ. Mycobacterium canariasense sp. nov. Int J Syst Evol Microbiol 2004; 54: 17291734.
  • 181
    Murcia MI, Tortoli E, Menendez MC, Palenque E, Garcia MJ. Mycobacterium colombiense sp. nov., a novel member of the Mycobacterium avium complex and description of MAC-X as a new ITS genetic variant. Int J Syst Evol Microbiol 2006; 56: 20492054.
  • 182
    Tortoli E, Rindi L, Goh KS et al. Mycobacterium florentinum sp. nov., isolated from humans. Int J Syst Evol Microbiol 2005; 55: 11011106.
  • 183
    Richter E, Niemann S, Gloeckner FO, Pfyffer GE, Rusch-Gerdes S. Mycobacterium holsaticum sp. nov. Int J Syst Evol Microbiol 2002; 52: 19911996.
  • 184
    Hughes MS, Ball NW, McCarroll J et al. Molecular analyses of mycobacteria other than the M. tuberculosis complex isolated from Northern Ireland cattle. Vet Microbiol 2005; 108: 101112.
  • 185
    Band JD, Ward JI, Fraser DW et al. Peritonitis due to a Mycobacterium chelonei-like organism associated with intermittent chronic peritoneal dialysis. J Infect Dis 1982; 145: 917.
  • 186
    Wilson RW, Steingrube VA, Bottger EC et al. Mycobacterium immunogenum sp. nov., a novel species related to Mycobacterium abscessus and associated with clinical disease, pseudo-outbreaks and contaminated metalworking fluids: an international cooperative study on mycobacterial taxonomy. Int J Syst Evol Microbiol 2001; 51: 17511764.
  • 187
    Wallace RJ Jr, Zhang Y, Wilson RW, Mann L, Rossmoore H. Presence of a single genotype of the newly described species Mycobacterium immunogenum in industrial metalworking fluids associated with hypersensitivity pneumonitis. Appl Environ Microbiol 2002; 68: 55805584.
  • 188
    Beckett W, Kallay M, Sood A, Zuo Z, Milton D. Hypersensitivity pneumonitis associated with environmental mycobacteria. Environ Health Perspect 2005; 113: 767770.
  • 189
    Loots MA, De Jong MD, Van Soolingen D, Wetsteyn JC, Faber WR. Chronic leg ulcer caused by Mycobacterium immunogenum. J Travel Med 2005; 12: 347349.
  • 190
    Sampaio JL, Junior DN, De Freitas D et al. An outbreak of keratitis caused by Mycobacterium immunogenum. J Clin Microbiol 2006; 44: 32013207.
  • 191
    Adekambi T, Reynaud-Gaubert M, Greub G et al. Amoebal coculture of ‘Mycobacterium massiliense’ sp. nov. from the sputum of a patient with hemoptoic pneumonia. J Clin Microbiol 2004; 42: 54935501.
  • 192
    Kim HY, Yun YJ, Park CG et al. Outbreak of Mycobacterium massiliense infection associated with intramuscular injections. J Clin Microbiol 2007; 45: 31273130.
  • 193
    Simmon KE, Pounder JI, Greene JN et al. Identification of an emerging pathogen, Mycobacterium massiliense, by rpoB sequencing of clinical isolates collected in the United States. J Clin Microbiol 2007; 45: 19781980.
  • 194
    Reischl U, Melzl H, Kroppenstedt RM et al. Mycobacterium monacense sp. nov. Int J Syst Evol Microbiol 2006; 56: 25752578.
  • 195
    Mohamed AM, Iwen PC, Tarantolo S, Hinrichs SH. Mycobacterium nebraskense sp. nov., a novel slowly growing scotochromogenic species. Int J Syst Evol Microbiol 2004; 54: 20572060.
  • 196
    Iwen PC, Tarantolo SR, Mohamed AM, Hinrichs SH. First report of Mycobacterium nebraskense as a cause of human infection. Diagn Microbiol Infect Dis 2006; 56: 451453.
  • 197
    Torkko P, Suomalainen S, Iivanainen E et al. Mycobacterium palustre sp. nov., a potentially pathogenic, slowly growing mycobacterium isolated from clinical and veterinary specimens and from Finnish stream waters. Int J Syst Evol Microbiol 2002; 52: 15191525.
  • 198
    Turenne CY, Thibert L, Williams K et al. Mycobacterium saskatchewanense sp. nov., a novel slowly growing scotochromogenic species from human clinical isolates related to Mycobacterium interjectum and Accuprobe-positive for Mycobacterium avium complex. Int J Syst Evol Microbiol 2004; 54: 659667.
  • 199
    Hamid ME, Maldonado L, Sharaf Eldin GS, Mohamed MF, Saeed NS, Goodfellow M. Nocardia africana sp. nov., a new pathogen isolated from patients with pulmonary infections. J Clin Microbiol 2001; 39: 625630.
  • 200
    Hattori Y, Kano R, Kunitani Y, Yanai T, Hasegawa A. Nocardia africana isolated from a feline mycetoma. J Clin Microbiol 2003; 41: 908910.
  • 201
    Kageyama A, Suzuki S, Yazawa K, Nishimura K, Kroppenstedt RM, Mikami Y. Nocardia aobensis sp. nov., isolated from patients in Japan. Microbiol Immunol 2004; 48: 817822.
  • 202
    Kageyama A, Torikoe K, Iwamoto M et al. Nocardia arthritidis sp. nov., a new pathogen isolated from a patient with rheumatoid arthritis in Japan. J Clin Microbiol 2004; 42: 23662371.
  • 203
    Yin X, Liang S, Sun X, Luo S, Wang Z, Li R. Ocular nocardiosis: HSP65 gene sequencing for species identification of Nocardia spp. Am J Ophthalmol 2007; 144: 570573.
  • 204
    Kageyama A, Poonwan N, Yazawa K, Mikami Y, Nishimura K. Nocardia asiatica sp. nov., isolated from patients with nocardiosis in Japan and clinical specimens from Thailand. Int J Syst Evol Microbiol 2004; 54: 125130.
  • 205
    Iona E, Giannoni F, Brunori L, De Gennaro M, Mattei R, Fattorini L. Isolation of Nocardia asiatica from cutaneous ulcers of a human immunodeficiency virus-infected patient in Italy. J Clin Microbiol 2007; 45: 20882089.
  • 206
    Yassin AF, Rainey FA, Steiner U. Nocardia ignorata sp. nov. Int J Syst Evol Microbiol 2001; 51: 21272131.
  • 207
    Kageyama A, Yazawa K, Ishikawa J, Hotta K, Nishimura K, Mikami Y. Nocardial infections in Japan from 1992 to 2001, including the first report of infection by Nocardia transvalensis. Eur J Epidemiol 2004; 19: 383389.
  • 208
    Rodriguez-Nava V, Couble A, Khan ZU et al. Nocardia ignorata, a new agent of human nocardiosis isolated from respiratory specimens in Europe and soil samples from Kuwait. J Clin Microbiol 2005; 43: 61676170.
  • 209
    Gurtler V, Smith R, Mayall BC, Potter-Reinemann G, Stackebrandt E, Kroppenstedt RM. Nocardia veterana sp. nov., isolated from human bronchial lavage. Int J Syst Evol Microbiol 2001; 51: 933936.
  • 210
    Kano R, Hattori Y, Murakami N et al. The first isolation of Nocardia veterana from a human mycetoma. Microbiol Immunol 2002; 46: 409412.
  • 211
    Pottumarthy S, Limaye AP, Prentice JL, Houze YB, Swanzy SR, Cookson BT. Nocardia veterana, a new emerging pathogen. J Clin Microbiol 2003; 41: 17051709.
  • 212
    Conville PS, Brown JM, Steigerwalt AG et al. Nocardia veterana as a pathogen in North American patients. J Clin Microbiol 2003; 41: 25602568.
  • 213
    Godreuil S, Didelot MN, Perez C et al. Nocardia veterana isolated from ascitic fluid of a patient with human immunodeficiency virus infection. J Clin Microbiol 2003; 41: 27682773.
  • 214
    Ansari SR, Safdar A, Han XY, O’Brien S. Nocardia veterana bloodstream infection in a patient with cancer and a summary of reported cases. Int J Infect Dis 2006; 10: 483486.
  • 215
    Agterof MJ, Van Der Bruggen T, Tersmette M, Ter Borg EJ, Van Den Bosch JM, Biesma DH. Nocardiosis: a case series and a mini review of clinical and microbiological features. Neth J Med 2007; 65: 199202.
  • 216
    Dewhirst FE, Paster BJ, Tzellas N et al. Characterization of novel human oral isolates and cloned 16S rDNA sequences that fall in the family Coriobacteriaceae: description of Olsenella gen. nov., reclassification of Lactobacillus uli as Olsenella uli comb. nov. and description of Olsenella profusa sp. nov. Int J Syst Evol Microbiol 2001; 51: 17971804.
  • 217
    Munson MA, Banerjee A, Watson TF, Wade WG. Molecular analysis of the microflora associated with dental caries. J Clin Microbiol 2004; 42: 30233029.
  • 218
    Rocas IN, Siqueira JF Jr. Species-directed 16S rRNA gene nested PCR detection of Olsenella species in association with endodontic diseases. Lett Appl Microbiol 2005; 41: 1216.
  • 219
    Bjorkroth KJ, Schillinger U, Geisen R et al. Taxonomic study of Weissella confusa and description of Weissella cibaria sp. nov., detected in food and clinical samples. Int J Syst Evol Microbiol 2002; 52: 141148.
  • 220
    Choi HJ, Cheigh CI, Kim SB et al. Weissella kimchii sp. nov., a novel lactic acid bacterium from kimchi. Int J Syst Evol Microbiol 2002; 52: 507511.
  • 221
    De Vuyst L, Schrijvers V, Paramithiotis S et al. The biodiversity of lactic acid bacteria in Greek traditional wheat sourdoughs is reflected in both composition and metabolite formation. Appl Environ Microbiol 2002; 68: 60596069.
  • 222
    Ennahar S, Cai Y, Fujita Y. Phylogenetic diversity of lactic acid bacteria associated with paddy rice silage as determined by 16S ribosomal DNA analysis. Appl Environ Microbiol 2003; 69: 444451.
  • 223
    Santos EM, Jaime I, Rovira J, Lyhs U, Korkeala H, Bjorkroth J. Characterization and identification of lactic acid bacteria in ‘morcilla de Burgos’. Int J Food Microbiol 2005; 97: 285296.
  • 224
    Graef EM, Devriese LA, Baele M et al. Identification of enterococcal, streptococcal and Weissella species in the faecal flora of individually owned dogs. J Appl Microbiol 2005; 99: 348353.
  • 225
    Srionnual S, Yanagida F, Lin LH, Hsiao KN, Chen YS. Weissellicin  110, a newly discovered bacteriocin from Weissella cibaria 110, isolated from plaa-som, a fermented fish product from Thailand. Appl Environ Microbiol 2007; 73: 22472250.
  • 226
    Coenye T, Vancanneyt M, Falsen E, Swings J, Vandamme P. Achromobacter insolitus sp. nov. and Achromobacter spanius sp. nov., from human clinical samples. Int J Syst Evol Microbiol 2003; 53: 18191824.
  • 227
    Coenye T, Vancanneyt M, Cnockaert MC, Falsen E, Swings J, Vandamme P. Kerstersia gyiorum gen. nov., sp. nov., a novel Alcaligenes faecalis-like organism isolated from human clinical samples, and reclassification of Alcaligenes denitrificans Ruger and Tan 1983 as Achromobacter denitrificans comb. nov. Int J Syst Evol Microbiol 2003; 53: 18251831.
  • 228
    Nemec A, Dijkshoorn L, Cleenwerck I et al. Acinetobacter parvus sp. nov., a small-colony-forming species isolated from human clinical specimens. Int J Syst Evol Microbiol 2003; 53: 15631567.
  • 229
    Nemec A, De Baere T, Tjernberg I, Vaneechoutte M, Van Der Reijden TJ, Dijkshoorn L. Acinetobacter ursingii sp. nov. and Acinetobacter schindleri sp. nov., isolated from human clinical specimens. Int J Syst Evol Microbiol 2001; 51: 18911899.
  • 230
    Dortet L, Legrand P, Soussy CJ, Cattoir V. Bacterial identification, clinical significance, and antimicrobial susceptibilities of Acinetobacter ursingii and Acinetobacter schindleri, two frequently misidentified opportunistic pathogens. J Clin Microbiol 2006; 44: 44714478.
  • 231
    Loubinoux J, Mihaila-Amrouche L, Le Fleche A et al. Bacteremia caused by Acinetobacter ursingii. J Clin Microbiol 2003; 41: 13371338.
  • 232
    Coenye T, Vanlaere E, Samyn E, Falsen E, Larsson P, Vandamme P. Advenella incenata gen. nov., sp. nov., a novel member of the Alcaligenaceae, isolated from various clinical samples. Int J Syst Evol Microbiol 2005; 55: 251256.
  • 233
    Johnson AS, Tarr CL, Brown BH Jr, Birkhead KM, Farmer JJ 3rd. First case of septicemia due to a strain belonging to enteric group 58 (Enterobacteriaceae) and its designation as Averyella dalhousiensis gen. nov., sp. nov., based on analysis of strains from 20 additional cases. J Clin Microbiol 2005; 43: 51955201.
  • 234
    Coenye T, Mahenthiralingam E, Henry D et al. Burkholderia ambifaria sp. nov., a novel member of the Burkholderia cepacia complex including biocontrol and cystic fibrosis-related isolates. Int J Syst Evol Microbiol 2001; 51: 14811490.
  • 235
    Matoba AY. Polymicrobial keratitis secondary to Burkholderia ambifaria, Enterococcus, and Staphylococcus aureus in a patient with herpetic stromal keratitis. Am J Ophthalmol 2003; 136: 748749.
  • 236
    Coenye T, Laevens S, Willems A et al. Burkholderia fungorum sp. nov. and Burkholderia caledonica sp. nov., two new species isolated from the environment, animals and human clinical samples. Int J Syst Evol Microbiol 2001; 51: 10991107.
  • 237
    Gerrits GP, Klaassen C, Coenye T, Vandamme P, Meis JF. Burkholderia fungorum septicemia. Emerg Infect Dis 2005; 11: 11151117.
  • 238
    Lawson AJ, Linton D, Stanley J. 16S rRNA gene sequences of ‘Candidatus Campylobacter hominis’, a novel uncultivated species, are found in the gastrointestinal tract of healthy humans. Microbiology 1998; 144 (Pt 8): 20632071.
  • 239
    Lawson AJ, On SL, Logan JM, Stanley J. Campylobacter hominis sp. nov., from the human gastrointestinal tract. Int J Syst Evol Microbiol 2001; 51: 651660.
  • 240
    Paster BJ, Falkler WA Jr, Enwonwu CO et al. Prevalent bacterial species and novel phylotypes in advanced noma lesions. J Clin Microbiol 2002; 40: 21872191.
  • 241
    Han XY, Meltzer MC, Woods JT, Fainstein V. Endocarditis with ruptured cerebral aneurysm caused by Cardiobacterium valvarum sp. nov. J Clin Microbiol 2004; 42: 15901595.
  • 242
    Hoover SE, Fischer SH, Shaffer R, Steinberg BM, Lucey DR. Endocarditis due to a novel Cardiobacterium species. Ann Intern Med 2005; 142: 229230.
  • 243
    Bothelo E, Gouriet F, Fournier PE et al. Endocarditis caused by Cardiobacterium valvarum. J Clin Microbiol 2006; 44: 657658.
  • 244
    Geissdorfer W, Tandler R, Schlundt C, Weyand M, Daniel WG, Schoerner C. Fatal bioprosthetic aortic valve endocarditis due to Cardiobacterium valvarum. J Clin Microbiol 2007; 45: 23242326.
  • 245
    Coenye T, Vandamme P, LiPuma JJ. Ralstonia respiraculi sp. nov., isolated from the respiratory tract of cystic fibrosis patients. Int J Syst Evol Microbiol 2003; 53: 13391342.
  • 246
    Vaneechoutte M, Kampfer P, De Baere T, Falsen E, Verschraegen G. Wautersia gen. nov., a novel genus accommodating the phylogenetic lineage including Ralstonia eutropha and related species, and proposal of Ralstonia [Pseudomonas] syzygii (Roberts et al. 1990) comb. nov. Int J Syst Evol Microbiol 2004; 54: 317327.
  • 247
    Vandamme P, Coenye T. Taxonomy of the genus Cupriavidus: a tale of lost and found. Int J Syst Evol Microbiol 2004; 54: 22852289.
  • 248
    Hoffmann H, Stindl S, Stumpf A et al. Description of Enterobacter ludwigii sp. nov., a novel Enterobacter species of clinical relevance. Syst Appl Microbiol 2005; 28: 206212.
  • 249
    Huys G, Cnockaert M, Janda JM, Swings J. Escherichia albertii sp. nov., a diarrhoeagenic species isolated from stool specimens of Bangladeshi children. Int J Syst Evol Microbiol 2003; 53: 807810.
  • 250
    Greenberg DE, Porcella SF, Stock F et al. Granulibacter bethesdensis gen. nov., sp. nov., a distinctive pathogenic acetic acid bacterium in the family Acetobacteraceae. Int J Syst Evol Microbiol 2006; 56: 26092616.
  • 251
    Greenberg DE, Porcella SF, Zelazny AM et al. Genome sequence analysis of the emerging human pathogenic acetic acid bacterium Granulibacter bethesdensis. J Bacteriol 2007; 189: 87278736.
  • 252
    Greub G, Raoult D. Rhodobacter massiliensis sp. nov., a new amoebae-resistant species isolated from the nose of a patient. Res Microbiol 2003; 154: 631635.
  • 253
    Helsel LO, Hollis D, Steigerwalt AG et al. Identification of ‘Haematobacter’, a new genus of aerobic Gram-negative rods isolated from clinical specimens, and reclassification of Rhodobacter massiliensis as ‘Haematobacter massiliensis comb. nov.’. J Clin Microbiol 2007; 45: 12381243.
  • 254
    Berger P, Barguellil F, Raoult D, Drancourt M. An outbreak of Halomonas phocaeensis sp. nov. bacteraemia in a neonatal intensive care unit. J Hosp Infect 2007; 67: 7985.
  • 255
    Melito PL, Munro C, Chipman PR, Woodward DL, Booth TF, Rodgers FG. Helicobacter winghamensis sp. nov., a novel Helicobacter sp. isolated from patients with gastroenteritis. J Clin Microbiol 2001; 39: 24122417.
  • 256
    Goto K, Jiang W, Zheng Q et al. Epidemiology of Helicobacter infection in wild rodents in the Xinjiang-Uygur autonomous region of China. Curr Microbiol 2004; 49: 221223.
  • 257
    Coenye T, Goris J, Spilker T, Vandamme P, LiPuma JJ. Characterization of unusual bacteria isolated from respiratory secretions of cystic fibrosis patients and description of Inquilinus limosus gen. nov., sp. nov. J Clin Microbiol 2002; 40: 20622069.
  • 258
    Wellinghausen N, Essig A, Sommerburg O. Inquilinus limosus in patients with cystic fibrosis, Germany. Emerg Infect Dis 2005; 11: 457459.
  • 259
    Schmoldt S, Latzin P, Heesemann J, Griese M, Imhof A, Hogardt M. Clonal analysis of Inquilinus limosus isolates from six cystic fibrosis patients and specific serum antibody response. J Med Microbiol 2006; 55: 14251433.
  • 260
    Chowdhury SP, Schmid M, Hartmann A, Tripathi AK. Identification of diazotrophs in the culturable bacterial community associated with roots of Lasiurus sindicus, a perennial grass of Thar Desert, India. Microb Ecol 2007; 54: 8290.
  • 261
    Herasimenka Y, Cescutti P, Impallomeni G, Rizzo R. Exopolysaccharides produced by Inquilinus limosus, a new pathogen of cystic fibrosis patients: novel structures with usual components. Carbohydr Res 2007; 342: 24042415.
  • 262
    Lau SK, Ho PL, Li MW et al. Cloning and characterization of a chromosomal class C beta-lactamase and its regulatory gene in Laribacter hongkongensis. Antimicrob Agents Chemother 2005; 49: 19571964.
  • 263
    Woo PC, Ma SS, Teng JL et al. Construction of an inducible expression shuttle vector for Laribacter hongkongensis, a novel bacterium associated with gastroenteritis. FEMS Microbiol Lett 2005; 252: 5765.
  • 264
    Woo PC, Ma SS, Teng JL, Li MW, Lau SK, Yuen KY. Plasmid profile and construction of a small shuttle vector in Laribacter hongkongensis. Biotechnol Lett 2007; 29: 15751582.
  • 265
    Woo PC, Teng JL, Ma SS, Lau SK, Yuen KY. Characterization of a novel cryptic plasmid, pHLHK26, in Laribacter hongkongensis. New Microbiol 2007; 30: 139147.
  • 266
    Han XY, Hong T, Falsen E. Neisseria bacilliformis sp. nov. isolated from human infections. J Clin Microbiol 2006; 44: 474479.
  • 267
    Daneshvar MI, Hollis DG, Weyant RS et al. Paracoccus yeeii sp. nov. (formerly CDC group EO-2), a novel bacterial species associated with human infection. J Clin Microbiol 2003; 41: 12891294.
  • 268
    Dabboussi F, Hamze M, Singer E, Geoffroy V, Meyer JM, Izard D. Pseudomonas mosselii sp. nov., a novel species isolated from clinical specimens. Int J Syst Evol Microbiol 2002; 52: 363376.
  • 269
    Meyer JM, Geoffroy VA, Baida N et al. Siderophore typing, a powerful tool for the identification of fluorescent and nonfluorescent pseudomonads. Appl Environ Microbiol 2002; 68: 27452753.
  • 270
    Clark LL, Dajcs JJ, McLean CH, Bartell JG, Stroman DW. Pseudomonas otitidis sp. nov., isolated from patients with otic infections. Int J Syst Evol Microbiol 2006; 56: 709714.
  • 271
    Chen WM, Laevens S, Lee TM et al. Ralstonia taiwanensis sp. nov., isolated from root nodules of Mimosa species and sputum of a cystic fibrosis patient. Int J Syst Evol Microbiol 2001; 51: 17291735.
  • 272
    Kampfer P, Avesani V, Janssens M, Charlier J, De Baere T, Vaneechoutte M. Description of Wautersiella falsenii gen. nov., sp. nov., to accommodate clinical isolates phenotypically resembling members of the genera Chryseobacterium and Empedobacter. Int J Syst Evol Microbiol 2006; 56: 23232329.
  • 273
    Song Y, Liu C, Finegold SM. Peptoniphilus gorbachii sp. nov., Peptoniphilus olsenii sp. nov., and Anaerococcus murdochii sp. nov. isolated from clinical specimens of human origin. J Clin Microbiol 2007; 45: 17461752.
  • 274
    Downes J, Wade WG. Peptostreptococcus stomatis sp. nov., isolated from the human oral cavity. Int J Syst Evol Microbiol 2006; 56: 751754.
  • 275
    Kononen E, Bryk A, Niemi P, Kanervo-Nordstrom A. Antimicrobial susceptibilities of Peptostreptococcus anaerobius and the newly described Peptostreptococcus stomatis isolated from various human sources. Antimicrob Agents Chemother 2007; 51: 22052207.
  • 276
    Huys G, Vancanneyt M, D’Haene K, Falsen E, Wauters G, Vandamme P. Alloscardovia omnicolens gen. nov., sp. nov., from human clinical samples. Int J Syst Evol Microbiol 2007; 57: 14421446.
  • 277
    Schwiertz A, Hold GL, Duncan SH et al. Anaerostipes caccae gen. nov., sp. nov., a new saccharolytic, acetate-utilising, butyrate-producing bacterium from human faeces. Syst Appl Microbiol 2002; 25: 4651.
  • 278
    Duncan SH, Louis P, Flint HJ. Lactate-utilizing bacteria, isolated from human feces, that produce butyrate as a major fermentation product. Appl Environ Microbiol 2004; 70: 58105817.
  • 279
    Song Y, Liu C, Molitoris DR et al. Clostridium bolteae sp. nov., isolated from human sources. Syst Appl Microbiol 2003; 26: 8489.
  • 280
    Ballard SA, Grabsch EA, Johnson PD, Grayson ML. Comparison of three PCR primer sets for identification of vanB gene carriage in feces and correlation with carriage of vancomycin-resistant enterococci: interference by vanB-containing anaerobic bacilli. Antimicrob Agents Chemother 2005; 49: 7781.
  • 281
    Finegold SM, Song Y, Liu C et al. Clostridium clostridioforme: a mixture of three clinically important species. Eur J Clin Microbiol Infect Dis 2005; 24: 319324.
  • 282
    Kitahara M, Takamine F, Imamura T, Benno Y. Clostridium hiranonis sp. nov., a human intestinal bacterium with bile acid 7alpha-dehydroxylating activity. Int J Syst Evol Microbiol 2001; 51: 3944.
  • 283
    Kitahara M, Sakamoto M, Benno Y. PCR detection method of Clostridium scindens and C. hiranonis in human fecal samples. Microbiol Immunol 2001; 45: 263266.
  • 284
    Korenek NL, Andrews FM, Maddux JM, Sanders WL, Faulk DL. Determination of total protein concentration and viscosity of synovial fluid from the tibiotarsal joints of horses. Am J Vet Res 1992; 53: 781784.
  • 285
    Roos S, Engstrand L, Jonsson H. Lactobacillus gastricus sp. nov., Lactobacillus antri sp. nov., Lactobacillus kalixensis sp. nov. and Lactobacillus ultunensis sp. nov., isolated from human stomach mucosa. Int J Syst Evol Microbiol 2005; 55: 7782.
  • 286
    De Angelis M, Siragusa S, Berloco M et al. Selection of potential probiotic lactobacilli from pig feces to be used as additives in pelleted feeding. Res Microbiol 2006; 157: 792801.
  • 287
    Duncan SH, Hold GL, Barcenilla A, Stewart CS, Flint HJ. Roseburia intestinalis sp. nov., a novel saccharolytic, butyrate-producing bacterium from human faeces. Int J Syst Evol Microbiol 2002; 52: 16151620.
  • 288
    Downes J, Munson MA, Radford DR, Spratt DA, Wade WG. Shuttleworthia satelles gen. nov., sp. nov., isolated from the human oral cavity. Int J Syst Evol Microbiol 2002; 52: 14691475.
  • 289
    Hall V, Collins MD, Lawson PA et al. Characterization of some actinomyces-like isolates from human clinical sources: description of Varibaculum cambriensis gen nov, sp nov. J Clin Microbiol 2003; 41: 640644.
  • 290
    Jumas-Bilak E, Carlier JP, Jean-Pierre H et al. Veillonella montpellierensis sp. nov., a novel, anaerobic, Gram-negative coccus isolated from human clinical samples. Int J Syst Evol Microbiol 2004; 54: 13111316.
  • 291
    Rovery C, Etienne A, Foucault C, Berger P, Brouqui P. Veillonella montpellierensis endocarditis. Emerg Infect Dis 2005; 11: 11121114.
  • 292
    Song Y, Kononen E, Rautio M et al. Alistipes onderdonkii sp. nov. and Alistipes shahii sp. nov., of human origin. Int J Syst Evol Microbiol 2006; 56: 19851990.
  • 293
    Bakir MA, Kitahara M, Sakamoto M, Matsumoto M, Benno Y. Bacteroides intestinalis sp. nov., isolated from human faeces. Int J Syst Evol Microbiol 2006; 56: 151154.
  • 294
    Kitahara M, Sakamoto M, Ike M, Sakata S, Benno Y. Bacteroides plebeius sp. nov. and Bacteroides coprocola sp. nov., isolated from human faeces. Int J Syst Evol Microbiol 2005; 55: 21432147.
  • 295
    Finegold SM, Vaisanen ML, Molitoris DR et al. Cetobacterium somerae sp. nov. from human feces and emended description of the genus Cetobacterium. Syst Appl Microbiol 2003; 26: 177181.
  • 296
    Langendijk PS, Kulik EM, Sandmeier H, Meyer J, Van Der Hoeven JS. Isolation of Desulfomicrobium orale sp. nov. and Desulfovibrio strain NY682, oral sulfate-reducing bacteria involved in human periodontal disease. Int J Syst Evol Microbiol 2001; 51: 10351044.
  • 297
    Downes J, Munson M, Wade WG. Dialister invisus sp. nov., isolated from the human oral cavity. Int J Syst Evol Microbiol 2003; 53: 19371940.
  • 298
    Rocas IN, Siqueira JF Jr. Detection of novel oral species and phylotypes in symptomatic endodontic infections including abscesses. FEMS Microbiol Lett 2005; 250: 279285.
  • 299
    Rocas IN, Baumgartner JC, Xia T, Siqueira JF Jr. Prevalence of selected bacterial named species and uncultivated phylotypes in endodontic abscesses from two geographic locations. J Endod 2006; 32: 11351138.
  • 300
    Jumas-Bilak E, Jean-Pierre H, Carlier JP et al. Dialister micraerophilus sp. nov. and Dialister propionicifaciens sp. nov., isolated from human clinical samples. Int J Syst Evol Microbiol 2005; 55: 24712478.
  • 301
    Morio F, Jean-Pierre H, Dubreuil L et al. Antimicrobial susceptibilities and clinical sources of Dialister species. Antimicrob Agents Chemother 2007; 51: 44984501.
  • 302
    Lawson PA, Falsen E, Inganas E, Weyant RS, Collins MD. Dysgonomonas mossii sp. nov., from human sources. Syst Appl Microbiol 2002; 25: 194197.
  • 303
    Matsumoto T, Kawakami Y, Oana K et al. First isolation of Dysgonomonas mossii from intestinal juice of a patient with pancreatic cancer. Arch Med Res 2006; 37: 914916.
  • 304
    Shukla SK, Meier PR, Mitchell PD, Frank DN, Reed KD. Leptotrichia amnionii sp. nov., a novel bacterium isolated from the amniotic fluid of a woman after intrauterine fetal demise. J Clin Microbiol 2002; 40: 33463349.
  • 305
    Goto M, Hitomi S, Ishii T. Bacterial arthritis caused by Leptotrichia amnionii. J Clin Microbiol 2007; 45: 20822083.
  • 306
    Boennelycke M, Christensen JJ, Arpi M, Krause S. Leptotrichia amnionii found in septic abortion in Denmark. Scand J Infect Dis 2007; 39: 382383.
  • 307
    Thilesen CM, Nicolaidis M, Lokebo JE, Falsen E, Jorde AT, Muller F. Leptotrichia amnionii, an emerging pathogen of the female urogenital tract. J Clin Microbiol 2007; 45: 23442347.
  • 308
    Song Y, Liu C, Lee J, Bolanos M, Vaisanen ML, Finegold SM. Bacteroides goldsteinii sp. nov.’ isolated from clinical specimens of human intestinal origin. J Clin Microbiol 2005; 43: 45224527.
  • 309
    Sakamoto M, Benno Y. Reclassification of Bacteroides distasonis, Bacteroides goldsteinii and Bacteroides merdae as Parabacteroides distasonis gen. nov., comb. nov., Parabacteroides goldsteinii comb. nov. and Parabacteroides merdae comb. nov. Int J Syst Evol Microbiol 2006; 56: 15991605.
  • 310
    Summanen PH, Durmaz B, Vaisanen ML et al. Porphyromonas somerae sp. nov., a pathogen isolated from humans and distinct from Porphyromonas levii. J Clin Microbiol 2005; 43: 44554459.
  • 311
    Finegold SM, Vaisanen ML, Rautio M et al. Porphyromonas uenonis sp. nov., a pathogen for humans distinct from P. asaccharolytica and P. endodontalis. J Clin Microbiol 2004; 42: 52985301.
  • 312
    Downes J, Sutcliffe I, Tanner AC, Wade WG. Prevotella marshii sp. nov. and Prevotella baroniae sp. nov., isolated from the human oral cavity. Int J Syst Evol Microbiol 2005; 55: 15511555.
  • 313
    Downes J, Sutcliffe IC, Hofstad T, Wade WG. Prevotella bergensis sp. nov., isolated from human infections. Int J Syst Evol Microbiol 2006; 56: 609612.
  • 314
    Hayashi H, Shibata K, Sakamoto M, Tomita S, Benno Y. Prevotella copri sp. nov. and Prevotella stercorea sp. nov., isolated from human faeces. Int J Syst Evol Microbiol 2007; 57: 941946.
  • 315
    Sakamoto M, Huang Y, Umeda M, Ishikawa I, Benno Y. Prevotella multiformis sp. nov., isolated from human subgingival plaque. Int J Syst Evol Microbiol 2005; 55: 815819.
  • 316
    Sakamoto M, Umeda M, Ishikawa I, Benno Y. Prevotella multisaccharivorax sp. nov., isolated from human subgingival plaque. Int J Syst Evol Microbiol 2005; 55: 18391843.
  • 317
    Collins MD, Hoyles L, Tornqvist E, Von Essen R, Falsen E. Characterization of some strains from human clinical sources which resemble ‘Leptotrichia sanguinegens’: description of Sneathia sanguinegens sp. nov., gen. nov. Syst Appl Microbiol 2001; 24: 358361.
  • 318
    De Martino SJ, Mahoudeau I, Brettes JP, Piemont Y, Monteil H, Jaulhac B. Peripartum bacteremias due to Leptotrichia amnionii and Sneathia sanguinegens, rare causes of fever during and after delivery. J Clin Microbiol 2004; 42: 59405943.
  • 319
    Wyss C, Dewhirst FE, Gmur R et al. Treponema parvum sp. nov., a small, glucoronic or galacturonic acid-dependent oral spirochaete from lesions of human periodontitis and acute necrotizing ulcerative gingivitis. Int J Syst Evol Microbiol 2001; 51: 955962.
  • 320
    Blackwood KS, Turenne CY, Harmsen D, Kabani AM. Reassessment of sequence-based targets for identification of bacillus species. J Clin Microbiol 2004; 42: 16261630.
  • 321
    Turenne CY, Tschetter L, Wolfe J, Kabani A. Necessity of quality-controlled 16S rRNA gene sequence databases: identifying nontuberculous Mycobacterium species. J Clin Microbiol 2001; 39: 36373648.
  • 322
    Patel JB, Wallace RJ Jr, Brown-Elliott BA et al. Sequence-based identification of aerobic actinomycetes. J Clin Microbiol 2004; 42: 25302540.